CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas

Abstract

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b⁺/Ly6C^hi/PD-L1⁺ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2⁺ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4⁺ and CD8⁺ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.

Keywords: glioblastoma; immunotherapy; myeloid-derived suppressor cells; programmed death-1; tumor microenvironment.

Fig. 1.

Distinct cell populations of CCR2- and CX3CR1-expressing myeloid cells in glioma-bearing mice. (A) Fluorescent images showing representative example of section of KR158 tumor-bearing Ccr2^RFP/WT/Cx3cr1^GFP/WT normal (N) and tumor (T) tissue. Red fluorescence denotes CCR2⁺ cells, while green fluorescence denotes CX3CR1⁺ cells. (Image magnification: 20×.) (B) Flow-cytometric analysis of tumor isolates from KR158 (n = 4) (Left) and 005 GSC (n = 3) (Right) tumor-bearing Ccr2^RFP/WT;Cx3cr1^GFP/WT mice. Higher CCR2 single-positive (P = 0.048) and CX3CR1 single-positive (P = 0.012) cell populations in KR158 vs. 005 GSC glioma models are noted. (C) Flow-cytometric analysis of bone marrow cell populations in CCR2/RFP vs. CX3CR1/GFP in naïve (n = 3) (Upper Left), mock PBS-injected (n = 6) (Upper Right), 005 GSC (n = 3) (Lower Left), and KR158 (n = 6) (Lower Right) tumor-bearing Ccr2^RFP/WT;Cx3cr1^GFP/WT animals. Quantification shows increase in CCR2 single-positive cells in KR158 (P = 0.032) and 005 GSC (P = 0.001) tumor-bearing animals. (D) Flow-cytometric analysis of tumor isolates from Ccr2^RFP/WT;Cx3cr1^GFP/WT mice. The left graphs represent forward scatter (FSC) vs. CD45 plots demonstrating 3 CD45 populations: negative (left), low (middle), and high (right). Blue arrows denote subpopulations plotted by expression of CCR2 and CX3CR1. CD45^low events (Upper) represent a primarily CX3CR1⁺ cell population, while CD45^hi events represent a heterogeneous cell population consisting of CCR2⁺, CX3CR1⁺, and CCR2⁻/CX3CR1⁻ cells. (E) Flow-cytometric analysis of tumor isolates from Ccr2^RFP/WT;Cx3cr1^GFP/WT mice. The left graphs represent Ly6C⁺ vs. Ly6G⁺ events and demonstrate 3 Ly6C populations: negative (bottom), intermediate (middle), and high (top). Blue arrows denote subpopulations plotted by expression of CCR2 and CX3CR1. Ly6C^hi events represent a cell population that is primarily CCR2⁺/CX3CR1⁺, while Ly6C⁻ events represent a heterogeneous cell population consisting of CCR2⁺, CX3CR1⁺, and CCR2⁻/CX3CR1⁻ cells. Representative plots are shown throughout. *P < 0.05; **P < 0.01.

Fig. 2.

Effect of Ccr2 deficiency on glioma-bearing mice. (A) Survival analysis of KR158 tumor-bearing Ccr2^RFP/WT and Ccr2^RFP/RFP mice treated with or without anti-PD-1. Ccr2 deficiency did not impact survival in IgG-treated Ccr2^RFP/WT mice (n = 8), while anti-PD-1 treatment (n = 10) enhanced survival (P = 0.035) in Ccr2-deficient mice only. Triangles mark anti-PD-1 administration. (B) Fluorescent imaging of CD11b (green stain) in Ccr2^RFP/WT and Ccr2^RFP/RFP mice. Representative images shown. (C) Fluorescent imaging of femur cross-section from Ccr2^RFP/WT and Ccr2^RFP/RFP naive and KR158 tumor-bearing mice. Loss of Ccr2 enhanced CCR2/RFP signal in bone marrow of naive mice (P = 0.029), which was further enhanced in tumor-bearing Ccr2^RFP/RFP animals (P = 0.036). Representative images are shown. Quantification: average pixel density/cross-sectional area from 3 consecutive sections, 3 mice/treatment group. *P < 0.05.

Fig. 3.

Impact of Ccr2 deficiency on peripheral and tumor MDSC populations. (A) Flow-cytometric analysis of RFP⁺ events in Ccr2^RFP/WT (n = 6) vs. Ccr2^RFP/RFP (n = 6) mice. Population of RFP⁺ cells within the tumor microenvironment (Upper) is reduced (P = 0.047) but increased (P = 0.024) in bone marrow (Lower) of Ccr2-deficient animals. (B) Flow-cytometric analysis of CD45⁺/CD11b⁺/Ly6C^hi events in Ccr2^RFP/WT (n = 5) vs. Ccr2^RFP/RFP (n = 5) mice. Population of CD45⁺/CD11b⁺/Ly6C^hi cells within the tumor microenvironment (Upper) was reduced (P = 0.039) but increased (P = 0.020) in bone marrow (Lower) of Ccr2-deficient animals. (C) Quantification of percentage of RFP⁺ cells that are CD45⁺, CD45⁺/CD11b⁺, and CD45⁺/CD11b⁺/Ly6C^hi within bone marrow (Upper) and tumor (Lower) in Ccr2^RFP/WT (n = 5) vs. Ccr2^RFP/RFP (n = 5) mice. Ratios remain unchanged in bone marrow but show a significant reduction (P = 0.007) of CD45⁺/CD11b⁺/Ly6C^hi cells in tumors of Ccr2^RFP/RFP vs. Ccr2^RFP/WT mice. Representative plots are shown throughout. *P < 0.05; **P < 0.01. FSC, forward scatter.

