NOX4 Inhibition Potentiates Immunotherapy by Overcoming Cancer-Associated Fibroblast-Mediated CD8 T-cell Exclusion from Tumors

Abstract

Determining mechanisms of resistance to αPD-1/PD-L1 immune-checkpoint immunotherapy is key to developing new treatment strategies. Cancer-associated fibroblasts (CAF) have many tumor-promoting functions and promote immune evasion through multiple mechanisms, but as yet, no CAF-specific inhibitors are clinically available. Here we generated CAF-rich murine tumor models (TC1, MC38, and 4T1) to investigate how CAFs influence the immune microenvironment and affect response to different immunotherapy modalities [anticancer vaccination, TC1 (HPV E7 DNA vaccine), αPD-1, and MC38] and found that CAFs broadly suppressed response by specifically excluding CD8⁺ T cells from tumors (not CD4⁺ T cells or macrophages); CD8⁺ T-cell exclusion was similarly present in CAF-rich human tumors. RNA sequencing of CD8⁺ T cells from CAF-rich murine tumors and immunochemistry analysis of human tumors identified significant upregulation of CTLA-4 in the absence of other exhaustion markers; inhibiting CTLA-4 with a nondepleting antibody overcame the CD8⁺ T-cell exclusion effect without affecting Tregs. We then examined the potential for CAF targeting, focusing on the ROS-producing enzyme NOX4, which is upregulated by CAF in many human cancers, and compared this with TGFβ1 inhibition, a key regulator of the CAF phenotype. siRNA knockdown or pharmacologic inhibition [GKT137831 (Setanaxib)] of NOX4 "normalized" CAF to a quiescent phenotype and promoted intratumoral CD8⁺ T-cell infiltration, overcoming the exclusion effect; TGFβ1 inhibition could prevent, but not reverse, CAF differentiation. Finally, NOX4 inhibition restored immunotherapy response in CAF-rich tumors. These findings demonstrate that CAF-mediated immunotherapy resistance can be effectively overcome through NOX4 inhibition and could improve outcome in a broad range of cancers. SIGNIFICANCE: NOX4 is critical for maintaining the immune-suppressive CAF phenotype in tumors. Pharmacologic inhibition of NOX4 potentiates immunotherapy by overcoming CAF-mediated CD8⁺ T-cell exclusion. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/9/1846/F1.large.jpg.See related commentary by Hayward, p. 1799.

Fig. 1. CAF suppress response to anti-cancer vaccination and αPD1 immunotherapy.

A) IHC for αSMA showing CAF content in murine and human tumors. Representative αSMA images, scale bar = 100 μm; area % of staining was quantified in human and murine lung, colorectal and breast tumors (human n=9 per group; murine 7-10 per group; mean ⁺/- s.e.m; *P < 0.05, ****P < 0.0001 One-Way ANOVA test). B) IHC showing αSMA⁺ CAF content in tumors with CAF co-injection; TC1, MC38 and 4T1 tumors respectively. Representative images from αSMA IHC are shown; scale bar = 50 μm (TC1), 100 μm (MC38, 4T1); area % of staining was quantified (mean ⁺/- s.e.m, *P < 0.05, **P < 0.01 two-tailed t test). C) TC1 tumor growth curves showing effect of CAF on response to HPV E7 vaccination (control tumors – red; CAF-rich tumors- blue). Control vaccine groups tumors are shown in black and grey respectively (mean ⁺/- s.e.m., from n = 7 - 8 mice per group, ns > 0.05, *P < 0.05 AUC analysis followed by two-tailed t test. D,E) Tumor growth curves from individual mice following HPV E7 vaccination (control tumors – red; CAF-rich tumors- blue respectively). F) MC38 tumor growth curves showing the effect of CAF on response to αPD-1 therapy (control tumors – blue; CAF-rich tumors- orange). Isotype control antibody-treated tumors are shown in black and grey respectively (mean +/- s.e.m., from n = 8 mice per group, ns > 0.05, *P < 0.05 AUC analysis followed by two-tailed t test). G,H) Tumor growth curves from individual mice following αPD-1 antibody treatment (control tumors – blue; CAF-rich tumors – orange respectively).

Fig. 2. CAF exclude CD8+ T-cells from tumors.

A) Flow cytometry analysis of CD8⁺ T-cell, CD4⁺ T-cell and macrophage infiltration in disaggregated TC1 control (CAF^low) and CAF-rich tumors, gating on CD8⁺, CD4⁺ and CD11b⁺ F480⁺ respectably from CD45⁺ viable singlets (mean ⁺/- s.e.m., n = 4 tumors per group, ns > 0.05, *P < 0.05, two-tailed t test). B,C,D) IHC on TC1 tumors showing the effect of on spatial distribution of CD8⁺ T-cells (B), CD4⁺ T-cells (C) and macrophages (D) at the tumor center/margin. Representative images are shown from tumor center (top panel) and margin (bottom panel). Scale bars = 100 μm and % staining area was quantified (mean ⁺/- s.e.m., *P < 0.05, two-tailed t test).

Fig. 3. Analysis of CAF and CD8⁺ T-cell spatial relationship in human head and neck cancers.

