Exploring Spatio-Temporal Signaling in Cancer Cells via 3D Immunofluorescence

Cancer metastasis is the spread of a tumour from a primary site to a secondary site in the body. Metastatic cancer cells grow into the surrounding tissue, intravisate into lymph nodes and blood vessels, travel the circulatory system and extravisate into a secondary site from where they begin to form another tumour. The exact mechanisms of how cancer cells migrate from the primary site are not clearly understood. One possible pathway implicated in cell migration is the IL-6/JAK/STAT pathway. Previous work has shown that this pathway allows cancer cells in a 3D environment and at high cell number density to switch to a more migratory phenotype compared to those at lower cell number density. This study aims to determine how cell number density regulates spatio-temporal pathway activation which is measured as a function of phosphorylated-STAT3 (pSTAT3) expression. To test the hypothesis that high cell number density of cancer cells lead to higher levels of pSTAT3, high throughput 3D immunofluorescence was performed on 3D collagen type 1 gels embedded with cancer cells at arbitrarily increasing cell number densities. The results showed no consistent upregulation in pSTAT3 with density, while validation with flow cytometry yielded negative results. Further there appears to be no spatial activation of the pathway while with time an increase in pSTAT3 is seen at low cell number density before falling back down. These results suggest that immunofluorescence might not be the optimum technique to study pSTAT3 signaling due to low sensitivity to change. Whereas this pathway could potentially reveal how cancer cells communicate with time and in space, pSTAT3 is not the best indicator of pathway activatio