Fig. 4.

Effect of combinatorial CCX872/anti-PD-1 treatment on survival of KR158 and 005 GSC glioma-bearing mice. (A) Schematic representation of CCX872 and anti-PD-1 treatment schedules. Survival analysis of KR158 (n= 8 to 10) (B) and 005 GSC (n = 8 to 10) (C) tumor-bearing WT mice treated with CCX872 and anti-PD-1. In KR158 glioma-bearing mice, CCX872 increased median survival (P = 0.002, 32 vs. 50 d). Combinatorial treatment increased durable survival (P = 0.001); 005 GSC-bearing animals had an increase in median survival (P = 0.005, 30 vs. 49 d) with combinatorial treatment. Triangles mark anti-PD-1 administration. The bracket indicates CCX872 administration. *P < 0.05; **P < 0.01.

Fig. 5.

Impact of combinatorial CCX872/anti-PD-1 treatment on peripheral and tumor myeloid cell populations. (A) Flow-cytometric analysis of Ly6C⁺ vs. Ly6G⁺ events in KR158 tumor isolates (Upper) and bone marrow cell populations (Lower) from vehicle-treated (n = 6) and CCX872-treated (n = 6) animals. Drug treatment resulted in a reduction (P = 0.038) of Ly6C^hi events within tumors and an increase (P = 0.028) in bone marrow. (B) Flow-cytometric analysis of Ly6C⁺ vs. Ly6G⁺ events in 005 GSC tumor isolates (Upper) and bone marrow cell populations (Lower) from vehicle-treated (n = 6) and CCX872-treated (n = 5) animals. Drug treatment resulted in a reduction (P = 0.015) in Ly6C^hi events within tumors and an increase (P = 0.028) in bone marrow. (C) Flow-cytometric analysis of tumor isolates from KR158 tumor-bearing Ccr2^RFP/WT/Cx3cr1^GFP/WT mice depicting CCR2⁺ vs. CX3CR1⁺ (Upper) and Ly6C⁺ vs. Ly6G⁺ events (Lower) from vehicle-treated (n = 5) and CCX872-treated (n = 7) animals. Drug treatment resulted in a significant reduction of CCR2⁺ (P = 0.024) and CCR2⁺/CX3CR1⁺ (P = 0.032) events. The lower graphs report a reduction (P = 0.004) in Ly6C^hi events within tumors. (D) Flow-cytometric analysis of tumor isolates from 005 GSC tumor-bearing Ccr2^RFP/WT/Cx3cr1^GFP/WT mice depicting CCR2⁺ vs. CX3CR1⁺ (Upper) and Ly6C⁺ vs. Ly6G⁺ events (Lower) from vehicle-treated (n = 6) and CCX872-treated (n = 6) animals. Drug treatment resulted in a reduction of CCR2⁺ (P = 0.003), CX3CR1⁺ (P = 0.003), and CCR2⁺/CX3CR1⁺ (P = 0.0419) events. The lower graphs report a reduction (P = 0.020) in Ly6C^hi events within tumors. Representative plots are shown throughout. *P < 0.05; **P < 0.01.

Fig. 6.

Impact of combinatorial CCX872/anti-PD-1 treatment on CD4⁺ and CD8⁺ T cells. (A) Flow-cytometric analysis of CD45⁺/CD3⁺/CD4⁺ and CD8⁺ events within tumor extracts from vehicle/IgG-treated (n = 7), CCX872/IgG-treated (n = 4), vehicle/anti-PD-1–treated (n = 6), or CCX872/anti-PD-1–treated (n = 4) 005 GSC glioma-bearing mice. The population of CD45⁺/CD3⁺/CD4⁺ cells (upper square) was significantly increased (P = 0.044) with combination CCX872/anti-PD-1 treatment as compared to vehicle/IgG, while the CD45⁺/CD3⁺/CD8⁺ population (lower square) trended toward increase (P = 0.056) between the same groups. (B) Flow-cytometric analysis of side scatter (SSC) vs. CD45⁺/CD3⁺/IFNγ⁺ events (denoted by square) within tumor extracts from vehicle/IgG-treated (n = 7), CCX872/IgG-treated (n = 4), vehicle/anti-PD-1–treated (n = 5), or CCX872/anti-PD-1–treated (n = 6) 005 GSC glioma-bearing mice. The population of CD45⁺/CD3⁺/IFNγ⁺ cells was significantly increased (P = 0.008) with combination CCX872/anti-PD-1 treatment as compared to vehicle/IgG. (C and D) Flow-cytometric analysis of CD45⁺/CD3⁺/PD-1⁺/Tim3⁺/CD4⁺ (C) and CD8⁺ (D) events within tumor extracts from vehicle/IgG-treated (n = 7), CCX872/IgG-treated (n = 4), vehicle/anti-PD-1–treated (n = 6), or CCX872/anti-PD-1–treated (n = 4) 005 GSC glioma-bearing mice. The population of CD45⁺/CD3⁺/PD-1⁺/Tim3⁺/CD4⁺ cells (circled population) was significantly decreased (P = 0.029) with combination CCX872/anti-PD-1 treatment as compared to vehicle/IgG. The population of CD45⁺/CD3⁺/PD-1⁺/Tim3⁺/CD8⁺ cells (circled population) also decreased (P = 0.011) between the same groups. Representative plots are shown throughout. *P < 0.05; **P < 0.01.