Soluble Factors Produced by Stromal Cells Greatly Influence Adenoviral Oncolytic Efficacy

MARIA VERÓNICA LOPEZ; DIEGO VIALE; EDUARDO G. CAFFERATA; OSVALDO L. PODHAJCER

San Diego Convention Center in San Diego, CA, USA

Congreso; 12th Annual Meeting of the American Society of Gene; 2009

American Society of Gene Therapy

Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter (F512Pr )-based CRAd (Ad(I)-F512-TK) since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. Next, we demonstrated that Ad(I)-F512-TK combined with GCV exhibited moderate efficacy on mixed melanoma tumors (Mel/endothelium or Mel/fibroblast). Remarkably, treatment of pancreatic tumors (Pan/endothelium) inhibited tumor growth in 5 out of 6 mice, demonstrating that in contrast to what occurred with melanoma tumors. The striking differences in CRAd activity observed between melanoma and pancreatic cancer when microendothelial cells were co-administered with malignant cells and the evidence that microendothelial cells and fibroblasts hampered CRAd activity irrespective of tumor architecture, led us to hypothesize that soluble factors might have been playing a role. Fibroblast- and endothelium-conditioned media induced a slight inhibition of F512Pr activity in SB2 melanoma cells. In clear contrast, both conditioned media strongly enhanced F512Pr activity in pancreatic MIA PaCa-2 cancer cells. In addition, fibroblast-conditioned media enhanced viral lytic activity on SB2 cells and other melanoma cells as well and both conditioned media enhanced at a different extent CRAd activity on MIA PaCa-2 cells. Surprisingly, preinfection of stromal cells with the CRAd completely obliterated the enhancement in CRAd lytic activity induced by the conditioned media on SB2 melanoma cells. But in clear contrast, conditioned media obtained from pre-infected stromal cells dramatically enhanced CRAd lytic activity on MIA PaCa-2 cells. Previous evidence demonstrated that oncolytic viruses increased the amount of cells in S-phase and that E1B mutant viruses, such as the present CRAds, exhibited better cytopathic effect on cells in S phase. To dissect CRAd’s effects, we first assessed whether arrested malignant cells infected with the CRAd showed an accelerated exit from quiescence compared to non-infected cells. Indeed, infected MIA PaCa-2 cells exhibited a higher cell number in S-phase at 20 hr compared to non-infected cells. Surprisingly, SB2 melanoma cells exhibited a retarded exit from G0/G1 compared to MIA PaCa-2 cells, regardless of whether SB2 cells were infected or not with the CRAd. Next, we assessed whether stromal cells-conditioned media might also accelerate MIA PaCa-2 cells exit from quiescence. Twenty four hours after MIA PaCa-2 cells release from G0/G1, we observed a clear increase in the amount of cells in S-phase when they were exposed to conditioned media obtained from fibroblast cells pre-infected with the CRAd, compared to MIA PaCa-2-own conditioned media obtained from cells infected also with the CRAd or the control media. This data suggest that soluble factors produced by stromal cells can play a dramatic and differential role in defining the therapeutic efficacy of a CRAd on specific tumor types.