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Marta Gabasa



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    OA08 - Advanced Models and "Omics" for Therapeutic Development (ID 133)

    • Event: WCLC 2019
    • Type: Oral Session
    • Track: Biology
    • Presentations: 1
    • Now Available
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      OA08.07 - Aberrant Epigenetic SMAD3 Signaling in Tumor-Associated Fibroblasts Modulates Fibrosis and Response to Nintedanib in NSCLC (Now Available) (ID 1972)

      11:00 - 12:30  |  Author(s): Marta Gabasa

      • Abstract
      • Presentation
      • Slides

      Background

      Tumor-associated fibroblasts (TAFs) exhibit a fibrotic phenotype in non-small cell lung cancer (NSCLC) that has beeen associated with critical steps of cancer progression. Paradoxically, we reported that the profibrotic TGF-β transcription factor SMAD3 was epigenetically downregulated through promoter hypermethylation in TAFs from NSCLC patients compared to patient-matched control fibroblasts. In addition, we reported that the antifibrotic drug nintedanib elicited a stronger inhibition of the fibrotic phenotype and its tumor-promoting effects in TAFs from adenocarcinoma (ADC) patients compared to squamous cell carcinoma (SCC) patients upon TGF-β1 stimulation in vitro, which was consistent with the selective therapeutic response to nintedanib observed in a clinical trial in ADC (but not SCC) patients. These previous results support the hypothesis that TGF-β1 signaling may be altered in lung TAFs according to their histologic subtype.

      Method

      In this study we tested our working hypothesis by determining the expression and activity of SMAD3 and its closely related homologue SMAD2 in patient-derived TAFs and paired control fibroblasts, and by dissecting their potential contribution to the differential therapeutic responses to nintedanib observed in ADC and SCC using in vitro and in vivo preclinical models.

      Result

      In vitro studies revealed a marked SMAD3 epigenetic repression through promoter hypermethylation, a low pSMAD3/pSMAD2 ratio and a limited fibrotic phenotype selectively in SCC-TAFs. In contrast, ADC-TAFs overexpressed a panel of fibrotic markers upon TGF-β1 stimulation concomitantly with a high pSMAD3/pSMAD2 ratio and a limited SMAD3 promoter methylation. Histologic analysis of a large patient cohort (112 ADC, 96 SCC) confirmed that the extent of fibrosis is larger in ADC than SCC patients. In addition, knocking-down SMAD3 in ADC-TAFs was sufficient to reduce the antifibrotic and antigrowth effects of nintedanib in vitro and in tumor xenografts in vivo. On the other hand, long-term exposure of pulmonary fibroblasts to cigarette smoke condensate was sufficient to hypermethylate the SMAD3 promoter. Since SCC and ADC tumors typically arise in the upper airways and distal pulmonary sites, respectively, it is conceivable that fibroblasts might be more exposed to the smoking epigenetic effects on SMAD3 in SCC.

      Conclusion

      We report for the first time that tumor fibrosis is higher in ADC than SCC patients, in association with a selective therapeutic response to the antifibrotic drug nintedanib in the former, and identify the subtype-specific extent of SMAD3 epigenetic repression in TAFs and the subsequent aberrant SMAD3/SMAD2 imbalance as major regulatory mechanisms of tumor fibrosis and response to nintedanib in NSCLC.

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    P1.03 - Biology (ID 161)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Biology
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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      P1.03-02 - Nintedanib Selectively Inhibits Angiogenesis Induced by the Conditioned Medium of Lung Adenocarcinoma TAFs in Vitro (ID 2193)

      09:45 - 18:00  |  Author(s): Marta Gabasa

      • Abstract
      • Slides

      Background

      Nintedanib is an antifibrotic and antiangiogenic drug that was clinically approved to treat EGFR-wild-type lung adenocarcinoma (ADC) patients based on the positive therapeutic results elicited in combination with docetaxel in the LUME-Lung1 clinical trial in ADC but not in squamous cell carcinoma (SCC) patients. However, the mechanisms underlying the selective therapeutic effects of nintedanib in ADC remain poorly understood. Tumor-associated fibroblasts (TAFs) are the most abundant stromal cell type and have been implicated in all steps of tumor progression, including growth, angiogenesis, invasion, metastasis and resistance to therapies. Of note, we recently reported that nintedanib elicits larger antifibrotic effects in ADC-TAFs compared to SCC-TAFs in vitro. However, the antiangiogenic effects of nintedanib in TAFs remain unknown.

