Cisplatin inhibits frequency and suppressive activity of monocytic myeloid-derived suppressor cells in cancer patients

Abstract

Cancer immunotherapies have induced long-lasting responses in cancer patients including those with melanoma and head and neck squamous cell carcinoma (HNSCC). However, the majority of treated patients does not achieve clinical benefit from immunotherapy because of systemic tumor-induced immunosuppression. Monocytic myeloid-derived suppressor cells (M-MDSCs) are implicated as key players in inhibiting anti-tumor immune responses and their frequencies are closely associated with tumor progression. Tumor-derived signals, including signaling via STAT3-COX-2, induce the transformation of monocytic precursors into suppressive M-MDSCs. In a retrospective assessment, we observed that survival of melanoma patients undergoing dendritic cell vaccination was negatively associated with blood M-MDSC levels. Previously, it was shown that platinum-based chemotherapeutics inhibit STAT signaling. Here, we show that cisplatin and oxaliplatin treatment interfere with the development of M-MDSCs, potentially synergizing with cancer immunotherapy. In vitro, subclinical doses of platinum-based drugs prevented the generation of COX-2⁺ M-MDSCs induced by tumor cells from melanoma patients. This was confirmed in HNSCC patients where intravenous cisplatin treatment drastically lowered M-MDSC frequency while monocyte levels remained stable. In treated patients, expression of COX-2 and arginase-1 in M-MDSCs was significantly decreased after two rounds of cisplatin, indicating inhibition of STAT3 signaling. In line, the capacity of M-MDSCs to inhibit activated T cell responses ex vivo was significantly decreased after patients received cisplatin. These results show that platinum-based chemotherapeutics inhibit the expansion and suppressive activity of M-MDSCs in vitro and in cancer patients. Therefore, platinum-based drugs have the potential to enhance response rates of immunotherapy by overcoming M-MDSC-mediated immunosuppression.

Keywords: Cancer; MDSC; cisplatin; immunotherapy; platinum-based chemotherapy.

Figure 1.

M-MDSC frequencies are increased in metastatic melanoma patients and associated to short survival after DC vaccination. Metastatic melanoma patient enrolled in clinical trials of DC vaccination were divided into short (<12 months) and long survivors (>24 months) based on overall survival. (a) Quantified frequency of M-MDSCs among freshly isolated total PBMCs in healthy donors (n = 10) and metastatic melanoma patients (n = 28) before DC vaccination. (b) Quantified frequency of M-MDSCs among frozen PBMCs in short (n = 14) and long (n = 8) survivors after DC vaccination. (c-d) Patients were divided into quartiles based on M-MDSCs frequency (low to high) and (e-f) divided into 2 groups based on above and below average M-MDSC frequency (12,5%). Increased M-MDSC frequencies correlated with significantly poor OS and PFS in 22 patients. Mann–Whitney test in panel b, unpaired t-test in panel c and log-rank (Mantel-Cox) test in panel c-f. Mean + SEM in panel a-b

Figure 2.

Platinum-based drugs inhibit the development and suppressive activity of melanoma-induced M-MDSCs in vitro.(a) Schematic overview of M-MDSC induction by THFR primary melanoma cells in the presence and absence of platinum drugs followed by autologous T cell co-culture. (b) Percentage of proliferated CD3 + T cell based on CFSE signal after 4 day co-culture with conditioned monocytes (n = 4). (c) Intracellular COX-2 expression by conditioned monocytes was quantified by flow cytometry. Data are depicted as fold increase of COX-2 MFI compared to isotype control (n = 4). One way ANOVA was used to test significance. Mean + SEM in both graphs *** p = <0.001, ** p = <0.01, * p = <0.05

Figure 3.

Cisplatin inhibits M-MDSC frequency and expression of STAT3-controlled enzymes in HNSCC patients. (a) Overview of treatment schedule for HNSCC patients undergoing cisplatin-based chemoradiotherapy and timepoints of blood collection for assessment of PBMCs by flow cytometry. Shown are (b) quantified frequency of M-MDSCs (n = 8–19) and (c) quantified frequency of monocytes (n = 8–19) of total viable PBMCs plus (d) a representative plot and quantified COX-2 positivity (n = 3–4) and (e) a representative plot and quantified ARG-1 positivity (n = 3–5) both within M-MDSCs and monocytes. One way ANOVA was used to test significance. Mean + SD in all graphs ****p = <0.0001 *** p = <0.001, ** p = <0.01, * p = <0.05, ns = non-significant

Figure 4.

Cisplatin inhibits suppressive activity of M-MDSCs in HNSCC patients. (a) Overview of treatment schedule for HNSCC patients undergoing cisplatin-based chemoradiotherapy and timepoints of blood collection for assessment of suppressive capacity by M-MDSCs. Subpanel indicates two experimental setups of suppression assays performed at each time point. (b) Absolute proliferation of activated T cells in counts per minute after 3 days of culture followed by 16 h thymidine incorporation. T cells from a paired HD were thawed and stimulated for a suppression assay at three timepoints for each patient isolation (in panel b) ensuring similar T cell responses. (c) Proliferation of activated HD T cells was measured by thymidine incorporation after 3 day co-culture with M-MDSCs isolated from patients before, during and after intravenous cisplatin treatment. Proliferation was normalized to monocultured activated HD T cells at every timepoint. (d) Absolute proliferation of activated T cells in counts per minute after 3 days of culture followed by 16 h thymidine incorporation. Autologous T cells were isolated in parallel with M-MDSCs for a suppression assay at three timepoints for each patient (in panel e) simulating in vivo T cell responses. (e) Proliferation of activated autologous T cells was measured by thymidine incorporation after 3 day co-culture with M-MDSCs isolated from patients before, during and after intravenous cisplatin treatment. Proliferation was normalized to monocultured activated autologous T cells at every timepoint. (f) Secreted IFNγ after 3 day co-culture of activated HD T cells and M-MDSCs (from panel C). (g) Secreted IFNγ after 3 day co-culture of activated autologous T cells and M-MDSCs (from panel E). One way ANOVA was used to test significance. Mean + SD in all graphs. ****p = <0.0001 *** p = <0.001, ** p = <0.01, * p = <0.05, ns = non-significant, na = not available, nd = not detected, value below detection limit ELISA, nd* = IFNγ could not be detected in any sample including monocultured activated T cells