Simultaneous, single cell, flow cytometric quantification of PD-L1/TILs/cell cycle in non-small cell lung cancer tissue biopsies: Concordance of PD-L1 expression detection between flow cytometry and immunohistochemistry.

159 Background: Tumor cell expression of PD-L1 leads to the inhibition of immune responses specifically inactivation of cytotoxic T-cells. PD-L1 expression on tumor cells by immunohistochemistry does not provide the complete picture of therapeutic (PD-L1) and prognostic (TILs, aneuploidy) markers. Here, we report a clinical assay that quantifies tumor infiltrating lymphocytes (TILs), cell cycle/DNA content as well as PD-L1 expression on tumor and aneuploid tumor cell populations. Methods: Punch biopsies were taken from 19 fresh tissues specimens and were processed into single cell suspensions using the IVD/CE-IVD IncellPREP Single Cell Preparation Kit. Cells were fixed and permeabilized in 1 mL IncellMax. Cells were tested with the OncoTect iO Lung Assay which contains antibodies directed against PD-L1 (clone 28-8), CD45, CD3, and CD8, and a cell cycle dye. Concordance to IHC was tested by the Dako PD-L1 IHC 28-8 PharmDx Kit. Immune cell populations were quantified and aneuploidy was determined using a DNA index > 1.05. Results: The number of CD8+ CTL were significantly increased (P = 0.02) in tumor compared to normal lung tissue (37.4% to 28.2%). The number of CTL in aneuploid tumors was twice the number of CTL in diploid tumors. The percentage of antigen presenting cells (CD45+, high SSC) was decreased in the tumor cell samples relative to the normal lung tissue (P = 0.01). PD-L1 expression varies in cells depending on the cell cycle. Conclusions: In this study, the concordance between Oncotect iO and IHC was 95% (NPA-97%, PPA-89%). PD-L1 expression varies with DNA content, PD-L1 expression decreases with increasing DNA content. Though CTL are increased in aneuploid tumors, PD-L1 expression decreasing with DNA content may contribute to the association of aneuploidy with distant metastases. PD-L1 expression is a powerful determinant of treatment and aneuploidy is a powerful prognostic factor that combined yield important clinical information.

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Phospholipid dynamics in ex vivo lung cancer and normal lung explants

Experimental & Molecular Medicine ◽

10.1038/s12276-020-00547-x ◽

2021 ◽

Author(s):

Julia Lesko ◽

Alexander Triebl ◽

Elvira Stacher-Priehse ◽

Nicole Fink-Neuböck ◽

Jörg Lindenmann ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Cells ◽

Lung Tissue ◽

De Novo ◽

Ex Vivo ◽

Normal Lung Tissue ◽

Normal Lung ◽

Cancer Tissue ◽

Glycerol Backbone ◽

Lung Cancer Tissue

AbstractIn cancer cells, metabolic pathways are reprogrammed to promote cell proliferation and growth. While the rewiring of central biosynthetic pathways is being extensively studied, the dynamics of phospholipids in cancer cells are still poorly understood. In our study, we sought to evaluate de novo biosynthesis of glycerophospholipids (GPLs) in ex vivo lung cancer explants and corresponding normal lung tissue from six patients by utilizing a stable isotopic labeling approach. Incorporation of fully 13C-labeled glucose into the backbone of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) was analyzed by liquid chromatography/mass spectrometry. Lung cancer tissue showed significantly elevated isotopic enrichment within the glycerol backbone of PE, normalized to its incorporation into PI, compared to that in normal lung tissue; however, the size of the PE pool normalized to the size of the PI pool was smaller in tumor tissue. These findings indicate enhanced PE turnover in lung cancer tissue. Elevated biosynthesis of PE in lung cancer tissue was supported by enhanced expression of the PE biosynthesis genes ETNK2 and EPT1 and decreased expression of the PC and PI biosynthesis genes CHPT1 and CDS2, respectively, in different subtypes of lung cancer in publicly available datasets. Our study demonstrates that incorporation of glucose-derived carbons into the glycerol backbone of GPLs can be monitored to study phospholipid dynamics in tumor explants and shows that PE turnover is elevated in lung cancer tissue compared to normal lung tissue.

