Abstract
Numerous studies have demonstrated a relationship between the extracellular matrix protein BIGH3 and variations in the malignant properties of different cancer cell types, including osteosarcoma cells. BIGH3 protein can suppress and promote tumor growth, even on the same cancer cell type, indicating that contextual cues regulate BIGH3-mediated divergent outcomes. We employed a multicellular tumor spheroid model to study the effects of BIGH3 with respect to physical and molecular features of three-dimensional tumor growth. The results demonstrated that exogenous recombinant BIGH3 blocked the development of multicellular large tumor spheroids so that only small spheroids formed. The effect was dependent on the BIGH3 concentration in the growth medium and the time of incubation of BIGH3 with the osteosarcoma cells in the spheroid model. TGF-β1 signaling induced multicellular tumor spheroids to synthesize a greater quantity of BIGH3 relative to non-treated spheroids. The TGF-β1-mediated increase in BIGH3 protein antagonized the development of multicellular large spheroids. Anti-BIGH3 antibody, and an inhibitor of TGF-β1 signaling, blocked the antagonistic effect induced through TGF-β1 stimulation and BIGH3 protein expression, resulting in the formation of multicellular large spheroids. Immunohistochemistry detected BIGH3 at cell bodies within the spheroid stroma, suggesting osteosarcoma cell-surface proteins bind BIGH3. Flow cytometry demonstrates that osteosarcoma cells interact with soluble BIGH3, and solid-phase cell adhesion assays show that osteosarcoma adhesion to BIGH3 substratum is mediated by integrin α4β1. However, anti-α4 antibody did not attenuate the BIGH3-mediated antagonism toward formation of multicellular large spheroids. We conclude that TGFβ1 and BIGH3 suppress the development of large osteosarcoma tumors.
Highlights
As an in vitro model of tumor progression, propagation of Multicellular Tumor Spheroids (MTS) provides a convenient means to study the effects of the extracellular matrix (ECM) on tumor formation
The primary objective of this study was to document the influence of BIGH3 on osteosarcoma cell MTS development
TGFβ1, BIGH3, integrins and apoptosis were highlighted as playing potential roles in osteosarcoma tumor biology
Summary
As an in vitro model of tumor progression, propagation of Multicellular Tumor Spheroids (MTS) provides a convenient means to study the effects of the extracellular matrix (ECM) on tumor formation. Apoptosis [11], chromosomal abnormalities [26], and cell motility and chemoresistance to lung and ovarian cancer cells [27] have been implicated in molecular mechanisms that underlie BIGH3’s divergent effects on tumors progression. Despite the accumulating evidence of differential roles that BIGH3 mediates in different cancer and cancer cell types, there has been a paucity of information on the effect of BIGH3 protein on cancer cell behavior in the context of a three-dimensional tumor environment. We previously reported BIGH3 mediates apoptosis in MG-63 osteosarcoma cell monolayers [11] To this end we sought to examine the effect of BIGH3 on developing osteosarcoma tumor spheroids by using a simple yet well-recognized MTS model system as a convenient method to examine tumor response to ECM protein. We utilized osteosarcoma MG-63 cells, MG-63 BIGH3, human recombinant BIGH3 protein, and TGF-β1 to test for BIGH3’ influence on osteosarcoma MTS
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