Expression pattern of immune- and cancer-associated genes in peripheral blood of mice bearing melanoma cells

G. V. Gerashchenko, I. M. Vagina, Yu. V. Vagin © 2019 G. V. Gerashchenko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.218+616.65


Introduction
Melanoma represents high malignant neoplasms. It occurs in a variety of tumor groups that differ in clinical and histological characteristics, profiles of metastasis, ethnic distribution, a causative role of UV radiation and mutational profile for each patient [1]. For now, such modern approaches to cure mela noma as immunotherapy [2] and combined methods [3] are widely used; however, none of them provides 100 % efficiency of treatment. Melanoma, like many solid tumors, is a quite heterogeneous disease with certain molecular features of individual tumors, hence, it is important, to define the markers for progno-Biomedicine ISSN 1993-6842 (on- sis, describing a status of immune system and specific characteristics of each tumor [4,5,6]. The least invasive methods to analyze markers are preferential. The analysis of liquid biopsies (blood, urine, saliva, etc.) is the most non-invasive for this purpose.
Many of these genes are expressed differently in various cell types, that is why it is quite difficult to correlate their RE levels with the tumor progression.
We hypothesized a possibility to find the correlation between the RE levels of abovementioned genes and the growth of melanoma cells in mice, using the peripheral blood of experimental animals to monitor the expression. We wanted to detect the specific changes in the RE levels and propose the novel noninvasive expression markers for the cancer diagnosis and prognosis.

Materials and Methods
Cell line. The mouse melanoma B16 cell line was obtained from the Bank of Cell Lines (R.E.Kavetsky IEPOR, NAS of Ukraine). The cells were cultured in DMEM (Sigma) medium with the addition of 10 % FBS (Sigma), 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in a CO 2 incubator. The cells were detouched by an EDTA/trypsin solution and rinsed in a phosphate buffered saline (PBS). Cells were counted, and suspension of 2x10 5 cells was injected into each mouse.
Experimental animals. Adult female mice of the C57BL/6j line were used. Suspension of the B16 mouse melanoma cell was subcutaneously introduced into the right posterior paw. Mice, not bearing melanoma cells, served as the control animals. Five mice were studied in each group. On the day 19 th following the injection of melanoma cells, the peripheral blood was collected for the analysis. All manipulations with animals were conducted in accordance with the rules of handling of experimental animals, approved by the Bioethic Committee of IMBG of NAS of Ukraine and the rules, described in "European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes" (Strasburg, 1986).
Total RNA isolation and cDNA synthesis. 100 μl of whole blood were thoroughly mixed with 300 μl of Trizol (Sigma). Total RNA was isolated, using a Direct-zol RNA MiniPrep total RNA kit (Zymo Research), according to the manufacturer's protocol. DNaseI treatment was performed on columns. The quality and concentration of total RNA samples were analyzed on spectrophotometer (NanoDrop Technologies Inc. USA) and agarose gel electrophoresis. cDNA synthesis was performed, using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, USA), according to the manufacturer's protocol.
Statistical analysis. A STATISTICA10 software was used to perform the statistical analysis. The Kruskal-Wallis and Fischer exact tests with correction on multiple comparisons, according to the Benjamini -Hochberg procedure with FDR = 0.2 [11] were used to calculate differences between groups.

