Tumor Organoid-Mediated Drug Testing and Clonality Analysis in Peritoneal Surface Disease of Intestinal Origin

项目摘要 该项目的总体目标是开发一个基于肿瘤有机物的药物筛选平台，该平台可以量化 肿瘤克隆异质性的差异性药物敏感性预测患者的治疗反应 在选择治疗之前。我们的研究针对转移性肠道腹膜表面恶性肿瘤。 (IPSM)，重点是结肠癌和阑尾癌。这些癌症的治疗和临床表现相似。 与不同的解剖结构相关的大量空间上不同的腹膜转移。临床部 这些癌症的病程通常遵循化疗耐药的路径，因此，体外模型可以识别药物 能够减轻化疗耐药性的组合和测序策略将具有重要的临床价值。 先天和后天的化疗耐药是由肿瘤克隆中的遗传改变引起的 建筑及其不断变化的克隆成分。在IPSM中，我们观察到了不同的克隆变体 同一患者体内空间上不同的病变表明不同患者之间药物反应的可能性 病人内部的损伤。我们的中心假设是患者对化疗的临床反应是成比例的 患者的主要癌症克隆所表现出的克隆反应的大小，这可能是 使用患者来源的肿瘤有机物(PTO)进行药敏试验和克隆性分析 从反映患者转移性克隆多样性的患者内部病变构建。在目标1中，我们将 从多个IPSM患者中构建多个病变PTO以研究肿瘤克隆之间的相互作用 组分与PTO药敏试验。我们将检验这一假设：化疗耐药的终结性措施和 与简单的有机化合物相比，敏感的克隆组分可以更好地预测患者的临床治疗反应 细胞存活率。此外，我们将寻求确定在化疗敏感的IPSM中频繁突变的基因。 和/或IPSM患者中的耐药克隆组分，并表征结肠癌候选基因功能 细胞系有机物和小鼠结肠癌异种移植模型。在目标2中，我们将努力扩大 PTO平台包括对免疫检查点抑制物(ICIS)的评估，但目前尚不能如实进行 在人类系统中建模，并在胃肠道癌症中发挥新的作用，但仍未被探索 IPSM管理。我们将检验这样一种假设，即保护患者的免疫增强型PTO(IPTO)- 癌细胞和细胞毒性T细胞(CTL)之间的特异性内环境平衡关系可以用来模拟ICI- 通过肿瘤反应性CTL诱导癌细胞杀伤，以及洞察肿瘤克隆性改变 影响ICI的响应能力。我们将使用已建立的小鼠免疫肿瘤模型(CT26和MC38)来 研究iPTO中肿瘤-CTL的动态平衡，并确认体外iPTO与原位iPTO之间ICI反应的平等性 小鼠肿瘤。我们将确认ICI治疗中抗原特异性CTL介导的癌细胞杀伤的参与 IPTOS。最后，与目标1平行，我们将确定在ICI敏感和/或耐药中频繁突变的基因 IPSM的克隆部分，并在免疫活性小鼠模型中表征其ICI调节功能。

Project Summary The overarching goal of this project is to develop a tumor organoid-based drug screening platform that quantifies the differential drug responsiveness of tumor clonal heterogeneity to predict a patient’s therapeutic response prior to treatment selection. Our research is directed at metastatic intestinal peritoneal surface malignancies (IPSM), with a focus on colon and appendiceal cancers. These cancers are similarly treated and present clinically with numerous spatially-distinct peritoneal metastases associated with different anatomic structures. The clinical course of these cancers typically follows a path of chemoresistance, thus, ex vivo models that can identify drug combinations and sequencing strategies able to mitigate chemoresistance would be of significant clinical value. Innate and acquired chemoresistance results from genetic alterations that manifest within the tumor’s clonal architecture and its changing clonal composition. In IPSM, we have observed distinct clonal variants among spatially distinct lesions within the same patient indicating the potential for differential drug responses among intrapatient lesions. Our central hypothesis is that a patient’s clinical response to chemotherapy is proportional to the magnitude of the clonal response exhibited by the patient’s predominating cancer clones, which can be assessed by chemosensitivity testing and clonality analysis using patient-derived tumor organoids (PTOs) constructed from intra-patient lesions that reflect the patient’s metastatic clonal diversity. In Aim 1, we will construct multi-lesion PTOs from a number of IPSM patients to investigate interactions between tumor clonal fraction and PTO chemosensitivity. We will test the hypothesis that summative measures of chemo resistant and sensitive clonal fractions will predict patient clinical treatment responses better than simple measures of organoid cell viability. Furthermore, we will seek to identify genes in IPSM that are frequently mutated in chemo sensitive and/or resistant clonal fractions across IPSM patients, and characterize candidate gene functions in colon cancer cell line organoids and mouse xenograft models of colon cancer. In Aim 2, we will seek to extend the reach of the PTO platform to include evaluation of immune checkpoint inhibitors (ICIs) which cannot yet be faithfully modeled in human systems, and that have emerging roles in gastro-intestinal cancers, yet remain unexplored in IPSM management. We will test the hypothesis that immune-enhanced PTOs (iPTOs) that preserve the patient- specific homeostatic relationship between cancer cells and cytotoxic T cells (CTLs) can be used to model ICI- induced cancer cell killing by tumor-reactive CTLs as well as to gain insight into tumor clonal alterations that influence ICI responsiveness. We will use established murine immune-oncology models (CT26 and MC38) to study tumor-CTL homeostasis in iPTOs, and to confirm ICI-response parity between ex vivo iPTOs and in situ mouse tumors. We will confirm the involvement of antigen specific CTL-mediated cancer cell killing in ICI-treated iPTOs. Finally, parallel to Aim 1, we will identify genes that are frequently mutated in ICI sensitive and/or resistant clonal fractions of IPSM, and characterize their ICI-modulatory functions in immunocompetent mouse models.