Targeting adenosine enhances immunotherapy in MSS colorectal cancer with EGFRvIII mutation

Abstract

Background: Patients with microsatellite stable (MSS) colorectal cancer (CRC) often display resistance to immunotherapy. Epidermal growth factor receptor (EGFR)-targeted therapies have shown potential in enhancing immunotherapy, yet clinical benefits remain unfulfilled, which may relate to inadequate patient stratification.

Methods: Circulating tumor cells and tumor tissues were collected from multicenter cohorts of patients with CRC receiving cetuximab to analyze EGFR variant type III (EGFRvIII) expression and immune infiltration. Syngeneic mouse models of EGFRvIII CRC were used to investigate the combined efficacy of adenosine inhibition and antiprogrammed cell death protein 1 (anti-PD-1).

Results: EGFRvIII mutations are found in about 10% of MSS CRC and are associated with poor response to cetuximab therapy. EGFRvIII-mutated patients with CRC exhibit an adenosine-mediated immunosuppressive tumor microenvironment (TME) subtype. Combination therapy with adenosine inhibitors remodels the TME, reversing cetuximab resistance and enhancing anti-PD-1 efficacy in EGFRvIII CRC.

Conclusions: Our findings identified EGFRvIII-positive CRC as a distinct subtype characterized by adenosine-mediated immunosuppressive TME. Targeting adenosine significantly improved the efficacy of anti-PD-1 in MSS CRC.

Keywords: adenosine; colorectal cancer; immunotherapy.

Figure 1. EGFRvIII mutation is associated with poor prognosis and an immunosuppressive TME. (A) Kaplan-Meier OS analysis using TCGA data showing that prognosis is markedly inferior in patients with EGFRvIII-positive CRC (cohort 2, n=192, p=0.011, log-rank test). (B) K-means clustering of CRC microenvironment phenotypes based on estimated numbers of 25 cell subsets conducted using TCGA CRC data (n=192) and calculated by ssGSEA. (C) Immune scores of EGFRvIII-negative and EGFRvIII-positive groups in TCGA CRC cohort data (n=192) calculated using the estimate package in R; p values were calculated using the Wilcoxon test. (D) Representative IF images of EGFRvIII and CD8 in human CRC specimens from cohort 4 (left panel) and correlation analysis of fluorescence intensity in tumor regions (right panel) (n=29). Scale bars=50 µm. (E) Representative IHC images showing differential CD8⁺ T-cell infiltration in EGFRwt or EGFRvIII subcutaneous tumors in C57BL/6 mice (left panel) and quantification of CD8⁺ T-cell density (right panel) (n=7). (F) Lymphocytes isolated from subcutaneous tumors from each group of C57BL/6 mice were analyzed for CD3 and CD8 expression by flow cytometry (left panel), and the percentage of CD3⁺CD8⁺ T cells summarized (right panel). (G) Expression of IFN-γ and granzyme B in CD3⁺CD8⁺ T cells detected using flow cytometry (left panel), and percentages of IFN-γ⁺ granzyme B⁺ cells as a proportion of CD3⁺CD8⁺ T cells (right panel). (H) IF staining showing CD8⁺ T-cell infiltration into multicellular spheroids built by EGFRwt or EGFRvIII cells in a three-dimensional system. Scale bars=25 µm. (I) Expression of IFN-γ and TNF-α in isolated human CD8⁺ T cells co-cultured with EGFRvIII DIFI cells in vitro detected by flow cytometry, along with statistical plots showing the proportions of positive cells. *P<0.05; ****P<0.0001. CRC, colorectal cancer; EGFRvIII, EGFR variant type III; EGFRwt, wild-type EGFR; IF, immunofluorescence; IFN, interferon; IHC, immunohistochemistry; OS, overall survival; ssGSEA, single-sample gene set enrichment analysis; TCGA, The Cancer Genome Atlas; TME, tumor microenvironment; TNF-α, tumor necrosis factor alfa.

