Cells In Pleural Effusion Research Articles

T Cell Receptor Repertoire Analysis Reveals Signatures of T Cell Responses to Human Mycobacterium tuberculosis.

Characterization of T cell receptor (TCR) repertoires is essential for understanding the mechanisms of Mycobacterium tuberculosis (Mtb) infection involving T cell adaptive immunity. The characteristics of TCR sequences and distinctive signatures of T cell subsets in tuberculous patients are still unclear. By combining single-cell TCR sequencing (sc-TCR seq) with single-cell RNA sequencing (sc-RNA seq) and flow cytometry to characterize T cells in tuberculous pleural effusions (TPEs), we identified 41,718 CD3+ T cells in TPEs and paired blood samples, including 30,515 CD4+ T cells and 11,203 CD8+ T cells. Compared with controls, no differences in length and profile of length distribution were observed in complementarity determining region 3 (CDR3) in both CD4+ and CD8+ T cells in TPE. Altered hydrophobicity was demonstrated in CDR3 in CD8+ T cells and a significant imbalance in the TCR usage pattern of T cells with preferential expression of TRBV4-1 in TPE. A significant increase in clonality was observed in TCR repertoires in CD4+ T cells, but not in CD8+ T cells, although both enriched CD4+ and CD8+ T cells showed TH1 and cytotoxic signatures. Furthermore, we identified a new subset of polyfunctional CD4+ T cells with CD1-restricted, TH1, and cytotoxic characteristics, and this subset might provide protective immunity against Mtb.

High-Throughput DNA Tensioner Platform for Interrogating Mechanical Heterogeneity of Single Living Cells.

Cell mechanical forces play fundamental roles in regulating cellular responses to environmental stimulations. The shortcomings of conventional methods, including force resolution and cellular throughput, make them less accessible to mechanical heterogeneity at the single-cell level. Here, a DNA tensioner platform is introduced with high throughput (>10 000 cells per chip) and pN-level resolution. A microfluidic-based cell array is trapped on "hairpin-structured" DNA tensioners that enable transformation of the mechanical information of living cells into fluorescence signals. By using the platform, one can identify enhanced mechanical forces of drug-resistant cells as compared to their drug-sensitive counterparts, and mechanical differences between metastatic tumor cells in pleural effusion and nonmetastatic histiocytes. Further genetic analysis traces two genes, VEGFA and MINK1, that may play deterministic roles in regulating mechanical heterogeneities. In view of the ubiquity of cells' mechanical forces in the extracellular microenvironment (ECM), this platform shows wide potential to establish links of cellular mechanical heterogeneity to genetic heterogeneity.

Single-cell immune profiling reveals functional diversity of T cells in tuberculous pleural effusion.

Orchestration of an effective T lymphocyte response at infection sites is critical for protection against Mycobacterium tuberculosis (Mtb) infection. However, the local T cell immunity landscape in human tuberculosis is poorly defined. Tuberculous pleural effusion (TPE), caused by Mtb, is characterized by an influx of leukocytes to the pleural space, providing a platform suitable for delineating complex tissue responses to Mtb infection. Using single-cell transcriptomics and T cell receptor sequencing, we analyzed mononuclear cell populations in paired pleural fluid and peripheral blood of TPE patients. While all major cell clusters were present in both tissues, their relative proportions varied significantly by anatomic location. Lineage tracking analysis revealed subsets of CD8 and CD4 T cell populations with distinct effector functions specifically expanded at pleural sites. Granzyme K-expressing CD8 T cells were preferentially enriched and clonally expanded in pleural fluid from TPE, suggesting that they are involved in the pathogenesis of the disease. The findings collectively reveal the landscape of local T cell immunity in tuberculosis.

Total Protein-Chloride Ratio in Pleural Fluid Independently Predicts Overall Survival in Malignant Pleural Effusion at the First Diagnosis.

