Tumor Cells In Pleural Effusion Research Articles

Arsenic Inhibits Proliferation and Induces Autophagy of Tumor Cells in Pleural Effusion of Patients with Non-Small Cell Lung Cancer Expressing EGFR with or without Mutations via PI3K/AKT/mTOR Pathway.

To clarify whether arsenic could exert inhibitory effects on tumor cells in pleural effusions of patients with non-small cell lung cancer (NSCLC), 36 NSCLC pleural effusion samples were collected from Changzheng Hospital and Ruijin Hospital, from 2019 to 2022. The genotype of epidermal growth factor receptor (EGFR) was identified. Tumor cells were isolated and treated with arsenic trioxide (ATO) or/and gefitinib. Additionally, six patients were intrapleurally administrated with ATO. Results showed that 25 samples bore EGFR wild type (WT) and 11 harbored EGFR mutations, including 6 with L858R, 3 with ΔE746-A750, and 2 with T790M. ATO diminished the number of tumor cells from patients with WT and mutant EGFR, down-regulated the expression or phosphorylation of EGFR, pmTOR, PI3K, PTEN, and p4E-BP1, and up-regulated the expression of LC3. Immunofluorescent experiments showed that ATO enhanced LC3 and P62. By contrast, gefitinib was only effective in those harboring EGFR sensitizing mutations. Notably, in patients with intrapleural ATO injection, the pleural effusion underwent a bloody to pale yellow color change, the volume of the pleural effusion was reduced, and the number of the tumor cells was significantly reduced. In conclusion, arsenic is effective against NSCLC with various EGFR genotypes in vitro and in vivo, and potentially circumvents gefitinib resistance.

A Microfluidic System for Detecting Tumor Cells Based on Biomarker Hexaminolevulinate (HAL): Applications in Pleural Effusion

Malignant pleural effusion is a common clinical problem, which often occurs in cases of malignant tumors, especially in lung cancer. In this paper, a pleural effusion detection system based on a microfluidic chip, combined with specific tumor biomarker, hexaminolevulinate (HAL), used to concentrate and identify tumor cells in pleural effusion was reported. The lung adenocarcinoma cell line A549 and mesothelial cell line Met-5A were cultured as the tumor cells and non-tumor cells, respectively. The optimum enrichment effect was achieved in the microfluidic chip when the flow rates of cell suspension and phosphate-buffered saline achieved 2 mL/h and 4 mL/h, respectively. At the optimal flow rate, the proportion of A549 increased from 28.04% to 70.01% due to the concentration effect of the chip, indicating that tumor cells could be enriched by a factor of 2.5 times. In addition, HAL staining results revealed that HAL can be used to identify tumor cells and non-tumor cells in chip and clinical samples. Additionally, the tumor cells obtained from the patients diagnosed with lung cancer were confirmed to be captured in the microfluidic chip, proving the validity of the microfluidic detection system. This study preliminarily demonstrates the microfluidic system is a promising method with which to assist clinical detection in pleural effusion.

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.

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.

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.

Liquid biopsy-based single-cell metabolic phenotyping of lung cancer patients for informative diagnostics

Accurate prediction of chemo- or targeted therapy responses for patients with similar driver oncogenes through a simple and least-invasive assay represents an unmet need in the clinical diagnosis of non-small cell lung cancer. Using a single-cell on-chip metabolic cytometry and fluorescent metabolic probes, we show metabolic phenotyping on the rare disseminated tumor cells in pleural effusions across a panel of 32 lung adenocarcinoma patients. Our results reveal extensive metabolic heterogeneity of tumor cells that differentially engage in glycolysis and mitochondrial oxidation. The cell number ratio of the two metabolic phenotypes is found to be predictive for patient therapy response, physiological performance, and survival. Transcriptome analysis reveals that the glycolytic phenotype is associated with mesenchymal-like cell state with elevated expression of the resistant-leading receptor tyrosine kinase AXL and immune checkpoint ligands. Drug targeting AXL induces a significant cell killing in the glycolytic cells without affecting the cells with active mitochondrial oxidation.

