Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti–PD-L1 immunotherapy in pancreatic cancer

In the KPC model, Cre-mediated expression of Trp53^R172H and Kras^G12D is targeted to the pancreas, causing the development of invasive and metastatic carcinoma that recapitulates many aspects of human PDA, including the loss of heterozygosity (LOH) of Trp53 in cancer cells but not in premalignant pancreatic intraepithelial neoplasia (PanIN) (10). KPC mice with appropriately sized tumors demonstrate consistent tumor growth, which permits robust analyses of experimental interventions (SI Appendix, Fig. S1). We examined whether blocking immunological checkpoints with α-CTLA-4 and α-PD-L1 would promote immune control of the tumor. Administering these antibodies over 6 d to mice bearing PDA did not diminish the ∼80% increase in tumor volume observed in mice receiving control IgG (Fig. 1A). We determined whether this could be explained by the absence of an immune response to the PDA. Splenic CD8⁺ T cells from PDA-bearing and non–tumor-bearing mice were stimulated with tumor cells, and IFN-γ–secreting CD8⁺ T cells reporting antigenic stimulation were detected in an enzyme-linked immunosorbent spot (ELISpot) assay. Tumor cells induced significantly more ELISpots among CD8⁺ T cells from tumor-bearing mice than from non–tumor-bearing mice (Fig. 1B). The frequency of IFN-γ–secreting CD8⁺ T cells was the same when the stimulating tumor cells were from the T-cell donor or from another PDA-bearing mouse (Fig. 1C). An established PDA cell line also was stimulatory, whereas dissociated cells from pancreata of LSL-Kras^G12D/+;Pdx-1-Cre mice with premalignant PanIN lesions expressing only Kras^G12D or from young KPC mice before the development of cancer, were not (Fig. 1D and SI Appendix, Fig. S2). Therefore, PDA-bearing mice have a spontaneous adaptive immune response to antigens that are shared by cancer cells from different PDA tumors, and the ineffectiveness of α-CTLA-4 and α-PD-L1 suggests an additional immunosuppressive mechanism.

We considered the possibility that the FAP⁺ stromal cell mediates this immunosuppression. FAP⁺ cells were present in PanIN and both cytokeratin-19⁺ (CK19⁺) and CK19⁻ PDA lesions (Fig. 2A). FAP⁺ cells in PanIN expressed CD34 (SI Appendix, Fig. S3 A and B) but rarely α-smooth muscle actin (αSMA) (Fig. 2B), whereas FAP⁺ cells among PDA cells were CD34⁻ (SI Appendix, Fig. S3B) and αSMA⁺ (Fig. 2B). All FAP⁺ cells were PDGF receptor-α⁺ (SI Appendix, Fig. S3A) and CD45⁻ (Fig. 2C), confirming their mesenchymal origin, and their frequency among dispersed tumor cells was 3.7% [95% confidence interval (CI): 1.1–6.3%; n = 8]. The expression of FAP by 92% of the αSMA⁺ fibroblasts (95% CI: 87.5–97.1%; n = 5) suggested that they are CAFs. This suggestion was supported by the transcriptomes of CD34⁺FAP⁺ (PanIN-associated) and CD34⁻FAP⁺ (PDA-associated) cells (Fig. 2D), which exhibited the “inflammatory gene signature” of CAFs (11). FAP⁺ cells from three normal tissues also displayed the signature and were clustered together by a principal component analysis of their transcriptomes (SI Appendix, Fig. S3C), indicating that FAP may identify a stromal cell lineage. The increased expression by tumoral FAP⁺ cells of genes encoding extracellular matrix proteins verifies their role in the desmoplasia of PDA, whereas their lower expression of decorin may relate to similar observations in human cancer (Fig. 2D).

