Cytolytic activity of mononuclear cells, T-lymphocytes and monocytes of the peripheral blood in patients with lung cancer

A comparative analysis of cytotoxic activity of mononuclear cells (MNC) from peripheral blood, T-lymphocytes and monocytes from patients with lung cancer has been performed. It has been shown that in 27% of cases MNC, T-lymphocytes and monocytes lyse freshly isolated autologous and allogenic tumor cells. In all the patients examined the effector cells were active in respect to culture cell line of lung adenocarcinoma (ACL). The decrease in NK activity of the cell population enriched by T-lymphocytes in comparison to the control group (p less than 0.05) was noticed. MNC and T-lymphocytes, in contrast to monocytes, had high killer activity identified by lectin-dependent cytotoxicity technique. The activity of the effector cells does not depend on the morphological structure of the tumor, but decreases with the disease progression. The results of the experiments show that in patients with lung cancer peripheral blood lymphocytes and monocytes are essential, independently functioning effectors involved in antitumor defense.     

Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody

Heteroaggregates containing anti-T3 cross-linked to anti-target cell antibodies have been shown to cause human T cells to lyse target cells that express antigens recognized by the anti-target cell antibody. In this study, we test targeted human T cells for the ability to lyse human tumor cells as a first step toward the application of this phenomenon to tumor immunotherapy. Several monoclonal anti-human tumor antibodies were assayed for binding to a number of human tumor lines and for the ability to promote specific tumor cell lysis when cross-linked with anti-T3. We found that anti-T3 cross-linked to anti-tumor monoclonal antibodies caused cloned human T cells and fresh peripheral blood T cells to lyse the tumor cells with the same specificity as predicted by the binding studies. Peripheral blood T cells were then tested in the presence of various heteroaggregates for the ability to lyse single cell suspensions prepared from fresh tumor or fresh normal tissue. These studies showed that heteroaggregates containing anti-T3 cross-linked to anti-tumor antibody cause fresh human T cells to specifically lyse fresh tumor cells, but not (with one exception) fresh normal cells.     

肺癌患者外周血血小板在肺癌血行转移中的作用研究

目的：观察肺癌患者外周血血小板对肺癌分期和血行转移的影响。方法：选择在我院呼吸科初诊的原发性肺癌患168例,分析其外周血小板计数与肺癌病理类型和分期的关系,并在模拟流体状态下,体外研究活化血小板对培养的肺癌细胞和内皮细胞相互作用的影响。结果：肺腺癌中外周血血小板计数增高现象最为明显,占37.09%（23/62）（P〈0.05）,鳞癌占22.64%,小细胞癌占22.70%,其他占14.20%。其中有远处血行转移者血小板增多（20.24%）较无明显转移者（7.14%）相差显著（P〈0.01）。同时体外研究显示流体状态对肺癌细胞粘附存在影响,而活化血小板增强了肺癌细胞与内皮细胞的相互作用。结论：血小板活化与肺癌尤其是肺腺癌的血行转移密切相关;活化血小板增强了肺癌细胞与内皮细胞的相互作用是血小板促进肺癌血行转移的重要机制之一。     

Lysis of human T cell leukemia virus infected T and B lymphoid cells by interleukin 2-activated killer cells

The human T cell leukemia (HTLV-1) retrovirus is the etiologic agent for adult T cell leukemia. Interleukin 2 (IL-2) activated killer (AK) cells have been shown to lyse freshly explanted tumor cells in vitro and have been used as a form of adoptive immunotherapy for the treatment of cancer. In this report, the ability of AK cells to lyse HTLV-1-infected targets was examined. Normal lymphocytes, when cultured in recombinant IL-2 for periods of 3 to 7 days, killed infected T and B cell lines. The precursor for these AK cells resided in the CD-16 antigen-positive subset (i.e., natural killer (NK) cells). Resting T cells, NK cells, or unfractionated lymphocytes did not lyse the infected targets. However, when isolated NK cells were incubated for 24 hr in IL-2, suboptimal cytolysis was induced whereas activation of NK cells with a four pulse of IL-2 was insufficient to generate effector cells. The results of performing cold target inhibition studies with Epstein-Barr virus-infected B cell lines and HTLV-1-infected T and B cell lines suggest that there are discrete subsets (i.e., clonotypic) in the AK population that preferentially lyse a given virally infected cell line. Thus to consider AK cells as true polyspecific killer cells may be inaccurate. Alternately AK cells may express a number of different receptors with variable affinities for the Epstein-Barr virus- and HTLV-1-infected cell lines. In addition, it was shown that HTLV-1-infected B cells are relatively resistant to AK cell-mediated lysis. These results clearly indicate that AK cells but not resting NK cells kill HTLV-1-infected cells.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.     

Establishment and characterization of a lung cancer cell line,SMC-L001, from a lung adenocarcinoma

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.     

Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines

Summary Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.     

NK cells infiltrating a MHC class I-deficient lung adenocarcinoma display impaired cytotoxic activity toward autologous tumor cells associated with altered NK cell-triggering receptors

NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking beta2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I- tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition.     

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.     

Decreased monocyte-mediated cytostasis of human cancer cell in patients with lung cancer

Summary In vivo animal studies support the concept that monocytes and macrophages are important in the immune surveillance of oncogenesis and that in vitro activated murine macrophages are cytocidal for tumour cells. In this study, the tumour cell cytotoxic activity of human peripheral blood monocytes was examined by measuring the inhibition of ³H-thymidine uptake in the human cancer cell line, established in our laboratory from human squamous cell lung cancer. The monocytes from 8 of the 31 lung cancer patients (26%) showed a percentage growth inhibition of less than 69.8%, which exceeded the 95% confidence limits of the percentage growth inhibition observed with healthy control monocytes. On the other hand, among the 16 sarcoidosis and the 8 tuberculosis cases no value was below 69.8%. However, there was no significant difference between the growth inhibition and the clinical stages or histological type. When OK-432, a Streptococal agent, was administered in vivo to patients with lung cancer, an elevation of the growth inhibition was observed in 7 out of 8 patients. It was confirmed that the tumour cell cytostatic activity of the monocyte is suppressed in patients with lung cancer, and these monocyte deficits hinder the inhibition of tumour growth and metastasis.     

The role of HLA class I antigens in recognition of melanoma cells by tumor-specific cytotoxic T lymphocytes. Evidence for shared tumor antigens

CTL lines were established in vitro by stimulating patient lymphocytes with autologous melanoma cells in the presence of IL-2. Resulting CTL lines lysed autologous melanoma and failed to lyse several allogeneic melanomas or K562. The mechanism of target cell recognition by autologous tumor-specific CTL was evaluated in this system, using several CTL lines: DT6, DT105, DT141, DT166, DT169, and DT179. Autologous melanoma lysis was inhibited by W6/32, mAb directed against HLA class I Ag, but not by L243, mAb directed against HLA class II Ag. CTL from DT6, DT141, DT166, DT169, and DT179 lysed fresh and cultured allogeneic melanomas, which shared the HLA-A2 Ag, but failed to lyse allogeneic melanomas, which shared B-region or C-region Ag, or shared no HLA class I Ag. CTL from DM141 lysed DM93, which shared A2 and Bw6, but failed to lyse DM105, which shared only Bw6. DM105 CTL failed to lyse allogeneic melanomas that shared HLA-A1, or that shared B or C region Ag, but they did lyse allogeneic melanoma DM49, which expressed an A region Ag that either was A10 or was serologically cross-reactive with A10. A T cell leukemia line, three EBV transformed B cell lines, and a pancreatic cancer line, all of which expressed HLA-A2, were not lysed by DM6 or DM179 CTL. Furthermore, HLA-matched nonmelanomas failed to inhibit autologous tumor lysis in cold target inhibition assays, whereas an HLA-A2+ allogeneic melanoma, DM93, inhibited autologous tumor lysis as effectively as the autologous tumor itself. HLA-A2, and possibly other HLA-A-region Ag, appear to function in HLA-restricted recognition of shared melanoma associated Ag by CTL.     

Increased tumor-specific CTL activity in human tumor-infiltrating lymphocytes stimulated with autologous tumor lines

Two long-term tumor-infiltrating lymphocyte (TIL) lines and their autologous tumor lines have been established from solid tumors derived from different patients with metastatic melanoma. In 4-hr 51Cr release assays, each TIL culture lysed only the autologous cryopreserved fresh or established melanoma line, but failed to lyse other melanoma tumors or K562 cells. Repeated stimulation of TIL with the autologous melanoma lines resulted in significant increases in anti-tumor CTL activity with no apparent loss in specificity. Stimulated cells have retained cytotoxic activity for up to 5 months in culture. Tumor cell CTL activity for both long-term TIL lines is inhibited by several mAbs, including those against CD3, CD8, and class I MHC molecules, indicating that the effector cells are class I-restricted CD8+, CTL. Furthermore, recognition of Ag on one of the established melanoma lines by TIL is restricted by HLA A-2. The availability of autologous tumor lines may prove clinically useful for the selective stimulation and expansion of cells with anti-tumor activity within a heterogeneous TIL population.     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen I (OFA-I)

Summary Oncofetal antigen I (OFA-I) has been identified by immunofluorescence and immune adherence (IA) as a membrane antigen on human tumor cells, which cross-reacts with fetal brain tissue. This antigen induces humoral antibody in patients with cancer. The present work was designed to evaluate the complement-dependent cytotoxic potential of anti-OFA-I antibody produced in melanoma patients against an OFA-I-positive melanoma cell line, UCLA SO M14 (M14). Patients' sera were chosen on the basis of anti-OFA-I activity by IA. Alloantibodies to M14 were removed by absorption of the sera with lymphoblastoid cells autologous to M14. Fourteen sera were tested, and all demonstrated cytotoxic activity in the presence of rabbit complement. Human complement was also shown to mediate cytotoxicity, although less effectively than rabbit complement. The specificity of the reaction was confirmed when the cytotoxic capability could be absorbed by fetal brain, but not by autologous fetal liver tissues. These results indicate that a patient's serum antibody to OFA-I can lyse tumor cells expressing this antigen.     

