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. 2023 Dec 21;25(1):148.
doi: 10.3390/ijms25010148.

Activation and Functions of Col6a1+ Fibroblasts in Colitis-Associated Cancer

Niki Chalkidi  1 , Maria-Theodora Melissari  1 , Ana Henriques  1 , Athanasia Stavropoulou  1 , George Kollias  2   3 , Vasiliki Koliaraki  1
Affiliations

Activation and Functions of Col6a1+ Fibroblasts in Colitis-Associated Cancer

Niki Chalkidi et al. Int J Mol Sci. .

Abstract

Cancer-associated fibroblasts (CAFs) comprise a group of heterogeneous subpopulations with distinct identities indicative of their diverse origins, activation patterns, and pro-tumorigenic functions. CAFs originate mainly from resident fibroblasts, which are activated upon different stimuli, including growth factors and inflammatory mediators, but the extent to which they also maintain some of their homeostatic properties, at least at the earlier stages of carcinogenesis, is not clear. In response to cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor (TNF), as well as microbial products, CAFs acquire an immunoregulatory phenotype, but its specificity and pathophysiological significance in individual CAF subsets is yet to be determined. In this study, we analyzed the properties of Col6a1-positive fibroblasts in colitis-associated cancer. We found that Col6a1+ cells partly maintain their homeostatic features during adenoma development, while their activation is characterized by the acquisition of a distinct proangiogenic signature associated with their initial perivascular location. In vitro and in vivo experiments showed that Col6a1+ cells respond to innate immune stimuli and exert pro-tumorigenic functions. However, Col6a1+-specific inhibition of TNF receptor 1 (TNFR1) or IL-1 receptor (IL-1R) signaling does not significantly affect tumorigenesis, suggesting that activation of other subsets acts in a compensatory way or that multiple immune stimuli are necessary to drive the proinflammatory activation of this subset. In conclusion, our results show that adenoma-associated CAF subsets can partly maintain the properties of homeostatic fibroblasts while they become activated to support tumor growth through distinct and compensatory mechanisms.

Keywords: colon cancer; heterogeneity; intestinal fibroblasts.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Col6a1+ CAFs and normal fibroblasts display transcriptional similarities. (A) Schematic showing the procedure for bulk RNA sequencing of fibroblasts isolated from AOM/DSS-induced adenomas (prepared using Biorender.com). A total of three samples were used for bulk RNA sequencing. Each sample originated from a pool of tumors from five to six mice. (B) FACS plot showing the sorting strategy for Col6a1+ and Col6a1− CAFs. (C) Heatmap showing gene expression signatures of Col6a1+ and Col6a1− fibroblasts in homeostasis (IMCs) and CAC (CAFs). Log2-transformed normalized read counts of genes for each replicate are shown. Red denotes high expression, and blue denotes low expression values. Read counts are scaled per column. (D) Immunohistochemistry for αSMA in AOM/DSS-induced adenomas of Col6a1-mTmG mice (n = 5 mice, Scale bar: 50 μm). (E) Representative FACS analysis of CD201 expression in Lin−GFP+ cells in AOM/DSS-induced adenomas of Col6a1-mTmG mice (n = 2 mice). (F) Venn diagram showing similarities in gene expression between Col6a1+ CAFs and mural cells [27].
Figure 2
Figure 2
Col6a1+ and Col6a1− CAFs are activated upon AOM/DSS colon carcinogenesis. Volcano plots of deregulated genes in (A) Col6a1+ CAFs versus normal Col6a1+ IMCs and (B) Col6a1− CAFs versus normal IMCs. (C) Venn diagram showing the differential and common upregulated genes in Col6a1+ and Col6a1− CAFs. (D) Network of enriched terms in Col6a1+ CAF and Col6a1− CAF common upregulated gene signature. (E) Network of enriched terms in Col6a1+ CAF unique gene signature. (F) Network of enriched terms in Col6a1− CAF unique upregulated gene signature. Networks are colored by cluster ID, where nodes that share the same cluster ID are typically close to each other (generated through metascape.org).
Figure 3
Figure 3
Co-culture experiments and allografts show similar effects of Col6a1+ and Col6a1− CAFs on cancer cell growth. (A) Average colony size per well, (B) size distribution, and (C) representative bright field images of Caco-2 colonies after 3 days of culture on sorted Col6a1+ and Col6a1− colonic IMCs. Data represents mean ± SD from one of three experiments performed, n = 10–12 wells, ** p < 0.01, Scale bar = 0.5 mm. (D) Tumoroid size per condition for each day of the co-culture, (E) size distribution of tumoroids at day 3 of the co-culture, and (F) representative bright-field images of AOM/DSS tumoroids at day three of their co-culture with sorted Col6a1+ and Col6a1− colonic IMCs. Data represents mean ± SD of tumoroids from one of three experiments performed. ns = not statistically significant, Scale bar = 1 mm. (G) Total volume of allografts after 15 days of growth with Col6a1+ and Col6a1− colonic IMCs. Data represents mean ± SD from one of three experiments performed (n = 6–8), ns = not statistically significant. (H) Representative fluorescent images of allografts with sorted Col6a1+ and Col6a1− colonic IMCs. Scale bar = 50 μm.
Figure 4
Figure 4
Deletion of IL-1R1 and TNFR1 in Col6a1+ IMCs is not sufficient to ameliorate CAC. (A) Proteome profiling of cultured unsorted IMCs upon LPS, IL-1β, and TNF stimulation. Only factors with differences in fold change > 2 in at least one condition are shown. Data represents mean ± SD from one experiment performed in duplicates. (B) Image showing the signal intensity of the proteome profile assay as obtained from the ChemiDoc XRS+ instrument. (C) MIP-2 quantification in the supernatants of Col6a1+ and Col6a1− IMCs stimulated for 24 h with LPS, TNF, and IL-1β. One representative of two independent experiments performed in triplicates is presented. (D) Number of tumors per mouse and (E) colon length in Il1r1IMCko mice (n = 6) and their littermate controls (n = 6) at the end of the AOM/DSS protocol (one representative experiment of four performed). (F) Number of tumors per mouse and (G) colon length in p55IMCko mice (n = 8) and their littermate controls (n = 9) at the end of the AOM/DSS protocol (one representative experiment of two performed). ns, not statistically significant.
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