Novelapproaches fororthotopic tumor engraftment in humanized immune system mice

Humanized immune system (HIS) mice support engraftment with human tumors, thereby allowing the assessment of drug candidates in preclinical oncology without needing to develop mouse-specific analogs. The site most often used for tumor engraftment in mice is the subcutaneous flank, which leads to imperfect vascularization, prevents metastasis, and does not capture organ-specific biology. We therefore set to develop and validate five protocols for orthotopic engraftment of cancer cell lines that can be reproducibly utilized in HIS mice. The five orthotopic engraftment sites include the femoral bone marrow (via knee-cap surgery), mammary fat pad, liver (via injection in the spleen), pancreas, and lung. All protocols led to an engraftment rate of 100% and were amenable to tumor monitoring via caliper measurements or bioluminescence imaging. Focusing on the orthotopic lung model, we found that 85% of engrafted mice developed liver metastases. The peripheries of both primary (lung) and metastatic (liver) tumors we marked by strong fibrogenesis depicted by picrosirius red staining) and were infiltrated by human T and myeloid cells. In the lungs of engrafted mice, CD4+ T and NK cells upregulated CD25 and CD16 expression, respectively, indicating significant activation compared to non-engrafted mice. Altogether, our results demonstrate that HIS mice can be orthotopically engrafted as robustly and reproducibly as syngeneic models. We also showed that lung orthotopic engraftment leads to an organ specific immune response and distant metastasis, thereby representing a valuable platform for the assessment of novel oncology therapies.