Fresh Human Tumor Cells Research Articles

Tumor-associated Macrophage-derived Interleukin-23 Interlinks Kidney Cancer Glutamine Addiction with Immune Evasion

BackgroundGlutamine addiction is a hallmark of clear cell renal cell carcinoma (ccRCC); yet whether glutamine metabolism impacts local immune surveillance is unclear. This knowledge may yield novel immunotherapeutic opportunities. ObjectiveTo seek a potential therapeutic target in glutamine-addicted ccRCC. Design, setting, and participantsTumors from ccRCC patients from a Shanghai cohort and ccRCC tumor data from The Cancer Genome Atlas (TCGA) cohort were analyzed. In vivo and in vitro studies were conducted with fresh human ccRCC tumors and murine tumor cells. Outcome measurements and statistical analysisImmune cell numbers and functions were analyzed by flow cytometry. Glutamine and cytokine concentrations were determined. Survival was compared between different subpopulations of patients using Kaplan-Meier and Cox regression analyses. Results and limitationsWe found that in ccRCC, high interleukin (IL)-23 expression was significantly associated with poor survival in both TCGA (overall survival [OS] hazard ratio [HR]=2.04, cancer-specific survival [CSS] HR=2.95; all p<0.001) and Shanghai (OS HR=2.07, CSS HR=3.92; all p<0.001) cohorts. IL-23 blockade prolongs the survival of tumor-bearing mice, promotes T-cell cytotoxicity in in vitro cultures of human ccRCC tumors, and augments the therapeutic benefits of anti-PD-1 antibodies. Mechanistically, glutamine consumption by ccRCC tumor cells results in the local deprivation of extracellular glutamine, which induces IL-23 secretion by tumor-infiltrating macrophages via the activation of hypoxia-inducible factor 1α (HIF1α). IL-23 activates regulatory T-cell proliferation and promotes IL-10 and transforming growth factor β expression, thereby suppressing tumor cell killing by cytotoxic lymphocytes. The positive correlations between glutamine metabolism, IL-23 levels, and Treg responses are confirmed in both TCGA cohort and tumors from Shanghai ccRCC patients. Study limitations include the unclear impacts of glutamine deprivation and IL-23 on other immune cells. ConclusionsMacrophage-secreted IL-23 enhanced Treg functions in glutamine-addicted tumors; thus, IL-23 is a promising target for immunotherapy in ccRCC. Patient summaryIn this study, we analyzed the immune components in glutamine-addicted clear cell renal cell carcinoma (ccRCC) tumors from two patient cohorts and conducted both in vitro and in vivo studies. We found that ccRCC tumor cell-intrinsic glutamine metabolism orchestrates immune evasion via interleukin (IL)-23, and IL-23–high patients had significantly poorer survival than IL-23–low patients. IL-23 should thus be considered a therapeutic target in ccRCC, either alone or in combination with immune checkpoint inhibitors.

Recognition of Fresh Human Tumor by Human Peripheral Blood Lymphocytes Transduced with a Bicistronic Retroviral Vector Encoding a Murine Anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.

In vivo activity of CHS 828 on hollow-fibre cultures of primary human tumour cells from patients

Fresh human tumour cells from patients with ovarian cancer and chronic lymphocytic leukaemia were cultured in semipermeable hollow fibres. The fibres were implanted on immunocompetent rats, which were treated with the cyanoguanidine N-(4-chlorophenoxyhexyl)-N′-cyano-N″-4-pyridylguanidine (CHS 828). CHS 828 showed high antitumour activity against all eight tumour samples; the fibres from control animals had a mean net growth of 73% while CHS 828 treatment induced a 37% mean reduction of cell density, without observable haematological toxicity. The results show a feasibility of using tumour cells directly from patients in the hollow-fibre rat model.

