RNA Analysis on XanoMatrix™ Scaffolds

Using MDA-MB-231 cancer cells as an example, we investigated the differential expression of certain genes when the cells were grown on scaffolds as compared to tissue culture (TC)-treated plastic.

Genes Investigated:

MMP-2: Matrix metalloproteinases are a complex system of proteins that when activated are involved in the breakdown and remodeling of extracellular matrix. MMP-2 is specifically are associated with degradation of Type IV collagen. These genes are normally expressed in cancer cells, especially MDA-MB-231, the overexpression of which may indicate the transition into metastasis.

Survivin: Survivin is an apoptosis inhibitor which is highly expressed in tumor tissues, yet not in terminally differentiated cells. Blocking survivin has been shown to increase apoptosis and decrease tumor growth.

VEGF: Vascular endothelial growth factor is a protein signaling angiogenesis and neovascularization of tissues, often seen upregulated in tumors and also used as a chemotactic factor for migration assays.

General Protocol:

MDA-MB-231 cells were seeded at a density of 2×104/cm2 in 6-well TC-treated plates or 6-well XanoMatrixTM scaffolds and grown for up to 7 days. Cells were lysed and RNA extracted from the cells using the TRIzol method and reverse transcribed to cDNA using a High Capacity cDNA kit (Invitrogen). mRNA expression was measured semi-quantitatively using gel PCR and ImageJ. Data was normalized to expression of mRNA in cells on TC-treated plates. Experiments were run in triplicate at least 3 times.

Results:

VEGF

Cells grown on XanoMatrix scaffolds showed no change in VEGF expression over the time course compared to TC-treated plastic.

Survivin

Survivin expression slightly increased over time, but was expressed generally similar to cells grown on TC-treated plastic over this time frame.

MMP2

Cells grown on scaffolds initially showed 7-fold upregulation of MMP-2 compared to TC-treated plastic which over 7 days stabilized at 2-fold increase in expression.

While ongoing studies will be required to understand more fully to the extent the scaffold changes expression of cancer-related genes, we have shown that MDA-MB-231 cells grown on XanoMatrixTM scaffolds highly overexpress MMP-2 initially in response to being grown in 3D, do not change expression of VEGF nor Survivin significantly over this time. The ease of RNA extraction from cells grown on the scaffold allow full ability to investigate these signaling pathways with no additional change in protocol.