**Fatty Acids and Membrane Lipidomics in Oncology: A Cross-Road of Nutritional, Signaling and Metabolic Pathways**

#### **Carla Ferreri 1,\*, Anna Sansone 1, Rosaria Ferreri 2, Javier Amézaga <sup>3</sup> and Itziar Tueros <sup>3</sup>**


Received: 1 August 2020; Accepted: 23 August 2020; Published: 25 August 2020

**Abstract:** Fatty acids are closely involved in lipid synthesis and metabolism in cancer. Their amount and composition are dependent on dietary supply and tumor microenviroment. Research in this subject highlighted the crucial event of membrane formation, which is regulated by the fatty acids' molecular properties. The growing understanding of the pathways that create the fatty acid pool needed for cell replication is the result of lipidomics studies, also envisaging novel fatty acid biosynthesis and fatty acid-mediated signaling. Fatty acid-driven mechanisms and biological effects in cancer onset, growth and metastasis have been elucidated, recognizing the importance of polyunsaturated molecules and the balance between omega-6 and omega-3 families. Saturated and monounsaturated fatty acids are biomarkers in several types of cancer, and their characterization in cell membranes and exosomes is under development for diagnostic purposes. Desaturase enzymatic activity with unprecedented *de novo* polyunsaturated fatty acid (PUFA) synthesis is considered the recent breakthrough in this scenario. Together with the link between obesity and cancer, fatty acids open interesting perspectives for biomarker discovery and nutritional strategies to control cancer, also in combination with therapies. All these subjects are described using an integrated approach taking into account biochemical, biological and analytical aspects, delineating innovations in cancer prevention, diagnostics and treatments.

**Keywords:** cancer cell membranes; fatty acid biosynthesis; essential fatty acids; desaturase enzymes; fatty acid signaling; fatty acid biomarker; sapienic acid; sebaleic acid; molecular nutrition; inflammation

#### **1. Introduction**

The development of lipid research in the last two decades has brought a fundamental contribution to the understanding of the main processes for cellular life, in all types of organisms as well as in plants [1]. In particular, fatty acids are the building blocks of the large majority of lipid structures, differentiated from lipids that have steroid and isoprenoid scaffolds. Fatty acids are known for their multiple roles, ranging from energy providers and gene regulators to precursors of signaling molecules and other important metabolites, but it is worth noting that fatty acids in phospholipids have specific structural and functional roles in order to create the envelope of all types of cells, i.e., the cell membrane [2]. In eukaryotes, fatty acids display structural diversity and, as represented in Figure 1 with the most important molecules for the organization of membrane phospholipids, are characterized by specific chain length and number of unsaturations. First of all, the length of the hydrocarbon

(hydrophobic) chains requires a certain number of carbon atoms (most often 16–22 carbon atoms) to create the membrane compartment and the thickness of the lipid bilayers. Biosynthesis is initiated with the formation of 16 carbon atoms containing palmitic acid, the first endogenous lipid which is a saturated fatty acid (SFA) (Figure 1) made by the enzymatic system of fatty acid synthase (FAS). Together with the chain length, another structural requirement present in unsaturated fatty acids is the geometry of cis double bonds. The enzymatic system of desaturases introduces the unsaturation in a precise position of the fatty chain (indicated with the carbon atom number; see Figure 1) and this creates a bend (angle of ca. 30 degrees), modifying profoundly the biophysical properties of the molecules [3]. The main endogenous formation of double bonds is due to delta-9 (Δ9) desaturase (also known as stearoyl CoA desaturase SCD-1) operating on palmitic and stearic acids, as shown in Figure 2A.

**Figure 1.** The fatty acid constituents of phospholipids: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) are shown with their most present structures in eukaryotic membranes.

On the other hand, the polyunsaturated fatty acid (PUFA) structures are necessary to eukaryotic cells but are not biosynthesized *de novo*, and the precursors of the omega-6 and omega-3 families must be taken from the diet. The structures of the omega-6 and omega-3 precursors are shown in Figure 1 (linoleic acid and α-linolenic acid, respectively) and, after their uptake, other PUFAs are formed and enter into the membrane composition, as shown in Figure 1. In Figure 2B, the two pathways followed for long-chain PUFA biosynthesis are shown, with formation of omega-6 di-homo-gamma linolenic (DGLA) and arachidonic (ARA) acids and omega-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. As a matter of fact, the transformation to mono- and polyunsaturated fatty acids (MUFA and PUFA) provides the precious building blocks of membrane phospholipids involved in the regulation of permeability and fluidity properties. MUFAs and PUFAs act in a manner opposite to SFA, which instead create the rigidity and the gel status of the lipid bilayer. The role of fats in cancer is generally recognized [4], and the SFA-MUFA pathway has been studied since it is one of the pieces of the puzzling scenario for tumoral cell development and invasion [5]. However, considering that the membrane is necessary for cell formation and reproduction, the ways in which the balance among SFAs, MUFAs and PUFAs influences these steps are still to be defined. MUFAs can be obtained totally by an endogenous process, whereas PUFAs cannot be biosynthesized in humans, as shown in Figure 2B. Due to this "dietary dependency", the effects of an impairment of both exogenous supply and endogenous metabolism needs a comprehensive approach in order to examine cellular metabolism, signaling and nutrition. This is why fatty acid-based membrane lipidomics drives important information in health and diseases and, in particular in cancer "-omics", it is needed for the comprehension of molecular mechanisms and for biomarker discovery.

**Figure 2.** Some metabolic transformations of fatty acids: (**A**) the saturated fatty acid (SFA), palmitic acid, is transformed into stearic acid and the monounsaturated fatty acid (MUFA), oleic acid; (**B**) omega-6 and omega-3 precursors taken from the diet are transformed into the other polyunsaturated fatty acids (PUFA) members of the two families.

Here we wish to remark that a multidisciplinary approach is necessary, where chemical, biological and clinical skills are required all at once. Indeed, in membrane lipids research and medical applications, all these skills are also necessary to address critical issues in protocols: are we fully conscious of the difference in monitoring circulating lipids from those entering the cell membrane composition? Can we make crucial decisions about what is the best sampling procedure for cell membrane lipids? Finally, can we make an effort to unify protocols in one accredited procedure, so that big data can be collected and results can be compared in multicentric studies? In our opinion, analytical and chemical competences here come first, since they are required in order to build up accurate and reliable protocols: the recognition of fatty acid structures must be unambiguous [6], as will be shown in this review, taking into account that fatty acids are tissue-specific and each tissue has its own distribution of these molecular components [7]. Quality control must involve the exact separation of fatty acids from the sample to be analyzed, and if membranes are the target, the procedure must isolate them. This accuracy is fundamental because, after analysis, fatty acids are interpreted for their biological effects, as precursors to lipid mediators and contributors to membrane fluidity.

The contribution of fatty acids to membrane properties has been recognized for a long time, particularly in cancer development [8]. More recently, it has been discussed as evidence that the application of membrane modification and manipulation as part of cancer therapeutical strategies is still not developed [9].

An interplay between biosynthesis and diet regulates fatty acid availability. We gathered the literature on how fatty acids are implicated in tumor onset and progression and how the cancer lipidome reflects the activation of the *de novo* synthetic pathways. In this overview, we wish also to highlight our own work on the discovery of a family of MUFA positional isomers, the n-10 family, as new biomarkers of the metabolic shift that allows human cells to build up the first endogenous PUFA component, sebaleic acid [10]. The review also covers the link between obesity and cancer in order to understand why and when lipid supply causes health complications, highlighting specific fatty acids for their biological effects, signaling and contribution to the membrane properties that influence cell growth and death. From this scenario, several hints emerge for innovative strategies in cancer prevention (primary and secondary) using fatty acid-based membrane lipidomics and fatty acid balance.

#### **2. Fatty Acids and Lipid Supply for Membrane Formation in Cancer**

Cancer is a very complex disease due to the large number of factors involved. Cells develop a great capacity to grow, proliferate and survive under stress conditions. They modify several processes to achieve favorable environments, such as the metabolism of lipids, carbohydrates, proteins and nucleotides, being able to maintain the functionality of the structures and functions [11,12]. Adapted metabolic pathways allow cancer cells to obtain energy, form metabolic intermediates and synthesize fatty acids, even when the exogenous availability of these compounds is reduced. For example, the hyperactivation of the phosphatidylinositol-3 kinase and AKT (PI3K-AKT) transduces the signal from the hormone insulin to drive glucose uptake and is one of the most frequently mutated pathways in cancer [13]. In this case, glycolysis is favored, leading the cells to form pyruvate, which could be used for ATP synthesis or for *de novo* lipogenesis [14]. The hyperactivation of PI3K-AKT also activates the glutamate pyruvate transaminase 2 (GPT2), favoring glutamine anaplerosis to supply sufficient metabolites for FA synthesis and, finally, remodel the cellular lipidome [15]. In the latter case, it has been shown that such remodeling makes lipids an important hallmark of cancer [16]. The overexpression of FA transporters, such as fatty acid translocase CD36, plasma membrane fatty acid-binding proteins (FABP) and the fatty acid transport protein family (FATP), elevates the uptake of exogenous FAs with their subsequent storage in lipid droplets (LDs), as is known in ovarian cancer, and this is in connection also with adipose tissue, as will be explained in Section 5 [17]. To fully evaluate the lipid supply and understand their role in cancer, we must distinguish between *de novo* synthesized and dietary fatty acids, as explained below.

#### *2.1. De Novo Synthesis of Saturated and Monounsaturated Fatty Acids*

Combined with a greater capacity for the biosynthesis of lipids, cancer cells are not only able to maintain lipid homeostasis but also to provide ATP and NADPH in conditions of metabolic stress and sufficient precursors to deal with the formation of lipid rafts that are essential for protein dynamics in membranes and cell survival [18,19]. Since phospholipids are the basic units of membranes, in cancer disease different enzymes involved in their endogenous synthesis are highly expressed, such as ATP-citrate lyase (ACLY), acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS) [20,21]. Each of them represents itself a target of study against cancer. Whereas in nutrient-unlimited and aerobic conditions, the glucose metabolism forms citrate through the tricarboxylic acid cycle (TCA), to later convert into acetyl CoA, being the key for *de novo* synthesis, cells also develop an alternative strategy to form FA when there is a lack of nutrients and hypoxia (Figure 3). Several groups have proposed that, in these mentioned cases, the TCA cycle can be modified to run in reverse and use glutamine from storage to act as source of acetyl CoA [22,23], whereas others describe that acetyl CoA can be obtained from histone deacetylation [24]. In the case of FAS, in addition, it responds to signals from the activation of the AKT and MAPK (mitogen-activated protein kinase) pathways, which, in turn, are also favored in cancer processes [25]. Besides the fact that the production of FAs is essential to sustain the structure and demands of membranes, their composition is also decisive in guaranteeing the functions of the dividing cells. The production of monounsaturated fatty acids (MUFA) from SFA provides fluidity, functionality and flexibility, which are essential for tumor cells. This step involves the action of delta-9 desaturase enzyme (known also as stearoyl CoA desaturase, SCD-1, and reported in Figure 3), which can act on both palmitic (16:0) and stearic (18:0) acids. As with the enzymatic complex

from *de novo* synthesis, SCD-1 is overexpressed in cancer and regulated by different signaling cascades such as MAPK and AKT or systems such as p53 [26], attracting interest in their inhibition [4,5].

**Figure 3.** The *de novo* synthesis of saturated fatty acids (SFA) starting from acetyl CoA and the transformation to monounsaturated fatty acids (MUFA) by two desaturase enzymes. Structures of some of these fatty acids are shown in Figure 1. ACC: acetyl CoA: carboxylase; FAS: fatty acid synthase; ELOVL: elongase enzyme; Δ6D: delta-6 desaturase (Δ6); SCD-1: stearoyl CoA desaturase.

Cancer cells modify lipid metabolism in order to respond to environmental modifications. Hypoxia, for example, affects acetyl CoA formation from glucose and SCD-1 activity, as they are oxygen dependent. However, in this case, tumoral cells escape the need for fatty acid synthesis by increasing the uptake of lysophospholipids as a shortcut to prepare phospholipids. FABPs are transcriptional targets of hypoxia-inducible factors (HIFs) that facilitate extracellular scavenging of long-chain unsaturated lysophospholipids, which can be used as a nutrient source under conditions of metabolic stress [15]. Interestingly, this effect can occur even in aerobic conditions after oncogenic RAS activation, making it independent from SCD-1, to achieve sufficient MUFAs [27]. It is worth highlighting the existing debate about whether fatty acids used by cancer cells are of endogenous or exogenous (dietary) origin, since some studies did not find differences [28]. Lipidomic studies have a fundamental role in the elucidation of the decisive contribution of fatty acid biosynthesis, evidencing storage, lipolysis and membrane remodeling implied in tumor onset, progression and metastasis. Table 1 summarizes the most important fatty acid-driven mechanisms and related biological effects.


**Table 1.** The main fatty acid-driven mechanisms and biological effects in cancer onset, growth and metastasis.

Readers are directed to the original references cited in Table 1 to elaborate on each subject appropriately. Among these mechanisms, a recent one (Entry 1) was individuated by some of our group, investigating the analytical protocols for efficient separation of the MUFA positional isomers, which will be addressed in Section 4. Knowledge of different mechanisms is necessary for research of new therapeutic targets that can act in a synergic manner, to disturb organization of membrane lipids, destabilize lipid rafts and activate apoptosis signaling [18,19].

