Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology

Abstract

We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells and this alters the cancer cell phenotype. Here, we report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles and to trace the transfer of fibroblast cytoplasm into SAOS-2 cells. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2 cells, as well as what we term ‘compensated fluorescence’, that numerically projects mother cell fluorescence post-mitosis into daughter cells. The comparison of absolute with compensated fluorescence allowed us to deduct if the phenotypic effects in mother SAOS-2 cells were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall’s tau with cell-profile area and without evidence of persistence in daughter cells (median tau = 0.51, p < 0.016); negatively and weakly with cell circularity and with evidence of persistence (median tau = −0.19, p < 0.05); and very weakly with cell migration velocity and without evidence of persistence (median tau = 0.01, p < 0.016). In addition, mitotic SAOS-2 cells had higher rates of prior fluorescence uptake (median = 64.9 units/day) than non-dividing cells (median = 35.6 units/day, p < 0.016) and there was no evidence of persistence post-mitosis. We conclude that there was an appreciable impact of cell-projection pumping on cancer cell phenotype relevant to cancer histopathological diagnosis, clinical spread and growth, with most effects being ‘reset’ by cancer cell mitosis.

A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping

“…We earlier described the exchange of membrane, organelles and cytoplasmic protein between cultured human fibroblasts and cancer cells [ 1 , 2 , 3 , 4 ], and others have made similar observations [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. Uptake of cellular contents by such transfers may be via exosomes, tunnelling nanotubes, or a mechanism we recently described and term ‘cell-projection pumping’, and these can significantly change the phenotype of acceptor cells [ 2 , 3 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ].…”

“…Uptake of cellular contents by such transfers may be via exosomes, tunnelling nanotubes, or a mechanism we recently described and term ‘cell-projection pumping’, and these can significantly change the phenotype of acceptor cells [ 2 , 3 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ]. Notably, a major role for either exosomes or tunnelling nanotubes is excluded in our cultures by video microscopy demonstrating highly localized, discrete and rapid transfer events independent of tunnelling nanotubes and inconsistent with exosomes, as well as in experiments with conditioned media containing putative exosomes [ 1 , 2 , 3 , 4 ], so that cell-projection pumping is responsible for the transfers we study. Transfer of mitochondria appears especially significant, and can confer chemotherapy resistance to cancer cells [ 5 , 7 , 8 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ].…”

“…Our doubts are further raised by recent work showing that most phenotypic effects in cancer cells of receipt of fibroblast contents, are lost upon cancer cell mitosis. This would not be expected if mitochondria were critical, because these organelles survive mitosis and would be expected to maintain in daughter cells any influence they may have had in the mother [ 4 ].…”

“…Most of our work has been with co-cultures of human fibroblasts with SAOS-2 osteosarcoma cells, because it has been helpful to build understanding with quantitative studies in a single cancer cell line; thus limiting the variability inherent to study of differing cancer cell types [ 1 , 2 , 3 , 4 , 29 , 30 ]. Nonetheless, we have also studied transfer between fibroblasts and a wide range of further cancer cell lines, including: osteosarcoma cell lines; melanoma cell lines; ovarian cancer cell lines; colon cancer cell lines; and lung cancer cells [ 1 , 2 , 3 ].…”

“…To examine the effect on cancer cell phenotype, we compared the cell-profile area and cell circularity of the population of SAOS-2 co-cultured with fibroblasts that were dual labelled by uptake of fibroblast fluorescence, with that of SAOS-2 without evident fibroblast fluorescence [ 1 ]. We also studied SAOS-2 and other cancer cells co-cultured with fibroblasts and then separated by fluorescence activated cell sorting (FACS) on the basis of the level of fibroblast label uptake [ 2 ]; while we further performed single-cell tracking analysis from time-lapse recordings of co-cultures of SAOS-2 with fibroblasts [ 4 ]. In those studies, we found that uptake of fibroblast contents by cancer cells increases cancer cell: proliferation; migration; cell-size; and internal cell complexity [ 1 , 3 , 4 ].…”