Using the Spleen as anIn VivoSystemic Immune Barometer Alongside Osteosarcoma Disease Progression and Immunotherapy withα-PD-L1

Abstract

Indications for immunotherapies are still unclear, and there is a great need for real-time patient immune status monitoring. In this study, we confirmed that the local and systemic immune profiles of an orthotopic osteosarcoma model with or without luciferase transfection were statistically equivalent. Next, we used flow cytometry to describe systemic immune cell populations influenced by osteosarcoma disease progression. When compared to vehicle-inoculated sham mice, it was found that tumor-bearing mice had significant immunophenotype disturbances at approximately 11 weeks after inoculation (at which time 90% of primary tumor-bearing mice have fulminant pulmonary metastases). Percent populations of natural killer cells and T regulatory cells were increased in the spleens of tumor-bearing mice (p < 0.0083) compared to shams. Additionally, T lymphocytes from spleens of tumor-bearing mice showed increased Tim-3/PD-1 exhaustion status (p < 0.0083). There were also increases in the percent populations of myeloid cells and overall M1/M2 macrophage marker expression on tumor-bearing mice spleens versus controls (p < 0.00714). Finally, treatment with 20 μg α-PD-L1 decreased T-cell exhaustion back to sham status, with a corresponding increase in CTLA-4 expression on cytotoxic T cells in the majority of mice tested. Checkpoint inhibition also increased splenic monocyte maturation and returned macrophage M1/M2 marker expression back to sham status. These data suggest that cancer induces systemic immune dysregulation and that these changes may be elucidated and utilized for treatment purposes by sampling the systemic immune environment via the spleen. In addition, treatment with the checkpoint inhibitor α-PD-L1 may neutralize but not overcome the systemic immunological changes induced by a progressing malignancy.

Macrophages in Osteosarcoma Immune Microenvironment: Implications for Immunotherapy

“…The progression and metastasis of osteosarcoma may induce an imbalance of macrophage subtype populations ( 36 ). It was reported that M2-like macrophage marker molecules, including CD206, Arg-1, and Ym-1, were significantly upregulated in the osteosarcoma tissues compared with non-tumor tissues ( 37 ).…”

“…Similarly, it has been reported that anti-PD-L1 treatment blocks the PD-L1 signaling pathway, promoting macrophage proliferation and activation, leading to pro-inflammatory macrophage phenotypes ( 141 ). In an osteosarcoma mice model, the PD-L1 inhibitor also promoted monocyte maturation and returned macrophage M1/M2 marker expression to nearly normal status ( 36 ).…”

PD‑L1/PD‑1 axis serves an important role in natural killer cell‑induced cytotoxicity in osteosarcoma

“…The association between the host immune system, the type of cancer and cancer treatment applied is extremely complex (29,30). Immunotherapies that include PD-1/PD-L1 blockade have shown prolonged clinical activity against various human malignancies excluding OS (31), despite the fact that there is evidence that PD-L1 contributes to OS progression in animal experiments (32). As the main components of the innate immune system, NK cells can kill tumor cells or infected cells directly, independent of antigen presenting cells.…”

“…Utilizing reverse transcription-quantitative polymerase chain reaction (RT-qPCR), PD-L1 mRNA expression was determined to be associated with lymphocyte infiltration (31). PD-L1 blockade in a mouse model of OS revealed the initial regression of the tumor followed by growth of PD-L1 antibody-resistant clones (32). PD-L1 expression was significantly linked to poor 5-year survival rates (33).…”

Cross‐talk between tumors at anatomically distinct sites

“…Thus, changes in the TME and immunity against a tumor are not just confined to the tumor, and/or the tumor‐draining lymph nodes, but can cause systemic immune dysfunction. In this light, a study of osteosarcoma found that T cells in the spleen of tumor‐bearing mice showed an exhaustion status [28]. In addition, there was an increase in the percent populations of Tregs and MDSCs in the spleens of the tumor‐bearing mice.…”