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Daisuke Matsubara
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OA 18 - Lung Cancer Pathology and Genetics (ID 687)
- Event: WCLC 2017
- Type: Oral
- Track: Biology/Pathology
- Presentations: 1
- Moderators:George R. Simon, Yoon-La Choi
- Coordinates: 10/18/2017, 14:30 - 16:15, Room 316
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OA 18.07 - 3D Low-Attachment Culture: A Putative Model for STAS And "Floating" Cancer Cells (ID 8320)
15:35 - 15:45 | Author(s): Daisuke Matsubara
- Abstract
- Presentation
Background:
Cancer cells “floating” in stroma, air space, or lymphovascular channels are often found in aggressive lung adenocarcinomas. Recently, the concept of STAS (spread through alveolar space) has been proposed, and studies have shown that STAS predicts early recurrence and poor patient prognosis. However, the biologic basis for the aggressive phenotype of the tumor with these histologic features remains elusive. In this study, we tested whether 3D (three-dimensional) low-attachment culture could be an in vitro model for STAS and “floating” cancer cells.
Method:
Lung adenocarcinoma cell lines harboring diverse driver mutations (EGFR, KRAS, BRAF, HER2, RET) were used. Cells were subjected to detached condition by using low-attachment culture dishes to generate “floating” cancer cells in vitro. Cell growth assay, immunohistochemistry and Western blotting were used to characterize the biologic properties of “floating” cancer cells. Cancer cells were inoculated in pleural cavity or left ventricle of the heart of NOD/SCID mice to test their metastatic potential in vivo.
Result:
Upon detachment, cancer cells formed solid, micropapillary, or hollow ring-like structures, admixed with single cells, recapitulating a histologic spectrum of STAS in primary tumors. Outlining with MUC1, a feature of micropapillary lung adenocarcinoma, was also demonstrated in the in-vitro-generated micropapillary clusters as well. Detached cells initially showed retarded cell growth, but some cell lines regained growth potential with time in culture. Expression analysis of various biomarkers revealed that detached cells showed features of cell stress and altered metabolism, as indicated by increased expression of phospho-p38 (a stress-activated MAP kinase) and GLUT-1 (glucose transporter-1), respectively, and these findings were confirmed by analysis of primary tumors. Finally, cancer cells that adapted to detached conditions exhibited increased metastasis in vivo. Figure 1
Conclusion:
3D low-attachment culture may be a convenient model to study the biology of aggressive lung cancer with STAS and “floating” cancer cells.
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P1.02 - Biology/Pathology (ID 614)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.02-041 - Mutation of SWI/SNF Complex Genes Is Frequent in Poorly Differentiated, Mesenchymal-Like Lung Cancer without Major Driver Mutation (ID 8100)
09:30 - 09:30 | Author(s): Daisuke Matsubara
- Abstract
Background:
Recently, components of the switch/sucrose non-fermenting (SWI/SNF) complex have emerged as novel tumor suppressors in non-small cell lung cancer (NSCLC). However, the detailed genotypic and phenotypic features of NSCLC with mutations of SWI/SNF subunits remain unclear. In this study, we intended to clarify detailed morphologic and phenotypic features of NSCLC with mutations of SWI/SNF subunits through analysis of a panel of NSCLC cell lines and primary NSCLC.
Method:
We used 38 NSCLC cell lines which had been analysed for mRNA profiles and major driver mutations in our previous studies (Matsubara et al. AJP 2010, JTO 2010). The genetic status of SWI/SNF subunits was interrogated and retrieved from the public databases (COSMIC, CCLE). Morphological features of these cell lines were examined by 3-D cell culture and xenografts in NOD/SCID mice. We also immunohistochemically analyzed the expressions of multiple SWI/SNF subunits (BRG1, BRM, BAF47, ARID1A, and ARID1B.) in primary 133 NSCLC consisting of 25 squamous cell carcinomas, 70 adenocarcinomas, 16 large cell carcinomas, and 22 pleomorphic carcinomas.
Result:
In a panel of 38 NSCLC cell lines, mutations of SWI/SNF subunits tended to be found in the cell lines without major driver mutations and with mesenchymal (E-cadherin-low, vimentin-high) phenotypes. Consistently, these cell lines formed grape-like or stellate spheroid in 3-D cell culture, and poorly differentiated, solid or medullary tumors in xenograft models. In the primary tumors, reduced expression of BRG1 and BRM was significantly more frequent in large cell carcinomas and pleomorphic carcinomas than in squamous carcinomas and adenocarcinomas. Loss of expression of ARID1A, ARID1B and BAF47 was only rarely found only in a fraction of NSCLC cases.
Conclusion:
Collectively, these results indicate that loss of the SWI/SNF complex components is found in driver mutation pauci tumors and correlates with dedifferentiation and mesenchymal phenotype in NSCLC.
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P3.02 - Biology/Pathology (ID 620)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 10/18/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P3.02-089 - Establishment of Highly Metastatic Lung Cancer Cell Sublines in Long-term Three-dimensional Low Attachment Cultures (ID 8135)
09:30 - 09:30 | Author(s): Daisuke Matsubara
- Abstract
Background:
Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. We investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential and examined the mechanisms underlying metastasis.
Method:
Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. A whole genomic analysis was performed to identify key molecular alterations in FL sublines.
Result:
Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3-4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo (Fig.). A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells.
Conclusion:
Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.
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P3.02-093 - Knockdown of BRM Causes Epithelial-Mesenchymal Transition in Lung Adenocarcinoma Cell Line H1975 (ID 8955)
09:30 - 09:30 | Presenting Author(s): Daisuke Matsubara
- Abstract
Background:
Recent genomic studies have identified mutations in the multiple subunits of the chromatin remodeling complex in various cancers. Expression of BRG1/BRM, the core subunit of the SWI/SNF complex, is frequently altered in lung cancer, and our group has previous shown that loss of BRG1/BRM is associated with the EMT phenotype and poor prognosis in lung adenocarcinomas (Cancer Sci 2013).
Method:
In this study, we explored the effect of BRM knockdown (KD) with lentiviral sh-RNA in lung cancer cell line H1975.
Result:
BRM KD with lentiviral sh-RNA caused profound changes in H1975; original polygonal cells became spindled shaped, and when injected subcutaneously into SCID mouse, BRM-KD cells formed tumors composed of uniform spindle cells, mimicking sarcomatoid carcinomas. qRT-PCR and Western blot analyses confirmed downregulation of E-cadherin and upregulation of vimentin. Analysis of signaling molecules showed that EMT occurred independent of the EGFR, MET, or TGF-β/SMAD pathway. Among the master regulators of EMT, ZEB-1 was most remarkably upregulated, as compared to snail or slug, by BRM knockdown.
Conclusion:
Loss of BRM caused EMT, which was due to upregulation of ZEB1. Figure 1