#### *2.2. PUFA Intake and Omega-6*/*Omega-3 Balance for Membrane Fatty Acid-Mediated Signaling*

On the basis of the importance of phospholipids for cell formation, the "membrane hypothesis" can be drawn, for which the initial steps of death or life of tumoral cells could be also driven by the quality of the membrane fatty acids. To create a fatty acid balance among SFA, MUFA and PUFA residues in the individual, it must be taken into account that the dietary intake of omega-6 and omega-3 regulates the presence of PUFA residues in lipid pools. Once the individual pool is formed, it exerts strong control upon the membrane composition and the types of fatty acids that will be detached from membrane phospholipids to determine the related cell fate. Indeed, the phospholipase A2 (PLA2)-induced release of fatty acids from membranes is a well-known process, involved in the membrane remodeling cycle, i.e., the Lands cycle [40]. It does not discriminate between omega-3 and omega-6 structures, thus highlighting the importance of the above-mentioned balance present in membranes for pro- and anti-inflammation signaling. Indeed, every time that the release in the cytoplasm of arachidonic acid from phospholipids occurs by PLA2, causing the subsequent formation of its eicosanoid mediators, other omega-6 and omega-3 fatty acids are released as well, such as di-homo gamma-linolenic acid (DGLA), eicosapentaenoic and docosahexaenoic acids (EPA and DHA). They are, in their turn, precursors of other lipid mediators with mainly anti-inflammatory properties, thus integrating the final inflammation and resolution responses [41]. Obviously, the result depends on the presence and balance of these fatty acids in membranes. Since recent data suggest inflammation as an important aspect in activating cancer proliferation pathways and resistance, it is evident that the membrane predisposition through its fatty acid composition is a piece of information to gather in the puzzling scenario of the cancer disease. Cancer is generated not only by genetic alterations, as a result of intrinsic or exogenous mutagens, but also by long-term exposure to acute or chronic inflammation. It is now becoming clear that the proliferation of cells alone does not cause cancer. However, sustained cell proliferation in an environment rich in inflammatory cells, growth factors and DNA-damage-promoting agents is necessary in the neoplastic process, promoting survival and migration. In this way, the causal relationship that exists between inflammation, innate immunity and cancer is more widely accepted [42]. Many of the molecular and cellular mechanisms that mediate this relationship are still unresolved, but the role that FAs play in inflammation processes related to cancer is increasingly relevant. Indeed, the role of dietary PUFAs omega-6 and omega-3 is a matter for discussion of their effects on cancer incidence and evolution [43]. The negative impact of Western diets, rich in omega-6, has recently been described in societies in which the intake of omega-6 fatty acids was traditionally in balance with that of omega-3. The number of cases with diseases associated with inflammatory processes, as well as their worse prognosis, has increased [44]. The scientific debate on the importance of the PUFA intake for cancer risk has not yet reached a conclusion. Large population studies are needed to address this task. For example, in a recent population-based (100,881 participants) prospective cohort study, using self-reported dietary data from the Västerbotten Intervention Programme, statistically significant associations have been described between a more anti-inflammatory or healthier diet and reduced risk of cancer [45]. In the development of inflammation mediated by PUFAs, both omega-6 and omega-3 FAs play crucial roles, since these two families of FAs are in constant competition with each other and, broadly speaking, they develop opposite effects. In this sense, omega-6 FAs are more related to inflammation (through the arachidonic acid (AA) cascade) and omega-3 to anti-inflammatory effects. Figure 2B shows how both omega-3 and omega-6 are closely related, by sharing the same enzymes for each step of their transformations. This fact implies that, from the beginning, there is a need for balance

between the families, since, if one is favored, it will hinder the synthesis of products from the other. It is worth adding that there are regulations also from the FA-derived mediators' formation and interactions: in the formation of prostaglandins (PG) and leukotrienes (LT) from omega-6 or omega-3 fatty acids, cyclooxygenases (COX-1, COX-2) respond more intensely for intermediates of omega-6 origin in the case of PG (PGD2, PGE2). Furthermore, not only enzymes but also receptors show different affinities, again being favored by PGs and some LTs of the omega-6 series [44]. Thus, in an environment in which omega-6 is biochemically favored, sufficient intake of omega-3 can have a key effect on the PUFA metabolism dynamics, as well as on the intensity of the action of the different eicosanoids. The involvement of PUFAs in cancer is demonstrated here by a few representative examples: regarding omega-6, AA modulates the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), involved in the immune response and altered in this disease. It also induces focal adhesion kinases, which promote progression and metastasis [46]. The signaling activity of AA is exerted through its transformation to PGE2, and, indeed, the overexpression of COX-2 protein was highlighted in several types of cancer, whereas human breast cancers frequently have high PGE2 levels, and breast tumors with high COX-2 protein levels are more likely to metastasize [47]. This led to consideration of COX inhibitors for cancer therapies, evaluating also their effects on angiogenesis. In this scenario, the PG interaction with EP receptors (E-series prostaglandin receptors), a family of G-protein coupled receptors designated as EP1–3 and EP4, was individuated, with the corresponding activation or deactivation of the c-AMP cascade or the extracellular signal-regulated kinase (ERK) 1 and ERK2 by way of PI3K [48]. As a matter of fact, not all the omega-6 FAs are equal in the tumor effects, and some studies suggest that, unlike the downstream omega-6 AA, the upstream omega-6s, such as linoleic acid (LA), γ-linolenic acid (GLA) and di-homo gamma-linolenic acid (DGLA), may possess anticancer effects. In fact, GLA and DGLA may exert anticancer properties via the production of PGE1. Although more work is needed to clarify the molecular basis of the anticancer effects of GLA and DGLA, it has been demonstrated that they are able to regulate gene and protein expression, disrupting cell-cycle progression and inducing apoptosis, a mechanism which implies also a direct effect on the lipid composition in cell membranes [48]. Regarding omega-3 PUFAs, they have the opposite effect to that mentioned for AA. For example, the combination of EPA and DHA decreases the production of eicosanoids formed from AA, leading to the inactivation of NF-κB and hindering proliferation [46]. They are also able to inhibit the activity of AKT protein, which is involved in cell survival and the inhibition of apoptotic processes [49]. Furthermore, PUFAs are involved in different processes such as lipid peroxidation, cell oxidative stress [50,51] and regulation of gene expression for controlling growth factor mediated carcinogenesis [52]. Moreover, there are other mechanisms involving lipid-based events that affect human health. The interest in ethanolamides has increased, as they are biological compounds that may have important beneficial actions by controlling inflammatory responses without being classical steroidal and non-steroidal anti-inflammatory drugs, that act by inhibiting the cascade of arachidonic acid. Palmitoyl ethanolamide (PEA) is an endogenous lipid mediator that can be found in foods (tomato, soybean, peanut) formed by palmitic acid and ethanolamide, with anti-inflammatory, neuroprotective and analgesic activities [53]. The suggested mechanisms of action involve various metabolic pathways in which some receptors seem to be activated directly (peroxisome proliferator activated receptor alpha (PPAR-α) and orphan G-protein coupled receptor 55 (GPR55)) or indirectly (cannabinoid receptors, CB1 and CB2) and transient receptor potential vanilloid type-1 channel (capsaicin receptor or TRPV1)) [53,54] to modulate mast cell activation and degranulation [53]. In colitis-associated cancer, more specifically, PEA inhibits angiogenesis through PPAR-α, suggesting a protective effect in both inflammation and cancer, being able to reduce mucosal damage, disease progression and carcinogenesis [55]. To conclude with the PUFA scenario, a brief mention of PUFA peroxidation products and the oxidative-based pathways to induce apoptosis in cancer, including ferroptosis, is made here, directing readers to reported works in these fields [56,57].

#### **3. The Membrane Fatty Acid-Based Profile in Cancer and the Relevance of Erythrocytes**

Considering the importance of fatty acids in membranes, a "bottom-up" approach can reveal the main changes in fatty acid profile between healthy controls and cancer patients. A systematic study of membrane fatty acid-based profiles in large populations is lacking, as is the agreement regarding the biological compartment in which fatty acids are measured. Therefore, there is large heterogeneity of data that does not currently allow us to draw conclusions about the most significant changes occurring in fatty acids in the human body. However, it is important to remark that, from the emerging scenario of fatty acids' involvement in cancer metabolism, it is reasonable to focus the efforts towards the cell membrane compartment. In this respect, the significance of the erythrocyte cell membrane and its fatty acid composition is highlighted for several reasons: (a) the numerosity of erythrocytes and the predominance among other tissues of these cells, which constitute 70–80% of the total cells formed each day [58], rendering them the best representatives of the availability of fatty acids to construct membrane phospholipids; (b) the continuous exchange of the erythrocyte membrane phospholipids with lipoproteins and tissues in order to reshape the molecular content and satisfy homeostatic requirements [59,60]; (c) the biological mission to reach tissues and organs during the erythrocyte's average life span of four months in humans, which requires the best performance of membrane properties in order to efficiently exchange gases; (d) the presence of the most representative SFA, MUFA and PUFA molecules and the preferred storage of arachidonic acid, known to be present in membrane phospholipids by 13–17%, as well as of other precious PUFAs [6]. Based on these considerations, the fatty acid profile can give information on the balance of these molecular components in erythrocyte phospholipids and help to establish the changes occurring under healthy and unhealthy conditions. Indeed, the fatty acid-based membrane lipidome monitoring used in different human conditions revealed how the endogenously and exogenously-derived fatty acids of erythrocytes are affected [61–64]. In Table 2, the relevant data relating to erythrocyte fatty acid monitoring from studies on cancer patients are gathered, highlighting the cancer types, the country and the number of patients and detailing the most important conclusions of each study. It is interesting to note that the SFA-MUFA transformation emerges as an important biomarker of cancer status, as well as the ratio between omega-6 and omega-3 PUFAs.



From these results, it is also clear that a large multicentric population study would definitely yield important results regarding the adoption of the fatty acid membrane profile for the follow-up of patients and therapies, assessing the importance of fatty acid biomarkers in primary and secondary prevention and discovering the molecular and clinical effects of personalized diets for cancer. We believe that the work in progress to map genetic alterations that control cell-cycle progression, apoptosis and cell growth in cancer [77] can be combined with molecular indicators such as membrane lipidomics in each tumor type to obtain more insights into the lipid pathways in cancer and clarify the epigenetic role of nutrition.

#### **4. The Study of the Cancer Lipidome and the Discovery of** *De Novo* **Pathways: Fatty Acid Positional Isomers as New Biomarkers of Metabolic Shift**

Lipidomics in cancer helps to clarify the connections between disease and lipidome, discovering novel lipid biomarkers for diagnosis as well as alternative and synergic strategies for therapy [78]. As mentioned before, the intake of essential fatty acids (EFA) with the omega-6/omega-3 PUFA balance is of crucial importance since membranes cannot be formed without this supply. The essentiality of PUFA derives from the fact that the insertion of a second double bond in the MUFA structure cannot occur in eukaryotic cells, which means that cells do not have desaturase enzymes able to convert oleic acid (as well as palmitoleic and vaccenic acids) into PUFA in the biosynthesis (see Figures 1 and 3). Since neither healthy nor cancerous cells can be formed without PUFA, it can be asked whether the dependence on dietary PUFA is a common feature and a limiting step of both types of cell metabolism. The answer to this question is not as straightforward as it seems, and in fact only recently have investigations been directed toward the study of the influence of metabolism and diet on the human lipidome. In lipidome analysis, it was also discovered that chemical skills are very important to create unambiguous protocols and distinguish fatty acid structures, especially those presenting unsaturations. A seminal example is provided by the report demonstrating for the first time of the presence of sapienic acid in various fractions of human plasma. This is a positional isomer of palmitoleic acid, which has the double bond in C6-C7 instead of C9-C10 [79]. The analytical approach for the unambiguous characterization and discrimination of positional and geometrical fatty acid isomers having the 16:1 structure is crucial for the determination of the sapienic acid presence. We described in detail the protocol of fatty acid analysis, which includes a crucial derivatization step to localize the double bond position, using the well-known dimethyl disulfide (DMDS) adducts and its diagnostic fragmentation in mass spectrometry [6,10,63,79]. It must be added that such derivatization procedure and mass spectra can be performed by regular equipment in chemical labs, and do not require specialized and expensive instrumentation. The quantitation of this fatty acid was performed in cholesteryl esters isolated from human plasma of healthy people (*n* = 5) (50.0 ± 4.0 ng/mL) and in commercially available human low density lipoprotein (LDL) samples (35.0 ± 2.0 ng/mL). How these levels are affected by health conditions in large cohorts remains to be thoroughly explored. These findings prompted us to understand in more detail the biosynthetic origin of sapienic acid. It is reported that, compared to all other types of cells that primarily form oleic acid (Figure 2A), sebocytes change their palmitic acid metabolism by the intervention of delta-6 (Δ6) desaturase enzyme (Figure 4) [80]. However, the systemic role of sapienic acid was not explored, and it was not highlighted the crucial step, that is the partition of palmitic acid between SCD-1 and delta-6 desaturase enzymes (see Figure 3). Whether this partition indicates a metabolic diversion with health significance is under current investigation. As a matter of fact, palmitic acid is an unusual substrate for delta-6 desaturase, which is an enzyme mostly involved with exogenous omega-6 and omega-3 EFA; therefore, the activation of sapienic acid biosynthesis could be attributed to several reasons, including (a) strong availability of the SFA substrate due to FAS activation, the latter well known in cancer [12,26,81]; (b) enzymatic activity competition or lack of normal intake/ presence of PUFA substrates [80,82]; (c) involvement of enzymatic polymorphism and competitive activity of desaturase for PUFA and SFA metabolisms [83]. Aiming at exploring sapienic acid and the other positional MUFA isomers in cell metabolism, we used the human colon carcinoma

cell line Caco-2 to compare the results of supplementation of sapienic and palmitoleic acids (150 and 300 μM), discovering that both are rapidly incorporated into membrane phospholipids and also that the former is converted to 8cis-C18:1 and 5cis, 8cis-18:2, as depicted in Figure 4, bringing also these two fatty acids in the cell membrane phospholipid composition. The n-10 fatty acid family has a still unexplored meaning for cancer cells and we were the first to demonstrate in a cancer cell line that it involves a unique type of endogenous PUFA biosynthesis (i.e., sebaleic acid; Figure 4) leading, more importantly, to its incorporation into membranes.

**Figure 4.** The metabolism of palmitic acid to sapienic acid (6cis-16:1) and its subsequent transformation to obtain the PUFA, sebaleic acid (5cis, 8cis-18:2).

