*Article* **The Assessment of IL-21 and IL-22 at the mRNA Level in Tumor Tissue and Protein Concentration in Serum and Peritoneal Fluid in Patients with Ovarian Cancer**

**Aleksandra Mielczarek-Palacz 1 , Celina Kruszniewska-Rajs 2 , Marta Smycz-Kuba ´nska 1 , Jarosław Strzelczyk 1 , Wojciech Szanecki 3 , Andrzej Witek <sup>3</sup> and Joanna Magdalena Gola 2, \***


**Abstract:** The aim of the analysis was for the first time to assess the expression of genes encoding IL-21 and IL-22 at the mRNA level in ovarian tumor specimens and the concentration of these parameters in serum and peritoneal fluid in patients with ovarian serous cancer. The levels of IL-21 and IL-22 transcripts were evaluated with the use of the real-time RT-qPCR. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of proteins. Quantitative analysis of IL-21 gene mRNA in the tumor tissue showed the highest activity in the G1 degree of histopathological differentiation and was higher in G1 compared to the control group. The concentration of IL-21 and IL-22 in the serum and in the peritoneal fluid of women with ovarian cancer varied depending on the degree of histopathological differentiation of the cancer and showed statistical variability compared to controls. The conducted studies have shown that the local and systemic changes in the immune system involving IL-21 and IL-22 indicate the participation of these parameters in the pathogenesis of ovarian cancer, and modulation in the IL-21/IL-22 system may prove useful in the development of new diagnostic and therapeutic strategies used in patients, which require further research.

**Keywords:** ovarian cancer; interleukin 21; interleukin 22

#### **1. Introduction**

Ovarian cancer is one of the gynecological cancers with the worst prognosis. Both the number of cases and the mortality caused by this disease in the world are constantly increasing [1,2]. In patients, the most important prognostic factor is the stage of clinical advancement, which determines the therapeutic strategy. Unfortunately, this cancer is detected in stage III or IV in more than 70% of women. It is associated with an unfavorable prognosis and a low five-year survival rate. It is caused by asymptomatic tumor growth, lack of characteristic clinical symptoms in the initial stage of the disease and diagnostic tests helpful in early diagnosis [3,4]. Currently conducted research indicates that the protein intercellular mediators—cytokines—play an important role in the formation and development of ovarian cancers. Cytokines interact with cells through characteristic surface receptors that transmit a signal to the interior of the cell. Altered expression of cytokines and their receptors in cancer cells affects the interaction in the tumor microenvironment, which may induce an anti-cancer response, promote tumor growth, participate in invasion and metastasis, and in immunosuppression [5,6].

**Citation:** Mielczarek-Palacz, A.; Kruszniewska-Rajs, C.; Smycz-Kuba ´nska, M.; Strzelczyk, J.; Szanecki, W.; Witek, A.; Gola, J.M. The Assessment of IL-21 and IL-22 at the mRNA Level in Tumor Tissue and Protein Concentration in Serum and Peritoneal Fluid in Patients with Ovarian Cancer. *J. Clin. Med.* **2021**, *10*, 3058. https://doi.org/10.3390/ jcm10143058

Academic Editors: Katarzyna Komosinska-Vassev and Paola Concolino

Received: 11 June 2021 Accepted: 8 July 2021 Published: 9 July 2021

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**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

Recent studies indicate that interleukin 21 (IL-21) and interleukin 22 (IL-22) play an important role in the pathogenesis of cancer and cancer therapy [7–12]. Winkler et al. showed, that Th17 cell sub-populations with expression of IL-21 and/or IL-22 infiltrate the tumor tissue of ovarian cancer [7]. Interleukin-21 is a cytokine secreted mainly by activated CD4 + T cells, including T helper 17 (Th17) and T follicular helper (Tfh) cells and NKT cells [8]. IL-21 is bound to the IL-21 receptor which contains the gamma chain [9]. It has an immunoregulatory function, has the ability to both promote and inhibit the immune response and induce an anti-cancer response, mainly by activating Tc lymphocytes, therefore, attempts are still being made to use it in cancer immunotherapy [10–12]. Studies have shown the effectiveness and safety of the therapy with the use of recombinant IL-21 in advanced melanoma, renal cell carcinoma, and non-Hodgkin's B cell lymphoma [13]. Subcutaneous administration of IL-21 augmented tumor regression and increased tumor infiltration by CD8 + T cells [11]. IL-21 can be also combined with other immunotherapies [14].

Interleukin-22 is a cytokine synthesized by lymphocyte T helper (Th) cells, including their Th17 and Th22 subpopulations as well as NKT lymphocytes, NK cells, and nonspecific lymphoid cells [15]. It is a pro-inflammatory cytokine, it induces the production of acute phase proteins and participates in the pathogenesis of many diseases, including cancer. Interestingly, IL-22 has also been shown to induce multiple cytoprotective mechanisms, participating in tissue regeneration following inflammatory lesions [16]. IL-22 is bound to the heterodimer receptor complex of IL–10R2 and IL-22R1 [17] and, by activating numerous signaling pathways, it influences such processes as cell survival, proliferation, migration, and angiogenesis [17]. These processes are crucial for the development of cancer. Protopsaltis et al. demonstrated that IL-22 directly stimulates angiogenesis through activation of the ERK and Stat3 pathways, and blockade of IL-22 inhibits tumor growth [18]. Interestingly, cancer cells release IL-1, which stimulates IL-22 production by memory T cells [17].

The analyses conducted so far on the evaluation of IL-21 and IL-22 in ovarian cancer have been interesting and induce further research. It has been shown that the determination of IL-21 with other cytokines, for example, IL-17a, may find clinical application as prognostic and therapeutic biomarkers [19]. Interleukin-22 has been shown to be an important factor in the ovarian cancer tumor microenvironment and may find application as a potential therapeutic target and/or biomarker [20]. Its prognostic value in other cancers has also been established, including pancreatic cancer [21] and hepatocellular carcinoma [22].

So far, no studies have been conducted to assess both the expression of IL-21 and IL-22 in tumor tissue, as well as the concentration of these cytokines in the serum and peritoneal fluid in women with ovarian cancer, and considering the fact that understanding the role of these parameters in the pathogenesis of ovarian cancer may be useful in developing new diagnostic and therapeutic regimens in patients with ovarian cancer in the future, the aim of the study was: assessment of IL-21 and IL-22 at the mRNA level in ovarian cancer tissue, analysis of the concentration of IL-21 and IL-22 in the serum and peritoneal fluid of patients with ovarian cancer, and determining whether there is a relationship between the concentration of IL-21 and IL-22 and the degree of histological differentiation of the cancer.

