Fibroblasts and macrophages cooperate to create a pro-tumorigenic and immune resistant environment via activation of TGF-ß/IL-6 pathway in neuroblastoma.

Tumor-associated macrophages (TAM) and cancer-associated fibroblasts (CAF) and their precursor mesenchymal stromal cells (MSC) are often detected together in tumors, but how they cooperate is not well understood. Here, we show that TAM and CAF are the most abundant nonmalignant cells and are present together in untreated human neuroblastoma (NB) tumors that are also poorly infiltrated with T and natural killer (NK) cells. We then show that MSC and CAF-MSC harvested from NB tumors protected human monocytes (MN) from spontaneous apoptosis in an interleukin (IL)-6 dependent mechanism. The interactions of MN and MSC with NB cells resulted in a significant induction or increase in the expression of several pro-tumorigenic cytokines/chemokines (TGF-ß1, MCP-1, IL-6, IL-8, and IL-4) but not of anti-tumorigenic cytokines (TNF-α, IL-12) by MN or MSC, while also inducing cytokine expression in quiescent NB cells. We then identified a TGF-ß1/IL-6 pathway where TGF-ß1 stimulated the expression of IL-6 in NB cells and MSC, promoting TAM survival. Evidence for the contribution of TAM and MSC to the activation of this pathway was then provided in xenotransplanted NB tumors and patients with primary tumors by demonstrating a direct correlation between the presence of CAF and p-SMAD2 and p-STAT3. The data highlight a new mechanism of interaction between TAM and CAF supporting their pro-tumorigenic function in cancer.

Anti-disialoganglioside antibody internalization by neuroblastoma cells as a mechanism of immunotherapy resistance.

Neuroblastoma (NBL) accounts for a disproportionate number of deaths among childhood malignancies despite intensive multimodal therapy that includes antibody targeting disialoganglioside GD2, a NBL antigen. Unfortunately, resistance to anti-GD2 immunotherapy is frequent and we aimed to investigate mechanisms of resistance in NBL. GD2 expression was quantified by flow cytometry and anti-GD2 antibody internalization was measured using real-time microscopy in 20 human NBL cell lines. Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were performed on a subset of the cell lines (n = 12), and results were correlated with GD2 expression and antibody internalization. GD2 was expressed on 19 of 20 NBL cell lines at variable levels, and neutrophil-mediated ADCC was observed only in GD2-expressing cell lines. We found no correlation between level of GD2 expression and sensitivity to neutrophil-mediated ADCC, suggesting that GD2 expression of many cell lines was above a threshold required for maximal ADCC, such that expression level could not be used to predict subsequent cytotoxicity. Instead, anti-GD2 antibody internalization, a process that occurred universally but differentially across GD2-expressing NBL cell lines, was inversely correlated with ADCC. Treatment with endocytosis inhibitors EIPA, chlorpromazine, MBCD, and cytochalasin-D showed potential to inhibit antibody internalization; however, only MBCD resulted in significantly increased sensitivity to neutrophil-mediated ADCC in 4 of 4 cell lines in vitro. Our data suggest that antibody internalization may represent a novel mechanism of immunotherapy escape by NBL and provide proof-of-principle that targeting pathways involved in antibody internalization may improve the efficacy of anti-GD2 immunotherapies.

Combined immune checkpoint blockade increases CD8+CD28+PD-1+ effector T cells and provides a therapeutic strategy for patients with neuroblastoma.

Immune checkpoint therapy has resulted in minimal clinical response in many pediatric cancers. We sought to understand the influence of immune checkpoint inhibition using anti-PD-1 and anti-CTLA-4 antibodies individually, in combination, and after chemotherapy on immune responses in minimal and established murine neuroblastoma models. We also sought to understand the role of the tumor microenvironment (TME) and PD-L1 expression and their alteration post-chemotherapy in our models and human tissues. PD-L1 expression was enriched in human tumor-associated macrophages and up-regulated after chemotherapy. In a murine minimal disease model, single and dual immune checkpoint blockade promoted tumor rejection, improved survival, and established immune memory with long-term anti-tumor immunity against re-challenge. In an established tumor model, only dual immune checkpoint blockade showed efficacy. Interestingly, dual immune checkpoint therapy distinctly influenced adaptive and innate immune responses, with significant increase in CD8+CD28+PD-1+ T cells and inflammatory macrophages (CD11bhiCD11c-F4/80+Ly6Chi) in tumor-draining lymph nodes. Adding chemotherapy before immunotherapy provided significant survival benefit for mice with established tumors receiving anti-PD-1 or dual immune checkpoint blockade. Our findings demonstrate anti-PD-1 and anti-CTLA-4 therapy induces a novel subset of effector T cells, and support administration of induction chemotherapy immediately prior to immune checkpoint blockade in children with high-risk neuroblastoma.

