Remodeling the Lung Tumor Microenvironment with Locally Administered Nano-Immunochemotherapies for Osteosarcoma Lung Metastases

Abstract

Osteosarcoma (OS) is the most common type of primary bone cancer in all ages1 and metastasizes exclusively to the lungs2. The majority of patients present with micrometastases at the time OS is first diagnosed. When lung metastasis (OSLM) develops, and gross lesions are detected, the curability of disease and the overall survival rate diminish drastically (<20%)2,3. OSLM is thus the leading cause of death in those patients1,4. The current standard of care with chemotherapy has failed to improve patients' therapeutic outcomes with OSLM2,3. The interplay between tumor cells and immune infiltrates in the tumor microenvironment (TME) is a critical determinant of metastasis growth. Systemically administered gemcitabine (GMT) chemotherapy is currently being used in the clinic to treat patients with OSLM, alone or in combination with other chemotherapies, but the response rates are very low. Early studies leading to current trials with GMT administered via pulmonary route have shown promise in decreasing tumor burden – but unfortunately not eradicating the tumor. New therapeutic strategies are needed to tackle this unmet need in clinical oncology. We propose the combination of orally inhaled GMT chemotherapy with colony-stimulating factor-1 receptor inhibitors (CSF-1Ris), macrophage immunotherapy, for the treatment of OSLM also via the pulmonary route. The benefits of CSF-1Ris have been recently shown in manipulating TME away from tolerance (but not for OSLM yet) and is now in the clinic 5. Immunotherapies for tumorassociated macrophages (TAMs) are particularly important in OSLM as immune checkpoint inhibitors (ICIs) have yielded very low response rates in such "cold tumors". Moreover, TAMs are the most abundant infiltrates in OSLM tumors. Our particular interest is PLX-3397 (PLX, or Pexidertinib), which has been recently approved by the FDA. As with systemic chemotherapy, potential limitations of CSF-1Ris are the poor biodistribution to the lungs upon systemic administration and the high off-target toxicity (mainly hepatotoxicity)6. Thus, there is an opportunity to develop novel combination immunochemotherapy and local lung delivery strategies to improve the therapeutic potential and decrease off-target toxicity of currently available therapeutics. In this work, we develop a reproducible, robust in vivo model of OSLM that enables screening of various therapeutics via local administration to the lungs and their influence on tumor growth in vivo and ex vivo. We also investigated the effect of gender as a variable in this model, given that tumor growth and treatment outcomes have been shown to be impacted by gender in the clinic. We evaluated the tolerability and efficacy of the local lung delivery of PLX TAM immunotherapy alone and in combination with GMT chemotherapy, and their effect in remodeling the TME to prevent the development of micrometastases to large lesions. We studied the impact of those therapies at the TME level using a combination of flow cytometry, immunofluorescence, and H&E staining, to assess levels of expression of upregulated Fas/FasL in both OS cells and infiltrating lymphocytes, and also the abundance of classically vs. alternatively activated TAMs with the CSF-1Ri treatment. We correlate those results with tumor burden and survival. As lung tissue retention and clearance limit this combination therapy's potential, we started our investigations of the effect of nanoformulation as a potential strategy to enhance drug efficacy by improving the lung pharmacokinetic profile of the treatments. We started with a translatable, lipid-based platform for the development of GMT nanoformulations that can be in the future delivered in nebulizer form; initial studies in the lung retention of GMT and optimization of the lipid formulation are presented. Our studies are clinically significant as they have the potential to introduce new translatable approaches to treat OSLM. They are scientifically significant and innovative, as for the first time, we are elucidating the tolerability and effect of TAM immunotherapy locally delivered to the lungs in combination with chemotherapy in the TME of an immunocompetent OSLM model.