#### **2. Materials and Methods**

The study group included 26 women, aged 41 to 82 (mean age: 62.80 ± 11.21 years) with the diagnosed ovarian cancer with the Cystadenocarcinoma papillare serosum IIIC (7 had G1, 5 had G2, and 14 had G3 staging) Staging employed the criteria recommended by the International Federation of Gynecology and Obstetrics (FIGO). The diagnosis of tumors was done on the basis of clinical symptoms, results of gynecological and histopathological examination and laboratory tests. The women qualified to the studied group were clinically diagnosed with ovarian tumor confirmed with a histopathological examination. Other coexisting disorders of the reproductive organs and autoimmune diseases were excluded. None of the examined women used pharmacological treatment in the last three months. The research was conducted on women hospitalized at the Clinical Department

of Gynecology and Obstetrics of the University Clinical Center in Katowice and the Department of Gynecology and Obstetrics with the Department of Pathology of Pregnancy and Gynecological Oncology of the Provincial Specialist Hospital in Cz ˛estochowa. Serum, peritoneal fluid, and tumor tissue were the material examined in all women. In the study group, blood was taken from women after establishing clinical diagnosis, before surgery. Blood was taken in the morning from the cubital vein, to a clot tube, in order to obtain the serum. Thirty minutes after taking the blood, it was centrifuged at 1500× *g* for 15 min. The serum obtained in this manner was kept in small portions at a temperature of −80 ◦C until the tests. Tumor tissue intended for molecular assessment was collected during the planned surgery and frozen at −80 ◦ C until the tests were performed. Peritoneal fluid was collected during laparoscopy for bacteriological examination, and then centrifuged at 2000 rpm for 10 min at 4 ◦C, and the obtained supernatant was partitioned and frozen at −80 ◦C until the remaining determinations were made.

The control group consisted of six women aged between 40–77 (mean age: 61.83 ± 12.48 years) who have been diagnosed with a benign lesion (Cystadenoma serosum). The concentration of antigen CA125 did not exceed 35 U/m in these patients. In all the women, the blood serum and tissue samples were the research material.

Total RNA was extracted from tissue samples with the use of TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Each tissue sample was placed in a tube containing TRIzol, and then homogenized using a Polytron® homogenizer (Kinematica AG, Malters, Switzerland). RNA extracts quantitative assessment was performed with the use of nanospectrophotometer MaestroNano MN-913 (MaestroGen Inc., Hsinchu City, Taiwan) and 2100 Bioanalyzer (Agilent Technologies, Inc. Santa Clara, CA, USA) (Figure S1, supplementary materials). The levels of IL-21 and IL-22 transcripts were evaluated with the use of the real-time RT-qPCR. The quantitative analysis was carried out using LightCycler® 480 System (Roche, Basel, Switzerland) and GoTaq® 1-Step RT-qPCR System (Promega, Madison, WI, USA), according to manufacturers' instructions. Amplification was performed using previously described oligonucleotide primers [23,24] and commercially available standards of β-actin cDNA (TaqMan™ DNA Template Reagents; Thermo Fisher Scientific, Waltham, MA, USA). All samples were tested in triplicate. The mRNA copy numbers of the gene examined were recalculated per 1 µg of the total RNA. Melting curve analysis and agarose gel electrophoresis were used to confirm the specificity of amplification and the absence of primer dimers (Figures S2–S4, supplementary materials). Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of the studied parameters. The following kits were used for this purpose: IL-21 Human Interleukin-21 ELISA and Human Interleukin-22 (BioVendor– Laboratorni medicina a.s., Brno, Czech Republic). Interleukin-21 Human ELISA, Sandwich ELISA, Biotin-labelled antibody, Calibration Range 78–5000 pg/mL, limit of detection 20.0 pg/mL and Interleukin-22 Human ELISA, Sandwich ELISA, Biotin-labeled antibody, Calibration Range 31.3–2000 pg/mL, limit of detection 5.0 pg/mL.

All the women who participated in the study consented to conducting the research. The approval of the Ethics Committee of the Medical University of Silesia in Katowice was obtained.

The obtained results were statistically analyzed using the Statistica 13.3 software (StatSoft Polska Sp. z o.o., Cracow, Poland). The normality of the distribution of the studied variables was checked using the Shapiro–Wilk test. The median and quartile range were determined for the parameters tested, and the obtained results were compared using the Mann–Whitney test. Correlations were tested by Spearman's rank correlation test and presented as correlation coefficient (*r*).

#### **3. Results**

Quantitative assessment of IL21 mRNA in tissue samples showed the highest gene activity in the degree of differentiation G1 (Figure 1). The expression of IL21 gene was higher comparing to control group (*p* = 0.002415), G2 (*p* = 0.033161), and G3 (*p* = 0.020397). No statistically significant difference was found between G2, G3, and a control group. Moreover, expression of IL21 mRNA did not differ between G2 and G3.

– Spearman's

**Figure 1.** Number of copies of IL21 mRNA/µg of total RNA in tissue samples depending on the degree of ovarian cancer differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated) compared to the control group (C).

Quantitative assessment of IL22 mRNA in tissue samples did not show statistically significant difference comparing to a control group. Moreover, expression of IL22 mRNA did not differ between degrees of differentiation G1, G2, and G3. Median values of the number of copies of IL21 mRNA/µg of total RNA in tissue samples were as follows: G1: Me = 6660, G2: Me = 5065, G3: Me = 7820, C: Me = 8555.

Il-21/IL-22 mRNA ratio was the highest in the degree of differentiation G1 (Figure 2). In all cancer tissues, regardless of degree of differentiation, the value of Il-21/IL-22 mRNA ratio was higher comparing to a control group (respectively: G1 vs. C *p* = 0.003405; G2 vs. C *p* = 0.008114; G1 vs. C *p* = 0.007473). Moreover, statistically significant difference was found between G1 and G3 (*p* = 0.032391).

The concentration of Il-21 was determined in the serum and peritoneal fluid of women with ovarian cancer. As the obtained values did not correspond to the normal distribution, the results were presented in the form of the median and the lower and upper interquartile range (Q1 and Q3).

In the serum of women with ovarian cancer, Q1 and Q3 were respectively: 314.25 and 736.00 with a median of 408.68 pg/mL. A statistically significantly lower concentration of IL-21 in the serum of women with ovarian cancer was demonstrated compared to the concentration in the control group (*p* < 0.001), where Q1 and Q3 were respectively: 722.19 and 909.60 with a median of 793.59 pg/mL.

In the peritoneal fluid of women with ovarian cancer, Q1 and Q3 were: 113.36 and 356.13, respectively, with a median of 175.63 pg/mL. A statistically significant higher concentration of the examined parameter was found in the serum of women with ovarian cancer as compared to the concentration in the peritoneal fluid (*p* < 0.0001).

