NECTIN4 regulates the cell surface expression of CD155 in non-small cell lung cancer cells and induces tumor resistance to PD-1 inhibitors

Abstract

The development of immune checkpoint inhibitors has changed treatment strategies for some patients with non-small cell lung cancer (NSCLC). However, resistance remains a major problem, requiring the elucidation of resistance mechanisms, which might aid the development of novel therapeutic strategies. The upregulation of CD155, a primary ligand of the immune checkpoint receptor TIGIT, has been implicated in a mechanism of resistance to PD-1/PD-L1 inhibitors, and it is therefore important to characterize the mechanisms underlying the regulation of CD155 expression in tumor cells. The aim of this study was to identify a Nectin that might regulate CD155 expression in NSCLC and affect anti-tumor immune activity. In this study, we demonstrated that NECTIN4 regulated the cell surface expression and stabilization of CD155 by interacting and co-localizing with CD155 on the cell surface. In a syngeneic mouse model, NECTIN4-overexpressing cells exhibited increased cell surface CD155 and resistance to anti-PD-1 antibodies. Of note, combination therapy with anti-PD-1 and anti-TIGIT antibodies significantly suppressed tumor growth. These findings provide new insights into the mechanisms of resistance to anti-PD-1 antibodies and suggest that NECTIN4 could serve as a valuable marker in therapeutic strategies targeting TIGIT.

Keywords: Nectin-like molecule-5 (Necl5); Non small cell lung cancer (NSCLC); Poliovirus receptor (PVR); T-Cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT).

Fig. 1

Cell surface expression of CD155 in NECTIN-overexpressing or -depleted NSCLC cells. A, Immunoblot analysis of total CD155 abundance in cell lysates in NECTIN1, 2, 3, or 4-overexpressing H322 and EBC1 cells. B, mRNA level of CD155 in NECTIN-overexpressing H322 and EBC1 cells by RT-qPCR. The relative quantification was performed using the ΔCt method with 18S rRNA as an internal control. C, Flow cytometric analysis of CD155 at the cell surface. D, Whole cell lysates and cell surface protein extracts were analyzed by immunoblot analysis. E, Localization of CD155 was evaluated by fluorescent immunostaining in EBC1 cells overexpressing each Nectin. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), and CD155 was labeled with red fluorescence. Representative images were captured using optical sectioning. Scale bars, 20 μm. F, NECTIN4 knockdown was performed using siRNA in NECTIN4-overexpressing H322 and EBC1 cells, and cell surface CD155 expression was evaluated by flow cytometry. G–H, NECTIN4 knockout was established in the H322 cell line using two different guide RNAs, and CD155 expression was assessed by immunoblotting (G) and flow cytometry (H). Experiments were performed at least in triplicate, and the data are presented as the mean ± standard deviation (SD). Western blot band intensities were quantified using Image Lab software (Bio-Rad). The relative protein expression levels were normalized to β-actin and are presented as fold changes relative to the control group. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s test (A, B, C), Dunnett’s test (G, H), or the Student’s t test (D, F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. OE, overexpression; KO, knockout

Fig. 2

Evaluation of NECTIN4 and CD155 interactions and colocalization. A, Co-immunoprecipitation (Co-IP) experiments were performed using HEK293T cells transfected with CD155 and Flag-NECTIN4 expression vectors. Cell lysates were immunoprecipitated using Flag antibody or CD155 antibody, and immunoblotting was performed. Mouse IgG was used as a control for immunoprecipitation. B, A proximity ligation assay (PLA) targeting CD155 and NECTIN4 was performed in empty vector control and NECTIN4 overexpressing H322 and EBC1 cell lines. The close colocalization of CD155 and NECTIN4 was detected by red fluorescent signals. Nuclei were stained with DAPI. Scale bars, 20 μm. The number of PLA signals indicating colocalization of CD155 and NECTIN4 was determined from z-projection images generated by z-stacking of optical sections for individual cells. C, Schematic diagram of domain-deleted Flag-NECTIN4. D, HEK293T cells were co-transfected with the CD155 expression vector and full-length Flag-NECTIN4 or domain-deleted Flag-NECTIN4 expression vectors, followed by co-immunoprecipitation. As references, input samples were analyzed by immunoblotting, and samples immunoprecipitated with CD155 antibody were detected with Flag antibody by immunoblotting. Statistical analysis was conducted using Student’s t test. *p < 0.05, **p < 0.01. OE, overexpression

Fig. 3

Identification of NECTIN4 domains involved in the cell surface expression of CD155. A, Schematic diagram of domain-deleted NECTIN4. B, Full-length NECTIN4 or NECTIN4 with deletions in each domain were transfected into EBC1 cells, and cell surface CD155 expression was evaluated. Experiments were performed in triplicate, and the data are presented as the mean ± SD. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s test (B). **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Overexpression of NECTIN4 confers resistance to PD-1 inhibitors. A, Schematic representation of the experiment: the subcutaneous transplantation of 4T1 EV control or NECTIN4-overexpressing cells, followed by treatment with anti-PD-1 antibody. The treatment was initiated on day 7 after tumor transplantation and administered every three days for a total of three times. Anti-PD-1 mAb, or IgG2a isotype were administered at a dose of 200 µg. B, Graph showing tumor volumes after transplantation. Tumor volumes were calculated using the formula: length × width × width/2. C, Individual tumor volume progression in each group. D, Analysis of tumor-infiltrating lymphocytes (TILs) by flow cytometry. Experiments were conducted with five mice per group and the data are presented as the mean ± SD. All in vivo experiments were performed in duplicates, with similar results. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s test (B) or Student’s t-test (D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. EV, empty vector; OE, overexpression

Fig. 5

Overcoming anti-PD-1 antibody resistance in NECTIN4-overexpressing 4T1 cells by combination therapy with anti-TIGIT antibody. A, Schematic representation of the experiment: the subcutaneous transplantation of 4T1 EV control or NECTIN4-overexpressing cells, followed by treatment with anti-PD-1 antibody alone or in combination with anti-TIGIT antibody. The treatment was initiated on day 7 after tumor transplantation and administered every three days for a total of three times. Anti-PD-1 mAb, or IgG2a isotype were administered at a dose of 200 µg, and anti-TIGIT mAb or IgG1 isotype were administered at a dose of 150 µg. B, Graph showing tumor volume progression after transplantation. C, Comparison of tumor weights in each group, measured post-excision. D, Photographs of tumors excised two days after the completion of treatment. E, Analysis of tumor-infiltrating lymphocytes (TILs) by flow cytometry. Experiments were conducted with five mice per group and the data are presented as the mean ± SD. All in vivo experiments were performed in duplicates, with similar results. Statistical analysis was conducted using one-way ANOVA followed by Tukey’s test (B, C) or Student’s t-test (E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. EV, empty vector; OE, overexpression