Data underlying the publication: Inter-Spheroid proximity and matrix remodelling determine CAF mediated cancer cell invasion

<strong>README</strong>Data Underlying: Inter-spheroid Proximity and Matrix Remodelling Determines CAF-mediated Cancer Cell InvasionStatus: Manuscript submitted and currently under review<br><strong>Overview</strong>This dataset supports the manuscript titled “Inter-spheroid Proximity and Matrix Remodelling Determines CAF-mediated Cancer Cell Invasion”. This work investigates the interaction between Cancer Associated Fibroblast (CAFs) and Luminal A breast cancer cell (MCF7s) spheroids in collagen matrices, with the objective of elucidating the interaction mechanisms that enable CAFs to promote cancer cell invasion. Timelapse imaging and analysis reveals a systematic interaction pattern between the two cell types. This dataset includes raw and processed data from 3D spheroid invasion assays of MCF7 breast cancer cell spheroids (mCherry-labelled), 19TT Cancer-Associated Fibroblast (CAF) spheroids (H2B-GFP-labeled), and heterospheroids consisting of a 1:1 ratio of MCF7 (mCherry-labelled) and 19TT Cancer-Associated Fibroblast (CAF) cells (H2B-GFP-labeled). All experiments were conducted in 3 mg/mL type 1 Collagen matrices. For invasion assays, experiments were run for a duration of 48h (Figs 2, 5, and 6) and 24h for MMP inhibition experiments (Figs 3 and 4). Imaging was performed using time-lapse fluorescence microscopy with reflection imaging to visualize collagen structure.<br><strong>Folder Structure</strong>Main folders are named Data_Fig1, Data_Fig2 through Data_Fig6. Within these folders, subfolders include Datasets, Fig Images, Graphs, and Movies. The Datasets folder contains the raw files used to analyse and generate images and graphs. Datasets used in Fig 1 (IF_pCK_aSMA and PSR_FL_slide_scans) can only be opened using the QuPath software. Datasets for the rest of the figures can be opened using FIJI/ImageJ2. Dataset folders are further divided into sub-folders by condition (e.g. Control, BB94). The Fig Images folder contains images used in manuscript figures. The Graphs folder contains vector image files of graphs (.pdf), Excel files (.xlsx) with data values, and Prism Graphpad files (.pzfx). The Movies folder contains video files (.avi) for datasets used in figures.Sample Analysis contains subfolders with sample analysis files and intermediates. Each type of analysis is shown once using data from a specific figure. For example, the Fig2C_Expansion_Analysis folder contains two datasets, intermediates, and results for the dArea graphs in Fig2C.Figure Files includes the graphical abstract, main text figures, and supporting submission figures in vector format (.pdf) and Keynote format (.key).Submission Videos contains all movies linked to the manuscript, organized by figure.Scripts contains all MATLAB codes used to analyse and quantify data. These include scripts for collagen fibre orientation analysis (colfibre_orientation.m, colfibre_mcf.m), CAF invasion index quantification (invasive_index.m), and collagen void fraction and pixel intensity measurement (Pxq_VF.m). To use these scripts, extract individual channels from composite tiff images and save them as image sequences via ImageJ. Scripts are modular, annotated, and require MATLAB’s Image Processing Toolbox. Some use custom fibre analysis functions. Custom functions saved in this folder include CAF_center.m, Centroid_3d.m, Circfit.m, Histwc.m, Imcrop.m, and PDF_function3.m. Supporting Information contains datasets and figure files used for supporting figures. The folder structure mirrors the main data folders. FigS2_CAF02-03-06 contains .mrxs files for CAF02, CAF03, and CAF06 tumors. Graphs are saved as Excel (.xlsx) and Prism Graphpad (.pzfx) files.Text contains various manuscript-related documents including the main manuscript (.docx), figure captions (.docx), electronic supporting information (.docx), declaration of competing interests (.docx), statement of significance (.docx), and supporting information text (.docx).Filetypes included in the dataset are text files (.docx), Excel spreadsheets (.xlsx) containing quantified data, image files (.tiff) of raw datasets and figures, MATLAB scripts (.m), native graph files (.pzfx) from Prism, slide scanner image files (.mrxs), Keynote files (.key), and high-resolution vector images (.pdf).

