Immune and Inflammatory Cell Composition of Human Lung Cancer Stroma.

Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+), cytotoxic-T cells (CD8+), T-helper cells (CD4+), B cells (CD20+), macrophages (CD68+), mast cells (CD117+), mononuclear cells (CD11c+), plasma cells, activated-T cells (MUM1+), B cells, myeloid cells (PD1+) and neutrophilic granulocytes (myeloperoxidase+) compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells) in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition

Immunohistochemical analysis and quantification of CD3-positive T lymphocytes in human lung cancer.

<p>Human lung cancer tissue array was stained with CD3 antibody to detect T lymphocytes. (A) Quantification of CD3⁺ cells in lung cancer vs. healthy donor specimens. (B–E) Quantification of CD3⁺ cells based on (B) their pathology, (C) cancer stage, (D) tumor size, and (E) nodal status. Cell numbers are given as CD3-positive cells per 1000 cells. (F) Representative images of human lung sections stained with CD3 antibody based on their pathology. Scale bar = 25 μm.</p

Immunohistochemical analysis and quantification of CD4-positive T lymphocytes in human lung cancer.

<p>Human lung cancer tissue array was stained with CD4 antibody to detect T helper cells. (A) Quantification of CD4⁺ cells in lung cancer vs. healthy donor specimens. (B–E) Quantification of CD4⁺ cells based on (B) their pathology, (C) cancer stage, (D) tumor size, and (E) nodal status. Cell numbers are given as CD4-positive cells per 1000 cells. (F) Representative images of human lung sections stained with CD4 antibody based on their pathology. Scale bar = 25 μm.</p

Immunohistochemical analysis and quantification of CD11c-positive cells in human lung cancer.

<p>Human lung cancer tissue array was stained with CD11c antibody to detect dendritic cells. (A) Quantification of CD11c⁺ cells in lung cancer vs. healthy donor specimens. (B–E) Quantification of CD11c⁺ cells based on (B) their pathology, (C) cancer stage, (D) tumor size, and (E) nodal status. Cell numbers are given as CD11c-positive cells per 1000 cells. (F) Representative images of human lung sections stained with CD11c antibody based on their pathology. Scale bar = 25 μm.</p

Antibody details.

<p>Antibody details.</p

Morphological analysis of human lung specimens.

<p>Representative images of human lung sections stained with hematoxylin and eosin based on their pathology. (A) Healthy donor, (B) squamous cell carcinoma, (C) adenocarcinoma, (D) adenosquamous carcinoma, (E) small cell carcinoma, (F) papillary adenocarcinoma, (G) metastatic adenocarcinoma, and (H) bronchioloalveolar carcinoma. Scale bar = 250 μm.</p

Immunohistochemical analysis and quantification of PD1–positive cells in human lung cancer.

<p>Human lung cancer tissue array was stained with PD1 antibody to detect activated T cells, B cells, myeloid cells, and a subset of thymocytes. (A) Quantification of PD1⁺ cells in lung cancer vs. healthy donor specimens. (B–E) Quantification of PD1⁺ cells based on (B) their pathology, (C) cancer stage, (D) tumor size, and (E) nodal status. Cell numbers are given as PD1–positive cells per 1000 cells. (F) Representative images of human lung sections stained with PD1 antibody based on their pathology. Scale bar = 25 μm.</p

Immunohistochemical analysis and quantification of CD8-positive T lymphocytes in human lung cancer.

<p>Human lung cancer tissue array was stained with CD8 antibody to detect cytotoxic T lymphocytes. (A) Quantification of CD8⁺ cells in lung cancer vs. healthy donor specimens. (B–E) Quantification of CD8⁺ cells based on (B) their pathology, (C) cancer stage, (D) tumor size, and (E) nodal status. Cell numbers are given as CD8-positive cells per 1000 cells. (F) Representative images of human lung sections stained with CD8 antibody based on their pathology. Scale bar = 25 μm.</p

Expression and Activity of Phosphodiesterase Isoforms during Epithelial Mesenchymal Transition: The Role of Phosphodiesterase 4

Epithelial–mesenchymal transition (EMT) has emerged as a critical event in the pathogenesis of organ fibrosis and cancer and is typically induced by the multifunctional cytokine transforming growth factor (TGF)-β1. The present study was undertaken to evaluate the potential role of phosphodiesterases (PDEs) in TGF-β1-induced EMT in the human alveolar epithelial type II cell line A549. Stimulation of A549 with TGF-β1 induced EMT by morphological alterations and by expression changes of the epithelial phenotype markers E-cadherin, cytokeratin-18, zona occludens-1, and the mesenchymal phenotype markers, collagen I, fibronectin, and α-smooth muscle actin. Interestingly, TGF-β1 stimulation caused twofold increase in total cAMP-PDE activity, contributed mostly by PDE4. Furthermore, mRNA and protein expression demonstrated up-regulation of PDE4A and PDE4D isoforms in TGF-β1-stimulated cells. Most importantly, treatment of TGF-β1 stimulated epithelial cells with the PDE4-selective inhibitor rolipram or PDE4 small interfering RNA potently inhibited EMT changes in a Smad-independent manner by decreasing reactive oxygen species, p38, and extracellular signal-regulated kinase phosphorylation. In contrast, the ectopic overexpression of PDE4A and/or PDE4D resulted in a significant loss of epithelial marker E-cadherin but did not result in changes of mesenchymal markers. In addition, Rho kinase signaling activated by TGF-β1 during EMT demonstrated to be a positive regulator of PDE4. Collectively, the findings presented herein suggest that TGF-β1 mediated up-regulation of PDE4 promotes EMT in alveolar epithelial cells. Thus, targeting PDE4 isoforms may be a novel approach to attenuate EMT-associated lung diseases such as pulmonary fibrosis and lung cancer