Then, the concentration of IL-21 in serum and the peritoneal fluid of women with ovarian cancer depending on the degree of histopathological differentiation of the cancer was analyzed. The analysis of the test results showed a statistical significance between G2 and G3 (*p* < 0.001) and between G1 and G3 (*p* < 0.0001) in the serum of the examined women. In the peritoneal fluid of women with ovarian cancer, a statistically significant difference was found only between G2 and G3 (*p* < 0.05). The obtained results are presented in Figures 3 and 4.

**Figure 2.** Il-21/IL-22 mRNA ratio in tissue samples depending on the degree of ovarian cancer differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated) compared to the control group (C). Ratio was calculated based on mRNA copies/µg of total RNA.

**Figure 3.** The concentration of IL-21 in the serum of women with ovarian cancer depending on the degree of differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated) compared to the control group.

**Figure 4.** The concentration of IL-21 in the peritoneal fluid in women with ovarian cancer depending on the degree of differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated).

The results of Il-22 concentration measurement in the serum and peritoneal fluid of women with ovarian cancer were presented in the form of the median and the lower and upper interquartile range (Q1 and Q3).

In the serum of women with ovarian cancer, Q1 and Q3 were respectively: 337.59 and 771.33 with a median of 572.64 pg/mL. A statistically significant higher concentration of IL-22 in the serum of women with ovarian cancer was demonstrated compared to the concentration in the control group (*p* < 0.001), where Q1 and Q3 were respectively: 66.01 and 140.04 with a median of 98.62 pg/mL.

In the peritoneal fluid of women with ovarian cancer, Q1 and Q3 were 132.40 and 275.70, respectively, with a median of 186.01 pg/mL. A statistically significant higher concentration of the examined parameter in the serum of women with ovarian cancer was demonstrated compared to the concentration in the peritoneal fluid (*p* < 0.0001).

Then, the concentration of IL-22 in serum and peritoneal fluid in women with ovarian cancer depending on the degree of histopathological differentiation of the cancer was analyzed. The analysis of the test results showed a statistical significance between G2 and G3 (*p* < 0.0001) and between G1 and G3 (*p* < 0.0001) in the serum of women. In the peritoneal fluid of women with ovarian cancer, no statistically significant differences were found between the degrees of histopathological differentiation of the cancer.

The obtained results are presented in Figures 5 and 6.

**Figure 5.** The concentration of IL-22 in the serum of women with ovarian cancer depending on the degree of differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated) compared to the control group.

**Figure 6.** The concentration of IL-22 in the peritoneal fluid in women with ovarian cancer in relation to on the degree of differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated).

Further analysis concerned the assessment of the relationship between the serum concentration of IL-21 and IL-22 and the peritoneal fluid concentration of IL-21 and IL-22. A statistically significant positive correlation was found between IL-21 and IL-22 in the serum of the examined women, and a positive, but not statistically significant correlation between the concentration of IL-21 and IL-22 in the peritoneal fluid of the examined women. The regression curves are shown in Figures 7 and 8. Ratio of IL-21/IL-22 in serum and peritoneal fluid in patients with ovarian cancer is shown in Figures 9 and 10.

**Figure 7.** Linear regression curve showing the relationship between IL-21 and IL-22 levels in the serum of women with ovarian cancer; *r*-correlation coefficient, *p*-probability value.

**Figure 8.** Linear regression curve showing the relationship between the concentration of Il-21 and IL-2 in the peritoneal fluid of women with ovarian cancer; *r*-correlation coefficient, *p*-probability value.

**Figure 9.** IL-21/IL-22 ratio in the serum of women with ovarian cancer depending on the degree of differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated) compared to the control group.

**Figure 10.** IL-21/IL-22 ratio in the peritoneal fluid of women with ovarian cancer depending on the degree of differentiation G1 (well differentiated), G2 (moderately differentiated), G3 (poorly differentiated).

#### **4. Discussion**

For years, the efforts of researchers have been focused on understanding the mechanisms of the formation and development of ovarian cancer in order to identify markers helpful not only in early diagnosis, but also in monitoring therapy. The challenge of contemporary gynecological oncology is to develop an effective targeted therapy that controls the expression of compounds that play a key role in the formation and development of

tumor's

the cancer. Numerous experimental studies and clinical observations indicate that chronic inflammation plays an important role in the pathogenesis of ovarian cancer, in which cells of the immune system participate, which, through secreted mediators in the tumor's environment, participate in the anti-tumor response and may promote tumor growth, which may be manifested by changes in tissue expression, serum concentrations, and peritoneal fluid.

Recent studies have shown that interleukin 21 and interleukin 22 play an important role in the pathogenesis of cancer, which role in the formation and development of ovarian cancer is still not fully understood. Therefore, the aim of the analysis performed in the study was to assess the expression of IL-21 and IL-22 in the tumor tissue and the concentration of these parameters in the serum and peritoneal fluid in patients with ovarian serous cancer, taking into account the degrees of histological differentiation of the tumor.

For the first time, the expression of genes encoding IL-21 and IL-22 was assessed at the mRNA level in ovarian tumor specimens. The number of mRNA copies of the studied genes was determined by RT-qPCR method (quantitative method real-time). The studies showed that in the samples of the G1 histological differentiation, the number of mRNA copies of IL21 gene was statistically significantly higher, not only compared to the control group, but also compared to the G2 and G3 histological differentiation samples. In the case of IL22, we did not observe any differences in the number of mRNA copies between the cancer specimens and the control group, as well as between the G1, G2, and G3 histologically differentiated groups. The studies by Fagerberg et al. [25] did not show the expression of IL21 and IL22 at the mRNA level in normal ovaries. Therefore, the presence of mRNA of these interleukins in the sections studied by us, both malignant and benign, may be caused by the presence of cells of the immune system. The differences observed by us suggest that the interaction with the cells of the immune system may have a different course in malignant and benign tumors, but this requires further detailed studies.

Our studies showed a statistically significantly lower concentration of IL-21 in the serum of women with ovarian cancer compared to the concentration in the control group, which indicates the participation of the tested cytokine in the immune response against ovarian cancer cells. In addition, statistical significance was demonstrated between the concentration of the tested cytokine and the degrees of histological differentiation in cancer: G2 and G3, and between G1 and G3, which indicates a relationship between IL-21 secretion and histological differentiation in cancer and may prove useful in the future in selecting the optimal therapeutic treatment and in the assessment of prognosis.

Interesting observations were also provided by the analysis of the concentration of IL-21 in the peritoneal fluid of the examined women. The concentration of this parameter was significantly lower in the peritoneal fluid as compared to the serum, which may indicate that the source of the tested cytokine in the serum may be cells of the immune system, which, through secreted mediators, show increased cytotoxic activity against cancer cells. Moreover, it was shown that the concentration of IL-21 in the peritoneal fluid of women with ovarian cancer differed between the grades of G2 and G3, which proves that the secretion of IL-21 is related to the histological differentiation of ovarian cancer also in this biological fluid.