The therapeutic effect of OK-432-combined adoptive immunotherapy against liver metastases from gastric or colorectal cancers

Twenty-four patients with liver metastases from gastric or colorectal cancer were treated with OK-432-combined adoptive immunotherapy (AIT). Lymphocytes isolated from regional lymph nodes or peripheral blood were cultured with medium containing T cell growth factor and sonicated tumor extract antigen (SE-Ag) for 9–13 days. The cultured lymphocytes were transferred mainly through the hepatic artery after the administration of OK-432, a streptococcal preparation. Sixteen of the 24 patients received a low dose of anti-cancer agents between the OK-432 injection and cell transfer. When cultured without SE-Ag, regional lymph node lymphocytes (RLNL) showed significantly (P<0.05) higher cytotoxic activity against autologous tumor cells and, on the contrary, lower cytotoxic activity against K562 than peripheral blood lymphocytes (PBL). When cultured with SE-Ag, cytotoxicity of RLNL against autologous tumor cells was nearly equivalent to that of PBL. The blastogenesis of fresh PBL to SE-Ag was significantly (P<0.05) augmented after the OK-432-combined AIT. Two patients showed complete response and 4 patients showed partial response among 19 patients who had evaluable lesions. Five patients whose liver metastases were resected were treated with OK-432-combined AIT as an adjuvant therapy. To date they are alive without recurrence in the liver.Abbreviations AIT adoptive immunotherapy - RLNL regional lymph node lymphocytes - SE-Ag sonicated tumor extract antigen     

Platelet cancer cell interplay as a new therapeutic target

The interaction between circulating tumor cells and platelets is a key factor in cancer metastasis. These interactions, driven by a variety of receptors, support circulating tumor cells by protecting them from immune detection, cushioning them from shear stress, and promoting their arrest at the endothelium. Additionally, platelets have been shown to accumulate in the primary tumors, promoting tumor growth and angiogenesis by releasing growth factors. Furthermore, tumor cells can interact with platelets by inducing aggregation, which further protects cancer cells. However, the platelet cancer cell interplay also offers new approaches to develop targeted therapies. The accumulation of platelets in tumors has successfully been leveraged to deliver chemotherapeutics and imaging agents. Likewise, these platelet-based interactions have been utilized to target cancer cells in circulation. Although these current systems have limitations including drug loading and storage, leveraging platelet-cancer cell interactions to effectively target circulating tumor cells and tumors shows great promise for future cancer treatments.     

Natural antibody to a human bladder carcinoma cell line

Summary Sera from 144 healthy blood donors were screened for the presence of antibody against the bladder carcinoma cell line RT4 by means of an immune adherence assay. Only one serum (9241) demonstrated high activity with a titer of 1/124. Qualitative and quantitative absorption analyses were carried out with a wide variety of cells to characterize the specificity of the reaction. Cells used for absorption were both cultured and non-cultured and normal and malignant. The reactivity was absorbed by the bladder cancer cell lines RT4 and 5637 and the lung cancer cell line SK-LC-LL. In addition, surgically obtained urothelium from a patient with transitional cell carcinoma abrogated reactivity. No other absorbing cells removed the reactivity of serum 9241 for RT4. The antigen defined by this serologic reaction is heat-stable and trypsin-resistant, as demonstrated by using heated or trypsinized RT4 cells for absorption.This study shows that naturally occurring antibody directed toward cell surface antigens of tumor cells is present in some normal individuals. This finding is important in the evaluation of a possible causal relationship between the presence of antibody specific for tumor cell antigens and the development of neoplasia.     

Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer

Lung cancer or pulmonary carcinoma is primarily derived from epithelial cells that are thin and line on the alveolar surfaces of the lung for gas exchange. ANO1/TMEM16A, initially identified from airway epithelial cells, is a member of Ca²⁺-activated Cl^- channels (CaCCs) that function to regulate epithelial secretion and cell volume for maintenance of ion and tissue homeostasis. ANO1/TMEM16A has recently been shown to be highly expressed in several epithelium originated carcinomas. However, the role of ANO1 in lung cancer remains unknown. In this study, we show that inhibition of calcium-activated chloride channel ANO1/TMEM16A suppresses tumor growth and invasion in human lung cancer. ANO1 is upregulated in different human lung cancer cell lines. Knocking-down ANO1 by small hairpin RNAs inhibited proliferation, migration and invasion of GLC82 and NCI-H520 cancel cells evaluated by CCK-8, would-healing, transwell and 3D soft agar assays. ANO1 protein is overexpressed in 77.3% cases of human lung adenocarcinoma tissues detected by immunohistochemistry. Furthermore, the tumor growth in nude mice implanted with GLC82 cells was significantly suppressed by ANO1 silencing. Taken together, our findings provide evidence that ANO1 overexpression contributes to tumor growth and invasion of lung cancer; and suppressing ANO1 overexpression may have therapeutic potential in lung cancer therapy.