Cancer-Associated Fibroblasts Facilitate Squamous Cell Carcinoma Lung Metastasis in Mice by Providing TGFβ-Mediated Cancer Stem Cell Niche.

Xueke Shi,Hongmei Zhou,Christian D Young,Danfeng Du,Spencer C Hall,Jingjing Luo,Fanglong Wu,Michael C Rudolph,Xiao-Jing Wang,Kelsey J Weigel

doi:10.3389/fcell.2021.668164

Abstract

Cancer-associated fibroblasts (CAFs) have been shown to enhance squamous cell carcinoma (SCC) growth, but it is unclear whether they promote SCC lung metastasis. We generated CAFs from K15.KrasG12D.Smad4–/– mouse SCCs. RNA expression analyses demonstrated that CAFs had enriched transforming growth factor-beta (TGFβ) signaling compared to normal tissue-associated fibroblasts (NAFs), therefore we assessed how TGFβ-enriched CAFs impact SCC metastasis. We co-injected SCC cells with CAFs to the skin, tail vein, or the lung to mimic sequential steps of lung metastasis. CAFs increased SCC volume only in lung co-transplantations, characterized with increased proliferation and angiogenesis and decreased apoptosis compared to NAF co-transplanted SCCs. These CAF effects were attenuated by a clinically relevant TGFβ receptor inhibitor, suggesting that CAFs facilitated TGFβ-dependent SCC cell seeding and survival in the lung. CAFs also increased tumor volume when co-transplanted to the lung with limiting numbers of SCC cancer stem cells (CSCs). In vitro, CSC sphere formation and invasion were increased either with co-cultured CAFs or with CAF conditioned media (which contains the highest TGFβ1 concentration) and these CAF effects were blocked by TGFβ inhibition. Further, TGFβ activation was higher in primary human oral SCCs with lung metastasis than SCCs without lung metastasis. Similarly, TGFβ activation was detected in the lungs of mice with micrometastasis. Our data suggest that TGFβ-enriched CAFs play a causal role in CSC seeding and expansion in the lung during SCC metastasis, providing a prognostic marker and therapeutic target for SCC lung metastasis.

Highlights

Squamous cell carcinomas (SCCs) arise from stratified epithelia, and the most relevant organ sites are the skin and oral cavity where high UV irradiation, tobacco carcinogens, or human papillomavirus (HPV) infection are initiating events
Cancer-associated fibroblasts (CAFs) and normal tissue-associated fibroblasts (NAFs) did not express keratins (Supplementary Figures 1B,C), which are still expressed in poorly differentiated squamous cell carcinoma (SCC) cells, indicating fibroblast cell lines are not contaminated with epithelial cells
While CAFs are reported to promote SCC growth (Orimo et al, 2005; Erez et al, 2010), the primary SCC growth in our model of transplantation of bulk SCC cells was not affected by CAFs

Summary

Introduction

Squamous cell carcinomas (SCCs) arise from stratified epithelia, and the most relevant organ sites are the skin and oral cavity where high UV irradiation, tobacco carcinogens, or human papillomavirus (HPV) infection are initiating events. Lung metastasis is the process of cancer cells disseminating from a primary SCC, entering into blood vessels or lymphatic vessels (intravasation), survival and traveling, and moving out of vessels (extravasation) into the lung, and survival and expansion in the lung thereafter (Chaffer and Weinberg, 2011). We previously generated an aggressive SCC mouse model driven by KrasG12D mutation and Smad deletion (Smad4−/−) in keratin 15 (K15)-positive stem cells of stratified epithelial tissues, i.e., hair follicle bulge or tongue papillae (White et al, 2013). We identified that cancer stem cells (CSCs) derived from mutant stem cells in these SCCs have a higher invasive ability than non-CSCs (White et al, 2013). How CSCs as SCC metastasis “seeds” interact with their “soil” during metastasis, remains to be assessed

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Conclusion