巨噬细胞是肿瘤微环境中数量最多的免疫细胞，因此成为肿瘤免疫治疗的一个有吸引力的靶点。我们之前的一项研究表明，羟基磷灰石纳米粒子（HANPs）增强了巨噬细胞Toll样受体4（TLR4）的信号传导。本研究旨在调查HANPs如何在存在或不存在低毒Toll样受体4（TLR4）激动剂MPLA的情况下，操纵巨噬细胞的表型和功能来对抗4T1肿瘤。结果表明，将HANPs加入到MPLA中明显促进了细胞因子分泌和巨噬细胞向抗肿瘤M1表型的极化。此外，HANPs/MPLA共刺激的巨噬细胞所产生的上清液，与单组分处理或PBS对照组相比，能够显著促进4T1肿瘤细胞的凋亡。特别地，我们发现HANPs诱导免疫原性细胞死亡（ICD），体现为4T1细胞中“危险信号”HMGB1、CRT和ATP表达的增加。随后，HANPs处理的4T1细胞中包含ICD衍生物的上清液激活了巨噬细胞，并将其表型转向M1型。此外，在肿瘤移植小鼠模型中，与PBS对照组相比，HANPs和MPLA的联用显著延迟了肿瘤生长，这与促进巨噬细胞极化和ICD诱导有关。因此，我们的研究结果证明了调节巨噬细胞功能的潜在平台，并为了解HANPs的免疫调节效应提供了新的见解。意义声明：通过利用Toll样受体（TLR）激动剂极化巨噬细胞以实现抗肿瘤表型已被证明对肿瘤免疫治疗有效。然而，由于TLR激活与免疫细胞中的免疫抑制或耐受有关，因此TLR激动剂的免疫调节效力受到限制。在本研究中，我们引入了羟基磷灰石纳米粒子（HANPs）来与TLR4激动剂MPLA联用。结果表明，将HANPs加入到MPLA中可以促进巨噬细胞极化为抗肿瘤M1表型，支持“热”肿瘤转化，并延缓4T1肿瘤的生长。此外，我们发现HANPs诱导了免疫原性细胞死亡，并从癌细胞中产生了“危险”信号，进一步促进了巨噬细胞的极化。本研究的意义在于指导HANPs的有理设计，其可以与或不与TLR激动剂联用，用于肿瘤免疫治疗。版权所有 © 2023 Elsevier Ltd.

Macrophages represent the most prevalent immune cells in the tumor micro-environment, making them an appealing target for tumor immunotherapy. One of our previous studies showed that hydroxyapatite nanoparticles (HANPs) enhanced Toll-like receptor 4 (TLR4) signal transduction in macrophages. This study was proposed to investigate how HANPs manipulated the phenotype and function of macrophage against 4T1 tumors in the presence or absence of MPLA, a low toxic Toll-like receptor 4 (TLR4) agonist. The results demonstrated that the addition of HANPs to MPLA significantly promoted cytokine secretion and macrophage polarization toward a tumoricidal M1 phenotype. Further, the resulting supernatant from HANPs/MPLA co-stimulated macrophages enhanced 4T1 tumor cells apoptosis compared to that from macrophages treated with a single component or PBS control. In particular, we found HANPs elicited immunogenic cell death (ICD) indicated by the increased expression of "danger signals", including HMGB1, CRT and ATP in 4T1 cells. Subsequently, the ICD derivatives-containing supernatant from HANPs-treated 4T1 cells activated macrophage and shifted the phenotype of the cells toward M1 type. Moreover, in a tumor-bearing mice model, HANPs and MPLA synergistically delayed tumor growth compared to PBS control, which was positively associated with the promoted macrophage polarization and ICD induction. Therefore, our findings demonstrated a potential platform to modulate the function of macrophages, and shed a new insight into the mechanism involving the immunomodulatory effect of HANPs for tumor therapy. STATEMENT OF SIGNIFICANCE: Polarizing macrophage toward tumoricidal phenotype by harnessing Toll-like receptor (TLR) agonists has been proven effective for tumor immunotherapy. However, the immunomodulatory potency of TLR agonists is limited due to immune suppression or tolerance associated with TLR activation in immune cells. Herein, we introduced hydroxyapatite nanoparticles (HANPs) to MPLA, a TLR4 agonist. The results demonstrated that the addition of HANPs to MPLA promoted macrophage shift toward tumoricidal M1 phenotype, supported a "hot" tumor transformation, and delayed 4T1 tumor growth. Moreover, we found that HANPs elicited immunogenic cell death that produced "danger" signals from cancer cells thereby further facilitated macrophage polarization. This work is significant to direct the rational design of HANPs coupled with or without TLR agonists for tumor immunotherapy.Copyright © 2023. Published by Elsevier Ltd.