Potential treatment benefits of a GLP-1R antagonist in combination with immune checkpoint inhibitors in colorectal cancer

Abstract

The clinical efficacy of immune checkpoint inhibitors (ICIs) in colorectal cancer (CRC) remains limited. Modulation of the glucagon-like peptide-1 receptor (GLP-1R) may enhance T-cell-mediated antitumor responses. The present study aimed to evaluate the antitumor effects of the GLP-1R antagonist Exendin 9-39 (Exe-9) combined with anti-programmed cell death protein-1 (PD-1) treatment in preclinical CRC models. Using in vitro co-culture assays, ELISA and in vivo murine models, alongside immunohistochemical and molecular analyses of clinical samples, HT-29 and MC38-OVA colon cancer cell lines were co-cultured in vitro with activated T cells in the presence of Exe-9. In vivo, male BALB/c mice were injected with MC38 to establish a CRC model and nude mice were used to assess T-cell dependency. To evaluate this synergistic effect, BALB/c mice with CRC were treated with Exe-9, anti-PD-1 or a combination. Additionally, clinical CRC samples were analyzed to assess the association of GLP-1R expression with the immunotherapy response. Exe-9 significantly enhanced T-cell-mediated cytotoxicity in CRC cell lines and reduced tumor growth in immunocompetent CRC mice; however, this effect was not observed in nude mice. Furthermore, combination therapy with the GLP-1R antagonist and anti-PD-1 yielded an improved antitumor effect compared with either treatment alone, and high GLP-1R ex2pression in clinical samples correlated with poor ICI response. These findings suggest that GLP-1R antagonism potentiates T-cell-mediated antitumor immunity and may provide a promising adjunctive therapeutic strategy for patients with CRC when combined with ICIs in the future.

Keywords: Exendin 9–39; T-cell-mediated cytotoxicity; colorectal cancer; glucagon-like peptide-1 receptor; immune checkpoint inhibitors.

Figure 1.

Glucagon-like peptide-1 receptor antagonist enhances T-cell-mediated cytotoxicity against colon cancer cells. Colon cancer cell lines HT-29 and MC38-OVA were cultured either individually or co-cultured with activated T cells for 48 h in the presence of Exe-9 at concentrations of 10 and 50 nM. (A) Viability of HT-29 colon cancer cells cultured alone or in co-culture with activated T cells for 48 h, treated with Exe-9 (10 or 50 nM) or control. Viability was assessed by crystal violet staining; quantitative spectrophotometric data are shown (right panel); (B) viability of MC38-OVA colon cancer cells under the same culture and treatment conditions as in, (A) quantified by crystal violet staining (right panel, spectrophotometric data); (C) IFN-γ levels in culture supernatants from the 48-h co-cultures described in (A) and (B), measured by ELISA; (D) TNF-α levels in the same supernatants as in (C), determined by ELISA.*P<0.05, **P<0.01 and ***P<0.001. Exe-9, Exendin 9–39; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α.

Figure 2.

Glucagon-like peptide-1 receptor antagonist enhances T-cell-mediated antitumor efficacy in mice. (A) A schematic representation of the experimental timeline. Mice were subcutaneously injected with MC38 tumor cells to generate a colorectal cancer model, after which Exe9 was administered intratumorally on days 10, 12, 14 and 16. (B) Representative images of excised tumor tissues collected on day 20. (C) Tumor volumes were measured at regular intervals during the treatment period. (D) Endpoint tumor volumes and (E) tumor weights are depicted to illustrate the difference between groups. (F) Immunohistochemical analysis was conducted to assess the infiltration of CD8⁺ T cells in tumor sections (scale bar, 100 µm). (G) In a parallel study using nude mice, tumor volumes of (H) individual mice and (I) their average value and (J) tumor weights, were similarly measured to determine the dependence of the antitumor effects on T-cell-mediated immunity. Data are presented as mean ± SEM. ***P<0.001. SC, subcutaneous; Exe-9, Exendin 9–39; i.t., intratumoral injections.

Figure 3.

Glucagon-like peptide-1 receptor antagonist induces tumor-specific CD8⁺ T-cell replication. (A) Number of IFN-γ-secreting cells quantified by ELISpot after restimulation of splenocytes from Exe-9-treated mice with MC38 tumor cells for 20 h; (B) number of IFN-γ-secreting cells quantified by ELISpot after restimulation of splenocytes from Exe-9-treated mice with the MHC-I-restricted AH1 peptide for 20 h; (C) Pearson correlation between the number of IFN-γ-secreting cells (from MC38-restimulated group) and tumor volume; (D) Pearson correlation between the number of IFN-γ-secreting cells (from AH1 peptide-restimulated group) and tumor volume. Data are presented as mean ± SEM. *P<0.05. Exe-9, Exendin 9–39; sec, secreting; IFN-γ, interferon-γ.

Figure 4.

A combination of glucagon-like peptide-1 receptor antagonist and immune checkpoint inhibitors synergistically suppresses colorectal cancer growth in vivo. Mice bearing MC38 tumors were administered intratumoral injections of the glucagon-like peptide-1 receptor antagonist Exe-9 on days 10, 12, 14 and 16 or intraperitoneal injections of the anti-PD-1 antibody on days 10, 13, 16 and 19 or both treatments as the (A) schematic representation of experimental design demonstrates. (B) Representative excised tumor images collected on day 20 post-treatments. (C) Tumor growth curves during the treatment period generated by regular measurements of tumor dimensions using digital calipers. (D) Tumor volumes and (E) weights quantified at the endpoint. Data are presented as mean ± SEM. ***P<0.001. Exe-9, Exendin 9–39; PD-1, programmed cell death protein-1; SC, subcutaneous; i.t., intratumoral injections; i.p., intraperitoneal.

Figure 5.

High GLP-1R expression correlates with reduced efficacy of anti-programmed cell death protein 1 immunotherapy in patients with CRC. Tumor specimens were collected from 11 patients with CRC treated with immune checkpoint inhibitors and subsequently categorized into responder (n=5) and non-responder (n=6) based on therapeutic outcomes. (A) Quantitative polymerase chain reaction analysis was performed to measure the mRNA expression levels of GLP-1R in tumor tissues. (B) Patients were further stratified into high and low receptor expression groups based on the median expression value. Pearson's correlation analysis was conducted to evaluate the relationship between receptor expression and treatment responsiveness. (C) Representative immunohistochemical images (scale bar, 100 µm) and (D) western blotting analysis illustrates the protein expression of GLP-1R in responders and non-responders. Data are presented as mean ± SEM. *P<0.05, **P<0.01 and ***P<0.001. GLP-1R, glucagon-like peptide-1 receptor; CRC, colorectal cancer.