Stromagenesis and cancer-associated fibroblast heterogeneity in primary tumors and metastasis: focus in non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) is the most common lung cancer type and one of the deadliest neoplasias worldwide. NSCLC is histologically classified into adenocarcinoma, squamous cell carcinoma, and other less frequent subtypes. Both subtypes and other solid tumors are increasingly regarded as abnormal organs, highlighting the critical role of the desmoplastic tumor stroma rich in cancer-associated fibroblasts (CAFs) in driving tumor progression and therapeutic resistance. This tumor stroma resembles a chronic fibrotic wound and is largely formed by activated/myofibroblast-like α-SMA+ CAFs (myCAFs), which are strongly associated with immunosuppression and poor prognosis. Despite the dominance of the myCAF phenotype, we reported a decade ago phenotypic alterations in NSCLC with a strong dependence on the histologic subtype. Subsequent studies using functional assays, single-cell techniques, and in vivo models have refined these initial observations, enhancing our understanding of the biology of both normal fibroblasts/myofibroblasts and CAFs in NSCLC and other cancer types, including their origins, subclassification, and physiopathologic functions. Notably, increasing evidence supports that CAFs can exhibit tumor-restraining or tumor-promoting effects, and current therapeutic efforts aim to shift the balance towards tumor-restraining phenotypes. Here, we review major advances in our understanding of tumor stromagenesis and CAF heterogeneity in both primary tumors and metastasis, including emerging consensus, with a special focus on NSCLC and its frequent dissemination to the brain. We also highlight the critical role of smoking through epigenetic reprogramming of the TGF-β/SMAD3 pathway. These advances are beginning to delineate how CAF heterogeneity depends on the stage and histologic subtype in NSCLC.

Keywords: cancer‐associated fibroblasts; desmoplasia; metastasis; myofibroblasts; non‐small cell lung cancer; stromagenesis.

Fig. 1

Histological hallmarks of the desmoplastic stroma in LUAD and LUSC. (A) Representative images of picrosirius red stains of fibrillar collagens imaged with polarized light (top) and corresponding single collagen fiber reconstructions by CT‐Fire software (bottom) of control pulmonary tissue, LUAD, and LUSC. Single collagen fibers are labeled with different colors. Reprinted from [95] with permission. Scale bar, 100 μm. (B) CT‐Fire quantification of collagen fibers straightness in LUAD and LUSC samples. Each dot represents the average of each patient. Reprinted from [95] with permission. ###, P < 0.001. (C) Representative histological stains of the proliferation marker ki‐67 in LUAD and LUSC tissue sections and zoom‐in selections. Black arrows point out individual CAFs. Reprinted from [56] with permission. (D) Representative histological stains of fibrocyte markers CD34 (left) and CD45 (right) in LUAD tissue sections and zoom‐in selections. Black arrows point out positive non‐endothelial mesenchymal cells. Reprinted from [60] with permission. (E) Quantification of CAF number density in LUAD and LUSC tissue sections. Black arrows point to CAFs identified by morphometric criteria in representative H&E images. Reprinted from [16] with permission. #, P < 0.05. (F) Representative histological images from tissue microarrays of picrosirius red stains of LUAD and LUSC tissue sections and quantification of the corresponding percentage of positive area, referred to as PSR%. LUAD and LUSC samples were further subclassified according to smoking status. Reprinted from [54] with permission. ***, P < 0.005 with respect to never‐smokers; ##, P < 0.01.

Fig. 2

Relevant molecular alterations between lung CAFs and paired control fibroblasts (CFs) or between histotypes in LUAD and LUSC. (A) Representative images of tumor‐free pulmonary tissue (left) and a tumor tissue sample (right) collected from surgical lung cancer patients. Surgical pieces were used to isolate control fibroblasts (CFs) and paired CAFs, respectively. Reprinted from [24] with permission. Scale bar, 50 μm. (B) Histogram showing the relative density of average DNA methylation levels (β‐value) in CAFs (red line) and control fibroblasts (blue line). Vertical dashed lines show the median β‐values. Reprinted from [60] with permission. ***P < 0.001. (C) Fold (CAF/CF) SMAD3 promoter methylation of primary CAFs and their paired CFs (6 LUAD, 6 LUSC) determined by pyrosequencing in three CpG sites (used as technical replicates). Reprinted from [54] with permission. ***, P < 0.005 with respect to CFs; ###, P < 0.005; (D) Fold SMAD3/SMAD2 mRNA ratio in LUAD and LUSC‐CAFs treated for 3 days with 2.5 ng·mL⁻¹ TGF‐β1. Reprinted from [63] with permission. #, P < 0.05. (E) Emerging model on the histotype‐dependent relationship between exposure to smoking particles, SMAD3 promoter methylation, fibrosis, and response to antifibrotic drugs in lung CAFs. Reprinted from [54] with permission. (F) Summary of major sources and molecular subtypes of CAFs in primary tumors and metastatic settings in NSCLC.

Fig. 3

Differential expression of major myCAF markers in LUAD and LUSC. Representative whole tissue and zoom images of α‐SMA (top), FAP (middle), and MYH11 (bottom panels) histological stains within tissue microarrays of LUAD and LUSC downloaded from the Human Protein Atlas (HPA) database. Scale bar, 200 μm.