Alícia Giménez

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ∼1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects

The design of 3D culture studies remains challenging due to the limited understanding of extracellular matrix (ECM)-dependent hindered diffusion and the lack of simple diffusivity assays. To address these limitations, we set up a cost-effective diffusivity assay based on a Transwell plate and the spectrophotometer of a Microplate Reader, which are readily accessible to cell biology groups. The spectrophotometer-based assay was used to assess the apparent diffusivity D of FITC-dextrans with molecular weight (4-70kDa) spanning the physiological range of signaling factors in a panel of acellular ECM gels including Matrigel, fibrin and type I collagen. Despite their technical differences, D data exhibited ∼15% relative difference with respect to FRAP measurements. Our results revealed that diffusion hindrance of small particles is controlled by the enhanced viscosity of the ECM gel in conformance with the Stokes-Einstein equation rather than by geometrical factors. Moreover, we provided a strong rationale that the enhanced ECM viscosity is largely contributed to by unassembled ECM macromolecules. We also reported that gels with the lowest D exhibited diffusion hindrance closest to the large physiologic hindrance of brain tissue, which has a typical pore size much smaller than ECM gels. Conversely, sparse gels (≤1mg/ml), which are extensively used in 3D cultures, failed to reproduce the hindered diffusion of tissues, thereby supporting that dense (but not sparse) ECM gels are suitable tissue surrogates in terms of macromolecular transport. Finally, the consequences of reduced diffusivity in terms of optimizing the design of 3D culture experiments were addressed in detail.

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

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 β1 integrin mechano-sensing through FAK (pY397), and were associated with a posttranscriptionally driven rise in β1 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), β1 expression, and ERK1/2 (pT202/Y204) than ADC–TAFs. 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 β1 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. Mol Cancer Res; 13(1); 161–73. ©2014 AACR.

Elastic properties of hydrogels and decellularized tissue sections used in mechanobiology studies probed by atomic force microscopy.

The increasing recognition that tissue elasticity is an important regulator of cell behavior in normal and pathologic conditions such as fibrosis and cancer has driven the development of cell culture substrata with tunable elasticity. Such development has urged the need to quantify the elastic properties of these cell culture substrata particularly at the nanometer scale, since this is the relevant length scale involved in cell‐extracellular matrix (ECM) mechanical interactions. To address this need, we have exploited the versatility of atomic force microscopy to quantify the elastic properties of a variety of cell culture substrata used in mechanobiology studies, including floating collagen gels, ECM‐coated polyacrylamide gels, and decellularized tissue sections. In this review we summarize major findings in this field from our group within the context of the state‐of‐the‐art in the field, and provide a critical discussion on the applicability and complementarity of currently available cell culture assays with tunable elasticity. In addition, we briefly describe how the limitations of these assays provide opportunities for future research, which is expected to continue expanding our understanding of the mechanobiological aspects that support both normal and diseased conditions. Microsc. Res. Tech. 80:85–96, 2017.

Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis

Myofibroblasts are key effector cells in tissue stiffening and fibrosis progression, but the contribution of cells undergoing epithelial–mesenchymal transition (EMT) is unclear. Unlike EMT, myofibroblasts contribute to tissue stiffening through their contractility and expression of fibrillar collagens, which is associated with aberrant FAK (Y397) hyperactivation.

Dysregulated Collagen Homeostasis by Matrix Stiffening and TGF-β1 in Fibroblasts from Idiopathic Pulmonary Fibrosis Patients: Role of FAK/Akt

Idiopathic pulmonary fibrosis (IPF) is an aggressive disease in which normal lung parenchyma is replaced by a stiff dysfunctional scar rich in activated fibroblasts and collagen-I. We examined how the mechanochemical pro-fibrotic microenvironment provided by matrix stiffening and TGF-β1 cooperates in the transcriptional control of collagen homeostasis in normal and fibrotic conditions. For this purpose we cultured fibroblasts from IPF patients or control donors on hydrogels with tunable elasticity, including 3D collagen-I gels and 2D polyacrylamide (PAA) gels. We found that TGF-β1 consistently increased COL1A1 while decreasing MMP1 mRNA levels in hydrogels exhibiting pre-fibrotic or fibrotic-like rigidities concomitantly with an enhanced activation of the FAK/Akt pathway, whereas FAK depletion was sufficient to abrogate these effects. We also demonstrate a synergy between matrix stiffening and TGF-β1 that was positive for COL1A1 and negative for MMP1. Remarkably, the COL1A1 expression upregulation elicited by TGF-β1 alone or synergistically with matrix stiffening were higher in IPF-fibroblasts compared to control fibroblasts in association with larger FAK and Akt activities in the former cells. These findings provide new insights on how matrix stiffening and TGF-β1 cooperate to elicit excessive collagen-I deposition in IPF, and support a major role of the FAK/Akt pathway in this cooperation.

