I recently performed subcutaneous (s.c.) injections for a CT26 syngeneic xenograft model.

The variation within the same experimental groups was very large. In some mice, we suspected ascites, but upon sacrifice and examination, we found tumors formed in the small intestine. In severe cases, tumors were also found in the spleen, liver, and ovaries. Is this something that can happen with subcutaneous xenografts? Upon further reflection, in the mice where intraperitoneal metastasis was confirmed, the subcutaneous tumor had either penetrated into the peritoneal cavity or was adhered to the peritoneum.

Additionally, each of my four experimental groups had n=7, and this intraperitoneal metastasis was observed in about 1/3 to 1/4 of the mice. In the remaining mice, we observed skin necrosis and wounds, but the tumor was confirmed to be within the subcutaneous fat layer.

My hypothesis is that the subcutaneous injection was not skillfully performed, leading to the cells entering the peritoneum or peritoneal cavity. I found a paper where CT26 cells were injected intraperitoneally (i.p.) to confirm metastasis. I'd like to know if metastasis to the peritoneal cavity can occur via lymphatic or blood vessels even when CT26 cells are injected subcutaneously, or if it's more likely due to an accidental injection into the intraperitoneal space instead of subcutaneously