Figure 6. Neutrophils and TGFβ activity associate with T‐cell suppression in human colon cancer.

• A–C
  Evaluation of S100A9⁺ neutrophil and CD8⁺ T‐cell infiltration in histologic sections of human CRC tumors. (A) Anti‐S100A9 immunostaining on a representative human CRC section. Arrowheads indicate neutrophil infiltration at the tumor boarder. (B) Co‐immunostaining for CD8 (brown) and S100A9 (gray‐blue) in the same CRC sample as shown in (A). (C) Quantification of S100A9⁺ cells (upper panel) and CD8⁺ T cells in different areas on CRC tumor sections containing tumor border with adjacent benign mucosa as determined by a specialized pathologist: “Tumor center” was tissue within tumor > 200 μm from tumor border, “Tumor border” was tissue within tumor < 100 μm from tumor border, and “Adjacent benign mucosa” was mucosa tissue outside tumor < 100 μm from tumor border. Per section, all identifiable tumor border and adjacent benign areas, as well as three arbitrary tumor center areas, were scored. Sections were derived from 10 individual patients.
• D, E
  Gene expression scores were generated as described in material and methods. Boxes are lower and upper quartiles with median as solid lines; horizontal lines define minimum and maximum; dots define outliers. (D) Neutrophil scores in tumor samples categorized according to CMS subtypes. (E) Comparison of T‐cell gene expression scores in CMS4 tumors within two publicly available CRC gene expression datasets. Samples are categorized according to the medians (high = >median; low = <median) of neutrophil and TGFβ gene expression scores. Upper panel: TCGA dataset. Lower panel GSE39582 dataset.
• F
  Proliferation of activated blood T cells in vitro in co‐culture with increasing ratios of CRC patient‐derived autologous blood neutrophils. CD3⁺ T cells and CD66b⁺ neutrophils were isolated and cultured individually for each patient. Bar graphs display relative numbers of proliferated T cells after 3 days of co‐culture. Each dot per condition represents a neutrophil sample from an individual patient (n = 13). Bars represent mean + SD.
• G
  Proliferation of activated blood T cells in vitro in co‐culture with autologous blood neutrophils. Cells were derived from both CRC patients (n = 2) and healthy volunteers (n = 5). T cells and neutrophils were cultured at a 1:1 ratio and treated with vehicle (T + Ne) or 10 μm Galunisertib (T + Ne + TGFBRi). Each line represents a neutrophil sample from an individual healthy volunteer.

Data information: In (C) and (G), statistical analysis was performed by paired two‐tailed Student's t‐tests. *P < 0.05; **P < 0.01. In (D–E), P‐values were calculated with Mann–Whitney test. *P < 1 × 10⁻³; **P < 1 × 10⁻⁶; ***P < 1 × 10⁻⁸; ****P < 1 × 10⁻¹⁰. In (F), statistical analysis was performed by using one‐way ANOVA with Dunnett's multiple comparison tests. *P < 0.05; **P < 0.01; ****P < 0.001. Exact P‐values are provided in Appendix Table S4.Source data are available online for this figure.