Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer

Abstract

Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T lymphocytes (CTLs). Unbiased analysis of the The Cancer Genome Atlas database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these, we focused on CXCL12 as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of the CXCL12/CXCR4 axis, we inhibited CXCR4 signaling pharmacologically and found that plerixafor decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. By deleting CXCR4 in αSMA⁺ cells, we confirmed that these immunosuppressive effects are dependent on CXCR4 signaling in αSMA⁺ cells, which include cancer-associated fibroblasts as well as other cells such as pericytes. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients.

Keywords: carcinoma-associated fibroblasts; immune checkpoint blockade; metastatic breast cancer; tumor desmoplasia; tumor microenvironment.

Fig. 1.

CXCR4 is strongly correlated with desmoplasia- and immune checkpoint-related gene and protein expression in human breast cancers. (A) Venn diagram of the number of genes associated with FAP, TGFB1, and COL1A1 from breast invasive carcinoma mined from the human breast cancer TCGA database. (B) Immunohistochemistry (IHC) staining of human formalin-fixed paraffin-embedded (FFPE) tissues for CXCR4 in primary tumor, metastatic lesions, and adjacent normal tissues. CXCR4 is overexpressed in both primary and metastatic human breast tumors, compared with normal tissues. (C, Left) Kaplan–Meier survival analysis of patients stratified by high CXCR4 expression (>70%) vs. low expression in cancer-cell–rich regions of the tumor (log-rank P = 0.084). (C, Right) Kaplan–Meier survival analysis of patients stratified by high stromal CXCR4 expression (>30%) vs. low expression (* log-rank P = 0.008; n = 17). (D) IHC images showing CXCR4 and PD-L1 in matched pairs of primary and metastatic human BC tissues (Left, lung metastases; Right, liver metastasis). Both primary and metastatic tissues show high levels of CXCR4 and PD-L1, enriched in tumor stromal regions. (E) Representative IHC staining of human FFPE tissues with CD31, CAIX, aSMA, and Collagen I in matched pairs of primary and metastatic BC tissues (Left, lung metastases; Right, liver metastases). Both primary and metastatic tissues show high level of fibrosis. H&E staining of corresponding regions are shown. (F) Representative IHC staining of CD8+ T cells in primary and metastatic BC tissues. A, adjacent normal tissue; T, tumor region (circled in white). (G–J) Pearson correlation coefficients of CXCR4 mRNA expression from the TCGA BRCA dataset with immune checkpoint markers (G) CTLA4 (r = 0.54, P < 0.0001) and (H) PDCD1 (r = 0.51, P < 0.0001), desmoplasia marker (I) COL1A1 (r = 0.23, P < 0.001), and Treg marker (J) FOXP3 (r = 0.53, P < 0.001), combined from all BC patients (n = 1,215). (Scale bar, 100 μm.)

Fig. 2.

Inhibition of CXCR4 reduces stromal αSMA+ cells in tumors. (A) αSMA-dsRed mice bearing mammary fat pad windows were implanted with MCa-M3C-CFP breast tumors. Representative time-lapse images from intravital multiphoton microscopy of cancer cells (blue) and αSMA+ cells (red) at days 1, 3, 7, and 13 postimplantation and during treatment of AMD3100 or saline (control). CXCR4 inhibition delays the accumulation of αSMA+ cells at both the center and periphery of the tumors. (Scale bar, 100 μm.) (B) Area fractions and representative histology images of tumor αSMA+ cells show that AMD3100 reduces density of αSMA+ cells in the tumors (*P < 0.05, Student’s t test). (Scale bar, 100 μm.)

Fig. 3.

Inhibition of CXCR4 reduces tumor desmoplasia and immunosuppression. (A–D) Histological and biomechanical quantification of orthotopic Mca-M3C tumors in mice treated with AMD3100 or saline (n = 7). (A) AMD3100 decreases relative solid stress level in BC tumors (*P < 0.05). (B) AMD3100 increases vessel decompression (*P < 0.01), as indicated by increased fractions of tumor blood vessels with open lumen, shown in representative images of tumor CD31+ vessels. (Scale bar, 100 μm.) (C) Quantification of hypoxic fractions in tumors measured by pimonidazole injection and staining shows that AMD3100 reduces tumor hypoxia (*P < 0.05). (D) Quantification of tumor collagen I area fractions shows that AMD3100 reduces expression of collagen I in the tumors (*P < 0.05). (E and F) Gene expression (qRT-PCR) analysis on whole tumors isolated from mice treated with AMD3100 or saline (control; n = 3–4). AMD3100 decreases fibrosis-related genes (E) and modulates expression of immune-related genes (F). Error bars indicate SEM. Analysis by unpaired two-sided Student’s t test.

Fig. 4.