ObjectivePre-treatment biomarkers to estimate overall survival (OS) for malignant pleural effusion (MPE) are unidentified, especially those in pleural fluid. We evaluated the relationship between OS and total protein–chloride ratio in malignant pleural effusion (PE TPClR).Materials and MethodsA retrospective study was undertaken to identify patients from 2006 to 2018 who had pathologically or cytologically confirmed MPE and received no tumor-targeted therapy. We recorded the pre-treatment clinicopathologic characteristics and follow-up status. OS was estimated by the Kaplan–Meier method, and the association between variables and OS was evaluated by Cox proportional hazards models.ResultsWe screened 214 patients who met the eligibility criteria. The optimal cutoff value for the PE TPClR was set at 0.53. The univariate analysis showed that there was a significant correlation between PE TPClR and OS (P < 0.001). The multivariate analysis between OS and the variables selected from the univariate analysis showed that the levels of neutrophil, alkaline phosphatase, neuron-specific enolase, platelets, albumin in peripheral blood, and white blood cells in pleural effusion were also independent predictors of OS.ConclusionIn patients with MPE, pre-treatment PE TPClR independently predicts OS. Although further research is necessary to generalize our results, this information will help clinicians and patients to determine the most appropriate treatment for MPE patients.

High-throughput concentration of rare malignant tumor cells from large-volume effusions by multistage inertial microfluidics.

On-chip concentration of rare malignant tumor cells (MTCs) in malignant pleural effusions (MPEs) with a large volume is challenging. Previous microfluidic concentrators suffer from a low concentration factor (CF) and a limited processing throughput. This study describes a low-cost multiplexed microfluidic concentrator that can enable high-throughput (up to 16 mL min-1) and high CF (over 40-fold for single run) concentration of rare cells from large-volume biofluids (up to hundreds of milliliters). The multiplexed device was fabricated using inexpensive polymer-film materials using a quick non-clean-room process within 30 min. The multiplexing and flow distribution approaches applied in the device achieved high-throughput processing. By adopting serial cascading, an ultrahigh CF of approximately 1400 was achieved. Moreover, the microfluidic concentrator was successfully applied for the concentration and purification of rare MTCs within MPEs collected from patients with advanced metastatic lung and breast cancers. The provision of concentrated samples with low background cells could improve the sensitivity of cytology and thus reduce the time required for cytological examination. This novel concentrator offers the distinct advantages of a remarkable CF, high throughput, low device cost, and label-free processing and can therefore be readily integrated with other on-chip cell sorters to enhance the identification of MPEs.

Immunohistochemical features of canine ovarian papillary adenocarcinoma and utility of cell block technique for detecting neoplastic cells in body cavity effusions.

Dogs with ovarian papillary adenocarcinoma occasionally present with ascites and/or pleural effusion. These aspirated fluids often contain a large number of cells, and distinction between neoplastic cells and activated mesothelial cells can be difficult. In this study, 7 cases of canine ovarian papillary adenocarcinoma, including 3 with ascites and pleural effusion, were immunohistochemically examined. Ovarian tumor cells were positive for cytokeratin CAM5.2 (CAM5.2), Wilms’ tumor 1 (WT-1) and progesterone receptor (PR) in all 7 cases. A metastatic lesion of the mediastinum in one case was also positive for CAM5.2, WT-1 and PR. Immunohistochemistry on cell blocks obtained from ascites and/or pleural effusion of 2 cases revealed the presence of PR-positive epithelial cells. Whereas, activated mesothelial cells in ascites or pleural effusion collected from dogs without neoplastic lesions were negative for PR. In addition, surface epithelium and subsurface epithelial structures (SES) of normal canine ovaries, that are considered to be the cell of origin for ovarian papillary adenocarcinoma, were also positive for CAM5.2, WT-1 and PR. These results indicate that, together with CAM5.2, WT-1 and PR is a useful diagnostic marker for canine ovarian papillary adenocarcinoma. Expression of PR may be associated with progesterone-dependent nature of canine ovarian papillary adenocarcinoma.

The Relevance of Ultrastructural Studies of Metastatic Cells from Women with Breast Cancer History.

