Gene expression analysis to investigate role of tumor-intrinsic hypoxia and tumor secretome in collective migration

We investigated two microenvironmental factors, tumor-intrinsic hypoxia, and tumor-secreted factors (secretome) as triggers of collective migration using a three-dimensional (3D) discrete-sized microtumor models that recapitulate hallmarks of Ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) transition. These two factors induced two distinct modes of collective migration: directional and radial migration in the 3D microtumors generated from the same breast cancer cell line model, T47D. Without external stimulus, large (>500µm) T47D microtumors exhibited tumor-intrinsic hypoxia and directional collective migration while small (<150 µm), non-hypoxic microtumors exhibited radial collective migration only when exposed to secretome of large microtumors. To investigate the differences in the underlying mechanism present between hypoxia- and secretome-induced directional versus radial migration modes, we performed differential gene expression analysis of hypoxia- and secretome-induced migratory microtumors vs. non-hypoxic, non-migratory small microtumors as controls. We used microarrays to detail the global programme of gene expression profiling to study tumor intrinsic hypoxia induced directional migration and secretom induced radial migrartion in large 600µm microtumors with small 150μm microtumors as controls in three dimensional (3D) breast microtumor model To understand the mechanism of two distinct directional and radial collective migratory phenotypes using our 3D microtumor models with controlled microenvironments we carried out RNA expression profiling using Affymetrix platform. To investigate mRNA expression changes induced by tumor-intrinsic hypoxia and secretome as two microenvironmental factors and also to understand the size effect and temporal effect over 6 day period. To that end, we harvested size specific microtumors at four time-points: i.e. small 150μm microtumors (day 6), large 600µm microtumors (day 1 and day 6) and secretome induced CM150μm microtumors(day 6).