Murine Sarcoma Cell Line Research Articles

Abstract 1096: Enhancing effector function in T cell adoptive cell therapy in murine models of pediatric sarcomas

Abstract New treatment approaches for high-risk pediatric sarcomas are desperately needed. Immunotherapy advances with adoptive cell therapy in hematologic malignancies have not yet translated to pediatric solid tumors including sarcomas. Hallmarks of the immune microenvironment in pediatric sarcomas include the presence of immunosuppressive cytokines and immune cells and a lack of potent T cell activation. We are seeking ways to overcome these barriers to effective adoptive cell therapy in pediatric sarcoma. We are exploring introduction of a novel synthetic co-receptor, CD8a:MyD88, that can augment tumor-specific T cell effector function and potentially improve outcomes in murine models of sarcoma. Tumor-antigen specific T cells or tumor-infiltrating T cell populations are transduced ex vivo to express the CD8a:MyD88 synthetic co-receptor resulting in MyD88 signaling when T cells encounter their cognate MHC I/epitope complex. We have studied the effects of CD8a:MyD88 expression on cytokine production and lytic activity against murine sarcoma cell lines in vitro. Initial experiments utilized the OT1 T cell model (specific to the model antigen chicken ovalbumin) which we introduced into our murine sarcoma cell lines (KPSarc pleomorphic sarcoma and M3-9M rhabdomyosarcoma). Using GFP as a marker of T cell transduction, the production of effector cytokine interferon gamma was measured following T cell activation compared with GFP-negative non-transduced T cells. CD8a:MyD88 expressing T cells expressed higher levels of interferon gamma at basal levels and following antigen-specific activation when co-cultured with Ova-expressing sarcoma cells. Improved tumor-specific lytic function in CD8a:MyD88 expressing T cells was also observed in co-culture experiments compared to untransduced or control vector- transduced T cells. We further hypothesized that skewing T cell differentiation toward an effector phenotype with the addition of interleukin-12 during T cell expansion would increase the anti-tumor effect of the CD8a:MyD88 expressing T cells. Co-culture with our murine sarcoma cell lines showed increased tumor-lytic function in T cells primed with IL-12. We have ongoing experiments studying the in vivo effects of the CD8a:MyD88 expressing T cells in model antigen-expressing murine sarcoma models. In addition, the ability of the CD8a:MyD88 co-receptor to augment the effector function of polyclonal sarcoma infiltrating T cells is being explored. These results will further define the obstacles and potential solutions to successful adoptive T cell therapy for pediatric sarcomas. Citation Format: Jessica P. Sanchez, Eduardo Davila, Brian H. Ladle. Enhancing effector function in T cell adoptive cell therapy in murine models of pediatric sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1096.

Abstract B50: A novel immunocompetent murine model for replicating oncolytic adenoviral therapy

Abstract Oncolytic adenoviruses are able to replicate selectively in cancerous but not normal human cells and are under investigation as potential cancer therapeutics. Unfortunately, mice are poor model systems for therapy with replication competent adenoviruses because murine cells are generally unable to produce infectious viral progeny. In order to model oncolytic adenovirus treatment, researchers have used human tumor xenografts that support viral replication in immunodeficient mice. Because xenografts require an immunodeficient host, xenograft model systems do not reflect a host's adaptive immune response against the virus and cannot model the potential for an immunostimulatory virus to induce an anti-tumor immune response. Using the tumor-selective oncolytic adenovirus TAV-255 previously developed in our laboratory, we find that the murine lung adenocarcinoma cell line LKR-13 supports adenoviral infection and generates infectious viral progeny. Western blotting showed that LKR-13 cells express the coxsackie and adenovirus receptor (CAR) to allow viral infection, and that infected cells express the viral protein E1A. MTT assays, crystal violet staining, and clonogenic assays showed that TAV-255 kills LKR-13 cells in a dose-dependent and time-dependent manner with similar potency and latency of infection as a panel of human cancer cell lines, with more than 50% cell death of murine LKR-13 cells and various human cancer cells (T1, 882 and A549) within 6 days of infection at a multiplicity of infection of 10. It produces infectious progeny from LKR-13 cells at titres similar to other human cancer cell lines. Other tested murine cancer cell lines do not share these features; for example, the CT-26 colon cancer cell line does not express CAR and is resistant TAV-255 infection, while the murine sarcoma cell line F244 is readily infected by adenovirus and produces the viral protein E1A but does not produce infectious progeny. Based on these in-vitro findings, we investigated LKR-13 cells as an in-vivo model system for replicating oncolytic adenoviruses. The LKR-13 cell line is derived from 129/Sv mice, and using fully immunocompetent 129/s4 mice, subcutaneous injection of 5x106 LKR-13 cells led to tumor formation in all injected mice. Intratumoral injection of 3x107 plaque forming units of TAV-255 in established tumors (greater than 200 mm3) caused a significant reduction in tumor growth: Eight days after treatment, tumors in mice treated with TAV-255 were 339 mm3 +/- 26 mm3 (n=3), and tumors in mice treated with PBS were 578 mm3 +/- 49 mm3 (n=5) (p=0.013). This model system represents the first fully immunocompetent mouse model for cancer treatment with replicating oncolytic adenoviruses, and will be useful to study the therapeutic effect of oncolytic adenoviruses in general and particularly immunostimulatory viruses designed to evoke an anti-tumor immune response. Citation Format: Lingzhi Zhang, Farah Hedjran, Christopher Larson, Min Yan, Tony Reid. A novel immunocompetent murine model for replicating oncolytic adenoviral therapy. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B50.

