Cisplatin plus anti-PD-1 antibody enhanced treatment efficacy in advanced esophageal squamous cell carcinoma

Abstract

Therapies for patients with advanced esophageal squamous cell carcinoma (ESCC) are limited and accompanied by dismal prognosis. Here we use ESCC cell line K30 and TE-1 to investigate the antitumor efficacy of cisplatin plus anti-PD-1 antibody. Enhanced antitumor effects and increased CD8⁺ tumor-infiltrating lymphocytes of combination therapy were observed in TE-1 cells bearing humanized mice model. Lower cell viability and more cell apoptosis were found in the combination therapy in vitro. We next analyzed clinical data from patients with advanced ESCC received cisplatin-based chemotherapy plus an anti-PD-1 antibody (Tislelizumab or Sintilimab) as first line therapy from two clinical trials (NCT03469557, NCT03748134). With the response rate of 81.8%, duration of response of 15.2 months, median progression-free survival of 15.5 months, median overall survival of 21.5 months and manageable toxicity in patients with advanced ESCC, we demonstrated that cisplatin-based chemotherapy plus anti-PD-1 antibody is an effective and safe option. We further confirmed sublethal cisplatin could induce PD-L1 expression in ESCC cells and cisplatin-treated ESCC cells suppressed the activation and function of immune cells while the addition of sintilimab prevented this process. These results highlight the effectiveness of cisplatin combining with anti-PD-1 antibody in patients with advanced ESCC, revealed its capability to promote the PD-L1 expression in ESCC cells and act synergistically with anti-PD-1 antibody to restore exhausted immune cells activities, thus providing a theoretical basis for further explorations in the mechanism of the combination treatment of cisplatin-based chemotherapy with immune checkpoint inhibitors in ESCC.

Keywords: Esophageal squamous cell carcinoma; PD-1; chemotherapy; cisplatin; immunotherapy.

Figure 1

CDDP plus anti-PD-1 antibody enhanced the antitumor effects and increased CD8⁺ TILs in ESCC in humanized mice model. (A) Schematic diagram of humanized mice establishment and experimental design. (B) Average tumor volumes. (C) Weights of tumors on day 15. (D) Average percentages of CD8⁺ TILs area in tumors per filed. (E) Representative immunohistochemistry images of tumors stained for CD8 (brown). Scale bar, 100 μm. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001; two-way analysis of variance (ANOVA) in (B), two tailed unpaired Student’s t test in (C) and (D). Data from two independent experiments were shown (means ± SEM).

Figure 2

Schematic diagram of co-culturing ESCC cell lines and PBMCs. K30 or TE-1 cells were cultured with CDDP (1 μg/ml) for 48 h. Thereafter, untreated or CDDP-treated K30 or TE-1 cells (5×10⁴ cells) were cultured with activated PBMCs (with 0.1 mg/ml IgG or with 0.1 mg/ml sintilimab) in 96-well round plates for 24 h, E:T=5:1. Subsequently, cell viability was detected by CCK8 assay and flow cytometry. The levels of IL-2 and IFN-γ in supernatants were measured.

Figure 3

CDDP plus anti-PD-1 antibody significantly inhibited the growth of ESCC cells in the ESCC cells/PBMCs co-culture system. A. Representative images of PBMCs before and after stimulation. B. FACS showing the ratio of CD25⁺ cells in CD8⁺ lymphocytes before and after stimulation. C. CCK8 assay showing K30 and TE-1 cells viability in responding to different dose of CDDP and plot against lg μg/ml of CDDP. In the concentration of 1 µg/ml (lg dose =0), the relative cell survival rate of K30 and TE-1 cells were between 50% and 60%. D and E. ESCC cells/PBMCs co-culture system was established. After co-culturing, supernatant was discarded and PBMCs were washed away. Cell viability of adherent K30 and TE-1 cells was measured by CCK8 assay. Data from three independent experiments were shown (means ± SEM). Scale bar, 100 μm. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

CDDP sensitizes K30 and TE-1 cells to PBMCs and the cell killing ability of immune cells can be enhanced by PD1 blockade. ESCC cells/PBMCs co-culture system was established. After co-culturing, whole cells were harvested and stained with anti-CD45-BV421, followed by PI and annexin V-FITC. CD45 negative cells were analyzed for apoptosis. (A and C) Representative dot plots of K30 (A) and TE-1 (C) cells from flow cytometry. (B and D) Percentages of annexin V⁺ of K30 (B) and TE-1 (D) cells. Data from three independent experiments were shown (means ± SEM). **P<0.01, ***P<0.001.

Figure 5

Treatment regimens.

Figure 6

Kaplan-Meier plots of median progression-free survival (A) and overall survival (B).

Figure 7

Change in sum of target lesion diameters over time for total patients (A). Best change from baseline in target lesion size for total patients (B).

Figure 8

Representative CT scan images of primary lesion (patient 1), lymph nodes (patient 2) and spine (patient 3) metastatic lesions after two cycles of FP+T regimen. Red arrows indicate the primary or metastatic lesions.

Figure 9

Representative CT scan images of lung (patient 1) and liver (patient 2) metastatic lesions after one and two cycles of TP+S regimen. Red arrows indicate the metastatic lesions.

Figure 10

CDDP increased ESCC cells PD-L1 expression. (A-E) PD-L1 expression of K30 (A) and TE-1 (D) cells, treated with CDDP (1 μg/ml) or PBS (control) for 48 h, detected by flow cytometry. Gates were set according to isotype control. Percentages of PD-L1 positive K30 (B) and TE-1 (E) cells from three independent experiments were shown (means ± SEM). (C, F) PD-L1 expression of K30 (C) and TE-1 (F) cells, treated with CDDP (1 μg/ml) or PBS (control) for 48 h, detected by western blot. (G-I) PD-L1 expression in ESCC from humanized mice treated by CDDP (5 mg/kg) for 15 days, detected by IHC staining (G) and western blot (I). Relative PD-L1 positive area (H) from two experiments were shown (means ± SEM). Scale bar, 50 μm. *P<0.05, **P<0.01.

Figure 11

The activation of CD8⁺ lymphocytes as well as secretion of IFN-γ and IL-2 can be suppressed by CDDP-treated ESCC cells in the co-culture system while the addition of anti-PD-1 antibody prevent this process. (A and B) ESCC cells/PBMCs co-culture system was established. After co-culturing, the levels of IL-2 and IFN-γ in supernatants were measured by ELISA. Data from three independent experiments were shown (means ± SEM). (C-F) PBMCs from K30 cells/PBMCs co-culture system (C) and TE-1 cells/PBMCs co-culture system (D) were collected and stained with CD8-PB and CD25-PE, the ratio of CD25⁺ cells in CD8⁺ lymphocytes were detected by flow cytometry. Percentages of CD25⁺ cells in CD8⁺ lymphocytes in K30 cells/PBMCs co-culture system (E) and TE-1 cells/PBMCs co-culture system (F) from three independent experiments were shown (means ± SEM). **P<0.01, ***P<0.001 and ****P<0.0001.