      Method

      ADC-TAFs and SCC-TAFs were activated with TGF-β1 in the presence or absence of nintedanib, and the corresponding conditioned medium was used to stimulate endothelial cells (HUVECs and HMVECs) to either migrate or form vascular networks on Matrigel.

      Result

      Our results showed that the conditioned medium from ADC-TAFs and SCC-TAFs induced an increase in human endothelial cell migration. In contrast, the conditioned medium from ADC-TAFs promoted an increase in angiogenesis that was selectively inhibited by nintedanib, whereas such inhibition was not observed in SCC-TAFs.

      Conclusion

      Our results reveal that nintedanib inhibits the pro-angiogenic effects elicited by factors secreted by ADC-TAFs but not SCC-TAFs, thereby supporting that angiogenesis may be regulated by TAFs through distinct mechanisms in ADC and SCC.

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    P2.03 - Biology (ID 162)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Biology
    • Presentations: 3
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.03-01 - MMP1 Secreted by Cancer Cells Induces a Pro-Tumorigenic Senescent Phenotype in Fibroblasts in Large Cell Carcinoma of the Lung (ID 2195)

      10:15 - 18:15  |  Presenting Author(s): Marta Gabasa

      • Abstract
      • Slides

      Background

      Tumor associated fibroblasts (TAFs) are key effector cells of cancer progression. Senescent TAFs have been reported in a growing list of aggressive cancer subtypes including the aggressive subtype large cell carcinoma (LCC) of the lung. We previously reported that LCC cells induce fibroblast senescence in normal fibroblasts after co-culture, revealing that paracrine signaling must be involved. Moreover, we found that senescent fibroblasts secrete factors that stimulate the growth and invasion of LCC cells beyond the stimulation elicited by non-senescent fibroblasts, supporting that fibroblast senescence may contribute to the aggressive nature of LCC. Whole-genome transcriptional profiling on a panel of non-small cell lung cancer (NSCLC) cell lines, including adenocarcinoma (ADC), squamous cell carcinoma (SCC) and LCC, identified MMP1 as highly overexpressed in LCC cells compared to non-LCC cells. Here we examined the role of MMP1 in LCC cells in the paracrine induction of fibroblast senescence.

      Method

      We silenced MMP-1 expression in LCC cancer cell lines by shRNA and analyzed common senescence markers after co-culture with normal fibroblasts, including β-galactosidase staining as well as the expression of common factors of the senescence-associated secretory phenotype (SASP) by qRT-PCR. In addition, the growth and invasion pro-tumorigenic effects elicited by the conditioned medium of fibroblasts co-cultured with shMMP1 or shscramble LCC cells was analyzed.

      Result

      Knocking-down MMP1 in LCC cells was sufficient to abrogate fibroblast senescence induction in co-cultures with LCC cells, as well as the growth and invasion enhancement of LCC cells elicited by the conditioned medium of fibroblasts. The addition of active recombinant MMP1 partially rescued the fibroblast senescent phenotype in co-culture, yet it was not sufficient to induce senescence when added to fibroblasts cultured alone.

      Conclusion

      Our results unveil a process of “niche construction” by LCC cells that is driven by the overexpression of MMP1, which induces senescence in adjacent fibroblasts. Moreover, our results unveil a novel biological function of MMP1 (i.e. paracrine senescence induction in fibroblasts), that is strikingly different from its well-known collagenolytic function. Our results also show that MMP1 is necessary but not sufficient to induce fibroblast senescence. Moreover, our findings support that the aberrant carcinoma cell-fibroblast crosstalk mediated by MMP1 may be a suitable therapeutic target in LCC.

