Boykin Libby A, Jayashankar Sneha, Budhwani Khidr Kishan K, Budhwani Brahma Mubarak K, Samal Diya, Crawford Chelsea L, Tsung Allan, Budhwani Karim I
CerFlux, Birmingham, AL, USA.
Department of Surgery, University of Virginia School of Medicine, Charlottesville, VA, USA.
bioRxiv. 2025 Jun 27:2025.06.25.661355. doi: 10.1101/2025.06.25.661355.
The extracellular matrix (ECM) plays a critical role in colorectal cancer (CRC) progression and therapeutic resistance. Accurate characterization of ECM composition and architecture is essential for understanding how CRC evades therapy, yet most protocols either assess ECM components in isolation or remain technically challenging. Here we present a robust yet simple protocol for spatial characterization of collagen and glycosaminoglycan (GAG) organization within the CRC tumor microenvironment (TME). Our method combines Alcian Blue and Picrosirius Red staining procedures with standardized tissue processing and imaging protocols. The protocol enables simultaneous visualization, assessment, and quantification of collagen and GAG distribution patterns in formalin-fixed, paraffin-embedded tissue sections. Key methodological advances include optimized dual-staining approach with distinct blue-red coloration for straightforward spectral separation on digital imaging systems, standardized reagent preparations, and validated imaging parameters. The complementary wavelengths facilitate both visual interpretation and potential digital separation, offering advantages over multi-component stains with overlapping spectral ranges. Validation across multiple CRC patient specimens demonstrates excellent reproducibility with consistent staining intensity using standard histology equipment. Because the resulting spatial maps can be compared directly with engineered or tumor models, the protocol also provides a practical benchmark for microenvironment validation. This standardized approach to ECM visualization will advance TME research, support morphological studies, and enable comparative analyses across CRC subtypes. The methodology can be adapted to other solid tumor types and integrated with complementary techniques including digital pathology workflows for comprehensive microenvironment characterization and enhanced analysis capabilities.
细胞外基质（ECM）在结直肠癌（CRC）进展和治疗耐药性中起关键作用。准确表征ECM的组成和结构对于理解CRC如何逃避治疗至关重要，但大多数方案要么单独评估ECM成分，要么在技术上仍具有挑战性。在这里，我们提出了一种强大而简单的方案，用于在CRC肿瘤微环境（TME）中对胶原蛋白和糖胺聚糖（GAG）的组织进行空间表征。我们的方法将阿尔辛蓝和天狼星红染色程序与标准化的组织处理和成像方案相结合。该方案能够在福尔马林固定、石蜡包埋的组织切片中同时可视化、评估和定量胶原蛋白和GAG的分布模式。关键的方法学进展包括优化的双重染色方法，具有独特的蓝红色，以便在数字成像系统上进行直接光谱分离、标准化的试剂制备和经过验证的成像参数。互补波长便于视觉解释和潜在的数字分离，比具有重叠光谱范围的多组分染色具有优势。对多个CRC患者标本的验证表明，使用标准组织学设备，染色强度一致，具有出色的可重复性。由于所得的空间图谱可以直接与工程或肿瘤模型进行比较，该方案还为微环境验证提供了一个实用的基准。这种标准化的ECM可视化方法将推动TME研究，支持形态学研究，并能够对CRC亚型进行比较分析。该方法可以适用于其他实体瘤类型，并与包括数字病理学工作流程在内的互补技术相结合，以进行全面的微环境表征和增强分析能力。
