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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.

The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non–small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFβ transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFβ1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFβ1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADC-TAFs and control fibroblasts increased TGFβ1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, because cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFβ1/SMAD3 activation. Significance: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFβ1/SMAD3.

The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in non-small cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs

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.

Background: ALK, ROS1 and RET fusions and MET exon 14 skipping variant (METex14) are present in 10-15% of advanced non-small-cell lung cancer (NSCLC) patients and their accurate identification is critical to guide targeted therapies. In a significant number of cancer patients, the tumor tissue available is insufficient for genetic analysis and repeated tissue biopsies for monitoring the course of the disease and the emergence of resistance are not feasible. Liquid biopsies constitute the only alternative available in these cases, but NGS techniques have shown insufficient sensitivity for fusion detection in blood samples. The nCounter technology has been adapted to detect fusions and skipping variants in FFPE tumor biopsies and we aimed to validate it for exosomes. Methods: Exosomes were purified using a miRCURY kit (Qiagen) and RNA was extracted using the TRI reagent (MRC Inc). A customized nCounter panel (Nanostring) for detection ALK, ROS1 and RET fusion transcripts and MET ex14 mRNA was used with a 10-cycles preamp step. First, proof-of-concept experiments were run by testing exosomes isolated from the culture medium of cell lines. Next, we tested exosomes isolated from the blood of NSCLC patients with know genotypes. Results: nCounter fusion probes successfully detected ALK, RET and ROS1 fusion transcripts in exosomes isolated from the culture medium of the cell lines H3122 (EML4-ALKv1), H2228 (EML4-ALKv3), HCC78 (SLC34A2-ROS1) and LC/2-Ad (CCDC6-RET). Exosomes from a cell line established from a patient progressing to alectinib were also positive for EML4-ALKv1and showed high MET expression levels, while exosomes from the fusion-negative cell lines A549 and H23 tested negative. Finally, fusion transcripts were detected in exosomes purified from the blood of fusion positive NSCLC patients but not in fusion negative cases. Conclusions: nCounter can detect ALK, RET and ROS1 fusion transcripts in exosomes purified from the blood of advanced NSCLC patients Citation Format: Ana Giménez Capitán, Jill Bracht, Chung-Ying Huang, Cristina Teixidó, Noemí Reguart, Rich Boykin, Sarah Warren, Joseph M Beechem, Santiago Viteri Ramirez, Juan José García, Andrés Aguilar, Rafael Rosell Costa, Jay Gerlach, Miguel Angel Molina-Vila. nCounter for detection of clinically relevant alterations in exosomes of non-small cell lung cancer cells and patients [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 809.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death, and its major histotypes are lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Nintedanib (Vargatef) is a multi-tyrosine kinase inhibitor of VEGF, FGF and PDGF receptors that has been approved as second-line treatment of advanced lung adenocarcinoma patients, owing to the positive therapeutic effects reported selectively on ADC but not SCC patients in the LUME-1 clinical trial. Nintedanib has been also approved to treat idiopathic pulmonary fibrosis due to its antifibrotic effects reported in the INPULSIS trial. Of note, we recently reported that tumor fibrosis is larger in ADC than SCC due to the stronger epigenetic repression of the pro-fibrotic TGF-β transcription factor SMAD3 in tumor-associated fibroblasts (TAFs) in SCC compared to ADC. Moreover, we also showed that nintedanib elicits a larger inhibition of TGF-β-induced fibrosis in ADC-TAFs compared to SCC-TAFs. However, the detailed antifibrotic mechanisms of nintedanib on the TGF-β1 pathway remain poorly understood. TGF-β1 signaling begins with its binding to type II TGF-β receptor, which phosphorylates type I TGF-β receptor ALK5, that subsequently phosphorylates SMAD2 and SMAD3, upon which they form heterotrimeric complexes with the co-factor SMAD4 that translocate to the nucleus to regulate gene expression. We found that nintedanib markedly inhibited both the activation of SMAD2 and SMAD3 as well as the increase in nuclear SMAD4 in response to TGF-β1 in pulmonary fibroblasts, thereby supporting that ALK5 may be an off-target of nintedanib. To examine this possibility, we performed a time-course analysis of phospho-ALK5 (pALK5) and pSMAD3 in fibroblasts in response to TGF-β1 with or without nintedanib. As expected, TGF-β1 elicited a peak in pALK5 before that of pSMAD3. Moreover, nintedanib downregulated pALK5 but not total ALK5, supporting that ALK5 is an unintended target of this drug. In addition, we examined the expression of total and phospho-Erk1/2 in the same samples, since Erk1/2 has been previously reported as a regulator of cellular responses to TGF-β. TGF-β1 elicited a peak in pErk1/2 within the same time-window than pSMAD3, which is consistent with previous observations on non-canonical TGF-β signaling. Interestingly, nintedanib abrogated both total and pErk1/2 expression in a time-dependent fashion. These results support that the antifibrotic effects of nintedanib may be mediated through their inhibition of the pro-fibrotic transcription factor SMAD3 as well as the downregulation of Erk1/2. In addition, our results support that Erk1/2 may be implicated in the limited responses of SCC-TAFs to nintedanib, since we previously showed that SCC-TAFs exhibit an enhanced activity of Erk1/2 compared to ADC-TAFs. Citation Format: Rafael Ikemori, Marta Gabasa, Paula Duch, Frank Hilberg, Noemí Reguart, Jordi Alcaraz. Unraveling the antifibrotic mode of action of nintedanib against the TGF-β pathway in tumor-associated fibroblasts in non-small cell lung cancer [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 2982.

