Comparison of the oncolytic activity of a replication-competent and a replication-deficient herpes simplex virus 1.

In 2015, the oncolytic herpes simplex virus 1 (HSV-1) T-VEC (talimogene laherparepvec) was approved for intratumoral injection in non-resectable malignant melanoma. To determine whether viral replication is required for oncolytic activity, we compared replication-deficient HSV-1 d106S with replication-competent T-VEC. High infectious doses of HSV-1 d106S killed melanoma (n = 10), head-and-neck squamous cell carcinoma (n = 11), and chondrosarcoma cell lines (n = 2) significantly faster than T-VEC as measured by MTT metabolic activity, while low doses of T-VEC were more effective over time. HSV-1 d106S and, to a lesser extent T-VEC, triggered caspase-dependent early apoptosis as shown by pan-caspase inhibition and specific induction of caspases 3/7, 8, and 9. HSV-1 d106S induced a higher ratio of apoptosis-inducing infected cell protein (ICP) 0 to apoptosis-blocking ICP6 than T-VEC. T-VEC was oncolytic for an extended period of time as viral replication continued, which could be partially blocked by the antiviral drug aciclovir. High doses of T-VEC, but not HSV-1 d106S, increased interferon-ß mRNA as part of the intrinsic immune response. When markers of immunogenic cell death were assessed, ATP was released more efficiently in the context of T-VEC than HSV-1 d106S infection, whereas HMGB1 was induced comparatively well. Overall, the early oncolytic effect on three different tumour entities was stronger with the non-replicative strain, while the replication-competent virus elicited a stronger innate immune response and more pronounced immunogenic cell death.

Nectin-1 Expression Correlates with the Susceptibility of Malignant Melanoma to Oncolytic Herpes Simplex Virus In Vitro and In Vivo.

Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus, is approved for intralesional injection of unresectable stage IIIB/IVM1a melanoma. However, it is still unclear which parameter(s) predict treatment response or failure. Our study aimed at characterizing surface receptors Nectin-1 and the herpes virus entry mediator (HVEM) in addition to intracellular molecules cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) as potential bio-markers for oncolytic virus treatment. In 20 melanoma cell lines, oncolytic activity of T-VEC was correlated with the expression of Nectin-1 but not HVEM, as evaluated via flow cytometry and immunohistochemistry. Knockout using CRISPR/Cas9 technology confirmed the superior role of Nectin-1 over HVEM for entry and oncolytic activity of T-VEC. Neither cGAS nor STING as evaluated by Western Blot and immunohistochemistry correlated with T-VEC induced oncolysis. The role of these biomarkers was retrospectively analyzed for the response of 35 cutaneous melanoma metastases of 21 patients to intralesional T-VEC injection, with 21 (60.0%) of these lesions responding with complete (n = 16) or partial regression (n = 5). Nectin-1 expression in pretreatment biopsies significantly predicted treatment outcome, while the expression of HVEM, cGAS, and STING was not prognostic. Altogether, Nectin-1 served as biomarker for T-VEC-induced melanoma regression in vitro and in vivo.

SOX9 Knockout Induces Polyploidy and Changes Sensitivity to Tumor Treatment Strategies in a Chondrosarcoma Cell Line.

As most chemotherapeutic drugs are ineffective in the treatment of chondrosarcoma, we studied the expression pattern and function of SOX9, the master transcription factor for chondrogenesis, in chondrosarcoma, to understand the basic molecular principles needed for engineering new targeted therapies. Our study shows an increase in SOX9 expression in chondrosarcoma compared to normal cartilage, but a decrease when the tumors are finally defined as dedifferentiated chondrosarcoma (DDCS). In DDCS, SOX9 is almost completely absent in the non-chondroid, dedifferentiated compartments. CRISPR/Cas9-mediated knockout of SOX9 in a human chondrosarcoma cell line (HTB94) results in reduced proliferation, clonogenicity and migration, accompanied by an inability to activate MMP13. In contrast, adhesion, apoptosis and polyploidy formation are favored after SOX9 deletion, probably involving BCL2 and survivin. The siRNA-mediated SOX9 knockdown partially confirmed these results, suggesting the need for a certain SOX9 threshold for particular cancer-related events. To increase the efficacy of chondrosarcoma therapies, potential therapeutic approaches were analyzed in SOX9 knockout cells. Here, we found an increased impact of doxorubicin, but a reduced sensitivity for oncolytic virus treatment. Our observations present novel insight into the role of SOX9 in chondrosarcoma biology and could thereby help to overcome the obstacle of drug resistance and limited therapy options.

Prospect of Plasmacytoid Dendritic Cells in Enhancing Anti-Tumor Immunity of Oncolytic Herpes Viruses.

The major type I interferon-producing plasmacytoid dendritic cells (pDC) surround and infiltrate certain tumors like malignant melanoma, head and neck cancer, and ovarian and breast cancer. The presence of pDC in these tumors is associated with an unfavorable prognosis for the patients as long as these cells are unstimulated. Upon activation by synthetic Toll-like receptor agonists or viruses, however, pDC develop cytotoxic activities. Viruses have the additional advantage to augment cytotoxic activities of pDC via lytic replication in malignant lesions. These effects turn cold tumors into hotspots, recruiting further immune cells to the site of inflammation. Activated pDC contribute to cross-presentation of tumor-associated antigens by classical dendritic cells, which induce cytotoxic T-cells in particular in the presence of checkpoint inhibitors. The modification of oncolytic herpes viruses via genetic engineering favorably affects this process through the enhanced production of pro-inflammatory cytokines, curbing of tumor blood supply, and removal of extracellular barriers for efficient viral spread. Importantly, viral vectors may contribute to stimulation of memory-type adaptive immune responses through presentation of tumor-related neo- and/or self-antigens. Eventually, both replication-competent and replication-deficient herpes simplex virus 1 (HSV-1) may serve as vaccine vectors, which contribute to tumor regression by the stimulation of pDC and other dendritic cells in adjuvant and neo-adjuvant situations.