Proteomic analysis of treatment-resistant, stem-like cancer cells.

Abstract

Valid data supports the cancer stem cell theory for solid tumors and it appears that tumor cells responsible for failure after successful initial therapy exhibit stem cell properties, including self-renewal, potent tumorigenic activity, an undifferentiated state and resistance to chemo or radiation therapy. We investigated the existence and characteristics of such cells in mouse neuroblastoma.

Mouse neuroblastoma and Neuro2A cells line were harvested and cultured in DMEM + 10% FBS or NeuroCult complete media specific for the proliferation of neural stem cells. As a positive control for normal stem cells, the adult mouse subventricular zone was dissected and adult neural stem cells isolated/cultured into neurospheres in NeuroCult complete media. Tumorigenicity was assessed in vivo with inoculation of 5x103 neuroblastoma tumorspheres and committed cells. Tumorspheres and committed cells were subjected to radiation (2.5Gy) followed by viability assays and Annexin-V/7-AAD staining to assess apoptosis. Similarly, cell types were treated with varying doses of Doxorubicin and Sunitinib malate to assess sensitivity. Total protein was extracted and subjected to mass spectrometry.

In vitro assays showed neuroblastoma stem-like cells grow as tumorspheres in serum free media and are capable of eliciting self-renewal capacity and multi-lineage differentiation. We show that tumorspheres are remarkably resistant to chemo, radiation and small molecule therapy.

However, compared to committed cells, tumorspheres proliferate slower in vitro and in vivo. Proteomic profiling revealed that tumorspheres differ significantly from murine subventricular zone (SVZ) stem cells and also lack the classic markers of cells undergoing EMT.

More importantly, compared to committed cells, tumorspheres showed up-regulation of proteins involved in small molecule biochemistry (indicating high metabolic activity), RNA post translational modification, and energy production. In addition, tumorspheres exhibited significant up-regulation of potent tumorigenic proteins including PDGFRB and survivin. Interestingly, survivin was significant up-regulation in both tumorspheres and adhered cells after radiotherapy.

Our pathway exploring analysis [Ingenuity Pathway Assist (IPA)] showed downstream targets of ERBB2 are indeed up-regulated in tumorspheres.

These observations define a stress induced (serum deprived) transitional tumor cell with stem-like qualities and enhanced malignant properties. We further propose that both tumorspheres and committed cells respond to the stress of radiotherapy by up-regulating survivin, perhaps as a survival mechanism to evade radiation-induced apoptosis. We propose that a stressor to the primary tumor will induce the transitional cell phenotype and may account for both the resistant late recurrences and metastatic deposits observed in high-risk tumors.