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.

Tumor associated fibroblasts (TAFs) are essential contributors of the progression of non-small cell lung cancer (NSCLC). Most lung TAFs exhibit an activated phenotype characterized by the expression of α-SMA and fibrillar collagens. However, the prognostic value of these activation markers in NSCLC remains unclear. To address this question we conducted a retrospective multicentric study of the prognostic value of the standard markers of activated fibroblasts. For this purpose, we conducted a quantitative image analysis of α-SMA immunostaining and picrosirius red staining of fibrillar collagens imaged by bright-field and polarized microscopy, respectively, using tissue microarrays with samples from 220 surgical patients, which elicited a percentage of positive staining area for each marker and patient. Kaplan-Meier curves showed that all TAF activation markers were significantly associated with poor survival, and their prognostic value was independent of TNM staging as revealed by multivariate analysis, which elicited an adjusted increased risk of death after 3 years of 129% and 94% for fibrillar collagens imaged with bright-field (p = 0.004) and polarized light (p = 0.003), respectively, and of 89% for α-SMA (p = 0.009). We also found a significant association between all TAF activation markers and tumor necrosis, which is often indicative of hypoxia, supporting a pathologic link between tumor desmoplasia and necrosis/hypoxia. Our findings identify patients with large histologic coverage of fibrillar collagens and α-SMA+ TAFs to be at higher risk of recurrence and death, supporting that they could be considered for adjuvant therapy. Moreover it supports that antifibrotic drugs aiming to target tumor fibrosis may be an effective therapeutic approach to improve survival in NSCLC. Citation Format: Jordi Alcaraz, Josep-Lluís Carrasco, Laura Millares, Iuliana-Cristiana Benchea, Francisco-Javier Fernández, Anabel Martínez, María José Pajares, Julio Sánchez de Cos, Ramon Rami-Porta, Luis Seijo, Josep Ramírez, Noemí Reguart, Esther Barreiro, Eduard Monsó. Stromal markers of activated tumor associated fibroblasts predict poor survival and are associated with necrosis in non-small cell lung cancer [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 3160.

