Reguart, Noemı́

Abstract Non-small cell lung cancer (NSCLC) exhibits strikingly different responses to antiangiogenic therapies between its two major histologic subtypes, lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC), suggesting an histotype-specific regulation of angiogenesis. Cancer-associated fibroblasts (CAFs) commonly exhibit an activated/myofibroblast-like phenotype in NSCLC, and are emerging as key modulators of tumor progression; however, their contribution to angiogenic control remains undefined. Here, we investigated angiogenesis and hypoxia signatures in NSCLC and integrated bulk RNA-seq, scRNA-seq, CAF secretome profiling, genetic perturbations, and functional in vitro and in vivo assays to dissect the histotype-dependent production of pro-angiogenic factors by CAFs. We observed greater angiogenesis and reduced necrosis/hypoxia in LUAD compared to LUSC across multiple patient cohorts. The LUAD-CAF secretome was primed for angiogenesis through SMAD3-dependent overproduction of key regulators, most notably TIMP-1 and VEGF-A. We also uncovered a previously unrecognized role for TIMP-1 in promoting endothelial hyper-branching. In contrast, LUSC-CAFs displayed attenuated angiogenic activity despite robust HIF-1α upregulation and an hypoxia-associated transcriptional program, due to their epigenetic repression of SMAD3 and compensatory increase in SMAD2. Collectively, these results reveal that CAFs critically shape the distinct angiogenic landscapes of LUAD and LUSC through opposing SMAD2/3 regulation of TIMP-1, VEGF-A, and hypoxia signaling. These results further highlight the therapeutic potential of targeting stromal SMAD3/TIMP-1 in LUAD or microenvironmental stressors such as hypoxia and acidosis in LUSC. In addition, these findings provide a biological framework for understanding histotype-specific patterns of dissemination, immune evasion, and response to antiangiogenic therapies in NSCLC. Citation Format: Jordi Alcaraz, Natalia Isabel Diaz Valdivia, Paula Duch, Marselina Arshakyan, Amelia Parker, Alejandro Bernardo Suarez, Danielle Park, Erik Sahai, Noemi Reguart, Derek C. Radisky, Oriol Casanovas. Antagonistic SMAD2/3 control of TIMP-1, VEGF-A, and hypoxia signaling in myofibroblasts shapes histotype-specific angiogenesis in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4797.

Abstract Background: Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are the most common lung cancer subtypes. SCC is traditionally resistant to anti-angiogenic therapies, though the mechanisms remain unclear. Nintedanib, a multi-angiokinase receptor inhibitor, is approved for treating ADC in combination with docetaxel, based on selective efficacy in the LUME-1 trial. It is also FDA-approved for idiopathic pulmonary fibrosis due to its antifibrotic properties. Both ADC and SCC feature fibrotic stroma rich in activated tumor-associated fibroblasts (TAFs). Our prior work showed Nintedanib has stronger antifibrotic effects on ADC-TAFs than SCC-TAFs. However, its impact on TAF-driven angiogenesis and the role of TAFs in SCC resistance to multi-angiokinase inhibitors remain unexplored. Methods: The pro-angiogenic activity of TAFs secretome from ADC and SCC patients was assessed. TAFs were stimulated with TGF-β1 to mimic their activated phenotype, then treated with Nintedanib. Conditioned media from treated TAFs was used to assess endothelial cell migration and vascular network formation. Pro-angiogenic factor secretion was analyzed using antibody arrays, and functional roles of selected proteins were validated in vitro and in vivo using genetic approaches. Results: Nintedanib inhibited the pro-angiogenic effects of ADC-TAF secretome but paradoxically enhanced angiogenesis in SCC-TAFs, increasing endothelial cell migration in vitro and in vivo. Notably, Nintedanib selectively upregulated CXCL-1 secretion in SCC-TAFs. Silencing CXCL-1 using siRNA in SCC-TAFs abrogated the Nintedanib-induced angiogenesis. Similar findings with other multi-angiokinase inhibitors suggest aberrant CXCL-1 production in SCC-TAFs may drive universal resistance to these therapies. Conclusion: SCC-TAFs are resistant to the anti-angiogenic effects of Nintedanib, which paradoxically promotes angiogenesis via CXCL-1 upregulation. This study provides the first mechanistic insight into SCC resistance to anti-angiogenic therapies and underscores the critical role of TAFs in modulating therapeutic responses. Targeting CXCL-1 or TAF-mediated pathways may inform alternative anti-angiogenic strategies in SCC, potentially in combination with immune checkpoint inhibitors. Citation Format: Natalia Isabel Diaz Valdivia, Paula Duch, Alejandro Bernardo, Marselina Arshakyan, Noemi Reguart, Derek Radisky, Oriol Casanovas, Jordi Alcaraz. Intrinsic resistance mechanism of squamous cell lung carcinomas to the multiangiokinase receptor inhibitor Nintedanib through CXCL-1 upregulation in tumor associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5554.

