Murine Tumor Lines Research Articles

Effect of culture media on lymphokine-activated killer effector phenotype and lytic capacity.

We compared the ability of murine lymphokine-activated killer (LAK) cells grown in either a serum-supplemented "standard" medium (alpha MEM plus fetal calf serum) or a serum-free medium (AIM-V) to lyse a range of tumour targets. LAK cells grown in either of the media killed a cultured murine tumour line (YAC-1 lymphoma) well and spared syngeneic "self" cells (concanavalin-A-stimulated splenocytes). However, a striking difference was noted in the ability of LAK cells grown in alpha MEM plus fetal calf serum (as opposed to AIM-V) to kill "modified self" cells (trinitrophenol-modified concanavalin A blasts); LAK cells grown in the former always killed modified self cells better than those grown in the latter. This pattern held under a broad range of experimental manipulations and was found to be related to a relative increase in CD3-bearing LAK cells grown in the standard medium. These data suggest that the two media cannot be used interchangeably. This conclusion may have clinical implications for the use of LAK cells, as animal studies have been done using LAK cells generated in serum-containing medium and clinical studies have used LAK cells generated in serum-free medium.

Interleukin-6 production by murine macrophage cell lines P388D1 and J774A.1: Stimulation requirements and kinetics

The murine macrophage tumor lines, P388D1 and J774A.1 were stimulated with LPS, IL-6, IL-1α, TNF-α, IFN-γ, PMA, or combinations of these reagents to induce the production of IL-6, IL-1, and TNF-α. Using Northern blot analysis and bioassays for the detection of cytokine production, it was noted that identical stimuli induced all three inflammatory mediators. However, unlike fibroblasts or endothelial cells, neither P388D1 nor J774A.1 macrophages respond to IL-1α or TNF-α by producing IL-6. The results imply that these cytokines are not autoregulatory for macrophages and may be tissue-specific in their stimulatory effects. IFN-γ and PMA synergize with LPS in the production of IL-6 as well as IL-1 and TNF-α. These observations suggest that IFN-γ may mediate amplification pathways important in the production of inflammatory mediators. Kinetic studies on the transcription of mRNA and secretion of IL-6, IL-1, and TNF-α revealed that TNF-α mRNA transcription and cytokine production occur very rapidly. TNF-α mRNA accumulation peaked 1–2 hr after stimulation and maximum concentrations of cytokine are found in supernatants collected after 2–4 hr of culture. IL-6 and IL-1α mRNA accumulation peaked simultaneously after 4–8 hr. However, IL-6 bioactivity peaks between 8 and 12 hr whereas maximum concentrations of IL-1 bioactivity are not detected in supernatants from stimulated cells collected prior to 12 hr of culture. Thus even though TNF-α production precedes that of IL-1 and IL-6, and stimulates IL-6 production in fibroblasts, there is no evidence that TNF-α acts to amplify the production of IL-6 or IL-1 by murine macrophage cell lines. The results suggest differential regulation of IL-6 expression between fibroblasts and macrophages.

Combination therapy of ACNU and ifosfamide in tumor bearing mice with M2661 breast cancer, B16 malignant melanoma or C38 colon cancer

Three murine tumor lines, B 16 melanoma, C 38 colon cancer and M 2661 breast cancer, were used to evaluate the therapeutic efficacy of the drug combination ACNU and ifosfamide. For each tumor type five treatment groups of 12 mice each were studied, which respectively received 16.5 mg/kg ACNU, 150 mg/kg ifosfamide, 33 mg/kg ACNU, 300 mg/kg ifosfamide or 16.5 mg/kg ACNU + 150 mg/kg ifosfamide. One group served as controls. Growth delay was measured as the endpoint. In the B 16 and C 38 tumor lines both drugs were active and showed additive antitumor effects. However, no synergism was observed. Neither ACNU or ifosfamide or the combination of both had any activity against the M 2661 tumor line.

