The treatment of breast cancer bone metastasis is an unresolved clinical challenge, mostly because currently therapeutic approaches cannot simultaneously block the tumor growth and repair the osteolytic bone injuries at the metastatic site. Herein, the study develops a novel nanomedicine to treat breast cancer bone metastasis. The nanomedicine is based on phosphate ion-responsive and calcium peroxide-based nanoparticles carrying the bone-targeting agent zoledronic acid on the surface and loaded with the photosensitizer indocyanine green. Following intravenous administration to a mouse model of breast cancer bone metastasis, the nanoparticles efficiently accumulate at the bone metastasis site, react with free phosphate ions, and form hydroxyapatite nanoaggregates and O2, while releasing the photosensitizer. Hydroxyapatite nanoaggregates elicit the remineralization of the collagenous bone matrix and trigger tumor cell apoptosis. Upon irradiating tumor-bearing legs with an 808 nm laser source, the O2 and free photosensitizer produced 1O2 by the reaction of the nanoparticles with phosphate ions, further boosting the anti-tumor effect. Tumor killing hampers the vicious cycle at the site of bone metastasis, translating to osteolysis blockade and further encouraging the remineralization of bone matrix. This work sheds light on the development of a novel, safe, and efficient approach for the treatment of breast cancer bone metastasis.

Breast cancer bone metastasis is a terminal-stage disease and is typically treated with radiotherapy and chemotherapy, which causes severe side effects and limited effectiveness. To improve this, Sonodynamic therapy may be a more safe and effective approach in the future. Bacterial outer membrane vesicles (OMV) have excellent immune-regulating properties, including modulating macrophage polarization, promoting DC cell maturation, and enhancing anti-tumor effects. Combining OMV with Sonodynamic therapy can result in synergetic anti-tumor effects. Therefore, we constructed multifunctional nanoparticles for treating breast cancer bone metastasis. We fused breast cancer cell membranes and bacterial outer membrane vesicles to form a hybrid membrane (HM) and then encapsulated IR780-loaded PLGA with HM to produce the nanoparticles, IR780@PLGA@HM, which had tumor targeting, immune regulating, and Sonodynamic abilities. Experiments showed that the IR780@PLGA@HM nanoparticles had good biocompatibility, effectively targeted to 4T1 tumors, promoted macrophage type I polarization and DC cells activation, strengthened anti-tumor inflammatory factors expression, and presented the ability to effectively kill tumors both in vitro and in vivo, which showed a promising therapeutic effect on breast cancer bone metastasis. Therefore, the nanoparticles we constructed provided a new strategy for effectively treating breast cancer bone metastasis.

Breast cancer is the most prevalent malignant tumor affecting women\'s health. Bone is the most common distant metastatic organ, worsening the quality of life and increasing the mortality of patients. Early detection of breast cancer bone metastasis is urgent for halting disease progression and improving tumor prognosis. Recently, extracellular matrix (ECM) with biomimetic tissue niches opened a new avenue for tumor models in vitro. Here, we developed a biomimetic decellularized ECM (dECM) system to recapitulate bone niches at different situations, bone mimetic dECM from osteoblasts (BM-ECM) and bone tumor mimetic dECM from osteosarcoma cells (OS-ECM). The two kinds of dECMs exhibited distinct morphology, protein composition, and distribution. Interestingly, highly metastatic breast cancer cells tended to adhere and migrate on BM-ECM, while lowly metastatic breast cancer cells preferred the OS-ECM niche. Epithelial-to-mesenchymal transition was a potential mechanism to initiate the breast cancer cell migration on different biomimetic dECMs. Importantly, in the nude mice model, the dECM system captured metastatic breast cancer cells as early as 10 days after orthotopic transplantation in mammary gland pads, with higher signal on BM-ECM than that on OS-ECM. Collectively, the biomimetic dECM system might be a promising tumor model to distinguish the metastatic ability of breast cancer cells in vitro and to facilitate early detection of metastatic breast cancer cells in vivo, contributing to the diagnosis of breast cancer bone metastasis.

