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

摘要 具有持续性微小残留病变的局部浸润性三阴性乳腺癌（TNBC）患者 (MRD)尽管进行了新辅助治疗，但仍有发生致命转移的显著风险。这代表了 乳腺癌治疗中尚未满足的需求。这种高故障风险的一个主要原因是存在罕见的，低- 增殖性播散性肿瘤细胞（lpDTC），其对治疗具有高度抗性。许多lpDTC可以持续存在， 在重新激活以形成转移之前，使远处器官存活多年。消除TNBC中的lpDTC的尝试已经 由于它们的稀有性而没有成功，使得难以分离单独的lpDTC以鉴定治疗靶点。到 最后，我们报告了IL-6、IL-6受体和p38（IL-6/R/p38）轴的鉴定和表征， 这是维持TNBC中绝大多数lpDTC所需的关键信号传导途径。培养的人 骨髓（BM）中的TNBC细胞和TNBC、lpDTC的小鼠模型可以简单地通过以下方法迫使其脱离静止： 抑制IL-6/R信号传导。更重要的是，一旦急性重新激活，lpDTC变得非常敏感， 化疗因此，IL-6/R途径是一个有吸引力的治疗靶点。在这个提案中，我们将测试一本小说 在TNBC患者的I期和II期试验中，通过特异性抑制IL-6/R通路， lpDTC进入增殖，然后使用常规化疗消除这些细胞。首先，我们将确定 在sar（一种IL-6 R抑制剂）的标准剂量探索I期研究中， 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， 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 目的是确定该组合的耐受性和安全性以及该组合的推荐剂量 对于第二阶段。接下来，我们将在患者中使用该推荐剂量方案进行II期单组研究。 新辅助治疗后乳腺组织或周围淋巴结中存在持续MRD的I-III期TNBC患者 治疗（目标2）。II期的目的是确定这种药物组合在清除BM方面的有效性 lpDTC，如果是，清除BM lpDTC的患者是否具有更高的两年无进展生存率 与历史上仅用卡培他滨治疗的患者相比。在目标3中，我们将从BM中分离单个lpDTC 本试验使用传统的基于磁铁的富集方法和当地开发的 1）在治疗之前和之后对lpDTC进行计数，以确定微流控装置的lpDTC清除功效。 测试药物组合;以及2）对单个lpDTC、原发性和转移性肿瘤样品进行基因组学分析 同样的病人。我们将使用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和肿瘤微环境的反应变化。 目标是：1）更深入地了解lpDTC是如何生成和维护的; 2）产生额外的 在不久的将来，可以与IL-6/R通路结合以改善DTC靶向策略的靶点。

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.