Low Dose Of 5-Fluorouracil Combined with A Dendritic Cell Vaccine Delays the Growth of Colorectal Cancer in Mice

Colorectal Cancer (CRC) ranks the 3rd most frequent type of
cancer in many developed countries, with estimated 147,950 new...


Short Communication
Colorectal Cancer (CRC) ranks the 3rd most frequent type of cancer in many developed countries, with estimated 147,950 new cases and 53,200 deaths in 2020 [1]. The standard treatment has been 5-Fluorouracil (5-FU) therapy alone or combined with other drugs, while combination with immunotherapies have also been used [2,3]. The potential role of the immune system in limiting the progression of colorectal cancer (CRC) is supported by the finding that patients with CRC who have high numbers of NK, CD4+ and CD8+ T cells in their peripheral blood or tumor tissue have higher survival rates compared with patients with lower numbers of these cells [4,5]. We previously observed that both human [6] and murine Dendritic Cells (DC) [7] sensitized with the lisate of tumor cells exposed to low concentrations of 5-FU, show increased ability to stimulate lymphoproliferation. Similarly, the pre-treatment of tumor cells with low concentrations of paclitaxel, increases their immunogenicity, facilitating the generation of specific cytotoxic T lymphocytes [8]. The generation of a T cell antitumor response is highly dependent on the role of DCs and we also have observed that sensitization of human DCs with tumor lysate is enhanced by previous incubation with low concentration of paclitaxel [9]. In addition, we have recently observed that treatment of tumor cells with low concentration of 5-FU in association with pharmacological blockage of autophagy, is able to improve tumor lisates to enhance DC activity to both improve the potential T cell responsiveness and DOI: 10.26717/BJSTR.2020.29.004826 the specific antitumor response [10]. Then, we were stimulated to investigate the feasibility of in vivo handling of antitumor resistance, combining low dose 5-FU therapy with DC-vaccine.
For the in vitro generation of DC, bone marrow was collected from the tibia and femurs of healthy C57BL/6 male mice.
Erythrocytes were lysed with Gey´s hemolytic solution and T and B lymphocytes were depleted with anti-CD4, CD8 and B220 magnetic beads (Miltenyi, Inc.). Resulting cells were cultured in complete culture medium supplemented with 80 mg/ml of both murine recombinant GM-CSF and IL-4 at 37°C in a 5% CO2 incubator. After 3 days, non-adherent and loosely adherent cells were harvested, split into 6-well culture plates and also cultured with complete culture medium supplemented with both cytokines. Three days later DC were loaded with tumor cell lysates (100 µg protein/106 cells) and harvested 24 h later. Control DC were cultured without MC38 lysate and referred as "DC" and those cultured with MC38 lysate are referred to as "VAX". Both VAX and DC preparations contained   Immunocompetent cells migration and homing is dependent on the expression of L-selectin (CD62L) by leukocytes. L-selectin actively take part in the T cell homing at lymphoid organs and infiltration into the tumor site [11] so that effector and memory cells differentially express CD62L [12]. Interestingly, both the treatment   On the other hand, L-selectin also take part in the infiltration of effector memory cells into the tumor site. Here, we observed that expression of L-selectin on CD8+ cells from 5-FU+VAX group is higher than in both DC and VAX groups suggesting that the combination 5-FU+VAX enhances the homing of T cells to lymphoid organs and tumor sites. According to Chao et al, [14] and Raffler et al, [12] murine CD8+ cells stimulated with anti-CD3 antibody presented initial loss of surface L-selectin due to cleavage and all cells became L-selectinlow/negative some days later. Activated cells rapidly divide and differentiate into L-selectinlow/positive effector memory cells that migrate to inflammatory sites. After that, most T cells undergo apoptosis but some of them evolve into memory cells.
Besides that, 5-FU at maximum dose, shows to be contradictory into its ability to favor the immune system and effectively helps downregulating tumor growth [15], suggesting low doses as a good alternative for cancer treatment.
We observed that the numbers of CD4+ and CD8+ circulating cells decreased as the tumor growth, while MDSC increased. Murine MDSC are described as CD11b+/Gr1+ cells, that are hypothesized to originate in the bone marrow of tumor bearing mice [16,17]. These cells populate the blood and peripheral lymphoid organs during the tumor growth and enter the tumor site where they play an immunomodulatory/immunosuppressive role [16,18]. Tongu et al. [19] observed that combination of low doses of cyclophosphamide and gemcitabine suppressed growing of CT26 colon cancer cells in Balb/c mice. This treatment decreased the percentages of tumorinfiltrating Gr-1high/CD11b+ MDSCs. In accordance with these results, depletion of this population prevents tumor growth in murine models [20]. In our study, contrasting with the findings in peripheral blood, analysis of tumor infiltrating cells showed

Competing Interests
Authors declare no conflict of interests.

Authors Contribution
MRC performed the experiments, analyzed the results and drafted the manuscript. SOR provided technical and infrastructural support for the study. PV helped to perform the in vitro studies. RK designed the study, analyzed the results, provided technical and infrastructural support, and wrote the final manuscript.