Differential activity of topotecan, irinotecan and SN-38 in fresh human tumour cells but not in cell lines

The topoisomerase I inhibitors topotecan irinotecan (CPT-11) and its metabolite SN-38 were studied in a panel of cell lines and in primary tumour cells from patients, using a non-clonogenic cytotoxicity assay. All three substances showed similar activity patterns in the panel of cell lines established to classify the drugs mechanistically. In the patient tumour cells the drugs had different effects. In haematological and ovarian cancer samples, SN-38 was much more potent than topotecan, followed by irinotecan, while in colorectal cancer samples only irinotecan showed substantial activity. This in vitro activity pattern seems to agree with clinical experiences to date. The inactivity of SN-38 in colorectal cancer suggests irinotecan may also have some other role in addition to being a prodrug to SN-38. This study raises questions as to the role and relevance of early preclinical model systems in anticancer drug development, and suggests that important information can be obtained from studies using primary cultures of human tumour cells.

P-glycoprotein-expressing tumor cells are resistant to anticancer drugs in human gastrointestinal cancer.

The resistance to doxorubicin (DOX) by some tumor cells is mainly due to the effect of P-glycoprotein encoded by the multidrug resistance-1 (mdr1) gene. We tried to prove the correlations between P-glycoprotein expression and the sensitivity for anticancer drugs including DOX and other cytotoxic drugs that are currently used for gastrointestinal cancer patients. We quantified the P-glycoprotein expression by flow cytometry techniques, and the sensitivity for anticancer drugs using a tetrazolium salt, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), assay in highly purified fresh human tumor cells obtained from 25 cancer patients. The inhibition rates were the lowest in DOX and mitomycin C (MMC), compared with other drugs. The most significant correlation between DOX and MMC was seen in the inhibition rates. A significant correlation was also seen between the inhibition rates for DOX and P-glycoprotein expression, whereas only a slight correlation between the sensitivity for MMC and P-glycoprotein expression was observed. We should therefore pay close attention to the effect of P-glycoprotein when treating cancer patients, especially if both the inhibition rates of DOX and MMC are low based on the findings of an MTT assay.

Relationship between diagnosis-specific activity of cytotoxic drugs in fresh human tumour cells ex vivo and in the clinic

The aim of this investigation was to evaluate the relationship between the disease-specific activity of cytotoxic drugs in the clinic and in fresh human tumour cells from patients, as detected by a non-clonogenic cytotoxicity assay. The activity of 18 different cytotoxic drugs in fresh human tumour cells ex vivo was analysed in up to 15 samples from each of 13 different diagnoses using the fluorometric microculture cytotoxicity assay (FMCA). For each drug and diagnosis, relative activity indices (RAIs) were calculated, defined as the fraction of samples within the diagnosis having a survival index (SI) less than the median SI for the drug in all tested samples. Clinical response rates for the drug in the same diagnoses were collected from published phase II trials and were compared with the RAIs using Spearman's rank correlation (Rho) and Pearson's correlation coefficient ( R). Correlation coefficients could be calculated for 12 drugs. The coefficients varied between 0.02 and 0.92 (Rho) and between 0.07 and 0.91 ( R), but for most drugs the correlation between RAI and clinical response rates was good, with Rho >0.6 and R>0.7. Weak correlations were observed for cyclophosphamide and vincristine (Rho=0.32 and 0.16, respectively), which might be due to old clinical data, and for paclitaxel (Rho=0.02), which perhaps could be explained by in vitro activity of the solvent, Cremophor EL. The 18 drugs were also categorised according to their suggested clinical use in solid or haematological tumours, and were then compared regarding the activity in solid compared with haematological tumours ex vivo, expressed as the ratio between the number of responders among solid and haematological tumours (S/H ratio). The mean ex vivo S/H ratios in the group of drugs registered for use in haematological tumours was only 0.09 and was significantly different ( P=0.05) from the mean S/H ratios for the drugs used in both haematological and solid tumours (0.31) and in solid tumours only (0.47). Furthermore, the FMCA could identify the 50% most and least sensitive diagnoses with respect to clinical phase II activity with 74% (78/106) correct classifications. The results indicate that the relative activity of cytotoxic drugs in different diagnoses may be detected by the FMCA. Thus, ‘phase II trials ex vivo’ using non-clonogenic cytotoxicity assays might be used to make clinical trials more effective by targeting trials to diagnoses in which a new agent is most likely to be active.

