Monitoring the Systemic Immune System to Understand and Improve the Efficacy of Immunotherapy for Metastatic Osteosarcoma

Osteosarcoma (OS) is a complex tumor with no effective targeted therapies due to its genomic heterogeneity and pleomorphism. The immune response it creates, particularly against metastatic lesions, is considerable; however, various suppressive mechanisms induced by the tumor prohibit its effectiveness. The presence of infiltrating lymphocytes suggests that therapeutic disinhibition through checkpoint blockade could increase antitumor immunity, though none have been successful in clinical trials. The complexities of the immune response to OS tumors have yet to be unraveled; however, there is evidence to suggest that cell-mediated immunity (CMI, specifically T cells, Natural Killer [NK] cells, and myeloid-lineage cells [MLCs]) plays an important role. New technologies have made it possilble to assess large numbers of cell antigens simultaneously, producing detailed information on the immune status of an individual. A snapshot of the immunological status of an organ or tumor can be conveyed by means of an immunophenotype (IPT), which uses cytometry, microarray, or gene set enrichment analysis to determine the relative percent populations and activation states of immune cells. The majority of data collected on the OS IPT are from primary tumors. Unlike metastatic tumros, primary tumors are easily removed by surgeons and do not account for aossicated high mortality rates. As such, we have investigated the systemic immune response to disease progression with and without immunotherapy. Using a highly metastatic luciferase(+) K7M2 (luc-K7M2) orthotopic OS model in an immunocompetent host, it was determined that the introduction of luciferase into tumor cells had little to no effect on the overall antitumor immune response and clinical disease progression. A systems-wide comparison of tumor-bearing and sham (surgery only) mice showed that there are significant changes in the IPTs of tumor-bearing mice at various time points in disease progression, and that these changes are consistent across different tissues. As disease progresses, T and NK cells are gradually depleted, and macrophages (Mφs) adopt intermediate phenotypes through increased expression of both M1 and M2 polarization markers. Moreover, NK cell depletion in blood is specific to disease progression. T cells, under constant exposure to OS tumor antigens, become overstimulated and upregulate exhaustion markers like programmed cell death protein 1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3). Further, a notable systemic immune biomarker was uncovered using a co-expression ratio of immunosuppressive programmed death ligand 1 (PD-L1) to immunostimulatory major histocompatibility complex class II (MHC-II) on monocytic-myeloid-derived suppressor cells (M-MDSCs), a specific marker of OS disease progression in blood. Monotherapy with monoclocal antibody anti(α)-PD-L1 reversed the malignancy-induced immunological disturbances but did coindice with increased survival. Conversely, treatment of OS tumor-bearing mice with poly(lactic-co-glycolic) acid (PLGA)-encapsulated interleukin(IL)-12 decreased metastatic rate and increased cure rate in a immunophenotype-dependent manner that coincided with treatment-induced NK cell proliferation. These results demonstrate that systemic immunophenotyping can be used to non-invasively monitor OS patient disease progression and response to immunotherapeutics, potentially offering clinicians the opportunity to modify treatment regimens in real-time