Spheroids represent an in vitro three-dimensional (3D) tissue structure that mimics in vivo tumor tissue organization and microenvironment. We compared the metastatic potential of spheroid- and monolayer-cultured CT26 colon carcinoma (CT26) cells. Because hypoxia is inherent to compact tumor cell colony development, the hypothesis was advanced that in vitro 3D-growth of cancer cells may upregulate their production of angiogenesis and stromagenesis-stimulating activities. CT26 spheroids were generated by the hanging-drop method and the hepatic metastasizing ability of cancer cells was studied following intrasplenic injection into BALB/c mice. Two-dimensional electrophoresis and mass spectrometry (2D-MALDI-TOF) were combined to study the protein profiles associated with spheroid- and monolayer-growing CT26 cells. Within 1-3 days, aggregated cancer cells had a high proliferation capacity until day 7 when the plateau phase of the growth curve was reached. In vivo, both metastasis density and volume significantly (P<0.01) increased in mice receiving spheroid-growing CT26 as compared to mice receiving monolayer-growing cells. Proliferating Ki67+ cancer cells, endothelial cells (as CD31-expressing cells) and smooth-alpha actin-expressing myofibroblasts significantly increased in metastases from mice receiving spheroid-growing CT26 cells as compared to metastases from monolayer-growing cells. Conditioned medium (CM) from spheroid-growing CT26 cells significantly (P<0.01) increased hepatic sinusoidal endothelial (HSE) cell migration compared to the effect of CM obtained from an equivalent number of monolayer-growing cells. The level of VEGF also significantly (P<0.01) increased in spheroid-growing CT26 cells compared to the level in monolayer-growing cells. Consistent with these data, subcutaneous injection of spheroid-growing CT26 cells produced a greater angiogenic response as compared to that produced by monolayer-growing cells. As shown by 2D-MALDI-TOF analysis of whole cell lysates, spheroid-growing CT26 cells had a differential protein profiling. Among overexpressed proteins, 60S acidic ribosomal protein, ferritin heavy chain, phosphoglycerate kinase-1 and estrogen-related receptor alpha have been involved in tumor-associated mechanisms. These results demonstrate that culture as spheroids alters the protein expression of colon cancer cells, increasing their hepatic metastasizing efficiency via induction of angiogenic and stromagenic-stimulating potential.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA