目的：乳腺癌作为全球女性发病率最高的恶性肿瘤，其伴随的骨代谢异常及免疫微环境失衡是影响患者治疗应答及预后的关键因素。CD8⁺T细胞耗竭是乳腺癌免疫治疗耐药的核心机制之一。本研究围绕乳腺癌骨-免疫微环境交互机制展开，通过临床回顾性研究与基础实验探索，揭示乳腺癌患者初诊时骨代谢紊乱特征，及骨代谢紊乱通过芳香烃受体（AhR）信号通路驱动CD8⁺T细胞耗竭的核心机制，并开发靶向干预策略，为优化乳腺癌全程管理提供新思路。

方法：本研究纳入636例初诊乳腺癌患者及268例健康女性做对照，通过横断面及化疗前后自身对照分析患者骨密度及骨代谢指标，了解乳腺癌患者骨代谢状况，评估骨量异常的危险因素及化疗对骨密度的动态影响。并基于TCGA数据库分析骨代谢、AhR信号通路与T细胞耗竭的相关性，结合4T1荷瘤小鼠模型，通过流式细胞术、双荧光素酶报告基因验证AhR对T细胞耗竭的调控机制。采用乙醇注入法搭配脂质体挤出器制备si-AhR/PFC@LNP-AbCD8纳米药物。

结果：乳腺癌患者存在显著骨代谢紊乱。92.3%初诊乳腺癌患者存在维生素D缺乏/不足（＜30 ng/ml），骨量异常率达74.7%（骨量减少48.0%，骨质疏松26.7%）。血清骨转换标志物（骨碱性磷酸酶、β-胶原蛋白特殊序列、总I型胶原N端前肽）随骨质流失显著升高，与骨密度呈负相关（P<0.001），提示乳腺癌患者存在骨破坏。年轻患者（<40岁）骨量异常率较同龄对照组高5倍（40.8% vs. 8.0%, P<0.001）。绝经前患者骨量异常风险为同龄对照组的9倍（OR=8.90）。三阴型乳腺癌（TNBC）骨量异常风险最高（OR=8.30），而激素受体阳性型骨质疏松风险突出（OR=3.58）。化疗会进一步加剧患者骨质流失，因化疗方案而异，且对绝经前患者影响显著（腰椎BMD下降1.46%，P<0.001），可能与化疗诱导闭经相关。加强钙/维生素D补充可减缓骨质流失。

骨代谢紊乱的产物犬尿氨酸是AhR的重要配体来源。TCGA数据库分析显示AhR通路（R=0.29, P<0.001）、缺氧相关基因（R=0.30, P<0.001）及骨吸收特征基因（R=0.41, P<0.001）均与T细胞耗竭特征基因呈显著正相关。体内外实验证实AhR激活可上调PD-1、TIM-3、LAG3等抑制性受体表达，使其转录活性提升2.48-5.07倍（P<0.001），并抑制IFN-γ/TNF-α分泌，敲低AhR或使用抑制剂CH-223191/StemRegenin 1可逆转此效应。外源性添加犬尿氨酸可激活AhR，加重T细胞耗竭。在4T1荷瘤小鼠模型中，肿瘤组织中HIF1α及抑制性受体表达较脾脏组织显著升高。基于此，我们通过靶向（AbCD8）递送siRNA抑制AhR表达，并利用全氟正己烷改善肿瘤氧供构建并制备多功能纳米药物si-AhR/PFC@LNP-AbCD，以期逆转TNBC的免疫治疗耐药，经初步验证有较好的物理表征及稳定性。

结论：本研究系统阐明乳腺癌初诊时的骨代谢状况（严重骨代谢异常和骨密度下降）及化疗对骨质的影响为乳腺癌患者的骨健康评估及个体化干预提供了重要依据。同时探索了骨代谢产物激活AhR信号通路与乏氧微环境协同诱导CD8⁺T细胞耗竭的机制，并创新性开发多功能纳米药物，为逆转TNBC免疫治疗耐药提供新策略。本研究成果为乳腺癌多维度全程管理及精准治疗奠定理论与实践基础。

Objective: As the most prevalent malignancy among women worldwide, breast cancer is critically influenced by concomitant bone metabolic disorders and immune microenvironment imbalance, which significantly impact patients’ treatment response and prognosis. CD8⁺T cell exhaustion is one of the core mechanisms of immunotherapy resistance in breast cancer. This study focused on the interaction between bone metabolism and the immune microenvironment in breast cancer, aiming to characterize bone metabolic abnormalities at initial diagnosis and elucidate the mechanism by which these disturbances drive CD8⁺T cell exhaustion via the aryl hydrocarbon receptor (AhR) pathway. Additionally, we developed targeted therapeutic strategies to optimize comprehensive breast cancer management.

