Combination therapies to improve the efficacy of immunotherapy in triple-negative breast cancer [ATAC-seq]. Combination therapies to improve the efficacy of immunotherapy in triple-negative breast cancer [ATAC-seq]

Immune checkpoint inhibitors combined with chemotherapy represent a promising treatment option in triple-negative breast cancer (TNBC). However, response rates are still relatively low necessitating the design of novel therapeutic strategies to improve clinical outcomes. Here, we describe a triple combination of anti-PDL-1 immune checkpoint blockade, epigenetic modulation thorough BET bromodomain inhibition, and chemotherapy with paclitaxel that effectively inhibits both primary and metastatic tumor growth in two different syngeneic murine breast cancer models. Detailed cellular and molecular profiling of tumors from single and combination treatment arms revealed increased T and B cell infiltration and macrophage reprogramming from M1 to a M2 phenotype in mice treated with triple combination. Overall design: 5- to 6-week-old BALBc mice were injected with 200,000 EMT-6 mammary tumors cells (ATTC, CRL-2755). Both inguinal mammary fat pads were injected in each mouse. Tumor development was monitored by palpation and once tumors were palpable, usually about 5-7 days after cancer cell inoculation, treatment was initiate. JQ1 was administered as single agents or in combination at the following doses: JQ1 at 50mg/kg by daily gavage,, anti-PD-L1 antibody (clone 10F.9G2, BioXCell) and paclitaxel each at 10mg/kg by i.p. injection twice per week. Tumor volumes were measured by caliper every three days until the end of the experiment. Once control tumors reached the study endpoint, all mice were euthanized, and primary tumors and various organs were collected. Tumors were then dissociated into single cells and separated into CD45+ and CD45- cell fractions using anti-CD45 antibody coupled to Dynabeads to enrich for leukocytes and tumor cells, respectively. Cells were counted and about 50,000 cells for each fraction were frozen for bulk ATAC-seq. The remaining cells were used for RNA extraction and bulk RNA-seq.

免疫检查点抑制剂联合化疗在三阴性乳腺癌（triple-negative breast cancer, TNBC）中是极具前景的治疗方案之一，但当前应答率仍相对偏低，亟需开发新型治疗策略以改善临床转归。

本研究报道了一种三联疗法，即抗PD-L1免疫检查点阻断、通过BET溴结构域抑制实现的表观遗传调控，联合紫杉醇化疗，该方案可在两种同基因小鼠乳腺癌模型中有效抑制原发性与转移性肿瘤生长。

对单药及联合治疗组的肿瘤进行详细的细胞与分子谱分析后发现，接受三联疗法处理的小鼠体内T细胞与B细胞浸润水平升高，且巨噬细胞由M1表型重编程为M2表型。

实验整体设计如下：选取5~6周龄的BALB/c小鼠，于双侧腹股沟乳腺脂肪垫接种200,000个EMT-6乳腺肿瘤细胞（美国典型培养物保藏中心，货号CRL-2755）。通过触诊监测肿瘤生长，待肿瘤可被触及（通常于癌细胞接种后5~7天）时启动治疗。JQ1可单药或联合给药，具体剂量方案为：JQ1以50mg/kg剂量每日灌胃；抗PD-L1抗体（克隆号10F.9G2，购自BioXCell）与紫杉醇均以10mg/kg剂量每周两次腹腔注射。每三日使用游标卡尺测量肿瘤体积，直至实验结束。当对照组肿瘤达到实验终点时，对所有小鼠实施安乐死，收集原发性肿瘤及各脏器组织。将肿瘤解离为单细胞悬液后，使用偶联抗CD45抗体的磁珠（Dynabeads）将细胞分为CD45+与CD45-细胞组分，分别富集白细胞与肿瘤细胞。对细胞进行计数后，取各组分约50,000个细胞冻存，用于批量转座酶可及性染色质测序（bulk ATAC-seq）；剩余细胞则用于RNA提取及批量RNA测序（bulk RNA-seq）。