Preclinical assessment of the efficacy and specificity of GD2-B7H3 SynNotch CAR-T in metastatic neuroblastoma.

The ability to utilize preclinical models to predict the clinical toxicity of chimeric antigen receptor (CAR) T cells in solid tumors is tenuous, thereby necessitating the development and evaluation of gated systems. Here we found that murine GD2 CAR-T cells, specific for the tumor-associated antigen GD2, induce fatal neurotoxicity in a costimulatory domain-dependent manner. Meanwhile, human B7H3 CAR-T cells exhibit efficacy in preclinical models of neuroblastoma. Seeking a better CAR, we generated a SynNotch gated CAR-T, GD2-B7H3, recognizing GD2 as the gate and B7H3 as the target. GD2-B7H3 CAR-T cells control the growth of neuroblastoma in vitro and in metastatic xenograft mouse models, with high specificity and efficacy. These improvements come partly from the better metabolic fitness of GD2-B7H3 CAR-T cells, as evidenced by their naïve T-like post-cytotoxicity oxidative metabolism and lower exhaustion profile.

Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC.

Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack MYCN amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking MYCN amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the MYC oncogene. Analysis of human neuroblastoma tumor specimens revealed that MYC up-regulation correlates with markers of TAM infiltration. In an IL6ko neuroblastoma model, the absence of IL-6 protein had no effect on tumor development and prevented neither STAT3 activation nor MYC up-regulation. In contrast, inhibition of JAK-STAT activation using AZD1480 or the clinically admissible inhibitor ruxolitinib significantly reduced TAM-mediated growth of neuroblastomas implanted subcutaneously in NOD scid gamma mice. Our results point to a unique mechanism in which TAMs promote tumor cells that lack amplification of an oncogene common to the malignancy by up-regulating transcriptional expression of a distinct oncogene from the same gene family, and underscore the role of IL-6-independent activation of STAT3 in this mechanism. Amplification of MYCN or constitutive up-regulation of MYC protein is observed in approximately half of high-risk tumors; our findings indicate a novel role of TAMs as inducers of MYC expression in neuroblastomas lacking independent oncogene activation.

Molecular subgroups of medulloblastoma identification using noninvasive magnetic resonance spectroscopy.

BACKGROUND: Medulloblastomas in children can be categorized into 4 molecular subgroups with differing clinical characteristics, such that subgroup determination aids in prognostication and risk-adaptive treatment strategies. Magnetic resonance spectroscopy (MRS) is a widely available, noninvasive tool that is used to determine the metabolic characteristics of tumors and provide diagnostic information without the need for tumor tissue. In this study, we investigated the hypothesis that metabolite concentrations measured by MRS would differ between molecular subgroups of medulloblastoma and allow accurate subgroup determination. METHODS: MRS was used to measure metabolites in medulloblastomas across molecular subgroups (SHH = 12, Groups 3/4 = 17, WNT = 1). Levels of 14 metabolites were analyzed to determine those that were the most discriminant for medulloblastoma subgroups in order to construct a multivariable classifier for distinguishing between combined Group 3/4 and SHH tumors. RESULTS: Medulloblastomas across molecular subgroups revealed distinct spectral features. Group 3 and Group 4 tumors demonstrated metabolic profiles with readily detectable taurine, lower levels of lipids, and high levels of creatine. SHH tumors showed prominent choline and lipid with low levels of creatine and little or no evidence of taurine. A 5-metabolite subgroup classifier inclusive of creatine, myo-inositol, taurine, aspartate, and lipid 13a was developed that could discriminate between Group 3/4 and SHH medulloblastomas with excellent accuracy (cross-validated area under the curve [AUC] = 0.88). CONCLUSIONS: The data show that medulloblastomas of Group 3/4 differ metabolically as measured using MRS when compared with SHH molecular subgroups. MRS is a useful and accurate tool to determine medulloblastoma molecular subgroups.

