Comparative Study of the Role of Interepithelial Mucosal Mast Cells in the Context of Intestinal Adenoma-Carcinoma Progression

Abstract

Mast cells (MCs) are crucial players in the relationship between the tumor microenvironment (TME) and cancer cells and have been shown to influence angiogenesis and progression of human colorectal cancer (CRC). However, the role of MCs in the TME is controversially discussed as either pro- or anti-tumorigenic. Genetically engineered mouse models (GEMMs) are the most frequently used in vivo models for human CRC research. In the murine intestine there are at least three different MC subtypes: interepithelial mucosal mast cells (ieMMCs), lamina proprial mucosal mast cells (lpMMCs) and connective tissue mast cells (CTMCs). Interepithelial mucosal mast cells (ieMMCs) in (pre-)neoplastic intestinal formalin-fixed paraffin-embedded (FFPE) specimens of mouse models (total lesions n = 274) and human patients (n = 104) were immunohistochemically identified and semiquantitatively scored. Scores were analyzed along the adenoma-carcinoma sequence in humans and 12 GEMMs of small and large intestinal cancer. The presence of ieMMCs was a common finding in intestinal adenomas and carcinomas in mice and humans. The number of ieMMCs decreased in the course of colonic adenoma-carcinoma sequence in both species (p < 0.001). However, this dynamic cellular state was not observed for small intestinal murine tumors. Furthermore, ieMMC scores were higher in GEMMs with altered Wnt signaling (active β-catenin) than in GEMMs with altered MAPK signaling and wildtypes (WT). In conclusion, we hypothesize that, besides stromal MCs (lpMMCs/CTMCs), particularly the ieMMC subset is important for onset and progression of intestinal neoplasia and may interact with the adjacent neoplastic epithelial cells in dependence on the molecular environment. Moreover, our study indicates the need for adequate GEMMs for the investigation of the intestinal immunologic TME.

Keywords: adenoma-carcinoma sequence; colorectal cancer; genetically engineered mouse models; human; interepithelial mucosal mast cells; tumor microenvironment.

Figure 1

Identification of interepithelial mucosal mast cells (ieMMCs; arrowheads) in murine (A–C) and human (D–F) intestine. (A) Immunohistochemical sequential double staining of basal lamina (collagen IV) and ieMMCs (MCPT1) clearly reveals interepithelial localization of murine ieMMCs; (B) PAS reaction and ieMMC IHC (MCPT1) confirms interepithelial location of MCPT1⁺ cells; (C) Prominent accumulation of small, round cells containing large, brightly eosinophilic granules located in intestinal hyperplastic epithelium of a hematoxylin-eosin (H.E.) section; (D) Immunohistochemical sequential double staining of basal lamina (collagen IV) and ieMMCs (MC-Chymase; MCC) clearly reveals interepithelial localization of human ieMMCs; (E) Serial sections of MCC and; (F) MC-Tryptase (MCT) immunohistochemistry (IHC) in a human intestinal adenoma.

Figure 2

Analysis of ieMMCs in murine colon rolls. (A) Intestinal lesions were graded and annotated (red line) in an H.E. section; (B) Accordingly, a serial section of MCPT1 IHC was annotated; (C) Hotspots of MCPT1⁺ mast cells (MCs) were identified in neoplastic mucosa (black frame) and normal mucosa (green frame); (D) Mast cell numbers were counted in one 40× field (high-power field, hpf) of the respective hotspot. A semiquantitative score was applied (Figure S4) (Table 1); (E) In the adjacent normal mucosa, MC numbers were counted and scored accordingly.

