Metastatic Melanoma Growth Research Articles

Targeting neutrophils potentiates hitchhiking delivery of drugs and agonists for postsurgical chemo-immunotherapy

Tumor resection is usually associated with tumor recurrence due to the residual tumor cells. Improving the localized accumulation of therapeutics at the surgical sites is a promising approach for the inhibition of tumor recurrence and metastasis. Herein, we report an in situ hitchhiking strategy for postsurgical tumor therapy via targeting of therapeutics-loaded liposomes to neutrophils (NEs). Anticancer drug doxorubicin (DOX) and non-nucleotide stimulator of interferon genes (STING) agonist SR-717 are encapsulated into liposomes followed by surface modification with anti-Ly6G antibodies, which can target NEs in situ in blood stream for hitchhiking delivery to the postsurgical inflamed tumor sites. The delivered DOX can inhibit the growth of residual tumor cells and trigger the tumor immunogenic cell death (ICD) for tumor-specific immunity, while SR-717 can facilitate dendritic cell maturation and enhance antitumor immunity. Consequently, the in situ NE-hitchhiking delivery of therapeutics can efficiently suppress tumor recurrence and metastatic melanoma growth after tumor resection, which circumvents the complex ex vivo processes of separation, engineering, and refusion of leukocytes and represents a targeted delivery strategy for postsurgical cancer chemo-immunotherapy.

Improved synthesis and anticancer activity of a potent neuronal nitric oxide synthase inhibitor

An improved synthesis of 4-methyl-7-(3-((methylamino)methyl)phenethyl)quinolin-2-amine (1) is reported. A scalable, rapid, and efficient methodology was developed to access this compound with an overall yield of 35%, which is 5.9-fold higher than the previous report. The key differences in the improved synthesis are a high yielding quinoline synthesis by a Knorr reaction, a copper-mediated Sonogashira coupling to the internal alkyne in excellent yield, and a crucial deprotection of the N-acetyl and N-Boc groups achieved under acidic conditions in a single step rather than a poor yielding quinoline N-oxide strategy, basic deprotection conditions, and low yielding copper-free conditions that were reported in the previous report. Compound 1, which previously was shown to inhibit IFN-γ-induced tumor growth in a human melanoma xenograft mouse model, was found to inhibit the growth of metastatic melanoma, glioblastoma, and hepatocellular carcinoma in vitro.

Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2−•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

A comprehensive review on phytochemistry, bioactivity and medicinal value of bioactive compounds of pomegranate (Punica granatum)

Pomegranate (Punica granatum) acts as a therapeutic fruit possessing different bioactive compounds. Different compartments of pomegranate (P. granatum) like seed, peel, juice and leaves are rich in potential bioactive compounds. Pomegranate contains sinapyl, coniferyl, ellagic acid, cinnamic acid, genistein, linoleic acid, anthocyanin, gallic acid, catechin, quercetin, rutin, kaempferol, cyanidin, punicalin, delphinidin, punicalagin, chlorogenic acid, coumaric acid, luteolin and pelletierine alkaloids, flavonoids, anthocyanin and caffeic acid. Pomegranate also exhibits significant anti-oxidative, anti-hypertensive, cardioprotective and anti-diabetic properties and anti-carcinogenic possessions including prevention of prostate cancer; reduction in colon inflammation, prevention of skin tumorigenesis, improvement in renal function and reduction in metastatic melanoma growth. This review summarizes the current available data on the phytochemistry, bioavailability and therapeutic importance of potential bioactive compounds of pomegranate.

Suppression of Metastatic Melanoma Growth in Lung by Modulated Electro-Hyperthermia Monitored by a Minimally Invasive Heat Stress Testing Approach in Mice.