Nuclear Thiols: Technical Limitations on the Determination of Endogenous Nuclear Glutathione and the Potential Importance of Sulfhydryl Proteins

Significant discrepancies were found between the values for glutathione levels determined by the Tietze enzymatic assay and those measured by labeling with monobromobimane followed by HPLC analysis when these methods were applied to proliferating and quiescent cells of the 66 murine mammary tumor line depleted of glutathione by buthionine sulfoximine or to nuclei prepared from these cells by permeabilization with Nonident detergent. The probable origin of the discrepancy was traced to the presence of acid-soluble sulfhydryl proteins in the extracts which are thought to lead to erroneous values in the Tietze assay method. Using the monobromobimane-HPLC method it was found that the low-molecular-weight thiol levels in nuclei prepared by detergent permeabilization equilibrate in less than 1 min with the permeabilizing medium, indicating that (i) endogenous nuclear glutathione levels cannot be determined reliably using conventional methods of cellular disruption and (ii) the endogenous nuclear glutathione level is likely to be the same as the cytoplasmic value. The levels of protein sulfhydryl associated with the nuclear preparations were found to be of the same magnitude as the cytoplasmic GSH level and must therefore be considered a potentially significant source of thiol capable of repairing DNA radicals.

Inhibition of Implantation of Murine Bladder Tumor by Thiotepa in Cauterized Bladder

This study was designed to determine the role of immediate intravesical instillation of single dose thiotepa post transurethral resection of bladder tumor in the prevention of recurrence by tumor implantation, using murine bladder tumor line 2 and 201 C3H/He mice. Previous studies have suggested implantation may take place as early as the first hour and reach its maximum in 24 hours after resection of bladder tumor. An in vitro dose response curve of MBT2 to thiotepa was established by treatment with various concentrations of thiotepa of 0.00, 0.01, 0.21, 0.44, and 1.91mg./ml. In a group of 201 mice, the bladder was catheterized with a 24G angiocatheter, and a fine copper wire was inserted through the lumen. The bladder was cauterized by touching the wire with a Bovie coagulator for four seconds at the lowest setting. All bladders were instilled with 1 × 106 cells of murine bladder tumor line 2, followed by instillation of 1.91mg./ml. of thiotepa with various time delays per treatment group. The bladder implantation rates were 30.4% (17/56), 3.4% (2/59), 6.5% (2/31) and 26.9% (7/26) in the control, immediate, one-hour delay and 24-hour delay groups, respectively. The urethral implantation rates were 21.4% (12/56), 0% (0/59), 6.5% (2/31) and 0% (2/26), respectively. The overall implantation rates (bladder, urethra, or both) were 42.9% (24/56), 3.4% (2/59), 6.5% (2/31) and 25.9% (7/27). respectively. Implantation rates were significantly higher in the control and 24-hour delay groups than in the immediate and one-hour instillation groups (p < 0.05, Fisher Exact Test). We conclude from this animal model that intravesical instillation of single dose thiotepa, to be effective, should be initiated within the first hour after tumor resection, since it dramatically decreased the incidence of bladder and urethral implantation.

Inherent adriamycin resistance in a murine tumour line: circumvention with verapamil and norverapamil.

Inherent adriamycin resistance in a murine tumour line: circumvention with verapamil and norverapamil

Unstable Expression of E‐Cadherin Adhesion Molecules in Metastatic Ovarian Tumor Cells

E‐Cadherin is a member of the cadherin family, which plays a key role in intercellular adhesion in various tumors as well as in normal tissues. Here, we examined the expression of this adhesion molecule in a murine ovarian tumor line OV2944, whose sublines show different degrees of spontaneous metastasis from subcutaneous sites; sublines LM‐1 and LM‐3 exhibit a low metastatic activity but a variant subline HM‐1 has a high metastatic activity. When the expression of E‐cadherin in these cells was examined by immunoblot analysis, the highly metastatic HM‐1 cells was found to express an extremely small amount of this molecule, as compared with a high level of E‐cadherin expression in the weakly metastatic LM‐1 and LM‐3 cells. Northern blot analysis showed that the amount of tanscripts from the E‐cadherin gene is proportional to the amount of proteins detected in these cells. Immunofluorescence staining revealed that cells of the highly metastatic line were heterogeneous, that is, their cultures contained both E‐cadherin‐positive and negative cells. In contrast, cells of the weakly metastatic lines homogeneously expressed E‐cadherin. When the highly metastatic line was subcloned, all the subclones consisted of E‐cadherin‐positive and negative cells. These results suggest that the expression of E‐cadherin gene is not stably controlled in the highly metastatic line.

The role of oxidant injury in tumor cell sensitivity to recombinant human tumor necrosis factor in vivo. Implications for mechanisms of action.