Processing of antibodies bound to B-cell lymphomas and lymphoblastoid cell lines

Previous experiments demonstrated that some human B-cell lymphoma cell lines were unusual in that antibodies bound to the cell surface dissociated at high levels. This did not occur with non-B-cell hematologic tumors or with carcinomas. In this study, additional B-cell lymphoma and lymphoblastoid (Epstein-Barr virus-transformed) cell lines were tested.The antibodies selected for most experiments, MA103 and anti-CD45, react with relatively high avidity to the cell surface. Antibodies to CD19, CD20, and CD22 also were tested on certain cell lines. The antibodies were labeled with 125I. After binding to the surface of viable cells, unbound antibody was washed away, and the fate of the bound antibody was investigated for 2-3 days.Of the eight B-cell lymphomas tested, three had high levels of dissociation, two had low levels of dissociation, and three had intermediate levels of dissociation. The six lymphoblastoid cell lines had only slightly elevated levels of dissociation, relative to non-B cell lines. Sublines of Raji and Ramos cells were identified that varied greatly in the level of antibody dissociation. The level of dissociation from lymphomas was correlated with the tendency of the cell lines to cluster, with single cells displaying less dissociation than clustered cells. However, some exceptions to this correlation were noted. Cell lines such as Ramos, which showed little dissociation of anti-CD20, displayed relatively rapid catabolism of this antibody.The level of antibody dissociation as well as the rate of antibody catabolism will affect the results of radioimmunotherapy strongly because these factors affect the time interval for which the cells are in contact with the radioisotope. Different B-cell lines display markedly different levels of dissociation. There is some evidence suggesting that antibody dissociation is high with fresh human tumor cells, but further investigation of this point is required.

Processing of antibodies bound to B-cell lymphomas and lymphoblastoid cell lines.

Previous experiments demonstrated that some human B-cell lymphoma cell lines were unusual in that antibodies bound to the cell surface dissociated at high levels. This did not occur with non-B-cell hematologic tumors or with carcinomas. In this study, additional B-cell lymphoma and lymphoblastoid (Epstein-Barr virus-transformed) cell lines were tested. The antibodies selected for most experiments, MA103 and anti-CD45, react with relatively high avidity to the cell surface. Antibodies to CD19, CD20, and CD22 also were tested on certain cell lines. The antibodies were labeled with 125I. After binding to the surface of viable cells, unbound antibody was washed away, and the fate of the bound antibody was investigated for 2-3 days. Of the eight B-cell lymphomas tested, three had high levels of dissociation, two had low levels of dissociation, and three had intermediate levels of dissociation. The six lymphoblastoid cell lines had only slightly elevated levels of dissociation, relative to non-B cell lines. Sublines of Raji and Ramos cells were identified that varied greatly in the level of antibody dissociation. The level of dissociation from lymphomas was correlated with the tendency of the cell lines to cluster, with single cells displaying less dissociation than clustered cells. However, some exceptions to this correlation were noted. Cell lines such as Ramos, which showed little dissociation of anti-CD20, displayed relatively rapid catabolism of this antibody. The level of antibody dissociation as well as the rate of antibody catabolism will affect the results of radioimmunotherapy strongly because these factors affect the time interval for which the cells are in contact with the radioisotope. Different B-cell lines display markedly different levels of dissociation. There is some evidence suggesting that antibody dissociation is high with fresh human tumor cells, but further investigation of this point is required.

Highly sensitive and specific detection of P-glycoprotein function for haematological and solid tumour cells using a novel nucleic acid stain.

