Oncolytic Herpes Simplex Virus Prevents Premalignant Lesions from Progressing to Cancer

Abstract

Early detection and timely treatment of precancerous lesions are hallmarks of successful strategies to prevent deaths due to cancer. Oncolytic viruses are a group of promising anti-cancer agents with wide-ranging experimental and clinical efficacy against solid tumors. Previously, we have shown that NV1066, an oncolytic herpes simplex-1 virus encoding enhanced green fluorescent protein, selectively infects, replicates in, and kills various cancer types. In this study, we sought to determine whether this oncolytic agent can treat precancerous lesions to prevent cancer formation. Using an oral chemical carcinogenesis model in hamsters, we assessed the ability of NV1066 to infect precancerous and cancerous lesions. NV1066 consistently infected dysplastic cells, carcinoma in situ, and squamous cell carcinoma. Animals receiving an intramucosal injection of NV1066 for 7 weeks showed significantly fewer (3-fold) and smaller (4-fold) lesions compared to animals that did not receive viral treatment. Results indicate that infectivity might be dependent on the herpes simplex virus 1 receptor, nectin-1. This study demonstrates that not only can NV1066 treat oral squamous cell carcinoma, but it can also infect and treat premalignant lesions, thus delaying cancer progression. Overall, our study shows the potential of the oncolytic virus NV1066 as a cancer prevention tool.

Keywords: cancer prevention; carcinogenesis; early cancer diagnosis; herpes simplex virus; oncolytic virotherapy; oral squamous carcinoma; viral tropism.

Figure 1

Stereoscopic Fluorescence Microscopy of Oral Lesions Helps Identify Areas of EGFP Expression (A) Schema representing a timeline of infection experiments. (B–E) Normal mucosa. (B) Bright-field stereoscope image of a normal cheek pouch that received NV1066. (C) The corresponding fluorescence stereoscope examination demonstrating absence of EGFP expression. (D) H&E staining of the tissue confirmed normal histology. (E) IHC for HSV-1 demonstrates absence of the virus in the lesion. (F and G) Lesion with dysplasia: a gross photograph of a polyp indicated by a black arrow (F), and area of corresponding EGFP expression under the fluorescent filter (G). (H and I ) H&E staining for a section of the polyp showing marked dysplasia (H), which corresponds to positive HSV-1 staining (I). (H) and (I) represent consecutive sections. (J–M) A heterogeneous cancerous lesion developed after DMBA treatment, shown with four white arrows (J), indicating extensive EGFP expression (K) corresponding to dysplasia as observed by H&E staining (L) and positive HSV-1 IHC staining (M). (L) and (M) represent consecutive sections. Size bars represent 16 μm. DMBA, 7,12-dimethylbenz[a]anthracene; EGFP, enhanced green fluorescent protein; H&E, hematoxylin and eosin; HSV-1, herpes virus 1; IHC, immunohistochemistry.

Figure 2

NV1066 Treatment Reduces the Number and Size of DMBA-Induced Lesions (A) Schema representing the treatment strategy for prevention. (B) Multiple large lesions were visible by gross examination on cheek pouch of PBS-treated group (lesions >5 mm were counted, as indicated by black circles). (C) Significantly fewer lesions >5 mm were present in the NV1066-treated group; (D and E) bar graphs show significantly decreased average nodule count (D) and average volume (E) in the NV1066-treated group compared to the PBS-treated group. A total of 123 lesions in 20 cheek pouches of the PBS-treated group were present as compared to 82 lesions in 36 check pouches in the NV1066-treated group. DMBA, 7,12-dimethylbenz[a]anthracene. The number of lesions was counted, and tumor volume was measured in each cheek pouch, and a paired t test was used to compare the two groups. A p value of 0.05 or less was considered statistically significant.

Figure 3

Changes in the Staining Pattern of the Adherens Junction Protein Nectin-1 Accompany Dysplasia (A–D) IHC for nectin-1 demonstrates the presence of free nectin-1 in dysplastic (A and B), but not in normal (C and D) mucosal cells. (A and C) are high magnifications of (B and D), respectively. Size bars represent 16 μm for all. H&E, hematoxylin and eosin staining; IHC, immunohistochemistry.