Human mesenchymal stem cells creating an immunosuppressive environment and promote breast cancer in mice

Abstract

Human mesenchymal stem cells (hMSC) can home to tumor sites and promote tumor growth. The effects of hMSC on tumor growth are controversial and involvement of hMSC in tumor immunology has not been adequately addressed. Therefore, we investigated whether injection of hMSC affects tumor appearance, growth and metastasis, and anti-tumor immunity in an experimental animal model of metastatic breast cancer. Injection of hMSC in BALB/c mice bearing mammary carcinoma promoted tumor growth and metastasis, which was accompanied by lower cytotoxic activity of splenocytes, NK cells and CD8⁺ T cells in vitro. Tumor-bearing mice that received hMSC had significantly lower percentages of CD3⁺NKp46⁺ NKT-like, higher percentages of CD4⁺Foxp3⁺ T cells, increased serum levels of Th2 and decreased serum levels of Th1 cytokines, and significantly higher number of CD4⁺ cells expressing IL-10. These results demonstrate that immunosuppressive environment created by hMSC promoted breast tumor growth and metastasis in mice.

Figure 1. hMSC migrated in tumor and promoted breast tumor growth and metastasis.

(a) Representative samples of PCR analysis showing migration and survival of hMSC. Human CYP1A1 gene, without cross-reactivity with mouse DNA, was detected by PCR analysis in tumor, blood, lymph node, spleen, liver, and lung samples at 1^st and 3^rd day of experiment. Photomicrographs showing the presence human mitochondrial marker in the tissues (b, c). Positive signals were detected in human ESC control group (b) and in the livers of tumor-bearing mice that received hMSC (c), but no compelling evidence of positively stained cells in lung mice from the same group (d). (e) Impact of 4T1: hMSC ratio on tumor growth. All animals received 2 × 10⁴ 4T1 cells. The highest incidence of tumor growth (e) and the largest tumor volume (f–g) was seen in tumor-bearing mice that received 1 × 10⁶ hMSC. There is a strong correlation between the number of injected hMSC and tumor volume (Fig. 1h; R² = 0.865). (i) Semi-quantitative analysis of lung and liver tissue sections. The incidence of lung and liver metastasis was significantly higher in tumor-bearing mice that received hMSC. Combined results of 3 experiments with total of 9 animals per group (mean ± s.e.m.; *P < 0.05).

Figure 2. hMSC decreased cytotoxic capacity of CD8⁺ T lymphocytes and NK cells in tumor-bearing mice.

(a) There was a significant increase in total number of splenocytes in tumor-bearing mice compared to mice from control group. The cytotoxic capacity of splenocytes derived from tumor-bearing mice was significantly lower than cytotoxic capacity of splenocytes derived from healthy animals. Combined results of 3 experiments with at least 9 animals per group (median ± s.e.m; *P < 0.05). (b) There was no significant difference in total number of CD8⁺ T lymphocytes in tumor-bearing mice compared to mice from control group. There was significant decrease in cytotoxic capacity of CD8⁺ T cells derived from spleens of tumor-bearing hMSC treated mice. Combined results of 3 experiments with at least 9 animals per group (median ± s.e.m.; *P < 0.05). (c) There was no significant difference in total number of CD3⁻NKp46⁺ NK cells in tumor-bearing mice compared to mice from control group. There was significant decrease in cytotoxic capacity of CD3⁻NKp46⁺ NK cells derived from spleens of tumor-bearing hMSC treated mice. Combined results of 3 experiments with at least 9 animals per group (median ± s.e.m.; *P < 0.05).

Figure 3. hMSC downregulated NKT-like cells and increase number of T regulatory cells.

(a) There was significant decrease in percentage and total number (b) of CD3⁺NKp46⁺ NKT-like cells in spleens of tumor-bearing mice that received hMSC. Isotype controls (c) and representative dotplots (d) confirm significant decrease in percentage of NKT cells in spleens of hMSC treated mice. Combined results of 4 experiments with at least 9 animals per group (mean ± s.e.m.; *P < 0.05). (e) There was significant increase in both percentage and total number (f) of Foxp3⁺ regulatory cells cells in spleens of tumor-bearing mice that received hMSC. Representative histogram (g) confirms significant increase in percentage of Foxp3⁺ T regulatory cells in spleens of hMSC treated mice. Histograms and mean values presented in the graphs are gated on CD4⁺ cells. Combined results of 4 experiments with at least 9 animals per group (mean ± s.e.m.; *P < 0.05).

Figure 4. hMSC stimulated production of Th2 and inhibited production of Th1 cytokines in tumor-bearing mice.

(a) Measurement of cytokines in serum. The level of TGF-β, IL-10 and IL-4 was significantly lower and level of IFN-γ was significantly higher in serum samples of tumor-bearing animals that received hMSC compared to tumor-bearing animals that were not treated with hMSC. Combined results of 4 experiments with at least 9 animals per group (mean ± s.e.m.; *P < 0.05; **P < 0.01). (b) Measurement of intracellular production of cytokines. There was significant difference in total number of IL-10 producing CD4+ splenocytes between hMSC treated and control tumor-bearing or healthy animals but no difference between groups in the total number of IFN-γ producing cells. The ratio between IL-10 and IFN-γ producing CD4⁺ splenocytes was significantly higher in tumor-bearing animals that received hMSC. Combined results of 4 experiments with 4 animals per group (median ± s.e.m.; *P < 0.05; **P < 0.01).