Tumor Infiltrating Neutrophils Are Enriched in Basal-Type Urothelial Bladder Cancer

Abstract

Background: Urothelial bladder cancers (UBCs) are distinct in two main molecular subtypes, namely basal and luminal type. Subtypes are also diverse in term of immune contexture, providing a rationale for patient selection to immunotherapy.

Methods: By digital microscopy analysis of a muscle-invasive BC (MIBC) cohort, we explored the density and clinical significance of CD66b⁺ tumor-associated-neutrophils (TAN) and CD3⁺ T cells. Bioinformatics analysis of UBC datasets and gene expression analysis of UBC cell lines were additionally performed.

Results: Basal type BC contained a significantly higher density of CD66b⁺ TAN compared to the luminal type. This finding was validated on TCGA, GSE32894 and GSE124305 datasets by computing a neutrophil signature. Of note, basal-type MIBC display a significantly higher level of chemokines (CKs) attracting neutrophils. Moreover, pro-inflammatory stimuli significantly up-regulate CXCL1, CXCL2 and CXCL8 in 5637 and RT4 UBC cell lines and induce neutrophil chemotaxis. In term of survival, a high density of T celsl and TAN was significantly associated to a better outcome, with TAN density showing a more limited statistical power and following a non-linear predicting model.

Conclusions: TAN are recruited in basal type MIBC by pro-inflammatory CKs. This finding establishes a groundwork for a better understanding of the UBC immunity and its relevance.

Keywords: CD3; CD66b; basal; bladder cancer; tumor-associated neutrophils.

Figure 1

Representative image analysis for CD66b⁺ TAN and CD3⁺ T cells in human MIBC. Sections are from four representative human MIBC (case #66, #31, #7 and #46) and stained as labeled and counterstained with hematoxylin (A,C,E,G). On the right column, the corresponding processed area is shown (B,D,F,H). Case with high and low density of CD66b⁺ TAN (A and C, respectively) and CD3⁺ T cells (E and G, respectively) are displayed. Images acquired from digital slides using 400× magnification have been digitally resized using Adobe Photoshop.

Figure 2

Density of CD66b⁺ TAN and CD3⁺ T cells and phenotype of TAN in human MIBC. Dot plots indicating the density of CD66b⁺ TAN and CD3⁺ T cells in the MIBC cohort is reported in (A); the correlation between the two variables is illustrated in (B). Sections are from two representative MIBC cases (case #70 in C, E and G; case #79 in D, F and H) and stained as labeled, illustrating CD66b⁺ TAN co-expressing neutrophil markers: CD11b, CD15 and ARG. Sections are counterstained with hematoxylin. Magnification 400× (scale bar 50 micron).

Figure 3

Clinical correlation of CD66b⁺ TAN and CD3⁺ T cells densities in human MIBC. Dot plots showing CD3⁺ T cells (A,B) and CD66b⁺ TAN (C,D) density distributions among different pT categories and Overall Stages in the MIBC cohort. p values were estimated by Kruskal-Wallis test and adjusted for multiple pairwise comparisons by Dunn’s method.

Figure 4

Clinical significance of CD66b⁺ TAN and CD3⁺ T cells in human MIBC. Kaplan-Meier curves illustrate the overall (A, C and E) and progression-free (B, D and F) survival in the MIBC cohort. p values are estimated by log-rank test and weighted for pT category (pT2/pT3-4).

Figure 5

Density of CD66b⁺ TAN and CD3⁺ T cells in basal and luminal MIBC. Dot plots indicating the density of CD3⁺ T cells (A) and CD66b⁺ TAN (B) in the MIBC cohort subdivided by molecular subtypes. Sections from two basal-type MIBC cases (case 70 and 49, in C–F) and two luminal-type MIBC cases (case 25 and 34, in G–L) are stained as labeled and counterstained with hematoxylin; magnification 200× (scale bar 100 micron). p values were estimated by Kruskal-Wallis test and adjusted for multiple pairwise comparisons.

Figure 6

Density of CD66b⁺ TAN and CD3⁺ T cells in human MIBC with hyper-activation of STAT3. Dot plots indicating the density of CD3⁺ T cells (A) and CD66b⁺ TAN(B) in the MIBC cohort based on pSTAT3 expression. Sections are from two pSTAT3 HIGH (case 49 and 2, in C–F) and two pSTAT3 LOW (case 68 and 57, in G–L) MIBC cases and stained as labeled and counterstained with hematoxylin. Magnification: 200× (C–L), scale bar 100 micron. p values were estimated by Kruskal-Wallis test.

Figure 7

Density of CD66b⁺ TAN and CD3⁺ T cells in MIBC in relation to FOSL1 expression. Dot plots indicating the density of CD3⁺ T cells (A) and CD66b⁺ TAN (B) in the MIBC cohort based on FOSL1 expression. Sections are from two FOSL1 HIGH (case 49 and 14, in C–F) and two FOSL1 LOW (case 68 and 23, in G–L) MIBC cases and stained as labeled and counterstained with hematoxylin. Magnification: 200× (C–L), scale bar 100 micron. p values were estimated by Kruskal-Wallis test.

Figure 8

Neutrophil gene signature in UBC datasets. Plots show estimated mean score values and corresponding 95% confidence intervals for neutrophil GSE scores in bladder subtypes (A TCGA, B GSE32894, C GSE124305). Pairwise comparisons p-values were computed from linear models and adjusted for multiple comparisons.

Figure 9

Chemokine signature in datasets. Plots show estimated mean score values and corresponding 95% confidence intervals for Chemokine GSE scores in bladder subtypes (A TCGA, B GSE32894, C GSE124305). Pairwise comparisons p-values were computed from linear models and adjusted for multiple comparisons.

Figure 10

Modulation of TAN-attracting CK in UBC cell line. Column bars (A–D) showing the mean ± SD of CKs mRNA relative gene expression (-ΔCt, n = 3), measured by qRT-PCR in luminal-type RT4 and basal-type 5637 UBC cell lines. After stimulation with a cocktail of proinflammatory cytokines, a significant induction of the CXCL1 and CXCL8 mRNA (A,C) is observed in both UBC cell lines. CXCL2 is significantly enriched in basal-type 5637 UBC cell line compared to luminal-type RT4 cells both at resting and after stimulation (B). Column bars (E,F) showing neutrophil chemotaxis towards tumor-conditioned supernatants from luminal-type RT4 or basal-type 5637 UBC cell lines untreated (E) or stimulated for 4 or 24 h with a pro-inflammatory cytokine cocktail (F) (n = 3). Chemotaxis is reported as increased percentage of migrated cells (expressed as % of total cell input, mean ± SD) induced by tumor-conditioned supernatants over the control medium. Floating bars (G–I) showing that CKs’ mRNA is not modulated by siRNA STAT3 in 5637 cell line stimulated with pro-inflammatory cytokine cocktail (mean, range; n = 3). A one-way ANOVA test was used for all statistical analysis and pairwise comparisons p values were adjusted for multiple comparisons; significant results were showed (* p < 0.05, ** p < 0.01, *** p < 0.001). INFL, with the pro-inflammatory cytokine cocktail (TNF-a, IL6, IL1b); supernatants, sup.