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F. Quaini
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ORAL 07 - Lung Cancer Pathogenesis (ID 91)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:J. Sage, E. Brambilla
- Coordinates: 9/07/2015, 10:45 - 12:15, 102+104+106
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ORAL07.05 - Differential Tumorigenic Properties of Mesenchymal Cells From Neoplastic and Non-Neoplastic Human Lung in NSCLC (ID 1006)
11:28 - 11:39 | Author(s): F. Quaini
- Abstract
- Presentation
Background:
Cancer Initiating Cells (CICs) and their niches may open new avenues in the pathogenesis and management of lung cancer. A relevant component of the niche is represented by supportive stromal cells that control the fate of CICs by a reciprocal cross-talk. The understanding of these cellular events could represent a significant advancement in cancer biology and treatment. Recent observations by our and other laboratories have suggested that mesenchymal stromal cells (MSC) regulate lung cancer growth and resistance, thus generating large expectations in novel anti-cancer strategies. The aim of our study was to determine whether MSC isolated from NSCLC and from non-neoplastic human lung samples possess different biologic properties and tumorigenic potential.
Methods:
Fresh samples of neoplastic and spared lungs from 58 male patients (80% smokers) affected by primary pulmonary adenocarcinoma undergoing surgical resection were processed. Stromal cells were separated from epithelial cells by negative selection using EpCAM (CD326)-based immunomagnetic sorting. After further enrichment, we could expand for at least 14 passages a population of CD90, CD105, CD73 and CD44 positive MSC from lung cancer (Lc-MSC) and non-neoplastic (Nn-MSC) lung tissue. The oncogenic potential of these cells from the same patient was tested on a Calu-3-based in vitro model of NSCLC by co-culture and conditioned media (CM) and in vivo by xenotransplantation in Balb/c Nude mice. In vivo cell tracking was achieved by pre-labeling MSC with Quantum dots 585 (Qdots). Morphometric assessment of tissue composition and immunofluorescence combined with FISH analysis of human X and Y chromosomes was performed on xenografted tumors.
Results:
Nearly 30x10[6] cells could be typically obtained after 3 passages in each case, however, compared to Nn-MSC, cultures of Lc-MSC displayed lower growth kinetic and mitotic index while higher survival and HIF-1-alpha (Hypoxia-inducible-factor-1) upregulation in response to hypoxia was observed. A larger fraction of Lc-MSC expressed transcription factors involved in stemness (Oct3/4, SOX2) and in bronchioalveolar (TTF1, ETS-1, CCL10) commitment. Co-cultures demonstrated that Lc-MSC significantly increased Calu-3 growth as compared to Nn-MSC in transwell assay and by contact. CM from Lc-MSC similarly promoted Calu-3 expansion as compared to Nn-MSC. When 2.5x10[6] Lc-MSC or Nn-MSC from the same patient were subcutaneously co-injected with Calu-3, a 38% and 17% increase in tumor volume was respectively observed, compared to the injection of an equal number of Calu-3 alone (CTRL). Lc-MSC or Nn-MSC injected alone did not generate tumors. Quantitative estimation of the in vivo expansion of neoplastic cells indicated that the addition of Lc-MSC increased by 6-fold and 29-fold Calu-3 replication compared to Nn-MSC and CTRL, respectively. Cell tracking documented that Qdots labelled MSC were located at the boundary of neoplastic epithelial glands generated by X-chromosome polysomic Calu-3 cells. A comparative molecular analysis of Lc-MSC and Nn-MSC is ongoing for the identification of distinctive signalling pathways implicated in the microenvironemental control of CIC on NSCLC development.
Conclusion:
Profound differences exist in the biology and oncogenic potential of intratumoral and normal lung MSC strongly supporting the notion that the tumor microenvironment may represent a potential target of new customized therapeutic strategies.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-059 - Structural and Functional Characterization of the Hemolymphangiogenic Microenvironment in Lung Cancer (ID 919)
09:30 - 09:30 | Author(s): F. Quaini
- Abstract
Background:
The hypothesis of the existence of a Lung Cancer Initiating Cell (LCIC) offers new pathogenetic and therapeutic options. CICs express vascular-related molecules in order to induce neoangiogenesis and establish an aberrant vascular niche. Conversely, tumor angiogenesis and formation of a cancer vascular niche contribute to the maintenance of CIC. The limited success of anti-angiogenic strategies in lung cancer imposes a better knowledge of the biology and architecture of the tumor vascular microenvironment. The aim of our study was to characterize the structural and functional changes of blood and lymphatic vasculature in human lung cancer.
Methods:
Fresh samples of the neoplastic (T) and spared distal (Dist) lung from 30 patients affected by NSCLC (21 Adeno and 9 Squamous) and 9 Neuroendocrine tumors were processed for immunohistochemical analysis and cell isolation. Control healthy lung (CTRL) was represented by 12 samples collected at autopsy from patients who died in the absence of respiratory diseases and 6 surgical specimens of pneumothorax. Immunofluorescence and confocal microscopy were employed using specific antibodies to detect blood (CD31, vWF, a-SMA) and lymphatic (Podoplanin Pdn, Lyve-1 and Prox-1) vessels. Moreover, the distribution of cells expressing stem/progenitor cell associated antigens (c-kit, CD133, CD34 and PDGFR) was assessed. Phenotypical and functional characterization on immunomagnetically sorted hematic (CD31[pos]) and lymphatic (CD31[pos]/Pdn[pos]) endothelial cells was performed on cells isolated from Dist and T lung samples.
Results:
Results indicated that, compared to CTRL capillary density increased by 77% and 74% in Dist and T portions of the lung, respectively. Conversely, the numerical incidence of venules did not show significant difference. These parameters were similarly represented in the three different tumor histotypes. A significant increase in arteriolar density was observed in all tumor types compared to CTRL. Moreover, the number of arterioles within T was increased by 3-fold compared to Dist portion. The quantitative analysis of lymphatic vessels showed similar values in all types of cancer specimens although rarefaction of these vascular structures was observed compared to CTRL. Moreover, lymphatic vessels density was 10-fold higher in Dist lung than in T. Immunofluorescence confocal analysis documented a positive gradient of c-kit[pos], CD34[pos] and PDGFR[pos] progenitor cells from Dist towards T in all cancer samples. However, compared to CTRL, cells expressing c-kit and CD34 were more numerous both in the Dist and T portion of the lung, while the increase in PDGFR[pos] cells was present only in T. Blood (BEC) and lymphatic (LEC) endothelial cells isolated from Dist and T samples of the lung, showed different growth properties and variable expression of Tumor Endothelial Marker (TEM) and receptor tyrosine kinases as VEGFR 2 and 3, PDGFRbeta, EGFR, IGF-1R, and c-met. Functional assays indicated that T derived LEC possess higher tube forming ability on matrigel than Dist LEC while this phenomenon was not observed in BEC. Moreover, wound healing assay showed a VEGF-C independent reduced migratory capacity of LEC isolated from T samples compared to the Dist counterpart.
Conclusion:
Specific changes in the composition and function of the tumor hemolymphangiogenic environment occur in lung cancer providing innovative pathogenetic and therapeutic approaches.
