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

肾上腺皮质癌是一种侵袭性很强的恶性肿瘤，患者的5年生存率很低， 转移性疾病没有针对这些患者的靶向治疗。不幸的是，目前的治疗方法 仅减缓疾病进展平均5个月，之后耐药性迅速发展。作为 因此，迫切需要确定新的干预靶点并开发新的ACC治疗方法。 可以通过患者之间的肿瘤间和患者内的肿瘤内的显著异质性来表征。 单个肿瘤，使得选择有效的治疗策略具有挑战性。几种机械 已经确定了可能与疾病进展相关的途径：IGF 2、Wnt/β-连环蛋白（Wnt）和细胞凋亡。 周期调节途径如p53/视网膜母细胞瘤蛋白（Rb）在90%的ACC中失调， 与转移性疾病有关。不幸的是，IGF 2、Wnt和p53/Rb的肿瘤内异质性 调节异常及其对ACC肿瘤进展的贡献是未知的，尽管这些途径中的每一个都有其独特的作用。 与肿瘤侵袭、基质金属蛋白酶（MMP）表达或转移有关， 其他癌症。这种缺乏了解可以部分归因于缺乏适当的临床前研究 模型大多数生成ACC模型的尝试都没有成功。现有的少量模型 不能充分反映人类ACC的致癌信号通路和肿瘤内异质性。 允许肿瘤功能和转录组学表征的基于人的ACC模型系统 不存在转移后的亚群。为了解决这些关键的研究差距，我们开发了 来自患者样品的第一患者来源的类器官（PTO）以及ACC转移芯片（MOC） 平台我们的MOC平台是一种体外微流体系统，它结合了肿瘤类器官，再循环 流体流动和下游组织类器官，这可以概括原发性肿瘤转移的各个方面 转移部位。此外，我们还开发了MMP肽生物传感器技术， 我们的类器官，能够近实时观察MMP介导的肿瘤细胞侵袭。我们将部署这个 将肿瘤细胞分选为转移性/运动性与非转移性/运动性较低亚群以进行分析的平台 ACC中表达失调的途径（IGF 2、Wnt和p53/Rb）和亚群异质性 通过单细胞RNA测序确定。我们假设IGF 2、Wnt和细胞周期失调 与在我们的MOC平台中部署的ACC PTO中增加的转移动力学和MMP活性相关。 针对这一假设：目的1将描绘转移动力学，MMP活性，和增殖的ACC PTO 目的2将使用单一的细胞模型来定义ACC关键致癌通路失调的瘤内异质性。 细胞RNA测序;目标3将确定每个驱动途径对转移潜能的相对重要性 通过药物抑制。在这个项目完成后，我们的目标是更好地了解ACC疾病， 生物学，并建立了ACC模型，可用于临床前研究。

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.