Recruitment of CCR2+ tumor associated macrophage to sites of liver metastasis confers a poor prognosis in human colorectal cancer

Abstract

The tumor microenvironment (TME) represents a significant barrier to creating effective therapies for metastatic colorectal cancer (mCRC). In several malignancies, bone marrow derived CCR2⁺ inflammatory monocytes (IM) are recruited to the TME by neoplastic cells, where they become immunosuppressive tumor associated macrophages (TAM). Here we report that mCRC expression of the chemokine CCL2 facilitates recruitment of CCR2⁺ IM from the bone marrow to the peripheral blood. Immune monitoring of circulating monocytes in patients with mCRC found this influx was a prognostic biomarker and correlated with worse clinical outcomes. At the metastatic site, mCRC liver tumors were heavily infiltrated by TAM, which displayed a robust ability to dampen endogenous anti-tumor lymphocyte activity. Using a murine model of mCRC that recapitulates these findings from human disease, we show that targeting CCR2 reduces TAM accumulation in liver metastasis and restores anti-tumor immunity. Additional quantitative analysis of hepatic metastatic tumor burden and treatment efficacy found that administration of a small molecule CCR2 inhibitor (CCR2i) improves chemotherapeutic responses and increases overall survival in mice with mCRC liver tumors. Our study suggests that targeting the CCL2/CCR2 chemokine axis decreases TAM at the metastatic site, disrupting the immunosuppressive TME and rendering mCRC susceptible to anti-tumor T-cell responses.

Keywords: CCL2; immunotherapy; inflammatory monocyte; tumor microenvironment.

Figure 1.

Elevated circulating CCR2+ inflammatory monocytes correlate with poor prognosis in mCRC patients. (A:) Patients with hepatic mCRC were stratified into high (≥8.9%; n = 111) and low (<8.9%; n = 110) cohorts based on percentage of monocytes in the peripheral blood obtained from preoperative CBC within 30 days of index operation and analyzed for recurrence-free survival following liver resection for mCRC. (B:) High and low preoperative monocyte cohorts analyzed for overall survival following index hepatic resection of mCRC (C:) Left panels, representative flow cytometry plots of inflammatory monocytes (CD11b+, CD14+, CCR2+) from the peripheral blood of healthy controls (Top) and mCRC patients (Bottom). Right panel, graph depicts inflammatory monocytes as percentage of CD45+ cells by flow cytometry analysis in healthy controls (n = 16) and mCRC patients (n = 30). (D:) Comparison of inflammatory monocytes as a percentage of CD45+ cells by flow cytometry analysis between mCRC patients surviving greater (n = 20) or less than (n = 10) 1–year following mCRC liver resection. Comparison between groups using a two-tailed unpaired t-test or Kaplan-Meier survival analysis used to calculate p-values and shown as mean ± SD.

Figure 2.

Human mCRC liver tumors express CCL2 and are infiltrated by immunosuppressive TAM (A:) Graph depicts serum CCL2 concentration (pg/ml) in healthy controls (n = 15) and mCRC patients (n = 44). (B:) Left panel, confocal microscopic images of human mCRC liver tumor showing CCL2 co-localization with Cytokeratin 20 (CK20) expressing neoplastic cells. Right panel, graph compares CCL2 gene expression by quantitative real time PCR between matched adjacent normal and mCRC liver tumors (n = 9). (C:) Left panel, representative immunohistochemistry of CD68+ TAM (brown) in a hepatic mCRC tumor. Right panel, graph represents TAM as a percentage of CD45+ cells by flow cytometry analysis in matched adjacent normal and mCRC liver tumor samples (n = 4). (D:) Left panel, graph depicts representative flow cytometry histogram of negative control (Blue) and CFSE labeled autologous CD8+ lymphocytes co-cultured alone (Orange), with CD14+ peripheral blood inflammatory monocytes (Green) or CD14+ TAM isolated from mCRC liver tumors (Red). Right panel, graph represents T-cell division index as determined between CD14+ cells isolated from matched pairs of peripheral blood and mCRC tumors (n = 3). Comparison between groups using a two-tailed unmatched t-test or a 2-tailed matched t-test for comparison of paired specimens were used to calculate p-values and shown as mean ± SD. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 3.

CCR2-/- mice with MC38 liver metastasis have reduced IM mobilization and prolonged survival (A:) Bone marrow CCR2+ inflammatory monocytes (CD11b+, Ly6High) as percentage of total cells determined by flow cytometry from MC38 mCRC liver tumor bearing wild type and CCR2-/- mice (n = 3 per group). (B:) Peripheral blood CCR2+ inflammatory monocytes (CD11b+, Ly6High) as percentage of total cells determined by flow cytometry from MC38 mCRC liver tumor bearing wild type and CCR2-/- mice (n = 6 per group). (C:) Graph represents CCR2+ TAM as a percentage of total cells from MC38 liver metastasis of wild type and CCR2-/- tumor bearing mice as determined by flow cytometry analysis (n = 9 per group). (D:) Survival analysis of wild type and CCR2-/- mice with established MC38 mCRC liver tumors (n = 9–15 per group). (E:) Graph represents tumor infiltrating CD8+ (Left) and CD4+ (Right) tumor infiltrating lymphocytes as a percentage of total cells determined by flow cytometry analysis from wild type and CCR2-/- mice with mCRC MC38 liver tumors (n = 9 per group). (F:) Graph depicts MC38 liver tumor weights of wild type and CCR2-/- animals with established mCRC hepatic MC38 tumors following treatment with a CD8 depleting antibody or control (n = 5 per group). Comparison between groups using a two-tailed unmatched t-test or ANOVA with Tukey post-test for comparison of multiple groups used to calculate p-values and shown as mean ± SD. Log-rank test used for survival analysis. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 4.

Treatment with a small molecule CCR2i alone and in combination with FOLFOX chemotherapy changes the mCRC tumor microenvironment (A:) Graphs representing CCR2+ inflammatory monocytes in the bone marrow (Left) and peripheral blood (Middle) as a percentage of CD45+ cells in animals with MC38 liver metastasis following treatment with a small molecule CCR2i alone and in combination with FOLFOX chemotherapy. Graph illustrates changes in the blood to bone marrow inflammatory monocyte ratio (Right) in tumor bearing animals following treatment. (B:) Graph represents mCRC liver tumor TAM as a percentage of total cells as determined by flow cytometry following treatment with a small molecule CCR2i alone and in combination with FOLFOX chemotherapy compared to ehicle. (C:) Graphs depict CD8+ (Left) and CD4+ (Right) tumor infiltrating lymphocytes as percentage of total cells determined by flow cytometry from metastatic MC38 liver tumors. Comparison between multiple groups using ANOVA with Tukey post-test used to calculate p-values and shown as mean ± SD. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 5.

CCR2i plus FOLFOX chemotherapy reduces established mCRC tumor burden and increases survival (A:) Representative BLI of in vivo and ex vivo livers from mice with established MC38 mCRC liver tumors. (B:) Graph represents region of intensity (ROI) as determined by BLI in mice with established MC38 hepatic metastasis following 15 days of treatment (n = 8–10 per group). (C:) Graph represents gross tumor weights from mice with established MC38 hepatic metastasis following 15 days of treatment (n = 10 per group). (D:) Overall survival following initiation of treatment in mice with established MC38 mCRC liver tumors (n = 9 mice per group). Comparison between multiple groups using ANOVA with Tukey post-test used to calculate p-values and shown as mean ± SD. Log rank test used for survival analysis. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.