Implementation of functional precision medicine for anaplastic lymphoma kinase-rearranged non-small lung cancer

Anaplastic lymphoma kinase (ALK)-rearranged kinase drives non-small cell lung cancer (NSCLC) in 3–7% of patients and treatment consists of a broad range of approved inhibitors which are administered sequentially in case of resistance developing invariably after approximately 1 year of therapy. Besides the former standard tyrosine kinase inhibitor (TKI) crizotinib, the second-line ALK inhibitors alectinib, ceritinib, brigatinib, as well as the third-line lorlatinib are approved for the treatment of ALK-positive NSCLC patients. The main challenge is to find individual schemes of ALK inhibitors therapy which provide the best benefit for the patients. A host of ALK fusion partners, rearrangement variants and ALK mutations prevent a direct correlation of ALK alterations and sensitivity to specific TKIs within the framework of genomic precision medicine. However, recurrent ALK-positive NSCLC is an aggressive disease resulting in accumulation of tumor cells in pleural effusions which may be collected for in vitro sensitivity testing in a manner designated as functional precision medicine. Provided that these cells are present in sufficient numbers, they can be exposed to all possible drug candidates and their chemosensitivity tested in short-term proliferation assays. In addition to ALK-directed TKIs, cytotoxic drugs and inhibitors of non-target bypassing pathways may be included in the tests. In contrast to other trials in genome-guided precision medicine, a range of suitable therapeutics is available for modified ALK proteins. In summary, ALK-positive NSCLC with pleural effusions offer a unique model to develop, test and validate concepts of functional precision medicine.

Identification and single-cell sequencing analysis of rare tumor cells in malignant pleural effusion of lung cancer patients

Tumor heterogeneity refers to distinct genomic or phenotypic characteristics of tumor cells. Under the environmental or drug stress, tumor cells exhibit different responses, corresponding to different properties of cell proliferation, invasion, metastasis and drug resistance. In particular, a small fraction of tumor cells are capable of detaching from primary tumor sites and initiating distant metastases. Thus, tumor heterogeneity sets the basis for tumor resistance and metastasis. Traditional methods in studying tumor heterogeneity are mainly based on bulk cells from different locations in primary tumors, lacking analysis at the single-cell level and of metastatic tumor cells. This study establishes a single-cell method to study metastatic tumor cells in malignant pleural effusions of lung cancer patients. Metabolically active tumor cells in malignant pleural effusions are firstly identified with a metabolic marker 2-NBDG, a fluorescent glucose analog. These metabolically active tumor cells are confirmed to harbor the same driver oncogenic mutations by Sanger sequencing, followed by high-throughput sequencing to analyze copy number variation profiles. Our results show metastatic tumor cells in pleural effusion have the same driver mutations but different features in copy number variation patterns. The study provides new insights to understand the mechanism of tumor metastasis.

High-throughput screening of rare metabolically active tumor cells in pleural effusion and peripheral blood of lung cancer patients

Malignant pleural effusion (MPE), the presence of malignant cells in pleural fluid, is often the first sign of many cancers and occurs in patients with metastatic malignancies. Accurate detection of tumor cells in pleural fluid is crucial because the presence of MPE denotes an advanced stage of disease and directs a switch in clinical managements. Cytology, as a traditional diagnostic tool, has limited sensitivity especially when tumor cells are not abundant, and may be confounded by reactive mesothelial cells in the pleural fluid. We describe a highly sensitive approach for rapid detection of metabolically active tumor cells in MPE via exploiting the altered glucose metabolism of tumor cells relative to benign cells. Metabolically active tumor cells with high glucose uptake, as evaluated by a fluorescent glucose analog (2-NBDG), are identified by high-throughput fluorescence screening within a chip containing 200,000 addressable microwells and collected for malignancy confirmation via single-cell sequencing. We demonstrate the utility of this approach through analyzing MPE from a cohort of lung cancer patients. Most candidate tumor cells identified are confirmed to harbor the same driver oncogenes as their primary lesions. In some patients, emergence of secondary mutations that mediate acquired resistance to ongoing targeted therapies is also detected before resistance is manifested in the clinical imaging. The detection scheme can be extended to analyze peripheral blood samples. Our approach may serve as a valuable complement to cytology in MPE diagnosis, helping identify the driver oncogenes and resistance-leading mutations for targeted therapies.

