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

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

The contribution of epithelial-to-mesenchymal transition (EMT) to the profibrotic stiff microenvironment and myofibroblast accumulation in pulmonary fibrosis remains unclear. We examined EMT-competent lung epithelial cells and lung fibroblasts from control (fibrosis-free) donors or patients with idiopathic pulmonary fibrosis (IPF), which is a very aggressive fibrotic disorder. Cells were cultured on profibrotic conditions including stiff substrata and TGF-beta 1, and analyzed in terms of morphology, stiffness, and expression of EMT/myofibroblast markers and fibrillar collagens. All fibroblasts acquired a robust myofibroblast phenotype on TGF-beta 1 stimulation. Yet IPF myofibroblasts exhibited higher stiffness and expression of fibrillar collagens than control fibroblasts, concomitantly with enhanced FAK(Y397) activity. FAK inhibition was sufficient to decrease fibroblast stiffness and collagen expression, supporting that FAK(Y397) hyperactivation may underlie the aberrant mechanobiology of IPF fibroblasts. In contrast, cells undergoing EMT failed to reach the values exhibited by IPF myofibroblasts in all parameters examined. Likewise, EMT could be distinguished from nonactivated control fibroblasts, suggesting that EMT does not elicit myofibroblast precursors either. Our data suggest that EMT does not contribute directly to the myofibroblast population, and may contribute to the stiff fibrotic microenvironment through their own stiffness but not their collagen expression. Our results also support that targeting FAK(Y397) may rescue normal mechanobiology in IPF

Research and development project assessment and social impact

Nowadays, organisations increasingly need to adapt to the fast evolution of markets and societies in our globalised world in order to be competitive. Therefore, it is essential to take the right decisions when it comes to invest in research and development (R & D) projects. However, an issue that has not been given much attention is how to measure the social impact (or return) of R & D projects. In this exploratory study, the findings of an analysis of how R & D projects are assessed and selected, including this social perspective, are presented. The methodology which has been used in this research includes both interviews and analysis of the data obtained through them. The major finding is that in the current situation the social impact is not taken into account, but is growing the awareness of this perspective among different types of organizations dealing with R & D activities.(undefined)info:eu-repo/semantics/publishedVersio

Evaluation of somatic mutations in cervicovaginal samples as a non-invasive method for the detection and molecular classification of endometrial cancer

Background The incidence of endometrial cancer is increasing worldwide. While delays in diagnosis reduce survival, case molecular misclassification might be associated with under- and over-treatment. The objective of this study was to evaluate genetic alterations to detect and molecularly classify cases of endometrial cancer using non-invasive samples. Methods Consecutive patients with incident endometrial cancer (N = 139) and controls (N = 107) from a recent Spanish case–control study were included in this analysis. Overall, 339 cervicovaginal samples (out of which 228 were clinician-collected and 111 were self-collected) were analysed using a test based on next-generation sequencing (NGS), which targets 47 genes. Immunohistochemical markers were evaluated in 133 tumour samples. A total of 159 samples were used to train the detection algorithm and 180 samples were used for validation. Findings Overall, 73% (N = 94 out of 129 clinician-collected samples, and N = 66 out of 90 self-collected samples) of endometrial cancer cases had detectable mutations in clinician-collected and self-collected samples, while the specificity was 80% (79/99) for clinician-collected samples and 90% (19/21) for self-collected samples. The molecular classifications obtained using tumour samples and non-invasive gynaecologic samples in our study showed moderate-to-good agreement. The molecular classification of cases of endometrial cancer into four groups using NGS of both clinician-collected and self-collected cervicovaginal samples yielded significant differences in disease-free survival. The cases with mutations in POLE had an excellent prognosis, whereas the cases with TP53 mutations had the poorest clinical outcome, which is consistent with the data on tumour samples. Interpretation This study classified endometrial cancer cases into four molecular groups based on the analysis of cervicovaginal samples that showed significant differences in disease-free survival. The molecular classification of endometrial cancer in non-invasive samples may improve patient care and survival by indicating the early need for aggressive surgery, as well as reducing referrals to highly specialized hospitals in cancers with good prognosis. Validation in independent sets will confirm the potential for molecular classification in non-invasive samples. Funding This study was funded by a competitive grant from Instituto de Salud Carlos III through the projects PI19/01835, PI23/00790, and FI20/00031, CIBERESP CB06/02/0073 and CIBERONC CB16/12/00231, CB16/12/00234 (Co-funded by European Regional Development Fund. ERDF: A way to build Europe). Samples and data were provided by Biobank HUB-ICO-IDIBELL, integrated into the Spanish Biobank Network, and funded by the Instituto de Salud Carlos III (PT20/00171) and by Xarxa de Bancs de Tumors de Catalunya (XBTC) sponsored by Pla Director d’Oncologia de Catalunya. This work was supported in part by the AECC, Grupos estables (GCTRA18014MATI). It also counts with the support of the Secretariat for Universities and Research of the Department of Business and Knowledge of the Generalitat de Catalunya, and grants to support the activities of research groups 2021SGR01354 and 2021SGR1112

