Abstract

Background: A hallmark of multiple myeloma (MM) is predominant localization in the BM and a propensity for progression from primary bone sites to new bone sites, even in distant bone. Recently, we discovered that MM cells can induce osteocyte apoptosis locally and at distant bone sites prior to the arrival of metastatic MM cells. To investigate whether osteocyte apoptosis at distant bone sites contributes to MM metastasis in these areas, we developed a unique syngenic animal model of murine MM in which osteocyte apoptosis can be induced through targeted expression of human diphtheria toxin receptor (DTR) in osteocytes and subsequent injection of DT. Our initial experiments demonstrated that 5TGM1 murine MM cells home to bone significantly faster and grow larger tumors in the bone of osteocyte-ablated (DTR+DT) mice than in control (DTR+PBS) mice, suggesting that osteocyte apoptosis in new bone sites is responsible for MM spread and progression in these areas. MM dissemination requires a receptive environment in bone. In normal bone marrow (BM), immune surveillance, especially dendritic cell (DC) identification and capture of tumor-specific antigens and presentation thereof to T cells, leads to anti-tumor-specific T cell activation and tumor killing. However, the function of DCs and cytotoxic T cells (CD8+ T cells) can be inhibited by immune suppressor cells, such as myeloid-derived suppressor cells (MDSCs) and regulatory T and B cells (Tregs and Bregs, respectively). In this study, we determined whether osteocyte apoptosis promotes MM metastasis to and progression in new bone sites by suppressing immune surveillance in BM.

Methods: Male and female 8-week-old syngenic DTR mice were initially injected intraperitoneally with DT (12.5 µg/kg) to induce apoptosis in osteocytes or with PBS as a control. After 7 days, 2×106 luciferase-expressing 5TGM1 MM cells were injected via lateral tail vein (n=12 per group). Tumor homing and growth were monitored by weekly bioluminescent imaging and ELISA for serum IgG2bκ (a soluble marker of 5TGM1 cells). Four weeks after tumor injection, we harvested the MM, BM, blood, and spleen cells from six mice in each group, stained the cells with fluorescent antibodies against MM cells and multiple immune cell populations, and analyzed the populations by flow cytometry. Bones (tibia and femurs) were collected from the other six mice in each group and sectioned for immunostaining with cell-specific antibodies. Because physical interaction between tumor cells and T cells is necessary for tumor killing, we stained for total T cells (CD3+), cytotoxic T cells (CD8+), and MDSCs in bone sections containing tumors to determine the localization of T cells and immunosuppressive MDSCs.

Results: Compared with DTR+PBS mice, DTR+DT mice had a much larger tumor burden and more tumor lesions in bone. Furthermore, the BM of DTR+DT mice had (a) significantly more MM cells (CD138-positive), which had a higher proliferation rate (Ki-67-positive) and a lower apoptotic rate (fewer cleaved caspase-3-positive MM cells and more BCL-2-positive MM cells); (b) substantially more immunosuppressive cells, including MDSCs, Tregs, and B regs; (c) fewer DCs and natural killer cells; and (d) significantly reduced production of granzyme B, perforin, and IFNγ (three critical factors for tumor killing) in T cells. The peripheral blood and spleen profiles of the same immune cells showed no significant differences between groups. Interestingly, DTR+DT mice had significantly fewer tumor-infiltrating CD3+ and CD8+ T cells and more MDSCs.

Conclusions: Osteocyte apoptosis in distant bone sites creates an immunosuppressive bone microenvironment and compromises immune reactivity and response to MM metastasis, thereby promoting MM dissemination and progression. This study provides novel insights into the mechanisms of MM progression and may inform the prognosis of outcomes in patients with MM.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.