The intracellular glutathione levels of two human tumor lines and seven murine tumor lines were determined in order to investigate the role of oxidant injury in tumor cell sensitivity to human rTNF (rhTNF). Correlations were found between high intracellular glutathione levels and in vivo tumor resistance to rhTNF, and on the other hand, low glutathione levels and rhTNF sensitivity. The transplantable murine fibrosarcoma, Meth A, a TNF-sensitive line in vivo, was less sensitive to rhTNF and host toxicity was reduced when the hosts were pretreated with uric acid, a major reactive oxygen scavenger in humans and certain other primates. Conversely, pretreatment of the tumor-bearing hosts with DL-buthionine-(S,R)-sulfoximine, an inhibitor of GSH biosynthesis, resulted in an increased sensitivity of Meth A to rhTNF. This effect was not limited to tumor-bearing mice, as rats pretreated with diethyl maleate, a compound which irreversibly binds glutathione, were more sensitive to rhTNF toxicity than control rats. On the other hand, pretreatment with N-acetyl cysteine, an oxidant scavenger, reduced the toxicity of rhTNF treatment in rats. The data are consistent with the hypothesis that tumor cell sensitivity to rhTNF in vivo is dependent on its capacity to buffer oxidative attack. In addition, host toxicity is also related to the production of reactive oxygen species. Activated effector cells such as granulocytes and macrophages are hypothesized to produce most of this damage by their respiratory burst and oxidant release, although the direct action of rhTNF may also contribute to oxidative injury in vivo.

Transiently Elevated Levels of c-fosand c-mycOncogene Messenger Ribonucleic Acids in Cultured Murine Leydig Tumor Cells after Addition of Human Chorionic Gonadotropin

The gonadotropic hormones LH and human CG (hCG) normally function to stimulate steroidogenesis in testicular and ovarian cells through receptor-mediated activation of adenylate cyclase. These hormones are also important in regulating the development and growth of responsive cells. Such regulation requires tightly controlled gene expression. Herein we demonstrate that hCG induces increases in mRNAs encoding the competence oncogenes c-fos and c-myc in a murine Leydig cell tumor line (MA-10). When stimulated by hCG (40 ng/ml), the mRNA levels of both genes increase rapidly, peaking at 30 min for c-fos and 1 h for c-myc. Both mRNAs fall to near control levels by 3-6 h. This response to hCG is dose-dependent with half-maximal stimulation of these genes occurring at a concentration of 3 ng/ml, approximating the level required for 50% occupancy of the LH/hCG receptors and the ED50 for steroidogenesis. (Bu)2 cAMP (2 mM) elicits responses similar to those produced by hCG. The observation of oncogene control by the gonadotropin hCG provides further insight regarding the pathways by which such hormones may regulate steroidogenesis, growth, and differentiation of endocrine and neoplastic cells.

Identification of anthracyclines and related agents that retain preferential activity over Adriamycin in multidrug-resistant cell lines, and further resistance modification by verapamil and cyclosporin A

A range of anthracyclines and related compounds were evaluated for activity against murine and human cell lines exhibiting multidrug resistance (MDR). Cell lines used were the NCI-H69 human small-cell lung cancer line and the EMT6 murine mammary tumour line, together with their multidrug-resistant counterparts produced by in vitro exposure to Adriamycin (ADM). Chemosensitivity testing was carried out using the tetrazolium (MTT) dye assay. Results were expressed as the ratio of the ID50 for the resistant line to that obtained in the parent, i.e. the resistance factor (RF). Compounds exhibiting much lower RF values than ADM in both resistant cell lines were identified as those anthracyclines with 9-alkyl substituents and those with certain changes to the amino sugar residue at position 3' and 4', together with the anthracenedione mitoxantrone (MIT). In a further attempt to overcome resistance, we used four of these compounds, Ro 31-1215, 4'-deoxy-4'-iodo-ADM (iodo-ADM), aclacinomycin A (ACL) and MIT (all yielding low RF values), in combination with the resistance modifiers verapamil (VRP) and cyclosporin A (CYA). Additional enhancement of chemosensitivity was achieved in the ADM-resistant sublines, as shown by the further decrease in RF values. At the concentrations used, the largest effects were generally seen with CYA, and the combination of this modifier with ACL and MIT was particularly effective. For the H69/LX4 resistant line, the latter combinations gave RF values approaching unity. These findings point to the use of analogues with the 9-alkyl substituent and/or specific changes to the sugar residue in combination with resistance modifiers as a therapeutic strategy for circumvention of the MDR phenotype.