Eupolyphaga sinensis Walker inhibits human chronic myeloid leukemia cell K562 growth by inducing G2–M phase cell cycle arrest and targeting EGFR signaling pathway and in S180 tumor-bearing mice

Eupolyphaga sinensis Walker is not only used as a food to enhance immunity, but is used as a traditional Chinese medicine and is known as the “preferred drug to regulate blood flow”. Previous studies have reported its potential biological activities including anticoagulation, antithrombotic, liver protective effect and antitumor effects. Our results indicated that E. sinensis Walker 70% ethanol extract exhibited anti-tumor effects on S180 (murine sarcoma cell line) cells implanted mice. It effectively inhibited K562 (human chronic myeloid leukemia cell line) cells proliferation and induced G2–M phase arrest accompanying through up-regulation of cyclin B1, cdc2 and down-regulation of cyclin D1, cyclin E1, cdc25c and p53. In addition, it inhibited EGF secretion and EGFR kinase activity. Western blotting analysis indicated that it also inhibited the phosphorylation EGFR and activation of its downstream signaling molecules AKT and ERK. These results suggested that the antitumor mechanism of E. sinensis Walker involved altering the cell cycle and inhibiting EGFR phosphorylation in the EGFR signaling pathway.

Proliferation and Tumorigenesis of a Murine Sarcoma Cell Line in the Absence of DICER1

MicroRNAs are a class of short ~22 nucleotide RNAs predicted to regulate nearly half of all protein coding genes, including many involved in basal cellular processes and organismal development. Although a global reduction in miRNAs is commonly observed in various human tumors, complete loss has not been documented, suggesting an essential function for miRNAs in tumorigenesis. Here we present the finding that transformed or immortalized Dicer1 null somatic cells can be isolated readily in vitro, maintain the characteristics of DICER1-expressing controls and remain stably proliferative. Furthermore, Dicer1 null cells from a sarcoma cell line, though depleted of miRNAs, are competent for tumor formation. Hence, miRNA levels in cancer may be maintained in vivo by a complex stabilizing selection in the intratumoral environment.

Effect of vegf gene knockdown on growth of the murine sarcoma cell line MS-K

The murine sarcoma cell line MS-K was previously established as a Ki-ras-positive cell line. Inoculation of this cell line under the flank of C3H/HeN mice results in the growth of large tumors with well-developed blood vessels within day 30 of transplantation without any metastasis because MS-K cells produce vascular endothelial growth factor (VEGF). To elucidate the role of VEGF in tumor formation in vivo, stable vegf-knockdown-MS-K clones were obtained using plasmid-based knockdown vectors. Interestingly, tumorigenesis was completely suppressed in a vegf-A-knockdown-MS-K clone [designated MS-K (A-KD)]. Proliferation and colony formation capacity of the MS-K (A-KD) cells in a semi-solid medium under low serum conditions was significantly lower than that of control MS-K (SCR) cells; however, the expression of vegf-receptor 1 (vegf-r-1) was not changed. Addition of the recombinant VEGF-A(165) partially restored the colony formation capacity of MS-K (A-KD) cells and caused the phosphorylation of VEGF-r-1 (Flt-1) in MS-K (Normal) cells. Furthermore, tumorigenicity of the vegf-r-1-knockdown-MS-K clone [designated MS-K (R1-KD)] had obviously delayed or strongly suppressed compared with the MS-K (Normal). These results indicate that Vascular endothelial growth factor-A, produced from MS-K, acts as a growth factor for MS-K cells itself and supports tumor formation in vivo by inducing the blood vessel formation.