A) Representative images of MxIHC staining in CAF^high and CAF^low tumors from the center and margin. B,C) Quantification of αSMA⁺ (B) and CD8⁺ (C) cells as a proportion of stromal/immune cells (non-epithelial PanCK- cells) at the tumor center or margin in CAF^high and CAF^low tumors. Plots show each independent data point (different patient samples) plus the mean and s.e.m., statistical comparisons are made using an ordinary one-way ANOVA multiple comparisons test. D) Representative images of MxIHC staining of a tumor where CAFs directly abutted the tumor and where CAFs are distant from the tumor border. E) Scatter plot showing the relationship between the WGCNA lymphocyte co-stimulation module eigengene and histo-cytometry measurements of the median distance between CAF and the nearest tumor cell. Pearson’s r and associated p values are shown. Scale bars represent 100μm.

Fig. 4. CD8⁺ T-cells in CAF-rich tumors upregulate CTLA4.

A) RNA-Seq analysis of differentially expressed genes (one per row) by CD8⁺ T-cells from TC1 control versus TC1 CAF-rich tumors (n = 4 mice per group) (adjusted P value of <0.05 (DESeq2 analysis; Benjamini-Hochberg test)), presented as row-wise z-scores of transcripts per million (TPM); each column represents an individual sample; right margin, examples of DEGs (vertical line groups genes upregulated in CD8⁺ T-cells in TC1 CAF-rich tumors relative to their expression in TC1 control tumors). B) GSEA of various gene sets (above plots) in the transcriptome of CD8⁺ T-cells from TC1 CAF-rich versus TC1 control tumors: top, running enrichment score (RES) for the gene set, from most enriched at the left to most under-represented at the right; middle, positions of gene set members (blue vertical lines) in the ranked list of genes; bottom, value of the ranking metric. Values above the plot represent the normalized enrichment score (NES) and P values, Kolmogorov-Smirnov test. C) Flow cytometry analysis of CTLA-4, IRF4 and TNFRSF9 (4-1bb) expression in CD8⁺ T-cells from TC1 control (CAF^low) and TC1 CAF-rich tumors (left and right plots respectively; mean ⁺/- s.e.m., from n = 6 mice per group, *P < 0.05, **P < 0.01 two-tailed t test). D) Flow cytometry analysis of PD-1, granzyme B and ki67 in CD8⁺ T-cells from TC1 control (CAF^low) and TC1 CAF-rich tumors (left and right plots respectively; mean ⁺/- s.e.m., from n = 6 mice per group, ns > 0.05 two-tailed t test). E) Analysis of cytokine expression in CD8⁺ T-cells isolated from E7 vaccinated TC1 control and CAF-rich tumors following peptide re-stimulation ex vivo. Example flow cytometry plots are shown from CAF-rich tumors with quantification, gating CD8⁺ cells expressing IFNy, TNFα and granzyme B from isotype controls (mean ⁺/- s.e.m., from n = 8 mice per group, ns > 0.05 two-tailed t test). F) Representative images from multiplexed IHC and histo-cytometry analysis of CTLA-4 expression on excluded CD8⁺ T-cells at the periphery of human HNSCC tumors (n= 7). i) Micrograph from tumor margin, showing Pan-Cytokeratin (PanCK; brown) and hematoxylin (blue) staining, scale bar represents 1mm. ii) Point Pattern plot (for the region shown in i), showing the slide position of cells identified through histo-cytometry analysis: each point represents the centroid of a cell, colored by classification shown in key below. iii) Micrograph showing a pseudo-coloured micrograph showing staining for PanCK, CD8 and CTLA-4 (as shown in the adjacent ‘single channel’ panels), in the region of interest (ROI) highlighted by the rectangle in ii. Scale bar represents 100μm. G) Tumor growth curves showing the effect of αCTLA-4 mAb treatment on CAF-rich and CAF-low TC1 tumors (mean +/- s.e.m., from n = 5 mice per group, ns > 0.05, *P < 0.05, AUC analysis followed by two-tailed t test. H) IHC showing CD8⁺ T-cell spatial distribution in TC1 CAF-rich tumors following treatment with αCTLA-4 mAb or isotype control mAb (tumor center [top panel]; tumor margin [bottom panel]), scale bars = 100 μm. Area % of staining is quantified (mean +/- s.e.m., ns > 0.05, *P < 0.05, two-tailed t test).

Fig. 5. NOX4 inhibition reverses the CAF phenotype and promotes CD8⁺ T-cell infiltration.

