Increased cell size, structural complexity and migration of cancer cells acquiring fibroblast organelles by cell-projection pumping

Abstract

We recently described a hydrodynamic mechanism for cytoplasmic transfer between cells, termed cell-projection pumping (CPP). Earlier image analysis related altered SAOS-2 osteosarcoma cell morphology, to what we now recognize as CPP uptake of fibroblast cytoplasm. We here examine SAOS-2 phenotype following co-culture with human dermal fibroblasts (HDF) in which organelles were pre-labelled with a fluorescent lipophilic marker. Fluorescence activated cell sorting (FACS) analysis was performed of HDF and SAOS-2, cultured either alone or together. FACS forward scatter is proportionate to cell size, and increased for SAOS-2 with high levels of HDF fluorescence uptake (p < 0.004). FACS side scatter is proportionate to internal cell complexity, and increased in SAOS-2 with increasing uptake of HDF fluorescence (p < 0.004), consistent with uptake of HDF organelles. Scratch migration assays revealed that HDF migrated more quickly than SAOS-2 in both isolated cell culture, and following co-culture (p < 0.004). Notably, SAOS-2 with high levels of HDF labelling migrated faster compared with SAOS-2 with low HDF labelling (p < 0.008). A slight and unconvincing reduction in SAOS-2 proliferation was seen (p < 0.02). Similar results were obtained in single additional experiments with A673 and H312 cancer cells. Forward and side scatter results suggest organellar transfer by CPP increases cancer cell morphological diversity. This may contribute to histological pleomorphism relevant to cancer diagnosis and prognosis. Also, increased migration of sub-populations of cancer cells with high CPP organellar uptake, may contribute to invasion and metastasis in-vivo. We thus suggest relevance of CPP to cancer diagnosis and progression.used the term 'cellular sipping' to convey our sense of cells sipping cytoplasm from one another [1]. However, our recent time-lapse recordings showed transfer in our co-cultures was not via either TNT or shed vesicles. Instead, transfer was by a mechanism not seemingly previously reported and for which we have proposed a hydrodynamic mechanism, 'cell-projection pumping' (CPP) [23].Details of CPP are available elsewhere [23], but in brief, CPP as observed by time-lapse fluorescence microscopy was mediated by highly mobile and often branching cell-projections in the size range of filopodia, that writhed adherent to the culture surface and alternately probed and retracted from neighboring cells [23]. Although the rapid movement and small size of these cellprojections obscured precise visualization, they were clearly different to TNT, which have a straight morphology, change little over prolonged periods of time, and are suspended above the culture surface as taught wire-like connections [2-10, 12, 13, 24, 25]. Increased hydrodynamic pressure in retracting cell-projections, normally returns cytoplasm to the cell body. We suggest, however, that in CPP, cytoplasm in retracting cell-projections equilibrates partially into adjacent recipient cells via temporary inter-cellular cytoplasmic continuities. Although the ...

Potential hydrodynamic cytoplasmic transfer between mammalian cells: Cell-projection pumping

“…One limitation of those earlier reports, was that we only studied cell phenotype in co-culture, and not cells separated after co-culture according to the extent of CPP uptake (1,18). This is addressed in more recent work submitted elsewhere but available as a pre-print (34), in which we study MC sub-populations separated by fluorescence activated cell sorting on basis of the level of fibroblast label uptake. We found that acceptance of fibroblast marker increased MC migration and cell size (34).…”

“…This is addressed in more recent work submitted elsewhere but available as a pre-print (34), in which we study MC sub-populations separated by fluorescence activated cell sorting on basis of the level of fibroblast label uptake. We found that acceptance of fibroblast marker increased MC migration and cell size (34). Internal complexity was also increased, as Simulations predicting fluorescence profiles for recipient cells similar to experimental results ( Fig.…”

“…expected from acquisition of additional organelles from fibroblasts (34). Because CPP generates sub-populations of MC with altered morphology, we suggest CPP might contribute to MC morphological diversity in-vivo, and hence to histopathological pleomorphism relevant to cancer diagnosis and prognosis (41).…”

“…Also, since MC migration is important in cancer invasion and metastasis (41), we suggest CPP contributes to these aspects of cancer. MC diversity is central to cancer progression (41), so CPP driven MC diversity may play a role (1,18,34). CPP seems mechanically more akin to intercellular exchange via TNT than via exosomes, because micro-fusions establish physical cytoplasmic continuity of neighboring cells in both TNT and CPP exchange, while exosome transfer does not require cell to cell contact.…”

“…We studied SAOS-2, because most of our earlier and subsequent work on phenotypic effects of transfer has been with this cell line (1,18,24,34). Fluorescence microscopy of a co-culture of Fib with SAOS-2, revealed the most evident transfer of fluorescent label was from Fib to SAOS-2, with less obvious fluorescence transfer from SAOS-2 to Fib (Fig.…”