thromboplastin and Adenocarcinoma-of-Lung

Chimeric antigen receptor (CAR)-modified T cell (CAR T) is a promising therapeutic option for patients with cancer. Such an approach requires the identification of tumor-specific antigen targets that are expressed in solid tumors. We developed a new third-generation CAR directed against tissue factor (TF), a surface molecule overexpressed in some types of lung cancer, melanoma and other cancers. First, we demonstrated by immunohistochemistry that TF was overexpressed in squamous cell carcinoma and adenocarcinoma of non-small cell lung cancer (NSCLC) and melanoma using a human tissue microarray. In the presence of TF-positive cancer cells, the CAR-modified T cells (TF-CAR T) were highly activated and showed specific cytotoxicity to TF-positive cancer cells in vitro. In established s.c. xenograft and lung metastasis models, TF-CAR T cells could significantly suppress the growth of s.c. xenograft and metastasis of TF-positive cancer cells. Additionally, the safety evaluation of TF-CAR T cells in vivo showed that the treatment did not cause obvious toxicity in mice. Taken together, these findings indicate that TF-CAR T cells might be a novel potential therapeutic agent for the treatment of patients with TF-positive cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Movement; Cell Proliferation; Cytokines; Cytotoxicity, Immunologic; Female; Humans; Immunotherapy, Adoptive; Lung Neoplasms; MCF-7 Cells; Melanoma, Experimental; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Receptors, Antigen, T-Cell; Skin Neoplasms; T-Lymphocytes; Thromboplastin; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Malignant pleural effusion (MPE) is a poor prognostic sign for patients with lung cancer. Tissue factor (TF) is a coagulation factor that participates in angiogenesis and vascular permeability and is abundant in MPE. We previously demonstrated that autocrine IL-6-activated Stat3 contributes to tumor metastasis and upregulation of VEGF, resulting in the generation of MPE in lung adenocarcinoma. In this study, we found IL-6-triggered Stat3 activation also induces TF expression. By using pharmacologic inhibitors, it was shown that JAK2 kinase, but not Src kinase, contributed to autocrine IL-6-induced TF expression. Inhibition of Stat3 activation by dominant negative Stat3 (S3D) in lung adenocarcinoma suppressed TF-induced coagulation, anchorage-independent growth in vitro, and tumor growth in vivo. Consistently, knockdown of TF expression by siRNA resulted in a reduction of anchorage-independent growth of lung adenocarcinoma cells. Inhibition of TF expression also decreased the adhesion ability of cancer cells in normal lung tissues. In the nude mouse model, both lung metastasis and MPE generation were decreased when PC14PE6/AS2-siTF cells (TF expression was silenced) were intravenously injected. PC14PE6/AS2-siTF cells also produced less malignant ascites through inhibition of vascular permeability. In summary, we showed that TF expression plays a pivotal role in the pathogenesis of MPE generation via regulating of tumor metastasis and vascular permeability in lung adenocarcinoma bearing activated Stat3.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Blotting, Western; Capillary Permeability; DNA Primers; Flow Cytometry; Fluorescent Antibody Technique; Gene Knockdown Techniques; Interleukin-6; Janus Kinase 2; Luciferases; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Metastasis; Pleural Effusion, Malignant; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; STAT3 Transcription Factor; Thromboplastin; Up-Regulation

