Novel Methods of Chemo-sensitizing Low-proliferative Disseminated Tumor Cells in Triple Negative Breast Cancer

摘要 局部浸润性三阴性乳腺癌(TNBC)患者有持续微小残留病变 (MRD)尽管进行了新的辅助治疗，但仍有发生致命转移的巨大风险。这代表了其中一个 乳腺癌治疗中未得到满足的突出需求。这种高故障风险的一个主要原因是存在罕见的、低成本的 增殖性播散性肿瘤细胞(LpDTCs)，对治疗具有高度耐药性。许多lpdt可以在 远处的器官在重新激活形成转移之前会持续很多年。在TNBC中消除LpDTC的尝试已经 由于LpDTCs的稀有，未能成功，因此很难分离出单个LpDTCs来确定治疗靶点。至 最后，我们报告了IL-6、IL-6受体和p38(IL-6/R/p38)轴作为一种 在TNBC中维持绝大多数LpDTCs所需的关键信号通路。在培养的人类中 TNBC细胞和小鼠模型骨髓(BM)中的TNBC、LpDTCs可以通过以下方法被迫脱离静止状态 抑制IL-6/R信号转导。更重要的是，一旦重新激活，lpdtc就会变得对 化疗。因此，IL-6/R通路是一个有吸引力的治疗靶点。在这个提案中，我们将测试一部小说 通过特异性抑制IL-6/R途径强制治疗TNBC患者的I期和II期试验的治疗策略 LpDTCs转化为增殖细胞，然后用常规化疗消除这些细胞。首先，我们将确定 这种治疗策略在IL-6R沙利单抗标准剂量发现I期研究中的患者是否安全 最近批准用于治疗类风湿性关节炎的抑制性抗体药物，与卡培他滨顺序联合，A 标准乳腺癌化疗药物，用于转移性乳腺癌患者(目标1)。第一阶段的 目的是确定联合用药的耐受性和安全性以及联合用药的推荐剂量。 对于第二阶段。接下来，我们将在患者中使用此推荐剂量方案进行第二阶段单臂研究 I-III期TNBC患者在新辅助治疗后乳腺组织或周围淋巴结有持续性MRD 治疗(目标2)。第二阶段的目标是确定这种药物组合在清除BM方面的效果 LpDTCs，如果是这样，清除BM lpDTCs的患者在两年内是否有更高的无进展存活率 与历史上仅接受卡培他滨治疗的患者相比。在目标3中，我们将从BM中分离出单个LpDTC 在这项试验中使用传统的磁基富集法和当地开发的 微流控装置，用于1)计数治疗前后的lpDTC，以确定lpDTC的清除效果 测试药物组合；以及2)对单个lpDTCs、原发和转移的肿瘤样本进行基因组分析 来自相同的病人。我们将使用最近开发的一个新的计算平台来分析基因网络 乳腺癌细胞在转移过程中对治疗的反应和肿瘤微环境的变化。 目标是1)更深入地了解LpDTC是如何生成和维护的；以及2)产生更多 可以与IL-6/R途径相结合的靶点，以在不久的将来改进DTC靶向策略。

ABSTRACT Patients with locally invasive triple negative breast cancer (TNBC) who have persistent minimal residual disease (MRD) despite neoadjuvant therapy are at significant risks of developing lethal metastasis. This represents one of the outstanding unmet needs in breast cancer therapy. A major cause of this high failure risk is the presence of rare, low- proliferative disseminated tumor cells (lpDTCs), which are highly resistant to treatment. Many lpDTCs can persist in distant organs for many years before reactivating to form metastasis. Attempts at eliminating lpDTCs in TNBC have not been successful due to their rarity, making it difficult to isolate individual lpDTCs to identify therapeutic targets. To that end, we report the identification and characterization of the IL-6, IL-6 receptor, and p38 (IL-6/R/p38) axis as a critical signaling pathway required for the maintenance of a significant majority of lpDTCs in TNBC. In cultured human TNBC cells and mouse models of TNBC, lpDTCs in the bone marrow (BM) can be forced out of quiescence simply by inhibiting IL-6/R signaling. More importantly, once acutely reactivated, lpDTCs become exquisitely sensitive to chemotherapy. Thus, the IL-6/R pathway is an attractive therapeutic target. In this proposal, we will test a novel therapeutic strategy in a Phase I and II trial in patients with TNBC by specifically inhibiting the IL-6/R pathway to force lpDTCs into proliferation, and then use conventional chemotherapy to eliminate these cells. First, we will determine whether this treatment strategy is safe in patients in a standard dose finding Phase I study of sarilumab, an IL-6R inhibiting antibody drug recently approved to treat rheumatoid arthritis, sequentially combined with capecitabine, a standard breast cancer chemotherapy drug, in patients with metastatic breast cancer (Aim 1). The Phase I’s objectives are to determine tolerability and safety of the combination and the recommended dose of the combination for the Phase II. Next, we will conduct a Phase II single-arm study using this recommended dose regimen in patients with stage I-III TNBC who have persistent MRD in breast tissue or surrounding lymph nodes after neoadjuvant therapy (Aim 2). The Phase II objectives are to determine how effective this drug combination is at clearing BM lpDTCs, and if so whether patients cleared of BM lpDTCs have a higher rate of progression-free survival at two years compared to patients historically treated with capecitabine alone. In Aim 3, we will isolate individual lpDTCs from BM aspirates collected in this trial using the conventional magnet-based enrichment method and a locally developed microfluidic device to 1) enumerate lpDTCs before and after treatment to determine the lpDTC clearing efficacy of the test drug combination; and 2) to perform genomics analysis of single lpDTCs, primary and metastatic tumor samples from the same patients. We will use a novel computational platform recently developed to analyze gene network changes in response to treatment and tumor microenvironments that breast cancer cells transition during metastasis. The goals are to 1) gain a deeper understanding of how lpDTCs are generated and maintained; and 2) yield additional targets that can be combined with the IL-6/R pathway to improve DTC-targeting strategies in the near future.