Progress in our understanding of the contribution of P-glycoprotein (P-gp)-mediated resistance to chemotherapy failure in haematological as well as solid tumours has been hampered by the lack of highly sensitive, reliable methods for the detection of P-gp function in fresh human tumour cells. The present study identifies the novel nucleic acid stain SYTO16 as a highly sensitive and specific dye to assess P-gp function. The effect of P-gp is expressed here as the ratio of dye fluorescence (RF) from cells incubated with dye with or without 2 microM of the P-gp inhibitor PSC 833. Using flow cytometric analysis, an RF of 0.9 was found for SYTO16 in the KB3-1 (P-gp-) and 1.6 in KB8 (P-gp+) cells. Three types of patients' cells were studied: (1) in haematopoietic CD34+ cells, which are known to express P-gp, the RF was 6.0 for SYTO16 compared with 2.5 for rhodamine 123 and 1.3 for daunorubicin (mean of five individuals); (2) in acute myeloid leukaemia cells, the RF for SYTO16 was 1.0 in P-gp- and 4.5 in P-gp+ samples; (3) for the first time, we have quantitated P-gp function in fresh human solid tumour (sarcoma) cells. We found, in a P-gp+ leiomyosarcoma, an RF of 16 for SYTO16 and 2.7 for daunorubicin. This means that complete inhibition of P-gp function in these sarcoma cells would lead to an increase of daunorubicin accumulation with 170% compared with 30% in the CD34+ cells. Next, we showed that SYTO16 could be fixed in nuclei by 3.6% formaldehyde treatment, allowing quantification of the nuclear fluorescence on cytospins by laser scanning microscopy. In conclusion, SYTO16 proved to have a combination of favourable properties: it can be excited at 488 nm and has large fluorescence enhancement upon binding to nucleic acids, allowing the use of low, nontoxic (< 10 nM) concentrations. Because the RF for SYTO16 is much higher than for daunorubicin, it can be applied for the determination of P-gp function in relatively small numbers of low-P-gp-expressing tumour cells by laser scanning microscopy. Individual sarcomas were found to have high P-gp function compared with CD34+ cells. This assay may be used to select patients for P-gp modulation protocols.

Modulation of multidrug resistance by cepharanthine in fresh human gastrointestinal tumor cells.

Resistance to doxorubicin (DOX) is mainly due to the effect of P-glycoprotein encoded by the multidrug resistance (MDR) gene. Cepharanthine (CEP) has been shown to circumvent multidrug resistance in P-glycoprotein-expressing cell lines. In the present study, we investigated the augmentation of DOX sensitivity by CEP using an MTT assay, and assessed the correlation between DOX sensitivity and P-glycoprotein expression by flow cytometry, in highly purified fresh human tumor cells obtained from 73 cancer patients. DOX sensitivity was decreased in proportion to P-glycoprotein expression. The cytotoxicity of DOX was increased by CEP in tumor cells possessing low DOX sensitivity. Moreover, there was a significant correlation between the effect of CEP on cytotoxicity and P-glycoprotein expression. Thus, CEP might be able to circumvent DOX resistance in cancer patients.

Tamoxifen Circumvents the Multidrug Resistance in Fresh Human Gastrointestinal Cancer Cells

The resistance to doxorubicin (DOX) is mainly due to the effect of P-glycoprotein encoded by the multidrug resistance-1 (mdr1) gene. Tamoxifen (TAM) has been shown to circumvent multidrug resistance in P-glycoprotein-expressing cell lines. In the present study, we clarified the augmentation of DOX sensitivity by TAM using MTT assay in highly purified fresh human tumor cells obtained from 85 cancer patients. Moreover, the correlation between DOX sensitivity and P-glycoprotein expression was assessed by flow cytometry. DOX sensitivity was decreased in proportion to P-glycoprotein expression. The cytotoxicity of DOX was increased by TAM in tumor cells possessing low DOX sensitivity. Moreover, there was a significant correlation between the effect of TAM on cytotoxicity and P-glycoprotein expression. The concentration of DOX in tumor cells was increased in combined exposure of TAM with DOX, compared with the exposure of DOX alone. Thus, TAM might be able to circumvent DOX-resistance for treatment in cancer patients.

Phospholipase C-γ 1 Is Present on the Surface of Human Colorectal Cancer Cells

Using murine monoclonal antibodies, we have detected the novel signal transducing enzyme, phospholipase C-γ 1, on the surface of cultured human colorectal cancer cells. We have also demonstrated the presence of this enzyme on the surface of fresh human tumor cells derived from primary and metastatic colorectal tumors. This enzyme has previously been described to be associated only with the inner face of the plasma membrane and the cell cytosol. The finding of an enzyme critical to the signal transduction pathway may have important implications for additional functions of this protein.

