Characterizing the regulation of PD-1 ligands in head and neck cancer stem cells using an autologous humanized model with T cell education capability

总结 研究人类癌症的一个障碍是具有人类间质和免疫原性的模型的有限可用性。 细胞这对于头颈部鳞状细胞癌（HNSCC）尤其重要，因为它在肿瘤的发生中起着关键作用。 免疫系统在它们的发育过程中起着重要作用。一个未满足的需求是调查癌症干细胞如何 （CSCs）调节免疫，因为了解CSCs如何利用致癌信号传导来调节免疫至关重要。 逃避免疫监视为了解决这些悬而未决的问题，我们将使用两种独特的工具：1）CSC 来源于在小鼠中繁殖的一组患者HNSCC，和2）人源化小鼠（HM）模型， 使研究肿瘤间质相互作用成为可能。我们在多个患者中将CSC定义为ALDH+ CD 44高 案例，并记录了SOX 2负责关键的CSC功能，如增长，入侵， 抗肿瘤治疗（1）。此外，我们观察到SOX 2和肿瘤表达之间的相关性， 配体PD-L1，其激活T细胞中的PD-1，导致免疫逃逸。另外，SOX 2调节SOX 9， 调节另一种PD-1配体PD-L2。在用PD-1抑制剂治疗HNSCC HM小鼠后，我们 记录的耐药与PD-L2增加相关;因此，我们建议使用替代配体 作为一种耐药机制。我们最初开发了一个不匹配的HM（mHM;肿瘤和 来自不同来源的造血干细胞[HSC]），并且现在已经产生了自体HM（aHM; 肿瘤、HSC和间充质干细胞[MSC]来自同一患者）。在HM中生长的肿瘤 与非HM小鼠中生长的肿瘤相比，其类似于原始肿瘤，并且基因表达的漂移由 先前的传代被部分逆转。在关键免疫和基质通路中的信号传导更为突出 并且在HM与非HM模型中与原始患者非常相似。免疫细胞有效地 肿瘤特异性免疫应答，由包括T细胞在内的人免疫细胞介导。两个突出的和下- 研究的问题限制了HM的广泛应用：肿瘤免疫错配和T细胞教育。肿瘤和 免疫匹配可以影响免疫应答的忠实性，这只能通过以下方式来理解： 比较mHM和aHM。HM通过小鼠胸腺细胞接受人T细胞的异种教育， 这是由于在这种模型中缺乏功能性的人胸腺。该提案的目标是：1）研究 SOX 2和SOX 9对PD-1配体调节; 2）了解SOX 2和SOX 9如何影响CSC 性质; 3）从10名HNSCC患者产生并表征aHM和mHM; 4）测试SOX 2和SOX 9是否 通过在HM上测试PD-1抑制剂，参与了对PD-1抑制剂的耐药性;最后，我们将5）产生胸腺 来自同一患者的HSC的上皮，其将导致具有完全自体HNSCC的HM，胸腺和 免疫系统，从而使免疫细胞教育在严格的人类背景下。该项目将导致一个 对HNSCC CSC与免疫/基质之间相互作用的更深入和更机械的理解 系统，并最终改善精准医疗。

SUMMARY An obstacle to studying human cancer is the limited availability of models with human stroma and immune cells. This is particularly relevant for head and neck squamous cell carcinomas (HNSCC) given the pivotal role the immune system plays in their development. An unmet need is an investigation of how cancer stem cells (CSCs) modulate immunity, because it is critical to understand how CSCs harness oncogenic signaling to evade immune surveillance. To address these outstanding questions, we will use two unique tools: 1) CSCs derived from a panel of patient HNSCCs propagated in mice, and 2) a humanized mouse (HM) model that enables studying tumor-stromal interactions. We have defined CSCs as ALDH+CD44high in multiple patient cases, and documented that SOX2 is responsible for key CSC features such as growth, invasion, and resistance to therapy (1). Further, we observed an association between SOX2 and tumor expression of the ligand PD-L1, which activates PD-1 in T cells leading to immune escape. Additionally, SOX2 modulates SOX9, which regulates the other PD-1 ligand, PD-L2. After treatment of HNSCC HM mice with a PD-1 inhibitor, we documented resistance associated with an increase in PD-L2; thus, we propose that alternate ligands are used by HNSCC as a resistance mechanism. We initially developed a mismatched HM (mHM; tumor and hematopoietic stem cells [HSCs] from different sources), and now have generated an autologous HM (aHM; tumor, HSCs and mesenchymal stem cells [MSCs] from the same patient). Tumors grown in HM more closely resemble the originator tumor than those grown in non-HM mice, and the drift in gene expression caused by prior passaging was partially reversed. Signaling in key immune and stroma pathways was more prominent and closely resembled the originator patient in HM vs. non-HM models. The immune cells mounted effective tumor-specific immune responses, mediated by human immune cells including T cells. Two salient and under- studied issues limit the wider application of HM: tumor-immune mismatch and T cell education. Tumor and immune matching can affect the faithfulness of immune response, and that can only be appreciated by comparing mHM and aHM. HM are subject to xenogeneic education of human T cells by mouse thymic cells, resulting from the lack of a functional human thymus in such models. The goals of this proposal are to: 1) study the regulation by SOX2 and SOX9 of PD-1 ligands; 2) understand how SOX2 and SOX9 affect CSC properties; 3) generate and characterize aHM and mHM from 10 HNSCC patients; 4) test if SOX2 and SOX9 are involved in resistance to PD-1 inhibitors by testing them on HM; and finally, we will 5) generate thymic epithelium from the same patient's HSCs that will result in HM with a fully autologous HNSCC, thymus and immune system, thus enabling immune cell education in a strictly human context. This project will lead to a deeper and more mechanistic understanding of the interplay between HNSCC CSCs and the immune/stroma systems, and ultimately, improve precision medicine.