PO.TB02.02 · 肿瘤生物学
一种多尺度成像流程识别结直肠癌中促进肿瘤存活的NMII激活区域
A multi-scale imaging pipeline identifies regions of NMII activation in colorectal cancer which promote tumor survival
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
本研究旨在界定结直肠癌（CRC）中非肌性肌球蛋白II（NMII）的空间激活，并确定其如何促进肿瘤存活。我们实验室已识别出在低黏附环境中于ROCK1/NMII驱动的膜泡（bleb）基部形成的感知曲率、促存活的信号中枢。采用这种弱黏附、高收缩性的阿米巴样表型与代谢转变和免疫抑制相关，会恶化预后。然而，这些观察主要来自小鼠模型，尚未在人类疾病中得到验证。我们假设，在人类CRC中，升高的肌动球蛋白收缩性通过激活ROCK1/NMII信号并减少肿瘤-免疫细胞接触，在黏附不良区域维持肿瘤存活。为检验这一假设，我们开发了一个循环多重免疫荧光组合（CODEX），并将其应用于CRC组织微阵列和全切片CRC标本。该组合包括细胞身份、代谢、增殖和免疫激活的标志物。空间分析使用SPACEMAP进行，这是一个整合多种表型分析算法以提高细胞类型分类准确性的共识机器学习平台。为表征纳米级结构，将每张CODEX成像切片相邻的30 μm切片包埋于水凝胶中、膨胀，并使用轴向扫描光片显微镜（ASLM）成像。膨胀的体积被配准到多重免疫荧光数据集，实现约80 nm各向同性分辨率的亚细胞分析。使用u-Segment3D进行3D分割和形态学定量。在25%的CRC病例中观察到肿瘤上皮中活性NMII（pMLC2）信号，定位于细胞间黏附减少的离散区域，包括坏死区、黏液池、腔面和孤立肿瘤细胞。pMLC2高表达的肿瘤细胞表现出增殖减少和线粒体丰度增加。引人注目的是，粒细胞和细胞毒性T细胞也表现出升高的pMLC2表达，而这些pMLC2高表达的免疫细胞显示出增加的免疫抑制标志物。CODEX与ExASLM数据集的配准实现了对膜结构的高分辨率评估，揭示了pMLC2高表达生态位内肿瘤细胞形态和细胞间界面的独特改变。总之，这些发现表明CRC肿瘤在低黏附微环境中上调pMLC2，通过增殖、代谢、膜结构和免疫接触的协调变化支持肿瘤存活。这项工作将空间调控的肌动球蛋白收缩性识别为CRC中一种关键的存活程序，并将其凸显为一个潜在的治疗易感靶点。
查看英文原文 English abstract
The aim of this study is to define the spatial activation of non-muscle myosin II (NMII) in colorectal cancer (CRC) and determine how it promotes tumor survival. Our lab has identified curvature-sensing, pro-survival signaling hubs that form at the base of ROCK1/NMII-driven blebs in low-adhesion environments. Adoption of this weakly adherent, highly contractile amoeboid phenotype is associated with metabolic shifts and immunosuppression that worsen prognosis. However, these observations come largely from mouse models and have yet to be validated in human disease. We hypothesize that, in human CRC, elevated actomyosin contractility sustains tumor survival in poorly adherent regions by activating ROCK1/NMII signaling and reducing tumor-immune cell engagement. To test this hypothesis, we developed a cyclic multiplexed immunofluorescence panel (CODEX) and applied it to a CRC tissue microarray and whole-slide CRC specimens. The panel included markers of cell identity, metabolism, proliferation, and immune activation. Spatial analysis was performed using SPACEMAP, a consensus machine-learning platform that integrates multiple phenotyping algorithms to improve cell-type classification accuracy. To characterize nanoscale architecture, a 30-µm section adjacent to each CODEX-imaged slide was embedded in a hydrogel, expanded, and imaged using Axially Swept Light-Sheet Microscopy (ASLM). Expanded volumes were registered to the multiplexed immunofluorescence dataset, enabling subcellular analysis with ~80-nm isotropic resolution. 3D segmentation and morphological quantification were performed using u-Segment3D. Active NMII (pMLC2) signal in the tumor epithelium was observed in 25% of CRC cases, localizing to discrete regions with reduced cell-cell adhesion, including necrotic zones, mucin pools, luminal surfaces, and isolated tumor cells. pMLC2-high tumor cells exhibited decreased proliferation and increased mitochondrial abundance. Strikingly, both granulocytes and cytotoxic T cells also demonstrated elevated pMLC2 expression, and these pMLC2-high immune cells showed increased markers of immunosuppression. Registration of CODEX and ExASLM datasets enabled high-resolution assessment of membrane architecture, revealing distinct alterations in tumor cell morphology and cell-cell interfaces within pMLC2-high niches. Together, these findings demonstrate that CRC tumors upregulate pMLC2 within low-adhesion microenvironments, supporting tumor survival through coordinated changes in proliferation, metabolism, membrane architecture, and immune engagement. This work identifies spatially regulated actomyosin contractility as a critical survival program in CRC and highlights it as a potential therapeutic vulnerability.
利益披露 Disclosure
D. H. Abbott, None..
T. Ngo, None..
K. M. Dean, None.