AbstractThe fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor‐associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase‐1 (TIMP‐1), a tumor‐promoting cytokine overproduced in ADC‐TAFs. To this aim, we combined genetic approaches with in vitro and in vivo preclinical models based on patient‐derived TAFs. Nintedanib reduced TIMP‐1 production more efficiently in ADC‐TAFs than SCC‐TAFs through a SMAD3‐dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC‐TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co‐injecting ADC cells with TIMP1‐knockdown ADC‐TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP‐1 in ADC‐TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP‐1 production in SCC‐TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP‐1 as a critical regulator of therapy response in solid tumors.

Abstract Adenocarcinoma (ADC) and Squamous Cell Carcinoma (SCC) are the most frequent histologic subtypes of lung cancer, and both are rich in activated/myofibroblast-like tumor-associated fibroblasts (TAFs). Nintedanib is a potent antifibrotic drug that targets the tumor stroma and is clinically approved to treat advanced lung ADC patients owing to the survival benefits observed in the LUME-1 clinical trial in ADC but not SCC patients. Although the mechanism underlying the ADC-selective therapeutic effects of nintedanib remained poorly understood, we have previously reported that nintedanib abrogates the pro-tumoral traits of the secretome of ADC-TAFs but not SCC-TAFs, suggesting that secreted factor(s) in ADC-TAFs may be implicated. In addition, we recently unveiled an ADC-specific tumor-promoting crosstalk between TAFs and cancer cells driven by TIMP-1 and CD63, whereas others have shown that TIMP-1 is a molecular target of nintedanib. However, it remains unknown if TIMP-1 is involved in the ADC-selective benefits of nintedanib. To address this question, we used patient-derived TAFs obtained with the patient’s informed consent and using protocols approved by the Ethics Committee of the Hospital Clinic. We treated TGF-β1-activated ADC-TAFs and SCC-TAFs with nintedanib and determined the content of TIMP-1 in their conditioned medium by ELISA. Moreover, we used cell-based functional assays and tumor xenografts after knocking-down TIMP-1 in TAFs by siRNA to examine how silencing TIMP-1 altered their response to nintedanib. By analyzing TCGA data and the human protein atlas, we found that TIMP-1 is consistently upregulated in ADC compared to SCC tumors both at the mRNA and protein levels. Moreover, TCGA data revealed that TIMP-1 is increased in tumors compared to paired control tissue in ADC but not SCC. In culture, we observed that TIMP-1 secretion was higher in ADC-TAFs than SCC-TAFs, and that this secretion was downregulated by nintedanib to a larger extent in ADC-TAFs compared to SCC-TAFs. In vivo analyses revealed that ADC cells co-injected with fibroblasts silenced for TIMP-1 as in SCC-TAFs into immunocompromised mice exhibited a less invasive growth pattern compared to tumors bearing control fibroblasts. In addition, we observed that the inhibition of the pro-tumoral secretome of ADC-TAFs elicited by nintedanib was abrogated upon knocking-down TIMP-1 in TAFs both in vitro and in vivo. Collectively, these results reveal that the high secretion of TIMP-1 in ADC-TAFs render them more responsive to nintedanib, whereas the low TIMP-1 secretion of SCC-TAFs may be a major contributor to the selective resistance of SCC patients to nintedanib. Moreover, our results identify TIMP-1 as a promising predictive biomarker of nintedanib responses. Citation Format: Paula Duch, Natalia Díaz-Valdivia, Marta Gabasa, Rafael Ikemori, Frank Hilberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Aberrant TIMP-1 secretion by tumor-associated fibroblasts is a major contributor to the selective positive response to nintedanib in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2356.

