How cells establish and dynamically change polarity are general questions in cell biology. Cells of the rod-shaped bacterium Myxococcus xanthus move on surfaces with defined leading and lagging cell poles. Occasionally, cells undergo reversals, which correspond to an inversion of the leading-lagging pole polarity axis. Reversals are induced by the Frz chemosensory system and depend on relocalization of motility proteins between the poles. The Ras-like GTPase MglA localizes to and defines the leading cell pole in the GTP-bound form. MglB, the cognate MglA GTPase activating protein, localizes to and defines the lagging pole. During reversals, MglA-GTP and MglB switch poles and, therefore, dynamically localized motility proteins switch poles. We identified the RomR response regulator, which localizes in a bipolar asymmetric pattern with a large cluster at the lagging pole, as important for motility and reversals. We show that RomR interacts directly with MglA and MglB in vitro. Furthermore, RomR, MglA, and MglB affect the localization of each other in all pair-wise directions, suggesting that RomR stimulates motility by promoting correct localization of MglA and MglB in MglA/RomR and MglB/RomR complexes at opposite poles. Moreover, localization analyses suggest that the two RomR complexes mutually exclude each other from their respective poles. We further show that RomR interfaces with FrzZ, the output response regulator of the Frz chemosensory system, to regulate reversals. Thus, RomR serves at the functional interface to connect a classic bacterial signalling module (Frz) to a classic eukaryotic polarity module (MglA/MglB). This modular design is paralleled by the phylogenetic distribution of the proteins, suggesting an evolutionary scheme in which RomR was incorporated into the MglA/MglB module to regulate cell polarity followed by the addition of the Frz system to dynamically regulate cell polarity.

Immunotherapies use components of the immune system, such as T cells, to fight cancer cells, and are changing cancer treatment, causing durable responses in some patients. Bone metastases are a debilitating complication in advanced breast and prostate cancer patients. Approved treatments fail to cure bone metastases or increase patient survival and it remains unclear whether immunotherapy could benefit patients. The bone microenvironment combines various immunosuppressive factors, and combined with T cell products could increase bone resorption fueling the vicious cycle of bone metastases. Using syngeneic mouse models, our study revealed that bone metastases from 4T1 breast cancer contain tumor-infiltrating lymphocyte (TILs) and their development is increased in normal mice compared to immunodeficient and T-cell depleted mice. This effect seemed caused by the TILs specifically in bone, because T-cell depletion increased 4T1 orthotopic tumors and did not affect bone metastases from RM-1 prostate cancer cells, which lack TILs. T cells increased osteoclast formation ex vivo and in vivo contributing to bone metastasis vicious cycle. This pro-osteoclastic effect is specific to unactivated T cells, because activated T cells, secreting interferon γ (IFNγ) and interleukin 4 (IL-4), actually suppressed osteoclastogenesis, which could benefit patients. However, non-activated T cells from bone metastases could not be activated in ex vivo cultures. 4T1 bone metastases were associated with an increase of functional polymorphonuclear and monocytic myeloid-derived suppressor cells (MDSCs), potent T-cell suppressors. Although effective in other models, sildenafil and zoledronic acid did not affect MDSCs in bone metastases. Seeking other therapeutic targets, we found that monocytic MDSCs are more potent suppressors than polymorphonuclear MDSCs, expressing programmed cell death receptor-1 ligand (PD-L1) + in bone, which could trigger T-cell suppression because 70% express its receptor, programmed cell death receptor-1 (PD-1). Collectively, our findings identified a new mechanism by which suppressed T cells increase osteoclastogenesis and bone metastases. Our results also provide a rationale for using immunotherapy because T-cell activation would increase their anti-cancer and their anti-osteoclastic properties.

Breast cancer (BCa) cells disseminating to the bone can remain dormant and resistant to treatments for many years until relapsing as bone metastases. The tyrosine kinase receptor TIE2 induces the dormancy of hematopoietic stem cells, and could also induce the dormancy of BCa cells. However, TIE2 is also a target for anti-angiogenic treatments in ongoing clinical trials, and its inhibition could then restart the proliferation of dormant BCa cells in bone. In this study, we used a combination of patient data, in vitro, and in vivo models to investigate the effect of TIE2 in the dormancy of bone metastases. In BCa patients, we found that a higher TIE2 expression is associated with an increased time to metastases and survival. In vitro, TIE2 decreased cell proliferation as it increased the expression of cyclin-dependent kinase inhibitors CDKN1A and CDKN1B and arrested cells in the G0/G1 phase. Expression of TIE2 also increased the resistance to the chemotherapeutic 5-Fluorouracil. In mice, TIE2 expression reduced tumor growth and the formation of osteolytic bone metastasis. Together, these results show that TIE2 is sufficient to induce dormancy in vitro and in vivo, and could be a useful prognostic marker for patients. Our data also suggest being cautious when using TIE2 inhibitors in the clinic, as they could awaken dormant disseminated tumor cells.