Chemosensitivity testing of fresh human gastric cancer with highly purified tumour cells using the MTT assay

A major problem associated with the chemosensitivity testing of fresh human tumour cells using the MTT assay is the contamination of nonmalignant cells in the tumour tissues. Highly purified fresh human gastric cancer cells could be obtained from 43 solid tumours and eight malignant ascites for the MTT assay. The success rate of the MTT assay was 87.9% (51 of the 58 cases), and the purity of tumour cells was greater than 90% after separation on Ficoll-Hypaque and Percoll discontinuous gradients in primary, or metastatic lesions, and also ascites. Cisplatin, mitomycin, and doxorubicin were more potent drugs than etoposide and 5-FU against gastric cancer cells. The chemosensitivity in differentiated cancer was equivalent to that in non-differentiated cancer. Twenty of the 51 patients with gastric cancer had evaluable lesions, and they received chemotherapy according to the results of the MTT assay using highly purified tumour cells. A clinical response was obtained in 12 of these 20 patients (response rate: 60.0%; five with complete response, seven with partial response).

Antiproliferative and antitumor activity of the 2-cyanoaziridine compound imexon on tumor cell lines and fresh tumor cells in vitro.

Imexon, a 2-cyanoaziridine, is therapeutic and reverses lymphadenopathy and splenomegaly in the LP-BM5 murine retrovirus-induced immunodeficiency disease (murine AIDS). It can restore chemotherapy-induced immunosuppression. Imexon reduced the incidence of lymphoma in severe combined immune deficient mice inoculated with human lymphocytes. To determine its antitumor activity, we screened imexon against fresh human tumor cells and tumor cell lines. To determine the time-concentration relationships of its cytotoxicity, we studied the effects of imexon on macromolecular synthesis and on the cell cycle. Imexon was incubated at 1-200 micrograms/mL with various tumor cell lines, mitogen-stimulated peripheral blood lymphocytes, and fresh tumor cells. Cell survival, macromolecular synthesis, and cell cycle progression were studied. The concentration of imexon that caused 50% inhibition of growth was under 10 micrograms/mL for lymphocytes stimulated with mitogens. It was about 3-10 micrograms/mL for B-cell lymphomas and both multi-drug-resistant and -sensitive myeloma cell lines. Imexon inhibited four of seven fresh lymphoma and 11 of 16 fresh myeloma biopsy specimens to less than 40% of the control. A 1-hour exposure of lymphoma cells to 50-100 microgram/mL followed by removal of drug by washing the cells and continuing culture resulted in greater than 95% inhibition during the next 48-72 hours. Imexon selectively inhibited protein synthesis during the first 24-48 hours of exposure of lymphoma and myeloma cells. Cells exposed to inhibitory concentrations of imexon were blocked in cell cycle progression. Imexon may be a potentially useful agent in the treatment of malignant disease, particularly lymphoid malignancies, and should be explored further.

A possible clinical application of multicytokine-producing cytotoxic mononuclear cell (MCCM) therapy

When peripheral blood mononuclear cells (PBMC) were incubated with a streptococcal preparation, OK-432, for 24 h, PBMC acquired cytolytic activity against cultured and fresh human tumor cells. Such PBMC were called OK-432-activated mononuclear cells (OK-MC). OK-MC produce several kinds of cytokines such as interferon alpha (IFN alpha), IFN gamma, and tumor growth inhibitory factor (TGIF) both in vitro and in vivo. OK-MC-produced cytokines also inhibited the growth of cultured and fresh human tumor cells. The growth inhibition was examined by human tumor clonogenic assay using a double-layer agar technique. The results indicate that two pathways of anti-tumor activity are induced in OK-MC, i.e., cell-mediated and cytokine-mediated.

Chemosensitivity testing with highly purified fresh human tumour cells with the MTT colorimetric assay

A major problem associated with the succinate dehydrogenase inhibition (SDI) test using tetrazolium dye (MTT) as a cancer chemosensitivity testing is the contamination of non-malignant cells in the tumour tissues. Highly purified fresh human tumour cells from 44 solid tumours and 24 malignant ascites were used for the MTT assay. The purity of tumour cells was greater than 90% after separation on Ficoll-Hypaque and Percoll discontinuous gradients. The OD 570 obtained from tumour cells alone was higher than that from non-malignant cells. The chemosensitivity of tumour cells was distinct from that of non-malignant cells. Moreover, the chemosensitivity of highly purified tumour cells was also distinct from that of non-purified cells just separated from tumour tissues. 31 of the 68 patients had evaluable lesions, and received cancer chemotherapy according to the results of MTT assay using highly purified tumour cells. A clinical response was obtained in 10 of the 31 patients (response rate = 32.3%, 5 complete responses, 5 partial responses).

