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Abstract
Background Patients with relapsed metastatic osteosarcoma have no effective treatments available to them,1 and immunotherapy thus far has not succeeded in improving outcomes.2–5 We aim to understand the immune architecture of the tumor microenvironment (TME) of osteosarcoma, with the goal of harnessing the immune system as a major therapeutic strategy for the treatment of patients with osteosarcoma.
Methods 66 osteosarcoma tissue specimens were stained and analyzed by immunohistochemistry. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional multiparameter flow cytometry (MFC). Distinct regions from 16 pulmonary metastases (PMs) were microdissected, and RNA was extracted to perform comparative transcriptomic studies. Clinical follow-up (median 24 months) was available from resection.
Results Digital image analysis of immunohistochemistry demonstrated significantly higher infiltrating immune cells in the PMs compared to primary bone tumors, concentrated at the tumor-normal lung ‘PM interface’ region, and elevated expression of multiple immune checkpoint molecules at the PM interface (figure 1). MFC confirmed the increased expression of the immune checkpoint molecules programmed cell death 1 (PD-1, p<0.01) and lymphocyte activation gene 3 (LAG-3, p<0.01), as well as the activation marker IFN-γ (p<0.05) in CD8+ TILs. Gene expression profiling provided further evidence for the presence of TILs with expression of activation markers and inhibitory immune checkpoint molecules at the PM interface compared to the PM interior (figure 2). A strong M2 macrophage signature was present in both regions. Further analysis revealed that genes related to neutrophil and myeloid cell chemotaxis and known to be associated with polymorphonuclear myeloid-derived suppressor cells were highly expressed at the PM interface, along with genes for multiple subsets of dendritic cells (figure 3). Expression of PD-L1, LAG-3, and CSF1R at the PM interface were associated with worse progression-free survival (PFS), while gene sets associated with productive T cell immune response were associated with improved PFS (figure 4).
Immunohistochemistry of osteosarcoma pulmonary metastasesA. H&E with demarcation of tumor-normal lung interface (center green line) and area quantified as the ‘PM interface’ (outer green lines). Pulmonary metastases demonstrate a higher concentration of immune cells (CD3 p<0.001, CD8 p<0.001, CD163 p<0.01) and PD-1 (p<0.001)/PD-L1 (p<0.05) at the PM interface.B. H&E with demarcation of PM interface as above. Pulmonary metastases demonstrating increased staining of TIM-3 (p<0.01), LAG-3 (p<0.01) and IDO1 (p<0.0001) at the PM interface (no significant concentration of CSF1R at PM interface).
Activated/exhausted lymphocyte signatures at PM interfaceA. Heatmap displaying significant genes that contribute to leading-edge of core enrichment subset via Gene Set Enrichment Analysis (GSEA) demonstrating higher expression of immune regulatory molecules at the PM interface compared to the PM interior. Expression levels were converted into heatmaps and colors quantitatively correspond to fold changes. FDR=GSEA false-discovery rate q-value.B. Heatmap illustrating coefficients of xCell analysis shows higher expression of markers of cytotoxicity and activation, as well as multiple checkpoint molecules, at the PM interface, with evidence that they are being contributed chiefly by T cells. Intensity represents xCell coefficient, which corresponds to the amount that a particular region (PM interior or PM interface) or cell population (T cells, B cells, or myeloid cells) contributes to the expression of a specific gene.
Genes related to dendritic cells and MDSCs at PM interfaceA. By GSEA, genes associated with multiple subclasses of antigen-presenting dendritic cells are significantly upregulated at the PM interface (cDC1=conventional type 1 dendritic cell; cDC2=conventional type 2 dendritic cell; pDC=plasmacytoid dendritic cell; moDC=monocyte-derived dendritic cell). FDR=GSEA false-discovery rate q-value.B. Heatmap shows heightened expression of cytokines, chemokines and endothelin transcripts associated with development, recruitment and maintenance of PMNs and granulocytic MDSCs at the PM interface compared to the PM interior.
Markers of immune TME at PM interface correlate with PFSA. Hazard ratios for immunohistochemistry markers at the PM interface as they relate to PFS. For absolute count biomarkers (CD3, CD8, Foxp3, PD-1, CD163, and LAG-3) the unit is per 100 cells, and for percentage biomarkers (PD-L1, CSF1R, TIM-3, and IDO1) the unit is per 1%.B. Hazard ratios for gene sets at the PM interface as they relate to PFS. NS=p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001
Conclusions In contrast to primary bone osteosarcoma ‘immune deserts,’ osteosarcoma PMs represent an ‘immune-excluded’ TME where immune cells are present but are halted at the PM interface. TILs can produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs that prevent TILs from penetrating into the PM interior, including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with PFS of patients, which could be potentially manipulated for successful immunotherapy.
Ethics Approval This study was approved by Johns Hopkins University’s Ethics Board, approval number FWA00005752.
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