Investigation of an oncolytic herpes simplex virus as a potential therapeutic agent for gastroenteropancreatic neuroendocrine neoplasms

Abstract

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) present unique challenges due to their heterogeneity and limited treatment options. Oncolytic virotherapy has emerged as a promising therapeutic for other NETs and thus, we sought to investigate the potential of an engineered oncolytic herpes simplex virus (oHSV), M002, for GEP-NETS. We employed an established long-term passage GEP-NET cell line and a unique, human pediatric patient-derived xenograft GEP-NET line. We found the virus to effectively infect, replicate within, and kill both cell lines in vitro. Similar effects were noted in vivo, with M002 decreasing tumor growth and improving overall survival in mice bearing tumors from both the established cell line and human GEP-NET PDX. Overall, these studies provide an evaluation of an oncolytic HSV in GEP-NETs, highlighting its therapeutic potential and considerations for clinical translation.

Keywords: Neuroendocrine tumors; Oncolytic virus; Patient derived xenografts; Translational research.

Fig. 1

QGP-1 and COA109 cells express HSV entry receptors. (A) The cell surface expression of viral entry receptors for M002 (CD111, CD112, syndecan, and HVEM) was assessed utilizing flow cytometry. QGP-1 (10⁶) or COA109 (10⁶) cells were cultured overnight in 6-well plates, collected, stained with fluorescent conjugated antibody, and evaluated with flow cytometry. Cells treated with FcR blocker alone served as negative controls. Receptors were expressed in variable quantities, but all four receptors were present on both cell types. (B) Representative histograms of flow cytometry for viral entry receptors in QGP-1 cells. Data represent three biologic replicates and are reported as mean ± standard error of the mean (SEM). *p ≤ 0.05, **p ≤ 0.01, COA109 vs QGP-1.

Fig. 2

M002 infects QGP-1 and COA109 cells and effectively replicates. (A,B) Single step viral recovery experiments assessed a single growth cycle of the virus. QGP-1 (3 × 10⁵) (A) or COA109 (3 × 10⁵) (B) cells were infected at a multiplicity of infection (MOI) of 10 PFU/cell for 2 h, resuspended in culture media, and harvested at 12 and 24 h post-infection. Titer of viral progeny was determined using plaque assays with Vero cells. By 24 h, viral progeny increased 2 log in the QGP-1 cells (A) and 11 log in the COA109 cells (B). (C,D) Multi-step viral recovery experiments were performed to assess multiple growth cycles of the virus. For multi-step experiments, QGP-1 (3 × 10⁵) (C) or COA109 (3 × 10⁵) (D) cells were infected a MOI of 1 PFU/cell for 2 h. Following infection, cells were centrifuged and replated. At 6, 12, 24, 48, or 72 h post-infection the cells and media were harvested, and titer of viral progeny were determined on monolayers of Vero cells. M002 successfully replicated in both cell lines leading to multi-log increase in viral progeny over initial infection MOI of 1 PFU/cell. Mean virion yields were determined in four replicates at each time point and standard error of the mean was determined. Data represent three biologic replicates. (E,F) The oncolytic virus, M002, has been genetically engineered to produce murine (m)IL-12. As corroboration that M002 was able to infect and replicate in the tumor cells, ELISA was used to measure mIL-12 after viral infection. QGP-1 (E) or COA109 (F) cells (1.5 × 10⁴) were plated in 96-well plates and allowed to attach. Cells were treated with M002 at a MOI of 0.1 or 1.0 PFU/cell for 24 and 72 h, collected, and flash frozen. There was a significant increase in mIL-12 concentration in both cell lines at both time points and doses compared to no virus. Data represent three biologic replicates and are reported as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 3

M002 treatment reduces viability in QGP-1 and COA109 cells. Cell viability was measured using colorimetric assay. (A) QGP-1 cells (1.5 × 10⁴) were plated in 96 well plates and treated with M002 at increasing MOI (0, 0.01, 0.05, 0.1, 0.5, 1, 5 PFU/cell) for 72 h. Treatment with M002 significantly decreased cell viability. (B) COA109 cells (1.5 × 10⁴) were plated in 96 well plates and treated with M002 at increasing MOI (0, 0.001, 0.01, 0.1, 1, 5 PFU/cell) for 72 h. Treatment with M002 significantly decreased cell viability. Data represent three biologic replicates. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 4

M002 treatment reduces tumor growth and improves animal survival in a QGP-1 model. (A) QGP-1 cells (3 × 10⁶ in 20% Matrigel) were implanted subcutaneously in the right flank of 6-week-old female athymic nude mice (n = 7 per group). Once tumors were palpable (100 mm³), animals were randomized to receive an intratumoral injection of either M002 (1 × 10⁷ PFU/50 μL) or an equal volume of PBS (50 μL, vehicle). Flank tumors were measured three times per week and tumor volumes calculated. Tumor growth was significantly decreased in animals treated with M002. (B) Animals treated with M002 had significantly improved survival compared to vehicle treated controls (median 30 vs 23 days, M002 vs vehicle, p = 0.003). Kaplan–Meier curves with log-rank statistics demonstrate data. (C) Immunohistochemistry detected M002 virus in tumor specimens with cells infected by virus staining positive (black arrows). Inset demonstrates negative IgG control. Scale bars represent 100 μm. *p ≤ 0.05, **p ≤ 0.01.

Fig. 5

M002 treatment reduces tumor growth and improves animal survival in a PDX model. (A) COA109 cells (5 × 10⁶ in 20% Matrigel) were implanted subcutaneously in the right flank of 6-week-old female athymic nude mice. Once tumors were palpable (100 mm³), animals were randomized to receive an intratumoral injection of either M002 (1 × 10⁷ PFU/50 μL, n = 6) or an equal volume of PBS (50 μL, vehicle, n = 5). Flank tumors were measured three times per week and tumor volumes calculated. Tumor growth was significantly decreased in animals treated with M002. (B) Animals treated with M002 had significantly improved survival compared to vehicle treated controls (median 24 vs 10 days, M002 vs vehicle, p = 0.018). Kaplan–Meier curves with log-rank statistics demonstrate data. C. Immunohistochemistry detected M002 virus in tumor specimens. Arrows indicate positive staining. Inset demonstrates negative IgG control. Scale bars represent 100 μm. *p ≤ 0.05, **p ≤ 0.01.