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Biomedical subjects

O Fodstad

Publications and source records attributed to O Fodstad.

At least 37 records · Page 2Linked to original sources
PubMed1999

Antitumor activity of P-4055 (elaidic acid-cytarabine) compared to cytarabine in metastatic and s.c. human tumor xenograft models.

The antineoplastic efficacy of P-4055, a 5'-elaidic acid (C18:1, unsaturated fatty acid) ester of cytarabine, a nucleoside antimetabolite frequently used in the treatment of hematological malignancies, was examined in several in vivo models for human cancer. In initial dose-finding studies in nude mice, the efficacy of P-4055 was highest when using schedules with repeated daily doses. In a Raji Burkitt's lymphoma leptomeningeal carcinomatosis model in nude rats, the control cytarabine- and saline-treated animals (five in each group) had a mean survival time of 13.2 days, whereas treatment with P-4055 resulted in three of five long-time survivors (>70 days). In a systemic Raji leukemia model in nude mice, 8 of 10 of the P-4055-treated animals survived (>80 days), compared with none of the cytarabine-treated animals (mean survival time, 34.2 days). In s.c. xenograft models, the effects of maximum tolerated doses of P-4055 and cytarabine, given in four weekly cycles of daily bolus i.v. injections for 5 subsequent days, against seven tumors (three melanomas, one lung adenocarcinoma, one breast cancer, and two osteogenic sarcomas) were investigated. P-4055 induced partial or complete tumor regression of the lung carcinoma, as well as of all three malignant melanomas. In two of the melanomas the activity was highly superior to that of cytarabine, and both P-4055 and cytarabine were, in general, more effective than several clinically established drugs previously tested in the same tumor models. In in vitro studies, inhibitors of nucleoside carrier-dependent transport, nitrobenzylmercaptopurine riboside and dipyridamol, reduced strongly the cellular sensitivity to cytarabine, but not to P-4055, indicating that P-4055 uses an alternative/additional mechanism of internalization into the cell compared with cytarabine. The results explain, at least in part, the observed differences between the two compounds in in vivo efficacy, and together the data strongly support the evaluation of P-4055 in clinical studies.

Animals↗
PubMed1999

Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.

The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.

Cytosol↗
PubMed1999

Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase-polymerase chain reaction assays used to detect micrometastatic tumor cells.

In many recent publications, it has been claimed that reverse transcriptase-polymerase chain reaction (RT-PCR) assays involving genes with tissue-restricted expression can be used for specific and sensitive detection of cancer cells in blood, bone marrow and lymph nodes. Many different target mRNAs have been evaluated for such purposes. One of the most extensively studied genes, CK19, is predominantly expressed in cells of epithelial origin and normally not at detectable levels in hematopoietic or lymphatic tissues. Based on previous reports on CK19 we wanted to establish a useful assay for detection of micrometastatic cells. RNA and DNA specimens extracted from peripheral blood nucleated cells of healthy volunteers, as well as cell lines positive and negative for CK19 expression, were used in nested RT-PCR assays. Using previously published primers, we found a novel pseudogene that shows a high degree of identity with the CK19 gene sequence, except for differences caused by 3 small deletions and a number of point mutations, resulting in termination codons and frameshifts. The gene has therefore no coding potential. Importantly, published primer sequences and reaction conditions used by several other groups to detect CK19 mRNA may have led to the amplification of this pseudogene. The data illustrate one of the problems that must be addressed in validating RT-PCR assays for micrometastasis detection, and it is suggested that previous work using CK19 as a marker should be reassessed in view of the present finding.

Artifacts↗
PubMed1999

Superior therapeutic efficacy of N-L-leucyl-doxorubicin versus doxorubicin in human melanoma xenografts correlates with higher tumour concentrations of free drug.

