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. 2013 Dec 10;110(50):20212-7.
doi: 10.1073/pnas.1320318110. Epub 2013 Nov 25.

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

Christine Feig  1 , James O Jones, Matthew Kraman, Richard J B Wells, Andrew Deonarine, Derek S Chan, Claire M Connell, Edward W Roberts, Qi Zhao, Otavia L Caballero, Sarah A Teichmann, Tobias Janowitz, Duncan I Jodrell, David A Tuveson, Douglas T Fearon
Affiliations

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

Christine Feig et al. Proc Natl Acad Sci U S A. .

Abstract

An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer cell-specific CD8(+) T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP(+) stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP(+) cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP(+) CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and inflammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP(+) CAF, may direct tumor immune evasion in a model of human PDA.

Keywords: KPC mouse; T cell exclusion; tumor immunogenicity; tumor stroma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Immunological characteristics of murine PDA. (A) Increase in PDA volume (mean ± SEM) following treatment of mice with α-PD-L1 (n = 6), α-CTLA-4 (n = 6), or control (n = 4) antibodies was measured by ultrasound. (B–D) Induction of IFN-γ secretion by splenic CD8+ T cells from various donor types following stimulation by different sources of pancreatic cells was measured by ELISpot assay (n ≥ 8 in B and D; Mann–Whitney test, n = 4 in C). *P < 0.05; ***P < 0.001.
Fig. 2.
Fig. 2.
Characterization of FAP+ stromal cells in murine PDA. (A) Tissue sections from PDA tumors were stained for FAP, CK19, and p53 and then analyzed by immunofluorescent (IF) microscopy. White arrows indicate examples of p53+ LOH cells. (B) Tissue sections from PDA tumors were stained for αSMA and FAP and analyzed by IF microscopy. (C) Single-cell suspensions of PDA tumor cells were analyzed by flow cytometry. (D) Heat map presents the reads per kilobase of transcript per million mapped reads of RNA-seq analyses of FACS-purified FAP+ cells.
Fig. 3.
Fig. 3.
Conditional depletion of FAP+ stromal cells and immune control of PDA. (A) KPCD mice with PDA received DTx or PBS; after 6 d, tumoral Fap mRNA was measured by quantitative RT-PCR (qRT-PCR) (PBS, n = 5; DTx, n = 7). (B) KPC mice with or without the DTR BAC transgene received DTx or PBS, and tumor volumes were measured by ultrasound (PBS, n = 6; DTx, n = 8; DTx to non-DTR transgenic, n = 4). (C) KPCD mice with PDA received CD4- and CD8-depleting antibodies or control IgG before and during treatment with DTx or PBS. Tumor volumes were measured (α-CD4/8 + PBS, n = 3; α-CD4/8 + DTx, n = 5; isotype IgG + DTx, n = 5). (D) KPCD mice with PDA received α-CTLA-4 or α-PD-L1 during treatment with DTx or PBS, and tumor volumes were measured [DTx, n = 13 (representing all DTx-treated KPCD mice presented in B and C); α-CTLA-4 + DTx, n = 6; α-PD-L1 + DTx, n = 4]. (E) Waterfall plots demonstrate the final tumor volume changes in individual mice. Dashed lines indicate the mean tumor volume for each treatment group. *P < 0.05; **P < 0.01.
Fig. 4.
Fig. 4.
FAP+ cell-derived CXCL12 and T-cell exclusion. (A) Tissue sections from PDA tumors were stained for CD3, p53, and CD11b and then analyzed by IF microscopy. White arrows demonstrate examples of CD3+ cells. (B) Tissue sections from PDA tumors were stained for CXCL12 and p53 and analyzed by IF microscopy. (C) Single-cell suspensions of PDA tumors (n = 3) were stained with antibodies for FAP, CD45, and CD11b. FAP+ cells, CD11b+ cells, and PDA/PanIN cells (FAP−CD45−CD11b−) were FACS-purified. Their levels of Cxcl12 mRNA were assessed by qRT-PCR. *P < 0.05.
Fig. 5.
Fig. 5.
Inhibition of CXCR4 by AMD3100 and immune elimination of PDA cells. (A) PDA-bearing mice, some of which had been pretreated with depleting CD4 and CD8 antibodies or control IgG, received PBS or AMD3100 by continuous infusion, and tumor volumes were measured by ultrasound (PBS, n = 5; AMD3100 + isotype IgG, n = 6; AMD3100 + α-CD4/8, n = 4). (B) PBS or AMD3100 (high dose) was given to PDA-bearing mice that were treated with α-CTLA-4, α-PD-L1, or control IgG (AMD3100 high + α-CTLA-4, n = 4; AMD3100 high + α-PD-L1, n = 7; AMD3100 high + isotype IgG, n = 6). (C) Waterfall plots present the final changes in tumor volumes in individual mice from A and B. Dashed lines indicate the mean tumor volume for each treatment group. (D) Tissue sections from PDA tumors taken from mice treated with PBS or AMD3100 high + α-PD-L1 for 6 d were stained for p53, and the entire cross-sections were imaged. White squares show the regions corresponding to the magnified images. Tissue sections from PDA tumors of mice treated with AMD3100 high + α-PD-L1 or PBS for 6 d were stained for Ki67 (E) or CD45 and CK19 (F) and analyzed by IF microscopy. *P < 0.05; **P < 0.01.
Fig. 6.
Fig. 6.
Accumulation of CD3+ T cells in cancer cell-containing regions of PDA induced by AMD3100 and α-PD-L1. Tissue sections from PDA tumors taken from mice that had been treated for 24 h with PBS, α-PD-L1, AMD3100 high, or AMD3100 high + α-PD-L1 were stained for CD3 and p53 and analyzed by IF microscopy.
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