Chemo-immunotherapy with oxaliplatin and interleukin-7 inhibits colon cancer metastasis in mice

Abstract

Combination of immunotherapy and chemotherapy has shown promise for cancer. Interleukin-7 (IL-7) can potentially enhance immune responses against tumor, while oxaliplatin (OXP), a platinum-based drug, can promote a favorable immune microenvironment and stimulate anticancer immune responses. We evaluated the anti-tumor activity of IL-7 combining OXP against a murine colon carcinoma in vitro and in vivo and studied the tumor immune microenvironment to investigate whether the combined treatment affects on the local immune cell populations. Utilizing lung and abdomen metastasis models by inoculation of CT26 mice colon cancer cells, we evaluated the anti-tumor efficacy of combining IL-7 and OXP in mice models. Tumor immune microenvironment was evaluated by flow cytometric analysis and immunohistochemical staining. Our study showed that the in vivo administration of IL-7 combined with OXP markedly inhibited the growth of tumors in lung and abdomen metastasis models of colon cancer. IL-7 alone had no effect on tumor growth in mice and IL-7 did not alter cell sensitivity to OXP in culture. The antitumor effect of combining IL-7 and OXP correlated with a marked increase in the number of tumor-infiltrating activated CD8+ T cells and a marked decrease in the number of regulatory T (Treg) cells in spleen. Our data suggest that OXP plus IL-7 treatment inhibits tumor cell growth by immunoregulation rather than direct cytotoxicity. Our findings justify further evaluation of combining IL-7 and chemotherapy as a novel experimental cancer therapy.

Figure 1. In vitro effect of OXP plus IL-7 on the growth of CT26 cells.

CT26 cells were cultured in vitro with IL-7 (25 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL) alone or in combination with OXP at three concentrations from 10 µmol/mL to 50 µmol/mL and the relative numbers of viable cells were assessed 24, 48 hours later respectively. Each bar represents the mean ± S.E. (n = 5). a. OXP induced the expected concentration-dependent cell growth inhibition. b. IL-7 alone had no effect on CT26 cell growth in vitro. The rate of tumor cell growth was not significantly different at any dose level of IL-7 from those observed in control cultures. c and d. The addition of IL-7 did not significantly alter CT26 cell growth or their sensitivity to OXP.

Figure 2. Combination with IL-7 increases the anti-tumor effect of OXP in vivo.

On day 6 post-tumor inoculation each model was randomized into 4 groups with 8 mice in each group for further administration as follows: PBS, IL-7, OXP, IL-7 combined with OXP. Metastatic tumor nodules in the sub-pleural regions of the lungs were counted under a dissection microscope. The metastatic tumor nodules in the abdominal cavity were counted by naked eyes and weighted. The results were expressed as mean ± S.E. P values: *, P<0.05, **, P<0.01; a. Metastatic tumor nodules number in lung metastasis model. b. Weight of lungs in lung metastasis model. c. Metastatic tumor nodules number in abdomen metastasis model. d. Weight of tumor nodules in abdomen metastasis model. All of a, b, c, d showed IL-7, when given in conjunction with OXP, increased the anti-tumor effect in vivo compared to PBS, IL-7 and OXP groups respectively.The administration of IL-7 alone resulted in a significant reduction in the lung weights and a small, not statistically reduction in the final tumor volume compared to PBS in the lung metastasis model. The administration of OXP alone did not provide any anti-tumor effect compared to PBS in the lung metastasis model. In the abdominal implantation model, the administration of OXP alone resulted in a significant reduction in the weights of tumor nodules and a small, not statistically reduction in the number of tumor nodules in the abdominal cavity compared to PBS. The administration of IL-7 alone did not provide any anti-tumor effect compared to PBS in the abdominal implantation model.

Figure 3. Combination of OXP with IL-7 inhibited proliferation in vivo.

The resected tumors of each group were analyzed by immunohistochemical staining for the proliferation marker Ki-67. a. The representative slides of each treatment group are shown. Original magnification, ×200. b. The proliferative activity was evaluated by Ki-67 staining. In each sample, a minimum of 1000 tumor cells were scored in the microscopic areas showing the highest degree of immunostaining, and the results were expressed as the percentage of positive cells. The results were expressed as mean ± S.E. (n = 3). P values: IL-7+OXP compared with the other treatment groups, *, P<0.05, **, P<0.01. The combined treatment with OXP and IL-7 presented fewer Ki-67 positive cells, compared by IL-7 or OXP alone and control.

Figure 4. Assay of apoptosis induced by combination therapy by TUNEL.

a. Paraffin sections were stained with the TUNEL analysis to detect apoptotic cells winthin CT26 tumor. The representative sections of each group are presented. Original magnification, ×200. b. Apoptotic index was determined by evaluating the average number of nuclei apoptotic cells in 5 high-power microscopic fields. The group of combination therapy showed the highest apoptotic index. Data represented apoptosic index were expressed as mean ± S.E. (n = 3). P values: IL-7+OXP compared with the other treatment groups, *, P<0.05, **, P<0.01.

Figure 5. Combination of OXP with IL-7 induced significant activated T cells into the tumors.

a. In cytometric analysis, we gate data using the FSC and SSC bivariate plot, then draw a region around the cluster of lymphocytes and then gate the fluorescence histogram on them. This gate, in the FSC vs SSC view, once established, is then applied to the analysis of all subsequent samples. Representative flow cytometry of each group was presented. b.IL-7 combined with OXP has significantly increased the CD8+/CD69+ positive cells in tumor compared with PBS controls and IL-7 or OXP alone lung model. The results were expressed as mean ± S.E. (n = 3). P values: IL-7+OXP compared with the other treatment groups, *, P<0.05, **, P<0.01.

Figure 6. Immunohistochemical staining of CD8 in tumor tissue.

a. The representative slides of immunohistochemical staining of CD8 each treatment group are shown. Original magnification, ×200. b. The degree of CD8+ infiltration was observed in more than 10 independent high-power (×200) microscopic fields for each tissue sample. The five microscopic fields with the most abundant distribution were selected within the cancer cell nest. The average number of CD8+ T cells was counted in the five microscopic fields. The results were expressed as mean ± S.E. (n = 3). P values: IL-7+OXP compared with the other treatment groups, *, P<0.05, **, P<0.01.

Figure 7. Combination of OXP with IL-7 had no effect the number of CD11b+F4/80 cells and DCs.

a. and b. Representative flow cytometry of each group was presented. c. and d. IL-7 combined with OXP had no effect on the number of CD11b+F4/80+ cells and DCs in tumors compared with those from IL-7 alone, OXP alone, and PBS treated mice in the lung metastasis model.

Figure 8. Combination of OXP with IL-7 reduces Treg cells in spleen.

a. Representative flow cytometry of each group was presented. b. IL-7 combined with OXP has significantly reduced the FoxP3+ population in CD4+ T cells in spleen compared with PBS controls and IL-7 or OXP alone in both lung and abdominal metastasis models. The results were expressed as mean ± S.E. (n = 3). P values: IL-7+OXP compared with the other treatment groups, *, P<0.05, **, P<0.01.

Figure 9. Combination of OXP with IL-7 had no effect on MDSCs.

a. Representative flow cytometry of each group was presented. b. IL-7 and OXP did not significantly decrease the number of MDSC in spleen compared with that of IL-7 alone, OXP alone or control in the lung metastasis model.