Figure 2. EGFRvIII mutation facilitates CET resistance in MSS CRC. (A) Representative co-immunofluorescence images of CTCs from one patient with CRC expressing EGFRvIII. Tumor cells were captured using fluorescently labeled probes for epithelial biomarkers (EpCAM, CK8/18/19) or mesenchymal biomarkers (vimentin and twist). Leukocytes were excluded by CD45 staining. Scale bars=5 µm. (B, C) The proportion of different treatment outcomes (B) and TTP (C) in groups with EGFRvIII-positive or EGFRvIII-negative CRC receiving CET-based therapy (cohort 5, n=80). (D) Longitudinal CT scans of two representative cases from the EGFRvIII-positive or EGFRvIII-negative groups showing changes in liver metastases after four cycles of CET-containing treatment. (E) Analysis of EGFRvIII expression on CTCs from two patients with CRC prior to therapy or at progression. (F, G) EGFRvIII expression levels before and after CET treatment evaluated by CTC detection in 25 patients from cohort 5. (H, I) Subcutaneous tumor model established in C57BL/6 mice using MC38 cells with EGFRwt or EGFRvIII overexpression. On day 4 after tumor implantation, mice were randomized into two groups: control and CET (n=4 per group). Representative photos of tumors on day 25 (H) and tumor growth curves on days 4–25 (I). P values were determined by two-way analysis of variance. **P<0.01 and ***P<0.001. CET, cetuximab; CR, complete response; CTC, circulating tumor cell; EGFRvIII, EGFR variant type III; EGFRwt, wild-type EGFR; MSS, microsatellite stable; ns, not significant; PD, progressive disease; PR. partial response; SD, stable disease; TTP, time to progression.

Figure 3. Tumor-derived ADO impairs T-cell infiltration and function in EGFRvIII-positive CRC. (A–B) In vitro IFN-γ and TNF-α expression in isolated human CD8⁺ T cells treated with CM from DIFI (A) or HCA7 (B) cells detected using flow cytometry (left panels), along with statistical plots of the proportions of positive cells (right panels). (C) Representative pictures reflecting the variation of multicellular spheroids established by DIFI or HCA7 tumor cells co-cultured with EGFRwt-CM-pretreated or EGFRvIII-CM-pretreated human CD8⁺ T cells in vitro. Scale bars=100 µm. (D) Representative flow cytometry plots (left panels) and proportions of apoptotic tumor cells (right panel) when DIFI cells were incubated with EGFRwt-CM-pretreated or EGFRvIII-CM-pretreated human CD8⁺ T cells at 1:1 or 1:5 ratios. (E) Expression of IFN-γ and TNF-α by isolated human CD8⁺ T cells detected by flow cytometry following exposure to CM depleted of proteins, lipids and small molecules (left panels), along with statistical plots of the proportions of positive cells (right panels). (F, G) Expression of IFN-γ and TNF-α by isolated human CD8⁺ T cells detected by flow cytometry following exposure to CM fractions <3 kDa or >3 kDa (F), along with statistical plots of the proportions of positive cells (G). (H) Untargeted metabolite analysis of EGFRwt-CM and EGFRvIII-CM by ESI-Q TRAP-MS/MS. Metabolites were considered differentially regulated based on threshold values of p<0.05 and VIP (based on the OPLS-DA model) >1. The top 15 differential metabolites are shown. (I) Adenosine levels in medium of cells with or without CET treatment determined using an Adenosine Assay Kit (Abcam). (J) Expression of IFN-γ and TNF-α in isolated human CD8⁺ T cells determined by flow cytometry following exposure to EGFRvIII-CM, with or without additional ADO (left panels), along with statistical plots of the proportions of positive cells (right panels). (K) Expression of IFN-γ and TNF-α in isolated human CD8⁺ T cells determined by flow cytometry following exposure to EGFRvIII-CM under A2AR inhibition using ZM241385 (left panels), along with statistical plots of the proportions of positive cells (right panels). Data are the mean±SEM values of at least three independent experiments, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ADO, adenosine; CM, conditioned medium; CRC, colorectal cancer; DCC, dextran-coated charcoal; EGFRvIII, EGFR variant type III; EGFRwt, wild-type EGFR; IFN, interferon; ns, not significant; OPLS-DA, orthogonal projections to latent structures discriminant analysis; TNF-α, tumor necrosis factor alfa; VIP, variable importance in the projectio.

