Background: Relapsed medulloblastoma (MB) poses a significant therapeutic challenge due to its highly immunosuppressive tumor microenvironment. Immune checkpoint inhibitors (ICIs) have struggled to mitigate this challenge, largely due to low T-cell infiltration and minimal PD-L1 expression. Identifying the mechanisms driving low T-cell infiltration is crucial for developing more effective immunotherapies. Methods: We utilize a syngeneic mouse model to investigate the tumor immune microenvironment of MB and compare our findings to transcriptomic and proteomic data from human MB. Results: Flow cytometry reveals a notable presence of CD45^hi/CD11b^hi macrophage-like and CD45^int/CD11b^int microglia-like tumor-associated macrophages (TAMs), alongside regulatory T-cells (T_regs), expressing high levels of the inhibitory checkpoint molecule VISTA. Compared to sham control mice, the CD45^hi/CD11b^hi compartment significantly expands in tumor-bearing mice and exhibits a myeloid-specific signature composed of VISTA, CD80, PD-L1, CTLA-4, MHCII, CD40, and CD68. These findings are corroborated by proteomic and transcriptomic analyses of human MB samples. Immunohistochemistry highlights an abundance of VISTA-expressing myeloid cells clustering at the tumor–cerebellar border, while T-cells are scarce and express FOXP3. Additionally, tumor cells exhibit immunosuppressive properties, inhibiting CD4 T-cell proliferation in vitro. Identification of VISTA’s binding partner, VSIG8, on tumor cells, and its correlation with increased VISTA expression in human transcriptomic analyses suggests a potential therapeutic target. Conclusions: This study underscores the multifaceted mechanisms of immune evasion in MB and highlights the therapeutic potential of targeting the VISTA–VSIG axis to enhance anti-tumor responses.

Osteosarcoma is the most common primary malignant bone tumor in childhood. Patients who present with metastatic disease at diagnosis or relapse have a very poor prognosis, and this has not changed over the past four decades. The Wnt signaling pathway plays a role in regulating osteogenesis and is implicated in osteosarcoma pathogenesis. DKK-1 inhibits the canonical Wnt signaling pathway, causing inhibition of osteoblast differentiation and disordered bone repair. Our lab previously demonstrated that an mAb against DKK-1 prevented metastatic disease in a mouse model. This study expands upon those findings by demonstrating similar results with a small-molecule inhibitor of DKK-1, WAY262611, both in vitro and in vivo. WAY262611 was evaluated in vitro on osteosarcoma cell lines, including proliferation, caspase activation, cell-cycle analysis, and signaling pathway activation. We utilized our orthotopic implantation/amputation model of osteosarcoma metastasis in vivo to determine the impact of WAY262611 on primary tumor progression and metastatic outgrowth of disseminated tumor cells. Differentiation status was determined using single-cell RNA sequencing. We show here that WAY262611 activates canonical Wnt signaling, enhances nuclear localization and transcriptional activity of β-catenin, and slows proliferation of osteosarcoma cell lines. We also show that WAY262611 induces osteoblastic differentiation of a patient-derived xenograft of osteosarcoma in vivo, as well as inhibiting metastasis. This work credentials DKK-1 as a therapeutic target in osteosarcoma, allowing for manipulation of the Wnt signaling pathway and providing preclinical justification for the development of new biologics for the prevention of osteosarcoma metastasis.

Supplementary Table S1: Top 10 GO Biological Processes Enriched in Treated vs Control

Osteosarcoma is the most common primary malignant bone tumor in childhood. Patients who present with metastatic disease at diagnosis or relapse have a very poor prognosis, and this has not changed over the past four decades. The Wnt signaling pathway plays a role in regulating osteogenesis and is implicated in osteosarcoma pathogenesis. DKK-1 inhibits the canonical Wnt signaling pathway, causing inhibition of osteoblast differentiation and disordered bone repair. Our lab previously demonstrated that an mAb against DKK-1 prevented metastatic disease in a mouse model. This study expands upon those findings by demonstrating similar results with a small-molecule inhibitor of DKK-1, WAY262611, both in vitro and in vivo. WAY262611 was evaluated in vitro on osteosarcoma cell lines, including proliferation, caspase activation, cell-cycle analysis, and signaling pathway activation. We utilized our orthotopic implantation/amputation model of osteosarcoma metastasis in vivo to determine the impact of WAY262611 on primary tumor progression and metastatic outgrowth of disseminated tumor cells. Differentiation status was determined using single-cell RNA sequencing. We show here that WAY262611 activates canonical Wnt signaling, enhances nuclear localization and transcriptional activity of β-catenin, and slows proliferation of osteosarcoma cell lines. We also show that WAY262611 induces osteoblastic differentiation of a patient-derived xenograft of osteosarcoma in vivo, as well as inhibiting metastasis. This work credentials DKK-1 as a therapeutic target in osteosarcoma, allowing for manipulation of the Wnt signaling pathway and providing preclinical justification for the development of new biologics for the prevention of osteosarcoma metastasis.

Supplementary Figure S1 shows results from toxicity evaluation of WAY262611, the effect of WAY262611 on the growth of the primary tumor and on mouse survival, as well as examples of metastatic lesions.