As one of the crucial steps in metastatic progression requires tumor to successfully interact with its local microenvironment, it follows that targeting this cross-talk may be an attractive adjuvant to standard treatment approaches. We are currently focused on developing therapies that target the associated tumor recruited host stromal cells. We have an IRB approved biological repository study to obtain blood, bone marrow, tumor and adjacent normal tissue when available from patients with malignancy and healthy donors. We continue our on-going studies of measuring and characterizing the circulating bone marrow-derived cells that may be altered in the setting of cancer and other chronic diseases. Utilizing both quantification and functional assays, including flow cytometry and colony forming unit assays, we are assessing the circulating bone marrow-derived progenitor cell populations in pediatric and adult patients with malignancies. We have broadened our investigations to better understand the changes in the hematopoietic stem cell niche that results in alterations in immune milieu in response to a growing primary tumor. These studies now include in addition to monitoring hematopoietic and endothelial progenitor cells but also CD4 and CD8 T cells and myeloid cells including MDSCs and M1 and M2 macrophages and stromal cell populations. Furthermore, we measure circulating microvesicles released by tumor cells and associated tumor hematopoeitic and stromal cells that may impact important cell behavior and are known to be critical to cell-cell communication. We have on-going investigations as to which cells make which microvesicles and their particular content and determining which would be most useful as a biomarker for metastatic risk. Our recent studies have determined host cell plasticity and cell state determine the microvesicles released from these cells and this plasticity in perivascular cells play key roles in regulating metastasis. We are currently investigating markers of this perivascular cell plasticity as a predictor of metastasis and response to conventional therapies and immune based therapies. We continue our collaboration with Dr. Sharon Savage to examine circulating bone marrow-derived cell populations in patients with Li Fraumeni syndrome, which is a high-risk cancer predisposition syndrome related to loss of tumor suppressor p53. We are enrolling patients in order to determine if changes in these bone marrow-derived cell populations predict tumor development in these patients. We are monitoring circulating levels of bone marrow-derived cells at the time of the yearly evaluation for cancer surveillance. We have also developed assays to examine biological correlates that can be measured in stored RNA samples in order to correlate outcome data with these biomarkers for metastatic risk. We have established a pre-clinical model system for testing microenvironment-targeting therapy in pediatric sarcomas. Utilizing a Ewings sarcoma (EWS) xenograft tumor cell line and a syngeneic rhabdomyosarcoma (RMS) cell line we have performed flow cytometry and immunofluorescence to demonstrate the influx of myeloid cells into the tumor and pre-metastatic tissues. We also monitor metastatic progression in a resection model using luciferase imaging. In this fashion, pre-metastatic, metastatic colonization and progression to visible metastasis can be followed and compared in treated and untreated groups without requiring multiple terminal end points. We are conducting pre-clinical investigations utilizing inhibitors targeting stromal cell plasticity specifically to assess impact on metastatic progression. We also now have a marker of tumor associated fibroblast activation and stromal cell lineage tracing mice in order to monitor activation of these cells in this process. We have performed serial in vivo mouse experiments examining targeting of myeloid cells and stromal cells to determine their impact on metastatic progression. We are using different investigational agents to determine their specific impact on each microenvironmental cell. These pre-clinical studies will answer whether this approach to treatment may likely be a good window for targeting the recruitment of these microenvironment tumor-associated cells that support tumor progression. Our studies using a colony stimulating factor -one receptor (csf1-R) inhibitor revealed that these models of RMS and Ewings sarcoma secrete a good deal of CSF-1 and lead to the recruitment of CSF-1R expressing cells. These cells are found in early metastatic sites and are immune suppressive and can protect disseminated tumor cells from cytotoxic T cell activity. These immune suppressive cells can be targeted with CSF-1R inhibitor and can reduce their immune suppressive nature and limit metastatic progression. We have determined that these immune suppressive myeloid cells populations are replaced by a population of antigen presenting cells in the metastatic tumor microenvironment. CSF1R targeting the adjuvant setting in our metastatic RMS resection model inhibits metastatic progression and when CSF1-R inhibition is combined with other immunotherapies have added therapeutic benefit to limit metastasis. We are preparing this work for publication. We have also established a good in vitro model to understand the role of tumor-secreted factors on myeloid cell development and function and investigating CSF1-R targeting in this process. These studies allow for investigating function of potential therapeutic inhibitors of the myeloid skewing and polarization process. This year, in collaboration with Brigitte Widemann, we continue to enroll in a phase I trial of tumor microenvironment targeting drugs in particular targeting CSF1R expressing cells by a new small molecule inhibitor of cfms in pediatric patients with relapsed solid tumors. This study has enrolled patients and based on early analysis of blood samples suggests we are lowering specific monocyte populations and may be enhancing immune competent cells that activate T cell cytotoxicity by presenting tumor associated antigens. We have completed the highest dose level and anticipate starting a CSF1R inhibitor and checkpoint inhibitor combined study in the near future.