The interaction between cancer cells and the surrounding microenvironment is determinant for metastasis success. In this study, the ultrastructural relevance of cells in the malignant pleural effusion (MPE) of women with breast cancer history was investigated. In MPE, it is possible to observe single cells and clusters. Women whose MPE presents carcinomas in aggregates have a better prognosis when compared to cases in which metastatic single cells are found. Samples were collected via fine-needle aspiration puncture (US-FNA). Subsequent to the material preparation and ultrathin cuts, they were observed using light and transmission electron microscopy (LM/TEM). LM and TEM images served as a basis for the creation of a digital sculpture using ZBrush® software. Clusters exhibited structural stability, en route vesicles allowing exocytosis of electron-dense fibrous elements, and cytoplasmic protrusions contributing to migratory and invasive skills. Single cells presented different necrotic phenotypes and many displayed leukocyte-like characteristics. Cluster cooperative relationships seem to be related to a long-term permanence in MPE. The absence of a collaborative network presumably triggers a more aggressive behavior of single cells. Its putative fusion with leukocytes can maximize the efficiency for transendothelial migration, increasing chances of metastatic success and, unfortunately, reducing survival of women with recidivism.

P8‐14: Development of an optimal protocol for molecular profiling of tumor cells in malignant pleural effusions at single‐cell level

P8‐14: Development of an optimal protocol for molecular profiling of tumor cells in malignant pleural effusions at single‐cell level

P68.05 Human Lung Carcinoma Primary Cultures From Malignant Pleural Effusions as a Tool for Drug Screening and Personalized Therapy

In recent years, new therapies have increased the number of treatment options available to the oncologist. However, the type and duration of response is still unpredictable in a significant number of patients and drug testing of patient-derived tumor cells can have clinical utility in this setting. A significant percentage of lung cancer (LC) patients show pleural effusion and viable malignant cells can be found in this fluid. Here, we present the results of the implementation of routine primary culture and drug testing from pleural effusions.

Anaphylatoxins orchestrate Th17 response via interactions between CD16+ monocytes and pleural mesothelial cells in tuberculous pleural effusion.

The complement system is activated in tuberculous pleural effusion (TPE), with increased levels of the anaphylatoxins stimulating pleural mesothelial cells (PMCs) to secrete chemokines, which recruit nonclassical monocytes to the pleural cavity. The differentiation and recruitment of naive CD4+ T cells are induced by pleural cytokines and PMC-produced chemokines in TPE. However, it is unclear whether anaphylatoxins orchestrate CD4+ T cell response via interactions between PMCs and monocytes in TPE. In this study, CD16+ and CD16- monocytes isolated from TPE patients were cocultured with PMCs pretreated with anaphylatoxins. After removing the PMCs, the conditioned monocytes were cocultured with CD4+ T cells. The levels of the cytokines were measured in PMCs and monocyte subsets treated separately with anaphylatoxins. The costimulatory molecules were assessed in conditioned monocyte subsets. Furthermore, CD4+ T cell response was evaluated in different coculture systems. The results indicated that anaphylatoxins induced PMCs and CD16+ monocytes to secrete abundant cytokines capable of only inducing Th17 expansion, but Th1 was feeble. In addition, costimulatory molecules were more highly expressed in CD16+ than in CD16− monocytes isolated from TPE. The interactions between monocytes and PMCs enhanced the ability of PMCs and monocytes to produce cytokines and that of monocytes to express HLA-DR, CD40, CD80 and CD86, which synergistically induced Th17 expansion. In the above process, anaphylatoxins enhanced the interactions between monocytes and PMCs by increasing the level of the cytokines IL-1β, IL-6, IL-23 and upregulating the phenotype of CD40 and CD80 in CD16+ monocytes. Collectively, these data indicate that anaphylatoxins play a central role in orchestrating Th17 response mainly via interactions between CD16+ monocytes and PMCs in TPE.

Pleural Fluid GSDMD Is a Novel Biomarker for the Early Differential Diagnosis of Pleural Effusion.

