Oncolytic Herpesvirus Effectively Treats Murine Squamous Cell Carcinoma and Spreads by Natural Lymphatics to Treat Sites of Lymphatic Metastases

Abstract

Oncolytic herpesviruses have significant antitumoral effects in animal models when delivered directly to established tumors. Lymphatic metastases are a common occurrence for many tumor types. This study investigates the potential of an attenuated, replication-competent, oncolytic herpes simplex virus (NV1023) both to treat a primary tumor by direct injection and to travel through the lymphatic system to treat metastatic tumor within the lymph nodes draining lymph from the site of primary cancer. Isosulfan blue dye was injected into murine auricles to determine normal lymphatic drainage patterns and demonstrated consistent blue staining of a group of ipsilateral cervical lymph nodes. Auricular injections of NV1023 resulted in viral transit to these lymph nodes as measured by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry and viral plaque assay. An oncolytic herpesvirus (NV1066) expressing green fluorescent protein also demonstrated viral transit from the auricle to the cervical lymph nodes on fluorescence microscopy. Using the SCC VII cell line, a novel murine model of auricular squamous cell carcinoma was developed with an approximately 20% incidence of cervical lymph node metastases. Delivery of NV1023 or NV1066 to the surgical beds after excision of auricular SCC VII tumors resulted in successful viral infection of metastatic SCC VII cells within the cervical lymph nodes. After a 7-week follow-up, significantly enhanced locoregional control (p < 0.05, Fisher exact test) and disease-free survival (p < 0.05, log rank test) were evident with NV1023 treatment. This study demonstrates that the delivery of an oncolytic herpesvirus to a primary tumor site after surgical excision may have a significant impact on reducing both primary site recurrence and regional nodal metastases.

Widespread intratumoral virus distribution with fractionated injection enables local control of large human rhabdomyosarcoma xenografts by oncolytic herpes simplex viruses

“…16 In order to better track virus infection following intratumoral injection, we tested the sensitivity of Rh18A cells to infection by NV1066, a mutant similar to NV1020 that expresses the enhanced green fluorescent protein (eGFP). 21 The cytotoxicty dose-response curves in Rh18A cells for each virus were essentially identical with a lethal concentration for 50% of the cells by 6 days after infection of o0.005 plaque forming units (PFU)/cell (Fig 1a). In addition, virus production in Rh18A cells following infection at a multiplicity of infection (MOI) ¼ 1 was also similar for the two viruses reaching levels B1.5 logs above baseline by 48 hours (Fig 1b).…”

“…The fact that oncolytic herpes viruses can spread via lymphatics to regional metastases is also encouraging for their use as a means of locoregional treatment. 21 While virus could be administered directly to a tumor, our study suggests that administration of virus might best be performed intraoperatively, in conjunction with a debulking procedure, to maximize geographic distribution of virus throughout the tumor. Alternatively, if an appropriate tumor vascular supply can be identified, widespread intratumoral distribution of virus might also be achievable through catheter-directed intra-arterial administration.…”

“…16,17 A number of different HSV mutants have been engineered to selectively replicate in tumor cells based on deletions of critical genetic functions that are dispensable for viral replication in cancer cells (for reviews, see Varghese et al 18 and Arceci and Cripe 19 ). For example, NV1020 20 and NV1066 21 are deleted for one copy of the diploid ''neurovirulence'' gene encoding ICP34.5. Viral infection activates the doublestranded RNA inducible protein kinase (PKR) pathway to terminate protein synthesis and prevent viral replication.…”

“…Most studies of oncolytic viruses have been performed on small tumors. We reviewed 16 studies of oncolytic HSVs in non-CNS tumors published between 1998 and 2003, and found that the average tumor size injected in 14 studies was o150 mm 3 , 17,21,[34][35][36][37][38][39][40][41][42][43][44][45] in two studies was 150-250 mm 3 , 46,47 and in no study was 4250 mm 3 . We used a high-risk alveolar rhabomyosarcoma model to determine the effect of oncolytic HSV injections on large human tumor xenografts.…”

Virally-directed fluorescent imaging (VFI) can facilitate endoscopic staging

“…A number of studies from our group and others have determined that these viruses are highly specific for tumor cells while sparing normal cells (13)(14)(15)(16)(17)(18)(19)(20)(21). Oncolytic HSV therapy has shown to be effective against multiple tumor types including lung, esophageal, gastric, colorectal, gallbladder, hepatic, pancreatic, bladder cancer and mesothelioma (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Given the specificity of herpes viral replication for tumor cells, we sought to determine whether NV1066, a HSV-1 mutant virus used in this study can delineate tumor tissue from normal tissue.…”