Figure 4. EGFRvIII tumor cells facilitate extracellular ATP-to-ADO transition via the CD39/CD73 pathway. (A) CD39 and CD73 expression in vector-transfected, EGFRwt and EGFRvIII cells determined by western blot analysis. (B) Representative flow cytometry plots showing CD39 and CD73 expression on the cell membranes of vector-transfected, EGFRwt and EGFRvIII cells. (C–F) Expression of EGFRvIII, CD39, and CD73 detected by IF in human CRC specimens from cohort 4 (n=40). Representative IF images of EGFRvIII-negative and EGFRvIII-positive tissues (C) and quantification of fluorescent intensity at each position along the indicated diagonal (D). CD39 and CD73 expression were positively correlated with EGFRvIII expression in CRC (linear regression) (E). Pearson’s correlation coefficient (left panel) and Mander’s overlap coefficient (right panel) values were calculated to analyze the colocalization of EGFRvIII with CD39/CD73 (n=18) (F). (G) ATP levels in the medium of EGFRvIII cells after silencing CD39, determined using an ATP Assay Kit (Beyotime). (H) Adenosine levels in the medium of EGFRvIII cells after silencing CD39, determined using an Adenosine Assay Kit (Abcam). (I) Expression of IFN-γ and TNF-α by isolated human CD8⁺ T cells following exposure to CM from EGFRvIII cells with CD39 silenced, tested by flow cytometry (left panels), along with statistical plots of the proportions of positive cells (right panels). (J, K) Subcutaneous tumor model in C57BL/6 mice established using MC38 EGFRvIII cells. On day 4 after tumor implantation, mice were randomized into six groups: control, CET, POM-1, AMP-CP, CET+POM-1, and CET+AMP CP. Following 3 weeks of treatment, lymphocytes isolated from subcutaneous tumors from each group were analyzed for CD3, CD8, IFN-γ, and granzyme B expression by flow cytometry. Percentages of CD3⁺CD8⁺ T cells (left panel) and of IFN-γ⁺ granzyme B⁺ cells as a proportion of CD3⁺CD8⁺ T cells are summarized (right panel) (J). CD8⁺ T-cell infiltration into subcutaneous tumors of each group analyzed by IHC staining. Representative IHC images (left panel) and quantification of the density of CD8⁺ T cells (right panel) (K). Scale bars=100 µm. Data are the mean±SEM values of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ADO, adenosine; CET, cetuximab; CM, conditioned medium; CRC, colorectal cancer; EGFRvIII, EGFR variant type III; EGFRwt, wild-type EGFR; IF, immunofluorescence; IFN, interferon; IFN, interferon; IHC, immunohistochemistry; TNF-α, tumor necrosis factor alfa.

Figure 5. p-STAT3 upregulates CD39/CD73 expression in EGFRvIII-positive CRC. (A) IF staining showing p-STAT3 was notably elevated in EGFRvIII cells compared with EGFRwt cells. Scale bars=25 µm. (B) Intracellular distribution of p-STAT3 detected by nuclear-cytoplasmic fractionation experiment followed by western blot analysis. (C, D) CD39 and CD73 protein levels detected by western blot analysis in EGFRwt or EGFRvIII tumor cells after inhibition of STAT3 using STA-21 (C) or siRNAs (D). (E, F) Representative IF images showing CD39 and CD73 expression in DIFI EGFRvIII tumor cells after STAT3 inhibition using STA-21 (E) or siRNAs (F) detected by western blot analysis. Scale bars=25 µm. (G) ATP levels in the culture medium of EGFRvIII cells after STAT3 inhibition using STA-21 (left panel) or siRNAs (right panel) determined using an ATP Assay Kit (Beyotime). (H) Adenosine levels in the culture medium of EGFRvIII cells after STAT3 inhibition using STA-21 (left panel) or siRNAs (right panel) determined using an Adenosine Assay Kit (Abcam). (I) Expression of IFN-γ and TNF-α by isolated human CD8⁺ T cells tested by flow cytometry following exposure to CM from EGFRvIII cells treated with STA-21 (top panels), along with statistical plots of the proportions of positive cells (bottom panels). (J) Expression of IFN-γ and TNF-α in isolated human CD8⁺ T cells tested by flow cytometry following exposure to CM from EGFRvIII with STAT3 silenced (top panels), along with statistical plots of the proportions of positive cells (bottom panels). (K, L) EGFRvIII cells were harvested for ChIP assay to detect p-STAT3 enrichment around the promoters of genes encoding CD39 (K) and CD73 (L). Immunoprecipitated DNA was analyzed by qRT-PCR using specific primers. Anti-IgG antibody served as the negative control. Data are presented as mean±SEM values of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ChIP, chromatin immunoprecipitation; CM, conditioned medium; CRC, colorectal cancer; EGFRvIII, EGFR variant type III; EGFRwt, wild-type EGFR; IF, immunofluorescence; IFN, interferon; p-STAT3, phosphorylated STAT3; qRT-PCR: quantitative real-time PCR; siRNA, small interfering RNA; TNF-α, tumor necrosis factor alfa.