Abstract The two most common lung cancer subtypes are adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Even though both subtypes are epithelial in origin, it is now clear that tumor associated fibroblasts (TAFs) are key regulators of tumor progression and response to therapies. Nintedanib is an antifibrotic drug that targets the tumor stroma and has been clinically approved to treat lung ADC patients owing to the therapeutic benefits exhibited by this drug selectively in ADC (but not in SCC) in the LUME-1 cinical trial. We have implicated recently both ADC-TAFs and ADC cancer cells in the selective effects of nintedanib in ADC, since this drug reduced the growth and invasion induction elicited by the secretome of TGF-β-activated ADC-TAFs on a panel of ADC cells, whereas such reduction was not observed in SCC-TAFs. However, the key molecules involved in the aberrant TAF-carcinoma crosstalk in ADC remain unknown. TIMP-1 is a multifunctional protein that has been associated with poor prognosis in lung cancer and is downregulated by nintedanib in a bleomycin model of pulmonary fibrosis. Our preliminary results revealed that the TIMP-1 receptor CD63 is overexpressed in ADC patients compared to SCC. Therefore, our working hypothesis was that nintedanib reduces ADC cells growth and invasion by abrogating the TAF-carcinoma crosstalk driven by TIMP-1 and CD63.To test this hypothesis, we used primary TAFs obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. TGF-β1-activated ADC-TAFs and SCC-TAFs were treated with nintedanib, and their secreted TIMP-1 was determined by ELISA. Two high-CD63 cell lines (H1437 and H23) were used in some experiments, and siRNA was used to knock-down either TIMP-1 in TAFs or CD63 in cancer cellsOur in vitro results showed that the secretion of TIMP-1 was significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher TIMP-1 downregulation in ADC-TAFs compared SCC-TAFs. Of note, TIMP-1 and CD63 were both implicated in the pro-tumorigenic crosstalk, since knocking-down TIMP-1 in ADC-TAFs or CD63 in ADC cells was sufficient to abrogate the growth and invasion enhancement elicited by the secretome of TAFs. Moreover, CD63 was necessary to enhance the invasion of ADC cells upon stimulation with recombinant TIMP-1. In addition, we found that knocking-down TIMP-1 in ADC-TAFs was sufficient to compromise the inhibitory effects of nintedanib on the growth and invasion enhancement elicited by the secretome of TAFs on ADC cells. Collectively, our results support a novel TAF-carcinoma crosstalk driven by TIMP-1 and CD63 in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib. Citation Format: Paula Duch, Natalia Díaz-Valdivia, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Stromal TIMP-1 drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3167.

Abstract Large cell carcinoma (LCC) is an aggressive lung cancer subtype with poor prognosis and no targeted therapies. We previously reported that tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we report that MMP1 is overexpressed specifically in LCC cell lines. Notably, silencing MMP1 expression in LCC cancer cell lines using shRNA revealed that MMP1 expression by LCC cells is necessary for induction of fibroblast senescence in coculture experiments with normal pulmonary fibroblasts, as revealed by the analysis of a panel of standard senescence markers, including β-galactosidase (SA-βgal) staining, permanent growth arrest and expression of senescence-associated secretory factors. Injecting control (shScr) or shMMP1 LCC cells into immunodeficient nude mice revealed that tumor growth, tumor take and cancer cell dissemination to the lung were reduced in shMMP1 H460 tumors compared to control tumors. We also observed fewer senescent fibroblasts in tumors from shMMP1 H460 cells using Sentragor staining, which allows identification of senescent cells in paraffin embedded tissues. Moreover, we found that recombinant active MMP1 in combination with TGF-β1 were sufficient to induce normal fibroblast senescence. In terms of the potential underlying mechanisms, treatment with the antioxidant n-acetyl cysteine (NAC) significantly attenuated the increase in SA-βgal+ fibroblasts elicited by co-stimulation with rMMP1 and TGF-β1, and its corresponding conditioned medium elicited a significantly lower growth and invasion in LCC cancer cells, revealing the oxidative stress implication in fibroblast senescence induction and associated pro-tumorigenic secretome. In summary, our results establish a new role for MMP1 in cancer and support that LCC cells elicit a tumor-supporting niche through the aberrant secretion of MMP1 and TGF-β1 to induce senescence in adjacent fibroblasts. Furthermore, we implicate oxidative stress in MMP1/TGF-β1-induced TAF senescence and support a novel therapeutic strategy in LCC based on targeting senescent TAFs. Citation Format: Marta Gabasa, Evette S. Radisky, Rafael Ikemori, Giulia Bertolini, Marselina Arshakyan, Alexandra Hockla, Paula Duch, Ornella Rondinone, Alejandro Llorente, Maria Maqueda, Alexandre Perera, Noemí Reguart, Luca Roz, Derek C. Radisky, Jordi Alcaraz. MMP1 and TGF-β1 cooperate to drive tumor progression in large cell carcinoma of the lung through fibroblast senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2515.