The concomitant isolation of cholesteryl esters and triglycerides from the cell line demonstrated that the n-10 fatty acids "invade" all lipid classes, and, even at high concentrations (300 μM) and at long time exposures, they are not harmful (sapienic acid EC50 232–265 μM for 96 h) [29]. In the same study, the biophysical properties of the cell membranes were monitored by two-photon fluorescent microscopy, using Laurdan as a dye, showing that the supplementation of sapienic acid, with respect to its positional isomer palmitoleic acid, increased fluidity in several regions, evidently correlated with the formation and distribution of n-10 MUFA and PUFA in lipid domains. Following the interest in extracellular vesicles EVs (exosomes) as relevant sites for cancer metabolism and diagnostics [84], we investigated the presence of the n-10 fatty acid family, comparing membrane phospholipids and EVs of prostate cancer cell lines with different degrees of aggressiveness: PC3 (prostate cancer) and LNCaP (prostate derived from metastatic site: left supraclavicular lymph node), the former being more aggressive [10]. We found that 12–13% of the membrane fatty acids of these cell lines were composed of n-10 fatty acids, with the sapienic acid content >7%. In EVs, n-10 fatty acids were 9% for PC3 EVs and 13% for LNCaP EVs, with statistically significant increases in 8cis-18:1 and 5cis, 8cis-18:2, which is relevant considering that the EV are involved also in the transport of biologically active lipids and lipid metabolites to feed cancer tissues. This discovery can have a strong impact also in cancer diagnostics and follow-up of intervention efficacy. We envisaged that the sapienic/pamitoleic ratio, found equal to 3.5 in prostate cancer cells, also provides a measure of the partition into two metabolic pathways, and, in these cell lines, the delta-6 desaturase transformation of palmitic acid was found to be unusually high. A parallel evaluation of gene expression for desaturase (FADS) and elongase (ELOVL) enzymes by qRT-PCR (quantitative real time polymerase chain reaction) evidenced significant increases in FADS expression in PC3 with respect to LNCaP cells, and the higher expression of ELOVL5 in PC3 compared to LNCaP cells with ELOVL6 significantly lower. We found interesting evidence of higher desaturase activity in the most aggressive PC3 cell line, and suggested deepening the study of FADS3 desaturase, which, so far, has an uncertain metabolic role [85]. Indeed, the role of desaturase enzymes represents an important aspect in cancer metabolism and is also considered as a target in anticancer therapy, as reported in reviews [86] showing these strategies applied in preclinical trials. Regarding elongases, most of them are tumor specific: for example, ELOVL1, ELOVL5, ELOVL6 and ELOVL2 are highly expressed in breast cancer [87,88] and ELOVL7 in prostate cancer [89]. In the new scenario of the n-10 fatty acid family, a recent work confirmed the presence of sapienic acid in different cancer cell lines, defining it as a contributor to cancer plasticity [30], and another paper reported an increase in the transformation of palmitic acid to sapienic acid induced by the increase in mammalian target of rapamycin (mTOR) and sterol regulatory element-binding protein 1 (SREBP-1) signaling in mouse embryonic fibroblasts (MEFs) and U87 glioblastoma cells [90]. In this report, the inhibition of the two signaling pathways led to a decrease in sapienic acid biosynthesis. On the other hand, it must be recalled that fatty acids' enzymatic activities can be influenced by dietary fats, as previously shown for the competition between palmitic acid and PUFA omega-6 and omega-3 precursors [91].

New pathways involving SFA and MUFA are going to be discovered, provided that analytical protocols are able to give satisfactory results, such as was recently shown by the transformation of oleic acid in MCF7 cell lines into an eicosanoic fatty acid (7cis, 11cis-20:2) obtained by the unusual activity of FADS1 desaturase introducing a double bond at the level of C7 and not C5 [92]. Considering all the published work on the subject, so far only our experiments with cancer cell lines demonstrated the new pathway that brings about endogenous PUFA synthesis (sebaleic acid) and determined the n-10 FA insertion at the level of membrane phospholipids. We believe that this outcome of the sapienate metabolism is the real contribution to cancer plasticity, strongly influencing fluidity changes that are deeply embedded in cancer signaling and metabolism. We envisage that the pathway of sapienic acid will have a strong development in metabolic, therapeutic and nutritional research; here, we have provided a careful literature summary of the various contributions available so far, that we hope will be useful to researchers interested in the field.

#### **5. Link between Obesity and Cancer: When the Lipid Supply Becomes Dangerous**

Despite the difficulty of definitively proving that obesity is one of the causes of cancer, it remains a recognized risk factor contributing to the development and progression of tumors [93]. Several observational studies evidenced that obese and overweight subjects have a higher risk of developing cancer than lean subjects; in 2016, the International Agency for Research on Cancer (IARC) declared that obesity was associated with an increased risk for 13 types of cancer, indicated in Table 3 with their corresponding epidemiological studies [94,95].


**Table 3.** Increased risk for 13 cancer types correlated to overweight/obesity (% increased risk OW/OB vs. lean) and their corresponding epidemiological studies.

The clarification of the mechanisms binding obesity to cancer is crucial for the diagnosis and implementation of effective therapies. Here, we have gathered the main molecular pathways connecting adipose tissue (AT) and adipocytes with cancer cells in the tumor microenvironment, as well as their impact on cancer growth, invasion and metastasis. We summarize relevant connections between adipose and cancer tissues in Figure 5.

**Figure 5.** Relevant metabolic connections between adipose and cancer tissues; the arrow ↑ means increase, the arrow↓ means decrease.

The close localization between adipose tissue and cancer cells, immoderately increased in obese subjects due to the effect of excess of calories not consumed, induces a deep modification of the phenotype and functioning of adipocytes, which become cancer-associated adipocytes (CAA), promoted for the induction of lipolysis by cancer cells. Adipocytes are decreased in number and size, showing delipidization and de-differentiation to fibroblast-like phenotype [128–131]. It is well known that the exposure of adipocytes to cancer cells for long periods, with consequent fibroblast morphology, induces the formation of cancer cell fibroblast populations that are involved in tumor invasiveness [132]. In this context, the transformation of adipocytes provides a total alteration of their secretory function involved in endocrine, metabolic and immune systems. The ways in which the specific fatty acid status of adipocytes is involved in the support of cancer cells growth and metastasis are not yet well defined. The identification of several fatty acid unbalances in the erythrocyte membranes of obese patients certainly highlights derangements of lipid metabolism, including the above mentioned sapienic acid pathway [63]. Increased release of free fatty acids accompanies altered levels of adipokines and pro-inflammatory cytokines, growth factors and hormones [133]. The signaling in several types of cancer cells is sustained by adipokines secreted by adipocytes, mainly including leptin, adiponectin, oestrogens, insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF). In particular, the leptin/adiponectin ratio can be an interesting value to examine, due to the opposite effects of these hormones. Leptin stimulates a cascade of signaling events inducing JAK2/STATs, MAPK/ERK 1/2, PI3K/AKT and PKC, JNK, p38 MAPK and AMPK pathways in diverse cellular types (see abbreviations) [134]. The mechanism implicates the interaction with transmembrane leptin receptor (LRb) that, if phosphorylated, mediates downstream LRb signaling controlling STAT3 (signal transducer and activator of transcription 3) and ERK activation [135,136]. Simultaneously, high levels of leptin induce

the stimulation of monocytes into macrophages, leading to chronic, obesity-associated inflammation. Leptin increases the expression of anti-apoptotic proteins, inflammatory markers (tumor necrosis factor, TNF-α, interleukin IL-6) and angiogenic factors (vascular endothelial growth factor, VEGF), all processes involved in cancer cell survival, proliferation and migration [137,138]. On the other hand, adiponectin is inversely correlated to the body mass and cancer, inducing apoptosis and decreasing tumor vascularization. It modulates multiple signaling pathways, exerting its physiological and protective functions through the receptors AdipoR1 and AdipoR2 [139–141]. It is also able to block angiogenesis, inhibiting endothelial cell proliferation induced by FGF2 (fibroblast growth factor 2) as well as the migration of endothelial cells by VEGF. Furthermore, adiponectin inhibits cancer growth and proliferation, interfering with several pathways like AMPK, MAPK and PI3K/AKT, ERK1/2-MAPK pathway and GSK3/catenin, inducing G0/G1 cell-cycle arrest [142–145]. Adiponectin-induced cell death is also accompanied by an increase in intracellular reactive oxygen species (ROS). As a matter of fact, adiponectin pre-treatment suppresses leptin-induced ERK and AKT signaling [146]. Here, we only mention the roles of insulin and glucose levels and their interactions with specific receptors, such as insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF), that correlate with increased risks of specific cancers, like ovarian and breast cancers, mainly through activation of PI3K/AKT and MAPK pathways [147], while the inhibition of IGF-IR kinase activity prevents the growth-promoting effect of adipocytes on breast cancer cells [148]. Additional factors are connected with the altered environment of adipose tissue and cancer for the increase in inflammatory conditions, with consequent liberation of pro-inflammatory mediators, among them TNF-α and IL-6, contributing to the growth and differentiation in tumors like lymphoma, pancreatic and liver cancers. TNF-α induces carcinogenesis, activating the nuclear transcription factor NF-κB that prevents apoptosis, allowing enhanced cell survival, growth and proliferation [134,149]. IL-6 is normally elevated in obesity, induces JAK-STAT3 signal transduction and, stimulates cell proliferation, differentiation and metastasis. It mediates cell proliferation through the MAPK pathway; in fact, in some studies, the inhibition of MAPK stopped proliferation in the presence of IL-6, evidencing the role of cytokines in cell proliferation connected with inflammation [150]. Inflammation signaling, as discussed in Section 3, is an important piece of information to acquire in order to estimate factors that trigger cancer and its progression. The need for an integrated metabolic scenario emerges, linking the balance of membrane fatty acid precursors of eicosanoids and other lipid mediators with the effects of fat accumulation and hormonal control.

The effect of transformation of adipocytes in CAAs is evidenced also by increased release of free fatty acids (FFA), with the immediate effect of generating energy to fuel tumor growth [151]. The mobilization of FFA from adipocytes is performed in three steps by lipase enzymes: ATGL (adipose triglycerides lipase), HSL (hormone sensitive lipase) and MAGL (mono acylglycerol lipase), enhancing their circulating levels [93]. Seminal experiments of co-culture of adipocytes with cancer cells showed that there is stimulation of lipolysis in adipocytes releasing FFA and glycerol, with a reduction in adipocyte size [152]. The amount of FFA promoted cancer progression, delivering building blocks for cancer cells but also stimulating lipid metabolism; in ovarian cancer cells, co-cultures with adipocytes induced upregulation of fatty acid β-oxidation (FAO), with a consequent large quantity of ATP [133,139,153,154], supporting the energy demand of the tumor mass. The transfer process of FFA between adipocytes and cancer cells is mediated by fatty acid-binding protein 4 (FABP4), which supplies energy to the cells and also active oncogenic pathways like IL-6/STAT3/ALDH1, leading to an enhanced stem cell-like phenotype and tumor progression [133]; FABP4 expression increased in cancer cells co-cultivated with adipocytes [155]. The interesting connections of disease development with the fatty acid structures and functions discussed in the previous sections should appear clear at this point, and this review has the scope of stimulating a constructive debate among scientists involved in cancer cell biology, metabolomics and lipidomics in order to use the substantial information available to develop lipid-based diagnostics and strategies for cancer.

The recent results obtained with EVs in cancer offer promising perspectives on mechanistic and diagnostic developments [156]. The transport of lipids by EV from AT can be an important player in the whole scenario. In a case study of melanoma, the biomolecular transfer process of EVs seems to increase in the presence of obesity. Incubation of melanoma cells with EVs deriving from AT caused the redistribution of lipid droplets close to mitochondria and the increase of fatty acid oxidation [157]. Research conducted on overweight subjects showed that exosomes derived from cancer cells were incorporated by adipocytes, modifying transcriptome and cytokine secretion; the exosomes obtained from adipocytes strongly helped tumor growth by angiogenesis and enhanced inflammation, recruiting macrophages, activating kinases and involving the NF-κB signaling pathway [158,159]. Together with the analysis of the fatty acid types contained in EVs, including sapienic and sebaleic acids, the EVs have enormous potential for unveiling new aspects of lipid supply to cancer.

Obesity also influences the effects of anticancer therapies, as shown in obese cancer patients compared with non-obese patients evaluated for the effects of the same drug treatment [160]. Besides the various aspects involved in these effects, it is important to highlight that the fatty acid constituents of adipose tissue assume a fundamental role in the modification of pharmacokinetics, conferring drug resistance [161,162].

In the scenario of lipid metabolism, the role of lipophagy (i.e., autophagic degradation of lipid droplets, the main lipid storage organelles of eukaryotic cells), discovered in 2009 to have important consequences on health [163], must be mentioned here, in connection with the ongoing debate concerning the role of fasting strategies in cancer treatment [164]. The ways in which calorie restriction/control impacts obesity and cancer treatment will be matter for further research and active debates from different perspectives [165].

#### **6. Some Considerations of Fatty Acid-Based Membrane Lipidomics and Lipid Therapy**

Tumoral cells develop accelerated *de novo* lipogenesis as well as strong lipid recruitment, also taking advantage of obesity, to sustain their needs. However, the quality of fatty acids contributes to their invasiveness, also due to their influence on the biophysical properties of membranes and signaling cascades. The proposal of the "membrane hypothesis" links the initial steps of death or life of tumoral cells with the moment of the phospholipid aggregation for membrane formation and the balance between the saturated and unsaturated fatty acid types present in the individual. This crucial balance is different from tissue to tissue, since each tissue has its own composition [7], and it is important to remark that the membrane formation is a completely spontaneous process of phospholipid aggregation, which in their turn are formed by the availability of fatty acids in the lipid pools. It could be said that, with respect to the adequate intake (AI) of lipids established by the main international agencies of health and food [166], the lipid pool should be able to reach a satisfactory balance, with scarce possibilities for impairment or excess. We are aware of the strong ongoing debate about the interplay between genetics and other causes of cancer [167–170]; however, we wish to highlight the importance of the environment (including nutrition), able to interfere with fatty acid levels and metabolic transformations, with strong impact on inflammatory responses and stress conditions, including on hormonal effects such as explained in obesity (Section 5), that can change the "normal" scenario and create unbalances. In our opinion, it is timely to introduce the monitoring of SFA, MUFA and PUFA membrane levels in clinical practice, in view of evaluating strategies that influence the formation of membranes in the individual. Fatty acid-based membrane lipidomics can give the necessary information to estimate the correctness of the molecular pool, which is the *conditio sine qua non* for the healthy behavior of this important compartment [61]. It is worth recalling that the erythrocyte membrane compositions of patients under parenteral nutrition reflected the lipid emulsions given to them. In particular, olive oil emulsion was able to induce statistically significantly higher levels of arachidonic acid and omega-6/omega-3 ratio compared to patients treated with a lipid emulsion containing a small percentage of fish oil [64]. This is an important message for those involved in patient care and nutrition and also for considering the exact dosage of fatty acid supplementations for therapeutic purposes. As a matter of fact, membrane homeostasis and related therapies are nowadays emerging, targeting cell membranes by dietary bioactive molecules able to obtain the remodeling of plasma membrane domains. The attenuation

of oncogenic protein activity by modulating the membrane organization of essential proteins and lipids was proven and this is a promising way to use such an approach to manage cancer expansion. It is worth underlining that omega-3 has a very potent influence on membrane organization and this ability, combined with the anti-inflammatory activity, should be developed toward successful cancer treatments [171,172]. However, it is also evident that, without assessing the membrane status in the individual, the assignment of the lipid strategy cannot be precise, in types and doses, thus even bringing about contrasting clinical outcomes since the membrane unbalance results or remains altered. Therefore, it will be necessary to develop a multidisciplinary approach, involving also clinicians, for the understanding of membrane molecular profiles and for creating protocols of membrane lipidomics and lipid therapy, gathering evidence-based results. In this direction, lipid replacement therapy (LRT) is described as a natural medicine approach to replace damaged lipids in cellular membranes and organelles; however, no personalization is proposed [173]. In the context of membrane therapy, we must also mention natural fatty acids with a structure able to interfere with lipid enzymes, such as sterculic acid, a cyclopropane-containing derivative of oleic acid (9,10-methylene-9-octadecenoic acid) found in plants of the genus *Sterculia*. This is an inhibitor of SCD-1, and of the related cascades, as previously explained, which has attracted interest for application in various diseases, including cancer [174]. As previously described, lipid enzyme inhibitors (fatty acid synthesis and desaturation) are attracting interest for innovative cancer treatments, and readers are directed to reviews to deepen the state-of-the-art of such therapeutic strategies [15,21,174].

