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

Abstract

We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of “cellular sipping” changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001), and this was increased by h-GF pre-treatment with TNF-α (p<0.001). TNF-α stimulated binding was dose dependent and maximal at 1.16nM (p<0.05) with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5hr pre-treatment (p<0.001). h-GF retained maximal binding up to 6hrs after TNF-α stimulation, but this was lost by 18hrs (p<0.001). FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04). Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003), and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we conclude that TNF-α stimulated cellular sipping likely reflects increased SAOS-2 binding as opposed to enhanced exchange mechanisms.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping

“…*randn(BNo+50,1)+MidExNum; %Rounding values ExNum=round(ExNum); %To remove values beyond the minimum and maximum, %values are tested for excursion beyond extremes and replaced † %with random values between minima and maxima. %Values are rounded for this variable a=(1:BNo+50); for a= (1:BNo+50) if ExNum(a,1)<MinExNum b=MinExNum+rand*(MaxExNum-MinExNum); ExNum(a,1)=round(b); end if ExNum(a,1)>MaxExNum b=MinExNum+rand*(MaxExNum-MinExNum); ExNum(a,1)=round(b); end end %Drawing a histogram subplot (3,5,2); histogram(ExNum); title ('Exchange Number'); %For Rate = Rate of cell-projection retraction for any given exchange event Rate=SDRate. *randn(BNo+50,1)+MidRate; %To remove values beyond the minimum and maximum, %values are tested for excursion beyond extremes and replaced † %with random values between minima and maxima.…”

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

“…Our earlier work showed CPP causes significant phenotypic change, including altered morphology and cytokine synthesis of recipient cells (1,18). The inflammatory cytokine Tumor Necrosis Factor-a increased transfer from fibroblasts to SAOS-2, and this seemed due to increased binding of SAOS-2 via ICAM-1 (1,24). 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).…”

“…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.…”

The effect of TNF-α on osteoblasts in metal wear-induced periprosthetic bone loss

“…This cell line resembles mature osteoblasts 26 is well characterized, commonly used for osteoblastic models, and has been used in studies on TNF-α-stimulation before. 7 , 27 …”