31P NMR spectroscopy measurements of human ovarian carcinoma xenografts: Relationship to tumour volume, growth rate, necrotic fraction and differentiation status

31P NMR spectroscopy was used to study lipid and energy metabolism as well as tumour pH in three human ovarian carcinoma xenograft lines with widely differing growth rate, necrotic fraction and differentiation status. Two of the lines showed decreasing PCr (phcsphocreatine) and NTPβ (nucleoside triphosphates β) resonances and an increasing P i (inorganic phosphate) resonance with increasing tumour volume in the volume range 100–4000 mm 3. This decrease in bioenergetic status was accompanied by a decrease in tumour pH from about 7.15 to about 6.95. The volume-dependence of these spectral parameters probably reflected increased nutritional deprivation and development of hypoxia and necrosis during tumour growth. The phosphomonoesters (PME) and phosphodiesters (PDE) resonances did not change significantly with tumour volume. The third xenograft line did not show changes in the intensity of any of the resonances during tumour growth, in agreement with the observation that necrotic fraction and tumour pH (about 7.0) remained constant over the entire volume range. The spectral parameters differed significantly among the xenograft lines at given tumour volumes, but no correlations with volume-doubling time, necrotic fraction or differentiation status were found. The xenograft lines showed less extensive volume-dependence of the spectral parameters than did the KHT and RIF-1 murine tumour lines under identical experimental conditions.

Plasminogen activator release from cultured murine mast cells

Mast cells from the Furth murine mastocytoma tumour line were found to contain significant levels of plasminogen activator (PA). Cultured cells released PA activity into the culture medium in parallel with the release of histamine, and both were proportionately increased following exposure to degranulating agents. Pretreatment of the mast cells with cycloheximide did not alter their total PA content or their ability to release PA. These studies suggest that PA is a prestored granule constituent. The ability of PA to generate plasmin from plasminogen suggests an important role for mast cell PA in fibrinolysis and tissue degradation, observations that have been associated with mast cell degranulation and infiltration in vivo.

Intracapillary HbO2 saturations in murine tumours and human tumour xenografts measured by cryospectrophotometry: relationship to tumour volume, tumour pH and fraction of radiobiologically hypoxic cells.

Frequency distributions for intracapillary HbO2 saturation were determined for two murine tumour lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) using a cryospectrophotometric method. The aim was to search for possible relationships between HbO2 saturation status and tumour volume, tumour pH and fraction of radiobiologically hypoxic cells. Tumour pH was measured by 31P NMR spectroscopy. Hypoxic fractions were determined from cell survival curves for tumours irradiated in vivo and assayed in vitro. Tumours in the volume range 100-4000 mm3 were studied and the majority of the vessels were found to have HbO2 saturations below 10%. The volume-dependence of the HbO2 frequency distributions differed significantly among the four tumour lines; HbO2 saturation status decreased with increasing tumour volume for the KHT, RIF-1 and MLS lines and was independent of tumour volume for the OWI line. The data indicated that the rate of decrease in HbO2 saturation status during tumour growth was related to the rate of development of necrosis. The volume-dependence of tumour pH was very similar to that of the HbO2 saturation status for all tumour lines. Significant correlations were therefore found between HbO2 saturation status and tumour pH, both within tumour lines and across the four tumour lines, reflecting that the volume-dependence of both parameters probably was a compulsory consequence of reduced oxygen supply conditions during tumour growth. Hypoxic fraction increased during tumour growth for the KHT, RIF-1 and MLS lines and was volume-independent for the OWI line, suggesting a relationship between HbO2 saturation status and hypoxic fraction within tumour lines. However, there was no correlation between these two parameters across the four tumour lines, indicating that the hypoxic fraction of a tumour is not determined only by the oxygen supply conditions; other parameters may also be important, e.g. oxygen diffusivity, rate of oxygen consumption and cell survival time under hypoxic stress.

Inhibition of experimental metastasis with indomethacin: Role of macrophages and natural killer cells

The metastatic capacity of murine mammary tumor line 410.4 is greatly increased by treatment of the host with asialo-GM 1 antiserum (5-fold), 2-chloroadenosine (4-fold) of k-carrageenan (2.5-fold). The suggests that both NK cells and macrophages contribute to control of metastatic dissemination. The metastatic potential of these cells is associated with the synthesis of high levels of prostaglandin E 2 (PGE 2) (1). When line 410.4 cells are cultured in vitro in the presence of the prostaglandin synthesis inhibitor indomethacin (INDO) (1 uM) their subsequent lung colonization ability (experimental metastasis) is reduced by 50–90% as compared to solvent-treated cells. The inhibitory effect of INDO is highly dependent on the presence of asGM 1 positive cells, and is compromised to a lesser extent by treatments directed towards macrophages. The INDO-mediated inhibition is neither due to differential arrest of tumor cells in the lung nor does it appear to be due to shifts in the replication cycle.