Differential growth suppression of human melanoma cells by tea (Camellia sinensis) epicatechins (ECG, EGC and EGCG).

We previously reported that catechins of green tea have different antiproliferative effects on cell lines derived from gender-dependent cancers; epicatechin 3-gallate (ECG) had the strongest inhibitory effect. In the present study, we examined the effects of epigallocatechin (EGC), epicatechin-gallate (ECG) and EGC 3-gallate (EGCG) on the viability, density, doubling time and cycle number of cell lines derived from melanoma metastasized to lymph nodes (MB-1133 and SE-0154) or distant organs (CH-0356, JK-0346, SA-1171, GE-0208, NS-1176 and LF-0023). These catechins have been documented to have no growth suppressive or apoptotic effects on normal melanocytes (Nihal et al., Int J Cancer 2005;114:513-21). EGCG (50 muM) showed greater inhibitory potency than EGC (50 muM) in SE-0154, NS-1176, GE-0208 and LF-0023 cell lines but the two catechins produced similar inhibitory effects in CH-0356, JK-0346 and SA-1171 cell lines. The IC(50) (50% inhibitory concentration) was lower for EGC than EGCG in MB-1133 and CH-0356 cells, higher for EGC than EGCG in GE-0208 cells and comparable (11-12 muM) for both the catechins in LF-0023 cells. When compared with EGC, the cytotoxic effect (% dead cell counts) and the suppression of the growth (change in cell number) of all melanoma cell lines tested were pronounced with EGCG. This investigation validates the hypothesis that anticancer action of the various catechins may vary with the type of malignancy and provides a model for tumor cell heterogeneity based on susceptibility and resistance of tumor cells to different green tea catechins. Therefore, this information is critical for undertaking chemopreventive or chemotherapeutic trials against melanoma and gender-based cancers.

Ganglioside signatures of primary and nodal metastatic melanoma cell lines from the same patient.

The primary cutaneous melanoma initially migrates to the regional lymph nodes (LNs). Human melanoma overexpresses gangliosides, the sialylglycosphingolipids. The ganglioside signatures may differ between primary and LN melanomas owing to the differences in the tumor microenvironments. The melanoma cells obtained from the primary and LN of the same patient might be useful to evaluate the above hypothesis. For this purpose, the cryopreserved cell lines from a primary cutaneous melanoma (IGR-39) and its nodal metastasis (IGR-37) from the same patient were used. We have also compared the ganglioside signatures of freshly obtained melanoma cells from primary, LN and organ metastases from different patients. Gangliosides were extracted, purified and identified by resorcinol and specific murine monoclonal antibodies. Comparison of the primary cell line with the nodal metastatic line obtained from the same patient distinctly showed the following features: (i) an increased production of gangliosides, (ii) O-acetylation of GM2 and GD3, (iii) an increased and altered O-acetylation of GD2 and (iv) possibly de-N-acetylation of GD3. These findings suggest that the nodal microenvironment might favor activation of O-acetyl-transferases capable of O-acetylating both alpha2, 3 and alpha2, 8 sialic acids of gangliosides. Supporting this, the primary melanoma cells obtained from different patients, showed no O-acetylation of GD3 or GD2. The cell line from groin LN showed the presence of O-acetyl (O-Ac)GD3. The cell lines from thyroid, spleen and jejunum expressed O-AcGD2. In all metastatic melanoma cell lines GD1a is more prevalent than GD3, suggesting that GD1a may be a major melanoma-ganglioside.

Significance of endogenous augmentation of antiganglioside IgM in cancer patients: potential tool for early detection and management of cancer therapy.

