TSG-6 affects anti-cancer drug resistance and angiogenesis in 3D culture system of canine mammary gland tumor cells mimicking the tumor microenvironment

Hypoxia affects tumor growth, angiogenesis, and anti-cancer drug resistance in the tumor microenvironment, and TSG-6 expression is known to influence HIF-1α expression in tumor tissues. In this study, we created TSG-6 knockdown spheroids of canine mammary gland tumor (MGT) cells (CIPp and CIPm) to investigate the effects of TSG-6 on angiogenesis and drug resistance in the tumor microenvironment. Using a siRNA transfection system, we induced TSG-6 knockdown in canine MGT cells and formed TSG-6 knockdown spheroids using an ultra-low adhesion plate. Cell viability and apoptosis were assessed by treating TSG-6 knockdown spheroids with doxorubicin. Angiogenesis was evaluated by forming vascularized spheroids with canine MGT and endothelial cells (ECs). TSG-6 knockdown led to reduced expression of tumor growth factors and multidrug resistance genes in canine MGT cells, as well as a significant reduction in hypoxic conditions within the spheroids. When treated with doxorubicin, TSG6 knockdown spheroids exhibited decreased viability and increased apoptosis. In a vascularized TSG-6 knockdown spheroid model, TSG6 knockdown significantly reduced the expression of CD31 and tube formation in canine ECs. In conclusion, we created a TSG-6 knockdown spheroid model to investigate the role of TSG6 in the tumor microenvironment. TSG6 knockdown significantly reduced anti-cancer drug resistance and angiogenesis in canine MGT cells. Therefore, TSG6 could be considered a potential therapeutic target for canine MGT.