The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent

Abstract

In spite of impressive response rates in multiple cancer types, immune checkpoint inhibitors (ICIs) are active in only a minority of patients. Alternative strategies currently aim to combine immunotherapies with conventional agents such as cytotoxic chemotherapies. Here, we performed a study of PD-1 or PDL-1 blockade in combination with reference chemotherapies in four fully immunocompetent mouse models of cancer. We analyzed both the in vivo antitumor response, and the tumor immune infiltrate 4 days after the first treatment. in vivo tumor growth experiments revealed variable responsiveness to ICIs between models. We observed enhanced antitumor effects of the combination of immunotherapy with chemotherapy in the MC38 colon and MB49 bladder models, a lack of response in the 4T1 breast model, and an inhibition of ICIs activity in the MBT-2 bladder model. Flow cytometry analysis of tumor samples showed significant differences in all models between untreated and treated mice. At baseline, all the tumor models studied were predominantly infiltrated with cells harboring an immunosuppressive phenotype. Early alterations of the tumor immune infiltrate after treatment were found to be highly variable. We found that the balance between effector cells and immunosuppressive cells in the tumor microenvironment could be altered with some treatment combinations, but this effect was not always correlated with an impact on in vivo tumor growth. These results show that the combination of cytotoxic chemotherapy with ICIs may result in enhanced, similar or reduced antitumor activity, in a model- and regimen-dependent fashion. The present investigations should help to select appropriate combination regimens for ICIs.

Keywords: PD-1; PD-L1; chemotherapy; combination therapy; oncology; preclinical mouse models; tumor microenvironment.

Figure 1

In vivo tumor growth in mice treated with ICI alone or in combination with chemotherapy. Anti-PD1 (clone RMP1.14) or anti-PDL1 (clone 10F.9G2) mAb (12,5 mg/kg, ip, q1wk) were administered in combination with (A,C) methotrexate (1 mg/kg, ip, q1wk), vinblastine (0.1 mg/kg, ip, q1wk), doxorubicin(1 mg/kg, ip, q1wk) and cisplatin(1 mg/kg,ip, q1wk, MVAC) in SC bladder cancer MB49 and MBT-2, and with (B) cyclophosphamide (CTX, 100 mg/kg, ip, q1wk) and doxorubicin (DOX, 2 mg/kg, ip, q1wk) in SC metastatic breast cancer 4T1, and with (D) capecitabine (CAPE, 250 mg/kg, po 5 days a week) and oxaliplatin (OXA, 5 mg/kg, ip, q1wk) in SC colorectal cancer MC38. Data shown are mean tumor volumes+ SEM. n = 5 to 6 mice/group (A), n = 6 mice/group (B), n = 6 mice/group (C), n = 3 to 6 mice/group (D). *p < 0.05 and ***p < 0.001 using Student's t-test.

Figure 2

Total leukocyte infiltrate in tumors from mice treated with ICI alone or in combination with chemotherapy. Effect of chemotherapies, anti-PD1 or anti-PDL1 mAbsand their combination on total leucocyte infiltrate in various preclinical tumor models. Flow cytometry analysis of total CD45⁺ cells of MBT-2 tumors (A), 4T1 tumors (B), MB49 (C), and MC38 tumors (D). Mice were treated as in Figure 1. Data are shown as mean values+ SEM. n = 5 to 6 mice/group (A), n = 5 to 6 mice/group (B), n = 6 mice/group (C), n = 5 mice/group (D). **p < 0.01 using Mann-Whitney test.

Figure 3

All the preclinical models studied are predominantly infiltrated with cells considered as ≪ immunosuppressive ≫, particularly MDSCs. Effect of chemotherapies, anti-PD1 or anti-PDL1 Mabs and their combination on immune cells subpopulations in various preclinical tumor models. Flow analysis of immune cells subpopulations of total CD45⁺ cells of MBT-2 tumors (A), 4T1 tumors (B), MB49 (C), and MC38 tumors (D). Mice were treated as in Figure 1. Data are shown as Mean of immune cells in each groups normalized from total CD45+ cells, n = 5 to 6 mice/group (A), n = 5 to 6 mice/group (B), n = 6 mice/group (C), n = 5 mice/group (D).