Focusing on mature erythrocytes, their membrane composition data can be gathered from cancer patients, accompanying the biological sample with an accurate food questionnaire. By this approach, we were able to highlight in a preliminary study of cancer patients that they have SFA-MUFA membrane levels which are significantly different from controls and independent of dietary intakes [69]. It is also straightforward that the analytical protocols used for membrane lipidomic analysis must be certified by international accreditation bodies, and it is advisable that such protocols are unified and automatized by high-throughput procedures, in order that clinical laboratories can gather reliable "big data" to depict cancer lipidomics in an incontrovertible manner.

#### **7. Conclusions**

The acquisition of a multidisciplinary vision of fatty acids' relevance to membrane formation and cancer development is necessary in order to go from the bench to the bedside and to the home of patients, associating nutrient choice with strategies to defeat cancer. The growing understanding of the response of cancer to diet will lead to new therapeutic opportunities but, at the same time, will have practical use in the everyday lives of patients, solving also contrasting effects reported in the literature for PUFA supplementation [175]. It is desirable to increase efforts for a larger understanding of molecular nutrition effects in combination with pharmacology and immunology to control this multifaceted disease [176]. Researchers of several disciplines are required in order to accomplish such goals. Specific effort is needed by clinical units to introduce fatty acid diagnostics tools and therapies to prove the validity of the concepts and translate them into medical practice. Indeed, previously reported clinical effects for some fatty acids, such as the omega-6 γ-linolenic acid (see Figure 2), of its antitumoral synergy with chemotherapy [177] must take into account its rare presence in foods and be evaluated in a personalized way, also determining the level of this fatty acid in the individual. Therefore, knowledge of molecular diagnostics, such as membrane lipidomics, is a fundamental step toward including endogenous and exogenous fatty acids in the cancer scenario.

**Author Contributions:** Contributed to data acquisition and writing of the initial version, C.F., A.S., I.T. and J.A.; revision performed, C.F. and R.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Centre for the Development of Industrial Technology (CDTI) of the Spanish Ministry of Science and Innovation under the grant agreement TECNOMIFOOD project, CER-20191010. This is contribution number 990 from AZTI.

**Conflicts of Interest:** C.F. is co-founder and scientific director of the company Lipinutragen srl, born as spin-off recognized officially by the National Council of Research and involved in research and development of the membrane lipidomic analysis. The company had no role in the design of the study; in the collection and interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**


#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Lipidomic-Based Advances in Diagnosis and Modulation of Immune Response to Cancer**

**Luis Gil-de-Gómez 1,\*, David Balgoma <sup>2</sup> and Olimpio Montero <sup>3</sup>**


Received: 30 June 2020; Accepted: 12 August 2020; Published: 14 August 2020

**Abstract:** While immunotherapies for diverse types of cancer are effective in many cases, relapse is still a lingering problem. Like tumor cells, activated immune cells have an anabolic metabolic profile, relying on glycolysis and the increased uptake and synthesis of fatty acids. In contrast, immature antigen-presenting cells, as well as anergic and exhausted T-cells have a catabolic metabolic profile that uses oxidative phosphorylation to provide energy for cellular processes. One goal for enhancing current immunotherapies is to identify metabolic pathways supporting the immune response to tumor antigens. A robust cell expansion and an active modulation via immune checkpoints and cytokine release are required for effective immunity. Lipids, as one of the main components of the cell membrane, are the key regulators of cell signaling and proliferation. Therefore, lipid metabolism reprogramming may impact proliferation and generate dysfunctional immune cells promoting tumor growth. Based on lipid-driven signatures, the discrimination between responsiveness and tolerance to tumor cells will support the development of accurate biomarkers and the identification of potential therapeutic targets. These findings may improve existing immunotherapies and ultimately prevent immune escape in patients for whom existing treatments have failed.

**Keywords:** immunotherapy; cancer; lipids; biomarkers; metabolism

#### **1. Introduction**

Following the discovery of the structure of DNA in 1953 [1], increasingly efficient technologies for the study of the whole genome (genomics) have enabled assessments of genome-based pathologies in large population cohorts [2]. However, since a broad number of factors, including environment, diet or lifestyle, are important in the etiology of diverse diseases such as cancer, a high-dimensional biological approach appears to be required [3]. A multi-omics/systems-level approach, which encompasses the combined analysis of data from genomics, RNA transcription (transcriptomics), proteins/peptides (proteomics) and metabolites (metabolomics), enables one to overlay gene information onto a complementary understanding of accrued molecular mechanisms [4]. Lipidomics represents an emerging discipline from metabolomics that connects lipid biology, technology and medicine, and that strives to build an all-inclusive atlas of the cellular/tissue lipidome [5]. In this regard, the role played by lipids in the etiology and treatment of cancer has loomed large over the last decades.

Early evidence that cancer cells undergo characteristic metabolic alterations was documented by Otto Warburg in the first half of the twentieth century. In a paradoxical process in terms of adenosine triphosphate (ATP) production, cancer cells increase the consumption of glucose to support aberrant cellular proliferation. Because proliferating tumor cells require cholesterol and other lipids, perturbations in the lipid metabolism are emerging as potential targets for therapeutic intervention in cancer [6,7]. Cancer immunotherapy has proven to have an unprecedented positive impact in clinical oncology. Increased evidence suggests that glycolytic metabolism not only rules cancer signaling but also the antitumor immune response where activated inflammatory immune cells display the same metabolic profile as tumor cells [8] (Figure 1). Multiple studies have separately reported the impact of lipids on immune cells and tumor progression. However, so far, little work has focused on reviewing how the lipid metabolism is associated with the immune response to tumors. Taking this shortfall into account, we aim to highlight the role of lipid mediators in the context of immune activation in order to explore potential biomarkers and therapeutic targets for cancer.

**Figure 1.** A metabolic shift is required by immune cells for them to respond actively to tumor cells. Inactive immune cells rely on oxidative phosphorylation (OxPhos) and fatty acid (FA) oxidation (left), while activated and responsive cells increase glucose uptake/glycolysis, resulting in an increased FA synthesis and lactate production (central panel). Lipogenesis, required for a robust cell proliferation, also characterizes tumor cell metabolism (right). Therefore, an untargeted lipid-based treatment to fuel effector immune cells may produce self-defeating effects inducing tumor cell growth. Many other lipid intermediates regulate inflammation, and exogenous lipids such as gut microbiota-derived short chain fatty acids (SCFAs) may impact the host immune response to tumor cells. Together, these findings indicate (1) the exhaustive regulation required to maintain immunity balance in the presence of tumor cells, and (2) the essential role of a large variety of lipids in this control. New precise lipidomic-based strategies may enhance therapeutic targeting and improve the capacity of existing immunotherapies to control tumor progression.

#### **2. Lipid Metabolism Impacts Immune Activation against Tumor Progression**

#### *2.1. Lipid Interplay with Immune Regulation*

Tumors impact immune cell function by supporting cancer stem cell survival, metastasis and immune evasion. The aggressiveness of tumor cells is linked to their capacity to store high levels of lipids and, in particular, cholesterol [6]. Metabolic challenges in the tumor microenvironment (TME), including hypoglycemia and hypoxia, induce changes in tumor cellular metabolism like aerobic glycolysis and fatty acid oxidation (FAO) [9]. In response, immune cells show the capacity to modulate lipid metabolism to better adapt to these special metabolic conditions.

The innate immune system is the first barrier against external stimuli, which are recognized via Toll-like receptors (TLR). TLR-dependent response, which regulates the activation of antigen-presenting cells (APC) (mainly macrophages or dendritic cells (DCs)), shifts the intracellular metabolism towards the glycolysis-fueled synthesis of fatty acid (FA) [10,11]. After the initial broad immune response, an adaptive immune response is initiated when APCs process and present antigens for recognition by certain lymphocytes such as T cells. Both phases of the immune response are characterized by a fragile equilibrium, whereas the heterogeneous groups of immune cells communicate and modulate each other via cytokine release. In this sense, cytokine production in activated DCs has been related to phospholipid remodeling to support FA demands [12]. Immune effector cells, such as T cells and macrophages, are induced by tumor-specific antigens and tumor-associated antigens. However, regulatory mechanisms of the immune system, such as immune checkpoints, make this cellular response incapable of preventing tumor progression. Immune check points are inhibitory regulators crucial for maintaining self-tolerance and controlling the duration of the immune response in order to prevent collateral tissue damage [13]. Since these key immune-regulatory molecules are used by tumor cells to promote evasion, immune checkpoint inhibitors have demonstrated their effectiveness as clinical targets for cancer immunotherapy [14]. This breakthrough is based on currently approved blocking monoclonal antibodies that inhibit cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the programmed cell death protein PD-1/PD-L1 axis [15].

Endogenous lipid reserves provide energy to T cells but may also regulate T cell function by an immune checkpoint such as PD-1 [16]. PD-1 is a member of the cluster of differentiation 28 proteins (CD28) superfamily that delivers negative signals upon interaction with its two ligands, the PD-L1 and PD-L2 proteins. PD-1 activation impairs glucose and glutamine uptake whilst promoting FAO and catabolism of endogenous esterified fatty acids in both cytotoxic (CD8+) and helper (CD4+) T cells [16,17]. Another lipid pathway that targets PD-1 is regulated by the members of the peroxisome proliferator-activated receptors (PPAR) subfamily. This subfamily of nuclear receptors might be modulated by fatty acid signals derived from exogenous sources, including diet [18]. PPAR is crucial in supporting the accumulation and function of immunosuppressive regulatory T cells (Tregs) [19]. In concordance, it has been reported that PPAR-γ inhibition increases the efficiency of anti-PD-1 antibody immunotherapy, leading to the suppression of tumor progression in colon adenocarcinoma and melanoma models [20,21]; likewise, an agonist for another isomer, PPARα, is able to restore the anti-melanoma effects of tumor-infiltrating lymphocytes (TILs) by blocking the reprogramming to fatty acid catabolism in mice [22].

TILs, largely comprised of CD8<sup>+</sup> and CD4<sup>+</sup> T cells, as well as natural killer (NK) cells, are key players in tumor cell death. This particular function of both cell subtypes has been shown to be dependent on the profile of polyunsaturated fatty acids (PUFAs) in the cell membrane [23]. However, current work on how PUFA supplementation may affect TIL function in humans is often contradictory. Whereas it has been reported that the percentage of NK cells in mouse blood is reduced after dietary supplementation of docosahexaenoic acid (DHA, 22:6 n-3) and eicosapentaenoic acid (EPA, 20:5 n-3) [24], a similar previous study using EPA-rich oil in the diet did not find such differences [25]. The discovery of the G-protein-coupled receptors (GPCRs) suggests that many of the effects of dietary FAs may be receptor-mediated. This family of cell-surface free-fatty acid receptors includes the long-chain fatty acid receptors FFA1 and FFA4. Anti-inflammatory effects of omega-3 PUFAs, especially EPA and DHA, have been related directly to the expression of these FFA receptors. Hence, FFA4 knock-out mice have shown a higher proportion of pro-inflammatory macrophages than the wild type [26]. In addition, agonists of FFA receptors have been connected with the suppression of the proliferation and migration of a large variety of tumor cells [27,28].

The phenotype and maturation of T cells is also regulated by the fatty acid metabolism. Differentiation of T cells is dependent on de novo FA synthesis and uptake. In tumor tissue, the inhibition of de novo fatty acid synthase (FAS) by different targets, such as acetyl-CoA carboxylase 1, promotes Tregs but suppresses memory T cell lineage (Th17) differentiation [29]. The challenge of maintaining T cell function in a nutrient-depleted environment like the TME is resolved by other effector T cells. Unlike naïve and central memory T cells, effector memory T cells are less dependent

on FA metabolism [30]. This feature plays an essential role in establishing immune equilibrium, since most effector T cells are removed after antigen elimination, whereas memory T cells remain for rapid response upon antigen re-exposure. The analysis of other molecules such as the mammalian target of rapamycin (mTOR) extends the list of lipid mediators that contribute to maintaining the immune balance. mTOR regulates Tregs differentiation, function and survival, ultimately defining the immunosuppressive profile of the TME [31]. Tregs are a dominant suppressive population that infiltrate the TME and dampen anti-tumor immune responses by inhibiting the effector T-cell function [32]. The singular metabolism of Tregs, including an increased FAO, provides them with critical advantages to survive and proliferate under hypoxia or low glucose conditions within the tumor [32,33].

The delivery and cellular distribution of PUFAs are indirectly regulated by desaturases, which perform the desaturation and elongation of essential fatty acids. However, phospholipases A2 (PLA2) are the main cellular regulators of PUFA release, maintaining the homeostatic levels of several free PUFAs, and in particular of those that are precursors of mediators with pro-inflammatory properties, such as arachidonic acid (AA, 20:4 n-6). In the inflammation process, AA is released by PLA2 activity, and prostaglandin E2 (PGE2) is subsequently generated from arachidonic acid by the enzyme cyclooxygenase-2 (COX-2) [34,35]. One of the mechanisms that Tregs uses to suppress T cell activity is PGE2 production, which can be reversed by COX-2 inhibitors [36]. PGE2 is essential in homeostasis, and while its pro-inflammatory role is crucial for host cell self-preservation, its immunosuppressive effects may support tumor progression [37]. Besides directly mediating inflammation, PGE2 might be used as an intermediate not only in the signaling between immune cells but also between immunity and tumors. Hence, PGE2 released from DCs affects the generation and proliferation of Tregs by immunosuppressive cytokines like IL-10, whereas PGE2 released from tumor cells is able to regulate DC maturation [37–39]. This COX2/PGE2 pathway is also involved in the regulation of the immune checkpoint enzyme expression, like PD-L1, in tumor-infiltrating macrophages and other myeloid cells [40]. Moreover, a recent study suggests that the combined blockade of PD-1 and PGE2 pathways is a promising therapeutic strategy for enhancing antitumor activity. This effect is due to an increased frequency of T cell-recognized tumor antigens, whose dysfunction is regulated by PD-1 [41].