So far, no studies have been conducted to evaluate the expression of IL-21 in tumor tissue and the concentration in serum and peritoneal fluid. However, the potential usefulness of using this cytokine in the diagnosis of ovarian cancer was analyzed. Such research was conducted by Chen YL. et al. [19], who showed the usefulness of the assessment of interleukins IL-17a and IL-21 as prognostic and therapeutic biomarkers. Studies on the role of IL-21 in the anti-tumor response were also conducted by Hermans et al. [26], who pointed to the therapeutic potential of the transient inhibition of LDH during adoptive T-cell immunotherapy with the anti-tumor effect of inhibiting LDH and IL-21. Similar studies were conducted by Dou et al. [27], who showed that SKOV3 cells genetically modified to secrete biologically active IL-21 and GM-CSF were effective in inducing anti-tumor immunity by increasing NK cytotoxicity, promoting the expression of MIC A/B, ICAM-1,

and NKG2D molecules, as well as increasing IFN-γ and TNF-α in the nude mouse model. On the other hand, Bhatt et al. [28] have shown that IL-21 has a strong anti-tumor activity against mantle lymphoma (MCL) cells through direct cytotoxic and indirect immunological effects. According to the authors, in vivo treatment with IL-21 leads to complete regression of FC-muMCL1 tumor in syngeneic mice through NK and T cell dependent mechanisms. Similar studies were also conducted by Li et al. [29], who showed that IL-21 may affect T cells, which are involved in the anti-tumor response, by fusing with an anti-PD-1 antibody. Wang et al. [30] assessed the role of interleukin 21 and its receptor in proliferation, migration, and invasion of breast cancer cells. The conducted research has shown that IL-21 promotes proliferation, invasion and migration of IL-21R + MDA-231 breast cancer cells, it does not show such strong properties against MCF-7 and ZR-75.1 cells, in which IL-21R expression is weak or negative. Moreover, the role of IL-21R in signaling pathways of matrix metalloproteinases, which are necessary for the processes of migration of MDA-231 cells, has been demonstrated. Zhang et al. [31] showed that reduction of the tumor and increased survival is accompanied by an increase of IFN-γ, IL-21, and TNF-α in serum, as well as the cytotoxic activity of the spleen. On the other hand, Gu et al. [32] showed that the co-expression of two members of the γ chain family of the cytokine receptor, IL-21 and IL-7, in anti-cancer vaccines increases anti-tumor immunity in a CD4 + and CD8 + T cell-dependent manner and generates an effective immune memory.

The increase of IL-21 mRNA in tumor tissue at the G1 stage may indicate that there are still efficient mechanisms aimed at limiting its growth, which is also reflected in the IL21/IL22 mRNA ratio value. In the G2 and G3 stages, the expression is reduced in tumor tissue, possibly as a result of disturbed intracellular signaling or epigenetic changes.

On the other hand, the increase in protein concentration, both in the serum and in the peritoneal fluid, suggests the activation of systemic mechanisms and a strong recruitment of the immune system. Due to blocked mechanisms in the tumor tissue itself, the antitumor effect of the immune system is probably ineffective, despite its strong stimulation. However, this hypothesis requires further investigation. It should also be taken into account that gene expression is a multi-step process and its regulation is complex. Strongly activated gene transcription in the tumor tissue does not mean that protein is synthesized and secreted. In our research, the protein source are likely cells of the immune system. If tumor tissue were the main source of interleukins, the protein concentration would be higher in the peritoneal fluid than in the serum.

An interesting observation is the fact that serum protein concentration is significantly decreased by the G1 stage and the concentration increases with the degree of histological differentiation of the cancer. In the G3 stage, the IL21 concentration increases to a level comparable to that in the control. Most likely, in the lower degree of histological differentiation of the cancer, the mechanisms responsible for "masking" the presence of cancer cells are activated. In our research, the study group was not very large, so the results should be interpreted with caution.

Further analysis included the evaluation of interleukin 22 expression at the mRNA level in tumor tissue and serum and peritoneal fluid concentration in women with ovarian cancer. The studies conducted so far have shown that the role of the interleukin IL-22 is complex. On the one hand, this cytokine promotes tumor development, is responsible in particular for the proliferation of malignant neoplasm of epithelial origin, but on the other hand, the intensification of its secretion may constitute a barrier against tumor development by suppressing inflammation due to the functions that protect against tissue damage, promote tissue repair, and prevent inflammation [33,34]. Our studies showed that the concentration of IL-22 in the serum of women with ovarian cancer was statistically significantly higher than that in the control group, which proves the participation of this cytokine in pro-cancer immune response through direct and indirect influence involving the immune system cells participating in the regulation of inflammation. In addition, a statistically significant higher concentration of the parameter in the serum of women with ovarian cancer was demonstrated compared to the concentration in the peritoneal fluid, which indicates increased secretion of Il-22 in the systemic immune response. Moreover, a relationship has been demonstrated between the concentration of IL-22 and the degrees of histological differentiation of cancer, including: G2 and G3, and between G1 and G3 in the serum of women, which indicates the relationship of this cytokine with tumor development and may in the future prove useful in selecting the optimal therapeutic treatment and in assessing the prognosis.

Further analysis concerned the assessment of the relationship between the concentration of IL-21 and IL-22 in the serum and in the peritoneal fluid. The demonstrated correlation between the concentrations of the tested parameters, both in the serum and in the peritoneal fluid, may indicate the need for further research in order to create complex diagnostic and therapeutic strategies targeting many different mechanisms of the immune response. Due to the fact that the interactions between these parameters may affect the direction of their biological activity, the stoichiometric relationship between the concentration of IL-21 and IL-22 and the number of IL21/IL22 mRNA copies was assessed. The determined IL-21/IL-22 ratio, both at the mRNA level in the tissue and at the protein level in the serum and in the peritoneal fluid in patients with ovarian cancer, showed a variable share of the tested cytokines in the IL-21/IL-22 system, depending on the histological differentiation of the cancer.

