Molecular and spatial dissection of endothelial cell heterogeneity in clear cell renal cell carcinoma

内皮细胞（ECs）排列在血管内壁上，在我们的器官中完成不同的任务。ECs具有显著的表型和功能异质性。然而，只有最近单细胞RNA测序（scRNA-seq）的进展，才能使研究人员能够真正全面地定义ECs的分子异质性。此外，scRNA-seq揭示了与许多主要疾病相关的ECs如何对压力和损伤做出反应的新原理。由于肿瘤生长依赖于持续的营养和氧气供应，新生血管形成（新生血管生成）是癌症的一个标志。对于许多类型的癌症，阻断血管内皮生长因子（VEGF）信号的抗血管生成治疗效果不如预期，但肾癌已被证明对靶向VEGF受体（VEGFR）的酪氨酸激酶抑制剂（TKIs）有相当大的反应。这与肾癌是高度血管化的，并且具有驱动新血管生成的突变的事实是一致的。透明细胞肾细胞癌（ccRCC）是最常见的亚型。VEGFR TKIs是目前ccRCC（晚期）联合免疫检查点阻断治疗的主流，尽管疗效和持久性是可变的。在这种情况下，ECs在VEGFR TKIs治疗的原发性或获得性耐药中的确切作用尚不清楚。因此，迫切需要对ccrcc的EC异质性进行更深入的分子和机制研究。肿瘤内部和周围血管的增生和萌芽是新生血管生成的典型组织学特征。该领域的大多数研究都集中在微血管密度或肿瘤细胞特征（如突变）上，但我们对ECs和ccRCC肿瘤细胞之间复杂的相互作用的理解远远落后。此外，病理学家认识到ccrcc具有非常不同的血管结构特征，大致可分为肾小球、低分支、高分支和窦状吻合。事实上，尚不清楚这些不同的血管模式是否与内皮细胞具有不同的分子相关性，以及这些表型是否本质上由内皮细胞驱动，受周围肿瘤细胞的指示，或两者兼而有之。本质上，这些都是我们在项目中要解决的问题。为此，我们将分离ECs并进行scRNA-seq，以探索人类ccrcc中ECs的表型空间。此外，我们将通过全外显子组测序和高复合免疫荧光将新的EC表型与ccrcc的基因组和空间背景联系起来。最后，我们将采用创新的患者来源的肿瘤片段平台和EC共培养分析来研究来自ccrcc的EC的表型多样性如何与功能多样性相关联。我们相信，我们将在ccrcc中建立EC异质性的新概念，并描绘EC表型如何指导基于VEGFR tki的治疗分层的新途径。

Endothelial cells (ECs) line the inner wall of blood vessels fulfilling different tasks in our organs. ECs are characterized by remarkable phenotypic and functional heterogeneity. However, only the recent advances in single-cell RNA sequencing (scRNA-seq) enabled researchers to define the molecular heterogeneity of ECs in a truly comprehensive manner. Also, scRNA-seq revealed novel principles how ECs respond to stresses and injuries being relevant for many major diseases. As tumour growth is dependent on constant nutrient and oxygen supply, de novo blood vessel formation (neoangiogenesis) is a hallmark of cancer. For many cancer types, anti-angiogenic therapy that blocks vascular endothelial growth factor (VEGF) signalling was less effective than hoped for, but renal cancers proved to be quite responsive to tyrosine kinase inhibitors (TKIs) targeting the VEGF receptor (VEGFR). This is in line with the fact that renal cancers are highly vascularized and harbour mutations that drive neoangiogenesis. Clear cell renal cell carcinoma (ccRCC) represents the most frequent subtype. VEGFR TKIs are the current mainstay of ccRCC treatment (advanced stage) combined with immune checkpoint blockade, though efficacy and durability are variable. In this context, the precise role of ECs in primary or acquired resistance to VEGFR TKIs treatment is poorly understood. Thus, more profound molecular and mechanistic insights into EC heterogeneity of ccRCCs are critically needed. Proliferation and sprouting of vessels within and around tumours are typical histological features of neoangiogenesis. Most studies in the field focused either on microvasculature density or on tumour cell characteristics like mutations, but our understanding of the complex interplay between ECs and ccRCC tumour cells lacks far behind. Moreover, pathologists appreciate that ccRCCs are characterized by very different blood vessel architectures, which can be roughly classified as glomeruloid, low branching, high branching and sinusoidal-anastomosing. As a matter of fact, it is unknown whether these distinct vascular patterns actually have distinct molecular correlates of ECs or not, and whether these phenotypes are intrinsically driven by ECs, instructed by the surrounding tumour cells or both. Essentially, these are the question that we aim to address in our project. For this, we will isolate ECs and perform scRNA-seq in order to explore the phenotypic space ECs in human ccRCCs. Further, we will link novel EC phenotypes to the genomic and spatial context of ccRCCs by whole-exome sequencing and high-plex immunofluorescence. Lastly, we will employ an innovative patient-derived tumour fragment platform and EC co-culture assays to study how phenotypic diversity of ECs from ccRCCs is linked to functional diversity. We believe that we will establish novel concepts of EC heterogeneity in ccRCCs and delineate new avenues how EC phenotypes may guide the stratification for VEGFR TKI-based therapies.