Suppressing tumor immune surveillance may lead to the exhaustion or inactivation of pro-inflammatory immune cells and may, subsequently, promote tumor growth and metastasis. Myeloid-derived suppressor cells (MDSC) and immunosuppressive type II (M2) tumor-associated macrophages (TAMs) are fueled by the ß-oxidation of lipids, rather than glycolysis, within the TME [42]. Recent studies have shown that the phenotype of M2-like TAMs is controlled by intracellular long-chain fatty acid (LCFA) homeostasis, specifically unsaturated fatty acids like oleate [43]. Additionally, lipid metabolism provides a mechanistic explanation for TAM polarization and differentiation [44]. The upregulation of lipogenesis by sterol regulatory element-binding protein-1 (SREBP1) promotes the transcriptional response of macrophages to TLR signaling by driving the synthesis of anti-inflammatory fatty acids [45]. SREBP1 signaling also impacts tumor cells by sustaining the high energetic demands required for their growth and survival, and has been shown to be important in melanoma and prostate cancer progression [46–49]. One of the metabolic effects of SREBP1 is the regulation of the de novo lipogenesis by the upregulation of, among others, fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) [50,51]. Consequently, the upregulation of SREBP1 entails the upregulation of saturated and monounsaturated fatty acids, both free and in glycerolipids. Regarding macrophages, stimulation by lipopolysaccharide (LPS), a component of cell wall of gram negative bacteria, upregulates SREPB1 expression which is required for the inflammatory response [52,53]. In contrast, the activation of liver X receptors (LXRs), which also upregulate SREPB1, decreases the inflammation level in macrophages [54]. Because of this opposed effect, it is expected that the level of de novo lipogenesis in TAMs presents a complex relationship with their activation state. LXRs are regulated by oxysterols and SREPB1 by sterols in the cell environment. Consequently, not only diet but also the tumor lipid microenvironment can regulate the metabolic/pro-inflammatory status of TAMs. In addition, external palmitic acid reprograms the microglia metabolism in a way that mimics LPS treatment [55], whereas

oleic acid reduces the pro-inflammatory response [56]. Furthermore, sexual hormones in the TME also play a key role in the lipid metabolism and the inflammatory state of TAMs. The androgen receptor decreases the LXR and SREBP1 activity, which decreases the de novo lipogenesis and remodels the lipid metabolism [57,58]. Interestingly, the interaction between prostate cancer cells and macrophages regulates the resistance to hormonal therapy [59]. This fact suggests an interplay in tumor growth among: (1) the activation of the androgen receptor, (2) the tumor microenvironment and (3) the LXR-mediated lipogenesis in both the tumor and TAMs. Altogether, these studies suggest that both the lipidic and hormonal microenvironment interact to reprogram the metabolic and inflammatory state of TAMs. This reprogramming is associated with therapy resistance and patient prognosis.

LXRs are major regulators of FA and cholesterol homeostasis. Cholesterol, a nonpolar lipid transported in plasma by low-density lipoproteins (LDL) and high-density lipoproteins (HDL), has been linked to the effect of IL-10 in immune regulation. The inhibition of cholesterol biosynthesis with atorvastatin or 25-hydroxycholesterol regulates IL-10 production by inducing human CD4<sup>+</sup> T cells to switch from an effector to an anti-inflammatory profile [60]. Furthermore, given the role of lipoproteins as cholesterol carriers, while they promote tumor growth by regulating T cell activation and functionality [61], recent studies have used them as anti-tumor drug delivery vehicles [62].

The impact of lipids on the immune response to cancer includes post-translational modifications. Palmitoylation has been found to be important in the context of cancer immunotherapy. This post-translational process involves the binding of palmitate (C16:0) to amino acid residues. Yao and colleagues identified palmitoyl transferase ZDHHC3, which contains a conserved Asp-His-His-Cys (DHHC) signature motif, as the main acyltransferase required for PD-L1 palmitoylation. This lipid modification stabilizes PD-L1 by blocking ubiquitination, which ultimately prevents lysosomal-driven degradation. Thus, DHHC3 targeting enhances T cell cytotoxicity against cancer cells in vitro, as well as the in vivo antitumor effect in a colon carcinoma model [63]. Other studies have related the ablation of ZDHHC3 in human mammary tumor cell xenografts to a reduced primary and lung metastasis infiltration. This effect correlates with an enhanced recruitment of macrophages and NK cells to the tumor, and its subsequent clearance [64].

#### *2.2. Short-Chain Fatty Acids from Gut Microbiota as E*ff*ectors of the Immune System*

FAs with chain lengths ranging from one to six carbon atoms are produced by trillions of harmless microorganisms that inhabit the human gastrointestinal tract. These short chain fatty acids (SCFAs) are the major end product derived from gut microbiota; very high concentrations are found in the colon [65]. The presence of SCFAs (propionic, butyric, acetic and valeric acids) regulates the intestinal microenvironment by reducing pH and impacting the microbial function and composition [66]. Besides various gut disorders, gut microbiota also play an important role in central nervous system disorders, the immune system and cancer malignancies [67]. Although the role of butyrate in fueling tumor cells proliferation has been described [68], SCFAs have been generally perceived as tumor suppressors because they induce cancer cell differentiation and apoptosis [69]. The ability of SCFAs to regulate effector immune cells is considered one of the essential mechanisms accounting for their anti-tumor properties [70]. SCFAs engage GPCRs such as FFA2 and FFA3, and act as histone deacetylases (HDACs) to regulate the activity of innate immune cells such as neutrophils, macrophages and DCs, and they also modulate antigen-specific adaptive immunity mediated by T cells and B cells [71,72].

SCFAs, particularly butyrate, directly impact the immune response to cancer through the reprogramming of the cellular metabolism. In activated CD8<sup>+</sup> T cells, butyrate increases glycolytic activity, mitochondrial mass and membrane signaling. Butyrate-stimulated CD8<sup>+</sup> T cells also show functional uncoupling of the TCA cycle from glycolysis, promoting additional sources of carbon such as glutamine and FAs [73]. An increased FA intake in butyrate-treated CD8<sup>+</sup> T cells serves to charge the TCA cycle, but triacylglycerides and phospholipids are other candidates that serve as suppliers [74,75]. The anti-inflammatory properties of SCFAs are also related to the ability of butyrate and propionate to abrogate IL-12 release from APCs, a cytokine with a primary role in effector T cell stimulation [76,77]. In contrast, both SCFAs are also associated with resistance to immune checkpoint CTLA-4 blockade and a higher proportion of Treg cells. These effects limit the clinical outcome of cancer patients treated with anti-CTLA-4 blocking monoclonal antibodies [78].

The capacity of butyrate to regulate T cell polarization and immune checkpoint blockade correlates with the diversity of commensal microbiota. In human bacterial communities, most butyrate-producing colon bacteria belong to the *Firmicutes* phylum. The equilibrium between species defines the therapeutic outcome, and a low Bacteroidetes/Firmicutes ratio has been used to identify lung cancer patients [79]. Moreover, the relative abundance of other specific bacteria, such as Bifidobacterium, increases anti-PD-L1 efficacy, promoting anti-tumor immunity [80]. Taken together, these findings point toward an alternative therapeutic strategy by targeting immune cells on a metabolic level. Augmenting the efficacy of the immune system by targeting the lipid metabolism could be useful for improving the antitumor immune response. However, as Chalmin et al. postulate, targeting the lipid metabolism may affect multiple immune populations and could have unpredictable outcomes [81]. Thus, since fatty acid oxidase is required not only for effector T cell development but also for Treg differentiation [82], its blockade limits Treg-dependent immunosuppression. Despite these drawbacks, data suggest that the capacity to define specific lipid reprogramming that correlates with disease stages will help to design new cancer treatments. The balance between immune activation and suppression is a critical feature of immunity, and lipids are able to alter this equilibrium. Therefore, targeting the lipid metabolism may be used to induce immune stimulation, which will ultimately determine the clinical success of cancer immunotherapy.

#### **3. Lipids as Biomarkers of Immune Response to Cancer**

Accurate and predictive biomarkers to diagnose early stages of disease are a critical objective of clinical and biomedical research. Lipids, among several other metabolites such as amino acids or sugars, have been described as potential predictors of systemic alterations that discriminate between healthy controls and patients [83]. Clinical success often hinges on an early diagnosis, especially in long and age-related malignances like Alzheimer's disease or cancer [84]. New technologies for the qualitative and quantitative analyses of metabolites can provide essential information on pathological conditions that can result in profound alterations in the architecture of the immune system. Identifying the metabolic profile associated with the immune response to tumor cells has emerged, parallel to immunotherapy, as a tool for obtaining an early and accurate diagnosis and for designing personalized treatments, both being essential for better clinical outcomes in cancer patients.

An increased de novo synthesis of fatty acids is required for membrane synthesis and, therefore, for the growth and proliferation of both immune and tumor cells. This makes fatty acids robust biomarker candidates. Recent studies have shown that genetic alterations observed in acute myeloid leukemia (AML) patients control lipid dynamics and metabolism [39,85]. Interestingly, patients with AML can be identified by specific lipid signatures in plasma [86] and bone marrow [87]. Whereas lipid biomarkers have been used to identify tumor progression, the relationship between a characteristic lipid profile and the immune response to cancer is still poorly understood. The major clinical advantages of immune checkpoint inhibitors have generated considerable interest in discovering biomarkers that predict the response to treatment [88]. Recent studies propose serum concentrations of very long chain fatty acids (VLCFA) as a way to identify the response to immune checkpoint inhibitors in urological cancer [42]. The rationale for this biomarker is motivated by the finding that lower serum VLCFA levels are associated with highly immunosuppressive TME with a high-VLCFA consumption rate.

As discussed previously, de novo lipogenesis is also associated in a complex manner with the metabolic/inflammatory state of TAMs. Consequently, the lipids associated with the de novo lipogenesis act as biomarkers of tumor growth and the activation of TAMs. The LXRs/SREBP1 pathway is the key player in the regulation of the de novo lipogenesis, and it is involved in tumor growth and in the inflammatory response [89]. LXRs/SREBP1 upregulation in tumor or inflammatory cells leads to an increase of saturated and monounsaturated fatty acids via the activation of FAS and SCD-1, which are incorporated into glycerolipids by acyltransferases. Consequently, the upregulation of glycerolipids with saturated and monounsaturated fatty acids acts as a biomarker for the tumor synthesis of membranes and the activation of macrophages [90,91]. In addition, LXRs/SREBP1 upregulate glycerol-3-phosphate acyltransferase 1 (GPAT-1), which has a strong preference for transferring palmitic acid to the *sn*-1 position of glycerol-3-phosphate. This leads to an enrichment of glycerolipids with palmitic acid in the *sn*-1 position of the glycerol backbone. Consequently, the triacylglycerides with palmitic acid in the external position of the glycerol act as a biomarker of LXRs/SREBP1 activation and the de novo lipogenesis [92,93]. In conclusion, these triacylglycerides have the potential to be used as biomarkers for (1) monitoring the metabolic reprogramming of TAMs in the TME, and (2) the effect or resistance to immunotherapy by evaluating the up- or downregulation of lipogenesis in the tumor.

Because SCFAs from gut microbiota have a wide-ranging impact on the host physiology, these metabolites are also increasingly studied as predictive biomarkers. SCFAs and microbiota composition have been used to determine the risk of cancer, and reduced levels of butyric acid in patients with colon cancer have been reported [94,95]. The levels of butyrate are also correlated with the responsiveness to melanoma in mice treated with antibiotics [96]. Recent results have reported a correlation between the relative abundance of certain SCFA-producing microbiota and the outcome of PD-1-based immunotherapy in melanoma patients [97]. These data correlate with those from a recent study that makes the case for the reduced serum content of SCFAs being a biomarker of refractory non-small cell lung cancer (NSCLC) [67]. According to Boticcelli et al., lower levels of SCFA are found in the fecal samples of patients with a poor prognosis treated with Nivolumab, a human PD-1-blocking antibody. Together, these results show that gut microbiota-induced immune effects are dependent on the specific cancer therapy and that certain blood lipid biomarkers are able to predict this relationship.

When cancer care is delayed, patient treatment is associated with greater clinical complications and a lower survival rate. In order to have the best chance for a successful treatment and prognosis, an early and precise diagnosis of cancer progression before and during the treatment is critical. Thus, identifying biomarkers that can monitor the tumor response in every stage of treatment has huge clinical implications. Further studies will be needed to correlate the lipid profile with the immune cell phenotype and immune checkpoint expression within the tumor. These data will help to discriminate between pro-inflammatory and immunosuppressive TME populations, resulting in more accurate biomarkers of cancer progression.

#### **4. Active Modulation of Lipid Metabolism to Improve CAR T Cell Therapy**

Chimeric antigen receptor—engineered T cell (CAR T) therapy has demonstrated its long-term clinical benefit for patients with advanced cancers [98]. CART therapy involves genetically modified patient T cells with chimeric antigen receptors that recognize specific antigens on the tumor cell surface. The antitumor efficacy of immunotherapy against hematologic cancers has been extended to other tumors [99–101]. Among diverse potential targets, such as CD19 for B-cell malignancies, GD2, a disialoganglioside glycolipid, was identified as a tumor antigen more than 30 years ago [102]. GD2 is normally present in developing brains and can be overexpressed in some tumors, with a greater recurrence in childhood cancer neuroblastoma, melanoma and diverse pediatric sarcomas [103]. However, while GD2-specific antibody therapies used in the treatment of neuroblastoma have been shown to be successful, the fatal neurotoxicity of GD2-specific CAR T cell therapy that has been observed in some studies suggests that GD2 may be a difficult target antigen for CAR T cell therapy [104].