Figure 3

(A–C) In murine intestinal neoplasia (n = 274), MCPT1⁺ ieMMC were generally more abundant in the apical two thirds of the lesion (luminal site; arrowheads) compared to the basal, lower third of tumor tissue (submucosal site). The dotted line divides the different zones; (D–E) Generally, ieMMC scores were higher in the apical (D) than in the basal (E) areas of intestinal neoplastic lesions; (D) Kruskal–Wallis test (KWT) of ieMMC scores in the apical aspects of tumors during adenoma-carcinoma progression. Scores for ieMMC were higher in precursors (atypical hyperplasia and low- and high-grade adenomas) than in carcinomatous lesions (KWT *** p < 0.001); (E) KWT of ieMMC scores in the basal aspects of tumors during adenoma-carcinoma progression (KWT *** p < 0.001). * = extreme statistical outliers; ° = mild statistical outliers.

Figure 4

(A–I) In the context of human adenoma-carcinoma progression (A–C), ieMMCs (arrowheads) positive for MCT (D–F) and MCC (G–I) decreased from low-grade (LG) to high-grade (HG) adenoma and adenocarcinoma (ACA) (serial sections); (J,K) Generally, median scores for ieMMCs in adenomas were higher for MCT (KWT, pairwise comparison of ACA-LG **** p < 0.0001; ACA-HG **** p < 0.0001) (J); than for MCC (KWT, pairwise comparison of LG-HG * p = 0.022; HG-ACA *p = 0.017; LG-ACA **** p < 0.0001) (K). * = extreme statistical outliers; ° = mild statistical outliers.

Figure 5

(A–D) Interepithelial mucosal mast cell (ieMMC) (arrowheads) in normal vs. neoplastic colonic mucosa of murine (A,C) and human (B,D) intestine; (E) Generally, scores of ieMMCs were higher in (pre-)neoplastic lesions of mice (Mann-Whitney U test (MWU), **** p = 0.000); (F) Contradictory, human ieMMC scores were higher in normal adjacent tissue (MWU, *** p < 0.001); (G) IeMMCs in the adjacent normal mucosa of mice increased in the course of adenoma-carcinoma progression (KWT, p = 0.690, not significant (ns)); (H) In humans, density of ieMMCs in adjacent normal mucosa decreased from LG adenoma to carcinoma to HG adenoma (KWT, * p = 0.047). * = extreme statistical outliers; ° = mild statistical outliers.

Figure 6

(A–D) In the context of murine adenoma-carcinoma progression (A) ieMMC scores generally decreased from low-grade adenoma to carcinoma (KWT, pairwise comparisons, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; not significant (ns) p > 0.05). IeMMCs were identifiable using MCPT1 (B), MCPT6 (C), and granzyme B (D) IHC; (E) Higher ieMMC scores were connected to a higher proliferation in terms of % Ki-67⁺ tumor cells in a neoplastic lesion (n = 271) (KWT, *** p < 0.001). * = extreme statistical outliers; ° = mild statistical outliers.

Figure 7

(A,B) Scores for ieMMCs over all tumor grades were compared according to genotype groups (KWT, **** p < 0.0001). For details on the pseudonymized genetically engineered mouse groups, please refer to Supplementary Table S1 (A); additionally, group comparison according to mode of tumor induction was performed (KWT, p < 0.0001) (B). (A) Scores assessed in lesions of Braf^V637E mice (BRAF) (n = 20; median score 0.5) were generally low, while scores were highest in lesions of the Catnb^Δex3 model (βCAT) (n = 26; median score 3.5); (B) Scores in Adeno-Cre-induced tumors were lower than in AOM-DSS-induced (KWT, pairwise comparison **** p < 0.0001) and in endogenous models’ scores (KWT, pairwise comparison **** p < 0.0001). There was no statistically significant effect between AOM-DSS and endogenous models (p = 0.636; not significant (ns)). * = extreme statistical outliers; ° = mild statistical outliers.

Figure 8

(A) Scores for ieMMCs were generally lower in murine colonic tumors (n = 162) than in murine small intestinal tumors (n = 112) (KWT, *** p < 0.001); (B) Scores for ieMMCs decreased from atypical hyperplasia to carcinoma in the colon (KWT, *** p < 0.001); (C) However, in the small intestine scores for ieMMCs did not decrease from precursors to invasive neoplasia (KWT, p = 0.222, not significant (ns)). ° = mild statistical outliers.