A matrix metalloproteinase biosensor-functionalized metastasis-on-a-chip platform for evaluating adrenocortical carcinoma progression

肾上腺皮质癌(ACC)是一种侵袭性的恶性肿瘤，患者的5年生存率很低，仅为6%。 患有转移性疾病。目前还没有针对这些患者的针对性治疗。不幸的是，目前的治疗方法 只能在平均5个月的时间内减缓疾病的进展，之后抗药性会迅速发展。AS 因此，迫切需要确定新的干预目标和开发新的ACC治疗方法。行政协调会 可表现为患者之间的肿瘤间和肿瘤内的显著异质性 单个肿瘤，使得选择有效的治疗策略具有挑战性。几种机械论 已确定可能与疾病进展相关的通路：IGF2、Wnt/β-catenin(Wnt)和细胞 在90%的ACC中，周期调节通路如P53/视网膜母细胞瘤蛋白(Rb)表达异常，以及 与转移性疾病相关。不幸的是，IGF2、Wnt和P53/Rb在肿瘤内的异质性 调控失调及其在ACC肿瘤进展中的作用尚不清楚，尽管这些途径中的每一条都 与肿瘤侵袭、基质金属蛋白酶表达或转移有关 其他癌症。这种缺乏理解的部分原因可以归因于缺乏适当的临床前研究 模特们。大多数生成ACC模型的尝试都没有成功。现有车型数量较少 不能充分反映人ACC的致癌信号通路和瘤内异质性。一个 允许肿瘤功能和转录特征描述的基于人的ACC模型系统 转移后的亚群不存在。为了解决这些关键的研究差距，我们开发了 首次从患者样本中提取患者衍生的有机化合物(PTO)以及一种ACC芯片上转移(MOC) 站台。我们的MOC平台是一个体外微流控系统，它结合了肿瘤有机物，再循环 体液流动和下游组织器官，可以概括原发肿瘤转移的各个方面 从一个部位到另一个转移部位。此外，我们还开发了基质金属蛋白酶多肽生物传感器技术，该技术集成到 我们的有机类化合物，能够近乎实时地观察基质金属蛋白酶介导的肿瘤细胞侵袭。我们将部署此功能 用于将肿瘤细胞分类为转移性/运动性与非转移性/较少运动性亚群以供分析的平台 癌组织中IGF2、Wnt和P53/Rb异常表达途径与亚群异质性 由单细胞RNA测序确定。我们假设IGF2、Wnt和细胞周期失调 与在我们的MOC平台中部署的ACC PTO的转移动力学和基质金属蛋白酶活性增加相关。 支持这一假设：目标1将描述ACC PTO的转移动力学、基质金属蛋白酶活性和增殖 Aim 2将用单一的方法定义ACC瘤内关键致癌途径调控失调的异质性 细胞-RNA-测序；AIM 3将确定每个驱动通路对转移潜能的相对重要性 通过基于药物的抑制。在这个项目完成后，我们的目标是更好地了解ACC疾病 并建立了可用于临床前研究的ACC模型。

Adrenocortical carcinoma (ACC) is an aggressive malignancy with a poor 5-year survival rate of 6% for patients with metastatic disease. There are no targeted therapies for these patients. Unfortunately, current treatments only slow disease progression for an average of 5 months, after which drug resistance quickly develops. As such, there is a critical need to identify new targets of intervention and develop new treatments for ACC. ACC can be characterized by significant heterogeneity both intertumorally between patients and intratumorally within an individual tumor, making choosing an effective therapeutic strategy challenging. Several mechanistic pathways have been identified that may correlate with disease progression: IGF2, Wnt/β-catenin (Wnt), and cell cycle regulating pathways such as p53/retinoblastoma protein (Rb) are dysregulated in 90% of ACC, and correlate with metastatic disease. Unfortunately, the intratumoral heterogeneity of IGF2, Wnt, and p53/Rb dysregulation and its contributions to ACC tumor progression is unknown, although each of these pathways has been implicated or correlated with tumor invasion, matrix metalloproteinase (MMP) expression, or metastasis in other cancers. This lack of understanding can partially be attributed to the lack of appropriate preclinical research models. Most attempts to generate ACC models have been unsuccessful. The small number of existing models do not adequately reflect the oncogenic signaling pathways and intratumoral heterogeneity of human ACC. A human-based ACC model system permitting functional and transcriptomic characterization of tumor subpopulations following metastasis does not exist. To address these critical research gaps, we developed the first patient-derived organoids (PTOs) from patient samples as well as an ACC metastasis-on-a-chip (MOC) platform. Our MOC platform is an in vitro microfluidic system that incorporates tumor organoids, recirculating fluid flow, and downstream tissue organoids, which can recapitulate aspects of metastasis from a primary tumor site to metastatic sites. In addition, we have developed MMP peptide biosensor technology that integrates into our organoids, enabling near-real time observation of MMP-mediated tumor cell invasion. We will deploy this platform to sort tumor cells into metastatic/motile versus non-metastatic/less motile subpopulations for analysis of expression of dysregulated pathways in ACC (IGF2, Wnt, and p53/Rb) and subpopulation heterogeneity determined by single cell RNA sequencing. We hypothesize that IGF2, Wnt, and cell cycle dysregulation correlates with increased metastasis kinetics and MMP activity in ACC PTOs deployed in our MOC platform. Towards this hypothesis: Aim 1 will delineate metastasis kinetics, MMP activity, and proliferation of ACC PTO cells; Aim 2 will define ACC intratumoral heterogeneity of critical oncogenic pathway dysregulation using single cell-RNA-sequencing; Aim 3 will determine the relative importance of each driver pathway on metastatic potential through drug-based inhibition. Upon completion of this project, we aim to both better understand ACC disease biology and have an established model of ACC that can be deployed for preclinical studies.