(82f) Implantable Bioengineered Microenvironments to Study Human Tumor-Immune Interaction

Introduction: Recent studies have revealed the important role of local stromal and immune cells in recruiting and regulating disseminated circulating tumor cells while direct investigation of natural metastatic niches represent multiple difficulties due to the vast stromal diversity and potential inaccessibility of organs like the bone marrow. Implantable â??humanizedâ? microenvironments in immunodeficient mice have emerged as a tunable surrogate for human tissues for studying functional interaction between local tissue cells and human tumor cells.^ Yet, an in vivo system that encompasses the human tumor-immune cell interaction has not been fully developed. In this work, we introduce a tissue engineering approach to generate an ectopic microenvironment and its application for studying the interaction of human immune cells and human cancer cells in an immunodeficient murine host.

Materials and Methods: Scaffolds fabricated as previously described were seeded with 0.5 x 10⁶ human bone marrow stromal cells. After 3 days in culture 4 scaffolds were subcutaneously implanted into an immunodeficient, NSG mouse. Two weeks following implantation 2 x 10⁶ PC-3 cells expressing luciferase and GFP were injected into the mouse prostate to develop an orthotopic prostate tumor xenograft that subsequently released circulating tumor cells. Four weeks after tumor injection, 10 x 10⁶ human peripheral blood mononuclear cells (PBMCs) were injected intravenously. One week later the scaffolds were retrieved from primary mice and re-implanted into new NSG mice at the same anatomical location. Bioluminescent images were taken weekly to non-invasively monitor tumor growth in vivofor up to 12 weeks. Finally mice were sacrificed and inter-scaffold vasculature development and recruitment of human tumor and immune cells was characterized via immunohistostaining. Tissue and scaffold samples from a small group of mice were prepared for the intact tissue clearing technique.

Results and Discussion:Immunohistostaining analysis revealed that both cancer and immune cells were successfully recruited to the scaffolds before transplantation and remained twelve weeks post-transplantation. Immune recruitment was also confirmed via FACS. Interestingly our implantable scaffolds displayed similar compositions to other immune related organs, the bone marrow and spleen. In the primary mice, none of the scaffolds displayed visible metastases under bioluminescent imaging. Following serial transplantation, six of the seventy-two scaffolds (8.3%) had visible metastases. Bioluminescence imaging revealed a steady growth in signal in control mice, while mice that received an injection of PBMCs had restricted growth.

Conclusions: Our results demonstrate a tissue engineering approach to study human cancer metastasis and immune cell interaction. We envision that implantable humanized microenvironments in NSG mice will be an invaluable tool for studying the role of tumor microenvironments and human disease.