ObjectiveGasdermin D (GSDMD), controlling pyroptosis in cells, has multiple physiological functions. The diagnostic role of GSDMD in pleural effusion (PE) remains unknown.MethodsSandwich ELISA kits that we developed were applied to measure the level of GSDMD for 335 patients with a definite cause of PE, including transudative PE, tuberculous pleural effusion (TPE), parapneumonic pleural effusion (PPE), and malignant pleural effusion (MPE). The diagnostic accuracy of Light’s criteria vs. the new marker GSDMD was performed. Clinical follow-up of 40 cases of PPE was conducted and divided into efficacy and non-efficacy groups according to the therapeutic outcome. Nucleated cells (NCs) in PE were isolated and further infected with bacteria to verify the cell source of GSDMD.ResultsThe diagnostic accuracy of GSDMD for the diagnosis of PE were 96% (sensitivity) and 94% (specificity). The receiver operating characteristic (ROC) curve indicated that GSDMD can be an efficient biomarker for the differential diagnosis of transudative PE and other groups (all AUC > 0.973). Noteworthily, the highest AUC belonged to tuberculosis diagnosis of 0.990, and the cut-off value was 18.40 ng/mL. Moreover, the same cut-off value of PPE and MPE was 9.35 ng/mL. The combination of GSDMD, adenosine deaminase (ADA), and lactate dehydrogenase (LDH) will further improve the diagnostic efficiency especially between TPE and PPE (AUC = 0.968). The AUC of GSDMD change at day 4, which could predict the therapeutic effect at an early stage, was 0.945 (P < 0.0001). Interestingly, bacterial infection experiments further confirm that the pleural fluid GSDMD was expressed and secreted mainly by the NCs.ConclusionGSDMD and its combination are candidates as a potentially novel biomarker not only to separate PEs early and effectively, but also monitor disease progression.

“CELL BLOCK AS A BETTER DIAGNOSTIC TOOL IN DIAGNOSIS OF LUNG CANCER FROM EXUDATIVE PLEURAL FLUID”

Introduction: The cytological examination of serous effusions helps in diagnosis, staging, management and prognostication of patients with various malignancies. But cytological method has disadvantage of lower sensitivity in differentiating reactive atypical mesothelial cells from malignant cells. Aim: The aim of this study is to compare the cytomorphology of pleural exudative uids by conventional smear (CS) method and cell block (CB) method and also to assess the utility of their combined approach. Furthermore, immunohistochemistry is used in differentiating the benign from malignant tumour cells in exudative pleural effusions. Materials and Methods: It is a cross sectional study. 38 pleural exudative uid samples were subjected to evaluation by both CS and CB methods over a period of 1 year. Cellularity, architecture patterns, morphological features and yield for malignancy were compared. Chi-square and P-value were calculated, p value &lt;0.05 considered as signicant relationship. Results: CB method provided higher cellularity, better architectural patterns and additional yield for malignancy as compared to CS method. pvalue found to be &lt;0.0318,and it is statistically signicant. Conclusion: The present study shows that it is advisable to routinely make CBs in every cases that were suspicious for malignancy by conventional smear examination method. Use of immunohistochemistry further aided in diagnosing and differentiating the benign cells from malignant cells in exudative pleural effusions.

Relative Expression of Voltage‐Gated Sodium Channels in Cancerous and Noncancerous Cells during the Cell Cycle

Upregulation of voltage gated sodium channels (VGSCs) is the target for many novel carcinoma therapies. The majority carcinomas over-express voltage gated sodium channels, including breast cancers, lung cancers and mesotheliomas. We found variability in VGSC expression in clonal cancer cell lines. There is some limited electrophysiologic evidence that there is an upregulation of VGSCs during cell division. We hypothesized that the doubling of DNA during the cell cycle would also double the cell surface expression of VGSC protein compared to nondividing cells. We tested this hypothesis using flow cytometry with the fluorescent DNA label, propidium iodide and a conjugated panspecific-VGSC antibody. For lung cells, we compared A549 lung cancer cells with MRC5 control cell line and KLN 205 mouse cancer line. We compared H28 mesothelioma cells with MeT-5a normal pleural effusion cells and AB1 mouse mesothelioma cells. For breast cancer, we compared MDA-MB 231 breast cancer cells, MCF 10a control cells, and 4T1 mouse breast cancer cells. We found that, amoungst all the cell lines, there is between 1.86 and 2.15 more VGSCs expressed in dividing cells than in nondividing cells. Additionally, in cells imaged with confoccal microscopy, VGSCs were also shown in abundance at the leading ends of cells in anaphase. With this information we can more effectively target novel cancer therapies and thus improve the efficacy in carcinomas that overexpress VGSCs.

Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69−CD8+ T cells expressed higher levels of CD45RO than CD103−CD69+CD8+ T cells did; CD103+CD69−CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103−CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69− memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-β extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single-cell level.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single‐cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic‐activated cell sorting system, and CD45‐negative and cytokeratin‐positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole‐genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4‐ALK fusion (17/20 cells, 85%) with an alectinib‐resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA‐associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single‐cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA‐associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.

