Abstract 6199: Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases
Authors: Arellano Danna L. , Corral-Avila Juan A. , Drescher Florian , Verdugo-Meza Andrea , Jimenez Samanta , Olvera-Rodriguez Felipe , Juarez Patricia , Fournier Pierrick 
Published: September 2020

Abstract
Bone metastases are a frequent complication in patients with advanced breast cancer and remain incurable. Thus, we need to explore new options for their treatment. Immunotherapy for the treatment of cancer is promising but remains untested for bone metastases. A potential limitation for such use is the ability of T cells to activate osteoclasts and bone resorption that releases pro-metastatic growth factors. Thus, we aim to characterize the effect of T cells in bone metastases. When inoculated in Balb/C SCID mice that lack T and B cells or in T cell-depleted Balb/C mice, 4T1 cells caused 72% and 38% less bone metastases, respectively (p&lt;0.05), when compared to control mice. Histology confirmed that mice with a functional immune system have an increased number of osteoclasts at the tumor/bone interface. Consistently, the addition of mouse T cells to bone marrow culture ex vivo increased the formation of osteoclasts. However, this behavior was limited to non-activated T cells. Upon activation with antibodies against CD3 and CD28, T cells inhibited the formation of osteoclasts in a dose-dependent manner. This anti-osteoclastic effect was not due to a cytotoxic effect of T cells, and was consistent with decreased levels of the pro-osteoclastic Rankl and increased expression of anti-osteoclastic Ifng and Il4. When measured in vivo, Ifng was not detected, and levels of pro-osteoclastic Rankl and Tnfa were higher in T cells from 4T1 bone metastases compared to splenic T cells, suggesting that T cells in the bone marrow are not activated. Flow cytometry confirmed that only up to 15% of T cells in bone metastases expressed the activation marker CD69. Interestingly, T cells in the bone marrow could not be further activated ex vivo, unlike T cells from the spleen. This could be due to increased levels of the immunosuppressive monocytic (CD11b+Ly6C+Ly6G−) and polymorphonuclear (CD11b+Ly6G+Ly6C−) Myeloid Derived Suppressor Cells (MDSC) in bone metastases. To activate T cells in vivo, we attempted to target MDSCs using the PDE-5 inhibitor, Sildenafil, in combination with zoledronate, an FDA-approved bone resorption inhibitor. Still, there were no effects on MDSCs or the development of bone metastases. Therefore, we sought other therapeutic targets, such as immune checkpoints. Although only ~27% of T cells in 4T1 bone metastases were CTLA-4+, &gt;70% of T cells were PD-1+, and ~80% of monocytic-MDSC were PD-L1+, which could trigger T cell suppression in bone. In conclusion, we found that the bone metastasis microenvironment suppresses T cells, making them pro-osteoclastic and increases the development of bone metastases. We also found that activation of T cells prevented the formation of osteoclasts. Therefore, the activation of T cells using immunotherapy, such as immune checkpoint inhibitors (anti-PD-1 or anti-PD-L1 antibodies), could be used for the treatment of patients with bone metastases.
Citation Format: Danna L. Arellano, Juan A. Corral-Avila, Florian Drescher, Andrea Verdugo-Meza, Samanta Jimenez, Felipe Olvera-Rodriguez, Patricia Juarez, Pierrick Fournier. Bone microenvironment-suppressed T cells increase osteoclast formation and breast cancer bone metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6199.