A shift from papillary to reticular fibroblasts enables tumour-stroma interaction and invasion

Abstract

Background: Tumour stroma consists of a heterogeneous population of fibroblasts and related mesenchymal cells, collectively dubbed cancer-associated fibroblasts (CAFs). These CAFs are key players in cancer invasion of cutaneous squamous cell carcinoma (SCC). As we have shown earlier, papillary and reticular fibroblasts (Pfs and Rfs, respectively) have distinct effects on epidermal and dermal homeostasis, but their role in cancer invasion and epithelial-to-mesenchymal transition (EMT) remains to be determined.

Methods: We used 3D cultures of human skin equivalents (HSEs) to analyse the effects of Pfs and Rfs on the invasive behaviour of SCC and EMT.

Results: We reveal for the first time the importance of Pfs versus Rfs in SCC invasion and EMT. Cell lines from different stages of SCC showed significantly more extensive invasion into a dermis composed of Rfs than of Pfs. In addition, Rfs-based HSEs showed increased cell activation and stained positive for CAF biomarkers α-SMA and vimentin. Further analysis revealed that invasively growing cancer cells in Rf-HSEs express markers of EMT transition, like SNAIL2, N-cadherin and ZEB1.

Conclusions: Conversely, our results show that Pfs contain cancer cells more within the epidermis. Rfs are clearly predisposed to differentiate into CAFs upon SCC signals, assisting invasion and EMT.

Fig. 1

Immunofluorescent and immunohistochemical analysis of vimentin, ZEB1 (green)/β-catenin (red), TGM2, α-SMA, PDPN and TCN expression in SCC biopsy cross sections (N = 8) Arrows in PDPN panel point at papillary fibroblasts, those in the alpha-SMA at evident CAFs, and in the vimentin and ZEB1 panels at EMT of tumour cells.

Fig. 2

a Cross-sections of Pf-FTMs and Rf-FTMs (HE). The epidermis was generated using MET1, MET2, MET4 or SCC12b2 SCC cell lines. b Quantification of the ingrowth; the ingrowth is significantly higher in Rf-FTMs of MET1 (P < 0.005), MET2 (P < 0.0001) and SCC12b2 (P < 0.05); N = 3

Fig. 3

a Cross-sections of Pf-FTMs and Rf-FTMs stained for K16, collagen type IV, laminin 332 and Ki67. The epidermis was generated with MET1, MET2, MET4 or SCC12b2 cell lines. Data are obtained for three independent experiments. b Quantification of Ki67 in FTMs seeded with MET1, MET2 and MET4. Results are expressed as the mean ± SD of three counts in three donors (*<0.05)

Fig. 4

qPCR analysis of the SCC-FTMs for EMT and invasion markers. Error bars represent ±SD *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001

Fig. 5

Shown are cross-sections of Pf-FTMs and Rf-FTMs that were stained for vimentin, α-SMA and ZEB/β-catenin. The epidermis was generated with MET1, MET2, MET4 or SCC12b2 cell lines. Arrows indicate the expression of these biomarkers surrounding the SCC ingrowth. Data are obtained for three independent experiments