A single-cell transcriptomic landscape of innate and adaptive intratumoral immunity in triple negative breast cancer during chemo- and immunotherapies

Breast cancer (BC) constitutes a major health problem worldwide, making it the most common malignancy in women. Current treatment options for BC depend primarily on histological type, molecular markers, clinical aggressiveness and stage of disease. Immunotherapy, such as anti-PD-1, have shown combinatorial clinical activity with chemotherapy in triple negative breast cancer (TNBC) delineating some therapeutic combinations as more effective than others. However, a clear overview of the main immune cell populations involved in these treatments has never been provided. Here, an assessment of the immune landscape in the tumour microenvironment (TME) of two TNBC mouse models (4T1 and EMT6 cell lines) has been performed using single-cell RNA sequencing (scRNA-seq) technology. Specifically, immune cells were evaluated in untreated conditions and after being treated with chemotherapy or immunotherapy used as single agents or in combination. A decrease of regulatory T cells, compared to the untreated TME, was found in treatments with in vivo efficacy as well as ?d T cells, which have a pro-tumoral activity in mice. Focusing on Cd8 T cells, across all the conditions, a general increase of exhausted-like Cd8 T cells was confirmed in pre-clinical treatments with low efficacy; on the other hand, an opposite trend was found for the proliferative Cd8 T cells. Regarding macrophages, M2-like cells were found enriched in treatments with low efficacy while opposite behaviour was associated with M1-like macrophages. For both cell lines, similar proportions of B cells were detected with an increase of proliferative B cells in treatments that involved cisplatin in combination with anti-PD-1. The fine-scale characterization of the immune TME in this work can lead to new insights on the diagnosis and treatment of TNBC for a possible application at the clinical level. Overall design: Eight treatments and one untreated control for two murine tumoral cell lines (4T1 and EMT6).

乳腺癌（BC）是全球范围内的重大公共健康问题，同时也是女性群体中发病率最高的恶性肿瘤。当前乳腺癌的临床治疗方案选择，主要取决于肿瘤的组织学类型、分子标志物特征、临床侵袭性以及疾病分期。诸如抗PD-1疗法的免疫治疗，在三阴性乳腺癌（TNBC）中已展现出与化疗联合的临床活性，且已有研究证实部分联合治疗方案的疗效优于单一疗法。然而，目前尚未有研究对这类治疗中涉及的核心免疫细胞群进行系统性的全面梳理。
本研究借助单细胞RNA测序（scRNA-seq）技术，针对两种三阴性乳腺癌小鼠模型（4T1与EMT6细胞系）的肿瘤微环境（TME）免疫图谱展开了系统性评估。具体而言，研究分别对未处理对照组，以及接受单一化疗、单一免疫治疗或二者联合治疗的小鼠模型的免疫细胞特征进行了检测分析。
相较于未处理的肿瘤微环境，在体内展现出抗肿瘤疗效的治疗方案中，调节性T细胞与在小鼠体内发挥促肿瘤活性的γδ T细胞的比例均出现显著下降。针对CD8⁺ T细胞的亚群分析显示，在所有实验条件下，疗效欠佳的临床前治疗组中，耗竭样CD8⁺ T细胞的比例普遍升高；而增殖性CD8⁺ T细胞则呈现完全相反的变化趋势。
在巨噬细胞亚群层面，疗效欠佳的治疗组中M2型巨噬细胞呈现富集状态，而M1型巨噬细胞则表现出与之相反的分布特征。两种小鼠肿瘤细胞系的B细胞比例整体相似，但在顺铂联合抗PD-1的治疗方案中，增殖性B细胞的比例均出现显著升高。
本研究对肿瘤微环境免疫组分的精细表征，可为三阴性乳腺癌的临床诊断与治疗提供全新的研究视角，有望推动相关成果向临床应用转化。
实验整体设计：针对两种小鼠肿瘤细胞系（4T1与EMT6），共设置8种治疗组与1个未处理对照组。