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

<strong>README</strong>Data Underlying: Inter-spheroid Proximity and Matrix Remodelling Determines Cancer Associated Fibroblast - 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 Cancer Associated Fibroblast (CAF) - mediated Cancer Cell Invasion”. This work investigates the interaction between 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 CAFs spheroids (H2B-GFP-labeled), and heterospheroids consisting of a 1:1 ratio of MCF7 (mCherry-labelled) and 19TT 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>README</strong> 支撑数据集：<strong>球体间邻近性与基质重塑决定癌相关成纤维细胞介导的癌细胞侵袭</strong> 稿件状态：已提交并处于审稿阶段
<strong>概述</strong>
本数据集支撑题为《球体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》的学术稿件。本研究聚焦癌相关成纤维细胞（CAF）与管腔A型乳腺癌细胞（MCF7）球体在胶原基质中的相互作用，旨在阐明CAF促进癌细胞侵袭的交互机制。通过延时成像与量化分析，本研究揭示了两种细胞类型间的系统性交互模式。本数据集包含三类样本的三维球体侵袭实验的原始与处理后数据：其一为标记有mCherry的MCF7乳腺癌细胞球体；其二为标记有H2B-GFP的19TT CAFs球体；其三为按1:1比例混合标记mCherry的MCF7细胞与标记H2B-GFP的19TT CAF细胞构建的混合球体。所有实验均在3 mg/mL的I型胶原基质中完成。侵袭实验的持续时长为48小时（对应图2、5、6），MMP抑制实验时长为24小时（对应图3、4）。成像采用延时荧光显微镜，并结合反射成像以可视化胶原纤维结构。
<strong>文件夹结构</strong>
主文件夹命名为Data_Fig1、Data_Fig2至Data_Fig6。各主文件夹内包含四个子文件夹：Datasets、Fig Images、Graphs及Movies。其中，Datasets文件夹存储用于分析并生成图表与图像的原始数据文件。图1所用数据集（IF_pCK_aSMA与PSR_FL_slide_scans）仅可通过QuPath软件打开；其余图表所用数据集可通过FIJI/ImageJ2软件打开。Datasets文件夹进一步按实验条件（如Control、BB94）划分次级子文件夹。
Fig Images文件夹存储学术稿件正图中使用的所有图像文件。Graphs文件夹包含图表的矢量图像文件（.pdf）、存储量化数据的Excel文件（.xlsx）以及GraphPad Prism原生图表文件（.pzfx）。Movies文件夹存储对应正图数据集的视频文件（.avi）。
Sample Analysis文件夹包含各类样本分析文件与中间产物，每种分析方法均使用特定图表的对应数据进行演示。例如，Fig2C_Expansion_Analysis文件夹包含用于生成图2C中面积变化（dArea）图表的两份数据集、中间产物与最终分析结果。
Figure Files文件夹存储矢量格式（.pdf）与Keynote格式（.key）的图形摘要、正文主图以及投稿用补充图表。Submission Videos文件夹存储所有与稿件相关的视频文件，按对应图表进行分类整理。
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）以及胶原空隙率与像素强度测量脚本（Pxq_VF.m）。使用此类脚本前，需先从复合TIFF图像中分离各通道，并通过ImageJ保存为图像序列。所有脚本均为模块化设计并带有详细注释，需依赖MATLAB的图像处理工具箱，部分脚本需使用自定义纤维分析函数。本文件夹内的自定义函数包括：CAF_center.m、Centroid_3d.m、Circfit.m、Histwc.m、Imcrop.m及PDF_function3.m。
Supporting Information文件夹存储用于补充图表的数据集与图表文件，其文件夹结构与主数据文件夹完全一致。FigS2_CAF02-03-06文件夹包含CAF02、CAF03与CAF06肿瘤样本的.mrxs格式玻片扫描图像文件。图表文件保存为Excel（.xlsx）与GraphPad Prism（.pzfx）格式。
Text文件夹存储各类与稿件相关的文档，包括正文稿件（.docx）、图表说明（.docx）、电子补充材料（.docx）、利益冲突声明（.docx）、研究重要性声明（.docx）以及补充材料文本（.docx）。
本数据集包含的文件类型如下：文本文件（.docx）、存储量化数据的Excel电子表格（.xlsx）、原始数据集与图表的图像文件（.tiff）、MATLAB脚本文件（.m）、GraphPad Prism原生图表文件（.pzfx）、玻片扫描图像文件（.mrxs）、Keynote演示文件（.key）以及高分辨率矢量图像（.pdf）。