Membrane and cytoplasmic marker exchange between malignant neoplastic cells and fibroblasts via intermittent contact: increased tumour cell diversity independent of genetic change

Abstract

We previously demonstrated that human osteosarcoma cells (SAOS‐2) induce contact‐dependent apoptosis in endothelium, and expected similar apoptosis in human gingival fibroblasts (h‐GF) using SAOS‐2 alkaline phosphatase (AP) to identify cells. However, h‐GF apoptosis did not occur, despite reduction in AP‐negative h‐GF number (p < 0.01) and enhancement of this by h‐GF TNFα pretreatment (p < 0.01). We suggest that TNFα‐enhanced transfer of membrane AP from SAOS‐2 to h‐GF would explain these data. This idea was investigated using fluorescence prelabelled cells and confocal laser scanning microscopy. Co‐cultures of membrane‐labelled h‐GF (marker‐DiO) and SAOS‐2 (marker‐DiD) generated dual‐labelled cells, primarily at the expense of single labelled h‐GF (p < 0.001), suggesting predominant membrane transfer from SAOS‐2 to h‐GF. However, opposite directional transfer predominated when membrane labels were reversed; SAOS‐2 further expressed green fluorescent protein (GFP) in cytoplasm and nuclei, and h‐GF additionally bore nuclear label (Syto59) (p < 0.001). Cytoplasmic exchange was investigated using h‐GF prelabelled with cytoplasmic DDAO‐SE and nuclear Syto59, co‐cultured with SAOS‐2 expressing GFP in cytoplasm and nuclei, and predominant cytoplasmic marker transferred from h‐GF to SAOS‐2 (p < 0.05). Pretreating h‐GF with TNFα increased exchange of membrane markers (p < 0.04) but did not affect either cell surface area profile or circularity. Dual‐labelled cells had a morphological phenotype differing from SAOS‐2 and h‐GF (p < 0.001). Time‐lapse microscopy revealed extensive migration of SAOS‐2 and cell process contact with h‐GF, with the appearance of SAOS‐2 indulging in ‘cellular sipping’ from h‐GF. Similar exchange of membrane was seen between h‐GF and with other cell lines (melanoma MeIRMu, NM39, WMM175, MM200‐B12; osteosarcoma U20S; ovarian carcinoma cells PE01, PE04 and COLO316), while cytoplasmic sharing was also seen in all cell lines other than U20S. We suggest that in some neoplasms, cellular sipping may contribute to phenotypic change and the generation of diverse tumour cell populations independent of genetic change, raising the possibility of a role in tumour progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

SAOS-2 Osteosarcoma Cells Bind Fibroblasts via ICAM-1 and This Is Increased by Tumour Necrosis Factor-α

“…In that study we observed the exchange of separate membrane and cytoplastmic fluorescent markers in the absence of nuclear exchange, between cultured h-GF and malignant cell lines including: SAOS-2 osteosarcoma; melanoma MeIRMu, NM39, WMM175, MM200-B12; and ovarian carcinoma cells PE01, PE04 and COLO316 [9]. Although studying a range of cell lines [9], our focus was on SAOS-2 cells because we wished to contrast h-GF interactions with our previous discovery of contact dependent endothelial cell apoptosis by SAOS-2 [6].…”

“…In that study we observed the exchange of separate membrane and cytoplastmic fluorescent markers in the absence of nuclear exchange, between cultured h-GF and malignant cell lines including: SAOS-2 osteosarcoma; melanoma MeIRMu, NM39, WMM175, MM200-B12; and ovarian carcinoma cells PE01, PE04 and COLO316 [9]. Although studying a range of cell lines [9], our focus was on SAOS-2 cells because we wished to contrast h-GF interactions with our previous discovery of contact dependent endothelial cell apoptosis by SAOS-2 [6]. Expression of mRNA for the inflammatory cytokine Tumour Necrosis Factor-α (TNF-αin malignant and stromal cells is associated with poor prognosis [10], [11], and fibroblasts respond to this cytokine with increased adhesion molecule expression and malignant cell binding [12], [13], hence we also investigated the effect of TNF-α and found that this cytokine significantly increased cellular sipping between h-GF and SAOS-2 [9].…”

“…With regard to the biological significance of cellular sipping, we observed that the morphology of neoplastic cells that have imbibed fibroblast material is intermediate to that of isolated fibroblasts and neoplastic cells cultured alone [9]. Since fibroblasts are the most prevalent non-vascular stromal cell type, we argue that uptake of fibroblast components by malignant parenchymal cells is an important source of tumour cell diversity [9], and that this may influence both tumour progression and responsiveness to anti-cancer therapies.…”

“…Of particular relevance to the current work is our recent paper describing the exchange of membrane and cytoplasm between cultured parenchymal malignant cells and human gingival fibroblasts (h-GF), a process we have termed ‘cellular sipping’ [9]. In that study we observed the exchange of separate membrane and cytoplastmic fluorescent markers in the absence of nuclear exchange, between cultured h-GF and malignant cell lines including: SAOS-2 osteosarcoma; melanoma MeIRMu, NM39, WMM175, MM200-B12; and ovarian carcinoma cells PE01, PE04 and COLO316 [9].…”

“…Although studying a range of cell lines [9], our focus was on SAOS-2 cells because we wished to contrast h-GF interactions with our previous discovery of contact dependent endothelial cell apoptosis by SAOS-2 [6]. Expression of mRNA for the inflammatory cytokine Tumour Necrosis Factor-α (TNF-αin malignant and stromal cells is associated with poor prognosis [10], [11], and fibroblasts respond to this cytokine with increased adhesion molecule expression and malignant cell binding [12], [13], hence we also investigated the effect of TNF-α and found that this cytokine significantly increased cellular sipping between h-GF and SAOS-2 [9]. In separate work, we demonstrated altered cytokine synthesis in response to TNF-α by h-GF permitted cellular sipping, compared with h-GF denied this contact dependent interaction [14].…”