In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation

The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were monitored by HSV1-TK or by the firefly luciferase reporter gene using HSV1-TK enzyme activity assay and luciferase assay, respectively. The results of dynamic gene expression studies showed that the luciferase gene is an optimal reporter gene for monitoring short-timescale, dynamic transcriptional events mediating a series of Dox inductions, whereas the HSV1-tk is not optimal to achieve this purpose. Furthermore, the enzyme half-life of HSV1-TK in NG4TL4 cells is about 35 h after cycloheximide-induced protein inhibition. On the other hand, the results of an efflux assay of [(131)I] FIAU and [(3)H] GCV revealed that the molecular probe phosphorylated by HSV1-TK can be trapped long term within HSV1-TK stably transformed cells. Therefore, a long half-life radionuclide is not suitable for dynamic gene expression studies. Based on these results, we suggest that the HSV1-TK reporter system is not optimal for monitoring short-timescale dynamic processes such as kinetic gene expression controlled by inducible promoters or a less stable protein with a more rapid turnover due to the limitations of the half-life of the HSV1-TK enzyme and the cellular retention time of their phosphorylated molecular probes.

In vitro and in vivo targeting of different folate receptor-positive cancer cell lines with a novel 99mTc-radiofolate tracer

For the assessment of folate-based radiopharmaceuticals, human nasopharyngeal KB carcinoma cells are traditionally used although nasopharyngeal cancer is rare. On the other hand, the folate receptor (FR) is frequently overexpressed on diverse cancer types, the highest frequency (>90%) being on ovarian carcinomas. The goal of our study was the in vitro and in vivo assessment of different FR-positive human carcinoma cells. In addition, a murine sarcoma cell line was assessed as a pre-clinical alternative to human xenograft models. FR-positive human nasopharyngeal, cervical, ovarian and colorectal cancer cell lines and the transgenic mouse sarcoma (24JK-FBP) cell line were targeted with a novel 99mTc-tricarbonyl folate derivative 2. Comparative in vitro cell binding studies were carried out under standardised folate-deficient conditions. In vivo studies were performed in nude mice and C6 black mice. The in vitro cell experiments revealed only FR-specific binding (unspecific <0.02%), ranging from 3.5% to 52% of complex 2 owing to variable levels of FR expression of the cell lines. In vivo tumour uptake of radiotracer 2 varied less than in vitro. It ranged from 0.66+/-0.17% ID/g (LoVo) through 1.16+/-0.64% ID/g (IGROV-1) and 1.55+/-0.43% ID/g (24JK-FBP) to 2.33+/-0.36% ID/g (KB) 4 h p.i. These pre-clinical studies indicate that in vitro data obtained in FR-positive cancer cells do not necessarily correspond with or predict in vivo radiofolate uptake in corresponding (xeno)grafts. In addition, the murine 24JK-FBP cell line proved to be a valuable pre-clinical alternative to human tumour models.

Evidence of dual function of macrophage migration inhibitory factor relevant to tumor progression and regression

Macrophage migration inhibitory factor (MIF) is known to play an important role in broad-spectrum inflammation and immune responses. To evaluate the role of MIF in tumor growth, we established transgenic (Tg) mice (ICR strain) driven by cytomegalovirus (CMV) enhancer and beta-actin promoter. We inoculated Tg mice in the back with murine sarcoma cell line S-180 cells. The tumor growth rate was more enhanced in Tg mice than in littermate non-Tg mice up to day 9 after tumor inoculation. Surprisingly, most tumors embedded on the back of Tg mice regressed at day 10 after inoculation and eventually disappeared. Tumor volumes of non-Tg mice incessantly increased until death. We reinoculated the Tg mice with S-180 cells, which had been recovered from the first challenge, and found that the tumor cells were completely rejected in all cases. To identify the effector cells that eradicated the tumor cells, we prepared spleen cells from tumor-bearing Tg mice and carried out cell lysis assay. The magnitude of cytolytic activity of spleen cells obtained from Tg mice was significantly higher against S-180 cells, as well as natural killer cell-sensitive YAC-1 cells, than was the activity of cells from non-Tg mice. Furthermore, we observed that CTL activity of Tg mice against S-180 cells was significantly decreased by the deletion of CD8+ T cells or NK cells. On the other hand, the deletion of CD4+ cells minimally affected the cytolytic activity. Taken together, these results suggest that MIF has the potential to promote tumor growth and angiogenesis in the early phase and, by contrast, this protein could activate CD8+ cytotoxic T cells and NK cells, leading to tumor regression.

