Fig. 5

Proximity between colorectal cancer cells and macrophages. A The representative CD68 immunohistochemistry-(IHC) in colorectal cancer (CRC) at depth 4; To evaluate tumor–stromal interactions between CRC cells and macrophages (Mφs), CRC cells and CD68-positive (CD68⁺) Mφs were annotated. The representative image of CD68-IHC identified 33 CRC cells as red dots and three CD68⁺ Mφs as green dots at depth 4 by HALO software. Bar, 30 μm. B Histological spatial analysis clipped the colored dots of A; Thirty-three red dots and three green dots at depth 4 were clipped and the extracted images (analytical images) could clarify the positioning of these dots. The gray-colored lines show the shortest distance between a single CRC cell (red dots) and CD68⁺ Mφs within 30 µm of the CRC cell (green dots). Bar, 30 μm. C The number of CD68⁺ Mφs in each interval of the distance between CRC cells and CD68⁺ Mφs at depth 4; The histogram revealed that 98.3% of CD68⁺Mφs were accumulated within 60 μm. The highest accumulation of CD68⁺Mφs was observed between 0 and 20 μm. D The number of CD163⁺ Mφs in each interval of the distance between CRC cells and CD163⁺ Mφs at depth 4; The histogram revealed that 99.5% of CD163⁺Mφs were accumulated within 60 μm. The highest accumulation of CD163⁺Mφs was observed between 0 and 20 μm. E The representative CD68-IHC in CRC at depth 5; similar to A, CRC cells and CD68-positive (CD68⁺) Mφs were annotated. The representative image of CD68-IHC identified 13 CRC cells as red dots and 37 CD68⁺ Mφs as green and light green dots at depth 5 by HALO software. Bar, 30 μm. F Histological spatial analysis clipped the colored dots from E; Thirteen red dots and 37 green and light green dots at depth 5 were clipped and the extracted images (analytical images) could clarify the positioning of these dots. The gray-colored lines show the shortest distance between a single CRC cell (red dots) and CD68⁺ Mφs within 30 µm of the CRC cell (green dots). Bar, 30 μm. G The number of CD68⁺ Mφs in each interval of the distance between CRC cells and CD68⁺ Mφs at depth 5; the histogram revealed that 98.2% of CD68⁺Mφs were accumulated within 60 μm. The highest accumulation of CD68⁺Mφs was observed between 0 and 20 μm. H The number of CD163⁺ Mφs in each interval of the distance between CRC cells and CD163⁺ Mφs at depth 5; The histogram revealed that 96.7% of CD163⁺Mφs were accumulated within 60 μm. The highest accumulation of CD163⁺Mφs was observed between 0 and 20 μm