Patients with lung adenocarcinoma undergoing surgery are in high risk for VTE and receive routine post-operative thromboprophylaxis with LWMH.. We investigated markers of hypercoagulability in patients with primary localized adenocarcinoma and the modifications induced by lobectomy and postoperative administration of enoxaparin.. Patients suffering from localised primary lung adenocarcinoma (n=15) scheduled for lobectomy were studied. The control group consisted of 15 healthy age and sex-matched individuals. Blood was collected before anaesthesia induction and after surgery, at several intervals until the 7th post-operative day. Samples were assessed for thrombin generation, phosphatidylserin expressing platelet derived microparticles expressing (Pd-MP/PS(+)), tissue factor activity (TFa), FVIIa and TFPI levels, procoagulant phospholipid dependent clotting time and anti-Xa activity.. At baseline, patients showed increased thrombin generation and Pd-MP/PS(+). After lobectomy thrombin generation significantly decreased. Administration of enoxaparin attenuated thrombin generation. In about 50% of samples collected post-operatively an increase of thrombin generation occurred despite the presence of the expected anti-Xa activity in plasma. At the 7th post-operative day, 3 out of 15 patients showed a significant increase of thrombin generation.. In patients with localized lung adenocarcinoma, hypercoagulability is characterized by high thrombin generation and increased concentration of Pd-MP/PS(+). Tumor mass resection is related with attenuation of thrombin generation, which is inhibited by postoperative thromboprophylaxis with enoxaparin. The response to enoxaparin is not predicted by the concentration of the anti-Xa activity in plasma. The assessment of thrombin generation during prophylaxis with enoxaparin allows to identify patients with high residual plasma hypercoagulability.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Anticoagulants; Blood Coagulation; Blood Coagulation Tests; Blood Platelets; Cell-Derived Microparticles; Enoxaparin; Factor Xa Inhibitors; Female; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Postoperative Period; Thrombin; Thrombophilia; Thromboplastin

The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers make TF an ideal target for cancer therapy. Here, we report an energized fusion protein, hlFVII-LDP-AE, which can be used for cancer therapy and is composed of a human Factor VII light chain (hlFVII) conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE). hlFVII-LDP-AE binds with specificity to TF expressed on tumor cells, resulting in internalization of the fusion protein and cytotoxicity induced by the LDM domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was examined in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and in vivo with a BALB/c nude mouse xenograft model of the human lung cancer line NCI-H292. hlFVII-LDP-AE caused chromatin condensation and cleavage of genomic DNA in NCI-H292 cells. In the MTT assays, the IC50 value of hlFVII- LDP-AE was 0.19 nM. In the in vivo tests, after two intravenous injections of hlFVII-LDP-AE at a dose of 0.6 mg/kg, the growth rate of the lung tumor xenograft was reduced to 15% of the control rate, and there was no excessive loss of body weight and inflammatory response in the mice. These findings suggest that hlFVII-LDP-AE is efficacious and tolerated in the mouse model of NCI-H292 human lung cancer examined and could have broad clinical applicability for treating cancer patients.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aminoglycosides; Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Proliferation; Chromatin Assembly and Disassembly; DNA Damage; Enediynes; Factor VII; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Protein Binding; Recombinant Fusion Proteins; Thromboplastin; Xenograft Model Antitumor Assays

The human coagulation trigger tissue factor (TF) is overexpressed in several types of cancer and involved in tumor growth, vascularization, and metastasis. To explore the role of TF in biological processes of lung adenocarcinoma, we used RNA interference (RNAi) technology to silence TF in a lung adenocarcinoma cell line A549 with high-level expression of TF and evaluate its antitumor effects in vitro and in vivo.. The specific small interfering RNA (siRNA) designed for targeting human TF was transfected into A549 cells. The expression of TF was detected by reverse transcription-PCR and Western blot. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The metastatic potential of A549 cells was determined by wound healing, the mobility and Matrigel invasion assays. Expressions of PI3K/Akt, Erk1/2, VEGF and MMP-2/-9 in transfected cells were detected by Western blot. In vivo, the effect of TF-siRNA on the growth of A549 lung adenocarcinoma xenografts in nude mice was investigated.. TF -siRNA significantly reduced the expression of TF in the mRNA and protein levels. The down-regulation of TF in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis in dose-dependent manner. Erk MAPK, PI3K/Akt pathways as well as VEGF and MMP-2/-9 expressions were inhibited in TF-siRNA transfected cells. Moreover, intratumoral injection of siRNA targeting TF suppressed the tumor growth of A549 cells in vivo model of lung adenocarcinoma.. Down-regulation of TF using siRNA could provide a potential approach for gene therapy against lung adenocarcinoma, and the antitumor effects may be associated with inhibition of Erk MAPK, PI3K/Akt pathways.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; RNA, Small Interfering; Signal Transduction; Thromboplastin; Tumor Burden; Xenograft Model Antitumor Assays