Efficient chromosomal mapping of a methylcholanthrene-induced tumor antigen by CTL immunoselection.

It has been difficult to genetically map the genes encoding tumor Ags because they arise as a consequence of somatic mutational events. CTL-mediated immunoselection can impose potent immunoselective pressure against tumor cells, resulting in the survival of rare tumor Ag-loss variants. We subjected a heterozygous 3-methylcholanthrene-induced murine sarcoma cell line to CTL immunoselection, selecting for the loss of a tumor-specific Ag, recognized antigen from MCA-induced tumor 1 (Ram1). Several variants eluded CTL recognition by genetic loss of the hemizygously expressed tumor-specific Ag epitope. A frequently observed genetic escape mechanism was spontaneous mitotic recombination resulting in loss of heterozygosity on chromosome 4. Higher density genetic analyses along with functional confirmation with an independently produced chromosome 4 loss of heterozygosity variant positioned the Ram1 locus to a distal 7.1 cM interval on chromosome 4. This region of the mouse genome is rich in tumor-modifier genes and this positioning of Ram1 may thus provide insight into the genetic basis of 3-methycholanthrene-induced tumor Ags.

Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector.

Our recent studies using IL-12 protein or fibroblasts genetically engineered to secrete IL-12 have demonstrated profound antitumor effects of IL-12 in murine models. The antitumor effects of local, high level IL-12 expression were examined using a retroviral vector, which can express both IL-12 subunits (p35 and p40) and the neomycin phosphotransferase (Neo)-marker gene from a polycistronic message utilizing internal ribosome entry site sequences. All animals intradermally (i.d.) receiving MCA207 murine sarcoma cell line nontransfected or Neo-transfected had progressively growing tumor, whereas all animals injected with MCA207 transfected with IL-12 were tumor free and were subsequently determined to be immune to a rechallenge of nontransfected MCA207 i.d. Similar results were obtained in experiments using the poorly immunogenic MCA102 murine sarcoma cell line. The inoculation of live MCA207-IL-12 tumor cells also caused the regression of contralateral nontransfected MCA207 inoculated either at the same time (80% protection) or up to 3 days before (33% protection) to the therapeutic tumor inoculation. In vivo depletion studies suggest that NK cells and IFN-gamma play important roles in the development of the early phase of the antitumor response, but that T cells (both CD4+ and CD8+) play the major role in the subsequent events, leading to long-term immunity. The potent antitumor effects observed for paracrine gene-delivered administration of IL-12 have thus been confirmed for multiple tumor cell types and in multiple murine strains. We believe that these results support the feasibility of IL-12 gene therapy for the treatment of human cancer.

A nonimmunogenic sarcoma transduced with the cDNA for interferon gamma elicits CD8+ T cells against the wild-type tumor: correlation with antigen presentation capability.

To be recognized by CD8+ T lymphocytes, target cells must process and present peptide antigens in the context of major histocompatibility complex (MHC) class I molecules. The nonimmunogenic, low class I-expressing, methylcholanthrene (MCA)-induced murine sarcoma cell line, MCA 101, is a poor presenter of endogenously generated viral antigens to specific CD8+ T lymphocytes and cannot be used to generate tumor infiltrating lymphocytes (TIL). Since interferon gamma (IFN-gamma) has been shown to upregulate three sets of molecules important for antigen processing and presentation, we retrovirally transduced wild-type MCA 101 (101.WT) tumor with the mIFN-gamma cDNA to create the 101.NAT cell line. Unlike 101.WT, some clones of retrovirally transduced 101.NAT tumor expressed high levels of class I, and could be used to generate CD8+ TIL. More importantly, these TIL were therapeutic in vivo against established pulmonary metastases from the wild-type tumor. Although not uniformly cytotoxic amongst several separate cultures, these TIL did specifically release cytokines (IFN-gamma and tumor necrosis factor-alpha) in response to 101.WT targets. 101.WT's antigen presentation deficit was also reversed by gene modification with mIFN-gamma cDNA. 101.NAT had a greatly improved capacity to present viral antigens to CD8+ cytotoxic T lymphocytes. These findings show that a nonimmunogenic tumor, incapable of generating a CD8+ T cell immune response, could be gene-modified to generate a therapeutically useful immune response against the wild-type tumor. This strategy may be useful in developing treatments for tumor histologies not thought to be susceptible to T cell-based immunotherapy.