Gangliosides expressed by solid malignancies are shed into the circulation at a rate that varies with tumor stage, burden, and progression. Gangliosides have an immunosuppressive effect; thus an increase in the total ganglioside (TG) serum level may coincide with tumor progression. However, circulating gangliosides also may induce an endogenous IgM response. Unlike conventional pentameric IgM antibodies against peptide antigens, antiganglioside IgM antibodies can be polymeric and may not have a J-chain. Because these antibodies can remove shed gangliosides from the tumor microenvironment and the circulation, therapy that actively or passively augments serum levels of IgM against tumor-derived immunosuppressive gangliosides might restore immunocompetence and thereby slow tumor progression. The success of this approach, in passive and active specific therapy of cancer patients, requires analysis of biopsy tissue or sera of therapy recipients to confirm the presence of target gangliosides, such as GM2 or GD3. A patient's response to active or passive immunotherapy against a specific ganglioside target(s) can be monitored by serial assessment of serum specimens for TG level and antiganglioside IgM titer(s). This tailored approach to immunotherapy could be incorporated in postoperative adjuvant protocols.

Immunogenic gangliosides in human ovarian carcinoma.

Ganglioside signatures of four poorly and three moderately differentiated ovarian epithelial cancer (OEC) cell lines reveal the presence of GM3, GM2, GD2, O-AcGD2, GD1a and GM1b. The expression of GM3, presence of GD1a and GM1b in the ascitic fluid and plasma, together with a positive correlation in the total-gangliosides levels between ascitic fluid and plasma of OEC patients support the earlier contention that the tumor-gangliosides may be released (or shed) into the tumor-microenvironment. The immunogenicity of OEC-gangliosides is determined by comparing anti-ganglioside-IgM titers in ascitic fluid (n = 14) and plasma (n = 23) of OEC-patients and age-matched healthy (n = 14). The titers were measured by ELISA. Strikingly, the level of anti-GD1a-IgM is significantly higher in ascitic fluid and plasma of patients than in the plasma of healthy volunteers. Paired sample analysis of ascitic fluid and plasma from the same patients confirmed the significant expression of anti-GD1a IgM in OEC patients, while no such difference was observed with other anti-ganglioside IgMs among different groups. The significance of the endogenous IgM response to GD1a may be to eliminate this immunosuppressive-ganglioside from the tumor-microenvironment.

Human antiganglioside autoantibodies: validation of ELISA.

Gangliosides have a hydrophilic sugar chain that contains antigenic determinants and a hydrophobic ceramide. In humans, gangliosides elicit a T-cell independent IgM response; antiganglioside IgM autoantibodies may be pentameric or polymeric. A correlation between specific neuropathies and antiganglioside autoantibodies has been confirmed. Although many neurologists attempt to lower titers of antiganglioside autoantibodies, oncologists are developing strategies to augment production of IgM antibodies that will remove immunosuppressive gangliosides from the circulation of patients and target gangliosides and kill tumor cells. Antiganglioside IgM antibodies can cause leakage of the blood-nerve barrier in a concentration-dependent and complement-independent manner, bind to neuronal gangliosides to create a neuromuscular block and serve as a marker of axonal damage in neuropathies such as multiple sclerosis. They are also a promising biomarker of early prostate cancer. There is a need to validate the protocol for enzyme-linked immunosorbent assay (ELISA) of antiganglioside IgM autoantibodies. This validation must consider the purity of gangliosides from different commercial sources, the coating of gangliosides onto a solid matrix in a manner that maximizes exposure of oligosaccharide epitopes to IgM paratopes, techniques to minimize background noise and eliminate nonspecific antibody binding, and carefully defined positive and negative controls. The validated protocol also must include a simple formula to estimate titers for several replicas. Finally, antibody titers must be converted to natural logs for statistical appraisal.

Endogenous immune response to gangliosides in patients with confined prostate cancer.

Our study investigated whether endogenous IgM antibodies to gangliosides occur in patients with early stages of prostate cancer (CaP) patients, after defining ganglioside profiles of CaP cell lines. Immune and resorcinol staining detected the presence of gangliosides GM3, GM2, GD3, GD2 and GD1a but not GM1a, GD1b or GT1b in the extracts of normal prostatic epithelial cells (PrEC) and neoplastic androgen-insensitive (PC-3, DU145) and -sensitive (LNCaP-FGC and LNCaP-FGC-10) CaP cells. Using a sensitive ELISA, developed and validated in our laboratory, the titers of IgM against 8 gangliosides from sera of patients with benign prostatic hyperplasia (BPH) (n = 11), organ-confined (T1/T2, n = 36) and unconfined (T3/T4, n = 27) CaP and age-matched healthy men (n = 11) were determined double-blinded. Using ANOVA and Fisher's least significant difference (LSD) methods, the log-titers among different groups were compared. CaP patients differed from healthy and BPH patients in increased titers against GD1a and decreased titers against GD3. Titers of antibodies to other gangliosides exhibited no difference between CaP patients and others. The specific augmentation of anti-GD1a IgM in patients with organ-confined CaP (stage T1/T2) but not in patients with unconfined CaP (stage T3/T4) or BPH or in healthy controls is striking. This finding together with identification of GD1a as a major ganglioside in CaP cell lines and with the accruing studies on the immunosuppressive nature of GD1a indicates that augmentation of anti-GD1a IgM in confined CaP may signify an early endogenous immune response to eliminate a "danger signal" from tumor microenvironment and circulation.

