Neoadjuvant in situ gene-mediated cytotoxic immunotherapy improves postoperative outcomes in novel syngeneic esophageal carcinoma models

Abstract

Esophageal carcinoma is the most rapidly increasing tumor in the United States and has a dismal 15% 5-year survival. Immunotherapy has been proposed to improve patient outcomes; however, no immunocompetent esophageal carcinoma model exists to date to test this approach. We developed two mouse models of esophageal cancer by inoculating immunocompetent mice with syngeneic esophageal cell lines transformed by cyclin-D1 or mutant HRAS(G12V) and loss of p53. Similar to humans, surgery and adjuvant chemotherapy (cisplatin and 5-fluorouracil) demonstrated limited efficacy. Gene-mediated cyototoxic immunotherapy (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir; AdV-tk/GCV) demonstrated high levels of in vitro transduction and efficacy. Using in vivo syngeneic esophageal carcinoma models, combining surgery, chemotherapy and AdV-tk/GCV improved survival (P=0.007) and decreased disease recurrence (P<0.001). Mechanistic studies suggested that AdV-tk/GCV mediated a direct cytotoxic effect and an increased intra-tumoral trafficking of CD8 T cells (8.15% vs 14.89%, P=0.02). These data provide the first preclinical evidence that augmenting standard of care with immunotherapy may improve outcomes in the management of esophageal carcinoma.

Figure 1

Cisplatin (Cis) and 5-fluorouracil (5-FU) in the murine esophageal carcinoma model, AKR. (a) Cis and 5-FU in AKR (no surgery). Cis/5FU was administered at days 13 and 19 in mice (n=9) bearing AKR flank tumors. Control mice (n=8) were administered normal saline at days 13 and 19 as a control. After chemotherapy protocol completion, tumors resumed normal growth rates and mice required being killed owing to tumor burden. (b) Bar graph demonstrating tumor volumes at day 19. (c) Postoperative Cis and 5-FU in AKR. Mice were administered cis/5-FU following surgery (n=10) on postoperative days 3 and 10. Control mice (n=10) received normal saline injections. As observed in mice not undergoing surgery, chemotherapy administration decreased tumor volume, but did not result in complete tumor eradication. All mice had recurrences, and ultimately were killed owing to excessive tumor burden. (d) Demonstration of flank tumor volumes at postoperative day 10. ^*P<0.05; ^**P<0.01; arrowheads signify time of chemotherapy administration.

Figure 2

In vitro transduction and efficacy of type 5 adenovirus (Ad.luciferase) and AdV-tk/GCV (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir) therapy for esophageal cancer models. (a) In vitro transduction of Ad.luciferase for esophageal cancer models. To determine in vitro transduction efficiency of human esophageal carcinomas (H-Eso) SEG-1, TE-1; murine esophageal carcinomas (M-Eso) AKR, HNM007; and A549 (human NSCLC line used as positive control) were transduced with Ad.luciferase at varying concentrations. Transduction efficiency was proportional to relative light units. SEG-1, TE-1 and HNM007 demonstrated high levels of transduction consistent with A549, whereas AKR was 10- to 100-fold more resistant to transduction. Next, in vitro efficacy of AdV-tk/GCV was investigated in the murine models of esophageal carcinoma (b) AKR and (c) HNM007. Cells were transduced at 1 × 10⁶ viral particles per ml with AdV-tk or Ad.LacZ (control), and then treated with varying concentrations of the prodrug, GCV. The half-maximal inhibitory concentration for AKR cells was 200 μ and for HNM007 cells was 20 μ.