The rate of catabolism of dATP to deoxyadenosine during the growth of different cell lines in vitro

A method has been developed for the determination of the rate of formation or deoxyadenosine from dATP in cultured cell lines. The lowest rate was found in the T-cell-derived Molt cell line while it was about 70-fold higher in Balb c/3T3 mouse fibroblasts. In the B-cell-derived Raji cells and in the murine sarcoma cell line SEWA it had intermediary values. It is concluded that in some cell types like the 3T3-cells the catabolism of dATP to deoxyadenosine may have a significant regulatory eftect on the cellular content of dATP.

Cross-resistance pattern of cell lines selected for resistance towards different cytotoxic drugs to membrane-toxic phospholipids in vitro

The synthetic ether lipids ET-18-OCH3 and BM41.440 and a derivative, hexadecylphosphocholine, were tested for inhibition of [3H]-thymidine uptake into a Chinese hamster ovarian cell line (AUXBl) and its multidrug-resistant subline selected for colchicine resistance (CHRC5). The activity of all three compounds against the multidrug-resistant subline was equal to or higher than that against the parent line. The same result was found for their activity against a human leukemic lymphoblastic cell line (CEM/O) and its methotrexate-resistant subline (CEM/MTX). In contrast, two multidrug-resistant cell lines selected for resistance to Adriamycin, the mouse leukemia cell line P388/ADR and the murine sarcoma cell line S180/ADR, expressed modest cross-resistance to the lipids as measured by thymidine uptake. Experiments performed using the trypan-blue dye-exclusion assay yielded comparable results, although this system revealed a slightly different sensitivity in showing the cytotoxicity of the drugs. By this assay, modest cross-resistance for ET-18-OCH3 and BM41.440 to Adriamycin was found only after 24 h incubation and decreased after 48 h incubation, with almost equal sensitivity to both drugs being shown by the parental (P388/W) and resistant lines (P388/ADR). Furthermore, findings from a human tumor-cloning assay were in accordance with these data, although they did not indicate cross-resistance for the P388/ADR cell line. These results suggest that certain ether lipids and derivatives might represent valuable anticancer drugs warranting further study in the setting of resistant disease.

Antitumour efficacy of human recombinant interleukin 2

Experiments were designed to test what percentage of experimental MC-induced murine sarcomas were sensitive to the local tumour inhibitory effect of IL-2 and whether any correlation existed between the sensitivity of these sarcomas to the immunotherapeutic effect of IL-2 and their susceptibility to the cytolytic effect of IL-2-activated killer cells. It was found that the sensitivity of MC-induced sarcomas to local IL-2 immunotherapy was a general phenomenon. Repeated peri-tumoural injections of RIL-2 inhibited the growth of five (MC11, MC13, MC14, MC15, MC16) out of six sarcomas in syngeneic mice. The sixth murine sarcoma (MC12) was found to be resistant to the tumour inhibitory effect of IL-2. Similarly, five (MC11, MC13, MC14, MC15, MC16) out of six murine sarcoma cell lines were sensitive to the cytolytic effect of IL-2-activated syngeneic killer spleen cells when examined in vitro, whereas the sixth (MC12) sarcoma cell line was resistant. These results suggest that LAK cells represent the effector cell mechanism responsible for the anti-tumour efficacy of local IL-2 immunotherapy and that in vitro testing of sensitivity to the LAK cell-mediated cytolysis may be used to detect tumours responding to IL-2 immunotherapy in vivo.

The anti-tumour efficacy of human recombinant interleukin 2. Correlation between sensitivity of tumours to the cytolytic effect of LAK cells in vitro and their susceptibility to interleukin 2 immunotherapy in vivo.

