*5.1. GAGs Roles in Tumor Immunology*

CD44-HA constitutes a molecular tandem that can affect tumor immunology by utilizing complex mechanisms [6]. Macrophages have a different expression of CD44 related to their functions, and their capacity to bind HA is variable. CD44 has the highest expression in M1 polarized macrophages, followed by M0 type of macrophages, whereas M2 type expression is similar to the latter. The higher CD44 expression in M1 induces increased binding of HA. On the other hand, the lower M2 expression favors a better internalization of HA. Therefore, the molecular mechanisms in the CD44-HA tandem exhibit subtleties to predict targeting behavior [245].

The presence of tumor-associated macrophages (TAMs) is correlated with the poor outcome in tumor-bearing organisms because these cells sustain the immune-suppression and enhance pro-tumoral mechanisms, and, last but not least, inhibit the actions of antitumoral drugs. Therefore, TAMs are preferred targets in tumor therapies [246]. Several years ago, a nanoparticle was designed comprising poly(lactic acid-co-glycolic acid-grafted HA (HA-g-PLGA) that carried a cytostatic, an active metabolite of irinotecan. At the acidic pH of the TME, HA is exposed, and by linking to the CD44 expressed on tumor cells and TAMs, it delivers the cytostatic intracellularly. Tumor cells continue to recruit TAMs that encounter carriers with the cytostatics; hence, the anti-proliferative effect is propagated [247].

In a recent study, novel carrier molecules were tested. Within the tested compounds, oligomeric HA (oHA) targeted CD44 receptors on TAMs for the delivery of curcumin (Cur) and baicalin (Bai) to overcome tumor resistance. The carrier had good cellular penetration and cytotoxicity upon tumor cells. In in vivo animal modelsof A549 tumor-bearing nude mice, the significant anti-tumoral effect was re-confirmed [248]. HA-based nanoparticles were tested as drug carriers in epithelial ovarian cancers to target TAMs specifically. Thus, HA nanoparticles that encapsulate miR-125b (HA-PEI-miR-125b) targeted TAMs in an experimental mouse model of syngeneic ID8-VEGF ovarian cancer and induced these cells to an immune-activating phenotype [249]. In the 4T1 breast cancer animal model, mesoporous Prussian blue (MPB) nanoparticles and LMWHA (LMWHA-MPB) were tested. This approach demonstrated that LMWHA-MPB penetrates M2 macrophages (pro-tumoral macrophages), which are subsequently diverted toward the M1 phenotype exhibiting anti-tumoral action. Therefore, LMWHA-MPB can induce TAMs pro-tumoral potential and can likewise be used in situ for microenvironmental tumoral regulation [250].

LMWHA per se was demonstrated to have an anti-tumoral effect in colorectal carcinoma. The immune response involves activated dendritic cells (DC). Authors have shown that preconditioning DC from tumors with LMWHA increased their ability to migrate in vitro and enhanced DC in vivo recruitment to regional lymph nodes. In a mouse animal model, tumor lysate-pulsed DC (DC/LMWHA) was administered, and a potent anti-tumor response was obtained. Splenocytes from animals treated with DC/LMWHA displayed higher proliferative capacity, enhanced IFN-γ production, and lower immunosuppressive cytokine levels. Therefore, LMWHA can be considered a new adjuvant candidate for DC-based anticancer vaccines [251].

Using HA's ability in reprogramming pro-tumoral M2 type TAMs to anti-tumor M1 macrophages, other nanoparticles with MnO2 were used to decrease tumor hypoxia chemoresistance in the breast cancer experimental model. Increased tumor oxygenation was obtained in conjunction with hypoxia-inducible factor-1 α (HIF-1α) and VEGF downregulation. When these nanoparticles were combined with classical cytostatic Dox, tumor growth/proliferation was inhibited [252].

As cancer immunotherapy has recently gained unprecedented momentum, HA's involvement as a drug carrier was tested. TC-1, a polymeric conjugate formed by HA and ovalbumin (OVA) as a foreign antigen, was tested using mouse lung tumor cells. This model showed that OVA257-264 peptide is presented complexed with MHC class I on the cells' surface. With this approach, the foreign antigen could induce an anti-tumor effect by enhancing the immune cells' attack. The mouse model's systemic administration showed that the conjugate is accumulated into tumor tissue and facilitates the cytotoxic T lymphocytes' (CTLs) attack of the tumor cells, thus inhibiting tumor proliferation [253].

OVA-loaded micelle consisting of PEGylated HA was tested for increasing the OVA uptake. The HA-coated micelle targeted CD44 on tumor cells and increased OVA cellular uptake more than 10 times. Loading tumor cells with a foreign antigen, such as OVA, would increase their recognition by CTLs, and thus, enhance destruction. In animal models, tumor growth was significantly inhibited, and the authors point out that in the case of cutaneous melanoma, this can be another approach to enhance immune-therapy [254]. The same principle was implemented in TC-1 mouse cells and lung cancer epithelial cells, using MMP9-responsive conjugates consisting of PEGylated HA and OVA. The complex was taken up through CD44-expressing cancer cells via receptor-mediated endocytosis. In an in vivo animal model, the tumor growth was significantly inhibited, antigen presentation on the tumor cells enhanced, and T cytotoxic anti-tumoral action increased [255]. A complex using HA and OVA on gold nanoparticles (AuNPs) was used to increase antigen uptake, by DC, via receptor-mediated endocytosis. The complex HA-OVA-AuNPs has enhanced nearinfrared (NIR) absorption and thermal energy translation, so after engulfment, the cytosolic antigen will be delivered through the photothermally targeted process. Proteasome activity is increased, and the MHC I antigen presentation is enhanced; thus, the CD8+ cytotoxic T-cell response is triggered. This protocol can be fruitfully expanded in the cancer vaccine development area [256].

As some of the tumor cells and primary lymphocytes have low HS expression, other carriers need to be utilized. Thus, proteins complexed with nanosize cholesteryl groupbearing pullulans (cCHP) can be efficiently delivered to myeloma cells and to primary CD4+ T cells by macropinocytosis. When using these new types of nanoparticles to deliver the anti-apoptotic protein Bcl-xL, T cells' functional regulation is achieved. These nanoparticles can bypass the lack of HS expression and deliver anticancer effectors and modulators of immune regulation [257].Figure 2 outlines the main mechanisms GAGs utilize to hinder immune anti-tumoral action.

β β β **Figure 2.** The main mechanisms through which GAGs hinder immune anti-tumoral action. A. HA binds t oCD44-expressing T suppresor cells and to the pool of tumor-associated macrophages contributing to the immuno-suppressive milieu in TME; B. Specific enzymes (e.g., β1,4-N-acetylgalactosaminyltransferase 3 and β1,4-galactosyltransferase 3) induce modification of β1 integrin expressed by tumor cells, triggering intracellular signaling that favor pro-tumorigenic effects in cell growth, cell cycle, and apoptosis. C. HA in the TME binds toCD44 expressed by tumor cells to physically block NK and T cytotoxic lymphocytes' access to tumor cells.

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