An Epstein-Barr virus susceptible immature T-cell line, WILL4, established from a patient with T-lymphoblastic lymphoma bearing CD21 and a clonal EBV genome

We managed a patient with an Epstein-Barr virus-associated T-cell lymphoblastic lymphoma. Mediastinal tumor cells at initial admission were positive for CD4, CD8, and TdT. Interestingly, a lymph node at necropsy was compatible for a CD4-positive peripheral T-cell lymphoma without CD8 and TdT expression, suggesting a different phenotype from the mediastinal tumor. Tumor cells in pleural effusion continued to proliferate in in vitro and were designated as WILL4. WILL4 cells were positive for CD3, CD4, CD8, CD21, T-cell receptor (TcR) αβ, and TdT, indicating a similar phenotype to thymocytes. Southern blot analyses showed that the pleural tumor and WILL4 cells shared a TcR gene rearrangement, and that both contained a clonal EBV genome in an episomal form. RT-PCR showed that EBNA1 and LMP1 were expressed in the fresh tumor and WILL4 cells. Southern blot analyses revealed that WILL4 cells were susceptible to EBV infection in vitro using B95-8 supernatant. Anti-CD21 antibody inhibited in vitro infection of EBV, suggesting that CD21 plays a role in EBV infection into WILL4 cells. In vitro infection of EBV did not affect latent gene expression in WILL4 cells. WILL4 is a useful tool for analyzing the roles of EBV in onocogenesis in immature T-lymphoid malignancies.

Abstract 2941: Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells

Abstract Alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, has shown promising activity in ALK-rearranged non-small cell lung cancer (NSCLC). The mechanisms of drug resistance to alectinib have been still unclear. To explore whether overexpression of ATP-binding cassette (ABC) transporters might provide a potential mechanism of alectinib resistance in lung cancer, we established alectinib-resistant cell lines from NCI-H2228 and primary ALK-rearranged cells and evaluated the expression of ABC transporters. We established ALK-rearranged NSCLC cell lines (KTOR 1, 2, and 3) from tumor cells in pleural effusion obtained from 3 patients with alectinib naïve ALK-rearranged NSCLC. In the 2 of 3 patients, tumor cells in pleural effusion were also obtained when disease progression was observed during alectinib treatment (KTOR1-RE and KTOR2-RE). Expression profiling of ABC transporters using quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the expression of ABCB1 was significantly increased in KTOR1-RE and KTOR2-RE compared with the parental cells (4.6 and 10.8 folds), but not other ABC transporters. Then, alectinib-resistant NCI-H2228 cell lines (H2228-AR) were established by exposing the parental cells to stepwise-increasing alectinib up to 5μM for 10 months. We found that the ABCB1/MDR1 expression in H2228-AR cells were significantly increased (2.09-16.6 folds) compared with the parental cells using qRT-PCR and immunoblotting. Then, we examined the alternation of alectinib cytotoxicity by inhibition of ABCB1. Knockdown of ABCB1 expression using small interfering RNA and inhibition of ABCB1 by verapamil enhanced alectinib cytotoxicity in H2228-AR cells. Next, the levels of ABCB1 expression after short-term alectinib exposure were examined in NCI-H2228 and the three alectinib naïve ALK-rearranged NSCLC primary cell lines (KTOR 1, 2, and 3). In all the four ALK-rearranged cell lines, exposure to 100 nM alectinib for 72 hours induced increased expression of ABCB1 compared with control medium. These data suggested that increased expression of ABCB1/MDR1 might be, in part, responsible acquired resistance and sensitivity to alectinib in ALK-rearranged NSCLC cells. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Yuichi Sakamori, Takashi Nomizo, Yoshitaka Yagi, Hiroki Nagai, Young Hak Kim, Michiaki Mishima. Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells. [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 2941.

