肿瘤相关巨噬细胞（TAMs）是乳腺肿瘤进展和转移中的重要因素。临床和临床前证据表明 zoledronate（ZOL）在乳腺癌转移预防中发挥了作用。此外，zoledronate 能够诱导单核细胞和巨噬细胞的炎症激活，这对癌症治疗可能是有利的。zoledronate 的固有骨向性限制了其在软组织和肿瘤中的可用性。本研究利用正位移小鼠乳腺癌模型评估了利用脂质体（EMP-LIP）将 zoledronate 定向靶向到肿瘤以修饰 TAM 激活的可能性。Triple-negative 乳腺癌4T1细胞接种于女性 Balb/c小鼠的第四乳腺脂肪垫中。根据治疗分为：车辆组、ZOL组、EMP-LIP组和脂质体封装 zoledronate (ZOL-LIP)组。治疗通过静脉注射（伴肿瘤切除）和腹腔注射（不伴肿瘤切除）进行。通过活体生物荧光成像 (IVIS) 和卡尺测量跟踪肿瘤生长。采用免疫组化和免疫荧光染色评估肿瘤浸润巨噬细胞。通过 Western Blotting 和 Taqman RT-qPCR 检测炎症转录因子和细胞因子的蛋白和 RNA 表达水平。脂质体封装 zoledronate (ZOL-LIP) 治疗抑制了 4T1 细胞的体外迁移。在体内，ZOL 和 ZOL-LIP 治疗均减少了肿瘤生长和血管生成标志物 CD34 的表达。长期的 ZOL-LIP 治疗导致转变为 M1 型巨噬细胞极化，增加 CD4 T 细胞浸润和 NF-κB 活化，表明肿瘤内炎症发生了变化，而 ZOL 治疗呈现类似但非显著的趋势。此外，与游离 ZOL 相比，ZOL-LIP 在骨中的双膦酸盐积蓄较低。结果表明，降低了骨中的双膦酸盐积蓄促进了 ZOL-LIP 的全身抗肿瘤效应，通过增加 TNBC 肿瘤中的炎症反应通过 M1 型巨噬细胞激活。

Tumour associated macrophages (TAMs) are important players in breast tumour progression and metastasis. Clinical and preclinical evidence suggests a role for zoledronate (ZOL) in breast cancer metastasis prevention. Further, zoledronate is able to induce inflammatory activation of monocytes and macrophages, which can be favourable in cancer treatments. The inherent bone tropism of zoledronate limits its availability in soft tissues and tumours. In this study we utilized an orthotopic murine breast cancer model to evaluate the possibility to use liposomes (EMP-LIP) to target zoledronate to tumours to modify TAM activation.Triple-negative breast cancer 4T1 cells were inoculated in the 4th mammary fat pad of female Balb/c mice. Animals were divided according to the treatment: vehicle, ZOL, EMP-LIP and liposome encapsulated zoledronate (ZOL-LIP). Treatment was done intravenously (with tumor resection) and intraperitoneally (without tumor resection). Tumour growth was followed by bioluminescence in vivo imaging (IVIS) and calliper measurements. Tumour-infiltrating macrophages were assessed by immunohistochemical and immunofluorescence staining. Protein and RNA expression levels of inflammatory transcription factors and cytokines were measured by Western Blotting and Taqman RT-qPCR.Liposome encapsulated zoledronate (ZOL-LIP) treatment suppressed migration of 4T1 cell in vitro. Tumour growth and expression of the angiogenic marker CD34 were reduced upon both ZOL and ZOL-LIP treatment in vivo. Long-term ZOL-LIP treatment resulted in shift towards M1-type macrophage polarization, increased CD4 T cell infiltration and activation of NF-κB indicating changes in intratumoural inflammation, whereas ZOL treatment showed similar but non-significant trends. Moreover, ZOL-LIP had a lower bisphosphonate accumulation in bone compared to free ZOL.Results show that the decreased bisphosphonate accumulation in bone promotes the systemic anti-tumor effect of ZOL-LIP by increasing inflammatory response in TNBC tumours via M1-type macrophage activation.Copyright © 2023. Published by Elsevier B.V.