Methods: A cross-sectional retrospective study was conducted on 636 newly diagnosed breast cancer patients and 268 healthy women to evaluate bone mineral density (BMD) and bone metabolism indexes, identifying risk factors for bone abnormalities and dynamic effects of chemotherapy on BMD. Bioinformatics analysis of TCGA data explored correlations among bone metabolism, AhR signaling, and T cell exhaustion. Mechanistic validation was conducted using 4T1 tumor-bearing mouse models, flow cytometry, and dual-luciferase reporter assays. The si-AhR/PFC@LNP-AbCD8 liposomal nanomedicine was prepared via ethanol injection and liposome extrusion.

Results: Breast cancer patients exhibit significant bone metabolism disturbances. At initial diagnosis, 92.3% of patients demonstrated vitamin D insufficiency/deficiency (<30 ng/ml), with 74.7% presenting abnormal bone mass (osteopenia 48.0%, osteoporosis 26.7%). Bone turnover markers - including bone-specific alkaline phosphatase, β-collagen telopeptide, and procollagen type I N-terminal propeptide - showed significant elevation with progressive bone loss, demonstrating an inverse correlation with BMD (all P < 0.001). Younger patients (<40 years) manifested a fivefold higher incidence of bone abnormalities compared to age-matched controls (40.8% vs. 8.0%, P < 0.001), while premenopausal patients exhibiting a ninefold increased risk (adjusted OR = 8.90). Molecular subtype analysis revealed triple-negative breast cancer carried the highest overall risk for bone abnormalities (adjusted OR = 8.30), whereas hormone receptor-positive subtypes demonstrated specific susceptibility to osteoporosis (adjusted OR = 3.58). Chemotherapy regimens exacerbated bone loss, particularly in premenopausal patients who experienced significant lumbar spine BMD reduction (-1.46%, P < 0.001), a phenomenon strongly associated with treatment-induced amenorrhea. Targeted calcium and vitamin D supplementation demonstrated efficacy in attenuating bone mass deterioration.

TCGA analysis revealed significant correlations among AhR signaling (R=0.29), hypoxia (R=0.30), bone resorption markers (R=0.41), and T cell exhaustion signatures (P<0.001). AhR activation upregulated inhibitory receptors (PD-1, TIM-3, LAG3; transcriptional activity increased 2.48–5.07-fold, P<0.001) and suppressed IFN-γ/TNF-α secretion. These effects were reversed by AhR knockdown or inhibitors (CH-223191/StemRegenin 1). Bone metabolite (kynurenine) acts as an AhR ligand and can increase T cell exhaustion. In 4T1 tumor-bearing mouse model, the expression of HIF1α and inhibitory receptors in tumor tissue was significantly higher than that in spleen tissue. Based on this, we developed and synthesized a multifunctional nanomedicine, si-AhR/PFC@LNP-AbCD8, by delivering siRNA targeting AhR and incorporating perfluoro-n-hexane to enhance tumor oxygenation. Preliminary evaluations demonstrated favorable physicochemical properties and stability.

Conclusion: This study systematically delineates the characteristics of bone metabolic disorders in newly diagnosed breast cancer patients and the detrimental effects of chemotherapy on bone health, providing a foundation for individualized bone management. Furthermore, we uncovered a mechanism by which bone-derived metabolites activate AhR signaling, synergizing with hypoxia to induce CD8⁺T cell exhaustion. A innovative nanomedicine developed herein offers a promising strategy to overcome immunotherapy resistance in triple-negative breast cancer. These findings advance multidimensional management and precision therapy for breast cancer.