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

摘要/项目摘要 乳腺癌(BC)转移是一种紧急特征，当肿瘤的新陈代谢能力 资源，避免免疫激活，并传播到远端超过周围正常能力的地方 组织来阻止这些过程。与此相一致，过去十年的工作证明了这一关键作用 由肿瘤微环境(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 它们产生细胞外基质(ECM)。此外，蛋白质的糖基化已被证明可以调节这些 互动。为了了解肿瘤糖基化和细胞外基质重塑如何相互作用以增强BC转移， 我们将使用空间转录和两种互补的质谱学方法，MIBI-TOF和MALDI， 确定糖依赖、细胞-细胞和细胞-ECM相互作用使TME向肿瘤允许方向转变 各州。所有这三种分析都将在来自相同组织块的空间共配准的连续切片上进行。 通过这样做，MIBI-TOF构建的每个组织样本的全面单细胞图谱可以直接 叠加了从头开始的蛋白质组和转录组数据。我们将绘制并列举世系和 肿瘤和基质细胞的主要功能亚群与相关的治疗和分子 参数以了解BC TME如何随着疾病的进展而演变。这些要素将被叠加 通过组织糖链的从头成像来确定涉及肿瘤的潜在免疫逃逸机制 唾液酸聚糖和巨噬细胞结合的SIGLECs。这些特征的频率和空间丰富程度将是 与空间转录数据相关联以识别促进这些的调节糖基转移酶 互动。接下来，ECM-MALDI和MIBI-TOF数据将被用来确定胶原类型、羟化和 与邻近细胞群的胶原蛋白结构和功能相协调的交联性变化。在……里面 特别是，我们将重点了解两个酶家族，Pro和赖氨酸的活性是如何 羟基酶，驱动ECM的结构变化，促进BC转移。这些指标的临床意义 提取的ECM重塑的细胞和分子定义将根据转移风险进行评估， 阶段，和IC亚型。综上所述，这项工作将提供一个前所未有的视角来了解TME的结构 肿瘤和间质之间的细胞间相互作用与肿瘤细胞外基质和糖蛋白的特定方面有关。

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.