Development of quantitative mass spectrometry assays and imaging for cancer metastasis

家长奖摘要。高级别浆液性卵巢癌(HGSC)是最常见和最致命的卵巢癌。 卵巢癌。新出现的证据表明，这些肿瘤发生在输卵管上皮(FTE)， 因此，它们在卵巢中的存在代表了原发转移。我们的初步数据显示 卵巢附近的异种移植增加了疾病的侵袭性。卵巢后 定植时，肿瘤细胞侵入腹膜器官，主要是大网膜。我们假设 HGSC原发和继发转移的生物学问题部分由化学物质介导 癌细胞和转移器官之间的通讯。我们的提案试图定义代谢物 以及驱动输卵管来源的高级别浆液性癌转移到卵巢的生物分子 还有大网膜。为此，我们的团队优化了卵巢和输卵管的3D共培养 肿瘤模型，并将其应用于成像质谱学技术以识别代谢组学驱动的 发生在卵巢原发定植期间和继发于卵巢的转移期间的通讯 大网膜。使用这项新兴技术，我们确定了几种代谢物，这些代谢物可以增强高级别血清 肿瘤向卵巢的迁移、侵袭和粘连。目标1的重点是揭示这些机制 允许FTE致瘤细胞劫持卵巢产生的去甲肾上腺素，以增强其侵袭和 在原发灶转移过程中附着在卵巢上。目标1将定义去甲肾上腺素在 对卵巢的侵袭和黏附，然后在体内用小鼠和 由FTE衍生的人体细胞模型。关键的肾上腺素能受体将通过CRISPR被删除以确认 这一途径在肿瘤转移中的重要性。荷瘤模型将用β-肾上腺素能受体治疗 拮抗剂试图将这些发现转化为阻止卵巢定植的新策略。焦点 目标2是关于一种新鉴定的蛋白质的鉴定和特性，这种蛋白质是由 致瘤性输卵管细胞与卵巢去甲肾上腺素驱动肿瘤的产生有关 细胞的侵袭和黏附。我们将使用蛋白质组学来确认分泌蛋白的身份，然后 从FTE模型中基因缺失该蛋白以研究其在卵巢定植中的作用。目标3将在此基础上发展 我们现有的3D器官和肿瘤细胞通讯模型技术并扩展到二次 转移。我们现在已经优化了大网膜与肿瘤细胞共培养的技术 并有一份代谢物清单，这些代谢物是独一无二的，不包括去甲肾上腺素。相反，一个 新的代谢物发现，当肿瘤细胞与 大网膜对应于叶酸，叶酸受体的配体在肿瘤细胞中过度表达。 综上所述，我们的创新实验方法将产生新的途径和目标，以减轻初级 高级别浆液性癌卵巢转移。

Parent award abstract. High grade serous ovarian cancer (HGSC) is the most common and deadliest form of ovarian cancer. Emerging evidence indicates that these tumors arise in the fallopian tube epithelium (FTE), and thus their presence in the ovary represents the primary metastasis. Our preliminary data identified that xenografting in close proximity to the ovary contributes to the aggressiveness of the disease. After ovarian colonization, tumor cells invade the peritoneal organs, primarily the omentum. We hypothesize that the biological problem of primary and secondary HGSC metastasis is partially mediated by chemical communication between the cancer cells and the metastatic organ. Our proposal seeks to define metabolites and biomolecules that drive the metastasis of fallopian tube derived high grade serous cancers to the ovary and the omentum. To this end, our teams optimized a 3D co-culture of the ovary and fallopian tube derived tumor models and adapted this to imaging mass spectrometry technology to identify the metabolomics-driven communication that occurs during primary colonization of the ovary and during secondary metastasis to the omentum. Using this emerging technology, we identified several metabolites that enhanced high grade serous tumor migration, invasion, and adhesion to the ovary. The focus of Aim 1 is to uncover the mechanisms allowing FTE tumorigenic cells to hijack NE produced by the ovary to increase their ability to invade and adhere to the ovary during primary metastasis. Aim 1 will define the signaling pathways mediated by NE during invasion and adhesion to the ovary and then confirm the importance of NE in vivo using both murine and human cell models derived from FTE. The key adrenergic receptor will be deleted using CRISPR to confirm the importance of this pathway in metastasis. Tumor bearing models will be treated with beta adrenergic receptor antagonists in an attempt to translate these findings for a new strategy to block ovarian colonization. The focus of Aim 2 is on the identification and characterization of a newly identified protein that is secreted from tumorigenic fallopian tube cells and is responsible for the production of ovarian norepinephrine driving tumor cell invasion and adhesion. We will use proteomics to confirm the identity of the secreted protein, followed by genetic deletion of the protein from FTE models to study the role in ovarian colonization. Aim 3 will build upon our existing technology of 3D organ and tumor cell communication models and expand into secondary metastasis. We have now optimized our technology for co-culture of the omentum together with tumor cell models and have an inventory of metabolites, which are unique and did not include norepinephrine. Instead a novel metabolite found to be produced in significantly more abundance when tumor cells were grown with the omentum corresponded to folate, the ligand for the folic acid receptor that is overexpressed in the tumor cells. Taken together, our innovative experimental approach will yield new pathways and targets to mitigate primary metastasis of high grade serous cancer to the ovary.