Experiments were designed to test what percentage of experimental MC-induced murine sarcomas were sensitive to the local tumour inhibitory effect of IL-2 and whether any correlation existed between the sensitivity of these sarcomas to the immunotherapeutic effect of IL-2 and their susceptibility to the cytolytic effect of IL-2-activated killer cells. It was found that the sensitivity of MC-induced sarcomas to local IL-2 immunotherapy was a general phenomenon. Repeated peri-tumoural injections of RIL-2 inhibited the growth of five (MC11, MC13, MC14, MC15, MC16) out of six sarcomas in syngeneic mice. The sixth murine sarcoma (MC12) was found to be resistant to the tumour inhibitory effect of IL-2. Similarly, five (MC11, MC13, MC14, MC15, MC16) out of six murine sarcoma cell lines were sensitive to the cytolytic effect of IL-2-activated syngeneic killer spleen cells when examined in vitro, whereas the sixth (MC12) sarcoma cell line was resistant. These results suggest that LAK cells represent the effector cell mechanism responsible for the anti-tumour efficacy of local IL-2 immunotherapy and that in vitro testing of sensitivity to the LAK cell-mediated cytolysis may be used to detect tumours responding to IL-2 immunotherapy in vivo.

Drug resistance of hypoxic tumour cells in vitro

In 1959 Thomlinson and Gray postulated that hypoxic cells might exist in the larger solid tumours ( 13), and some evidence to support this has been obtained in trials of hyperbaric oxygen (3). Hypoxic cells in solid tumours, however, appear to be not only relatively resistant to the cytotoxic effect of ionizing radiation, but also to some chemotherapeutic agents (12). Cytotoxic drugs which may be at least as active under hypoxic as under euoxic conditions are therefore of considerable clinical interest. Lin et al. (8) postulated that bioreductive drugs like Mitomycin C should have a particular toxicity for hypoxic tumours. The decreased oxygen tension in hypoxic tumour cells should create conditions conducive to reductive processes thought to be necessary for activation of Mitomycin C. The reduction of the benzoquinone ring of the Mitomycin molecule to dihydrobenzoquinone has been proposed as an essential step for the biological activity of this drug (8), and it has therefore been classed as a “bioreductive alkylating agent”. Using the murine sarcoma cell lines EMT6 and S180, Kennedy et al. (7) have demonstrated that Mitomycin C is preferentially activated and metabolized by hypoxic murine tumour cells. We have studied the cytotoxic effect ofMitomycin C on human carcinomas under euoxic and hypoxic conditions in the tumour clonogenic assay (HTCA) of Hamburger et al. (6). We also measured the rate of metabolism of Mitomycin C by EMT6 cells and the human colon tumour cell line WIDR, under both euoxic and hypoxic conditions. Additionally we have studied the cytotoxic effect of Bisantrene, a new anthracene bishydrazone derivative, on human carcinomas under euoxic and hypoxic conditions in the HTCA. This drug has shown antitumour activity in phase I trials (1, 10, 14) and in the HTCA (1). In contrast to Mitomycin C or Adriamycin, the drug has no quinone ring and in cardiac studies carried out in beagle dogs, it showed no cardiotoxicity (11). In addition to the clonogenic experiments, the induction of protein associated DNA breaks by Bisantrene has also been investigated under euoxic and hypoxic conditions in the murine L12 10 cell line, using the alkaline elution technique (2). For our experiments, cells were rendered hypoxic by gassing the suspension containing the medium and 10% serum in completely gas tight tubes, designed to obtain and maintain

Synthesis of adriamycin-coupled polyglutaraldehyde microspheres and evaluation of their cytostatic activity.

Adriamycin was coupled to polyglutaraldehyde microspheres having an average diameter of 4500 A. The coupled microspheres remained stable during incubation with cells. Full cytostatic activity was observed when the coupled adriamycin was tested with murine or human leukemia and murine sarcoma cell lines. A 10-fold increase in sensitivity was obtained with drug-resistant human leukemia cell lines. Repeated use of the coupled microspheres in the cytostatic assays did not increase their activity, indicating that these complexes can be recycled. The results suggest that coupled adriamycin sufficiently perturbs the plasma membrane to lead to cytostatic activity. It is proposed that this mode of drug delivery provides multiple and repetitious sites for drug-cell interactions. In addition, the drug-polymer complexes may overcome those forms of resistance that are the result of decreased drug binding at the cell surface.