Combination of LIGHT (TNFSF14)-Armed Myxoma Virus Pre-Loaded into ADSCs and Gemcitabine in the Treatment of Experimental Orthotopic Murine Pancreatic Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a deadly neoplasm. Oncolytic viruses have tumorolytic and immune response-boosting effects and present great potential for PDAC management. We used LIGHT-armed myxoma virus (vMyx-LIGHT) loaded ex vivo into human adipose-derived mesenchymal stem cells (ADSCs) to evaluate murine PDAC treatment in conjunction with gemcitabine (GEM). The cytotoxicity of this treatment was confirmed in vitro using human and murine pancreatic cancer cell cultures, which were more sensitive to the combined approach and largely destroyed. Unlike cancer cells, ADSCs sustain significant viability after infection. The in vivo administration of vMyx-LIGHT-loaded ADSCs and gemcitabine was evaluated using immunocompetent mice with induced orthotopic PDAC lesions. The expression of virus-encoded LIGHT increased the influx of T cells to the tumor site. Shielded virus followed by gemcitabine improved tumor regression and survival. The addition of gemcitabine slightly compromised the adaptive immune response boost obtained with the shielded virus alone, conferring no survival benefit. ADSCs pre-loaded with vMyx-LIGHT allowed the effective transport of the oncolytic construct to PDAC lesions and yielded significant immune response; additional GEM administration failed to improve survival. In view of our results, the delivery of targeted/shielded virus in combination with TGF-β ablation and/or checkpoint inhibitors is a promising option to improve the therapeutic effects of vMyx-LIGHT/ADSCs against PDAC in vivo.

Keywords: adipose tissue-derived stem cells (ADSCs); gemcitabine; immune response; myxoma virus; oncolytic virotherapy; oncolytic virus; pancreatic ductal adenocarcinoma.

Figure 1

Early and late gene expression following infection of various cell lines with LIGHT-encoding myxoma construct. Cultures of ADSCs, Pan02, RK13, AsPC-1 and Panc-1 were infected with vMyx-mLIGHT-FLuc/tdTr (MOI = 5). (a) Constitutive expression of LIGHT gene in infected ADSCs and pancreatic cancer cell lines at 24 h p.i. LIGHT gene transcript was measured using RT-qPCR, and expression was rendered as a ratio of target gene (LIGHT) vs. reference gene (glyceraldehyde 3-phosphate dehydrogenase /GAPDH/). The data show mean ± SD of two independent experiments. (b) Infection visualized at 24 h p.i. by fluorescence microscopy (magn. 20×; scale bar = 50 µm; Zeiss LSM 710 confocal Workstation); blue: DAPI staining (nuclei); red: tdTr fluorescence.

Figure 2

Examination of apoptosis and necrosis in cell lines: RK-13, Pan02, AsPC-1, Panc-1 and ADSCs infected with vMyx-LIGHT (MOI = 5) at 24 and 48 h after infection; flow cytometry, Annexin V (FITC channel) and 7-AAD (PerCP-Cy5.5 channel). The data show mean ± SD of two independent experiments.

Figure 3

Viability of ADSC and pancreatic cancer cells after GEM treatment. (a) Determination of optimized GEM doses. Cell lines: ADSCs, Pan02, Panc-1, AsPC-1 treated with various GEM concentrations for 24, 48 and 72 h and analyzed for cell viability using MTS. (b) Timeline of MYXV and GEM experimental combination. (c) Combination of MYXV infection and GEM. Pancreatic cancer cells (Panc-1, AsPC-1 and Pan02) and ADSC (1 × 10⁴ cells/well) were infected with vMyx-LIGHT at MOI = 5 and/or treated with GEM and then analyzed for cell viability using MTS assay at 24, 48 and 72 h post-GEM treatment. The assays were performed in triplicate; error bars shown are mean ± SD.

Figure 4

Combined therapy of experimental pancreatic adenocarcinoma using armed MYXV construct. C57Bl/6NCrl mice (n = 12) with induced orthotopic lesions were injected ip. (days 4, 6, 8, 10 and 12) with LIGHT-expressing MYXV—either ADSC-shielded (5 × 10⁵ cells/100 µL PBS¯) or unshielded (2.5 × 10⁶ FFU/100 µL PBS¯), and were then injected ip. (days 14, 17 and 20) with GEM (66.5 mg/kg in 100 µL PBS¯). (a) Timeline of experiment; (b) size and (c) weight of pancreata and spleens on days 14 and 21 (n = 3); (d) representative micrographs of H&E-stained sections derived from the indicated treatment groups (scale bars: 50–1000 µm); (e) mouse survival (n = 6): log rank test (Mantel–Cox). The data (mean ± SD) were analyzed with one-way ANOVA; statistically significant differences are indicated (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Figure 5

Antitumor immune response after combined therapy of experimental pancreatic adenocarcinoma using LIGHT-armed MYXV construct and GEM. C57Bl/ C57Bl/6NCrl mice (n = 6) with induced orthotopic lesions were injected ip. (days 4, 6, 8, 10 and 12) with LIGHT-expressing MYXV—either ADSC-shielded or unshielded; then, they were injected ip. (days 14, 17 and 20) with GEM. (a) Flow cytometry data (n = 3) showing CD4+ and CD8+ cell percentage among CD3+ lymphocytes in blood and pancreas on days 14 and (b) 21; (c) analysis of gene expression (RT-qPCR) in pancreata for: TNFα, INFγ, IL-2, IL-15, IL-10, TGF-β, CD4, CD8, PD-L1, PD-1, LIGHT, HVEM and LTβR (day 14) and (d) for: CD4, CD8, PD-L1, PD-1, TGF-β, LIGHT, HVEM and LTβR (day 21). Changes in the gene expression were rendered as a ratio of target gene vs. reference gene (S18) relative to expression in control samples. The data (mean ± SD) were analyzed with one-way ANOVA; statistically significant differences are indicated (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).