Bone Microenvironment-Suppressed T Cells Increase Osteoclast Formation and Osteolytic Bone Metastases in Mice

Abstract

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. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Keywords: BONE METASTASIS; IMMUNOSUPRESSION; IMMUNOTHERAPY; MDSC; OSTEOCLAST; T CELLS.

Fig. 1

T cells infiltrate the osteolytic bone metastases of MHC‐I⁺ 4T1 breast cancer cells in mice. (A) Flow cytometry analysis of MHC‐I expression in mouse PBMCs, breast (4T1, PyMT‐R221A), and prostate (RM‐1, TRAMP‐C1) cancer cells, and melanoma cells (B16‐F1). The gray histogram corresponds to cells stained with an isotype control antibody and the pink one to cells stained with the antibody against MHC‐I. (B) Bone metastasis caused by B16‐F1 or 4T1 cancer cells inoculated in the left cardiac ventricle of C57BL/6 or Balb/C mice, and of RM‐1 cells inoculated in the tibia of C57BL/6 mice. Results are shown as representative radiographs (arrows indicate osteolytic lesions), H&E‐stained sections (arrows indicate bone metastases), sections with TRAP staining (arrows indicate osteoclasts), and immunostaining with an anti‐CD3 antibody to detect TILs (T = tumor; B = bone matrix; arrows indicate T cells). H&E = hematoxylin and eosin; PBMC = peripheral blood mononuclear cell; TIL = tumor infiltrated lymphocyte.

Fig. 2

Bone metastases from 4T1 cells are increased in mice with a functional immune system. 4T1 cells were inoculated in the left cardiac ventricle of Balb/C SCID (n = 12) or Balb/C mice (n = 11). (A) Representative radiographs from hindlimbs, 10 days after the inoculation (arrows indicate osteolytic lesions) and quantification of the osteolysis area on radiographs. (B) Representative H&E‐stained sections of tibias (T, tumor) and histomorphometric analysis of tumor area (left graph) and bone area (right graph). (C) Representative images of bone sections after TRAP staining (arrows indicate osteoclasts) and quantification of the number of osteoclasts at the tumor bone interface. Results are shown as box plots, and were compared using an unpaired, two‐tailed Student's t test. H&E = hematoxylin and eosin.

Fig. 3

Bone metastases from 4T1 are increased in mice with T cells, whereas orthotopic 4T1 tumor are decreased in mice with T cells. 4T1 cells were inoculated in the left cardiac ventricle or the 4th left mammary fat pad of Balb/C mice to cause bone metastases or orthotopic tumors. Mice received then a treatment with anti‐CD4 and anti‐CD8 antibodies (anti‐CD4 and anti‐CD8) or an isotype control antibody (Ctrl Ab). (A) Representative radiograph from forelimbs, 10 days after the inoculation (arrows indicate osteolytic lesions) and quantification of the osteolysis area on radiographs (anti‐CD4 and anti‐CD8, n = 6; control antibody, n = 8). (B) Histomorphometric analysis of tumor area, bone area, and osteoclast number at the tumor‐bone interface of H&E stained sections of tibia of mice with 4T1 bone metastases with or without T‐cell depletion. (C) Representative picture of 4T1 orthotopic tumors grown in mice with or without T‐cell depletion and quantification of their volume (n = 7 per group). Results are shown as box plots (B,C) or as mean ± SEM, and were analyzed using an unpaired, two‐tailed Student's t test (A,B) and a two‐way ANOVA with Bonferroni's post‐test (C). H&E = hematoxylin and eosin.

Fig. 4

T cells from bone metastases increase osteoclast formation ex vivo. (A) T cells were isolated from the BM of mice with or without 4T1 bone metastases and co‐cultured with bone marrow cells in the presence of M‐CSF (25 ng/mL) and RANKL (25 ng/mL) to induce osteoclastogenesis. Results are presented as (left) representative pictures the cells and (right) the number of osteoclasts (multinucleated TRAP⁺ cells). Results are presented as box plots, and were compared using a two‐way ANOVA with a Bonferroni post‐test. (B,C) The expression of the pro‐osteoclastic genes Tnfa and Rankl and the anti‐osteoclastic genes Ifng and Il4 was measured by quantitative real‐time PCR in B. T cells isolated from the spleen or the bone marrow of mice with or without 4T1 bone metastases, or in (C) splenic T cells activated or not using anti‐CD3 and anti‐CD28 antibodies ex vivo. Results are presented as box plots, and were compared using a one‐way ANOVA with a Tukey's post‐test (B) or an unpaired, two‐tailed Student's t test (C). BM = bone marrow.