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      P2.03-17 - Optimization of an Ex-Vivo Preclinical Model for Drug Testing (ID 2385)

      10:15 - 18:15  |  Author(s): Marta Gabasa

      • Abstract
      • Slides

      Background

      Predicting drug response in advanced lung cancer patients remains a major challenge. A promising approach to tackle this challenge is based on testing drugs in an ex-vivo preclinical model using cultured precision-cut fresh tumour slices, which maintain not only the tumour itself but also its microenvironment. However, even though different protocols have been reported for ex-vivo models with different time-windows in a growing list of cancer types, no standard protocols are currently defined in lung cancer. To address this limitation, we have begun to optimize a protocol for an ex-vivo preclinical model for drug testing in lung cancer, using samples from the standard in vivo model of bleomycin-induced lung fibrosis, which resembles the fibrotic stroma of non-small cell lung cancers.

      Method

      Bleomycin-induced fibrotic and control rat lungs were precision-cut with a tissue-chopper and cultured on cell culture inserts up to 12 days in a sterile setting. Two main variables were tested: the coating of the cell culture insert (bare or collagen-I coated), and the interaction with the culture medium (submerged or floating). Small lung pieces were collected at days 0, 2, 6, 9 and 12 and examined by hematoxylin-eosin staining to assess their integrity and viability.

      Result

      Tissue architecture was maintained equally well in all culture conditions up to 12 days. In contrast, cellular content appeared to decline after day 6; since only subsets of cells in the periphery of the explants remained viable at day 9-12. No contamination was detected at any culture time.

      Conclusion

      Our preliminary results suggest that our protocols for an ex-vivo preclinical model maintain the integrity and viability of lung cells and their microenvironment up to one week, which is comparable to time-windows previously reported in other cancer types. This time-window is expected to be sufficient to test drug responses, thereby underlying the potential of this preclinical model for drug testing as well as for the study of drug resistance mechanisms.

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      P2.03-35 - Stromal TIMP-1 Drives Tumor Progression in Lung Adenocarcinoma Through CD63 Interaction (ID 2196)

      10:15 - 18:15  |  Author(s): Marta Gabasa

      • Abstract
      • Slides

      Background

      Tumor associated fibroblasts (TAFs) are important regulators of tumor growth and resistance to therapies. We have recently shown that TAFs in vitro from lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) respond positively to the antifibrotic drug nintedanib in the former only, thereby mimicking the selective therapeutic effects of nintedanib in ADC reported in the LUME-Lung1 clinical trial. We also showed that the tumor-promoting effects of TAFs are driven by different mechanisms in ADC and SCC. However, it remains to be elucidated the key signaling molecules involved in the aberrant fibroblast-carcinoma crosstalk in ADC. Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a multifunctional protein that has been associated with poor prognosis in lung cancer and other cancer types, and is downregulated by nintedanib in a bleomycin model of pulmonary fibrosis. Moreover, our preliminary analysis revealed that the TIMP-1 cell surface binding protein CD63, is overexpressed in ADC compared to SCC. Therefore, our objective was to study whether the selective tumor-promoting effects of ADC-TAFs are mediated by the interaction of stromal TIMP-1 with epithelial CD63.

      Method

      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 reduced in ADC-TAFs by siRNA, and the corresponding conditioned medium was used to assess the impact of TIMP-1 on the growth, invasion and survival of the CD63-high ADC cell line H1437. Likewise, CD63 expression in H1437 cells was reduced, by siRNA. In addition we performed immunohistochemical analyses of CD63 in tissue sections from lung cancer patients.

      Result

      Our results showed that TIMP-1 secretion induced by TGF-β1 is significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a larger downregulation of TIMP-1 secretion in ADC-TAFs compared to SCC-TAFs. We also confirmed that CD63 expression is higher in ADC patients than SCC, and revealed that knocking-down CD63 in H1437 ADC cells is sufficient to reduce the growth and invasion elicited by the conditioned medium of activated ADC-TAFs. Likewise, knocking-down TIMP-1 in ADC-TAFs was sufficient to downregulate the growth, invasion and survival of H1437 elicited by the conditioned medium of these TAFs.

      Conclusion

      Collectively, our results unveil a novel stroma-carcinoma interaction driven by TIMP-1 and CD63 selectively in lung ADC, and support that such crosstalk is a major regulator of the aberrant tumor-promoting effects of ADC-TAFs that are downregulated selectively by nintedanib.

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