Bone metastasis (BM) is one of the main manifestations of advanced breast cancer (BC), causing complications such as pathological fractures, which seriously affects the quality of life of patients and even leads to death. In our study, a global single-cell landscape of the tumor microenvironment was constructed using single cell RNA sequencing data from BM. BC cells were found to be reduced in the BM, while mesenchymal stem cells (MSCs), Fibroblasts and other cells were significantly more abundant in the BM. The subpopulations of these cells were further identified, and the pathways, developmental trajectories and transcriptional regulation of different subpopulations were discussed. The results suggest that with the development of BM, BC cells were vulnerable to oxidative damage, showing a high level of oxidative stress, which played a key role in cell apoptosis. Fibroblasts were obviously involved in the biological processes (BPs) related to ossification and bone remodeling, and play an important role in tumor cell inoculation to bone marrow and growth. MSC subpopulations were significantly enriched in a number of BPs associated with bone growth and development and oxidative stress and may serve as key components of BC cells homing and adhesion to the ecological niche of BM. In conclusion, our research results describe the appearance of tumor microenvironment cell subpopulations in breast cancer patients, reveal the important role of some cells in the balance of BM bone remodeling and the imbalance of BM development, and provide potential therapeutic targets for BM.

Bone is one of the prone metastatic sites of patients with advanced breast cancer. The \"vicious cycle\" between osteoclasts and breast cancer cells plays an essential role in osteolytic bone metastasis from breast cancer. In order to inhibit bone metastasis from breast cancer, NIR-II photoresponsive bone-targeting nanosystems (CuP@PPy-ZOL NPs) are designed and synthesized. CuP@PPy-ZOL NPs can trigger the photothermal-enhanced Fenton response and photodynamic effect to enhance the photothermal treatment (PTT) effect and thus achieve synergistic anti-tumor effect. Meanwhile, they exhibit a photothermal enhanced ability to inhibit osteoclast differentiation and promote osteoblast differentiation, which reshaped the bone microenvironment. CuP@PPy-ZOL NPs effectively inhibited the proliferation of tumor cells and bone resorption in the in vitro 3D bone metastases model of breast cancer. In a mouse model of breast cancer bone metastasis, CuP@PPy-ZOL NPs combined with PTT with NIR-II significantly inhibited the tumor growth of breast cancer bone metastases and osteolysis while promoting bone repair to achieve the reversal of osteolytic breast cancer bone metastases. Furthermore, the potential biological mechanisms of synergistic treatment are identified by conditioned culture experiments and mRNA transcriptome analysis. The design of this nanosystem provides a promising strategy for treating osteolytic bone metastases.

Background: During a developmental process, embryos employ varying tactics to remove unwanted cells. Using a procedure analogous to some of the embryonic cells, we generated a tumor-eliminating conditioned medium (CM) from AMPK-inhibited lymphocytes and monocytes in peripheral blood mononuclear cells (PBMCs). Methods: AMPK signaling was inhibited by the application of a pharmacological agent, Dorsomorphin, and the therapeutic effects of their conditioned medium (CM) were evaluated using in vitro cell cultures, ex vivo breast cancer tissues, and a mouse model of mammary tumors and tumor-induced osteolysis. The regulatory mechanism was evaluated using mass spectrometry-based proteomics, Western blotting, immunoprecipitation, gene overexpression, and RNA interference. Results: While AMPK signaling acted mostly anti-tumorigenic, we paradoxically inhibited it to build induced tumor-suppressing cells and their tumor-eliminating CM. In a mouse model of breast cancer, the application of AMPK-inhibited lymphocyte-derived CM reduced mammary tumors additively to a chemotherapeutic agent, Taxol. It also prevented bone loss in the tumor-bearing tibia. Furthermore, the application of CM from the patient-derived peripheral blood diminished ex vivo breast cancer tissues isolated from the same patients. Notably, proteins enriched in CM included Moesin (MSN), Enolase 1 (ENO1), and polyA-binding protein 1 (PABPC1), which are considered tumorigenic in many types of cancer. The tumor-suppressing actions of MSN and ENO1 were at least in part mediated by Metadherin (Mtdh), which is known to promote metastatic seeding. Conclusion: We demonstrated that PBMCs can be used to generate tumor-suppressive proteomes, and extracellular tumor-suppressing proteins such as MSN, ENO1, and PABPC1 are converted from tumor-promoting factors inside cancer cells. The results support the possibility of developing autologous blood-based therapy, in which tumor-suppressing proteins are enriched in engineered PBMC-derived CM by the inhibition of AMPK signaling.