Similar studies were conducted by Balint et al. [20], who assessed the expression of IL-22 in human ovarian cancer tissues and in ascites samples. Expression of both IL-22 and the IL-22 receptor was higher in cancer tissues compared to the control tissue, which correlated with poor prognosis. Studies have shown that interleukin-22 is an important factor in the ovarian cancer tumor microenvironment because it stimulates tumor growth by increasing proliferation and serves as a protective factor for ovarian cancer during TNFinduced apoptosis. According to the authors, IL-22 is a potential therapeutic target and/or biomarker in human ovarian cancer. Wang et al. [35] showed an increased expression of Th22 lymphocytes, which produce IL-22 in the tumor microenvironment, which, in their opinion, may stimulate tumor growth and affect the patient's further prognosis. Katara et al. [36] demonstrated the presence of IL-22 at all stages of the breast cancer, and that blocking of IL-22 gene expression resulted in inhibition of tumor invasion and reduction of metastasis. On the other hand, Rui et al. [37] showed an increased expression of the interleukin-22 receptor 1 in breast cancer. IL-22 binds to the IL-10R2 and IL-22R1 receptor complexes, activating the transcription factor STAT3, thereby promoting tumor progression. IL-22R1 is expressed exclusively on the surface of epithelial and tissue cells. Moreover, IL-22 promoted the expression of pro-tumor HOXB-AS5 proteins in breast cancer. Khosravi et al. [38] showed that high expression of IL-22RA1 in KRAS mutated lung adenocarcinoma is associated with a short remission time. Genetic ablation of IL-22 resulted in a significant decrease in tumor mass and a reduction in the number of tumor cells, their proliferation, and the ability to activate STAT3, and it was also associated with a reduction in angiogenesis and the number of inflammatory cells that infiltrated the lung tissue.

#### **5. Conclusions**

Local and systemic changes in the immune system with the participation of soluble mediators IL-21 and IL-22 indicate the participation of these parameters in the development of ovarian cancer and may participate in pro and anti-inflammatory activation involving the tested cytokines.

Modulation in the IL-21 and IL-22 system may affect the course of the inflammatory process that accompanies the development of cancer, which may prove useful in the development of new diagnostic and therapeutic strategies used in patients with ovarian cancer, which requires further studies.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jcm10143058/s1, Figure S1: Quality of total RNA, Bioanalyzer Agilent electrophoresis run, Figure S2: Melting peak analysis showing specificity of RT-qPCR reaction, Figure S3: Agarose

gel electrophoresis of IL22 PCR-amplified product, Figure S4: Agarose gel electrophoresis of IL21 PCR-amplified product.

**Author Contributions:** A.M.-P.—conceptualization, supervision of immunological research, investigation, writing (original draft preparation, review and editing), funding acquisition; J.M.G. conceptualization, supervision of molecular research, investigation, writing—review and editing; M.S.-K.—methodology (immunological research), data analysis and interpretation; C.K.-R. methodology (molecular research), data analysis and interpretation; J.S., W.S., A.W.—clinical research conceptualization and data interpretation. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Medical University of Silesia in Katowice, Poland, grant number PCN-1-100/N/0/O.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Medical University of Silesia in Katowice, Poland (protocol code KNW/0022/KB1/49/19).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data is contained in the article.

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

#### **References**


### *Article* **Eotaxins and Their Receptor as Biomarkers of Colorectal Cancer**

**Monika Zajkowska 1, \* , Agnieszka Kulczy ´nska-Przybik 1 , Maciej Dulewicz 1 , Kamil Safiejko 2 , Marcin Juchimiuk 2 , Marzena Konopko 2 , Leszek Kozłowski <sup>2</sup> and Barbara Mroczko 1,3**


**Abstract:** Colorectal cancer (CRC) is one of the most common malignancies. Despite the availability of diagnostic tests, an increasing number of new cases is observed. That is why it is very important to search new markers that would show high diagnostic utility. Therefore, we made an attempt to assess the usefulness of eotaxins, as there are few studies that investigate their significance, in patients with CRC. The study included 80 subjects (CRC patients and healthy volunteers). Serum concentrations of all eotaxins were measured using a multiplexing method (Luminex), while CCR3 was measured by immunoenzymatic assay (ELISA). CRP levels were determined by immunoturbidimetry and classical tumor marker levels (CEA and CA 19-9) and were measured using chemiluminescent microparticle immunoassay (CMIA). The highest usefulness among the proteins tested showed CCR3. Its concentrations were significantly higher in the CRC group than in healthy controls. The diagnostic sensitivity, specificity, positive and negative predictive value, and the area under the ROC curve (AUC) of CCR3 were higher than those of CA 19-9. The maximum values for sensitivity, negative predictive value, and AUC were obtained for a combination of CCR3 and CRP. Our findings suggest the potential usefulness of CCR3 in the diagnosis of CRC, especially in combination with CRP or CEA.

**Keywords:** CCL11; CCL24; CCL26; CCR3; CRC

#### **1. Introduction**

Colorectal cancer (CRC) is one of the most common malignancies not only in Europe, but also around the world. According to the World Health Organization (WHO), in 2020, the global incidence of all cancers was 19.3 million new cases, with approximately 9.9 million deaths. CRC comprises about 10% and 9.4% of all cases, respectively. Unfortunately, the incidence of this cancer is slowly but steadily increasing [1]. Currently, as a part of preventive diagnostics of colorectal cancer, tests such as FOBT (fecal occult blood test), FIT (fecal immunochemical test), colonoscopy, sigmoidoscopy, computed tomographic (CT) colonography, or multi-target stool deoxyribonucleic acid (mt-sDNA) test are used [2]. Also, markers such as CEA (carcinoembryonic antigen) or CA 19-9 (carbohydrate antigen 19-9) are routinely determined in patients with CRC. However, their diagnostic sensitivity and specificity are not satisfactory. That is why establishing new, more accurate markers for CRC detection at its earliest stage is vital [3].

Eotaxins belong to a group of small proteins called chemokines. They were discovered relatively recently—less than 30 years ago in London by Williams et al. [4,5]. Therefore, the number of papers on these parameters is still insufficient to fully understand their potential usefulness in the diagnosis or monitoring of certain diseases. The most widespread and so far the best-known usefulness of these proteins has been found in the course of allergic

Kulczy ´nska-Przybik, A.; Dulewicz, M.; Safiejko, K.; Juchimiuk, M.;

**Citation:** Zajkowska, M.;

Konopko, M.; Kozłowski, L.; Mroczko, B. Eotaxins and Their Receptor as Biomarkers of Colorectal Cancer. *J. Clin. Med.* **2021**, *10*, 2675. https://doi.org/10.3390/jcm10122675

Academic Editors: Katarzyna Komosinska-Vassev, Pawel Olczyk and Hidekazu Suzuki

Received: 12 April 2021 Accepted: 14 June 2021 Published: 17 June 2021

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**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

diseases. It is closely related to their influence on cells such as eosinophils and basophils, which are the most important cells in the development and course of those diseases. All eotaxins have the ability to bind to CCR3 (C-C chemokine receptor type 3) [6,7].

