Matrix Stiffening and β1 Integrin Drive Subtype-Specific Fibroblast Accumulation in Lung Cancer

Abstract

The crucial role of tumor-associated fibroblasts (TAF) in cancer progression is now clear in non-small cell lung cancer (NSCLC). However, therapies against TAFs are limited due to a lack of understanding in the subtype-specific mechanisms underlying their accumulation. Here, the mechanical (i.e., matrix rigidity) and soluble mitogenic cues that drive the accumulation of TAFs from major NSCLC subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were dissected. Fibroblasts were cultured on substrata engineered to exhibit normal-or tumor-like stiffnesses at different serum concentrations, and critical regulatory processes were elucidated. In control fibroblasts from nonmalignant tissue, matrix stiffening alone increased fibroblast accumulation, and this mechanical effect was dominant or comparable with that of soluble growth factors up to 0.5% serum. The stimulatory cues of matrix rigidity were driven by b1 integrin mechanosensing through FAK (pY397), and were associated with a posttranscriptionally driven rise in b1 integrin expression. The latter mechano-regulatory circuit was also observed in TAFs but in a subtype-specific fashion, because SCC-TAFs exhibited higher FAK (pY397), b1 expression, and ERK1/2 (pT202/Y204) than ADCTAFs. Moreover, matrix stiffening induced a larger TAF accumulation in SCC-TAFs (>50%) compared with ADC-TAFs (10%-20%). In contrast, SCC-TAFs were largely serum desensitized, whereas ADC-TAFs responded to high serum concentration only. These findings provide the first evidence of subtype-specific regulation of NSCLC-TAF accumulation. Furthermore, these data support that therapies aiming to restore normal lung elasticity and/or b1 integrin-dependent mechano regulation may be effective against SCC-TAFs, whereas inhibiting stromal growth factor signaling may be effective against ADC-TAFs.Implications: This study reveals distinct mechanisms underlying the abnormal accumulation of tumor-supporting fibroblasts in two major subtypes of lung cancer, which will assist the development of personalized therapies against these cells.

Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung

“…Likewise, SCC-TAFs failed to enter into the cell cycle upon serum stimulation (Figure 1E–1F and Supplementary Figure S1). However, it is worth noting that we recently reported a marked mitogenic activity in SCC-TAFs in the absence of exogenous growth factors [18] that, in line with the lack of SA-βgal positivity, indicates that serum desensitization in SCC-TAFs is indicative of fibroblast dysfunction rather than senescence. In contrast, the percentage of growth arrested cells stimulated with 10% FBS drop by ~20% in CFs, and slightly less in ADC-TAFs (Figure 1F).…”

“…TAFs from the two major NSCLC subtypes (ADC, SCC) and other solid tumors exhibit an activated/myofibroblast-like phenotype in culture and in vivo [7, 18, 19]. Here we extended these observations by showing that LCC-TAFs are also activated and exhibit a statistically significant 3-fold increase in α-SMA expression with respect to paired CFs similar to that observed in ADC- and SCC-TAFs as shown by immunofluorescence analysis (Figure 1A, 1B).…”

“…However, we recently challenged this assumption by reporting strikingly distinct responses to extracellular matrix stiffening and growth factor stimulation between ADC- and SCC-TAFs, which were associated with subtype-specific β1 integrin expression and FAK activity [18]. In the present study we expanded these observations further by reporting for the first time two new subtype-specific aberrant phenotypes in lung TAFs: (i) poor growth response in the absence of senescence markers in SCC-TAFs, and (ii) premature cellular aging or senescence in LCC-TAFs.…”

“…The human fibroblast cell line CCD-19Lu obtained from normal pulmonary tissue (ATCC) and primary fibroblasts were maintained in DMEM-based fibroblast culture medium described elsewhere [18, 39]. Fibroblasts were used up to either passage 10 (CCD-19Lu) or 6 (primary) to prevent replicative senescence [28].…”

“…Immunofluorescence detection and quantification of α-SMA in cultured fibroblasts were carried out as described elsewhere [18, 19]. …”