BACKGROUND: Bone metastasis of breast cancer could lead to serious osteolysis and severe pain. This study is aimed to investigate the existence of necroptosis, a new type of programmed cell necrosis pathway, in breast cancer-induced osteoblast cell death.
METHODS: In this study, conditioned medium (CM) of breast cancer cells was prepared to simulate the micro-environment of bone metastasis in breast cancer in vitro and co-cultured with osteoblast. Then the percentage of cell survival and death was detected via cell viability and flow cytometry. Western blot and PCR were taken to measure protein and mRNA expression level of RIPK 3, MLKL and caspase 3 respectively.
RESULTS: CM could induce osteoblasts death, including apoptosis and necroptosis and necrostatin-1 plus Z-IETD-FMK could decrease the percentage of death cells significantly in the flow cytometry detection. Moreover, CM could increase cleaved caspase 3, RIPK 3 and p-MLKL significantly, while RIPK 3 and p-MLKL was reduced statistically when osteoblasts were treated with Necrostatin-1 (Nec-1). In addition, the mRNA level of three proteins was not consistent with the change of their corresponding protein level.
CONCLUSIONS: In conclusion, the necroptosis pathway exists in osteoblast cell death pathway induced by breast cancer cells and could be inhibited by Necrostatin-1 (Nec-1).

Breast cancer bone metastases may block normal bone remodeling and promote bone degradation, during which several signaling pathways and small non-coding miRNAs might all play a role. miRNAs and target mRNAs that might be associated with breast cancer bone metastasis were analyzed and selected using bioinformatics analyses based on online data. The 3\' untranslated region of key factors associated with breast cancer metastasis were examined for candidate miRNA binding site using Targetscan. The predicted binding was validated. The specific effects of single miRNA and dynamic effects of the miRNA-mRNA axis on breast cancer cell metastasis were investigated. miR-556-5p was downregulated in breast cancer samples according to online datasets and experimental analyses. In breast cancer cells, miR-556-5p overexpression inhibited, whereas miR-556-5p inhibition promoted cancer cell invasion and migration. Among key factors associated with breast cancer bone metastasis, parathyroid hormone related protein (PTHrP) 3\'UTR possessed miR-556-5p binding site. Through direct binding, miR-556-5p negatively regulated PTHrP expression. In breast cancer cell lines, miR-556-5p inhibition promoted, whereas PTHrP silencing suppressed cancer cell migration, invasion, and epithelial-mesenchymal transition; the effects of miR-556-5p inhibition were partially reversed by PTHrP silencing. In summary, miR-556-5p targets PTHrP to modulate the cell migration and invasion of breast cancer.

Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes\' resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.

Osteolytic bone destruction is found in approximately 60% of advanced breast cancer patients. With the pathogenesis of bone metastasis being unclear, traditional antiresorptive therapeutic strategies might not be ideal for treatment. The Wnt pathway is a highly organized cascade involved in multiple stages of cancer bone metastasis, and Wnt-targeted therapeutic strategies have shown promise in achieving favorable outcomes. In this review, we summarize the current progress of pharmacological Wnt modulators against breast cancer bone metastasis, discuss emerging therapeutic strategies based on Wnt pathway-related targets for bone therapy, and highlight opportunities to better harness the Wnt pathway for bone metastasis therapeutics to further reveal the implications of the Wnt pathway in bone metastasis pathology and provide new ideas for the development of Wnt-based intervention strategies against breast cancer bone metastasis.