Several studies and clinical trials reveal that CAR T cell therapy for leukemia achieved high rates of complete remission, but therapy-relapsed leukemia remains a significant source of mortality [105]. Because T cell exhaustion elevates the risk of relapse [106], additional research on how to avoid this detrimental effect is urgently needed. Differentiated effector T cells use glycolysis for proliferation, and after activation they ultimately succumb [21]. Only a small proportion of long-surviving memory T cells with OXPHOS-mediated ATP production contributes to a favorable and durable antitumor response in the TME [107]. Since Notch signaling, a conserved cellular interaction mechanism,

promotes mitochondrial biogenesis and FA synthesis, recent studies have evaluated the impact of the manipulation of this metabolic pathway on the success of CAR T cells. According to Kondo et al. [108], the overexpression of Notch and its downstream gene Forkhead box M1 (FOXM1) results in enhanced anti-tumor effects as compared with conventional CART cells, suggesting a novel strategy to improve CART-based therapy [108].

The success of CAR T cell therapy in treating hematological malignancies is limited in solid tumors, where finding, entering and surviving in the tumor are extra challenges [109]. Other restrictions are driven by constraints from the on-target off-tumor toxicity of CAR T cells, where the lack of tumor specificity increases the potential risk for normal tissues to be attacked by CAR T cells [109,110]. In order to avoid these limitations, new strategies have focused on providing an anti-tumor effect with an absence of side-effects. Besides the enormous ability of PUFAs, such as AA, EPA and DHA, to regulate the immune responses, as presented above, gamma-linolenic acid (GLA, 18:3 n-6) has shown a selective effect against tumor cells [111]. According to an open-label clinical study that included 21 patients with stage IV glioma, the intra-tumor injection of GLA enhanced the sensitivity of tumor cells to chemotherapeutic drugs and radiation, producing tumor regression without harming normal cells [112]. Additionally, together with AA, EPA and DHA, GLA has been reported to regulate the antioxidant properties of glutathione peroxidase 4 (GPX4), as well as the levels of cytokines such as IL-1, IL-6 and tumor necrosis factor alpha (TNF-α) that play essential roles in inflammation [113,114]. These data suggest that the combination of PUFAs as an adjuvant may help immunotherapy block tumor progression.

Lipid metabolism has a dual impact on CAR T cell therapy. Lipids can systematically fuel tumor cells and immune cells. However, enhancing the immune response via CAR T cells presents evident advantages beyond the described obstacles. T cells can be successfully designed and prepared for the restricted metabolic conditions within the TME [115]. Identifying and reprogramming the mechanisms involved in the dysfunction of CAR T cells may help support more proliferative and ultimately successful CART cell-based therapies [109]. Therefore, metabolic targets that include the lipid metabolism may generate improved CAR T cells, so as to avoid cancer relapses related to T cell disability.

#### **5. Conclusions**

The complexity and variability of tumors still constitute a challenge for physicians and researchers. Although immunotherapy has attained ambitious milestones and improved prognoses for cancer patients, the systemic character and self-regulation capacity of immunity should be considered in order to obtain improvements. A multi-focal anti-tumor strategy, in combination with other treatments, appears to be required in order to avoid relapses; moreover, it should draw from diverse perspectives: First, a global intervention, by for instance modulating the gut microbiota, which could have positive effects on the immune cell activity; second, an early and precise diagnosis so as to achieve better clinical outcomes; and third, targeted treatments, where genetically engineered patient CAR T cells have already shown clinical benefits.

Current treatment limitations are related to an immunosuppressive TME, which modifies the T cell function in terms of differentiation and exhaustion. Combining CAR T cells with checkpoint inhibitors and the depletion of suppressive factors in the microenvironment via lipid targets may mitigate this phenomenon. Although new studies will be necessary to characterize specific metabolic pathways implicated in the immune response to tumor cells, data suggest that lipid reprogramming will be key to generating a favorable metabolic environment to avoid tumor evasion.

In conclusion, the modulation of the immune system has been extensively demonstrated to be an effective cancer treatment. However, further investigations should focus on reducing treatment limitations that ultimately lead to tumor relapse. Several studies are currently focusing on therapy improvements by facilitating energy influx to T cells, where lipids play an essential role. Targeting

lipid reprogramming in the immunity setting may generate new tools to create lasting, robust and personalized therapies against cancer.

**Author Contributions:** L.G.-d.-G. wrote the manuscript. D.B. and O.M. contributed to writing, reviewing and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The elaboration of this manuscript received no external funding.

**Acknowledgments:** We thank Sue Seif and Julián Abad for their excellent figure editing assistance. We also thank Rodrigo Gier and Dinesh Dilip Thakur for improving the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


leads to mitochondrial dysfunction in human CD8+ T lymphocytes. *J. Immunother. Cancer* **2019**, *7*, 151. [CrossRef] [PubMed]


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **The Cardiac Lipidome in Models of Cardiovascular Disease**

#### **Mateusz M. Tomczyk 1,2,3 and Vernon W. Dolinsky 1,2,3,\***


Received: 22 April 2020; Accepted: 11 June 2020; Published: 17 June 2020

**Abstract:** Cardiovascular disease (CVD) is the leading cause of death worldwide. There are numerous factors involved in the development of CVD. Among these, lipids have an important role in maintaining the myocardial cell structure as well as cardiac function. Fatty acids (FA) are utilized for energy, but also contribute to the pathogenesis of CVD and heart failure. Advances in mass spectrometry methods have enabled the comprehensive analysis of a plethora of lipid species from a single sample comprised of a heterogeneous population of lipid molecules. Determining cardiac lipid alterations in different models of CVD identifies novel biomarkers as well as reveals molecular mechanisms that underlie disease development and progression. This information could inform the development of novel therapeutics in the treatment of CVD. Herein, we provide a review of recent studies of cardiac lipid profiles in myocardial infarction, obesity, and diabetic and dilated cardiomyopathy models of CVD by methods of mass spectrometry analysis.

**Keywords:** cardiovascular disease; heart failure; myocardial infarction; obesity; diabetic cardiomyopathy; dilated cardiomyopathy; lipids; lipidomics; mass spectrometry

#### **1. Introduction**

Cardiovascular disease (CVD) is the leading cause of death worldwide [1]. CVD encompasses stroke, cardiomyopathy, coronary artery disease (CAD), and other disorders that can lead to myocardial infarctions and heart failure. The pathophysiological processes in each of these diseases can differ, but lipids play a significant role in every model of CVD [2]. Lipid molecules are important structural components of cardiomyocyte plasma and organelle membranes. For example, a specific phospholipid molecular species composition is necessary for the assembly of the electron transport chain in the mitochondria [3]. In addition, fats are the primary fuels utilized for cardiac energy production [4–6]. Therefore, lipids have a direct role in cardiovascular function. On the other hand, regarding lipid excess experienced when diets high in fat are consumed, hyperlipidemia and hypercholesterolemia can result, which puts patients at risk for developing atherosclerosis and cardiometabolic disease [7,8].

The development of mass spectrometry (MS) technology such as high performance liquid or gas-chromatography MS (HPLC-MS/GC-MS) separation techniques and ionization methods such as electrospray ionization MS (ESI-MS), and matrix assisted laser desorption/ionization MS (MALDI-MS) have enabled the detailed analysis of chemically complex lipids from biological tissues, which contain heterogeneous pools of lipid species [9]. These MS methods are increasingly utilized to analyze multiple lipid species from a single sample in a methodology termed lipidomics. This has been an important advance for identifying potential biomarkers of disease. A number of studies have analyzed the

changes in serum lipid profiles of patients with CVD [10–16]. However, information about lipid profiles in cardiac tissues in models of CVD is more limited and has not been reviewed. The following review will focus on recent lipidomic research findings about lipid profiles in cardiac tissue in experimental models of CVD that has contributed novel information about lipid biomarkers for myocardial infarction, obesity, and diabetic and dilated cardiomyopathies by MS methods. Furthermore, this review will focus on current and novel therapies that alter cardiac lipid profiles.

#### *1.1. Importance of Lipids in the Development of Cardiovascular Disease*

Lipids are a class of amphipathic molecules that are characterized as being insoluble in water [17]. They consist of a wide array of structures including some that are depicted in Table 1. Lipids play an important role in CVD development. Beyond their well recognized structural function in lipid bilayers [18], lipids can also act as signalling molecules and secondary messenger molecules such as those involved in G protein coupled receptor signalling [19,20]. An excess of deleterious lipid species can also contribute to CVD progression [7]. Obesity is a growing epidemic and patients characterized as obese are at risk for developing cardiovascular complications that could be linked to 3.4 million deaths worldwide in 2010 [21]. High fat and high cholesterol diets found in Western countries contribute to the development of cardiovascular risk factors such as hyperlipidemia and hypercholesterolemia [8]. High levels of these circulating lipids can lead to the accumulation of lipid plaques in arterial walls, which are also known as atherosclerosis [22]. Specifically, high quantities of low-density lipoprotein (LDL) increase the likelihood of LDL translocating from the arterial lumen to the endothelial intima [23]. LDL oxidation results in the release of cytokines, which signal uptake of the modified lipoproteins by macrophages [24]. Macrophage-filled particles or foam cells can efflux cholesterol out of the arterial wall into the blood stream or undergo apoptosis, which results in fatty streaks [25]. Fatty streaks are then converted to fibrous plaques, which can block arterial blood flow. Furthermore, macrophages release growth factors, which initiates smooth muscle cell proliferation from the media across the internal elastic membrane and into the intima. This results in further bulging and blockage of blood flow [25]. CAD is a result of atherosclerotic plaques that occur in the micro vessels, which supply blood to the heart. When these arteries are blocked, it results in ischemic injury as a result of hypoxic conditions [26]. In this environment, the heart relies on anaerobic respiration. Further cardiovascular compensation and influx of blood flow can result in reperfusion injury since sudden increases in oxygen leads to increased reactive oxygen species (ROS) and calcium flux, which causes cardiomyocyte damage (e.g., myocardial infarction (MI)) and death [26].

High density lipoprotein (HDL) and LDL cholesterol are standard measurements for patients at risk for the development of myocardial infarctions and CVD in the clinic [27]. Troponin I and creatine kinase are used as markers for cardiac damage [28]. However, new biomarkers for earlier disease diagnosis are needed to prevent CVD progression. Recent advances in MS technology have enabled the determination of lipid quantities and composition in serum as well as in myocardial tissues. In order to analyze the large datasets that accompany lipidomic analyses, researchers must apply consistent computational and statistical approaches. The lipidomics standard initiative, launched in 2018, aims to overcome challenges presented by working with lipidomic data [29,30]. Specifically, using software for lipid annotation, overreporting and using arbitrary units rather than concentrations when reporting lipid species. For example, according to this initiative, when quantifying lipids from tissue internal standards must be added prior to lipid extraction, standards should not be present in samples and tissue samples should be normalized to wet weight or protein [29]. Standardization is critical in order to determine clinical reference values, which can bring the lipid biomarkers identified at the lab bench to clinical use at the bedside for patient diagnosis.

**Table 1.** Lipid Classes and Examples of General Structures.

Table is limited to lipids discussed in the review. Structures are examples or general structures. R1, R2, R3, and R4 indicate unspecified fatty acid groups. X specifies phosphatidyl head group. Structures made with MarvinSketch Version 20.11 [31].

#### *1.2. Cardiac Lipid Composition*

The heart is composed of numerous cell and tissue types. Cardiomyocytes account for the largest percentage (30–40%) of cells within the heart, which occupy ~70–85% of heart volume [32,33]. Studies investigating the lipid composition in the heart began in the 1950s. Gray and colleagues performed the first studies to isolate and determine the composition of phospholipids from the ox heart by chromatography separation [34]. These studies established that lipids, which constitute cardiac tissues include free fatty acids (FA), triglycerides (TG), diglycerides (DG), cardiolipin (CL) phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) [35]. A recent large scale lipidomic study in rats has been able to decipher tissue-specific lipid composition [36]. These data reinforce the classical cardiac lipid composition and compare the lipid composition of major tissue types. The heart exhibited a high abundance of PC, PE, PS, phosphatidylinositol (PI), phosphatidylglycerol (PG), and CL species [36]. PG is a precursor for CL synthesis and was enriched in the heart when compared to other tissues, which may be indicative of the high mitochondrial content in cardiac tissue [36]. Mitochondria are closely linked to cardiac function since cardiomyocyte contractility require an abundance of ATP production.

Cardiac muscle contains high numbers of mitochondria in order to produce sufficient amounts of ATP to supply the heart with abundant energy needed for the mechanical action of pumping blood throughout the body. CL is abundant in the heart and is a major phospholipid of the inner mitochondrial membrane [37]. It is a unique phospholipid since it composed of two glycerol phosphatidyl moieties. This means CL is comprised of four fatty acyl molecules (Table 1) [38]. Tetra-linoleic acid is the predominant form of CL in the mature heart [38]. It is responsible for the mitochondrial structure and the function of inner mitochondrial membrane proteins. For example, it is required for the efficient transfer of electrons and the formation of super complexes in the respiratory chain [39]. Therefore, the cardiac lipid composition and the lipids in the mitochondria play an important role in cardiac energy production and, as a result, are implicated in cardiac function.

#### *1.3. Cardiac Lipid Utilization*

FA and glucose are the major fuel sources of the heart [5,40]. Specifically, the utilization of FA through beta-oxidation and subsequent oxidation-reduction reactions within the tricarboxylic (TCA) cycle are responsible for the majority of ATP production in the heart [41]. FA are transported into the cardiomyocyte through the plasma membrane by the FA binding protein. CD36 and FA transport proteins (FATPs) [42,43]. The FA are acylated by some transporters (e.g., FATPs) or through acyl-Coenzyme A synthetase. Carnitine palmitoyltransferase I (CPT-I) then converts the acyl-CoA derivatives into long-chain acylcarnitine molecules on the outer side of the outer mitochondrial membrane. The acyl-carnitine molecules are transported through the inner membrane space and then across the inner mitochondrial membrane by carnitine-acylcarnitine translocase [42]. On the inner membrane, CPT-II is responsible for transferring the acyl residue from carnitine back onto a CoA molecule. The FA acyl-CoA molecules can then enter beta-oxidation for the conversion of FA into acetyl-CoA, which enters the TCA cycle [42]. The TCA cycle produces nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) molecules, which can be utilized by the electron transport chain for the production of ATP [44].

In CVD including MI and pathological cardiac hypertrophy, cardiac metabolism changes from a state of primarily relying on FA and glucose through oxidative phosphorylation to utilizing anaerobic energy production such as glycolysis [41,45,46]. Glycolysis is an inefficient means of energy production in the failing heart [41]. Inefficient ATP production can lead to increased ROS and further oxidation of phospholipids and cardiotoxicity [47,48]. Lipid molecules play an important role in cardiac energy production, the plasma membrane, and organelle composition as well as the progression and pathogenesis of CVD such as the development of atherosclerotic plaques. Mass spectrometry methods are now being used in order to determine lipid levels and examine lipid composition in models of CVD.