<strong>说明文档</strong>底层数据集：类器官球间邻近性与基质重塑决定癌相关成纤维细胞（Cancer Associated Fibroblast，CAF）介导的癌细胞侵袭<br>状态：已投稿，目前处于审稿阶段<br><strong>概述</strong>本数据集支撑题为《类器官球间邻近性与基质重塑决定癌相关成纤维细胞（CAF）介导的癌细胞侵袭》的学术论文。本研究探究了胶原基质中癌相关成纤维细胞（Cancer Associated Fibroblast，CAF）与管腔A型乳腺癌细胞（Luminal A breast cancer cell，MCF7s）类器官球之间的相互作用，旨在阐明CAF促进癌细胞侵袭的相互作用机制。延时成像与分析揭示了两种细胞间的系统性相互作用模式。本数据集包含三类样本的三维类器官球侵袭实验原始与处理后数据：标记有mCherry的MCF7乳腺癌细胞类器官球、标记有H2B-GFP的19TT癌相关成纤维细胞（CAF）类器官球，以及按1:1比例混合的MCF7（mCherry标记）与19TT癌相关成纤维细胞（CAF）细胞构成的混合类器官球。所有实验均在3 mg/mL的I型胶原基质中完成。侵袭实验的培养时长为48小时（对应图2、5、6），而基质金属蛋白酶（Matrix Metalloproteinase，MMP）抑制实验的培养时长为24小时（对应图3、4）。成像采用延时荧光显微镜结合反射成像技术，以可视化胶原基质结构。<br><strong>文件夹结构</strong>主文件夹命名为Data_Fig1、Data_Fig2至Data_Fig6。各主文件夹内包含以下子文件夹：Datasets、Fig Images、Graphs与Movies。Datasets文件夹存储用于分析并生成图表的原始文件。图1所用数据集（IF_pCK_aSMA与PSR_FL玻片扫描文件）仅可通过QuPath软件打开。其余图表对应的数据集可通过FIJI/ImageJ2软件打开。数据集文件夹还会根据实验条件进一步细分子文件夹（例如对照组Control、BB94处理组）。Fig Images文件夹存储论文图表所用的图像文件。Graphs文件夹包含图表的矢量图像文件（.pdf格式）、存储量化数据的Excel文件（.xlsx格式）以及GraphPad Prism原生文件（.pzfx格式）。Movies文件夹存储对应图表数据集的视频文件（.avi格式）。<br>Sample Analysis文件夹包含各类样本分析文件与中间产物的子文件夹。每种分析类型均会使用特定图表的数据集进行一次演示。例如，Fig2C_Expansion_Analysis文件夹包含用于生成图2C中dArea图表的两个数据集、中间产物与分析结果。<br>Figure Files文件夹存储矢量格式（.pdf）与Keynote格式（.key）的图形摘要、正文配图以及投稿用补充图表。Submission Videos文件夹存储所有与论文相关的视频文件，并按图表进行分类整理。<br>Scripts文件夹存储所有用于数据分析与量化的MATLAB代码，包括胶原纤维取向分析脚本（colfibre_orientation.m、colfibre_mcf.m）、CAF侵袭指数量化脚本（invasive_index.m）以及胶原孔隙率与像素强度测量脚本（Pxq_VF.m）。使用这些脚本前，需通过ImageJ从复合TIFF图像中分离各通道，并保存为图像序列。所有脚本均为模块化设计并带有注释，且需要使用MATLAB的图像处理工具箱。部分脚本使用自定义纤维分析函数，本文件夹内存储的自定义函数包括：CAF_center.m、Centroid_3d.m、Circfit.m、Histwc.m、Imcrop.m与PDF_function3.m。<br>Supporting Information文件夹存储补充图表所用的数据集与图像文件，其文件夹结构与主数据文件夹一致。FigS2_CAF02-03-06文件夹包含CAF02、CAF03与CAF06肿瘤样本的.mrxs格式文件。图表保存为Excel（.xlsx）与GraphPad Prism（.pzfx）格式文件。<br>Text文件夹存储各类与论文相关的文档，包括正文手稿（.docx）、图表说明（.docx）、电子版补充材料（.docx）、利益冲突声明（.docx）、重要性声明（.docx）以及补充材料文本（.docx）。<br>本数据集包含的文件格式如下：文本文件（.docx）、存储量化数据的Excel表格（.xlsx）、原始数据集与图表的图像文件（.tiff）、MATLAB脚本（.m）、GraphPad Prism原生图表文件（.pzfx）、玻片扫描图像文件（.mrxs）、Keynote文件（.key）以及高分辨率矢量图像（.pdf）。