Abstract 1490: Matrigel rescues breast cancer cells from the growth inhibitory effects of HIF-1α and HIF-2α silencing

Abstract

Abstract Tumor microenvironments are frequently hypoxic and result in the stabilization of hypoxia inducible factors (HIF-1/2) that transcriptionally activate genes involved in invasion, metastasis, metabolism and angiogenesis [1]. The role of hypoxia and the contribution of HIF in the angiogenic switch leading to tumor progression and resistance to treatment are well documented. This angiogenic response to HIF activity is largely mediated through activation of vascular endothelial growth factor (VEGF). Noninvasive characterization of the loss of both isoform of HIFs (HIF-1α & HIF-2α) on tumor vascularization is relatively unexplored. Here we investigated the effect of HIF silencing on tumor growth in the presence or absence of Matrigel that resembles the complex extracellular matrix (ECM) found in most tumors and determined its effect on tumor vasculature using noninvasive MRI. MDA-MB-231 human breast cancer cells expressing shRNA against both HIF-1α and HIF-2α (231-DK) were established as previously described [2]. In vivo studies were performed using MDA-MB-231 breast cancer cells expressing an empty vector control (231-EV) and 231-DK cells implanted in the mammary fat pad of female SCID mice. Tumor growth curves were obtained from cells inoculated either in 0.05 ml of Hanks balanced salt solution (HBSS) or together with Matrigel solution (8.8 mg/ml). All MRI studies were performed on a 9.4T Bruker Biospec horizontal bore scanner. 3D maps of vascular volume (V/V) and permeability (VP) were obtained using a rapid gradient-echo sequence and albumin-GdDTPA (0.5 g/kg) as the contrast agent (CA). A proton density (PD) image was acquired prior to CA injection, using a 3D gradient echo sequence, with TE/TR = 1.5/10 ms and 3° flip angle and analysis of the images were performed as previously described [3]. Exposure to hypoxia showed no increase in HIF-1 or 2α protein expression in 231-DK compared to 231-EV cells. A significant growth advantage of the 231-DK cells in vivo was observed when inoculated in the presence of Matrigel compared to 231-DK in HBSS. Growth advantage of tumors in the presence of Matrigel was less dramatic for 231-EV cells. When inoculated with Matrigel, 3D reconstructed maps of 231-DK tumors showed significantly increased VP compared to 231-EV tumors with no difference in the VV. Increased vascular permeability in tumors derived from 231-DK cells compared to tumors derived from 231-EV cells can be attributed to VEGF in the Matrigel that is known to exhibit paracrine effects. These data suggest that ECM components may modulate molecular targeting and highlight the importance of the tumor microenvironment in modifying HIF silencing effects. Work is under way to analyze the effects on the metastatic burden in these systems.