Figure 3

AdV-tk/GCV (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir) treatment in murine model of esophageal cancer, AKR. (a) AdV-tk/GCV in AKR (no surgery). AdV-tk/GCV (n=8) or Ad.LacZ/GCV (control) (n=8) was administered intratumorally at day 13, and volume was recorded. AdV-tk/GCV provided a temporary, but significant reduction in tumor volume. (b) At day 31 after tumor cell inoculation, tumors treated with AdV-tk/GCV were less than 50% of the size of controls. The bystander effect. (c) AKR cells were infected with AdV-tk in vitro and mixed with uninfected AKR cells in varying ratios, and then injected into the flanks of NSG mice (n=6 for each group). At day 6 following inoculation, GCV was administered for 7 days. Mice bearing tumors originating from AdV-tk-infected cells grew slower in the presence of GCV. (d) Tumor volumes demonstrating a strong bystander effect at day 20. AdV-tk works in a CD8 T-cell-dependent manner. (e) C57Bl/6 mice bearing flank tumors were randomized into four groups: (1) Ad.LacZ/GCV treatment (control), (2) Ad.LacZ/GCV with CD8 T-cell depletion, (3) AdV-tk/GCV, or (4) AdV-tk/GCV with CD8 T-cell depletion. Effects of AdV-tk were eliminated in the absence of CD8 T cells. (f) Chart displaying the tumor volumes at day 26 following treatments evaluating AdV-tk/GCV dependence on CD8 T cells. ^*P<0.05; ^**P<0.01; shaded bar designates time over which AdV-tk/GCV therapy took place.

Figure 4

Neoadjuvant AdV-tk/GCV (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir) for AKR. (a) AdV-tk/GCV (n=8) or Ad.LacZ/GCV (control) (n=7) was started on at day 10. Suboptimal surgery was performed on day 17, and postoperative local recurrent volumes were monitored. As in non-surgical models, administration of AdV-tk/GCV resulted in decreased tumor volumes at postoperative periods; however, all tumors recurred and ultimately grew to a point requiring animals to be killed. (b) Bar graph demonstrating tumor volumes at postoperative day 14. ^*P<0.05; ^**P<0.01; shaded bar designates time over which AdV-tk/GCV therapy took place.

Figure 5

Combination of neoadjuvant AdV-tk/GCV (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir) with postoperative cisplatin (cis)/5-fluorouracil (5-FU) is effective in AKR. (a) Flank tumor growth with combination adjuvant therapy. AdV-tk/GCV or Ad.LacZ/GCV (control) was begun at day 10. Surgery was completed on day 17, followed by cis/5-FU administration (days 20 and 27). Seven mice were randomized to each of the four treatment protocols. Postoperative volumes were recorded, and significant decreases were observed in mice randomized to treatment arms, with combination therapy providing the most significant reduction at day 31 (postoperative day 14); P=0.007. (b) Bar graph further describing dramatic reduction in recurrent tumor burden following protocols incorporating AdV-tk/GCV with postoperative cis/5-FU. (c) Kaplan–Meier analysis with log-rank comparison demonstrating postoperative survival benefits associated with adjuvant combination therapy administration. ^*P<0.03; ^**P<0.01; shaded bar designates time over which AdV-tk/GCV therapy took place; arrowheads designate timing of cis/5-FU.

Figure 6

Neoadjuvant AdV-tk/GCV (adenoviral vector carrying the herpes simplex virus thymidine kinase gene in combination with the prodrug ganciclovir) with postoperative cis/5-FU increases CD8 T-cell lymphocyte infiltration and systemic populations more effectively than Ad.LacZ/GCV with cis/5-FU. (a) Representative immunohistochemistry staining for CD8 T-cell trafficking (tissue was obtained from tumors of two different mice per group). Flank tumors were harvested, sectioned and stained for CD8 T cells following the first dose of chemotherapy. Tumor immunohistochemical staining revealed increased CD8 cells in mice receiving combination AdV-tk/GCV with cis/5-FU vs Ad.LacZ/GCV with cis/5-FU (13.6 vs 4.2 cells per high power field (HPF); P=0.009). (b) Representative fluorescence-activated cell sorter (FACS) tracings for intratumoral CD8 T cells (three representative samples per group). Tumors infiltrates were analyzed by flow cytometry following the administration of the first dose of chemotherapy. Flow cytometry tracing revealed an increased percentage of intratumoral leukocytes composed of CD8 T cells in those mice receiving AdV-tk/GCV with cis/5-FU vs Ad.LacZ/GCV with cis/5-FU (P=0.02). (c) Winn assay results reveal that combination of AdV-tk/GCV with chemotherapy functions in a CD8 T-cell-dependent manner. CD8 T cells from mice receiving combination treatments were superior in neutralizing fresh tumor cells in vivo when compared to those CD8 T cells harvested from mice receiving postoperative chemotherapy and surgery alone, and tumor cells alone (n=5 per group). ^*P<0.05; ^**P<0.01; shaded bar designates time over which AdV-tk/GCV therapy took place.