306. Imaging HSV-1 Viral Oncolysis of Colon Cancer Cells by microPET
Authors: Darshini Kuruppu, Aijun Zhu, Meixiang Yu, Anna-Liisa Brownell, Kenneth K. Tanabe
Journal: Molecular Therapy (2006)

Background: The destruction of cancer cells by replicating viruses (viral oncolysis) is evolving as a potentially effective treatment for cancer. Herpes simplex virus-1 (HSV-1) mutant hrR3 has its genome altered by insertion of the b-galactosidase gene into the UL39 locus to generate a replication conditional oncolytic mutant. This mutant directs its cytotoxicity towards cancer cells by replicating preferentially in cancer cells rather than in normal cells. Oncolysis of colon cancer by hrR3 was studied by microPET using the HSV-1 thymidine kinase (HSV-TK) as a reporter gene and [18F]FHBG as a reporter probe. Phosphorylated [18F]FHBG co-localizes with HSV- TK gene expression and can be imaged by microPET. We report imaging of hrR3 viral oncolysis in murine tumor by microPET.Methods: Bilateral flank tumors were established in balb/c mice by direct injection of MC26 murine colon cancer cells. hrR3 (1×109 PFU) was injected into the right flank tumor and one week later imaged by microPET using [18F]FHBG at 2, 6, 24, 48, and 72 hours after virus injection. Tumor destruction by viral oncolysis was also studied by microPET imaging for proliferating cells with [18F]FLT. The left flank tumor was left untreated and served as a control. Tumors were excised at the specified time points after virus injection to study the molecular changes resulting from viral oncolysis. Serial tumor sections were prepared for histological analysis by H&E, immunohistochemical staining of HSV-TK and PCNA, and TUNEL labelling for apoptosis. Frozen sections were stained for b-galactosidase expression. Protein lysates prepared from tumors were assayed for HSV-TK protein expression by Western blot.Results: Radiolabelled FHBG localization in tumors is observed 2 hours after virus infection. The intensity of the microPET image peaks at 6 hours and diminishes thereafter to a nearly undetectable intensity at 72 hours. HSV-TK and b-galactosidase expression in these tumors increase over the study duration to 72 hours. However, this expression is observed to occur together with tumor cell lysis characterized by cellular degradation, necrosis and apoptosis, all of which are observed to increase over time. In concordance with the tumor cell destruction observed in harvested tumors, in vivo [18F]FLT imaging with microPET showed that cell viability decreases over these time points as a result of viral oncolysis.Conclusion: MicroPET imaging of [18F]FHBG permits detection of hrR3 infection of colon carcinoma cells during viral oncolysis. This observation has direct clinical applicability in current clinical trials of HSV-1 oncolysis. However, intracellular retention of HSV-TK-phosphorylated [18F]FHBG appears to be limited by cell lysis resulting from lytic replication (viral oncolysis), which permits diffusion of the radiolabel away from the focus of viral replication. Accordingly, this system may not be ideal for imaging later stages of viral oncolysis. Background: The destruction of cancer cells by replicating viruses (viral oncolysis) is evolving as a potentially effective treatment for cancer. Herpes simplex virus-1 (HSV-1) mutant hrR3 has its genome altered by insertion of the b-galactosidase gene into the UL39 locus to generate a replication conditional oncolytic mutant. This mutant directs its cytotoxicity towards cancer cells by replicating preferentially in cancer cells rather than in normal cells. Oncolysis of colon cancer by hrR3 was studied by microPET using the HSV-1 thymidine kinase (HSV-TK) as a reporter gene and [18F]FHBG as a reporter probe. Phosphorylated [18F]FHBG co-localizes with HSV- TK gene expression and can be imaged by microPET. We report imaging of hrR3 viral oncolysis in murine tumor by microPET. Methods: Bilateral flank tumors were established in balb/c mice by direct injection of MC26 murine colon cancer cells. hrR3 (1×109 PFU) was injected into the right flank tumor and one week later imaged by microPET using [18F]FHBG at 2, 6, 24, 48, and 72 hours after virus injection. Tumor destruction by viral oncolysis was also studied by microPET imaging for proliferating cells with [18F]FLT. The left flank tumor was left untreated and served as a control. Tumors were excised at the specified time points after virus injection to study the molecular changes resulting from viral oncolysis. Serial tumor sections were prepared for histological analysis by H&E, immunohistochemical staining of HSV-TK and PCNA, and TUNEL labelling for apoptosis. Frozen sections were stained for b-galactosidase expression. Protein lysates prepared from tumors were assayed for HSV-TK protein expression by Western blot. Results: Radiolabelled FHBG localization in tumors is observed 2 hours after virus infection. The intensity of the microPET image peaks at 6 hours and diminishes thereafter to a nearly undetectable intensity at 72 hours. HSV-TK and b-galactosidase expression in these tumors increase over the study duration to 72 hours. However, this expression is observed to occur together with tumor cell lysis characterized by cellular degradation, necrosis and apoptosis, all of which are observed to increase over time. In concordance with the tumor cell destruction observed in harvested tumors, in vivo [18F]FLT imaging with microPET showed that cell viability decreases over these time points as a result of viral oncolysis. Conclusion: MicroPET imaging of [18F]FHBG permits detection of hrR3 infection of colon carcinoma cells during viral oncolysis. This observation has direct clinical applicability in current clinical trials of HSV-1 oncolysis. However, intracellular retention of HSV-TK-phosphorylated [18F]FHBG appears to be limited by cell lysis resulting from lytic replication (viral oncolysis), which permits diffusion of the radiolabel away from the focus of viral replication. Accordingly, this system may not be ideal for imaging later stages of viral oncolysis.