Anticancer therapeutic potential of soy isoflavone, genistein.

Genistein (4'5, 7-trihydroxyisoflavone) occurs as a glycoside (genistin) in the plant family Leguminosae, which includes the soybean (Glycine max). A significant correlation between the serum/plasma level of genistein and the incidence of gender-based cancers in Asian, European and American populations suggests that genistein may reduce the risk of tumor formation. Other evidence includes the mechanism of action of genistein in normal and cancer cells. Genistein inhibits protein tyrosine kinase (PTK), which is involved in phosphorylation of tyrosyl residues of membrane-bound receptors leading to signal transduction, and it inhibits topoisomerase II, which participates in DNA replication, transcription and repair. By blocking the activities of PTK, topoisomerase II and matrix metalloprotein (MMP9) and by down-regulating the expression of about 11 genes, including that of vascular endothelial growth factor (VEGF), genistein can arrest cell growth and proliferation, cell cycle at G2/M, invasion and angiogenesis. Furthermore, genistein can alter the expression of gangliosides and other carbohydrate antigens to facilitate their immune recognition. Genistein acts synergistically with drugs such as tamoxifen, cisplatin, 1,3-bis 2-chloroethyl-1-nitrosourea (BCNU), dexamethasone, daunorubicin and tiazofurin, and with bioflavonoid food supplements such as quercetin, green-tea catechins and black-tea thearubigins. Genistein can augment the efficacy of radiation for breast and prostate carcinomas. Because it increases melanin production and tyrosinase activity, genistein can protect melanocytes of the skin of Caucasians from UV-B radiation-induced melanoma. Genistein-induced antigenic alteration has the potential for improving active specific immunotherapy of melanoma and carcinomas. When conjugated to B43 monoclonal antibody, genistein becomes a tool for passive immunotherapy to target B-lineage leukemias that overexpress the target antigen CD19. Genistein is also conjugated to recombinant EGF to target cancers overexpressing the EGF receptor. Although genistein has many potentially therapeutic actions against cancer, its biphasic bioactivity (inhibitory at high concentrations and activating at low concentrations) requires caution in determining therapeutic doses of genistein alone or in combination with chemotherapy, radiation therapy, and/or immunotherapies. Of the more than 4500 genistein studies in peer-reviewed primary publications, almost one fifth pertain to its antitumor capabilities and more than 400 describe its mechanism of action in normal and malignant human and animal cells, animal models, in vitro experiments, or phase I/II clinical trials. Several biotechnological firms in Japan, Australia and in the United States (e.g., Nutrilite) manufacture genistein as a natural supplement under quality controlled and assured conditions.

Gangliosides of organ-confined versus metastatic androgen-receptor-negative prostate cancer.

Prior development of a unique androgen-receptor (AR)-negative cell line (HH870) from organ-confined (T2b) human prostate cancer (CaP) enabled comparison of the gangliosides associated with normal and neoplastic prostate epithelial cells, organ-confined versus metastatic (DU 145, PC-3), and AR-negative versus AR-positive CaP cell lines. Resorcinol-HCl and specific monoclonal antibodies were used to characterize gangliosides on 2D-chromatograms, and to visualize them on the cell surface with confocal-fluorescence microscopy. AR-negative cells expressed GM1b, GM2, GD2, GD1a, and GM3. GM1a, GD1b, and GT1b were undetectable. GM1b and GD1a were more prominent in AR-negative than in AR-positive cells. PC-3 and HH870 cells were unique in the expression of O-acetylGD2 (O-AcGD2) and two alpha2,3-sialidase-resistant, alkali-susceptible GMR17-reactive gangliosides. Expression of GD1a, GM1b, doublets of GD3, GD2, and O-AcGD2, and the presence of an additional alkali-labile-14.G2a-reactive ganglioside, two alkali-susceptible, and three alkali-resistant GMR17-reactive gangliosides makes HH870 a potential component of a polyvalent-vaccine for active-specific immunotherapy of CaP.

