Fig 1.
Original and post-processed images of α-SMA-positive cancer-associated fibroblasts.
(A) α-SMA-positive cancer-associated fibroblasts at x40 HPF. (B) A post-processed image in which the brown area of α-SMA-positive cancer-associated fibroblasts was changed to a red color using ImageJ.
Fig 2.
Original and post-processed images of Masson’s trichrome staining.
(A) Collagen fibers stained blue on Masson’s trichrome staining at x40 HPF. (B) A post-processed image in which the blue area of collagen fibers was changed to a red color using ImageJ.
Fig 3.
Regarding the evaluation of the distribution of TILs in the high-fibrosis area and that in the low-fibrosis area, the median value of the area ratios of collagen fibers in peritoneal metastases was defined as the cut-off value (15.64%).
Areas with an area ratio of collagen fibers greater than the cut-off value (≥15.64%) were defined as high-fibrosis areas, and those with an area ratio of collagen fibers lower than the cut-off value (<15.64%) were defined as low-fibrosis areas. (A) The distribution of tumor-infiltrating lymphocytes in fibrotic areas on double-staining using immunohistochemical staining of anti-CD3 and anti-CD8 and Masson’s trichrome staining at x200 HPF. (B) A post-processed image in which the blue area of collagen fibers changed to a red color and were able to be manually separated into high- and low-fibrosis areas (C) according to the density of collagen fibers. (D) To verify the accuracy of the division, the area ratios of collagen fibers were calculated in each area using the ImageJ software program (area ratios of high-fibrosis areas of a sample image: 41.0% and 17.1%; area ratio of a low-fibrosis area of a sample image: 3.1%) and contrasted with the definitions. If inaccurate division was suspected, the division of the fibrosis area selected by the ImageJ software program was adjusted until the defined conditions were met. (E) The area ratios of the high- and low-fibrosis areas were calculated using the ImageJ software program, and the numbers of tumor-infiltrating lymphocytes in the high- and low-fibrosis areas were counted. The densities of CD3+/CD8+TILs in the high- and low-fibrosis areas were then calculated.
Fig 4.
CD3-positive tumor-infiltrating lymphocytes and positive controls.
(A) The specimens of lymph nodes containing CD3-positive T lymphocytes were used as positive controls for immunohistochemical staining of anti-CD3 (x400 HPF). (B) Immunohistochemical detection of CD3-positive tumor-infiltrating lymphocytes (x400 HPF).
Fig 5.
CD8-positive tumor-infiltrating lymphocytes and positive controls.
(A) The specimens of lymph nodes containing CD8-positive T lymphocytes were used as positive controls for immunohistochemical staining of anti-CD8 (x400 HPF). (B) Immunohistochemical detection of CD8-positive tumor-infiltrating lymphocytes (x400 HPF).
Fig 6.
α-SMA-positive cancer-associated fibroblasts and internal positive controls.
(A) α-SMA expression in vascular smooth muscle (red arrow) was used as an internal positive control for immunohistochemical staining of anti-α-SMA (x40 HPF). (B) Immunohistochemical detection of α-SMA-positive cancer-associated fibroblasts (x40 HPF).
Fig 7.
Detection of collagen fibers stained blue on Masson’s trichrome stain (x40 HPF).
Fig 8.
Tumor-infiltrating lymphocytes and collagen fibers using double-staining.
Detection of tumor-infiltrating lymphocytes (brown) and collagen fibers (blue) double-stained using immunohistochemical staining and Masson’s trichrome staining (x200 HPF).
Table 1.
Number of primary tumors and metastases used for the analysis.
Fig 9.
Relationship between the Immunoscore of primary tumors and the degree of TIL infiltration in the peritoneal metastases.
(A) (B) The density of tumor-infiltrating lymphocytes in peritoneal metastases with high-Immunoscore primary tumors was significantly higher than that with low-Immunoscore primary tumors (CD3: p = 0.0045, CD8: p = 0.0015).
Fig 10.
Relationship between the Immunoscore of primary tumors and the degree of TIL infiltration in the liver metastases.
(A) (B) The density of tumor-infiltrating lymphocytes in liver metastases with high-Immunoscore primary tumors was significantly higher than that with low-Immunoscore primary tumors (CD3: p = 0.0009, CD8: p = 0.0002).
Fig 11.
Relationship between the Immunoscore of primary tumors and the degree of TIL infiltration in the lung metastases.
(A) (B) There was no significant difference between the Immunoscore of primary tumors and the density of tumor-infiltrating lymphocytes in lung metastases.
Fig 12.
The comparison of the density of tumor-infiltrating lymphocytes and intratumoral fibrosis in each organ.
(A) The density of CD3-positive tumor-infiltrating lymphocytes in peritoneal metastases was significantly lower than that in liver metastases (p<0.0001) and lung metastases (p<0.0001). (B) The density of CD8-positive tumor-infiltrating lymphocytes in peritoneal metastases was significantly lower than that in liver metastases (p = 0.0007) and lung metastases (p = 0.0014). (C) The ratio of the α-SMA-positive cancer-associated fibroblast area in peritoneal metastases was significantly greater than that in liver metastases (p<0.0001)and lung metastases (p<0.0001). (D) The ratio of the collagen fiber area in peritoneal metastases was significantly greater than that in liver metastases (p<0.0001) and lung metastases (p = 0.0001).
Fig 13.
The comparison of the density of tumor-infiltrating lymphocytes in high-/low-fibrosis areas.
(A) The density of CD3-positive tumor-infiltrating lymphocytes in the high-fibrosis area was significantly lower than that in the low-fibrosis area (p<0.0001). (B) The density of CD8-positive tumor-infiltrating lymphocytes in the high-fibrosis area was significantly lower than that in the low-fibrosis area (p<0.0001).