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A Trial on the Overexpression of Epidermal Growth Factor Receptor and Proliferating Cell Nuclear Antigen in NSCLC of Similar Differentiation

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.881-883.410 ◽

2014 ◽

Vol 881-883 ◽

pp. 410-413

Author(s):

Feng Wu Lin ◽

Chuan Zhang ◽

Kun Peng Cheng ◽

Qiang Zhang ◽

Yan Zhao

Keyword(s):

Lung Cancer ◽

Lymph Node ◽

Lung Tissue ◽

Normal Lung Tissue ◽

Nuclear Antigen ◽

Normal Lung ◽

Cancer Tissue ◽

Metastatic Tissue ◽

Significant Difference ◽

Lung Adenocarcinomas

Objective: To explore the expression of EGFR and PCNA in lung squamous cell carcinomas and lung adenocarcinomas, and the relationships between their overexpression and cancer tissue typing or lymphatic metastasis. Method: Detect the expression of EGFR and PCNA in 44 cases of similarly differentiated cancer tissue and 16 cases of normal lung tissue by immunohistochemistry. Result: The expression of EGFR and PCNA in lung cancer tissue was significantly higher than that in normal lung tissue. Meanwhile, no significant difference existed between expression of PCNA and EGFR in lung squamous carcinomas and that in lung adenocarcinomas (P>0.05), nor between lymph node metastatic tissue and negative lymph node metastatic tissue (P>0.05). Conclusion: EGFR and PCNA were involved in the onset and development of lung cancer, and had some correlation with lung cancer. No significant correlations were observed between EGFR or PCNA overexpression and lymph node metastasis.

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Cellular Localization of Enzymatically Active Thrombin in Intact Human Tissues by Hirudin Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653870 ◽

1995 ◽

Vol 73 (05) ◽

pp. 793-797 ◽

Cited By ~ 60

Author(s):

Leo R Zacharski ◽

Vincent A Memoli ◽

William D Morain ◽

Jean-Marc Schlaeppi ◽

Sandra M Rousseau

Keyword(s):

Tumor Cell ◽

Cell Carcinoma ◽

Cellular Localization ◽

Thrombin Generation ◽

Normal Lung ◽

Cancer Tissue ◽

Immunohistochemical Techniques ◽

Tumor Types ◽

Negative Controls

SummaryCellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inihibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

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Metformin Protects against Radiation-Induced Acute Effects by Limiting Senescence of Bronchial-Epithelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22137064 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7064

Author(s):

Christine Hansel ◽

Samantha Barr ◽

Alina V. Schemann ◽

Kirsten Lauber ◽

Julia Hess ◽

...

Keyword(s):

Epithelial Cells ◽

Lung Tissue ◽

Cellular Stress ◽

Lung Epithelial Cells ◽

Normal Lung Tissue ◽

Normal Lung ◽

Metformin Treatment ◽

Acute Effects ◽

Lung Epithelial ◽

Radiation Induced

Radiation-induced damage to normal lung parenchyma remains a dose-limiting factor in thorax-associated radiotherapy (RT). Severe early and late complications with lungs can increase the risk of morbidity in cancer patients after RT. Herein, senescence of lung epithelial cells following RT-induced cellular stress, or more precisely the respective altered secretory profile, the senescence-associated secretory phenotype (SASP), was suggested as a central process for the initiation and progression of pneumonitis and pulmonary fibrosis. We previously reported that abrogation of certain aspects of the secretome of senescent lung cells, in particular, signaling inhibition of the SASP-factor Ccl2/Mcp1 mediated radioprotection especially by limiting endothelial dysfunction. Here, we investigated the therapeutic potential of a combined metformin treatment to protect normal lung tissue from RT-induced senescence and associated lung injury using a preclinical mouse model of radiation-induced pneumopathy. Metformin treatment efficiently limited RT-induced senescence and SASP expression levels, thereby limiting vascular dysfunctions, namely increased vascular permeability associated with increased extravasation of circulating immune and tumor cells early after irradiation (acute effects). Complementary in vitro studies using normal lung epithelial cell lines confirmed the senescence-limiting effect of metformin following RT finally resulting in radioprotection, while fostering RT-induced cellular stress of cultured malignant epithelial cells accounting for radiosensitization. The radioprotective action of metformin for normal lung tissue without simultaneous protection or preferable radiosensitization of tumor tissue might increase tumor control probabilities and survival because higher radiation doses could be used.

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