Abstract Tumor associated fibroblasts (TAFs) are important regulators of tumor growth and resistance to therapies. We have recently shown that lung TAFs in vitro respond to the antifibrotic drug nintedanib in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). We also showed that the tumor-promoting effects of TAFs are driven by different mechanisms in ADC and SCC, which remain to be elucidated. Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been associated with poor prognosis in lung cancer, its expression is downregulated by nintedanib, and our preliminary results reveal that its putative receptor, CD63, is overexpressed in ADC patients compared to SCC, supporting a selective crosstalk between TAFs and cancer cells in ADC through TIMP-1 and CD63. The aim of this study was to test this hypothesis using in vitro preclinical models. Primary fibroblasts were obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. ADC-TAFs and SCC-TAFs were stimulated with TGF-β1 in the presence or absence of nintedanib, and the TIMP-1 content in their conditioned medium was determined by ELISA. TIMP-1 was knocked-down in ADC-TAFs by siRNA, and the corresponding conditioned medium was used to stimulate growth and invasion of the high-CD63 ADC cell line, H1437. Likewise, CD63 expression in H1437 cells was reduced by siRNA. Our in vitro results showed that TIMP-1 secretion induced by TGF-β1 is significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher downregulation of secreted TIMP-1 in ADC-TAFs compared to SCC-TAFs. Of note, TIMP-1 from ADC-TAFs was necessary to induce growth and invasion of H1437 cells. Likewise, knocking-down CD63 in H1437 ADC cells was sufficient to reduce the growth and invasion elicited by the conditioned medium of TGF-β1 activated ADC-TAFs. Collectively, our results unveil a novel stroma-carcinoma crosstalk driven by TIMP-1 and CD63 selectively in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib. Citation Format: Paula Duch, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5091.

Abstract Tumor associated fibroblasts (TAFs) are key effector cells of cancer progression. Intriguingly, senescent TAFs have been reported in a growing list of agressive cancer subtypes including large cell carcinoma (LCC) of the lung. We previously reported that LCC cells induce fibroblast senescence in indirect co-cultures with transwells, revealing that paracrine signaling must be involved. A follow-up study identified MMP1 as an important regulatory protein in fibroblast senescence, since knocking-down MMP1 in LCC cells was sufficient to abrogate the induction of fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. Here we examined the potential role of the protease activated protein 1 (PAR-1) in fibroblast senescence by MMP1, since PAR-1 can be activated by MMP1 and has been reported in the stroma of lung cancer patients. We found that PAR-1 expression was down-regulated in fibroblasts that become senescent upon co-culture with LCC cells. In addition, forcing a reduced PAR-1 expression in fibroblasts by siRNA increased their expression of markers of the senescence associated secretory phenotype (SASP) during co-culture with LCC cells, whereas the percentage of senescence-associated beta-galactosidase positive fibroblasts remained largely unaffected. These results suggest that fibroblast senescence elicited by MMP1 secreted by LCC cells is mediated by processes other than PAR-1 activation and/or overexpression in fibroblasts. Citation Format: Marta Gabasa, Rafael Ikemori, Evette Radisky, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Role of MMP1-PAR-1 crosstalk in the pro-tumorigenic senescent fibroblasts in large cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2021.

Abstract Large cell carcinoma (LCC) is among the most aggressive histologic subtypes of non-small cell lung cancer, but the mechanisms underlying such aggressive nature remain unknown. We recently showed that fibroblasts from LCC patients exhibit premature senescence in vitro, and that co-culturing LCC cells (but not cancer cells from other lung cancer subtypes) with normal fibroblasts in transwells is sufficient to induce senescence in the latter in an oxidative stress-dependent manner, supporting that fibroblast senescence is induced by a secreted factor(s) from LCC cells. Remarkably, we also found that senescent fibroblasts secrete factors that stimulate the growth and invasion of LCC cells beyond the stimulation elicited by nonsenescent fibroblasts, revealing that fibroblast senescence may contribute to the aggressive nature of LCC. Whole-genome transcriptional profiling of a panel of lung cancer cell lines identified MMP1 among the genes with larger expression in LCC compared to other lung cancer subtypes. Since MMPs can induce oxidative stress, we examined whether the excessive MMP1 expression in LCC cells was involved in the induction of fibroblast senescence in co-cultures. Knocking down MMP1 in LCC cells was sufficient to abrogate fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. In contrast, autophagy, which has been previously associated with fibroblast senescence in breast cancer, was not upregulated in fibroblasts upon co-culture with LCC cells. These results support that the selective aberrant expression of MMP1 in LCC cells plays a major role in their ability to induce a protumorigenic senescent phenotype in adjacent fibroblasts through a mechanism that is independent of autophagy. Moreover, our observations identify MMP1 as a potential therapeutic target against the aberrant cancer cell-fibroblast crosstalk in LCC. Citation Format: Jordi Alcaraz, Marta Gabasa, Rafael Ikemori, Noemí Reguart, Evette Radisky, Derek Radisky. Cancer cells induce a protumorigenic senescent phenotype in fibroblasts through MMP1 but not autophagy in large cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 994.