Correlations between therapeutic response of leukaemias and in-vitro drug-sensitivity assay

To develop the differential staining cytotoxicity (DiSC) assay, an in-vitro drug sensitivity test designed specifically for use with fresh human haematological tumour cells, into a predictive test for response to therapy as well as a tool for identifying new treatment strategies, test results have been correlated with response in patients with haematological malignancies. 22 of 119 tests indicated extreme drug resistance (tumour cell survival >55%) in vitro. None of these patients responded to chemotherapy. The proportion of patients responding to therapy and 50% patient survival rose with in-vitro drug sensitivity. 11 specimens showing extreme drug resistance to the agents prescribed for the patient were subjected to drug sensitivity tests of drugs not prescribed; patients showing some sensitivity to these drugs had better survival than did those with extreme drug resistance to all drugs tested. The DiSC assay can also be used to identify new agents worth testing in clinical trials, and cross resistance profiles of new agents may indicate what combinations might be useful.

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines.

During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) alpha, IFN gamma, interleukin-1 alpha (IL-1 alpha) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h 51Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN alpha, rIFN gamma, rIL-1 alpha and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN alpha, rIFN gamma, or rIL-1 alpha did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.

In vitro Synergistic Activity of 5-Fluorouracil with Low-Dose Ozone against a Chemoresistant Tumor Cell Line and Fresh Human Tumor Cells

We followed the concept that the chemical reactivity of ozone depends upon its oxidative properties. Activated oxygen species are causally involved in toxicity of certain chemotherapeutic drugs. We have tested this prediction in human cell cultures either to overcome chemoresistance and/or to increase chemical cytotoxicity. Our results indicate that ozone in combination with 5-fluorouracil (5-FU) makes a 5-FU-resistant cell line susceptible for the combined treatment modality. Furthermore, ozone acts synergistically or at least additive to chemotherapy in different tumor cell suspensions, derived from the breast and the colon.

Generation of cytotoxic factor by human large granular lymphocytes during interaction with autologous tumor cells: lysis of fresh human tumor cells.

Large granular lymphocytes (LGL) obtained by centrifugation (Percoll gradient) of blood from patients with carcinomatous pleural effusions and solid tumors lysed autologous, freshly isolated tumor cells in a 4-hour 51Cr-release assay. When cocultured with autologous tumor cells, LGL released soluble cytotoxic factor(s), large granular lymphocyte-derived cytotoxic factor (LGL-CF). In contrast, small T lymphocytes were unable to kill autologous tumor cells or to produce a cytotoxic factor. The LGL-CF demonstrated cytotoxicity against autologous fresh tumor cells but also against allogeneic fresh tumor cells in a 48-hour microcytotoxicity assay and in an 18-hour 51Cr-release assay in the presence of dactinomycin. Binding of LGL-CF to autologous tumor cells occurred within 2 hours and reached the maximum by 6 hours; this binding was sufficient to cause subsequent lysis of the target cells without the continued presence of LGL-CF. Neither the supernatants produced by culture of LGL alone nor the lysates of LGL had detectable cytolytic activity. In addition, treatment of LGL with cytochalasin A inhibited both direct cell-mediated autologous tumor lysis and generation of LGL-CF. Production of LGL-CF required active cell metabolism and protein and RNA syntheses in LGL, but not DNA synthesis. Addition of dactinomycin to LGL-CF in assays augmented the lysis. LGL-CF was stable at 56 degrees C, but it was reduced at 70 degrees C and destroyed at 100 degrees C. Treatment of LGL-CF with trypsin or proteinase K reduced or abrogated the lytic effect, respectively, while the lytic effect was not affected by papain, catalase, or superoxide dismutase. These results indicate that during interaction with autologous tumor cells, LGL release a soluble cytotoxic factor that mediates lysis of fresh human tumor cells.