CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response

Abstract

Inhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells.

Keywords: AMD3100; CXCR4; colorectal cancer; immunotherapy; pancreatic cancer.

Fig. 1.

The CXCL12-coat of human pancreatic and colorectal cancer cells. Sections of human pancreatic (PDA) and colorectal (CRC) adenocarcinoma were stained with fluorescent antibodies to CXCL12, and to KRT19 to reveal cancer cells. The ratios shown in the top right corners of the photomicrographs indicate the frequency of the observed staining relative to the total number of independent tumors that were assessed. (Scale bar, 50 µm.)

Fig. 2.

The effect of CXCL12-stimulated CXCR4 on chemokine receptor-mediated migration of human immune cells. (Left) The coexpression of CXCR4 with (A) CXCR1, (B) CXCR3, (C) CXCR5, (D) CXCR6, and (E) CCR2 on human immune cell lines was evaluated by flow cytometry after staining with antibodies specific for the relevant chemokine receptors. Gray peaks indicate isotype controls. (Center) The effect of CXCL12-stimulation of CXCR4 on the chemotactic responses of A CXCR1-coexpressing Jurkat T lymphoblastoid cells to CXCL8, (B) CXCR3-coexpressing HSB2DP T lymphoblastoid cells to CXCL10, (C) CXCR5-coexpressing Raji B lymphoblastoid cells to CXCL13, (D) CXCR6-coexpressing Jurkat T lymphoblastoid cells to CXCL16, and (E) CCR2-coexpressing Molm13 monocytoid cells to CCL2 was assessed by including CXCL12 in the upper chamber (blue) and the other chemokines in the lower chamber (red) in the Boyden two-chamber assay. (Right) The chemotaxis assays were performed with the five cell lines when the placement of the chemokines in the Boyden chambers was reversed. Bar diagrams display mean and SEM (n = 3-4). Statistical analysis by Student’s t test: ***P < 0.001; ****P < 0.0001; ns, not significant.

Fig. 3.

Inhibition by AMD3100 of the suppression mediated by CXCL12-stimulated CXCR4 of the function of other chemokine receptors. (A–E) The chemotactic responses were assessed of the dual chemokine receptor-expressing human immune cells to the relevant chemokines in the absence or presence of CXCL12, with increasing concentrations of AMD3100. The results are presented as percent of the chemotactic response in the absence of CXCL12 and AMD3100. The mean and SEM are indicated.

Fig. 4.

The immunological effects in human CRC and PDA of treatment with AMD3100. Paired biopsy tissues were obtained from the same metastasis in each patient before (pre-Rx) and after 7 d of continuous infusion of AMD3100. (A) Tissue sections were stained with fluorescent antibodies to pan-keratin (pan-CK) to reveal cancer cells, and to CD8 to reveal cytotoxic T cells. White arrowheads designate CD8⁺ T cells within cancer cell islets, and red arrowheads designate CD8⁺ T cells outside of cancer cell islets. (Scale bar, 50 µm.) (B) The presence of CD8⁺ T cells within cancer cell islets, assessed by staining with anti-CD8 antibody, correlates with the CD8A mRNA levels, assessed by RNA-seq analysis, in tissues obtained from different pass biopsies of the same metastatic lesions. (C) Immunological gene sets that identify T and NK cell accumulation, T and NK cell effector cells, activated B cells (germinal center B cells) and plasma cells are enriched in genes up-regulated after treatment with AMD3100. (D) The expression of CCL19 and FAP in sections from paired biopsies was analyzed by FISH using specific probes for mRNA of FAP and CCL19. The total counts of FAP⁺/CCL19⁺ cells, FAP⁺/CCL19⁻ cells, and FAP⁻/CCL19⁺ cells are displayed. (Scale bar, 50 µm.) (E) The enrichment analysis for a TLS gene set (13) is shown. (F and G) Enrichment analyses are shown for those genes that (F) are differentially expressed in rejecting compared to nonrejecting kidney allografts (28, 29) and (G) MSI compared to MSS CRC (30). (A–G) n = 14 comprising of PDA (n = 4) and CRC (n = 10) Statistical comparisons by Spearman’s rank correlation test (B), and by Fisher’s exact test (D): ****P < 0.0001.

Fig. 5.

Comparative analyses of the INTIRE induced by AMD3100. (A) The heat map is shown of the enrichment analyses of nine gene sets representing different immune components that characterize the INTIRE in different immunological contexts, along with E2F target genes and genes involved in the G2M checkpoint. Gene set enrichment analyses demonstrating that genes up-regulated by treatment with AMD3100 are significantly enriched in B genes associated with longer overall survival in cancer according to the PRECOG (25) study, (C) genes up-regulated in pretreatment biopsies from patients with melanoma who responded to anti–PD-1 treatment vs. nonresponding patients (32, 33), and (D) genes down-regulated in biopsies of patients with melanoma treated with anti-CD20 antibodies to induce B cell depletion (34). (A–D) n = 14 comprising of PDA (n = 4) and CRC (n = 10).

Fig. 6.

Analyses of the anticancer effect induced by AMD3100 treatment. (A) Changes of mRNA expression in the paired biopsies obtained from each patient with CRC (n = 10) and with PDA (n = 4) before and after treatment with AMD3100 for granzymes A, B, H, K, and M and perforin negatively correlate with changes in the expression of CEACAM 5, 6, and 7. (B) Plasma ctDNA levels (n = 15) and (C) serum concentrations of CXCL8 (n = 18) pretreatment (pre-Rx) and after 7 d of continuous infusion of AMD3100 are shown. Statistical comparisons by Spearman’s rank correlation test (A), by paired Wilcoxon signed-rank test (B), and by paired t test (C): *P < 0.05; ****P < 0.0001.