N-L-leucyl-doxorubicin (Leu-DOX), a prodrug of doxorubicin (DOX), has previously shown antitumour activity against human ovarian, breast and lung carcinomas in nude mice. In the present study, the efficacy of Leu-DOX was compared with free DOX in inhibiting the growth of four DOX-sensitive and -resistant malignant melanoma xenografts. In an attempt to elucidate mechanisms underlying any differential effect, a sensitive high-performance liquid chromatography (HPLC) method was established for measuring plasma and tumour concentrations of the two drugs and their main metabolites. Leu-DOX was more effective than free DOX in inhibiting xenograft growth. At equitoxic intravenous doses of Leu-DOX (28 mg/kg) and DOX (8 mg/kg) administered to tumour-bearing nude mice, comparable levels of DOX were found in plasma, whereas differences were seen in tumour tissue concentrations. Thus, in animals carrying highly sensitive (LOX) and resistant (THX) melanomas, higher tumour concentrations of free DOX were observed in the Leu-DOX treated group from 24 up to 240 h after drug injection. Notably, the difference in drug-induced tumour growth inhibition correlated with differences in tumour exposure to free DOX, assessed as area under the curve (AUC) calculated over the first 48 h. In conclusion, the results confirm the prodrug nature of Leu-DOX and provide a possible explanation for its increased antitumour efficacy.

Animals↗
PubMed1999

Sensitive fluorescent in situ hybridisation method for the characterisation of breast cancer cells in bone marrow aspirates.

AIM: The presence of malignant cells in the blood and bone marrow of patients with cancer at the time of surgery may be indicative of early relapse. In addition to their numbers, the phenotypes of the micrometastatic cells might be essential in determining whether overt metastases will develop. This study aimed to establish a sensitive method for the detection and characterisation of malignant cells present in bone marrow. METHODS: In spiking experiments, SKBR3 cells were mixed with mononuclear cells in known proportions to mimic bone marrow samples with micrometastatic cells. Tumour cells were extracted using SAM-M450 Dynabeads coupled to the MOC-31 anti-epithelial antibody, and were further analysed for amplification of erbB2 and int2 by fluorescent in situ hybridisation (FISH). erbB2 and int2 copy numbers were also determined in 15 primary breast cancers, and bone marrow samples from patients with amplification were analysed for micrometastatic cells by immunomagnetic enrichment and FISH. RESULTS: In model experiments, cells with amplification could be detected in bead selected fractions when ratios of tumour cells (SKBR3) to mononuclear cells were as low as 10:10(7). Among the tumour samples, eight showed increased copy numbers of erbB2 and/or int2, and three of these patients had detectable numbers of tumour cells in their bone marrow: 4000, 540, and 26 tumour cells/10(7) mononuclear cells, respectively. The patient with 540 tumour cells/10(7) mononuclear cells showed high level amplification of erbB2 and suffered from a particularly aggressive disease, whereas the patient with 4000 tumour cells/10(7) mononuclear cells had favourable disease progression. CONCLUSION: These results demonstrate the feasibility and advantage of combining immunomagnetic selection and FISH characterisation of cancer cells in bone marrow samples. It is possible that molecular characterisation of such cells could provide prognostically valuable information.

Biopsy, Needle↗
PubMed1999

Growth of precultured human glioma specimens in nude rat brain.

OBJECT: The aim of this study was to develop an improved animal model for brain tumor study. The need for better and more relevant brain tumor models is generally acknowledged. Glioma tissue can be cultured directly from the biopsy specimen as tumor spheroids. Using such precultured tissue, a new in vivo model for studying human gliomas was established. METHODS: Precultured small tumor spheroids (< 300 microm) prepared from cell lines or tumor biopsy fragments were injected into the brains of immunodeficient rats by using a 5-microl Hamilton syringe that had a piston in the needle. Tumors could be established by injecting a single spheroid derived from the U-87MG cell line, whereas inoculation of 10 spheroids resulted in a tumor take comparable to that attained with injection of 10(6) single cells. Biopsy specimens obtained from six patients who underwent surgery for glioblastoma multiforme were cultured as organotypic spheroids for 11 to 18 days before inoculation into the rats. The animals were killed 3 months after spheroid implantation. Microscopic examination revealed tumor growth in 87.5 to 100% of the animals inoculated with tumor spheroids from all but one of the tumor biopsy specimens. Extensive invasion and cell migration along the nerve tracts of the corpus callosum was found in tumors that originated from four of the six biopsy specimens. CONCLUSIONS: This approach, in which spheroids from precultured biopsy specimens are injected into the brains of immunodeficient animals, provides new means for experimental studies of human malignant brain tumors in a clinically relevant animal model.

