Modeling and Reprogramming the Diseased Bone Marrow

Abstract

Disseminated breast cancer cells that take up residence in the bone marrow (BM) can disrupt the tightly regulated process of bone turnover, leading to painful pathological fractures. Current therapies for bone metastases include antiresorptives, which prevent excess resorption of the bone matrix and improve quality of life but do little to deter the growth of metastatic disease. Despite being a primary lymphoid organ, the marrow’s resident immune cells fail to recognize and eliminate disseminated tumor cells. At steady state, the BM assumes an immunosuppressive milieu through high concentrations of regulatory T cells and suppressor myeloid populations to protect resident hematopoietic stem cells (HSCs). To elicit an immune response and inhibit tumor growth, we hypothesized that therapeutic activation of the pro-inflammatory stimulator of interferon genes (STING) pathway would polarize the BM microenvironment to an antitumor phenotype through immune cell activation. In a mouse model of metastatic mammary carcinoma, we systemically administered an existing endosomolytic nanoparticle platform containing STING agonist cGAMP. Treatment improved both bone and tumor outcomes. An open-source automated liquid handler was designed to increase the reproducibility of the quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analysis of the inflammatory cell populations in the BM, which demonstrated a transient inflammatory landscape induced by STING activation. Observing transient phenomena in the bone marrow is difficult due to the opacity of bone. To enable future studies aimed at investigating cellular dynamics, we developed a subcutaneous model of BM that is amenable to longitudinal intravital imaging. The novel scaffold design was fabricated from an indirect inkjet 3D printing method and when implanted stimulated ectopic bone growth and recruited circulating HSCs to form marrow. Flow cytometry revealed a similar hematopoietic progenitor composition as femoral marrow, and fluorescently-labeled progenitors were able to home to the scaffolds after adoptive transfer. To increase the throughput of scaffold fabrication, a selective laser sintering 3D printer modification was devised for the direct printing of polymer-ceramic composites. This work demonstrates that the diseased BM is a candidate for immunotherapies. It also provides accessible models for evaluating the spatiotemporal heterogeneity of BM.