Modulation of fibronectin extracellular matrix enhances anti-tumor efficacy of immune checkpoint blockade

Abstract

The success of immune checkpoint inhibitors is limited by multiple factors, including poor T cell infiltration and function within tumors, partly due to a dense extracellular matrix (ECM). Here, we investigate modulating the ECM by targeting integrin α5β1, a major fibronectin-binding and organizing integrin, to improve immunotherapy outcomes. Use of a function-blocking murinized α5β1 antibody reduces fibronectin fibril formation, enhances CD8⁺ T cell transendothelial migration, increases vascular permeability, and decreases vessel-associated collagen. These changes culminate in improving the effectiveness of PD-L1 blockade, alone or with chemotherapy, in the E0771 breast cancer model. Clinically, high integrin alpha 5 (ITGA5) expression correlates with worse survival in patients treated with atezolizumab as monotherapy or combined with chemotherapy or anti-angiogenic therapies in numerous clinical trials. Overall, our studies suggest that ECM-modulating approaches could be used as a future strategy to increase the proportion of patients who respond to immune checkpoint inhibition and other immunotherapies.

Keywords: atezolizumab; cancer; combination; extracellular matrix; fibronectin; integrin a5b1; tumor immunotherapy.

Graphical abstract

Figure 1

Fibronectin/ECM disruption using α5β1 antibodies increases CD8⁺ T cell adhesion and transendothelial migration (A) Western blot of DOC-soluble or insoluble fractions of HUVEC lysates blotted for fibronectin (anti-FN), treated with isotype control or α5β1 antibodies. (B) Immunofluorescence microscopy of HUVECs cultured on fibronectin-coated plates treated with isotype control or α5β1 antibodies and stained with fibronectin antibodies, with cells intact or cells removed (decellularized), revealing disruptions in mature fibronectin matrix formation after 20 h of α5β1 antibody treatment. Scale bars represent 50 μm. (C) HUVEC permeability increased with addition of α5β1 antibodies, TNF-α (100 ng/mL) as positive control, unpaired t test of α5β1 10 μg/mL conditions in comparison to control treatment. ∗∗∗p < 0.0001 n = 6 biological replicates per group, data point represents mean, error bars represent standard error of the mean (SEM). (D) Schematic diagram of endothelial-immune cell adhesion and transmigration assays (made using BioRender). (E) CD8⁺ T cells stimulated with CD3e antibodies and interleukin-2 (IL-2) adhere more strongly with α5β1 antibody treatment, when HUVECs are untreated or treated with CXCL12, TNF-α, or both CXCL12 and TNF-α. Unpaired t test, ∗∗∗p < 0.0001, ∗∗p = 0.0060, n = 12 biological replicates, error bars represent SEM. (F) CD8⁺ T cells stimulated with CD3e antibodies and IL-2 undergo enhanced endothelial transmigration with α5β1 antibody treatment, when HUVEC are untreated or treated with CXCL12, TNF-α, or both CXCL12 and TNF-α. Unpaired t test, ∗∗∗p < 0.0001, n = 12, error bars represent SEM.

Figure 2

α5β1 antibody targeting reduces fibronectin and collagen deposition and increases intratumoral vascular permeability in mice (A) Immunofluorescence microscopy of mouse lung endothelial cells stained with fibronectin antibodies after treatment in vitro with α5β1 antibody shows reduced formation of fibronectin fibrils. (B) Quantification of percentage fibronectin per region of interest (ROI), n = 12, unpaired t test, ∗∗∗p < 0.0001, error bars represent SEM. (C) α5β1 antibody increased vascular permeability in mice bearing 66cl4 breast tumors, visualized by injection of near-infrared AngioSense dye after 24 h. Scale bars, 5 mm. (D) Quantification of fluorescence intensity after 24-h time period, n = 9–10 mice per group, unpaired t test, ∗∗∗p = 0.0001, error bars represent SEM. (E) Representative western blot of tumor lysates from mice treated with IgG isotype antibody or α5β1 antibody bearing 66cl4 breast tumors; fibronectin was detected in deoxycholate (DOC)-soluble and insoluble fractions, normalized to β-actin and a ratio determined. (F) Statistical analysis of fibronectin insoluble/soluble ratios, n = 6 lysates of tumors from individual mice per group, unpaired t test, ∗p = 0.0144, n = 6 per group, error bars represent SEM. (G) Immunofluorescence microscopy of breast tumors demonstrates reductions in vessel (endomucin⁺)-associated collagen I with α5β1 antibody treatment. Scale bars represent 50 μm. (H) Quantification of vessel-associated collagen I H-score, unpaired t test, n = 9 per group, ∗p = 0.0296, error bars represent SEM.