Nintedanib is a clinically-approved multikinase receptor inhibitor that, in combination with docetaxel, provides clinical benefits to advanced lung adenocarcinoma (ADC) patients but not to lung squamous cell carcinoma (SCC) patients. However, the mechanisms underlying the selective therapeutic effects of Nintedanib in ADC remain poorly understood. Of note, Nintedanib is also approved to treat patients with idiopathic pulmonary fibrosis (IPF), a rare disease characterised by an abundant desmoplastic stroma rich in pathologically activated fibroblasts. Since the tumor stroma in lung cancer is also desmoplastic, and we recently showed that tumor-associated fibroblasts (TAFs) derived from ADC and SCC patients exhibit different phenotypes in vitro, we hypothesized that TAFs may underlie the selective effects of Nintedanib in ADC. To test this hypothesis we activated TAFs in culture with the pro-fibrotic cytokine TGF-β1 in the presence of increasing concentrations of Nintedanib, and collected the corresponding conditioned medium. Remarkably SCC-TAFs showed very modest inhibition of a panel of fibrotic markers including α-SMA, P4HA2 and fibrillar collagens (COL1A1, COL3A) in response to Nintedanib, in striking contrast to ADC-TAFs and paired lung parenchyma fibroblasts, which were markedly affected. This was matched by a significant reduction in the abilities of the conditioned medium of ADC-TAFs but not SCC-TAFs to promote cancer cell growth and invasion in a panel of lung cancer cell lines after Nintedanib treatment. These results reveal that Nintedanib is an effective inhibitor of stromal fibrosis and its associated tumor-promoting effects in ADC, and that the poor antifibrotic response of SCC-TAFs to Nintedanib may contribute to the differential clinical benefit observed in both subtypes. Our findings also support that TGF-β signalling and aberrant TAF-carcinoma cross-talk are regulated by different mechanisms in ADC and SCC. In addition they support that preclinical models based on carcinoma-TAF interactions may help defining the mechanisms of the poor antifibrotic response of SCC-TAFs to Nintedanib and testing new combined therapies to further expand the therapeutic effects of this drug in solid tumors. Citation Format: Jordi Alcaraz, Marta Gabasa, Rafael Ikemori, Frank Hilberg, Noemí Reguart. Nintedanib abrogates the activation and tumor-promoting effects of fibroblasts from lung adenocarcinoma patients [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 2869.

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.

Nintedanib (BIBF 1120) is a multikinase inhibitor recently approved to treat lung adenocarcinoma (ADC) patients based on the therapeutic benefits reported in clinical trials on ADC but not on squamous cell carcinoma (SCC), which are the two most common lung cancer subtypes. However, the biological processes underlying the selective anticancer function of nintedanib in ADC remain obscure. To shed light on this gap of knowledge, we examined the role of tumor-associated fibroblasts (TAFs) in the differential effects of nintedanib in ADC and SCC. Because TAFs are largely quiescent and activated in vivo, we focused on the antifibrotic effects of nintedanib on TAFs stimulated with TGF-β1, which is a potent fibroblast activator frequently upregulated in lung cancer. Nintedanib dose-dependently inhibited the TGF-β1-induced expression of activation markers in both ADC-TAFs and control fibroblasts derived from uninvolved parenchyma, whereas such inhibiton was very modest in SCC-TAFs, thereby suggesting that TAF activation is regulated by different mechanisms in ADC and SCC. Remarkably, nintedanib abrogated the stimulation of growth and invasion in a panel of carcinoma cell lines induced by secreted factors from activated TAFs in ADC but not SCC. These results reveal that nintedanib is a potent inhibitor of fibrosis and its associated tumor-promoting effects in ADC, and strongly suggest that the subnormal antifibrotic response of SCC-TAFs to nintedanib may underlie the lack of therapeutic benefits of this drug in SCC. Our findings also support that preclinical models based on carcinoma-TAF interactions may help unraveling the resistance mechanisms of SCC-TAFs to nintedanib and testing new combined therapies to further expand the therapeutic effects of this drug. Citation Format: Jordi Alcaraz, Marta Gabasa, Rafael Ikemori, Noemí Reguart. Nintedanib selectively inhibits the activation and tumor-promoting effects of fibroblasts from lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1228. doi:10.1158/1538-7445.AM2017-1228

Permanent cell cycle arrest through senescence has been previously regarded as a protective mechanism against tumor progression. In contrast, there is evidence that senescence in tumor-associated fibroblasts (TAFs) enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of selected cancer types. However, the presence of senescent TAFs in lung cancer remains undefined. To address this gap of knowledge, we examined common markers of senescence in primary TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent cell collections were used. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Of note, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those provided by control (non-senescent) fibroblasts. These results expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors. Citation Format: Roberto Lugo, Marta Gabasa, Francesca Andriani, Marta Puig, Federica Facchinetti, Josep Ramírez, Abel Gómez-Caro, Ugo Pastorino, Pere Gascón, Albert Davalos, Noemí Reguart, Luca Roz, Jordi Alcaraz. Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4103.