Increased levels of gangliosides in the plasma and ascitic fluid of patients with advanced ovarian cancer.

OBJECTIVES: To assess the expression of total gangliosides in primary ovarian cancer cell lines, ascitic fluid and plasma of advanced ovarian cancer patients. DESIGN: A prospective study. SETTING: The Department of Obstetrics and Gynecology at the University of Arkansas for Medical Sciences and the Laboratory of Glycolipid Immunotherapy, John Wayne Cancer Institute. POPULATION: Twenty-two women diagnosed with advanced ovarian cancer and seven normal female controls. METHODS: Total gangliosides shedding from primary ovarian cancer cell lines were measured by estimating lipid-associated sialic acids (LASAs test) and compared with the ganglioside levels shed by primary cervical and uterine cancer cell lines. In addition, plasma and ascitic samples from advanced ovarian cancer patients were collected at the time of surgery and analysed for the presence of total gangliosides. MAIN OUTCOME MEASURES: Levels of total ganglioside in plasma and ascites fluid samples drawn from ovarian cancer patients, relative to total gangliosides levels in plasma from normal female controls. RESULTS: All primary ovarian tumours secreted high levels of total gangliosides (mean 4 mg/mL, range between 2.7 and 4.8 mg/mL/10(5) cells/24 h) when compared with primary cervical cancers (mean 1.4 mg/mL, range between 0.7 and 2.2 mg/mL/10(5) cells/24 h) (P < 0.008) and uterine carcinoma cell lines (mean 1.4 mg/mL, range between 1.3 and 1.6 mg/mL/10(5) cells/24 h) (P <.004). Elevated levels of total gangliosides were detected in the plasma [mean (SD) 31 (12) mg/mL, range between 18 and 57 mg/mL] (P <.001), and in the peritoneal fluid [mean (SD) 27 (9) mg/mL, range between 14 and 40 mg/mL] (P <.003) of ovarian cancer patients when compared with the levels detectable in the plasma samples of normal female controls tested [mean (SD) 15 (2) mg/mL, range between 12 and 18 mg/mL]. CONCLUSIONS: Increased serum levels of total gangliosides may reflect shedding or release of gangliosides from the surface of ovarian tumour cells. Secretion of gangliosides may play an important role in the inhibition of anti-tumour immune function commonly observed in advanced ovarian cancer.

Cryosurgical ablation of liver tumors in colon cancer patients increases the serum total ganglioside level and then selectively augments antiganglioside IgM.

Cryosurgical ablation (CSA) of tumors induces disruptive necrosis. Necrosis may release tumor gangliosides into circulation and they may augment serum antiganglioside antibodies depending on the nature of gangliosides released. The hypothesis is tested by determining the level of serum total gangliosides (STG) and their antibody titers in the sera of colon cancer patients with cryoablated liver tumors. As controls, we examined the sera of patients who underwent radiofrequency ablation (RFA) and regular surgery (RS), none of which cause disruptive necrosis. The STG level (expressed as lipid-bound sialic acids, LBSA) is higher (p(2)<0.001) in 35 patients (stage IV) than in 38 healthy case-controls (median 23.48 mg/dL, Q-range 7.1 vs 16.04 mg/dL, Q-range 4.5). The mean STG level increased significantly to 31.2+/-6.0mg/dL (p(2)<0.03) after CSA. Concomitantly, the IgM titer against colon cancer-associated gangliosides (GM(2), GD(1a), GT(1b)), increased significantly, but no increase was observed against normal tissue gangliosides (GM(3) or GM(1)). Also after RFA and RS, no such increase was observed either in the level of STG or in IgM titer against tumor gangliosides. The results suggest that CSA-induced necrosis might have acted as an adjuvant, because purified gangliosides without exogenous adjuvants even after repeated immunization failed to elicit antibody response. The post-CSA decline in the STG level correlated with the increase in the antibodies, suggesting a homeostatic role of the antibodies.