Adenoviral oncolytic capacity is defined by tumor cell-stroma interaction

M. VERÓNICA LOPEZ; DIEGO VIALE; EDUARDO G. CAFFERATA; DAVID GOULD; YUTI CHERNAJOVSKY; OSVALDO L. PODHAJCER

boston

Congreso; 11th Annual Meeting of the Society for Gene Therapy; 2008

<!-- /* Font Definitions */ @font-face {font-family:Batang; panose-1:2 3 6 0 0 1 1 1 1 1; mso-font-alt:"Arial Unicode MS"; mso-font-charset:129; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 151388160 16 0 524288 0;} @font-face {font-family:"@Batang"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:129; mso-generic-font-family:auto; mso-font-format:other; mso-font-pitch:fixed; mso-font-signature:1 151388160 16 0 524288 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-fareast-language:ES;} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells. Tumor-associated endothelial cells and fibroblasts are not merely a scaffold but are actively engaged in tumor growth. Therefore, we designed two novel CRAds to target both the malignant and stromal cell compartments. We selected SPARC promoter since SPARC is overexpressed in malignant and tumor associated-stromal cells in human melanoma and other cancers. By using luciferase expression as a reporter gene, we performed a detailed analysis of the activity and specificity of different fragments of the SPARC promoter. A promoter sequence extending from –513 bp to +35 bp named F512 showed the best ratio of activity vs. specificity in human melanoma cells compared to non-melanoma malignant and normal cells. We constructed a CRAd in which the E1A expression was driven by F512 promoter (Ad-F512) and a second CRAd, Ad(I)-F512-TK that was additionally armed with the Herpes Simplex thymidine kinase gene. Both CRAds were cytotoxic on a panel of melanoma cells but were unable to replicate in normal cells from different sources (melanocytes, keratinocytes, mammary and colon). CRAds exhibited also a certain cytotoxic effect on transformed-human microendothelial cells HMEC-1 and fetal fibroblasts, but had no lytic effect on adult fibroblasts. Ad(I)-F512-TK combined with GCV significantly enhanced the in vitro cytotoxic capacity of the CRAd both in melanoma and in HMEC-1 cells that express SPARC. Ad-F512 treatment of nude mice carrying established human melanomas inhibited tumor growth in 7 out of 9 mice compared to none in the control group treated with Ad-β-gal. However, Ad-F512 was much less effective on established tumors composed of a mix of melanoma and human stromal cells expressing SPARC (endothelial cells or fibroblasts) demonstrating that the presence of stromal cells imposed a restriction on viral activity. Interestingly, Ad(I)-F512-TK combined with GCV exhibited better efficacy than Ad-F512 on mixed tumors suggesting that adding TK/GCV can improve viral lytic activity. Remarkably, treatment of pancreatic tumors made of a mix of MIA PaCa-2 cells and HMEC-1 cells inhibited tumor growth in 5 out of 6 mice, demonstrating that in contrast to what occurred with melanoma tumors, the presence of stromal cells improved viral therapeutic efficacy on pancreatic tumors. Preliminary evidence indicate that conditioned media obtained from stromal cells enhanced F512 activity in pancreatic cancer cells, while reduced F512 activity in melanoma cells. This data suggest that stromal cells can play a key role in defining the therapeutic efficacy of a CRAd. Moreover, based on the paradigm of targeting malignant and stromal cells we propose that this novel CRAd might be considered as a potential therapeutic tool for different cancer types.