However, there are only a few studies that would indicate the usefulness of these parameters in the diagnosis, monitoring, or staging of neoplastic diseases, such as colorectal cancer. Extending the availability of information on this topic is quite important due to the discrepancy of the available data. Some of the studies show a decrease in the concentration of CCL11, while others, on the contrary, report an increase in the concentration of this parameter in the course of CRC. On the other hand, for the other parameters (CCL24, CCL26, and CCR3), no work is available on their concentration in patients with CRC. However, there are works that describe the increased expression of eotaxins in neoplastic tissues. Some authors believe that those parameters increase the proliferation of neoplastic cells and their migration, and increases the expression of the receptor for eotaxins (CCR3). Activation of this receptor on endothelial cells leads to increased angiogenesis and tumor development. On the other hand, increased tissue expression of eotaxins can also lead to the recruitment of eosinophils (cells with anti-tumor activity) into the tumor environment [8]. Therefore, we made an attempt to clarify and assess the usefulness of eotaxins in patients with colorectal cancer compared to the healthy control. We have investigated serum levels, diagnostic utility (sensitivity, specificity, predictive values of positive and negative test results), and power (ROC curve analysis) of all eotaxins (CCL11, CCL24, CCL26), their receptor (CCR3), comparative tumor markers (CA 19-9, CEA), and inflammatory parameter such as C-reactive protein (CRP) in colorectal cancer detection. The data obtained in this study may prove the usefulness of the analyzed parameters in the detection of CRC.

#### **2. Results**

Table 1 shows the serum levels of CCL11, CCL24, CCL26, CCR3, CA 19-9, CEA, and CRP in patients with colorectal cancer and in the control group. Concentrations of CCR3, CEA, and CRP in the total cancer group were statistically significantly higher when compared to the control group (in all cases *p* < 0.05).


**Table 1.** Serum levels of tested parameters in cancer and control groups.

\* U Mann–Whitney test.

In order to carry out a more detailed analysis of the obtained results, we divided the study group into patients with colon cancer and rectal cancer. Performed statistical analysis with use of the Mann–Whitney U test showed similar results. In the case of the results of colon cancer vs. control group patients, statistical significance was demonstrated for CEA and CRP (*p* < 0.001 in both cases) (Supplementary Table S1). For rectal cancer,

statistical significance was demonstrated for CEA, CRP, and CCR3 (*p* = 0.003; *p* < 0.001; *p* = 0.012, respectively) (Supplementary Table S2). We did not perform a statistical analysis for sigmoid cancer patients due to an insufficient number of patients in this subgroup. We also compared the results between the two subgroups (colon cancer vs. rectal cancer), but there were no statistical differences (Supplementary Table S3). Additionally, we performed a more detailed analysis with division into advancement groups. We divided the colorectal cancer group of patients into Early (TNM stages 0+I+II) and Advanced (TNM stages III+IV) CRC. Statistical analysis with use of Mann–Whitney U test in this case also revealed similar results. In analysis of Early CRC vs. control group, statistical significance was observed in the case of CEA and CRP (*p* = 0.006, *p* < 0.001, respectively) (Supplementary Table S4). In analysis of Advanced CRC vs. control group, statistical significance was observed in the case of CCR3, CEA, and CRP (*p* = 0.024, *p* = 0.002, *p* = 0.001, respectively) (Supplementary Table S5). We also tried to compare results between two TNM subgroups (Early vs. Advanced CRC), but none of the results were significant (Supplementary Table S6). Differences in the statistical significance of CCR3 in the case of rectal cancer and its absence in the case of colon cancer may be related to the differences in the biology of these two neoplasms. However, due to the insufficient number of patients with colon cancer, further analysis was carried out on the entire study group of CRC patients. The differences obtained between the two histological types should be repeated in a larger study group, which could significantly influence the development of knowledge about eotaxins, and we assume that at this point the obtained results should be treated as a pilot, preliminary study.

Table 2 shows the sensitivity (SE), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of all tested parameters. We indicated that the highest SE from all tested parameters revealed CCR3 (68%) and this value is comparable to SE of C-reactive protein (72%) and higher than SE of commonly used tumor marker CA 19-9 (40%). Only CEA showed higher SE (80%). In the case of SP, the highest value was observed for CCL26 (92.31%). SP for CCR3 (62.07%) was comparable to results of both tumor markers (CA 19-9 and CEA) but lower than SP observed for CRP (79.31%). Positive predictive value was highest in the case of CCL24 and CCR3 (72.97% and 75.56%, respectively). These values were slightly lower than the PPV of CEA and CRP. Interestingly, almost all tested parameters (CCL11, CCL24 and their receptor) showed higher PPV than CA 19-9. The NPV was highest for CCR3 (52.94%), but slightly lower than the NPV of CEA and CRP. What is more, similar to PPV, the NPV of all tested parameters was similar or even higher than the NPV of commonly used tumor marker—CA 19-9.

According to promising results obtained for CCR3, we decided to check whether the simultaneous analysis of two parameters would significantly change the value of the diagnostic criteria. Therefore, we created two panels consisting of CCR3 with a comparative marker—CEA and CCR3 with CRP. Combined analysis of CCR3 with both parameters resulted in an increase of SE (92%; 94%, respectively) and NPV (76.47%; 82.35%, respectively) in both cases. The most favorable combination proved to be CCR3 + CRP, what may indicate the significance of the inflammatory component in the course of this malignancy.


**Table 2.** Diagnostic criteria of tested parameters in patients with colorectal cancer.

SE—sensitivity; SP—specificity; PPV—positive predictive value; NPV—negative predictive value.

The ROC curve is an illustrated relationship between the diagnostic SE and SP. The area under the ROC curve (AUC) indicates the clinical usefulness of a tumor marker and its diagnostic power. All data related to AUC has been shown in Table 3. We noticed that the CCR3 area under the ROC curve (0.683) in the total group of colorectal cancer was the highest from all the tested parameters but lower than the AUC for CEA and CRP. Moreover, similarly to previously mentioned statistical parameters, in the case of all eotaxins, the AUC was similar or even higher than the AUC of CA 19-9. Likewise, in the case of diagnostic criteria, the AUC for the simultaneous analysis of CCR3 with CEA or CRP showed a marked increase in the area under the ROC curve value (0.779; 0.846, respectively). Graphical versions of all significant ROC analysis results are shown in Figure 1. The AUCs for the tested parameters, similar to commonly used tumor markers and combined analysis, were statistically significantly larger in comparison to AUC = 0.5 (borderline of the diagnostic usefulness of the test) (*p* < 0.05 in all cases).


**Table 3.** AUC of tested parameters in patients with colorectal cancer.

*p*—statistically significantly larger AUC's compared to AUC = 0.5. AUC—area under curve; SE—standard error; C.I.—confidence interval.

**Figure 1.** Receiver operating characteristics for all significant ROC analysis results.

#### **3. Discussion**

Currently, little is known about the concentration and diagnostic usefulness of eotaxins and their receptor in the course of CRC. Available literature describes results that are fragmentary, characterized by a high discrepancy, or, in some cases, not entirely clear. Therefore, we decided to conduct a confirmation study on whether the examined parameters may be useful in the detection, screening, or prognosis and monitoring of the applied treatment.