Precision immunotherapy; dynamics in the cellular profile of pleural effusions in malignant mesothelioma patients

ObjectivesClinical studies have proven the potential of immunotherapy in malignancies. To increase efficacy, a prerequisite is that treatment is tailored, so precision immune-oncology is the logical next step. In order to tailor treatment, characterization of the patient’s tumor environment is key. Pleural effusion (PE) often accompanies malignant pleural mesothelioma (MPM) and is an important part of the MPM environment. Furthermore, the composition of PE is used as surrogate for the tumor. In this study, we provide an insight in the dynamics of the MPM environment through characterization of PE composition over time and show that the immunological characteristics of PE do not necessarily mirror those of the tumor. Materials and methodsFrom 5 MPM patients, PE and tumor biopsies were acquired at the same time point. From one of these patients multiple PEs were obtained. PEs were acquired performing thoracocenteses and total cell amounts were determined. Immunohistochemistry was performed to quantify immune cell composition (T cells, macrophages) and tumor cells in PE derived cytospins and tumor biopsies. ResultsThe PE amount and (immune) cellular composition varied considerably over time between multiple (n=10) thoracocenteses. These dynamics could in part be attributed to the treatment regimen consisting of standard chemotherapy and dendritic cell (DC)-based immunotherapy. In addition, the presence of T cells and macrophages in PE did not necessarily mirror the infiltration of these immune cells within tumor biopsies in 4 out of 5 patients. ConclusionsIn this proof-of-concept study with limited sample size, we demonstrate that the composition of PE is dynamic and influenced by treatment. Furthermore, the immune cell composition of PE does not automatically reflect the properties of tumor tissue. This has major consequences when applying precision immunotherapy based on PE findings in patients. Furthermore, it implies a regulated trafficking of immune regulating cells within the tumor environment.

The negative enrichment by immunomagnetic beads for tumor cells from malignant pleural effusions

To establish a method (negative enrichment by immunomagnetic beads) for detection of tumor cells in pleural effusions and to evaluate the sensitivity and specificity of the method for clinical application. Five, 10, 20, 50 and 100 A549 (lung adenocarcinoma) cells were labeled with DAPI and added into 20 ml pleural effusions [containing (1 - 10)×10(6)cells] from heart failure patients, followed by immunomagnetic negative enrichment method. Recovered cancer cells were enumerated using a fluorescent microscope. Tumor cells were enriched from pleural effusion samples by means of density gradient centrifugation and negative enrichment by immunomagnetic beads method, followed by identification with cytology analysis (Wright's Giemsa's staining), immunofluorescence staining (IF) and fluorescence in situ hybridization (FISH) using centromere DNA probes of chromosome 7 and 8. Cytology, IF and FISH evaluations were performed in 53 pleural effusion samples, including 36 cases of malignant disease (25 male and 11 female patients aging 40 to 78 years, mean age (63 ± 9) and 17 cases of benign disease (8 male and 9 female patients aging 25 to 81 years, mean age (53 ± 18). After DAPI staining and mixing with pleural effusions from heart failure patients, the cell recovery rates of A549 cells evaluated under fluorescence microscope were 75%, 78%, 82%, 85%, 88%, and the average recovery rate was 81.6%. Using negative enrichment method and density gradient centrifugation combined with cytology analysis, the positive rates of tumor cells in 36 malignant pleural effusion samples were 81% (29/36) and 61% (22/36), respectively (χ(2) = 4.00, P = 0.039). Using negative enrichment method combined with IF, the positive rate of CK18(+), DAPI(+), CD(45)(-) cells was 100%. Moreover, using negative enrichment method combined with FISH analysis, the positive rate of tumor cells was 86% (31/36), much higher than that using density gradient centrifugation combined with cytology analysis (χ(2) = 5.818, P = 0.012). In 17 cases of benign pleural effusions, using negative enrichment method combined with IF, the positive rate was 100%. But other methods didn't find cancer cells from benign pleural effusions. It was applicable to enrich tumor cells from pleural effusions using negative enrichment method by immunomagnetic beads. This method combined with cytology analysis or FISH significantly enhanced the sensitivity and specificity of tumor cell detection in pleural effusions. But it was difficult to distinguish cancer cells from mesothelial cells using immunofluorescence staining with CK18, DAPI and CD(45) label. More specific markers were needed to recognize tumor cells from pleural effusions.