Results and Discussion
After the RE levels of 56 cancer-and immuneassociated genes were assessed in peripheral blood of experimental animals, we could divide the genes in three groups, (Figure 1 A,  B). Thus, the genes such as Ccl5, Il1b, Mif, Rnasel, S100a1 and Tgfb1 were highly expressed, whereas the genes Erbb2, Ifnb1, Il6, Pdcd1 and Prom1 were low expressed genes. The RE levels differ between the highly and low expressed genes more than 100-fold.
13 genes were differently expressed, when the mice bearing melanoma cells were compared with the control animals, as was calculated by a statistical analysis. 11 genes were upregulated (p<0.05) (Table 1A) and 2 genes were downregulated (p<0,05) (Table 1B).
Difference in the RE levels was considered significant, when the change was 2-fold or more. Several genes showed the tendency in changes, for example, Xdh (p=0.055) and Arg2 (p=0.058) were upregulated, and three genes, namely Ifng, Erbb2 (p=0.055) and Il12a (p=0.057) were downregulated. We believe that in larger groups the changes in the RE levels could be significant.
The protein, encoded by Lbp, is involved in the TLR signaling pathway and in the acute phase of the immunological response to gramnegative bacterial infections and also is a marker of fibril neutropenia and a poor state in cancer patients [12,13]. Tlr8 and Tlr3, receptors of the TLR family, were upregulated in the mice bearing melanoma cells. It was shown, that an increased expression of TLR8 leads to a faster proliferation of tumor cells and also promotes chemoresistance of the pancreatic tumors. NF-kB and COX2 are upregulated with the TLR8 increase [14]. Moreover, it is known that TLR3 can stimulate invasiveness of cancerous cells [15].
Noteworthy, the expression levels and a role of Cxcl9 in the development of various tumor types is controversial [16]. Even so, Cxcl9 is a promising target for the creation of new approaches to treat cancer [17].
Among the interferon (INF alpha) targeted genes, Oas3, Oas1a and Rnasel were upregulated in the group of mice bearing melanoma cells. Functioning of OAS1 and OAS3 in the immune pathways and response to RNA viruses are closely related to RNASEL [18]. It is known that downregulation of these genes is associated with the poor prognosis for cancer patients and also with the resistance to chemotherapy [19].
It could be that the mice still have the immune system reserves to fight the tumor growth at this stage after the melanoma cells were inoculated. However, several symptoms of the immunosuppressive state were detected in experimental animals, as was evident by the expression data.
Next, we observed a group of genes that showed moderate upregulation in the mice, bearing melanoma cells, i.e. RE changes were 2-3 fold. The Cd14 upregulation could be due to the infiltration of monocytes into tumor, and could serve as a marker for cancer progression and metastasizing [20,21].
It has been reported already that GSTP1, encoding Glutathione S-transferase PI, is overexpressed in many types of cancers [22], for example, is associated with the K-ras mutation in colorectal cancer [23] and with downregulation of miR-133α in head and neck squamous cell carcinoma [22]. An increased GSTP1 expression is also indicated in an enhanced detoxification activity, protecting cancer cells against cytotoxic and cytostatic drugs [24,25]. However, there are no data on the Gstp1 expression in the blood of cancer patients. Noteworthy, an increased GSTP1 expression enhances oncogenicity of breast cancer by regulating glycolytic and lipid metabolism as well as the energy and oncogenic signaling pathways, resulting in the activation of glyce-ral dehyde-3-phosphate dehydrogenase [26]. Moreover, the genetic or pharmacological inactivation of GSTPI worsens the survival of tumor cells, due to abnormalities in the underlying signaling pathways. PROM1 (CD133) is a marker of cancer stem cells, for lung and colorectal tumors [27,28]. High levels are correlated with the poor prognosis in colorectal cancer [29]. Importantly, the increased RE levels of Prom1 were detected in the mice, bearing melanoma cells. This could be an evidence of the presence of circulating tumor cells in the mouse bloodstream as well as of an elevated expression of Cd14 and Gstp1, described above.
Additionally, the Il1b expression increased in the mice, bearing melanoma cells. This interleukin can be expressed by tumor cells and different stromal elements, such as myeloidderived suppressor cells and tumor-associated macrophages [30]. IL1B enhances metastazing [31]. Importantly, the experimental animals, bearing tumor cells, showed lower RE levels of Ifnb1, Pdcd1 and Ifng. This indicates the immunosuppressive state of the mice [32,33].
Summarizing, several studied genes showed significant differences in the RE levels in the mice, bearing melanoma cells, compared with the control animals. The detected alterations refer to the genes, associated with the immune and cancer cells and could serve as putative noninvasive biomarkers of tumor growth. These genes are Lbp, Tlr3, Tlr8, Prom1 and Cd14. Additionally, the Oas3, Rnasel, Gstp1 and Cxcl9 genes could be the markers of sensitivity to chemotherapy.

Acknowledgments
This work was supported by the Scientific Program of National Academy of Science of Ukraine "Molecular and cell biotechnology for medicine, industry and agriculture" №41/19.

Conclusions
Assessment of the expression of cancer-and immune-associated genes in the peripheral blood of the experimental animals upon the growth of malignant cells in vivo may result in the discovery of the effective noninvasive expression markers for the prognosis of the outcome of the cancer disease and effectiveness of the chemotherapy.