Animals↗
PubMed1998

Intracellular metabolism of a 2'-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis.

The uptake and cellular metabolism of a fluorescein-labelled synthetic ribozyme stabilized by 2'- O -methyl modification and a 3' inverted thymidine have been studied, employing capillary gel electrophoresis as a novel and efficient analytical method. After internalization by DOTAP transfection, electrophoretic peaks of intact ribozyme and different degradation products were easily resolved and the amount of intracellular intact ribozyme was quantified to >10(7) molecules/cell at the peak value after 4 h transfection. On further incubation the amount of intracellular intact ribozyme decreased due to both degradation and efflux from the cell. However, even after 48 h incubation there were still >10(6) intact ribozyme molecules/cell. Clear differences both in uptake and in metabolism were seen when comparing DOTAP transfection with the uptake of free ribozyme. Fluorescence microscopy studies indicated that the ribozyme was mainly localized in intracellular granules, probably not accessible to target mRNA. This implies that agents able to release the intact ribozyme from intracellular vesicles into the cytosol should have a considerable potential for increasing the biological effects of synthetic ribozymes.

Base Sequence↗
PubMed1998

Invasion potential and N-acetylgalactosamine expression in a human melanoma model.

Reactivity of the N-acetylgalactosamine-binding Helix pomatia agglutinin (HPA) in tumours has been associated with poor prognosis and metastasis development. In our LOX/FEMX-I human melanoma model, the binding of HPA correlates with experimental lung metastasis formation in athymic nude mice. In the present study, the metastatic potential of 2 human melanoma cell lines (LOX and FEMX-I) was assessed in relation to carbohydrate and invasive phenotype. Immunocytological and invasion assays highlighted significant differences between these 2 cell lines. Immuno-cytochemical analysis confirmed the widespread expression of HPA-binding glycoconjugates on LOX but not FEMX-I cells. One of these HPA-binding glycoconjugates, the Tn antigen, was expressed highly on the surface of LOX cells but only weakly in the cytoplasm of FEMX-I cells. The sialyl Tn antigen was expressed in FEMX-I but not in LOX cells. There was no difference between the cell lines in adhesion/rate of trapping in athymic nude mouse lung tissues. In Matrigel invasion assays, LOX cells demonstrated an invasion potential more than 6 times greater than that observed with FEMX-I cells. Matrigel invasion of LOX cells was inhibited after incubation with HPA (89%) compared to controls with HPA and GalNAc blocking sugar or without HPA (p < 0.0005 at 5 df). In contrast, there was no inhibitory effect with the anti-Tn antibody IE3. Invasion of FEMX-I cells was not affected by the lectin and the IE3 antibody. Immuno-cytochemical analysis revealed expression of the terminal galactose- and polylactosamine-binding lectin galectin 3 (Mac-2) in these melanoma cell lines. Expression of both the lectin and its receptor may be a contributory feature in the pulmonary invasion of LOX melanoma cells. Overall, our findings suggest that HPA-binding glycoconjugates other than the alphaGalNAc-O-Ser/Thr of the Tn antigen may be important in the extracellular matrix invasion of LOX melanoma cells.

Acetylgalactosamine↗
PubMed1998

The antitumour activity of the prodrug N-L-leucyl-doxorubicin and its parent compound doxorubicin in human tumour xenografts.