Aberrant TIMP-1 regulation in tumor-associated fibroblasts by the TGF-β1/SMAD3 pathway in lung cancer: implications in tumor progression and resistance to antifibrotic therapies

[eng] Increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types, including lung cancer. However, how TIMP-1 is regulated in lung cancer and how it drives tumor progression is poorly defined. In the first part of this thesis, we analyze the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). We report that TIMP-1 is aberrantly overproduced in tumor-associated fibroblasts (TAFs) in ADC compared to SCC-TAFs, and identify the selective hyperactivity of the pro-fibrotic TGF- β1/SMAD3 pathway in ADC-TAFs as a major mechanism driving the excessive secretion of TIMP-1 in ADC-TAFs. In contrast, CD63 was markedly downregulated in SCC cells compared to ADC cells. In the second part of this thesis, we define the tumor-promoting effects of the heterotypic crosstalk, specific for ADC patients, between TIMP-1 secreted by TAFs and CD63 in cancer cells. Functional assays revealed that TIMP-1 within the pro- tumoral secretome of TGF-β1-activated ADC-TAFs is necessary and sufficient to promote the growth and invasion of ADC cells in culture. Concomitantly, tumor cell expression of CD63 was required for the tumor-promoting effects of TIMP-1. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced TIMP-1 expression into immunocompromised mice exhibited a lower tumor volume together with a less invasive growth pattern compared to tumors bearing parental fibroblasts. Finally, we studied the relationship of TIMP-1/CD63 crosstalk with the resistance mechanism of SCC patients to nintedanib, which is an antifibrotic drug that has been selectively approved for the treatment of ADC patients. Of note, nintedanib strongly inhibited TIMP-1 secretion in TAFs. Moreover, we observed that TIMP-1 deficient TAFs are resistant to the nintedanib inhibition of their pro-tumoral traits, both in vitro and in vivo, compared to parental TAFs. This work identifies the first tumor-promoting TAF- carcinoma crosstalk that is selective for lung ADC, provides new insights on the pathologic role of stromal TIMP-1 in lung cancer, identifies a novel resistance mechanism to nintedanib in SCC, and points to the TIMP-1/CD63 interaction as a novel therapeutic target.[cat] L’expressió elevada de TIMP-1 està associada a un mal pronòstic en gairebé tots els tipus de càncer, inclòs el càncer de pulmó. No obstant, es desconeix com es regula l’expressió de TIMP-1 en càncer de pulmó ni mitjançant quins mecanismes promou la progressió tumoral. En la primera part d’aquesta tesi, s’han analitzat els nivells de TIMP-1 i CD63, el seu receptor de membrana, en els dos subtipus majoritaris de càncer de pulmó: l’adenocarcinoma (ADC) i el carcinoma escatós (CE). Hem trobat que en els fibroblasts associats al tumor (TAFs de l’anglès) del subtipus ADC hi ha una secreció aberrant de TIMP-1 en comparació amb els TAFs de CE. Aquesta excessiva secreció de TIMP-1 en ADC es deu a la hiperactivació selectiva de la via del TGF-β1/SMAD3 en aquest subtipus de TAFs. D’altra banda, els nivells de CD63 són més baixos en les cèl·lules canceroses de CE que en les d’ADC. En la segona part d’aquesta tesi, hem descrit els efectes pro- tumorals d’un mecanisme de comunicació creuada entre el TIMP-1 secretat pels TAFs i el CD63 de les cèl·lules canceroses que és específic per als pacients amb ADC. El TIMP- 1 secretat pels TAFs d’ADC és necessari i suficient per promoure el creixement i la invasió en les cèl·lules de càncer. Simultàniament, l’expressió de CD63 en les cèl·lules tumorals es necessària per observar aquests efectes pro-tumorals del TIMP-1. De manera consistent, in vivo hem observat que la inhibició de la secreció del TIMP-1 redueix tant el volum tumoral com el creixement invasiu en ratolins co-injectats amb cèl·lules canceroses i TAFs. Finalment, hem estudiat la relació d’aquesta comunicació creuada amb la resistència al nintedanib trobada en pacients amb CE, el qual és un fàrmac antifibròtic aprovat selectivament en pacients amb ADC. Hem observat que en els TAFs en els quals s’ha inhibit genèticament la secreció de TIMP-1, hi ha una menor resposta a nintedanib. Aquest treball identifica per primer cop un mecanisme de comunicació creuada entre TAFs i cèl·lules canceroses que és específic per pacients amb ADC, aporta nous coneixements sobre el rol patològic del TIMP-1 en càncer de pulmó, descriu un nou mecanisme de resistència al nintedanib en CE i apunta la interacció TIMP-1/CD63 com a nova diana terapèutica

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

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