We introduced into the KPC line the bacterial artificial chromosome (BAC) transgene containing a modified Fap gene that drives the expression of the human diphtheria toxin receptor (DTR) selectively in cells that are FAP⁺ (9). Administering diphtheria toxin (DTx) to PDA-bearing BAC transgenic mice depleted ∼55% the tumoral FAP⁺ cell content (Fig. 3A). DTx appeared to deplete FAP⁺ cells throughout the tumor. Depleting FAP⁺ cells slowed PDA growth (Fig. 3B), but not when CD4⁺ and CD8⁺ T cells were removed (Fig. 3C). Combining depletion of FAP⁺ cells with administration of α-CTLA-4 or α-PD-L1 further diminished tumor growth (Fig. 3 D and E), indicating that the FAP⁺ cell contributes to the resistance of murine PDA to these checkpoint antagonists. The absence of an increase in IFN-γ–secreting CD8⁺ T cells from the spleens of DTx- and α-PD-L1–treated mice indicates that immune control was not accomplished by enhanced priming of cancer-specific CD8⁺ T cells (SI Appendix, Fig. S2).

Therapy involving the depletion of FAP⁺ cells is precluded by their essential roles in normal tissues (12), and a therapeutic target that accounts for their immunosuppression had to be identified. We noted a paucity of CD3⁺ T cells in the vicinity of cancer cells (Fig. 4A), a characteristic of human PDA that is associated with FAP⁺ cells (SI Appendix, Fig. S4) and other carcinomas (13, 14). This T-cell trafficking problem directed attention to the chemokine, CXCL12, which localizes to cancer cells in both human (15) (SI Appendix, Fig. S4) and murine (Fig. 4B) PDA. We identified the source of CXCL12 as the tumoral FAP⁺ cell (Fig. 4C), as has been previously reported for CAFs (16). FAP⁺ cells in the s.c. Lewis lung carcinoma (LL2) model also are the tumoral source of CXCL12 (SI Appendix, Fig. S5A). CXCR4 is unlikely to mediate the uptake of the chemokine because cancer cell expression of CXCR4 is low (SI Appendix, Fig. S6). We hypothesize that high mobility group box 1 (HMGB1), which is overexpressed and secreted by metabolically stressed cancer cells (17), captures CXCL12 by forming a high-affinity heterocomplex (18), thus explaining the paradoxical localization of CXCL12 on cancer cells despite their not producing CXCL12 (Fig. 4C).

To assess the role of CXCL12 in tumoral immunosuppression, we administered AMD3100, a specific CXCR4 inhibitor that is licensed for clinical use (19), to mice bearing PDA in the presence or absence of depleting antibodies to CD4⁺ and CD8⁺ T cells. Tumor growth was slowed by AMD3100 in a T cell-dependent manner (Fig. 5A). AMD3100 also induced T cell-dependent control of s.c. immunogenic LL2 tumors (SI Appendix, Fig. S5 B and C). We administered a higher dose of AMD3100 that almost completely arrested PDA growth and combined it with immunological checkpoint antagonists (Fig. 5 B and C). The combination of AMD3100 with α-PD-L1 led to a significant 15% decline in PDA volume by 48 h and no further decrease over the following 4 d. α-CTLA-4 did not augment the antitumor effect of AMD3100.

Rapid, therapy-induced, clinically relevant reductions in cancer cell number are often not associated with comparable decreases in tumor volume (20). To evaluate more definitively the response of the PDA to immunotherapy, we examined tumors after 6 d of treatment for the presence of cancer cells. These can be unambiguously identified because their loss of the wild type p53 allele results in the appearance and stabilization of the protein product of the mutant p53^R172H allele (10). These p53⁺ PDA cells were abundant in tumors from mice that had received PBS or α-PD-L1 but were greatly diminished in tumors from mice that had received AMD3100 alone or with α-PD-L1 (Fig. 5D and SI Appendix, Fig. S7). The marked decrease in Ki67⁺ cells reflects this diminution in proliferating cancer cells (Fig. 5E and SI Appendix, Fig. S4). Serial sections demonstrate that the residual tumors were composed of CK19⁺ epithelial cells and CD45⁺ inflammatory cells (Fig. 5F). The selective elimination of p53⁺ LOH cells is consistent with the finding that the CD8⁺ T-cell response is cancer cell-specific (Fig. 1D).

