Fig. 1

Image capture and quantitative evaluation of collagen, elastin, and immune cells. (a) For the image acquisition of EVG staining and the quantitative evaluation of collagen and elastin, five representative images were captured at 100× magnification. The percentage of collagen and elastin area was assessed using the Hybrid Cell Count system, and the calculation was performed by dividing the extracted area by the total area of each image. (b) For image acquisition of immunohistochemical staining and immune cell quantification (CD8⁺, CD3⁺, CD86⁺, and CD163⁺), four representative areas, each composed of nine images, were captured at 200× magnification. The number of stained immune cells per mm2 was semiautomatically counted using the Hybrid Cell Count system. Scale bar, 100 μm. (c) Representative images of EVG staining showing the distribution of collagen and elastin in adjacent normal and cancerous tissues. Images were captured at 100× magnification. Scale bar, 100 μm. (d) Comparison of collagen and elastin distribution between adjacent normal tissue and primary tumor tissue from 19 patients from whom the sections were available to evaluate cancerous and noncancerous areas on EVG-stained CRC sections simultaneously. The Wilcoxon matched-pairs tests analyzed the differences. (e) Categorization of samples according to collagen and elastin deposition. The categorization process of tumors was based on intratumoral collagen and elastin according to the cutoff values determined based on ROC curves for cancer recurrence. (f) The EVG scores were determined based on collagen and elastin deposition. CRC samples with high collagen and elastin levels were categorized as EVG-high. CRC samples were classified as EVG-low when collagen, elastin, or both were detected at low levels. The upper panel shows representative images of patients with low and high EVG scores.