Colorectal cancer (CRC) is the third cause of cancer-related mortality in 
industrialized countries. Local invasion and metastasis formation are events 
associated with poor prognosis for which today there are no effective 
therapeutic options. Invasion and metastasis are strongly modulated by cells of 
the tumor microenvironment (TME), in particular fibroblasts and endothelial 
cells. Unraveling interactions between tumor cells and cells of the TME may 
identify novel mechanisms and therapeutic targets to prevent or treat 
metastasis. We report here the development and in vivo validation of a 3D tumor 
spheroid model to study the interactions between CRC cells, fibroblasts and 
endothelial cells in vitro. Co-cultured fibroblasts promoted SW620 and HCT116 
CRC spheroid invasion, and this was prevented by the SRC and FGFR kinase 
inhibitors Dasatinib and Erdafitinib, respectively. To validate these findings 
in vivo, we injected SW620 cells alone or together with fibroblasts 
orthotopically in the caecum of mice. Co-injection with fibroblasts promoted 
lung metastasis growth, which was fully reversed by treatment with Dasatinib or 
Erdafitinib. Co-culture of SW620 or HCT116 CRC spheroids with endothelial cells 
suppressed spheroid growth while it had no effect on cancer cell migration or 
invasion. Consistent with this in vitro effect, co-injected endothelial cells 
significantly inhibited primary tumor growth in vivo. From these experiments we 
conclude that effects on cancer cell invasion and growth induced by co-cultured 
TME cells and drug treatment in the 3D spheroid model in vitro, are predictive 
of in vivo effects. The 3D spheroid model may be considered as an attractive 
model to study the effect of heterotypic cellular interactions and drug 
activities on cancer cells, as animal testing alternative. This model may be 
adapted and further developed to include different types of cancer and host 
cells and to investigate additional functions and drugs.