CSPG4 Protein as a New Target for the Antibody-Based Immunotherapy of Triple-Negative Breast Cancer

The cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)-based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb. CSPG4 protein expression was assessed in 44 primary TNBC lesions, in TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435, and SUM149, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. The effect of CSPG4-specific mAb 225.28 on growth, adhesion, and migration of TNBC cells was tested in vitro. The ability of mAb 225.28 to induce regression of tumor metastases (n = 7 mice) and to inhibit spontaneous metastasis and tumor recurrence (n = 12 mice per group) was tested in breast cancer models in mice. The mechanisms responsible for the antitumor effect of mAb 225.28 were also investigated in the cell lines and in the mouse models. All statistical tests were two-sided. CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. CSPG4-specific mAb 225.28 statistically significantly inhibited growth, adhesion, and migration of TNBC cells in vitro. mAb 225.28 induced 73.1% regression of tumor metastasis in a TNBC cell-derived experimental lung metastasis model (mAb 225.28 vs control, mean area of metastatic nodules = 44590.8 vs 165950.8 μm(2); difference of mean = 121360.0 μm(2), 95% confidence interval = 91010.7 to 151709.4 μm(2); P < .001). Additionally, mAb 225.28 statistically significantly reduced spontaneous lung metastases and tumor recurrences in an orthotopic xenograft mouse model. The mechanisms responsible for antitumor effect included increased apoptosis and reduced mitotic activity in tumor cells, decreased blood vessel density in the tumor microenvironment, and reduced activation of signaling pathways involved in cell survival, proliferation and metastasis. This study identified CSPG4 as a new target for TNBC. The antitumor activity of CSPG4-specific mAb was mediated by multiple mechanisms, including the inhibition of signaling pathways crucial for TNBC cell survival, proliferation, and metastasis.

Correlation between DNA Content, Expression of Ki-67 Antigen of Tumor Cells and Immunophenotype of Lymphocytes from Malignant Pleural Effusions

Malignant cells isolated from 26 malignant pleural effusions, collected from patients bearing metastatic carcinoma, were examined using flow cytometry. Expression of Ki-67 antigen, DNA ploidy, S phase fraction, DNA index of tumor cells and the percentage of aneuploid cell populations were determined. Lymphoid cells from the same malignant effusions and from 10 nonmalignant ones were examined using a panel of monoclonal antibodies (mAb) versus several CD antigens. While the percentages of T cells (CD3+, CD4+, CD8+) and B cells (CD19+) did not differ significantly in malignant versus nonmalignant effusions, those of CD56+ lymphocytes were found to be significantly depressed in malignant diploid and malignant aneuploid effusions as compared to benign ones. Expression of IL-2 receptors on lymphocytes, demonstrated by anti-CD25 mAb, was significantly higher in malignant diploid and malignant aneuploid effusions as compared with nonmalignant ones. An increase in the percentage of Ki-67-positive malignant cells was significantly correlated with the decrease of NK cells. S phase fraction, DNA index and the percentage of aneuploid populations did not correlate with the picture of lymphoid cells from pleural effusion. On the other hand, the percentage of Ki-67-positive tumor cells in pleural effusions was inversely proportional to the patients’ survival time, although it was not statistically significant. Our data suggest that the poor prognosis of cancer characterized by the presence of an aneuploid cell population of malignant cells and the high percentage of proliferating cells in pleural effusions can be linked to the depression of NK cells.

Binding capacities of two immunomodulatory lectins, carrier-immobilized glycoligands and steroid hormones in lung cancer and the concentration of nitrite/nitrate in pleural effusions.