The stability, binding, and tissue penetration of variable new-antigen receptor (VNAR) single-domain antibodies have been tested as part of an investigation into their ability to serve as novel therapeutics. V13 is a VNAR that recognizes vascular endothelial growth factor 165 (VEGF165). In the present study V13 was used as a parental molecule into which we introduced mutations designed in silico. Two of the designed VNAR mutants were expressed, and their ability to recognize VEGF165 was assessed in vitro and in vivo. One mutation (Pro98Tyr) was designed to increase VEGF165 recognition, while the other (Arg97Ala) was designed to inhibit VEGF165 binding. Compared to parental V13, the Pro98Tyr mutant showed enhanced VEGF165 recognition and neutralization, as indicated by inhibition of angiogenesis and tumor growth. This molecule thus appears to have therapeutic potential for neutralizing VEGF165 in cancer treatment.

Bone metastases is a frequent (>75%) complication of advanced breast cancer (BCa). BCa cells in bone are supported by cytokines released during the osteoclastic resorption, and remain untreatable. T cells during immunotherapy could turn against BCa cells in bone but could also increase osteoclasts and bone metastases. Comparing the development of bone metastases from 4T1 BCa cells between Balb/C and SCID or T cell-depleted mice, the absence of T cells decreased bone metastases (−72% and −38%, respectively), while orthotopic tumors were increased. Histology confirmed that the osteoclast number was decreased at the tumor-bone interface of mice lacking T cells. Ex vivo addition of non-activated T cells increased osteoclast formation, and flow cytometry confirmed that >85% of T cells in bone metastases were not expressing activation markers. In sharp contrast, activated T cells were potent inhibitors of osteoclast formation, which could be beneficial for the treatment of patients. Although activated T cells expressed the anti-osteoclastic Ifng and Il4, their effect was not reversed by neutralizing antibodies, or due to a bystander effect by cytotoxic T cells. Thus, we sought to activate T cells of bone metastases. However, it was not possible to activate T cells in bone marrow cultures ex vivo. This could be due to an increase of MDSCs in 4T1 bone metastases, including M-MDSCs that were PD-L1+ (>85%) and could suppress PD-1+ T cells (>70%) in bone. Our results suggest that while T cells under the influence of the bone microenvironment promote BCa bone metastases, immunotherapy-activated T cells could suppress bone resorption and eliminate BCa cells in patients with bone metastases.

Bone metastases are a frequent complication of breast cancer and cannot be cured. Immunotherapy could be used for their treatment. However, its efficiency could be limited by the ability of T cells to increase bone resorption that supports cancer cell proliferation and bone metastases. To characterize the effect of T cells on bone metastases, we used a syngeneic mouse model with 4T1 breast cancer cells. When inoculated in normal mice, 4T1 caused more osteolysis as measured on x-rays, compared to SCID and T cell-depleted mice (253% and 61% more, respectively). Histology confirmed that normal mice have an increased tumor burden and number of osteoclasts at the tumor/bone interface compared to SCID mice. T cells isolated from bone metastases suppressed osteoclast formation ex vivo, in contrast with in vivo data. However, this effect was due to the ex vivo activation of T cells, consistent with decreased levels of Rankl and increased expression of anti-osteoclastic Ifng and Il4 mRNA in activated T cells. In vivo, Ifng was not detected, and levels of pro-osteoclastic Rankl and Tnfa were higher in T cells from bone metastases, suggesting they are pro-osteoclastic, unlike activated T cells. Culturing bone metastasis T cells ex vivo with ConA or anti-CD3/CD28, failed to increase the expression of the activation marker CD69, confirming the presence of suppressive factors in this microenvironment. Accordingly, there was an increase of MDSCs, including monocytic MDSCs that are PD-L1+ (87%) in 4T1 bone metastases. They could suppress the activation of T cells that are PD-1+ (71%) in bone metastases. These results suggest that unactivated T cells increase bone metastases and their activation by immunotherapy could be used for the treatment of bone metastases.

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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<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+, >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.