The antitumour activity of the investigational agent N-L-leucyl-doxorubicin (Leu-DOX) was compared with that of doxorubicin (DOX) in human tumour xenografts growing subcutaneously in athymic nude mice. Leu-DOX was developed as a prodrug of DOX, and may be converted into the clinically active parent compound by hydrolytic enzymes present in or on tumour cells. It has been suggested that a better therapeutic index with a reduced cardiac toxicity and higher efficacy might be obtained. Both compounds were administered intravenously weekly for 2 weeks, each at maximum tolerated doses of 8 mg/kg and 28 mg/kg for DOX and Leu-DOX, respectively. The panel of xenografts represented three different tumour types. Leu-DOX showed antitumour activity, defined as tumour growth inhibition > 50% and specific growth delay > 1.0, in 10 of the 16 tumours, including two of five breast, five of seven small cell and three of four non-small cell lung carcinomas. In comparison, DOX was active in one breast, four small cell lung and two lung adenocarcinoma xenografts. In all the DOX sensitive lung tumours, Leu-DOX showed higher efficacy than the parent compound. Based on the results of the present study, and since phase I clinical trials with Leu-DOX have already been performed, phase II clinical evaluation of Leu-DOX in patients with breast and lung cancer is recommended.

Animals↗
PubMed1998

Regulation of tissue-degrading factors and in vitro invasiveness in progression of breast cancer cells.

Hormone-independent growth and invasiveness represent phenotypic properties acquired during early progression of breast cancer. We compared human mammary adenocarcinoma cells, MCF-7, which are estrogen-dependent and poorly metastatic, with the estrogen-independent and highly metastatic subline, MCF7/LCC1, with regard to expression of tissue-degrading factors of the matrix metalloproteinase (MMP)-and urokinase (uPA)-dependent degradative pathways, as well as for their in vitro invasive properties. Both cell lines showed low constitutive mRNA expression of the MMP inhibitor TIMP-1. Baseline expression of TIMP-2 mRNA was also very low in MCF-7 cells, whereas the MCF7/LCC1 level was much higher (approximately 10-fold). Furthermore, both cell lines revealed low constitutive capacity to migrate in an in vitro invasion assay. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nM) induced the mRNAs for TIMP-1 as well as for MMP-1, MMP-9, the uPA receptor, and the uPA inhibitor PAI-1, amongst which only the responses of MMP-9 and PAI-1 were cell-specific. The mRNA levels of MMP-9 and PAI-1 were approximately 10-fold and approximately 15-fold higher in MCF7/LCC1 cells compared to MCF-7 cells. The secretion of immunoreactive PAI-1 was considerably elevated (> 20-fold) in TPA-treated MCF7/LCC1 cells, whereas the TPA-dependent level of 92-kDa MMP-9 was only approximately 2-fold higher in MCF7/LCC1 cells than in MCF-7 cells. In both cell lines treatment with TPA was associated with an increase (approximately 10-fold) in in vitro migration, which in the MCF7/LCC1 cells was significantly attenuated by a reconstituted basement membrane extract (Matrigel). These data suggest that TPA-responsive in vitro invasive properties that are probably associated with PAI-1 expression may co-vary with progression from hormone-dependent to -independent breast cancer.

Breast Neoplasms↗
PubMed1998

Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas.

In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours. Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively. Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples. FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones. However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each. We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between D1S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples. Six samples had amplification of the YAC containing D1S3620 and, in three, 764a1 was also included. Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close. Two samples had high copy numbers of the most distal YACs. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near D1S3620/789f2 and one more distal.

Biomarkers↗
PubMed1998

Human hepatoma cells rich in P-glycoprotein display enhanced in vitro invasive properties compared to P-glycoprotein-poor hepatoma cells.