Eotaxins are closely related to cells such as eosinophils and basophils. These cells are mainly involved in allergic reactions, which can be included in the group of inflammatory reactions. Neoplastic changes also begin as a result of inflammatory reactions leading to the formation of cancer. Therefore, it can be assumed that similar cytokines may appear and change their concentration as a result of both of these phenomena (allergic reactions and onset of neoplastic changes). The first references to the relationship between eosinophils and neoplastic changes were described over 100 years ago, but so far, their role in the carcinogenesis [7–9] has not been precisely explained. There are suspicions that the involvement of eotaxins in CRC may be related to the large number of circulating eosinophils that appear and accumulate in neoplastic tissues. TATE (tumor-associated tissue eosinophilia) has also been connected with improved prognosis in CRC and some other types of cancer such as esophageal, oral squamous cell, bladder, and prostate cancer [10–16]. Moreover, there are some studies that clearly indicate that tissue eosinophilia in the course of neoplastic changes may be closely related to the factors secreted directly by tumor cells. These factors can certainly include eotaxins. That is why it is so important to extend the research carried out so far on these parameters not only in the course of CRC but also in other cancers [17,18].

We indicated that serum concentrations of CCL11 in the tested group (CRC) were lower than in the control group. Despite the lack of statistical significance, similar results were obtained in the course of other experiments carried out by Wagsater et al. [19]. These researchers obtained significantly lower concentrations of CCL11 in CRC patients (which can be connected with a larger test group). In addition, they also performed IHC (immunohistochemistry) staining and examined the concentration of CCL11 in tissue homogenates. This revealed that CCL11 may accumulate in tissues, which is why its concentration can be lowered in the serum of CRC patients. By contrast, Mir et al. [20], Yamaguchi et al. [21], and Komura et al. [22] reported that CCL11 levels are higher in CRC and inflammatory bowel disease patients than in control groups. This discrepancy might be correlated with some differences between the composition of study groups, i.e., receiving corticosteroids in the case of the Mir et al. [20] study and different ethnicity [21] or smaller study group in the case of the last paper [22].

The concentration of CCL24 (Eotaxin-2) in the CRC group was higher than in the control group, but the results were not significant. It may also be connected with the small size of the tested group. Unfortunately, there were no papers that could confirm or contradict the obtained results. In the case of tissue expression of CCL24, the results of available papers were similar to those concerning CCL11. Cheadle et al. [23] revealed that in biopsy samples of CRC and samples of adjacent liver metastases (with CRC origin), the levels of CCL24 were elevated.

In the case of CCL26 (Eotaxin-3), the medians did not differ between the groups which may indicate that this parameter is not related to the ongoing neoplastic changes. However, some authors [24] indicated that, similar to CCL11 and CCL24, tissue expression of Eotaxin-3 was higher in CRC patients. What is more, its expression increased among those with TNM stage and strongly correlated with lymph node metastasis. Perhaps the small number of patients with distant metastasis (stage IV of TNM classification) in our tested group was too low to affect the entire CRC group and demonstrate statistical significance. It can be associated with a generally small number of patients at this stage. In our department, screening diagnostics for CRC are well developed. In addition, the available research methods are very sensitive, which is why cancer detection occurs at a relatively early stage. Even if a patient arrives already in stage IV, when distant metastases are detected, not every patient can be included in the study group (high BMI, no consent to research, palliative treatment). Therefore, we believe that further studies performed on a larger study group (additional patients with distant metastasis) are vital, and research on the concentrations of this eotaxin should not be discontinued.

The most interesting results were obtained for CCR3, which showed a significantly higher concentration in the serum of CRC patients when compared to the control group (*p* = 0.012). Unfortunately, we have not found any studies that would concern the concentrations of this parameter in the course of CRC. However, there are several studies that clearly indicate the usefulness of this parameter. Lan et al. [24], Cheadle et al. [23], and Cho et al. [25] unequivocally showed that the tissue expression of CCR3 in the course of this tumor is much higher when compared to healthy tissues. Interestingly, Devaud et al. [26] demonstrated that CCR3 has an anti-tumor effect correlated with the delayed growth of tumor cells. This information may be very important when we compare this study with the study by Steegenga et al. [27] showing that the tissue expression of this receptor was higher in female than in male mice. This could explain the differences in morbidity and mortality between the sexes for this type of cancer. That is why, further studies on CCR3 concentration and tissue expression should be performed.

We also measured the commonly used tumor markers (CEA, CA 19-9) and C-reactive protein concentrations in CRC patients and in the control group. CEA and CRP revealed significantly higher concentrations in the tested group when compared to healthy subjects. In the case of CA 19-9, we did not observe any significance. This is in accordance with different results obtained in the course of other experiments concerning CRC [28,29].

According to our knowledge, the present study is the first that assesses the diagnostic significance of serum CCL11, CCL24, CCL26, and CCR3 in CRC patients. We have found only one paper concerning diagnostic SE, SP (without PPV and NPV), and AUC for CCL11. In this paper, Yamaguchi et al. [20] reported that diagnostic sensitivity for CCL11 was 75.80%, specificity 66.70%, and AUC 0.714. These values were higher than ours, but the discrepancy might be connected with the size of the tested groups and their ethnicity (Japanese patients). Almost all tested parameters (CCL11, CCL24, and CCR3) showed higher SE than commonly used tumor marker CA 19-9. In the case of CEA and CRP, none of the newly tested parameters showed higher SE. In the case of SP, CCL26 showed the highest value. From the rest of tested parameters, CCR3 and CCL24 showed similar values to CEA and CA 19-9, but lower than the value for CRP. The PPV similar to SE, was higher in the case of almost all tested parameters than the PPV of CA 19-9, but lower than the PPV of CEA and CRP. The NPV, similar to SP, was highest for CCR3 but slightly lower than the NPV of CEA and CRP. What is more, similar to PPV, the NPV of all tested parameters was similar or even higher than the NPV of CA 19-9. In addition, simultaneous analysis of CCR3 + CEA and CCR3 + CRP revealed a marked increase in diagnostic SE and NPV. We noticed that the CCR3 area under the ROC curve in CRC was highest from all tested parameters but lower than AUC for CEA and CRP. Moreover, similar to previously mentioned statistical parameters, in the case of all tested proteins, AUC was comparable or even higher than AUC for CA 19-9. Similar to the diagnostic criteria (SE, SP, PPV, NPV), we observed a higher AUC value for simultaneous analysis of CCR3 with CEA and CRP. Due to the absence of appropriate data, it is impossible to discuss the above-described results.

