DHA-SBT-1214 Taxoid Nanoemulsion and Anti-PD-L1 Antibody Combination Therapy Enhances Antitumor Efficacy in a Syngeneic Pancreatic Adenocarcinoma Model

Abstract

The goal of this study was to evaluate combination of a novel taxoid, DHA-SBT-1214 chemotherapy, in modulating immune checkpoint marker expression and ultimately in improving antibody-based checkpoint blockade therapy in pancreatic adenocarcinoma (PDAC). DHA-SBT-1214 was encapsulated in an oil-in-water nanoemulsion and administered systemically in Panc02 syngeneic PDAC-bearing C57BL/6 mice. Following treatment with DHA-SBT-1214, expression levels of PD-L1 were measured and anti-PD-L1 antibody was administered in combination. The effects of combination therapy on efficacy and the molecular basis of synergistic effects were evaluated. PD-L1 expression was lower on Panc02 pancreatic tumor cells in vitro, which significantly increased after exposure to different chemotherapy drugs. Administration of DHA-SBT-1214, gemcitabine, and PD-L1 antibody alone failed to increase CD8+ T-cell infiltration inside tumors. However, combination of anti-PD-L1 therapy with a novel chemotherapy drug DHA-SBT-1214 in nanoemulsion (NE-DHA-SBT-1214) significantly enhanced CD8+ T-cell infiltration and the therapeutic effects of the anti-PD-L1 antibody. Furthermore, in the Panc02 syngeneic model, the NE-DHA-SBT-1214 combination therapy group reduced tumor growth to a higher extend than paclitaxel, nab-paclitaxel (Abraxane), gemcitabine, or single anti-PD-L1 antibody therapy groups. Our results indicate that NE-DHA-SBT-1214 stimulated immunogenic potential of PDAC and provided an enhanced therapeutic effect with immune checkpoint blockade therapy, which warrants further evaluation.

Conflict of interest statement

Conflict of interest: The authors declare that they have no conflicts.

©2019 American Association for Cancer Research.

Figures

Figure 1.

Characterization of DHA-SBT-1214 nanoemulsion formulations. (A) – Transmission electron microscopy image of DHA-SBT-1214 encapsulated nanoemulsion. (B) – The oil droplet particle size determination in nm. (C) – The measurement of zeta potential or surface charge on the oil droplets in mV; and (D) – The uptake of rhodamine-encapsulated nanoemulsion formulation in Panc02 cells. Fluorescence microscopy images showing the blue (nucleus), red (rhodamine encapsulated nanoemulsion) and overlay images in purple color. The images were acquired at 63x magnification. The image scale bar is 100 μm.

Figure 2.

The activity of different anti-cancer agents against Panc02 cells in vitro. The percentage maximal response as a function of anti-cancer agents when administered to Panc02 cells. The cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 96 h of incubation at 37 ºC. Data represent mean ± standard deviation (n=3). Significant differences are indicated as follows: *p < 0.05, and **p < 0.01.

Figure 3.

In vivo efficacy of the PD-L1 antibody in combination to different therapeutic drugs including gemcitabine solution, Abraxane™, paclitaxel in solution, DHA-SBT-1214 in solution and NE-DHA-SBT-1214 against Panc02 induced syngeneic mice tumors. (A)– Graph summarizing all treatment modalities. The values are means ± SD (n=3). Significant differences are indicated as follows: *p < 0.05, and **p< 0.01. (B)– Tumor images taken at the time of harvest from different treatment modalities. (3b-A)– Tumors from mice treated with vehicle; (3b-B)– Three tumors each from PD-L1 (200μg) treated mice; (3b-C, D)–Tumors from Abraxane™ plus IgG or PD-L1 (200μg) treated mice respectively; (3b-E)– Tumors from NE-DHA-SBT-1214 (10mg/kg) plus IgG (200μg) treated mice; (3b-F, G)– Tumors from gemcitabine plus IgG or PD-L1 (200μg) treated mice respectively; (3b-H)– Tumors from NE-DHA-SBT-1214 (10mg/kg) plus PD-L1 (200μg) treated mice; (3b-I, J)– Tumors from NE-DHA-SBT-1214 (25mg/kg) plus IgG or PD-L1 (200μg) treated mice respectively. (C)– Graph for all the tumors from (3B) to show their progression over time.

Figure 4.

In vivo PD-L1 surface protein expression in response to different therapeutic modalities. (A)– mRNA expression of PD-L1 from different mouse tumor treatment groups analyzed using RT-PCR. Relative gene expression for RT-PCR data was calculated relative to murine β-actin. (B)– Tumor tissue lysate from different treated groups was prepared and protein level of different proteins was analyzed using western blotting. (C)– The bands corresponding to PD-L1 were quantified using Image J software and was normalized relative to band intensities for the corresponding Histone 3 loading controls. The bar represents the mean ± standard deviation of data from at least 3 independent experiments; *p<0.05, **p<0.01.

Figure 5.

Histopathological evaluation of the Panc02-induced tumor tissues collected from control and different combination treated mice (hematoxylin & eosin staining). Significant reduction in tumor stroma observed with combination of NE-DHA-SBT-1214 and anti-PD-L1 treated groups. The images were taken at 63x magnification.

Figure 6.

Immunohistochemical analysis of infiltrating CD4 or CD8 cells by immunohistochemistry and their quantification. (A &Supplementary Figure S3a)–Tumor tissue from all treatment groups were fixed in PFA and stained with anti-CD4 antibody, (B &Supplementary Figure S3b)– anti-CD8 antibody, and (C &Supplementary Figure S3c)– anti-PD1 antibody according to vendors protocol. The images were taken at 63x magnification.