A-D) Effect of NOX4 inhibition (GKT137831) on established BALBneuT breast CAF. A) Q-PCR analysis of NOX4 gene expression. B) Intracellular ROS analysis. C,D), Immunofluorescence showing αSMA stress fiber formation (red; C) and collagen I (green; D). Representative images are shown; scale bars = 50 μm and 200 μm respectively and mean fluorescence intensity was quantified (all data represents mean ⁺/- s.e.m, from 2 or 3 independent experiments, *P < 0.05 two-tailed t test with Welch’s correction). E,F) IHC for αSMA showing the effect of GKT137831 treatment on CAF content in TC1 CAF-rich and MC38 CAF-rich tumors respectively. Representative images are shown, scale bar = 100 μm and area % of staining was quantified (mean ⁺/- s.e.m, *P <0.05 two-tailed t test). G,H) IHC showing the effect of GKT137831 treatment on CD8⁺ T-cell spatial distribution in TC1 CAF-rich and MC38 CAF-rich tumors respectively. Representative images are shown from tumor center (top panel) and margin (bottom panel). Scale bars = 100 μm and % staining area was quantified (mean ⁺/- s.e.m, *P < 0.05 two-tailed t test). I,J) Tumor growth curves showing the effect of GKT137831 treatment on TC1 CAF-rich and MC38 CAF-rich tumors respectively (mean ⁺/- s.e.m., from n = 6 - 7 mice per group, *P < 0.05, **P < 0.01 AUC analysis followed by two-tailed t test. K) Tumor growth curves showing effect of CAF shRNA NOX4 knockdown on the growth of TC1 CAF-rich tumors (mean ⁺/- s.e.m., from n = 8 mice per group, **P < 0.01 AUC analysis followed by two-tailed t test. L, M) IHC showing the effect of CAF shRNA NOX4 knockdown on αSMA expression (L) and CD8⁺ T-cell infiltration (M) in TC1 CAF-rich tumors Representative images are shown from tumor center (top panel) and margin (bottom panel). Scale bars = 100 μm and % staining area was quantified (mean ⁺/- s.e.m, *P < 0.05, **P < 0.01 two-tailed t test).

Fig. 6. NOX4 inhibition promotes CD8+ T-cell infiltration and re-sensitises CAF-rich tumors to anti-HPV E7 vaccination.

A) Tumor growth curves of TC1 CAF-rich tumors showing the effect of HPV E7 vaccine (blue), GKT137831 (grey) and combination (purple) treatments; control tumors are shown in black (mean ⁺/- s.e.m., from n = 10 mice per group, *P < 0.05 AUC analysis followed by two-tailed t test. B) IHC showing the effect of vaccine vs vaccine/GKT137831 on CD8⁺ T-cell infiltration in TC1 CAF-rich tumors. Representative images are shown from tumor center (top panel) and margin (bottom panel). Scale bars = 100 μm and % staining area was quantified (mean ⁺/- s.e.m, *P < 0.05, **P < 0.01 two-tailed t test). C) Flow cytometry analysis of % CD8⁺ E7 tetramer⁺ staining in control, vaccine, GKT137831 and vaccine/GKT137831 treated tumors, gated from CD45⁺ viable single cells (mean +/- s.e.m., n = 8 mice per group), ns > 0.05, two-tailed t test). D) Tumor growth curves of TC1 CAF-rich tumors showing relapse following withdrawal of GKT137831 at day 31. Individual mouse tumor volume measurements are shown from vaccine monotherapy (blue) and combination (purple) n = 6 mice per group. Dotted line represents when GKT137831 was stopped. (E) Kaplan-Meier survival curves showing effect of HPV E7 vaccine (blue), GKT137831 (grey) and combination (purple); control mice are shown in black (n=6 mice per group *P < 0.05 Log-rank (Mantel-Cox) test). F,G) Tumor growth curves (F) and Kaplan-Meier analysis (G) of TC1 CAF-rich tumors showing the effect of single dose HPV E7 vaccination combined with either short-term or long-term treatment with GKT137831 (black and grey respectively); long-term GKT137831 treatment was also tested in combination with a second dose of HPV E7 vaccination administered following initial response (purple). Individual mouse tumor volume measurements are shown; GKT137831 removed (black), continued GKT137831 only (grey) and continued GKT137831 + additional vaccine (purple) n = 7-8 mice per group, **P < 0.01 Log-rank (Mantel-Cox) test).

Fig. 7. NOX4 inhibition re-sensitizes CAF-rich tumors to anti-PD1 checkpoint therapy

A) Tumor growth curves of MC38 CAF-rich tumors showing the effect of αPD-1 (orange), GKT137831 (grey) and combination (blue) assessed by tumor volume measurements; control tumors are shown in black (mean ⁺/- s.e.m., from n = 11 mice per group, *P < 0.05 AUC analysis followed by two-tailed t test. B) IHC showing the effect of αPD-1 vs αPD-1/GKT137831 on CD8⁺ T-cell infiltration in MC381 CAF-rich tumors. Representative images are shown from tumor center (top panel) and margin (bottom panel). Scale bars = 100 μm and % staining area was quantified (mean ⁺/- s.e.m, *P < 0.05 two-tailed t test. C) Growth curves showing individual mouse tumor volume measurements of CAF-rich MC38 tumor growth following αPD-1 treatment (orange) or αPD-1 + GKT137831 treatment (blue) n = 8 mice per group. D) Kaplan-Meier survival curves showing effect of αPD-1 (orange), GKT137831 (grey) and combination (blue) in CAF-rich MC38 tumors; control mice are shown in black (n = 8 mice / group *P < 0.05 Log-rank (Mantel-Cox) test).