Whether the phenotypes of drug resistance and metastatic activity in cancer are dependent on each other or not is controversial. We compared in vitro invasive properties of human hepatoma cells resistant to epirubicin and rich in P-glycoprotein (Pgp) (HB8065/R) with the parental epirubicin-sensitive, Pgp-poor cells (HB8065/S). The HB8065/R cells displayed elevated capacity to migrate in a transwell chamber assay (three- to fourfold compared to the HB8065/S cells), both in the absence and presence of a reconstituted basement membrane extract (Matrigel). In the presence of the P-gp inhibitor PSC 833 (1.5 micrograms/ml) the capacity of the HB8065/R cells to cross Matrigel-coated filters was attenuated by approximately 25%. Compared to the HB8065/S cells, the resistant cell line expressed higher level of plasminogen activator inhibitor (PAI)-1 mRNA (approximately threefold), which was reflected by a approximately fivefold increase in secreted PAI-1 immunoactivity (approximately 50 ng/10(6) HB8065/R cells). Furthermore, treatment with PSC 833 was associated with upregulation of PAI-1 mRNA (approximately 3.5-fold) and immunoactivity (approximately twofold) in the HB8065/R cells. Level of tissue inhibitor of metalloproteinases (TIMP)-1 was also significantly increased in the HB8065/R cells compared to the HB8065/S cells, whereas both cell lines showed low constitutive expression of TIMP-2. Levels of TIMPs were not altered by PSC 833. These data suggest that overexpression of Pgp in these hepatoma cells may covariate with the phenotypes of both enhanced in vitro invasiveness and high PAI-1 expression, whether randomly acquired or not.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Differential expression patterns of S100A2, S100A4 and S100A6 during progression of human malignant melanoma.

Three members of the S100 gene family, S100A2, S100A4 and S100A6, have been suggested to be associated with cancer development and metastasis. To study their involvement in the tumorigenesis of human melanoma, we examined the mRNA expression levels of the 3 genes in 45 melanoma metastases and in 20 benign nevi. Interestingly, whereas none of the metastases expressed S100A2 mRNA, and the expression level was low in 6 cell lines established from primary melanomas, all nevi showed moderate to high expression levels. Our results suggest that loss of S100A2 gene expression may be an early event in melanoma development. A significant correlation was found between the expression of S100A6 in melanoma metastases and both the survival time of the patients and the thickness of the corresponding primary tumors. For the S100A4 gene, however, no relationship was found between gene expression and clinical parameters of melanoma malignancy. The observed differences in expression patterns of the 3 S100 genes suggest distinct roles of their products in melanoma tumorigenesis and/or metastasis, and the results encourage studies to evaluate the potential value of using S100A2 and S100A6 expression levels as markers in the clinical management of melanoma.

Blotting, Southern↗
PubMed1997

Ectopic expression of target genes may represent an inherent limitation of RT-PCR assays used for micrometastasis detection: studies on the epithelial glycoprotein gene EGP-2.

Our objective was to develop and study the feasibility of a quantitative, nested reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of micrometastatic, epithelial tumor cells using the epithelial glycoprotein EGP-2 gene as a target. Several carcinoma cell lines and peripheral blood samples of 10 healthy volunteers were screened for levels of EGP-2 mRNA. The assay included EGP-2 competitor molecules, carrying an internal deletion, that had been titrated by limiting dilution. Seven carcinoma cell lines showed a wide spectrum of EGP-2 mRNA expression levels, with the highest values (20-100 molecules/cell) seen in 3 breast-cancer cell lines. Unexpectedly, a consistent low level of EGP-2 mRNA expression (0.0004 molecules/cell) was observed in peripheral blood mononuclear cells, probably representing ectopic non-functional expression. Because of this background level, spiking experiments with T47D breast-carcinoma cells added to blood mononuclear cells exhibited a detection limit that was not better than approximately one tumor cell in 2 x 10(4) normal cells. Together with the considerable variation of EGP-2 transcript levels that is observed in different carcinoma cell lines, the extent of expression in normal blood cells would prevent a reliable estimation of low numbers of carcinoma cells in clinical samples. A similar situation might well apply for other target genes. This emphasizes the need for a critical evaluation of the different steps involved in the methods used for RT-PCR detection of micrometastatic tumor cells.

Adolescent↗
PubMed1997

HMGIC, the gene for an architectural transcription factor, is amplified and rearranged in a subset of human sarcomas.