Novel Patient Metastatic Pleural Effusion-Derived Xenograft Model of Renal Medullary Carcinoma Demonstrates Therapeutic Efficacy of Sunitinib.

BackgroundRenal medullary carcinoma (RMC) is a rare but aggressive tumor often complicated by early lung metastasis with few treatment options and very poor outcomes. There are currently no verified RMC patient-derived xenograft (PDX) mouse models established from metastatic pleural effusion (PE) available to study RMC and evaluate new therapeutic options.MethodsRenal tumor tissue and malignant PE cells from an RMC patient were successfully engrafted into 20 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. We evaluated the histopathological similarity of the renal tumor and PE PDXs with the original patient renal tumor and PE, respectively. We then evaluated the molecular integrity of the renal tumor PDXs between passages, as well as the PE PDX compared to two generations of renal tumor PDXs, by microarray analysis. The therapeutic efficacy of sunitinib and temsirolimus was tested in a serially-transplanted generation of 27 PE PDX mice.ResultsThe pathologic characteristics of the patient renal tumor and patient PE were retained in the PDXs. Gene expression profiling revealed high concordance between the two generations of renal tumor PDXs (RMC-P0 vs. RMC-P1, r=0.865), as well as between the first generation PE PDX and each generation of the renal tumor PDX (PE-P0 vs. RMC-P0, r=0.919 and PE-P0 vs. RMC-P1, r=0.843). A low number (626) of differentially-expressed genes (DEGs) was seen between the first generation PE PDX and the first generation renal tumor PDX. In the PE-P1 xenograft, sunitinib significantly reduced tumor growth (p<0.001) and prolonged survival (p=0.004) compared to the vehicle control.ConclusionsA metastatic PE-derived RMC PDX model was established and shown to maintain histologic features of the patient cancer. Molecular integrity of the PDX models was well maintained between renal tumor and PE PDX as well as between two successive renal tumor PDX generations. Using the PE PDX model, sunitinib demonstrated therapeutic efficacy for RMC. This model can serve as a foundation for future mechanistic and therapeutic studies for primary and metastatic RMC.

ES07.02 Role of Pleural Fluid Molecular Markers and PD-L1 Testing

Patients with pleural metastases or lymphatic metastases in 50-60% develop pleural effusions (PE). Conventional cytology is negative (CNPE) in up to 40% of malignant PEs. The cells in PE are in a different microenvironment, affecting biology and biomarkers' expression considering the impact of tumor-host cell interactions. Obtaining PE, an early diagnostic step provides important biomarkers; when other tissue samples might not be available. PE represents tumor heterogeneity; tumor cells in pleural effusions might represent more lesions than biopsies. There are studies involving genomic, transcriptomic, proteomic, and metabolomics. Circulating cell-free DNA from PF supernatants may provide relevant information to clinicians that might complement or replace invasive diagnostic procedures. Cytology negative and positive PE is useful in early decision-making and when no additional time is available for later diagnostics steps due to disease aggressiveness. Biomarkers show high specificity, and the low sensitivity limits the diagnostic value that might increase with the PE amount available. Molecular testing on PE specimens is becoming a feasible alternative to tissue biopsy for phenotyping malignant PEs in lung cancer patients. PE might be suitable for assessing PD-L1 expression in malignant cells, and we compare the results of those reported for histological specimens.

Clinical value of the detection of EGFR gene mutations in pleural effusion cell blocks among patients with non-small-cell lung cancer.

To evaluate the clinical value of epidermal growth factor receptor (EGFR) detection in pleural effusion cell blocks among patients with non-small-cell lung cancer (NSCLC). From July 2016 to September 2018, EGFR gene mutations in 40 lung tumor tissue samples and pleural fluid samples from NSCLC patients in Jinhua Municipal Central Hospital were assessed by the amplification refractory mutation system method. The EGFR results of the two types of samples were compared using the paired Chi-square test, and the mutation positive rates in EGFR exons 18, 19, 20 and 21 were compared between the two types of specimens using the four-grid Chi-square test. Among the 40 tissue samples and pleural effusion samples, 21 and 18 cases of EGFR mutations were detected, respectively, and the mutation positive rates were 52.5% and 45%, respectively. The κ value of the consistency test of the two specimens was 0.851. There were no significant differences in the mutation positive rates in EGFR exons 18, 19, 20, and 21 between the two types of specimens. The EGFR results of pleural fluid and tissue samples were in good agreement. Therefore, we can use pleural fluid samples to detect EGFR mutations to guide tyrosine kinase inhibitor treatment for NSCLC patients in whom tumor tissue samples cannot be obtained.