#### **2. Models of Cardiovascular Disease**

One of the first MS studies performed in cardiac tissue was reported in 1968 by Funasaki and Gilbertson who isolated and identified cholesteryl alkyl ethers from bovine cardiac muscle [49]. Advancements in MS methods [50] have enabled researchers to use these technologies to investigate how lipid species are being altered in the cardiac lipidome (the entire lipid composition of the heart) and how it is altered in different models of CVD.

#### *2.1. Cardiac Lipid Profiles in Experimental Myocardial Infarction Models*

MI is the loss of blood flow that leads to myocardial damage [26]. As described above, atherosclerotic plaques are the main cause for MIs. Hsueh et al. were the first to identify that ischemia stimulates fatty acid release in rabbit heart tissue by chromatography separation [51]. The following study identified increases in arachidonic acid in ischemic canine myocardium using high pressure LC separation [52]. Researchers also began using MS methods to examine molecular changes that occur post-MI. The first study to utilize MS technology to examine lipids after a MI was in 1979, where Epps and colleagues identified an increase in N-acylethanolamine 24 h after a canine heart was subjected to ligation of the left descending artery [53]. Since then, numerous studies have examined the effect of MIs on cardiac lipids. For example, in a recent paper utilizing an ischemia and/or starvation model in the H9c2 rat cardiomyocyte cell line, differences in lipid levels including PC (34:1), PC (36:2), lyso-phosphatidylcholine lysoPC (16:0), lysoPC (18:1), lysoPC (18:0), PE (34:1), PS (36:1), PI (36:2), PI (38:3), PI (38:5), sphingomyelin (SM) (34:1), CL (68:4), CL (72:5), and CL (74:7) were observed [54]. Increases in lyso-PCs and decreases in CL were observed in ischemic/starvation conditions compared to controls [54]. Similar results were reported by Nam and colleagues who performed metabolomic and lipidomic analysis of rat hearts, which followed ligation of the left anterior descending coronary artery by ultra-HPLC-MS. In this animal model, gradual increases of free FA, ceramides, PE (40:6), lysoPE(16:0), PC (30:0), PC (32:1), lysoPC (16:0), lysoPC (o-16:0), lysoPC (o-18:0), PG (40:8), PG(42:9), lysoPG (18:2), PS (38:4), SM, and mono and triacylglycerides were observed [55]. Alterations in acylcarnitines, adenine, S-adenosyl methionine, adenosine monophosphate, NAD+, and succinic acid were reported, which suggested a disruption in lipid metabolism. Additional lipidomic studies have also described increased de novo ceramide synthesis and accumulation of long chain ceramides in human serum myocardial tissue [56]. Using an animal model of ischemic left ventricular dysfunction and the serine palmitoyl transferase (SPT) inhibitor, myriocin, this study also showed that reduced ceramide accumulation (C16, C24:1, C24) prevented ventricular remodelling post-MI [56].

A study using a rat MI model reported increases in lysoPC (16:0), lysoPC (18:0), lysoPC (18:2), lysoPC (18:1), lysoPC (20:4), and lysoPE (18:0) in heart tissue by MALDI-MS imaging technology similar to that of the cell and animal studies discussed previously [57]. However, in a comprehensive lipidomic analysis of post-MI cardiac mouse tissue by Halade and colleagues, strong increases in lysophospholipids were not observed [58]. This discrepancy could be due to species' specific differences but warrants further investigation. MALDI imaging MS technology has been used to identify spatial distribution of lipids within cardiac tissue such as the even distribution of tetralinoleic acid CL within healthy heart sections [54,55]. The study by Halade et al. is unique as it identified FA substrates such as arachidonic, docosahexaenoic, eicosapentaenoic acid, and their bioactive lipid mediators (e.g., hydroxydocosahexaenoic acid, hydroxyeicosapentaenoic acid) in infarct LV post MI tissue using MALDI-imaging [53]. The study also reported an increase in PC (36:4), PC (40:8), PC (40:6), and oxidized PC (O-32:0), PC (O-34:0), and PC (O-42:2), which could be indicative of oxidative stress as a result of ischemic conditions that occur during MIs. More specialized studies have focused on the lipid composition of organelles' membranes such as the nucleus. Williams and colleagues employed ESI-MS methods and identified the loss of choline and ethanolamine glycerophospholipids in the nuclear membrane from ischemic and reperfused rat myocardial tissue [59].

Novel studies are now using transgenic and knockout (KO) animal models to decipher pathways, which contribute metabolic signalling in CVD. In a follow-up study, researchers used lipoxygenase

(LOX−/−) deficient mice to study its effect on ischemic heart failure [60]. LOX enzymes are a class of FA metabolizing enzymes and, therefore, play an important role in regulating bioactive lipid mediators and FA utilization during myocardial injury [60]. Hearts from LOX deficient mice displayed both increases and decreases in certain PC and lysoPCs species with specific fatty acyl compositions [60]. Additionally, they exhibited increases in sphingolipids in comparison to wild-type mice. Ultimately, the LOX−/<sup>−</sup> mice showed altered lipidomic and metabolomic profiles and exhibited delayed heart failure progression and improved survival. However, additional studies are needed to decipher important protein, enzyme, and lipid targets that contribute to lipidome alterations during CVD progression and how these alterations can be prevented.

Lipidomic studies of cardiac tissue post MI allow for a greater understanding of how the cardiac lipidome is altered as well as the changes of the fatty acyl chains within these lipid classes. This is notable because it can provide insights into the molecular mechanisms of the pathogenesis of CVD. Understanding these changes may help the development of better therapies to prevent and treat MIs and could be translated to other models of CVD.

#### *2.2. Cardiac Lipid Profiles in Animal Models of Obesity*

Weight gain puts patients at risk for developing dyslipidemia and lipotoxicity [61]. In addition to hypertension and diabetes, patients that are characterized as obese are at twice the risk for developing cardiovascular complications [62]. Thus, analysis of the lipidome provides a wealth of information about mechanisms of disease progression. In a study comparing standard, high fat, or high fat/high sucrose (western) diets in rat hearts, researchers identified increases in C16, C18, C20, and C24 ceramides in the western diet group by HPLC-ESI-MS methods [63]. As expected, increased cardiac TG levels were also observed [63]. In heart tissue of mice fed a high fat diet, increases in polyunsaturated fatty acyl chains were observed in ceramides, glycosphingolipids, and sphingomyelins whereas decreases in monounsaturated fatty acyl chains were observed in phospholipids and sphingomyelins [64]. In another study, researchers determined that feeding mice a diet enriched in polyunsaturated fatty acids (arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid supplemented) for two weeks decreased cardiac phospholipids containing linoleic acid when compared to control mice on a fish meal free diet [65]. This study also went on to describe differences in the oxylipin profiles of tissues in a targeted lipidomics approach.

Peroxisome proliferator-activated receptor-gamma coactivator 1β (PGC1β) is a transcriptional co-activator, which has a role in regulating mitochondrial biogenesis genes and is thought to have a role in the development of obesity and diabetes [66,67]. McCombie et al. utilized a PGC1β KO mouse model to investigate lipidomic changes induced by a high fat diet [68]. In this study, LC-MS lipidomics of cardiac tissue revealed alterations in polar lipid composition and increases in TG. The preceding study focused on results from a combined dataset of male and female mice. However, they did report larger differences in male datasets in KO mice fed a high fat diet when compared to females using partial least squares discriminant analysis (PLS-DA) models, which indicated the importance of performing sex-specific lipidomic studies.

Using cardiac specific diacylglycerol O-acyltransferase 1 (DGAT1) transgenic mice as a model of cardiac steatosis, LC-MS analysis of myocardial tissue revealed no changes in ceramides [62]. In contrast, exposure of these mice to angiotensin II resulted in increased ceramide levels [62]. Increased ceramide ratios (C16:0/24:0) in plasma have been associated with increased cardiac remodelling and cardiac dysfunction in a human study, which examined 2652 Framingham Offspring Study participants [69]. Therefore, activation of the renin-angiotensin system exacerbates the risk of cardiac lipid remodelling. This could be a rationale for investigating whether angiotensin converting enzyme (ACE) inhibitors prevent increased ceramides in models of CVD.

More comprehensive models are now being developed where diets are coupled with models of CVD and aging. A recent study investigated the effect of high-unsaturated fatty acid diet (HUFA) on rats subjected to supra-valvar aortic stenosis (SVAS). The study reported decreases in unsaturated (oleic and linoleic) free fatty acids as well as diacylglycerol and triacylglycerol molecules in SVAS heart tissue. However, the HUFA diet did not restore these lipids to normal levels [70]. In one model, mice on a PUFA diet had impaired wound healing post-MI [71]. The study specifically observed an increase in plasma arachidonic acid by LC-MS analysis, which implicated a high PUFA diet that increases pro-inflammatory lipid metabolites capable of affecting post-MI tissue [71]. In another study combining obesity and MI, researchers examined mitochondrial lipid species from cardiac tissue [72]. Decreases in PCs, PEs, and increases in TGs, lysoPCs, and lysoPEs were reported in mitochondrial lipids from total cardiac tissue of rats subject to MI. In contrast, MI rats fed high fat diets did not exhibit such drastic changes in cardiac mitochondrial glycerophospholipids [72]. This study also reported decreases in total CL. Under closer inspection, the researchers reported decreases in CL (18:2) but increases in CL (20:4, 22:6) in high fat fed groups post MI. This interesting finding suggests that CL composition was altered from a form enriched with linoleic acid to one that is increased in arachidonic acid [72]. The study went on to show an association between levels of fibrosis with cardiac lipids such as TG, CL, ceramide, and several plasma microRNA (miRNA) species including 194-5p, 301a-3p, 144-5p, and 15b-5p [72]. These findings could be significant since miRNA play an important role in transcriptional regulation. Studies such as these could bridge the gap between lipidomic alterations and epigenetic regulation. Complex models of obesity and MI are more representative of cardiac lipid changes that occur in patients in the clinic. Furthermore, they can be used to more accurately decipher the molecular pathways and epigenetic changes that occur in these diseased states.

#### *2.3. Cardiac Lipid Profiles in Diabetic Cardiomyopathy Models*

Diabetic cardiomyopathy is characterized by structural and functional changes that occur in the myocardium as a result of diabetes mellitus [73]. Specifically, these changes occur without the presence of CAD or hypertension but are a direct result of diabetes [73]. Hypertrophy or thickening of ventricular walls is a characteristic of diabetic cardiomyopathy and leads to diastolic dysfunction typically with conserved systolic function [74,75]. Ultimately, these structural and functional changes can lead to heart failure. The first study to use MS technology (by ESI-MS) was performed by Han and colleagues who examined alterations in the lipid profile of the diabetic myocardium [76]. Utilizing a rat model and a single injection of streptozotocin, they identified alterations to ethanolamine glycerophospholipids. Specifically, a 24% increase in PE and a 44% increase in plasmenylethanolamine could be restored by insulin treatment [76]. The study identified a 60% decrease in TG, which was not prevented by insulin treatment. Furthermore, a 44% increase in PI and small increases in PG and PS molecular species were also observed. No changes in CL were identified in the heart tissue from this streptozotocin-induced diabetes model. Reminiscent of the PUFA MI model, this group also identified a predominance of PC molecules with arachidonic acid FA moieties in their lipid fractions. However, no statistically significant differences between the diabetic and control rats were observed [76]. The same group followed up with a separate study to examine the acylcarnitine species from cardiac tissue in the streptozotocin-induced diabetes rat model using ESI-MS approaches. They identified a four-fold increase in long-chain acylcarnitines (16:0, 18:2, 18:1, 20:4) in diabetic myocardium compared to controls that could be partially or fully reversed with insulin treatment [77]. These data suggest that impaired FA transport or β-oxidation of FA leads to the accumulation of acylcarnitine species in diabetic cardiomyopathy. The same group also performed a study in streptozotocin-induced diabetic mice using a shotgun MS approach. Unlike rats, CL depletion as well the CL precursor PG was observed in diabetic myocardium mice (7.2 nmol/mg to 3.1 nmol/mg in diabetic hearts) [78]. These findings suggest that CL depletion occurs through ineffective FA utilization, which leads to lipotoxicity that manifests into diabetic cardiomyopathy.

Using leptin receptor deficient mice as a model of diabetic cardiomyopathy increases in TGs and DGs as determined by MS analysis, which were observed in cardiac tissue. There were increases in C14:1, C16:1, C16:0, C18:1, and C20:4 free fatty acid molecular subspecies in the leptin receptor deficient mice compared to controls [79]. Similar to the MI models reviewed above, leptin receptor deficient mice also exhibited increases in ceramides, SM, PC, lysoPC, and PE. In a more recent paper using UPLC/QTOF/MS with ESI positive and negative modes to distinguish acyl chains at the sn-1 and sn-2 positions in myocardial tissue revealed down regulation of PC (22:6/18:2), PC (22:6/18:1), PC (20:4/16:1), PC (16:1/18:3), PE (20:4/18:2), PE (20:4/16:0) and an increase in PC (20:2/18:2), PC (18:0/16:0), PC (20:4/18:0) in a streptozotocin-induced rat model [80]. This study revealed that diabetic cardiomyopathy also induced differences in the fatty acyl chain composition of glycerolipids such as PC and PE. Reporting these changes allows for a more reliable comparison of changes in lipid profiles between models of CVD such as MI and other cardiomyopathies. The power of MS technology is now allowing researchers to decipher how lipid species are being affected at a global scale and at the chemical level. However, more investigation needs to occur in other cardiomyopathy models.

#### *2.4. Lipid Profiles in Cardiac Hypertrophy*

Cardiac hypertrophy is characterized by ventricular wall thickening, which can be accompanied by both reduced systolic and diastolic function [81]. Pathological causes for cardiac hypertrophy include hypertension and valvular disease [82]. A common experimental model of cardiac hypertrophy is transverse aortic constriction (TAC) in which surgical ligation of the transverse aorta leads to a pressure-overload induced hypertrophy [83]. A recent study used this model of TAC to investigate molecular changes in mice with cardiac restricted acyl-coenzyme A synthetase-1 overexpression (ACSL1) [84]. ACSL1 is responsible for mediating the activation of long-chain fatty acids to acyl-CoA substrates, which can undergo further β-oxidation for energy production within the heart [85]. LC-ESI-MS/MS of TAC heart tissue revealed increases in ceremide levels (C16, C24:1, C24), which were not observed in the ACSL1 overexpressing hearts subject to TAC. ACSL1 TAC hearts exhibited increases in C20 and C22 ceramides. The authors suggest that ACSL1 overexpression could, therefore, mitigate TAC-induced cardiac hypertrophy through mitochondrial oxidative metabolism.

