Project 2: Cellular topography and function of the breast cancer tissue microenvironment

摘要/项目摘要 乳腺癌（BC）转移是一个紧急的特点，发生时，肿瘤的能力，招募代谢， 资源，避免免疫激活，并传播到遥远的网站超过周围正常的能力 组织以防止这些过程。与此相一致，过去十年的工作已经证明了它的关键作用 肿瘤微环境（TME）在促进或阻止BC进展中发挥作用。肿瘤细胞迁移 和免疫募集都被证明受到成纤维细胞和周围环境的严重影响。 细胞外基质（ECM）是它们产生的。此外，蛋白质糖基化已被证明可以调节这些 交互.为了了解肿瘤糖基化和ECM重塑如何相互作用以增强BC转移， 我们将使用空间转录组学和两种互补的质谱方法，MIBI-TOF和MALDI， 确定聚糖依赖性、细胞-细胞和细胞-ECM相互作用，这些相互作用使TME向肿瘤允许的方向转变， states.所有三项分析均将对来自相同组织块的空间配准连续切片进行。 在这样做时，通过MIBI-TOF构建的每个组织样本的全面单细胞图谱可以直接用于 与从头蛋白质组学和转录组学数据叠加。我们将绘制并列举血统， 肿瘤细胞和基质细胞的主要功能亚群， 参数，以了解BC TME如何随疾病进展而演变。这些要素将被叠加 通过组织聚糖的从头成像来识别涉及肿瘤的免疫逃避的潜在机制， 唾液酸聚糖和巨噬细胞结合的SIGLEC。这些特征的频率和空间富集将是 与空间转录组学数据相关，以鉴定促进这些的调节性糖基转移酶。 交互.接下来，将使用ECM-MALDI和MIBI-TOF数据来确定胶原类型、羟基化和 交联转移与相邻细胞群的胶原结构和功能相协调。在 特别是，我们将重点了解两个家族的酶，脯氨酰和赖氨酰的活性如何 羟化酶驱动ECM中的结构变化，促进BC转移。这些临床意义 ECM重塑的提取的细胞和分子定义将相对于转移风险进行评估， 阶段和IC亚型。总的来说，这项工作将提供一个前所未有的观点，如何TME结构 肿瘤和间质之间的细胞-细胞相互作用涉及肿瘤ECM和糖组的特定方面。

Abstract/Project Summary Breast cancer (BC) metastasis is an emergent feature that occurs when the tumor’s ability to recruit metabolic resources, avoid immune activation, and disseminate to distant sites exceeds the capacity of surrounding normal tissue to prevent these processes. In line with this, work over the last decade has demonstrated the crucial role played by the tumor microenvironment (TME) in promoting or deterring BC progression. Tumor cell migration and immune recruitment have both been shown to be heavily influenced by fibroblasts and the surrounding extracellular matrix (ECM) they produce. Additionally, protein glycosylation has been shown to modulate these interactions. To understand how tumor glycosylation and ECM remodeling interact to potentiate BC metastasis, we will use spatial transcriptomics and two complementary mass spectrometry methods, MIBI-TOF and MALDI, to identify glycan-dependent, cell-cell, and cell-ECM interactions that shift the TME toward tumor permissive states. All three analyses will be performed on spatially-coregistered serial sections from the same tissue blocks. In doing so, comprehensive single-cell maps of each tissue sample constructed by MIBI-TOF can be directly superimposed with de novo proteomic and transcriptomic data. We will map and enumerate the lineage and major functional subsets of tumor and stromal cells with respect to relevant therapeutic and molecular parameters to understand how the BC TME evolves with disease progression. These features will be overlaid with de novo imaging of tissue glycans to identify potential mechanisms of immune evasion that involve tumor sialoglycans and macrophage-bound SIGLECs. The frequency and spatial enrichment of these features will be correlated with spatial transcriptomics data to identify regulatory glycosyltransferases promoting these interactions. Next, ECM-MALDI and MIBI-TOF data will be used to identify how collagen type, hydroxylation, and crosslinking shift in coordination with the collagen structure and function of neighboring cell populations. In particular, we will focus on understanding how the activity of two families of enzymes, prolyl and lysyl hydroxylases, drive structural changes in the ECM that promote BC metastasis. The clinical significance of these extracted cellular and molecular definitions of ECM remodeling will be assessed with respect to metastatic risk, stage, and IC subtype. Taken together, this work will provide an unprecedented view into how TME structure and cell-cell interactions between tumor and stroma relate to specific facets of the tumor ECM and glycome.