In vivo and in vitro cell-based model of lung adenocarcinoma from patients with pleural effusion.

Lung adenocarcinoma (LAC) is a common and aggressive form of lung cancer that is increasing in incidence among never smokers at a younger age. Current treatment of patients with LAC is insufficient and there is a need for identification of effective biomarkers and development of therapeutic targets. These demands require also improved models for in vivo and in vitro experimentation. In this study, we describe the establishment of two LAC cell lines, named LuCa-3 and LuCa-6. Both were derived from pleural effusion (PE) cells of LAC patients (L3 and L6) and readily propagated as tumor xenografts in immunodeficient mice. PE cells from the patient L6 exhibited also the capacity for in vitro growth and were cultured in two forms: (i) as a suspension growing cell population, labeled LuCa-6S, composed of non-clumping single cells; and (ii) as a monolayer-like culture, labeled LuCa-6A, exhibiting tight cell-to-cell and to culture surface adherence. Unique features of these two sublines and their cell clones are the capacity to convert from a non-clumping single-cell suspension into the adherent growth pattern and vice versa. Immunostaining of patients' tumor tissue xenografts and cultured subline cells displayed markers specific for the phenotype of human LAC. LuCa-6S and LuCa-6A cells did not reveal a noticeable disparity in quantitative growth characteristics. However, a number of differences were detected between these two cell populations manifested in detection or intensities of antigen expressions on the cell surface (CD133, SFTPC) and in the nucleus (TTF-1) including pluripotent (OCT-4, SOX-2, NANOG) genes in cancer stem-like cells (CSCs). Dissimilarities between these two sublines were also detected in N-glycan profiles and in the sensitivity to natural killer cells. Salient features of these subline cell populations are responsiveness to selective upregulation of the pluripotent genes in subsets of CSCs via conversion of their growth patterns and/or by using culture stem media with growth factors. The described in vivo/in vitro model enables broader experimental approaches in studies of lung adenocarcinoma.

Molecular Mechanisms of Resistance to Osimertinib from Liquid Biopsies of Plasma and Pleura Effusion in EGFR Mutant NSCLC Patients

In the past decade, tyrosine kinase inhibitors (TKI) have been developed and widely-approved by countries to treat advanced lung cancer patients with sensitizing epidermal growth factor (EGFR) mutations, and substantially improved patients’ prognosis compared to traditional chemotherapies. However, a large proportion of patients who initially experienced ostensibly remarkable therapeutic outcomes still developed inevitable drug resistance eventually, including those received newly developed third-generation TKIs. Previous studies have demonstrated the value of plasma cell-free DNA (cfDNA) in monitoring patients’ response to therapies in lung cancer. However due to several physiological reasons and DNA degradation, DNA released from tumor cell apoptosis in lung was hard to maintain at a high concentration and good quality before being collected from venous blood, leading to the loss of pivotal mutational information. Pleural effusion is a potential substitute to overcome these problems. However, limited studies have been reported to characterize the consistency of genetic alterations found in these two sample types, and due to the modest sample sizes, the mechanisms underlying third-generation TKI resistance remains elusive. Herein we drew 8ml venous blood and collected 20ml pleural effusion by pleural effusion drainage from 8 lung cancer patients, and performed hybrid-capture based next generation sequencing assays on 18 matched plasma cfDNA, pleural effusion cells genomic DNA and pleural effusion supernatant cfDNA samples, we have shown that only sequencing either plasma or pleural effusion sample resulted in the missing of mutations in key cancer drivers. Sequencing both pleural effusion and plasma yielded a more concrete genetic landscape for monitoring tumor response. We have also exhibited that somatic mutations and copy number changes in PI3K/AKT pathway, NOTCH signaling pathway, hedgehog signaling pathway and pathways involved in DNA damage repair might be potential mechanisms underlying TKI resistance, and might serve as candidate targets for developing novel therapies to circumvent patients’ resistance.