Figure 3

Combination of α5β1 and PD-L1 antibody treatment in the primary E0771 orthotopic breast cancer model extends median survival (A) Waterfall plots demonstrating percentage tumor growth from time of treatment initiation. CRs were observed in 0/18 mice for isotype control antibody (black), 3/20 mice for α5β1 antibody (blue), 1/20 mice for PD-L1 antibody (green), and 8/21 mice for PD-L1 + α5β1 antibody (red). (B) Survival analyses were generated from mice that reached primary tumor endpoint from (A). The same isotype control antibody group is included in each for comparison. (C) Table displaying hazard ratios (Cox proportional hazards model) and p values from log rank (Mantel-Cox) analyses of survival.

Figure 4

Analysis of the E0771 primary tumor microenvironment exposed to short-term therapy (A) Individual growth plots showing tumor volumes of E0771 treated with isotype control antibody, α5β1, PD-L1 antibody, or the α5β1 + PD-L1 antibody combination. CRs were observed in 0/8 for IgG isotype, 0/8 for α5β1 antibody, 1/8 for PD-L1 antibody, and 2/12 for PD-L1 + α5β1 antibody. Arrows represent antibody administration. (B) Contour flow cytometry plots of CD8⁺ T cells stained with PD-1 and TIM-3, revealing PD-1⁺ TIM-3⁺ terminally exhausted CD8⁺ T cells. (C–E) Flow cytometry analysis of (C) pan-T cells (CD3⁺) as a percentage of live cells, (D) terminally (Term) exhausted CD8 T cells (PD-1⁺ TIM3⁺) as a percentage of live cells, n = 7–10, unpaired t test, ∗p = 0.0217, and (E) ratio of terminally exhausted CD8 T cells (PD-1⁺ TIM3⁺) to non-exhausted CD8 T cells (PD-1⁺ TIM3⁻, PD-1⁻ TIM3⁻), unpaired t test, ∗p = 0.0185, error bars represent SEM.

Figure 5

Combination α5β1 and PD-L1 antibody blockade with PTX chemotherapy increases survival in mice bearing E0771 tumors that are less responsive to α5β1 and PD-L1 antibody treatment alone (A) Mice bearing E0771 tumors less than 90 mm³ at the start of treatment displayed increased survival especially in the PD-L1 and α5β1 antibody combination treatment group (red). (B) E0771 tumors larger than 90 mm³ at start of treatment responded poorly to each therapy. (C) Median OS, hazard ratios (cox proportional hazards model), log rank Mantel-Cox test, p values. Black arrows indicate treatment time points of antibodies, and gray arrows indicate PTX. (D) Percentage tumor growth inhibition of large (>90 mm³) E0771 tumors treated with PTX + PD-L1 (green), PD-L1 + α5β1 antibody (red), or PD-L1 antibody + α5β1 + PTX (purple). (E) Survival plots from (D). (F) Table displaying hazard ratios (Cox proportional hazards model) and p values from log rank (Mantel-Cox) analyses of survival.

Figure 6

Elevated ITGA5 gene expression is associated with poor OS of patients in groups treated with atezolizumab in multiple phase 2 and phase 3 clinical trials (A–C) Data from patient tumors were pooled from all trials and separated into above median or below median expression of ITGA5 for patients in groups treated with (A) atezolizumab monotherapy, (B) atezolizumab combination (anti-angiogenic agent or chemotherapy), and (C) no atezolizumab treatment (control chemotherapy, control anti-angiogenic, or observation), which revealed significantly worse median OS in patients with high ITGA5 expression in the context of atezolizumab monotherapy or atezolizumab combination therapy. (D) Swimmer plots depicting hazard ratios using univariate Cox proportional hazards model and p values using log rank (Mantel-Cox test) of ITGA5 expression for all trials listed. Data represent hazard ratio, and error bars represent 95% confidence intervals of the hazard ratios.