Using natural killer cells to prevent breast cancer metastases

项目总结/摘要 大多数乳腺癌相关死亡率与转移性疾病有关。虽然NK细胞在免疫系统中发挥着重要作用， 转移性乳腺癌的控制，我们不完全了解NK细胞的关键机制和时间事件， 细胞免疫监视为了满足这一需求，我创建了NK细胞和侵袭性NK细胞的新型3D共培养试验。 乳腺癌类器官和细胞簇。使用这些模型，我证明了NK细胞限制乳腺癌 通过诱导侵袭性乳腺癌细胞的凋亡来抑制转移。我们发现了微转移癌 癌细胞重新编程NK细胞以促进转移性生长。使用批量RNA-seq，我们鉴定了KLRG 1 作为teNK细胞表达的关键基因。阻断KLRG 1可恢复抗转移活性。teNK细胞还 表达高水平的DNA甲基转移酶（DNMT）。在teNK细胞中抑制DNMT实现抗肿瘤作用。 转移结果。KLRG 1阻断和DNMT抑制的组合进一步减少肿瘤生长。 基于这些令人兴奋的数据，我们假设：1）重编程的teNK细胞KLRG 1表达是 由DNMT调节并联合靶向两者将逆转NK细胞重编程; 2）重编程 人NK细胞增加人乳腺癌转移并阻断优先的NK细胞抑制性受体 恢复人NK细胞的细胞毒活性。 目的1将确定阻断DNMT和KLRG 1如何协同逆转NK细胞重编程。 我们已经观察到在teNK细胞中一起阻断KLRG 1和DNMT协同地减少了集落 阵我们将首先在体内转移模型中测试这些离体发现。接下来，我们将确定 NK细胞重编程和DNMT之间的联系，使用新的功能性离体和体内模型， 使用CRISPR-Cas9分子工具进行转移。 目的2：探讨乳腺癌中重组NK细胞的功能。第一、 我们发现人NK细胞可以在培养物中重编程以积极促进转移性生长。我们 将利用重新编程的NK细胞的初步转录组学分析来识别转移的标志物- 重新编程的NK细胞我们将这些工具应用于转移性乳腺癌患者的FFPE切片 来确定重编程NK细胞的丰度。与患者样本一起，单细胞RNA-seq， 捕获从抗转移NK细胞到促进肿瘤转移的NK细胞的进展的离体和体内模型 NK细胞，我们将进一步研究人类重编程NK细胞的功能和分子特征。 乳腺癌最后，使用初步的目标列表，我们将测试有效的个体或组合 治疗可以恢复人类NK细胞对转移性乳腺癌细胞的细胞毒性。 影响：完成这些目标将加深我们对NK细胞生物学的基本理解， 为我们的患者带来新的NK细胞定向免疫疗法的潜力。

Project Summary/Abstract Most of breast cancer related mortality is related to metastatic disease. While NK cells play an essential role in the control of metastatic breast cancer we do not fully understand key mechanistic and temporal events of NK cell immunosurveillance. To address this need, I created novel 3D co-culture assays of NK cells and invasive breast cancer organoids and cell clusters. Using these models I demonstrated NK cells limit breast cancer metastasis through induction of apoptosis in invasive breast cancer cells. We discovered micrometastatic cancer cells reprogram NK cells to promote metastatic outgrowth. Using bulk RNA-seq, we identified KLRG1 as a key expressed gene by teNK cells. Blocking KLRG1 restores anti-metastatic activity. teNK cells also express high levels of DNA methyltransferases (DNMTs). Inhibition of DNMTs in teNK cells achieves anti- metastatic results. Combination KLRG1 blockade and DNMT inhibition further reduces tumor growth. Building on these exciting data, we hypothesize: 1) reprogrammed teNK cell KLRG1 expression is regulated by DNMTs and targeting both in combination will reverse NK cell reprogramming; 2) reprogrammed human NK cells increase human breast cancer metastasis and blocking prioritized NK cell inhibitory receptors restores the cytotoxic activity of metastases-reprogrammed human NK cells. Aim 1 will determine how blocking DNMTs and KLRG1 synergistically reverses NK cell reprogramming. We have observed blocking KLRG1 and DNMTs together in teNK cells synergistically reduces colony formation. We will first test these ex vivo findings in in vivo models of metastasis. Next, we will determine the link between NK cell reprogramming and DNMTs using novel functional ex vivo and in vivo models of metastasis using CRISPR-Cas9 molecular tools. Aim 2 will determine the function of metastases-reprogrammed NK cells in human breast cancer. First, we showed that human NK cells can be reprogrammed in culture to actively promote metastatic outgrowth. We will leverage preliminary transcriptomic analysis of reprogrammed NK cells to identify markers of metastases- reprogrammed NK cells. We will apply these tools to FFPE sections of patients with metastatic breast cancer to define the abundance of reprogrammed NK cells. Together with patient samples, single-cell RNA-seq, and ex vivo and in vivo models that capture the progression from anti-metastatic NK cells to metastases-promoting NK cells, we will further characterize the function and molecular profile of reprogrammed NK cells in human breast cancer. Finally, using a preliminary list of targets, we will test effective individual or combination treatments that can restore human NK cell cytotoxicity against metastatic breast cancer cells. Impact: Completing these aims will deepen our fundamental understanding of NK cell biology and have the potential to bring new NK cell directed immunotherapies to our patients.