A Bioengineered Model of Tumor Vessel Interactions in Pancreatic Cancer

胰腺导管腺癌(PDAC)是导致恶性肿瘤死亡的主要原因之一。PDAC是 高度侵袭性，在肿瘤进展的非常早期就在远处器官形成转移。为了更好地 了解PDAC的转移，肿瘤-血管的相互作用需要进一步评估，作为肿瘤细胞 主要通过血液循环传播。然而，PDAC如何与血管相互作用并建立 人们对远处转移知之甚少。最近，我们的研究使用了三维(3D)仿生 PDAC芯片和多个活体小鼠模型显示，PDAC细胞侵入血管和 通过ALK7信号主动替换内皮细胞，导致肿瘤-血管杂合结构的形成 PDAC肿瘤。我们将这种现象称为肿瘤血管替代。尽管这一发现很新颖，但它 目前尚不清楚PDAC中肿瘤血管置换的生物学后果是什么。了解 肿瘤血管置换的表型后果对于确定临床相关性和 阻断ALK7在PDAC中的意义。我们假设PDAC肿瘤血管置换术增加肿瘤 血管通透性；然后通过促进肿瘤细胞进入血管内和转移促进肿瘤的转移 血液通过渗漏的血管循环。为了检验这些假设，我们的目标是确定-在这两个方面 体内外研究--(I)肿瘤血管置换是否导致肿瘤血管渗漏并促进转移 (Ii)如果ALK7抑制或ALK7敲除(KO)改善或逆转肿瘤血管渗漏 和转移。在目标1中，我们将评估ALK7在周细胞覆盖的血液中PDAC血管通透性中的作用。 微血管内皮细胞和周细胞共培养模拟生理性血管的芯片上血管 被周细胞包围(目标1.1)。接下来，我们将评估ALK7在体内PDAC血管功能障碍中的作用。 我们将使用野生型或ALK7-KO PDAC细胞建立原位PDAC模型，并检测PDAC肿瘤 静脉注射葡聚糖分子的血管通透性(目标1.2)。在目标2中，我们将研究ALK7 通过建立转移前肝脏微环境来实现PDAC的体外转移 连接到工程血管上。多个PDAC线路将被评估以测试ALK7介导的 肿瘤血管替代影响转移扩散(目标2.1)。然后我们将评估ALK7在PDAC中的作用 与曼努埃尔·伊达尔戈博士合作，使用人类患者来源的异种移植(PDX)模型进行体内转移。 将评估对照组与ALK7KO组的转移肿瘤负荷，以及循环肿瘤的数量 将确定细胞和总存活率(目标2.2)。总而言之，我们的3D PDAC芯片系统将提供 一个独特的平台，可以更好地研究PDAC与血管的相互作用和转移进展。我们会 破译ALK7信号在介导肿瘤血管调节失调和转移中的作用；并评估 我们是否能够通过靶向ALK7来减少PDAC的进展和转移。

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths among malignancies. PDAC is highly invasive and forms metastases in distant organs at the very early stage of tumor progression. To better understand PDAC metastasis, tumor-blood vessel interactions need to be evaluated further, as tumor cells spread primarily through the blood circulation. However, how PDAC interacts with blood vessels and establishes distant metastases are poorly understood. Recently, our study using both three-dimensional (3D) biomimetic PDAC-on-chip and multiple in vivo mouse models showed that PDAC cells invaded blood vasculatures and actively replaced endothelial cells via ALK7 signaling, leading to a formation of tumor-vessel hybrid structure in PDAC tumors. We refer to this phenomenon as tumor vessel replacement. Despite the novelty of the finding, it is unknown what the biological consequences of the tumor vessel replacement in PDAC are. Understanding the phenotypic consequences of the tumor vessel replacement is critical to determine the clinical relevance and significance of blocking ALK7 in PDAC. We hypothesize that PDAC tumor vessel replacement increases tumor vessel permeability; then promotes tumor intravasation and metastasis by facilitating tumor cells’ entering the blood circulation through the leakier vessels. In order to test these hypotheses, we aim to determine—in both in vitro and in vivo—(i) if tumor vessel replacement induces tumor vessel leakiness and promotes metastatic dissemination and (ii) if ALK7 inhibition or ALK7 knock out (KO) ameliorates or reverses tumor vessel leakiness and metastasis. In Aim 1, we will assess the role of ALK7 in PDAC vessel permeability in pericyte-covered blood vessel on-chip by co-culturing microvascular endothelial cells and pericytes to mimic physiological blood vessels surrounded by pericytes (Aim 1.1). Next, we will evaluate the role of ALK7 in PDAC vessel dysfunction in vivo. We will generate an orthotopic PDAC model using wild-type or ALK7-KO PDAC cells, and examine PDAC tumor vessel permeability by intravenously injecting dextran molecules (Aim 1.2). In Aim 2, we will examine ALK7 in PDAC metastasis in vitro by establishing pre-metastatic liver microenvironment in the reservoirs that are connected to the engineered blood vessel. Multiple PDAC lines will be assessed to test whether ALK7-mediated tumor vessel replacement affects metastatic spreading (Aim 2.1). We will then evaluate the role of ALK7 in PDAC metastasis in vivo using human patient-derived xenograft (PDX) models in collaboration with Dr. Manuel Hidalgo. Metastatic tumor burdens in control vs. ALK7 KO groups will be assessed, and the number of circulating tumor cells and overall survival rate will be determined (Aim 2.2). In summary, our 3D PDAC-on-chip system will provide a unique platform to better investigate PDAC interactions with blood vessels and metastatic progression. We will decipher the roles of ALK7 signaling in mediating tumor vessel dysregulation and metastasis; and assess whether we will be able to reduce PDAC progression and metastasis by targeting ALK7.