Interaction of immune checkpoint PD-1 and chemokine receptor 4 (CXCR4) promotes a malignant phenotype in pancreatic cancer cells

Abstract

Despite recent therapeutic advances, pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with limited therapeutic options. Immune checkpoint inhibitors (ICIs) have demonstrated promising results in many cancers, but thus far have yielded little clinical benefit in PDAC. Based on recent combined targeting of programmed cell death protein-1 (PD-1) and C-X-C chemokine receptor 4 (CXCR4) in patient-derived xenografts (PDXs) and a pilot clinical trial, we sought to elucidate potential interactions between PD-1 and CXCR4. We observed concomitant expression and direct interaction of PD-1 and CXCR4 in PDAC cells. This interaction was disrupted upon CXCR4 antagonism with AMD3100 and led to increased cell surface expression of PD-1. Importantly, CXCR4-mediated PDAC cell migration was also blocked by PD-1 inhibition. Our work provides a possible mechanism by which prior studies have demonstrated that combined CXCR4 and PD-1 inhibition leads to decreased tumor growth. This is the first report investigating PD-1 and CXCR4 interactions in PDAC cells and our results can serve as the basis for further investigation of combined therapeutic targeting of CXCR4 and PD-1.

Fig 1. PD-1 and CXCR4 expression in PDAC cells and PDOs.

(A) PD1 and CXCR4 were detected in PDAC cells and PDOs by western blot. Notably, different cell and PDO lines had unique expression patterns. MOLT-4 was used as a positive control for PD-1 and CXCR4. (B) IF staining shows co-expression of PD-1 and CXCR4 in PDAC cells (magnification 40X). (C) IF staining shows co-expression of PD-1 and CXCR4 in PDAC PDOs. Consistent with western blot results, hPT1 PDOs had lower expression of CXCR4 compared to hPT4 PDOs (magnification 20X).

Fig 2. PD-1 and CXCR4 expression in PDAC tumors and PDOs.

Operative human primary PDAC specimens and corresponding PDOs for hPT26 were serially sectioned and stained with multiplex IF. Both primary PDAC tissues and PDOs showed co-expression of PD-1 and CXCR4 (green + violet→ teal). These regions corresponded to areas consistent with pancreatic duct cells in primary PDAC tumors and PDOs. α-SMA was used as a marker for CAFs, which are known components of the PDAC TME that express PD-1 and CXCR4 (red + green→ yellow, red + violet→ pink, red + green + violet→ white). In primary tumors, CAFs with PD-1 and CXCR4 expression were predictably in regions of desmoplasia characteristic of the TME. α-SMA+ CAFs were similarly noted in areas typical of the TME in PDOs, along the periphery of the 3D organoid structures. Altogether, these results show concomitant expression of PD-1 and CXCR4 in human PDAC.

Fig 3. Co-immunoprecipitation studies in MIAPaCa-2 and PANC-1 cell lines.

Serum-starved PDAC cells were pre-treated with solvent (-) or AMD3100 (1 μM) (+) for 45 min. Cell lysates were immunoprecipitated with IgG control or anti-PD-1 antibodies. (A, B) Immunoprecipitates were immunoblotted (IB) with anti-PD-1 and anti-CXCR4 antibodies, revealing successful pull down of PD-1 and resultant co-IP of CXCR4; 5% of IB lysate (lower panel) was used as input control. Treatment with AMD3100 revealed reduced levels of PD-1-bound CXCR4 in PDAC cells. (C, D) Quantification of CXCR4 immunoprecipitated with PD-1 in MIAPaCa-2 (B) and PANC-1 (D) cells normalized to β-actin as shown in A and C, respectively. *p < 0.05, **p < 0.01 compared with IgG. ^#p < 0.05, ^##p < 0.01 compared with solvent control.

Fig 4. CXCR4 inhibition leads to PD-1 translocation to the cell membrane.

(A, B) Immunofluorescence analysis of MIAPaCa-2 and PANC-1 PDAC cells. Treatment with AMD3100 resulted in increased PD-1 cell surface expression compared to controls (magnification 100x). (C, D) Flow cytometry analysis of PDAC cells. Cells were treated with AMD3100, isotype antibody, or solvent control and then prepared for flow cytometry analysis of PD-1 membrane expression. Treatment with AMD3100 revealed increased surface expression of PD-1 compared to controls. (E, F) Quantification of flow cytometry analysis demonstrated 156% and 53.3% increase in cell surface expression of PD-1 after AMD3100 treatment.

Fig 5. Transwell migration assays of PANC-1 and MIAPaCa-2 cells.

(A) Treatment with AMD3100 or pembrolizumab alone did not alter cell migration. However, exposure to CXCL12 promoted cell migration in both lines as expected. The addition of AMD3100 or pembrolizumab to CXCL12-treated cells resulted in inhibition of cell migration in both cell lines, demonstrating that inhibition of PD-1 or CXCR4 can block CXCL12-induced migration. All images at 10x magnification. (B) Quantification of transwell migration assays revealed that migration was significantly inhibited in CXCL12-treated cells when exposed to AMD3100 and pembrolizumab, demonstrating that combined CXCR4 and PD-1 inhibition abrogated CXCL12-induced migration. ***p<0.001 vs. control; ##p<0.01, ###p<0.001 vs. CXCL12.

Fig 6. PD-1 knockdown in MIAPaCa-2 cells attenuates CXCR4 downstream pathways.

(A) MIAPaCa-2 PD-1 KD was most successful in decreasing PD-1 in construct #2. (B) PD-1 KD cells demonstrated decreased migration even when cells were exposed to CXCL12 (100 ng/mL). (C) Quantification of transwell migration assays demonstrated a 107% decrease in migration in PD-1 KD cells compared to KD control. When treated with CXCL12, PD-1 KD cells had a 99% less migration than KD controls. (*** p<0.001 vs. KD control; ### p <0.001 vs. PD-1 KD; &&& p <0.001 vs. KD control + CXCL12).