#### *2.5. Lipid Profiles in Dilated Cardiomyopathy*

Dilated cardiomyopathy is characterized by an enlarged ventricle, ventricular wall thinning, reduced ejection fraction, and decreased cardiac output [86]. In contrast to diabetic cardiomyopathy, it is characterized by both systolic and diastolic dysfunction. It can be caused by genetic mutations (e.g., Tafazzin, β-Myosin heavy chain, α-Tropomyosin, Cardiac troponin T, Lamin/C (LMNA)) or chemical toxicity (e.g., anthracycline chemotherapeutics). However, it is often idiopathic [87]. One study performed lipidomic analysis of serum from control individuals and patients with dilated cardiomyopathy as a result of an LMNA mutation. In the serum, changes in PC (38:5e, 38:2) and TGs were identified [10]. Sparagna and colleagues performed a study examining CL in human left ventricular tissue samples and in the spontaneously hypertensive heart failure (SHHF) rat model, which exhibits idiopathic dilated cardiomyopathy (IDC) [88]. The human tissue in this study was isolated from the left ventricle of explanted hearts of patients diagnosed with IDC (*n* = 10) and exhibited decreases in tetra-linoleic CL [88]. Similar decreases in tetra-linoleoyl CL in subsarcolemmal and interfibrillar cardiac mitochondria isolated from 5-month rats and 15-month rats were observed in parallel with increases in CL species with oleic and arachidonic acid side chains. Additionally, a positive relationship with decreased tetra-linoleic CL and impaired cytochrome oxidase activity was observed [88]. This shows the importance that cardiac tissue lipid composition plays in mitochondrial function. In the same study, a rat model of heart failure using SHHF rats subject to thoracic aortic banding (TAB) surgery was also used and decreased tetra-linoleic CL. Increased CL species containing oleic and arachidonic acid side chains were observed in the rat heart tissue. While, in a follow-up study, LC-MS/MS analysis of cardiac tissue explants from eight human patients with dilated cardiomyopathy revealed lower levels of linoleic acid and also reported similar increases in arachidonic and docosahexaenoic acid phospholipid species [89]. These elevated polyunsaturated fatty acid product/precursor ratios suggested that delta-6-desaturase enzyme activity was elevated in dilated cardiomyopathy. Notably, inhibition of the delta-6-desaturase enzyme (with SC-26196 for four weeks) reversed these changes

in polyunsaturated fatty acid composition in two different rat models of heart failure (SHHF and TAC). Inhibition of delta-6-desaturase also attenuated elevations in pathogenic eicosanoids and lipid peroxides and normalized the CL fatty acyl chain composition in the rat heart [89]. Another study performed LC-ESI-MS in left ventricular tissue from pediatric patients with IDC and reported similar decreases in total and tetra linoleic CL. The authors do, however, report a unique pediatric cardiac CL profile attributed to differences in the expression of CL biosynthesis genes with age [90].

Doxorubicin (DOX) is an anthracycline chemotherapeutic used in treating pediatric leukemias and lymphomas, but its utility is limited since high dosages of DOX put patients at risk for developing a dilated cardiomyopathy [91,92]. In animal models, DOX is frequently used to induce dilated cardiomyopathy. There has been a modest number of published studies examining cardiac tissue profiles by MS methods in DOX models of dilated cardiomyopathy. In one study, male and female rats were injected with 2 mg/kg of DOX weekly for seven weeks and lipidomic analysis was performed [93]. This study uncovered sex-specific differences in the cardiac lipid profile with response to DOX treatment. Male rats exhibited decreased phospholipid content in cardiac tissue after DOX treatment. Specifically, sex-specific fatty acid composition of PE and PC were different in males and females prior to and after DOX treatment. Furthermore, analysis of CL species revealed no sex differences, but DOX treatment induced a decrease in the most abundant tetra-linoleic CL and an increase in every other CL species [93]. In another study, rats were injected with 2.5 mg/kg of DOX for two weeks and MS analysis of ceramides revealed an increase in C16 and C18 ceramide levels in heart tissue [63]. These two studies illustrate some of the similarities of cardiac lipid profile alterations to other models of CVD discussed above including the depletion of CL in models of diabetic cardiomyopathy and obesity and the increase in ceramides seen in MI models.

#### *2.6. Similarities in Cardiac Lipid Profiles in Models of Cardiovascular Disease*

CVD encompasses a wide range of cardiac diseases that have different underlying causes. Cardiac lipid profiles in these models share many similarities. Specifically, most models (whether they be MI, obesity, diabetes, or dilated cardiomyopathy) show increases in ceramide, sphingomyelin, and lyso-phospholipids in cardiac tissue, which suggests these may be molecular markers of disease progression. Increases in ceramides have also been linked to increases in apoptosis in a variety of models including neonatal rat cardiomyocytes [94–96]. Furthermore, ceramides have been shown to modulate lipotoxic cardiomyopathy in mice through interactions with proteins involved in cardiac contractility, apoptosis, and lipogenesis (myosin chaperone, annexin, and fatty acid synthase) [97]. Other similarities in the findings from lipidomic studies in different models of CVD was increases in arachidonic acid fatty acid acyl chains in the failing heart. Specifically, in most models of CVD, when measured, there appears to be decreases in tetralinoleoyl CL species and increases in other forms of CL such as those containing arachidonic. Since CL is so closely linked to the electron transport chain, changes in lipid composition of CL could be related to disrupted oxidative phosphorylation super-complex formation and, thus, decreases in cardiac energy production.

Where the lipidomic studies differ is in changes to phospholipid fatty acyl composition. Specifically, phospholipid fatty acid molecules are shown to be increased and decreased in different models of CVD. This could be an indication of different alterations to FA metabolism, which may be present in different models (e.g., diabetic vs. dilated cardiomyopathies). Other differences include changes in glycerolipids. For example, obesity and diabetic models frequently cite increases in TG and DG lipid species. Specifically, DG lipid accumulation has been linked to impaired insulin-stimulated glucose oxidation in the heart [98] and incomplete oxidation of fatty acids in skeletal muscle, which leads to insulin resistance and mitochondrial dysfunction [99]. In contrast, MI and dilated cardiomyopathy models do not exhibit altered DG species or did not report them altogether. The lipidomic studies discussed are summarized in Table 2.



#### *Metabolites* **2020** , *10*, 254



*Metabolites* **2020** , *10*, 254

227

Phosphatidylserine.

 PG:

PS: Acylcarnitine.

 AA: Arachidonic Acid. ↑ increase. ↓ decrease. increase or decrease depending on FA chain. -: No change. Blank: Not reported.

Phosphatidylglycerol.

 LysoPL:

Lyso-phospholipids.

 OxPL: Oxidized

phospholipids.

 CL: Cardiolipin.

 CER: Ceramides.

 SM:

sphingomyelin.

 AC:

#### **3. The E**ff**ect of Current and Novel Therapies on Cardiac Lipid Profiles**

Extensive efforts have gone into examining how cardiac lipid profiles are altered in different models of CVD. The next area of lipidomic research reviewed focuses on understanding how current therapeutics used in treating cardiovascular and lipid disorders affect cardiac lipids.

#### *3.1. The E*ff*ect of Non-Pharmacological Interventions on Cardiac Lipid Profiles*

Non-pharmacological interventions such as diet and lifestyle changes are often the front line to prevent CVD in patients who are at risk [100]. A recent study utilized the power of MS technology to examine how cardiac lipid profiles are altered in models of exercise and CVD [101]. Specifically, they were interested in examining the differences between physiological hypertrophy that occurs as a compensatory mechanism in response to exercise and the pathological hypertrophy that occurs during CVD. Using a swim model of exercise and a four-week model of pressure overload TAC, LC-MS/MS technology was utilized to perform lipidomic analysis of cardiac tissue. A total of 104 lipid species were significantly altered in swimming mice compared to controls, and 100 lipid species in the severe TAC model. Lipid concentrations in this study were determined by internal standards and normalized to levels of PC rather than protein concentrations or tissue weight. In these models, differences between PC lipids were not observed. However, phospholipids such as alkylphosphatidylcholine (PC(O)), alkylkphosphatidylethanolamine (PE(O)), and phosphatidyl-ethanolamine plasmalogens (PE(P)) were decreased in the hearts of exercised mice and unchanged in the TAC mice. Furthermore, sphingolipids were decreased in cardiac tissue from the exercise model and increased in the TAC model of CVDs. This study suggests that differences in cardiac sphingolipid levels could distinguish between physiological and pathological hypertrophy, which are indicative of damage to cardiomyocyte cell membranes. Identification of how non-pharmacological interventions affect myocardial lipids is important since it may provide information on the actionable mechanism of classic and novel therapeutics used in treating CVD.

#### *3.2. The E*ff*ect of Commonly Prescribed CVD Medications on Cardiac Lipids*

There is a modest amount of literature that focuses on how common drugs (e.g., statins, fenofibrates) used to treat cardiovascular and lipid disorders affect the cardiac tissue lipidome. Statins prevent cholesterol synthesis by inhibiting 3-hydroxy-3-methyl-glutaryl–CoA reductase and, in turn, reduce circulating levels of LDL. There are several studies that examine serum lipidomics in patients treated with statins [27,102–105]. They report decreased plasma TGs and circulating sphingomyelins in patients treated with statins. However, to date, no study has used MS technology to examine the effect of statin therapy on the cardiac tissue lipidome in obesity models.

Other commonly used therapeutics in treating CVD such as atherosclerosis are fibric acid derivatives. Drugs such as gemfibrozil, fenofibrate, and clofibrate lower TG and LDL levels by increasing lipoprotein lipase activity and inhibiting synthesis of very low-density lipoprotein by activating peroxisome proliferator activated receptor α (PPARα)[46,106]. The Fibrate Intervention and Event Lowering in Diabetes (FIELD) study identified that patients treated with fenofibrates did not benefit the primary endpoint of coronary heart disease events [107]. A substudy of the FIELD assessed serum from patients treated with fenofibrates and identified decreases in lysoPCs and increases in SM. Consistent with the paucity of research surrounding the cardiac lipidome in response to statin therapy in models of CVD, there is also a lack of studies that examine how these tissues are affected by other classic drugs used in treating CVD such as fibric acid derivates. Future studies should aim to examine how drugs already used in treating CVD affect cardiac lipids.

#### *3.3. The E*ff*ect of Natural Health Products and Novel Drugs on Cardiac Lipid Profiles*

Resveratrol is a polyphenolic molecule derived from plants shown to improve myocardial lipid oxidation and cardiac function in rats [108]. We have shown that, in the spontaneously hypertensive rat model of cardiac hypertrophy as well as the Wistar control rat strain, resveratrol attenuates pathological cardiac hypertrophy and using mass spectroscopy increases total CL mass as well as the tetra-linoleic CL species [109]. Therefore, resveratrol-induced increases in cardiac CL could be linked to improved mitochondrial function. Berberine is a naturally occurring alkaloid extracted from various plants and used in traditional Chinese medicine. It is also available at health food markets [110]. A recent study examined the effect of berberine on myocardial lipid profiles in a high fat, high sucrose diet and a streptozotocin-induced rat model of diabetic cardiomyopathy [110]. Berberine partially reversed alterations to PC (16:0/20:4), PC (18:0/18:2), PC (18:0/18:2), PC (18:0/22:5), PC (20:4/0:0), PC (20:4/18:0), PC (20:4/20:2), PE (18:2/0:0), and SM (d18:0/16:0) in diabetic heart tissue. Berberine also decreased SM, which is a lipid species often reported as upregulated in other models of CVD (obesity, dilated cardiomyopathy). Resveratrol and berberine are thought to have antioxidant capabilities as indicated by decreased ROS levels [111,112]. However, when compared to placebos in clinical trails, antioxidants have had little success in treating CVD [113]. This could be due to improper timing or dosing. Other concerns regarding natural health products such as resveratrol or berberine is their lack of specificity. These compounds have multiple targets, which means they can have a broad impact on metabolism. However, initial studies suggest that some natural health products could be broadly protective in CVD by modifying lipid profiles. This result merits further investigation [114–116]. Therefore, it may be efficacious to investigate novel drugs that have specific protein or lipid targets.

Another novel therapeutic that is gaining attention in treating CVD is elamipretide (a.k.a. Bendavia, MTP-131 and SS-31). Elamipretide is a cell permeable tetrapeptide, which is targeted to the mitochondria by binding directly to CL and reducing ROS formation while increasing mitochondrial function [117]. It has been shown to have cardioprotective effects in animal models of atherosclerotic renovascular disease [118], ischemic-reperfusion injury [119], myocardial infarction [120], hypertension [121], DOX-induced cardiomyopathy models [122], and improvement in mitochondrial function in failing human myocardium [123]. One study has employed MS approaches to examine how elamipretide alters lipids. Specifically, they examined a decrease in tetra-linoleic CL in explanted failing heart tissue from pediatric and adult patients. Treatment with elamipretide prevented changes in CL when compared to untreated controls [123]. The study reports coupling of oxidative phosphorylation supercomplex activity as the mechanism of action. However, more comprehensive lipidomics studies are needed to assess the effect of elamipretide on the entire cardiac lipidome.

#### **4. Conclusions**

Lipidomic analysis by MS technology is an expanding field of research. Lipids play an important role in cardiac structure, function, and disease progression. Utilizing this sensitive technique to determine changes that occur in cardiac lipid profiles in models of CVD (MI, obesity, diabetic, or dilated cardiomyopathies, etc.) is important in understanding the pathology behind each disease. Furthermore, performing lipidomic studies in experimental models of CVD holds the promise of increasing our understanding of how novel therapeutics affect the heart. New challenges facing the ever-growing field of lipidomics will include data standardization to generate comparable and reproducible results. Future cardiac lipidomic studies should also focus on sorted cell populations from cardiac tissue to address heterogenous cell populations found in cardiac tissue during CVD. Ultimately, the intention of utilizing MS approaches will be to integrate lipidomics data with other -omics technology to get a better understanding of how the cardiovascular system is affected in its entirety in disease models.

**Author Contributions:** Conceptualization: M.M.T. and V.W.D. Writing—original draft preparation: M.M.T. Writing—review and editing: M.M.T. and V.W.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** Thank you to Grant Hatch for his expert insights during the paper writing process. M.M.T. is the recipient of a Research Manitoba Studentship in Partnership with CHRIM. V.W.D. is the Allen Rouse-Manitoba Medical Services Foundation Basic Scientist.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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