Figure 6. Blocking ADO generation reignites the established immunologically ‘cold’ TME to synergize with immunotherapy. (A–E) C57BL/6 mice carrying subcutaneous MC38 EGFRvIII tumors received four-drug combination therapy, including POM-1 administered using different dosing schemes (n=4 per group). Representative images of subcutaneous tumors from each group harvested on day 28 (A). Tumor growth curves on day 4–28 postinjection with MC38 EGFRvIII cells. Tumor volumes were calculated every 3 days (B). P values were determined by two-way ANOVA. ADO levels in the supernatants of subcutaneous tumor tissues were detected using an Adenosine Assay Kit (Abcam) (C). Expression of IFN-γ and TNF-α in extracted infiltrating CD8⁺ T cells tested by flow cytometry (D), and the summarized percentages of IFN-γ⁺ TNF-α⁺ cells as proportions of CD3⁺CD8⁺ T cells (E). (F–J) Transplanted subcutaneous MC38 EGFRvIII tumors from C57BL/6 mice were treated with CET+IRI, POM-1, CET+IRI+POM-1, CET+IRI+α-PD-1 or CET+IRI+POM-1+α-PD-1, respectively. In all regimens, the POM-1 dosing scheme comprised pretreatment of mice at a 5 mg/kg dose daily for 3 days, before initiation of combination treatment. Representative images of subcutaneous tumors from each group harvested on day 28 (F). Tumor growth curves on day 4–28 postinjection with MC38 EGFRvIII cells. Tumor volumes were calculated every 3 days (n=5 per group) (G). P values were determined by two-way ANOVA. Expression of IFN-γ and TNF-α in extracted infiltrating CD8⁺ T cells analyzed by flow cytometry (H), and summarized percentages of IFN-γ⁺ TNF-α⁺ cells as a proportion of CD3⁺CD8⁺ T cells (I–J). (K–M) BALB/c mice carrying subcutaneous CT26 EGFRvIII tumors were treated with POM-1 or α-PD-1, alone or in combination. Representative images of subcutaneous tumors from each group harvested on day 22 (K). Tumor growth curves on day 4–22 postinjection with CT26 EGFRvIII cells. Tumor volumes were calculated every 3 days (n=4 per group) (L). P values were determined by two-way ANOVA. Expression of IFN-γ and TNF-α in extracted infiltrating CD8⁺ T cells determined by flow cytometry. Percentages of CD3⁺CD8⁺ T cells (left panel) and IFN-γ⁺ TNF-α⁺ cells (right panel) as proportions of CD3⁺CD8⁺ T cells (M). Data are mean±SEM values of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. ADO, adenosine; ANOVA, analysis of variance; CI, cetuximab plus irinotecan; CRC, colorectal cancer; CET, cetuximab; EGFRvIII, EGFR variant type III; EGFRwt, wild-type EGFR; GZMB, granzyme B; IFN, interferon; IRI, irinotecan; ns, not significant; TME, tumor microenvironment; TNF-α, tumor necrosis factor alfa.