Fig. 5

Activated T cells prevent osteoclast formation ex vivo, while T cells from bone metastases are not and cannot be activated ex vivo. (A,B) T cells isolated from the spleen of normal mice were activated or not ex vivo using either ConA or anti‐CD3 and anti‐CD28 antibodies. Seven days later, T cells were added to a bone marrow culture in the presence of M‐CSF (25 ng/mL) and RANKL (25 ng/mL). The number of osteoclasts (multinucleated TRAP⁺ cells) was assessed 5 days later. (A) Representative pictures of the cells and quantification of the osteoclasts. Results are presented as the median and range number of osteoclasts and compared by using a two‐way ANOVA with a Dunnett's post‐test. (B–E) T cells from the bone marrow of Balb/C mice inoculated or not inoculated with 4T1 breast cancer cells in the left cardiac ventricle were analyzed using flow cytometry. Results are presented as (B) representative density plots of bone marrow cells to identify T cells (CD3⁺ CD90.2⁺ events), and the quantification of T cells (CD3⁺ CD90.2⁺ events) in the bone marrow and of the amount of CD62L⁻ (C) and CD69⁺ T cells (D) from in the bone marrow. (E) Splenocytes and bone marrow cells of mice with 4T1 bone metastases were treated or not treated overnight with the activator ConA or the agonistic antibodies anti‐CD3 and anti‐CD28 before analyzing the expression of CD69 on T cells, using flow cytometry. Quantitative results are presented as box plots and compared using a Mann‐Whitney test (C,D) or a two‐way ANOVA with Bonferroni post‐test (E). ConA = concanavalin A.

Fig. 6

Polymorphonuclear and monocytic MDSCs are increased in mice with 4T1 bone metastases. Mice were inoculated or not with 4T1 cells to cause bone metastases and cells were analyzed using flow cytometry 10 days later. (A) Representative pseudocolor dot plots of CD11b, Ly6G, and Ly6C expression on bone marrow cells. (B) Quantification of the amount of CD11b⁺Ly6G⁺Ly6C^lo and CD11b⁺Ly6G⁻Ly6C^hi cells in spleen and bone marrow cells (Control n = 13; 4T1 n = 14). (C,D) Quantification of the levels of (C) ROS and (D) NO as the MFI in CD11b⁺Ly6G⁺Ly6C^lo and CD11b⁺Ly6G⁻Ly6C^hi cells (Control n = 3; 4T1 n = 4). Quantitative results are presented as box plots and compared using a two‐way ANOVA with Bonferroni's post‐test. (E) Pseudocolor dot plots of Edu and CD8 levels in CD8⁺ T cells co‐cultured with CD11b⁺Ly6G⁺Ly6C^lo or CD11b⁺Ly6G⁻Ly6C^hi cells isolated from 4T1 bone metastases to assess their T‐cell suppressive function. Representative dot plots of two independent experiments. MFI = mean fluorescence intensity; NO = nitric oxide; ROS = reactive oxygen species.

Fig. 7

Expression of immune checkpoints in T cells and MDSCs from bone marrow. Bone marrow cells from mice inoculated or not inoculated with 4T1 to cause bone metastases were analyzed using flow cytometry to quantify the amount of T cells expressing (A) CTLA‐4 and (B) PD‐1 of (C) bone marrow cells, (E) PMN‐MDSCs, and (F) M‐MDSCs expressing PD‐L1 (Control n = 13; 4T1 n = 14). (D) Representation of the distribution of cells expressing PD‐L1 in 4T1 bone metastases. Results are presented as box plots and compared using a Mann‐Whitney U test (B,E) or an unpaired, two‐tailed Student's t test (A,C,F).