31P NMR spectroscopy in vivo of two murine tumor lines with widely different fractions of radiobiologically hypoxic cells.

Energy and lipid metabolism as well as tumor pH in two murine tumor lines, the KHT and RIF-1 sarcomas, were studied using 31P NMR spectroscopy. Possible relationships between spectral parameters on the one hand and volume fraction of necrosis and fraction of radiobiologically hypoxic cells on the other were investigated. For both tumor lines the PCr and NTP beta resonances decreased and the Pi resonance increased significantly with increasing tumor volume in the volume range 100-4000 mm3. This decrease in bioenergetic status was accompanied by a decrease in tumor pH from about 7.2 to about 6.8. The NTP beta resonance and the tumor pH tended to be somewhat higher and the Pi resonance somewhat lower for the KHT than for the RIF-1 tumors. Linear relationships were found between tumor pH and Pi or (PCr + NTP beta)/Pi for both tumor lines (P much less than 0.05). The PME resonance increased slightly and the PDE resonance decreased slightly during tumor growth and were not significantly different for the KHT and the RIF-1 tumors. The volume fraction of necrosis was about 5 per cent in both lines at a tumor volume of 100 mm3 and increased to about 30 per cent (KHT) and 50 per cent (RIF-1) at a tumor volume of 4000 mm3. The fraction of radiobiologically hypoxic cells was found to increase from 12 to 23 per cent for the KHT line and from 0.9 to 1.7 per cent for the RIF-1 line when tumor volume was increased from about 200 to about 2000 mm3. The volume-dependence of the 31P NMR spectral parameters indicated increased nutritional deprivation and development of hypoxia and necrosis during tumor growth, and was thus qualitatively in good agreement with the changes observed in necrotic and hypoxic fraction. However, quantitative relationships between any spectral parameter and necrotic or hypoxic fraction across tumor lines were not found, implying that other physiological parameters and/or cellular characteristics may contribute significantly to a 31P NMR tumor spectrum. Consequently, 31P NMR spectra of untreated tumors have to be supplemented with other tumor data, e.g. rate of oxygen consumption, cell survival time under hypoxic stress and/or fraction of metabolically active, non-clonogenic hypoxic cells, to be useful in quantitative determination of tumor hypoxia and hence prediction of tumor radioresistance caused by hypoxia.

Molecular identification of lectin binding sites differentiating related low and high metastatic murine lymphomas.

We have previously shown that differences in cell surface carbohydrates can be detected in related murine tumor lines of varying metastatic capacity using plant lectins such as soybean agglutinin (SBA) or Vicia villosa (VV) but not concanavalin A (ConA). Here we show that weakly metastatic Eb cells bind SBA via four glycoproteins and one GL2-like glycolipid. The major high-affinity SBA binding component of weakly metastatic ESb-MP cells was a glycoprotein of 210-220 kd. Highly metastatic ESb cells also expressed this protein but the oligosaccharide side chains were altered in such a way that SBA-binding was completely lost while ConA and peanut agglutinin (PNA) binding remained similar. Quantitative binding studies using iodinated lectins indicated that SBA binding of ESb cells could only be detected at lectin concentrations greater than 75 micrograms/ml. The role of altered carbohydrates in metastasis is discussed.

Tumor progression in vitro: tumor-promoter-induced reversible decrease in natural immune susceptibility.