Combined analysis of the binding properties of inflammatory and tumor cells in pleural effusion, and tumor imprints for various carrier-immobilized types of ligands and lectins, and of a biochemical feature of the effusions is performed to extend the characterization of these cells and their activity. In detail, the binding of Viscum album agglutinin (VAA), Urtica dioica agglutinin (UDA), and of carrier-immobilized N-acetyl-D-glucosamine (GlcNAc), lysoganglioside GM1, estradiol, progesterone, testosterone, and hydrocortisone to native specimens consisting of 46 tumor imprints from surgically treated patients with lung cancer and 74 smears of pleural effusion (PE) cells from cancer or non-cancer patients was studied using fluorescence microscopy with Texas red-labeled streptavidin. Among the tested ligands, VAA was found to provide the most effective staining of cells (60-78.1% of positive cases). When compared with inflammatory cells from PE, cancer cells were seen to bind more frequently only two ligands, namely UDA and estradiol. Significant (P < 0.001) difference between patients with bronchial carcinoma and non-cancer patients were found, when the content of NO2-/NO3- in PE fluids was measured. Whereas the level of NO2-/NO3- in PE of non-cancer patients was 12.6 +/- 10.7 microM (n = 12), it was 37.7 +/- 19.4 microM (n = 14) in cancer patients without pleural metastases and 37.5 +/- 16.0 microM (n = 26) in patients with pleural metastases. The level of NO2-/NO3- in PE appeared to correlate with extent of staining with GM1 and GlcNAc: in non-cancer patient groups it was significantly higher (P = 0.032) for negative subjects than those binding the ligand GlcNAc, whereas in the patient group with adenocarcinoma it was significantly lower (P = 0.032) for patients without binding capacities for GlcNAc and GM1. The results obtained suggest that the combined analysis of increased levels of NO2-/NO3- in PE and of glycohistochemical properties of cancer and inflammatory cells may be useful in exploring the interrelationship of functionally important cellular characteristics.

胸水中に出現し，化学療法により完全寛解したＫｉ‐１ ｌｙｍｐｈｏｍａの１例

胸水中に出現し，化学療法により完全寛解したＫｉ‐１ ｌｙｍｐｈｏｍａの１例

Monoclonal antibodies to human squamous cell carcinoma of the lung and their application to tumor diagnosis

Three immunoglobulin G1 monoclonal antibodies, LuCa2, LuCa3, and LuCa4, were produced by fusing murine myeloma NS1 cells with splenocytes obtained from a BALB/c mouse immunized with SK-MES1 cells derived from human squamous cell carcinoma of the lung. These three monoclonal antibodies were shown to recognize different protein antigens on SK-MES1 cells by indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. While the pattern of cell line distribution of antigens recognized by these antibodies was not tumor type specific, their reactivity with tissue and pleural effusion was much more informative than with cell lines. The presence of target antigens in vivo was analyzed by immunoperoxidase staining of frozen tissue sections and immunofluorescence staining of tumor cells in pleural effusions. LuCa2 antibody was reactive with lung squamous carcinoma and adenocarcinoma tumor tissues and pleural effusions, but only infrequently with those of small cell carcinoma. This antibody was also reactive with many tumor tissues from other organs as well as with various normal tissues, including alveoli and bronchus. LuCa3 and LuCa4 antibodies reacted with lung squamous carcinoma in tissues and pleural effusions, but not with lung adenocarcinoma nor with small cell carcinoma. These two antibodies reacted only weakly with normal squamous tissues of the esophagus, skin, and cervix uteri, but not with various other normal tissues. Moreover, LuCa3 had weak reactivity with squamous cell carcinoma tissue of tongue and esophagus, whereas LuCa4 had no reactivity with nonpulmonary tumor tissues. LuCa3 and LuCa4 antibodies should be of clinical interest, because our data suggest that these antibodies may be potentially useful for the diagnosis of the histological type of lung tumor cells in both cancer tissue and pleural effusions.