Background: Targetable rearrangements in anaplastic lymphoma kinase (ALK), ROS1, and RET genes are present in approximately 7% of patients (p) with advanced NSCLC. Current methods for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHC) or RT-PCR. However, these tests have disadvantages in terms of sensitivity, cost and throughput, and often show discrepancies. The nCounter platform allows simultaneous detection with no enzymatic reaction, within 72 hours, of several fusion genes in formalin-fixed paraffin-embedded (FFPE) samples using a transcript-based method. Methods: A custom set of 5′and 3′ probes and fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was designed and evaluated. A panel of ALK-ROS1-RET positive cell lines (H2228, H3122 [EML4-ALK], SUDHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS1], BaF3 pBABE [CD74-ROS1], LC2/ad [RET]) and negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) was used to validate the technique. Then, a total of 70 FFPE samples was analyzed, 49 of them positive by FISH, IHC and/or qRT-PCR for ALK (n = 30), ROS1 (n = 17) and RET (n = 2). Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and four house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative translocations were defined by two criteria: (1) a 3’/5’ ratio score of > 2.0 and ≤ 2.0 respectively or/and (2) a signal for a fusion-specific probe above background. Response to crizotinib by RECIST criteria was retrospectively collected in p with ALK-positive NSCLC by any technique. Results: nCounter sensitivity to detect fusion transcripts ALK, ROS1 and RET in cell lines was 100% using the two criteria (3’/5’ and direct probes) and specificity was also 100%. Among 20 ALK-FISH-positive p, ALK 3’-5’ scoring was positive in 18 (95%). One p was non-evaluable (NE) by ALK 3’-5’ scoring. Among 48 ALK-FISH-negative p, nCounter score was positive in 13 (27%). All p positive for ALK by nCounter were either positive or NE for ALK by IHC. A total of 17 p were treated with crizotinib, 16 of whom responded to treatment and were positive by nCounter. Regarding FISH, five p responding to crizotinib were negative and one was NE. Finally, one p not responding to crizotinib was positive by RT-PCR but negative by nCounter. Conclusion: The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening assay that identifies ALK-FISH-negative/NE NSCLC patients who could benefit from treatment with ALK inhibitors. Citation Format: Cristina Teixido, Noemí Reguart, Ana Giménez-Capitán, Miguel Ángel Molina-Vila, Patricia Galván, Sonia Rodriguez, Laia Paré, Santiago Viteri, Vicente Peg, Zaira Yeste, Núria Viñolas, Rafael Rosell, Aleix Prat. Comparison of nCounter, immunohistochemistry, RT-PCR and FISH to detect ALK, ROS1 and RET rearrangements in advanced non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4344.

There is growing interest in defining the aberrant molecular differences between normal and tumor-associated fibroblasts (TAFs) that support tumor progression. For this purpose, we recently conducted a genome-wide DNA methylation profiling of TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer (NSCLC) patients, and reported a widespread hypomethylation concomitantly with focal gain of DNA methylation; in addition, we found evidence that a fraction of lung TAFs are fibrocytes in origin. Of note, the aberrant epigenome of lung TAFs had a global impact in gene expression and a selective impact on the TGF-β pathway. To get insights on the functional implications of the latter impact, we analyzed the response of lung TAFs to exogenous TGF-β1 in terms of activation and contractility. We found a larger expression of a panel of activation markers including α-SMA and collagen-I in TAFs compared to control fibroblasts. Likewise, TGF-β1 elicited a larger contractility in TAFs than in CFs as assessed by traction force microscopy. These findings reveal that lung TAFs are hyperresponsive to TGF-β1, which may underlie the expansion and/or maintenance of the tumor-promoting desmoplastic stroma in lung cancer. Citation Format: Miguel Vizoso, Marta Puig, F. Javier Carmona, María Maqueda, Antonio Gomez, Anna Labernardie, Marta Gabasa, Saioa Mendizuri, Rafael Ikemori, Xavier Trepat, Sebastian Moran, Enrique Vidal, Noemí Reguart, Alexandre Perera, Manel Esteller, Jordi Alcaraz. DNA methylation profiling unveils TGF-β hyperresponse in tumor associated fibroblasts from lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2763.