Growth of established murine tumor lines in media containing the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), was associated with reversible reductions in sensitivity to in vitro and in vivo parameters of natural resistance. L5178Y-F9 cells exposed to 100 ng TPA/ml for 2 days and returned to culture without TPA for 0-2 days, exhibited reductions in sensitivity to complement-mediated lysis by natural antibodies (Nab), activated macrophages and hypotonic lysis. The natural killer (NK) cell sensitive SL2-5 lymphoma was less sensitive to NK cells, complement-dependent NAb and hypotonic lysis after 2 days growth in 2 or 3 micrograms TPA/ml. Although TPA-treated L5178Y-F9 cells could acquire higher levels of serum NAb in vitro, this was complicated by the instability of the binding at 37 degrees C resulting in an effectively reduced capacity to bind NAb which was also demonstrated by TPA-treated SL2-5 cells. The tumor frequency of threshold s.c. inocula and the i.v. metastatic potential of the TPA-treated tumors was increased in syngeneic DBA/2 mice revealing possible correlations between reductions in the cellular characteristics assayed in vitro and decreased susceptibility to host-mediated defenses in vivo. Continued growth of the TPA-treated cells for a total of 2-8 days without TPA produced a reversal in the in vitro parameters, in the tumor frequency and in the metastatic potential, indicating the requirement for TPA to maintain the resistant phenotype. These data are consistent with the initial reversible nature of the promotion phase of multistage carcinogenesis. The reversible TPA-induced reductions in sensitivity to mediators of natural resistance may be an integral component of promotion, contributing to tumor survival in vivo and increasing the probability that the tumor will progress to a more malignant phenotype.

Study of cell kinetics by computer-analyzed flow cytometric histograms

A mathematical procedure for analyzing the cell proliferation kinetics from DNA content histograms, measured by flow cytometry, is presented. This procedure is based on a cell cycle model which uses a continuity equation for the S-phase transit. Four tumor celllines in culture were studied. A human melanoma line (JR01) was examined in exponential growth. For another human melanoma (M14) and a human astrocytoma (DF) lines, a sequence of flow cytometric histgrams with the corresponding growth curve was processed, determining the rate of DNA synthesis and the S-phase cell influx. The same kinetic parameters were evaluated also in a murine tumor line (3LL C108) treated with an antineoplastic drug (ICRF 159).

Characterization of cellular and extracellular plasmamembrane vesicles from a low metastatic lymphoma (Eb) and its high metastatic variant (ESb): inhibitory capacity in cell-cell interaction systems

Spontaneously shed extracellular plasma membrane vesicles (ECM) of a highly metastatic murine tumor line (ESb) were compared (i) with plasma membrane vesicles (PM) of the same cells prepared by the nitrogen cavitation method and (ii) with ECM and PM preparations of the related low metastatic tumor line Eb. From a previous biochemical analysis it was concluded that the exfoliation of ECM vesicles, which is very pronounced in metastatic ESb cells, is not a random process. This conclusion is further corroborated by the present functional analysis. Compared to ESb PM, ESb-derived ECM were selectively enriched for Fc receptors and depleted in glycoproteins with affinity for hepatocytes. Tumor-derived ECM carried the same tumor antigen as the corresponding tumor line and showed in comparison to PM material an increased inhibitor capacity in a T-cell-mediated tumor-specific cytotoxicity test.

Glycoconjugates of murine tumor lines with different metastatic capacities. II. Diversity of glycolipid composition.

A syngeneic tumor system in DBA/2 mice consisting of a methyl-cholanthrene-induced, weakly metastatic lymphoma, L5178YE (= Eb), its spontaneous strongly metastatic variant, L5178YES (= ESb), and an unrelated, methylcholanthrene-induced, metastasizing tumor, MDAY-D2, were used to study the relationship between metastatic behavior and composition of GSLs. The D-1-14C galactose and D-1-14C glucosamine-labelled neutral GSL and gangliosides of these tumor cells, and additionally ConA-stimulated spleen T cells from normal mice, were analysed by thin-layer chromatography. Unlabelled GSLs of the tumors were also characterized by (HPLC) after perbenzoylation. Results obtained with the radioactively labelled GSLs correlated with those of the HPLC analysis of unlabelled GSLs. All tumors contained neutral GSL of the ganglio-series. Weakly metastatic tumor Eb showed neutral GSL patterns comparable to those from ConA-stimulated spleen cells, whereas strongly metastatic tumors ESb and MDAY-D2 had an enhanced expression of lactosylceramide. Gangliosides of metastatic ESb and MDAY-D2 had a higher degree of polarity than those of weakly metastatic Eb. Eb cells expressed primarily GM1. Metastasizing ESb and MDAY-D2 had significantly higher amounts of GM3, GM2 and GD1a. An unusual ganglioside, IV3GalNAc-GM1, was found in MDAY-D2 cells and ConA blasts. When the extent of label was compared in neutral GSLs and gangliosides, metastasizing ESb and MDAY-D2 were more heavily labelled in the ganglioside fraction (62%, 58%) than Eb (39%). ESb and MDAY-D2 also contained larger amounts of gangliosides than Eb.