To determine if this immunotherapeutic effect was caused by enhanced T-cell accumulation among cancer cells, we treated mice for 24 h with AMD3100 and α-PD-L1. α-PD-L1 alone had no effect, whereas AMD3100 increased the accumulation of T cells (Fig. 6 and SI Appendix, Fig. S9A). The combination of α-PD-L1 with AMD3100 amplified this effect and led to an apparent decrease in the frequency of p53⁺ LOH cancer cells (Fig. 6 and SI Appendix, Fig. S9A). Local T-cell proliferation could not account for the increase in tumoral T cells, because there were few Ki67⁺ CD3⁺ cells in AMD3100- and α-PD-L1–treated tumors (mean of 1.58%; range: 0.75–2.99%; n = 3) (SI Appendix, Fig. S9B). Furthermore, because Foxp3⁺ cells also were increased in tumors of mice given AMD3100 and α-PD-L1 (SI Appendix, Fig. S9C), we conclude that regulatory T (Treg) cells are not critically involved once immunosuppression by CXCR4/CXCL12 and PD-1/PD-L1 is overcome.

These studies reveal a hierarchy of immunosuppression in murine PDA, with that mediated by the FAP⁺ CAF being dominant over two T-cell checkpoints. Depleting the FAP⁺ stromal cell or inhibiting the interaction of its chemokine, CXCL12, with CXCR4 uncovers the antitumor activity of α-CTLA-4 and α-PD-L1. We do not know whether CXCR4-mediated exclusion of T cells reflects T-cell apoptosis, as occurs with HIV gp120 (21), or a chemorepulsive effect of CXCL12 (22). A direct effect of AMD3100 on cancer cells, however, is excluded by their not expressing CXCR4 (SI Appendix, Fig. S6). The absence of a synergistic interaction between AMD3100 and α-CTLA-4 may indicate that inhibiting CXCR4 so strongly promotes the accumulation of T cells among cancer cells such that any augmented priming by α-CTLA-4 is superfluous. We excluded the possibility that other mechanisms may have accounted for the absence of an effect of α-CTLA-4 in combination with AMD3100. The α-CTLA-4 clone, 9H10, that was used in this experiment depletes Treg cells (23) and was effective when combined with a suboptimal reduction of FAP⁺ CAFs in PDA-bearing mice (Fig. 3A). Furthermore, macrophages that are capable of engaging in Fc receptor-mediated Treg cell clearance (23) were abundant in tumors treated with AMD3100 (SI Appendix, Fig. S10). Finally, the finding that Treg cells even accumulated in tumors along with other CD3⁺ cells during treatment with AMD3100 and α-PD-L1 (SI Appendix, Fig. S9C) suggests that the inhibitory function of Treg cells only plays a minor role once the immune suppressive effect of CXCL12 is neutralized. Hence, it is plausible that immune control of PDA may be achieved without incurring the severe adverse effects of α-CTLA-4 that have been observed in human patients with melanoma, especially when coadministered with another T-cell checkpoint antagonist (6).

Because T cells are also excluded from other carcinomas (14, 15) in which FAP⁺ stromal cells are abundant (8) and CXCL12 is associated with cancer cells (24), our findings may be widely relevant to tumor immunotherapy. Finally, this study strongly supports the use of autochthonous, genetically engineered models of cancer to study the mechanisms of escape from immune surveillance. These models have permitted the demonstration that cancers may maintain the expression of antigens despite the occurrence of systemic immune responses (25) and that metastatic lesions may be immunologically controlled despite outgrowth of primary tumors (26), observations that focus attention on the need to understand fully the rules of tumoral immunosuppression.

We thank Frances Connor, Paul Mackin, and Lisa Young for providing KPC mice. We also thank the staff of the Cancer Research UK Cambridge Institute core facilities. This work was supported by Cancer Research UK (D.A.T., D.T.F.), the Wellcome Trust (D.T.F., T.J.), the Ludwig Institute for Cancer Research (D.T.F.), The Anthony Cerami and Anne Dunn Foundation for World Health (D.T.F.), the Medical Research Council (D.T.F.), Addenbrooke’s Charitable Trust (D.T.F.), and GlaxoSmith Kline (D.T.F.). This work has also been supported by the National Institute for Health Research Cambridge Biomedical Research Centre. C.F. was supported by a European Molecular Biology Organization long-term fellowship and a Marie Curie Intra European Fellowship within the Seventh European Community Framework Programme. R.J.B.W. received funding from the University of Cambridge School of Clinical Medicine, Rosetrees Trust, and Frank Edward Elmore Fund.