Conditional deletion of CXCR4 in aSMA+ cells reduces immunosuppression and improves animal survival. (A) Schematic of generation of a αSMA-CreERT2/Cxcr4^flox/flox mouse. Mice with CXCR4 alleles flanked by LoxP sites (Cxcr4^flox/flox) were bred with αSMA-CreERT2 mice expressing CreERT2 specifically in the αSMA+ cells to generate αSMA-CreERT2/Cxcr4^flox/flox mice. (B–F) Conditional knockout of CXCR4 expression was induced by daily injection of tamoxifen (10 mg/kg) for 2 wk before tumor implantation (n = 5–8). Control mice also received tamoxifen. (B) Flow cytometry analysis of CXCR4+ αSMA+ expression level in E0771 breast tumors implanted in αSMA-CreERT2/Cxcr4^{flox/flox mice}, αSMA-CreERT2-negative (control), or wild-type mice. The wild-type mice were treated with AMD3100 via osmotic pumps for 2 wk. AMD3100 reduces the CXCR4+αSMA+ cell population (*P < 0.05), and the genetic deletion (αSMA-CreERT2) further decreases the population (**P < 0.01). (C) Flow cytometry analysis of cytotoxic lymphocytes (CD8) and regulatory T-lymphocyte (Treg) populations from orthotopic E0771 breast tumors. The αSMA-CreERT2/Cxcr4^flox/flox mice have increased CD8+ cell fractions and decreased Treg cell fractions. *P < 0.05, by one-way ANOVA. (D, Left) Immunohistochemical analysis of hypoxia (pimonidazole injection) from orthotopic E0771 breast tumors is quantified. Both the AMD3100 treatment and genetic deletion (αSMA-CreERT2/Cxcr4^flox/flox) reduce hypoxic fractions of the tumors (*P = 0.049, **P = 0.0034, Student’s t test). (D, Right) Linear regression analysis shows strong negative correlation between infiltration of CD8+ T cells and hypoxia (r = −0.55, P < 0.05; Pearson correlation). (E, Left) Quantification of the number of spontaneous lung metastatic nodules after primary (mammary fat pad) tumor resection at day 21. Both the AMD3100 treatment and genetic deletion reduce spontaneous metastasis formation in the lung (*P < 0.05, **P < 0.001, Student’s t test). (E, Right) Representative gross images of lungs stained with Bouin’s solution. Black arrow points to example of lung nodules. (F) Kaplan–Meier survival analysis of metastatic setting studies in mice with spontaneous lung metastases arising from orthotopic E0771 tumors. The mice were treated with saline (Cre-mice) or AMD3100 (wild-type) using an osmotic pump for 2 wk. Both AMD3100 treatment and genetic deletion improve animal survival (P < 0.05, by log-rank test). Error bars indicate SEM.

Fig. 5.

Inhibition of CXCR4 reduces desmoplasia and increases effector to regulatory T-lymphocyte ratio in the lung metastases. (A–D) Representative histology images of lung metastases derived from orthotopic E0771 breast tumors. Immunofluorescence (IF) images of lung metastases were field-of-view images from the mosaic confocal imaging. After primary tumor resection, the mice were treated with AMD3100 in combination with an ICB mixture of α-CTLA-4 and α-PD-1 for one cycle, and the lung metastases were collected for analysis. (A) H&E stainings of spontaneous lung metastases from E0771 tumors. The metastatic nodules (red arrows) are significantly larger in the control, compared with all three treatment groups. (Scale bars, 100 μm.) (B) Representative images of IF staining of hyaluronan. Treatment with AMD3100 reduces the hyaluronan fraction in the metastatic TME. (C) Representative images of IF staining of collagen I. Treatment with AMD3100 reduces collagen I in the metastases. (D) Representative IF images showing aSMA (red), collagen I (white), and CD3 (green) in the metastases. Combination of AMD3100 and ICB increases infiltration of CD3+ cells into the TME. (E and F) Flow cytometry analysis of lung metastases derived from orthotopic E0771 breast tumors. (E) CD3+CD4+Foxp3+ regulatory T cell population. Both the monotherapy and combination therapy decrease Foxp3 T cells in the tumors (*P = 0.017, by one-way ANOVA). (F) CD8+ to Foxp3+ Treg ratio. Both AMD3100 groups increase the ratio of effector CD8+ T cells to Treg, and the combination of AMD3100 with immunotherapy mixture further extends the ratio (*P = 0.026, by one-way ANOVA). n = 5. Error bars indicate SEM. (Scale bar, 100 μm.)

Fig. 6.

CXCR4 inhibition improves outcome of ICBs. (A–C) Quantification of lung nodules in mice with spontaneous lung metastases arising from orthotopic breast tumors. Mice were treated with AMD3100 or saline (control) through an osmotic pump for 2 wk and with or without immune checkpoint blockades (α-CTLA-4 and α-PD-1) on days 2, 5, and 8. Lungs were collected and counted at the end point of the metastatic survival studies. Both AMD100 or combination therapy of AMD310 with immunotherapy mixture reduces metastatic nodules (E0771: *P = 0.011, MCa-M3C: *P = 0.0018, 4T1: *P = 0.001). By Student’s tests. Error bars indicate SEM. n = 7–10. (D–F) Kaplan–Meier survival analyses of metastatic setting study in mice with spontaneous lung metastases arising from orthotopic breast tumors, by log-rank tests. (D) Animal survival in mice with spontaneous E0771 lung metastases. The immunotherapy mixture improves median animal survival time by day 43 (**P < 001), and the combination with AMD3100 greatly extends the animal survival by curing four of seven mice (*P < 0.0001). n = 7. (E) Animal survival in mice with spontaneous MCa-M3C lung metastases. The immunotherapy mixture does not improve median animal survival time, but the combination with AMD3100 extends the animal survival by 76%, curing 2 of 10 mice (*P < 0.001). n = 9–10. (F) Animal survival in mice with spontaneous 4T1 lung metastases. The immunotherapy mixture does not improve median animal survival time, but the combination with AMD3100 extends the animal survival by 35% (*P = 0.055), curing 2 of 10 mice. n = 9–10.