The observed diagnostic usefulness of the parameters tested indicates that CCR3 could potentially prove to be the best of all tested proteins, especially with the combined analysis with CEA or CRP as a diagnostic panel. As CCR3 is present on the surface of endothelial cells and eosinophils, a significant increase in its concentration and tissue expression may contribute to both the intensification of angiogenesis and the influx of eosinophils, which can lead to tissue eosinophilia (important in the development of neoplastic changes). Considering the current results, it would be necessary in the future not only to expand the study group to a larger number of patients, but also to compare the obtained results with the number of circulating eosinophils, their expression in tissues, and the concentration of other parameters involved in the angiogenesis process, i.e., VEGF (vascular-endothelial growth factor).

#### **4. Materials and Methods**

#### *4.1. Patients*

The study included 50 colorectal cancer patients (CRC) diagnosed by the oncology group (Table 4). The patients were treated in the Department of Oncological Surgery with Specialized Cancer Treatment Units, Maria Sklodowska-Curie Oncology Center, Bialystok, Poland. Tumor classification and staging were conducted in accordance with the International Union Against Cancer Tumor-Node-Metastasis (UICC-TNM) classification. Colorectal cancer histopathology was based on the microscopic examination of tissue samples. Moreover, all patients were grouped according to not only tumor stage (TNM), but also depth of tumor invasion (T factor), the presence of lymph node (N factor), and distant metastases (M factor) as well as the histological grade (G factor) of the tumor. The pretreatment staging procedures included physical and blood examinations, computed tomography (CT), and, in case of patients with rectal cancer, magnetic resonance imaging (MRI) of the small pelvis. Additionally, all patients were assessed according to the Eastern Cooperative Oncology Group (ECOG) score. The blood was collected the day before the treatment (surgery, radio, or chemotherapy). The control group included 30 healthy volunteers. For each of the patients qualified for the control group, the following exclusion criteria were applied: active infections and symptoms of an infection (both bacterial and viral), other comorbidities that can affect cytokine concentrations (respiratory diseases, digestive tract diseases) or systemic diseases such as lupus, rheumatoid arthritis, or collagenosis. In addition, none of the patients included in the control group abused alcohol, smoked, or had a personal or familial history of cancer. None of the patients included (both in the study and control group) had a BMI > 35 to fully exclude the influence of an increase in obesity-related inflammatory factors.


**Table 4.** Characteristics of colorectal cancer and healthy patient groups.

TNM—Tumor Node Metastasis classification.

#### *4.2. Biochemical Analyses*

Venous blood samples were collected from each patient into a tube with clot activator (S-Monovette, SARSTEDT, Numbrecht, Germany), centrifuged to obtain serum samples, and stored at −80 ◦C until assayed. The tested chemokines were measured with a multiplexing method (Luminex Human Discovery Assay (3-Plex), R&D Systems, Abingdon, UK). The CCR3 receptor was measured with the enzyme-linked immunosorbent assay (ELISA) (Aviva Systems Biology Corp., San Diego, CA, USA). Serum levels of classical tumor markers were measured with chemiluminescent microparticle immunoassay (CMIA) (Abbott, Chicago, IL, USA), and for the analysis of CRP concentration, immunoturbidimetric method (Abbott, Chicago, IL, USA) was used according to the manufacturer's protocols. In Luminex and ELISA, according to the manufacturer's protocols, duplicate samples were assessed for each standard, control, and sample.

#### *4.3. Statistical Analysis*

Statistical analysis was performed by RStudio (Boston, MA, USA). The preliminary statistical analysis (using the Shapiro–Wilk test) revealed that the tested parameters and tumor marker levels did not follow normal distribution. Consequently, statistical analysis between the groups was performed by using the U-Mann–Whitney test, the Kruskal– Wallis test, and a multivariate analysis of various data by the post-hoc Dwass–Steele– Crichlow–Flinger test. The data were presented as a median and a range. Diagnostic sensitivity (SE), specificity (SP), and the predictive values of positive and negative test results (PPV and NPV, respectively) were calculated by using the cut-off values which were calculated by the Youden's index (as a criterion for selecting the optimum cut-off point) from the control group, and for each of the tested parameters were as follows: CCL11— 12.75 pg/mL, CCL24—1401.83 pg/mL, CCL26—29.15 pg/mL, CCR3—0.17 ng/mL, CA 19-9—5.44 U/mL, CEA—1.11 ng/mL, CRP—2.5 mg/L. We also defined the receiveroperating characteristics (ROC) curve for all the tested parameters, tumor markers, and CRP to evaluate the diagnostic accuracy. Statistically significant differences were defined as comparisons resulting in *p* < 0.05.

#### **5. Conclusions**

According to our knowledge, the present study is the first that compares the diagnostic characteristics of all eotaxins and their receptor with the well-established tumor markers (CEA and CA 19-9) and the marker of inflammation (CRP) in CRC patients. In addition, due to a limited number of papers, it is extremely difficult to determine the direction in which changes in their concentrations are progressing in the course of colorectal cancer, especially if the available literature is limited to the concentrations of only one of the tested parameters. It is certain that further studies on the concentrations of eotaxins in the course of CRC are necessary to confirm and clarify their diagnostic usefulness and clinical application as potential tumor markers of CRC. However, the most promising factor seems to be CCR3, especially in combined use with CRP or CEA.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/jcm10122675/s1, Table S1: Serum levels of tested parameters in colon cancer subtype and control, Table S2: Serum levels of tested parameters in rectal cancer subtype and control, Table S3: Serum levels of tested parameters in cancer subtype groups, Table S4: Serum levels of tested parameters in Early TNM and control groups, Table S5: Serum levels of tested parameters in Advanced TNM and control groups, Table S6: Serum levels of tested parameters in different TNM stages of CRC.

**Author Contributions:** Conceptualization, M.Z. and B.M.; methodology, M.Z. and A.K.-P.; formal analysis, M.Z., A.K.-P. and M.D.; investigation, M.Z., K.S., M.J., L.K., M.K. and A.K.-P.; resources, M.Z.; data curation, M.Z. and M.D.; writing—original draft preparation, M.Z.; writing—review and editing, M.Z. and B.M.; supervision, B.M.; project administration, M.Z. and B.M.; funding acquisition, M.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Medical University of Bialystok, Poland, grant numbers: SUB/1/DN/20/001/1198 and SUB/1/DN/21/004/1198.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Medical University of Bialystok (R-I-002/564/2019; 28 November 2019).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. Key data are stated in the text.

**Acknowledgments:** This study was conducted with the use of equipment purchased by Medical University of Bialystok as part of the RPOWP 2007–2013 funding, Priority I, Axis 1.1, contract No. UDA-RPPD.01.01.00-20-001/15-00 dated 26 June 2015. B.M. has received consultation and/or lecture honoraria from Abbott, Wiener, Roche, Cormay and Biameditek. M.Z. has received lecture honoraria from Roche.

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

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