Abstract 1089: Matrix stiffening and β1 integrin promote fibroblast accumulation in lung squamous cell carcinomas but not in adenocarcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The mechanisms underlying the abundance of tumor associated fibrobasts (TAFs) in non-small cell lung cancer (NSCLC) remain poorly understood. To address this gap of knowedge, we examined the relative contribution of mechanical (i.e. matrix rigidity) and soluble biochemical (i.e. serum) cues in the accumulation of primary TAFs from either adenocarcinoma (ADC) or squamous cell carcinoma (SQC) NSCLC patients. For this purpose, fibroblasts were cultured on collagen-I coated gels engineered to exhibit either normal- or tumor-like stiffnesses at different serum concentrations. In fibroblasts from non-malignant tissue, matrix stiffening alone was sufficient to increase fibroblast accumulation by 20%. In addition, matrix stiffening-induced accumulation was dominant or comparable to that due to soluble growth factors up to moderate serum concentrations. These mechanical stimulatory effects were β1 integrin-dependent, and were associated with both increased integrin mechanosensing through FAKpY397and a post-transcriptionally-driven rise in β1 integrin expression. The latter mechanoregulatory circuit could be extended to TAFs in a subtype-dependent fashion, since SQC-TAFs exhibited higher FAKpY397 and β1 expression than ADC-TAFs. Remarkably, SQC-TAF density was enhanced > 50% in response to matrix stiffening, whereas they were largely desensitized to serum. Conversely, substantial accumulation of ADC-TAFs required large serum concentration, whereas it was poorly induced by either matrix stiffening or low serum concentration. These results reveal that a therapeutic strategy aiming to inhibit soluble growth factors may be effective against ADC-TAFs, whereas a strategy aiming to rescue normal lung elasticity may be more efficient against SQC-TAFs. Citation Format: Jordi Alcaraz, Marta Puig, Roberto Lugo, Alicia Gimenez, Adriana Velasquez, Roland Galgoczy, Josep Ramirez, Abel Gomez-Caro, Pere Gascon, Noemi Reguart. Matrix stiffening and β1 integrin promote fibroblast accumulation in lung squamous cell carcinomas but not in adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2014-1089

Abstract 1103: An abnormally stiff microenvironment supports the overabundance of fibroblasts in non-small cell lung cancer.

Breathing demands our lungs to be soft and elastic. In contrast, lung tumors exhibit an abnormal tissue hardening concomitantly with an altered lung architecture and function, which brings the mechanical microenvironment of the tumor closer to that of muscle or bone. This tissue hardening has been associated with the abundant reactive stroma rich in activated fibroblasts (also referred to as tumor associated fibroblasts or TAFs) that is a hallmark of non-small cell lung cancer (NSCLC). However, it remains unknown whether tissue hardening alone is sufficient to drive the abnormal abundance of fibroblasts in the stroma of NSCLC. To address this question, we cultured the human lung fibroblast cell line CCD-19Lu or primary human lung fibroblasts on collagen-coated polyacrylamide gels exhibiting normal- or tumor-like stiffness in the absence (0%) or presence (0.1%) of serum for 5 days. Primary cells were isolated from both tumor-free regions (referred to as control fibroblasts) or tumors of NSCLC patients diagnosed with either Adenocarcinoma (ADC, n=5) or Squamous Cell Carcinoma (SCC, n=5) histological subtypes. We observed a significantly higher density of CCD-19Lu fibroblasts in tumor-like gels compared to normal-like gels regardless the presence of serum. Similar results were obtained in both primary control fibroblasts and SCC-TAFs. In contrast, tumor-like gels induced a weaker density increase in ADC-TAFs. The increased fibroblast density in the tumor-like gels was associated with increased survival rather than proliferation, as revealed by a downregulation of caspase-3 and a rise in AKT phosphorylation. Our results indicate that extracellular hardening alone is sufficient to induce an increased density in lung fibroblasts by activating pro-survival pathways and suggest an abnormal response of ADC-TAFs to extracellular mechanical cues. Collectively these findings underline a major role of tissue hardening in the abnormal abundance of TAFs in NSCLC, particularly in the SCC subtype. These data may be useful in developing novel targeted therapies against the pro-survival effects of tissue hardening in SCC-TAFs, which act as co-conspirators of tumor progression. Citation Format: Marta Puig, Alicia Gimenez, Roland Galgoczy, Roberto Lugo, Josep Ramirez, Abel Gomez-Caro, Pere Gascon, Noemi Reguart, Jordi Alcaraz. An abnormally stiff microenvironment supports the overabundance of fibroblasts in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1103. doi:10.1158/1538-7445.AM2013-1103