Figure 4

Activated T cells are increased by chemotherapies in breast and bladder cancer. Effect of chemotherapies, anti-PD1 or anti-PDL1 Mabs and their combination on T effector cells activation in various preclinical tumor models. Flow cytometric analysis of TNFα+ CD8+ T cells of total CD45⁺ cells of 4T1 tumors (A), MB49 (B), and MC38 tumors (C). Mice were treated as in Figure 1. Data are shown as Mean + SEM, n = 5 to 6 mice/group (A), n = 6 mice/group (B), n = 5 mice/group (C), Mann–Whitney test: *P < 0.05 and **P < 0.01. Results not shown for MBT-2 bladder cancer (Figure S4).

Figure 5

Combination of MVAC with anti-PDL1 significantly reduces T reg cells infiltrate compared with anti-PDL1 single treatment in bladder cancer MB49. Effect of chemotherapies, anti-PD1 or anti-PDL1 Mabs and their combination on T regulator cells in various preclinical tumor models. Flow cytometric analysis of FoxP3+ CD4 T cells of total CD45⁺ cells of 4T1 tumors (A), MB49 (B), and MC38 tumors (C). Mice were treated as in Figure 1. Data are shown as Mean + SEM, n = 5 to 6 mice/group (A), n = 6 mice/group (B), n = 5 mice/group (C), Mann–Whitney test: *P < 0.05 and **P < 0.01. Results not shown for MBT-2 bladder cancer (Figure S4).

Figure 6

Combination of cyclophosphamide and doxorubicine increase inhibitory checkpoint on immune CD4+ T cells in 4T1 breast cancer model. Effect of chemotherapies, anti-PD1 or anti-PDL1 Mabs and their combination on alternative immune checkpoints expression on CD4 T cells in various preclinical tumor models. Flow cytometric analysis of CD279 (PD-1), CD223 (LAG-3), TIM-3, CD278 (ICOS), CD274 (PDL-1), TIGIT on CD4 T cells infiltrate of total CD45⁺ cells of MBT-2 tumors (A), 4T1 tumors (B), MB49 (C) and MC38 tumors (D). Mice were treated as in Figure 1. Data are shown as Mean percentage of CD4 T cells which express the different immune checkpoint, n = 5 to 6 mice/group (A), n = 5 to 6 mice/group (B), n = 6 mice/group (C), n = 5 mice/group (D).

Figure 7

Chemotherapies decrease inhibitory checkpoint expression on immune CD8+ T cells. Effect of chemotherapies, anti-PD1 or anti-PDL1 Mabs and their combination on alternative immune checkpoints expression on CD8+ T cells in various preclinical tumor models. Flow cytometric analysis of CD279 (PD-1), CD223 (LAG-3), TIM-3, CD278 (ICOS), CD274 (PDL-1), TIGIT on CD8 T cells infiltrate of total CD45⁺ cells of MBT-2 tumors (A), 4T1 tumors (B), MB49 (C), and MC38 tumors (D). Mice were treated as in Figure 1. Data are shown as Mean percentage of CD8+ T cells which express the different immune checkpoint, n = 5 to 6 mice/group (A), n = 5 to 6 mice/group (B), n = 6 mice/group (C), n = 5 mice/group (D).

Figure 8

MVAC chemotherapy regimen induces a large decrease of TIGIT expression on tumor cells in MB49 bladder cancer. Effect of chemotherapies, anti-PD1 or anti-PDL1 Mabs and their combination on alternative immune checkpoints expression on tumor cells in various preclinical tumor models. Flow cytometric analysis of CD279 (PD-1), CD223 (LAG-3), TIM-3, CD278 (ICOS), CD274 (PDL-1), TIGIT on tumor cells of total CD45⁺ cells of MBT-2 tumors (A), 4T1 tumors (B), MB49 (C), and MC38 tumors (D). Mice were treated as in Figure 1. Data are shown as Mean percentage of tumor cells which express the different immune checkpoint, n = 5 to 6 mice/group (A), n = 5 to 6 mice/group (B), n = 6 mice/group (C), n = 5 mice/group (D).