Amplified segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate amplified regions around the MDM2 and CDK4 genes. Here we show recurrent amplification of a third region encompassing HMGIC, a human architectural transcription factor gene. Reduced amplification frequency of sequences flanking the gene was observed, indicating that inclusion of this third region in the amplicons is also selected for. In three samples only the 5' part of HMGIC was amplified, suggesting preferential loss of the 3' part of the gene preceding or during amplification. In several other samples rearrangement of the gene was observed. Expression analysis showed transcripts of aberrant sizes, lacking 3' sequences, and 3' RACE of one sample revealed replacement of exons 4 and 5 with ectopic sequences. This truncation of HMGIC resembles that reported for translocations of HMGIC in benign tumors, including lipomas, and it is striking that the gene was frequently amplified or rearranged in well differentiated liposarcomas, the malignant counterpart of lipomas. It seems conceivable that high levels of either full length or truncated hmgic could be relevant for the etiology of these tumors.

Amino Acid Sequence↗
PubMed1997

Zilascorb(2H), a new reversible protein synthesis inhibitor: clinical study of an oral preparation.

The new anti-cancer drug zilascorb(2H) has shown promising activity in preclinical models. Its putative mechanism of action is reversible protein synthesis inhibition and long-term treatment is required. As a clinical treatment modality, long-term daily zilascorb(2H) infusions, as used in previous studies, are not regarded feasible. Therefore, an oral formulation of the drug was developed, and pharmacokinetic profile, toxicity and antitumor activity of zilascorb(2H) tablets were studied. Thirteen patients with advanced solid cancer not amenable to established therapy, but with adequate performance status and organ functions, were included. The treatment was given as a daily i.v. zilascorb(2H) infusion for 5 days, followed by zilascorb(2H) tablets twice daily for 3 months. Blood and urine sampling was performed when estimated plasma steady-state level was reached for each formulation, respectively. Analyses of drug concentrations in plasma and urine were performed by high performance liquid chromatography. Zilascorb(2H) in tablet formulation had a bioavailability of 32%, was quickly absorbed and slowly eliminated. Concomitant use of the H2-blocker ranitidine possibly enhanced bioavailability. Zilascorb(2H) was well tolerated. Two patients experienced drug-related fever, disturbing the treatment schedule for one of them. Moderate nausea was reported. One objective response was obtained. The bioavailability of zilascorb(2H) tablets was satisfactory. The principle of oral administration of zilascorb(2H) is feasible for long-term treatment and the side effects are acceptable. The mechanisms of action and the very low toxicity of the drug makes it a candidate for combination with other anticancer agents.

Adolescent↗
PubMed1997

Immunobead filtration: a novel approach for the isolation and propagation of tumor cells.

We have developed a method to facilitate the isolation and expansion of tumor cells from body fluids and tissue biopsies. Antibody-conjugated magnetic beads (immunobeads) were used to isolate tumor cells from blood, bone marrow, ascitic/pleural fluids, and enzyme-digested tissue biopsies. Filtration of the resulting cell suspension through a 20-micron nylon monofilament filter secured to the base of polystyrene 96-well strips purged the bead-rosetting cell fraction of contaminating normal cells and unbound beads. Tumor cells that bound the magnetic beads were retained on the membrane due to their increased size and concentrated into a small area (0.332 cm2), thus maintaining a high cell density. The filters provided a stable and uniform three-dimensional matrix for cell growth, with a total surface area of 1.42 cm2 available for cell attachment. The filters could be easily removed from the base of the 96-well strips to facilitate handling and transfer between culture vessels. Tumor cells grown on the filters could subsequently be harvested using trypsin/EDTA or left in situ for immunostaining with conventional immunohistochemical procedures. Filter-grown cells have shown extended passage in conventional cell culture in six cases. In two of five cases, the orthotopic implantation of confluent filters that contained approximately 10(4) cells/8 x 8 mm filter successfully produced tumors in nude mice after only 4 weeks. Our new approach may be of value in improving the success rate of generating long-term cultures from previously unproductive sources of tumor cells and thus may yield a greater variety of cell lines/strains for the study of malignant disease.

Animals↗