Development of a 3D coculture method for studying the effect of stromal cells on tumor cells response to drugs.

Konference: 2015 XI. Dny diagnostické, prediktivní a experimentální onkologie

Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie

Téma: Nové technologie a metody

Číslo abstraktu: 032

Autoři: Mgr. Světlana Miklíková (Školeková), Ph.D.; Mgr. Viswanath Das, Ph.D.; Mgr. Lucia Kučerová, Ph.D.; Mgr. Jan Gurský, Ph.D.; doc. MUDr. Marián Hajdúch, Ph.D.


Colorectal cancer (CRC) is one of the most frequent malignancies affecting individuals across the world. Epigenetic alterations have been identified to contribute to the complexity of pathogenesis of CRC. The accumulating findings for epigenetic defects in cancer could be potentially useful for cancer diagnosis and treatment. Although epigenetic therapy is very promising in cancer prevention, there are many question that need to be answered before these agents can be used routinely. For example, it is yet to be elucidated the precise role of tumor stroma in CRC progression, and how the intra-tumor stroma percentage alters the efficacy of CRC therapy. In this study, we analyzed the impact of different tumor-stroma ratios on tumor cells response to epigenetic drugs in 3D spheroid culture models that closely correspond to in vivo tumors.


HCT116-EGFP-FLJ-H2B reporter cells stably expressing red fluorescent protein, and based on the methylation status of the FLJ gene,green fluorescent protein were maintained in McCOY´s medium supplemented with 10% FBS and 2mM L-glutamine. Mesenchymal stromal cells (MSC) were isolated from healthy individuals undergoing elective lipoaspiration, cultivated in low-glucose DMEM supplemented with 10% FBS and characterized based on the established minimum definition criteria. Each subject provided informed consent. Activity of epigenetic drugs was examined in spheroid cultures of different tumor-stroma ratios using confocal microscopy, flow cytometry and MTT analyses.

Results and conclusions

Our preliminary results show an increase in the demethylating activity of epigenetic drugs in spheroids containing higher ratio of stromal cells. Additionally, we have established a flow cytometrybased protocol that distinguishes tumor cells from stromal cells based on fluorescence, and permits determination of percentage of tumor cells responding to therapy. The response of tumor cells to epigenetic therapy in 3D cultures seems to differ in high-and lowstroma spheroids. These 3D coculture models can serve as potential screening tools for studying the influence of stroma on tumor cell response to anticancer drugs.

Datum přednesení příspěvku: 3. 12. 2015