Simple SummaryThe lung is the most frequent site of distant melanoma metastases. Metastases of melanoma in the lungs offer a very poor prognosis, with a 5-year survival rate of below 10%. Hyperthermic therapies including modulated electro-hyperthermia (mEHT) in clinical settings have been used to improve the efficacy of radiotherapy, chemotherapy, and immunotherapy of tumors. In this study, we focused primarily on the optimization of mEHT for targeted lung treatment of mice lungs burdened with B16F10 melanoma pulmonary metastases, with a particular focus on elucidating the mechanism of action of mEHT on treated melanoma cells while investigating any potential treatment-related side effects on normal lung tissue. mEHT showed evidence of significant anti-tumor effects as demonstrated by the reduced number of pulmonary metastatic nodules, DNA damage response, downregulation of Ki67 expression, higher immune cell infiltration, and upregulation of p21waf1 expression in mEHT-treated tumors.Modulated electro-hyperthermia (mEHT) is a novel complementary therapy in oncology which is based on the higher conductivity and permittivity of cancerous tissues due to their enhanced glycolytic activity and ionic content compared to healthy normal tissues. We aimed to evaluate the potential of mEHT, inducing local hyperthermia, in the treatment of pulmonary metastatic melanoma. Our primary objective was the optimization of mEHT for targeted lung treatment as well as to identify the mechanism of its potential anti-tumor effect in the B16F10 mouse melanoma pulmonary metastases model while investigating the potential treatment-related side effects of mEHT on normal lung tissue. Repeated treatment of tumor-bearing lungs with mEHT induced significant anti-tumor effects as demonstrated by the lower number of tumor nodules and the downregulation of Ki67 expression in treated tumor cells. mEHT treatment provoked significant DNA double-strand breaks indicated by the increased expression of phosphorylated H2AX protein in treated tumors, although treatment-induced elevation of cleaved/activated caspase-3 expression was insignificant, suggesting the minimal role of apoptosis in this process. The mEHT-related significant increase in p21waf1 positive tumor cells suggested that p21waf1-mediated cell cycle arrest plays an important role in the anti-tumor effect of mEHT on melanoma metastases. Significantly increased CD3+, CD8+ T-lymphocytes, and F4/80+CD11b+ macrophage density in the whole lung and tumor of treated animals emphasizes the mobilizing capability of mEHT on immune cells. In conclusion, mEHT can reduce the growth potential of melanoma, thus offering itself as a complementary therapeutic option to chemo- and/or radiotherapy.

Anti-angiogenic effects of mangiferin and mechanism of action in metastatic melanoma.

Advanced metastatic melanoma, one of the most aggressive skin malignancies, is currently without reliable therapy. The process of angiogenesis is crucial for progression and metastasis of the majority of solid tumors including melanomas. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone which exerts many pharmacological activities against cancer-inflammation. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we demonstrate that mangiferin interferes with inflammation, lipid and calcium signaling which selectively inhibits multiple NFkB target genes including interleukin-6, tumor necrosis factor, interferon gamma, vascular endothelial growth factor receptor 2, plasminogen activator urokinase, matrix metalloprotease 19, C-C Motif Chemokine Ligand 2 and placental growth factor. This abrogates angiogenic and invasive processes and capillary tube formation of metastatic melanoma cells as well as human placental blood vessel explants in-vitro and blocks angiogenesis characteristic of the chicken egg chorioallantoic membrane assay and in melanoma syngeneic studies in vivo. The results obtained in this research illustrate promising anti-angiogenic effects of the natural glucosylxanthone mangiferin for further (pre)clinical studies in melanoma cancer patients.

Inhibition of ER stress-related IRE1α/CREB/NLRP1 pathway promotes the apoptosis of human chronic myelogenous leukemia cell

Endoplasmic reticulum (ER) stress is induced in chronic myelogenous leukemia (CML) cells. As an important sensor of ER stress, inositol-requiring protein-1α (IRE1α) promotes the survival of acute myeloid leukemia. NLRP1 inflammasome activation promotes metastatic melanoma growth and that IRE1α can increase NLRP1 inflammasome gene expression. This study aimed to investigate the role and molecular mechanism of IRE1α in CML cell growth. We found that overexpression of IRE1α or NLRP1 significantly promoted the proliferation and decreased the apoptosis of CML cells, whereas downregulation of these two genes showed the opposite effects. 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, reduced the expression of IRE1α and NLRP1. IRE1α elevated NLRP1 expression via cAMP responsive element binding protein (CREB) phosphorylation. NLRP1 inflammasome was activated in CML cells and its activation partly reversed ER stress inhibitor-induced cell apoptosis. Furthermore, inhibition of IRE1α/NLRP1 pathway sensitized CML cells to imatinib-mediated apoptosis. Additionally, IRE1α expression was elevated and NLRP1 inflammasome was activated in primary cells from CML patients. Downregulation of IRE1α or NLRP1 suppressed the proliferation and elevated the apoptosis of primary CML cells. Collectively, this study demonstrated that the IRE1α/CREB/NLRP1 pathway contributes to the progression of CML and the development of imatinib resistance. Hence, targeting ER stress-related IRE1α expression or NLRP1 inflammasome activation may block CML development.

Mangiferin, a novel nuclear factor kappa B-inducing kinase inhibitor, suppresses metastasis and tumor growth in a mouse metastatic melanoma model

Advanced metastatic melanoma, one of the most aggressive malignancies, is currently without reliable therapy. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone and exerts many beneficial biological activities. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we evaluated the effect of mangiferin on metastasis and tumor growth in a mouse metastatic melanoma model. We found that mangiferin inhibited spontaneous metastasis and tumor growth. Furthermore, mangiferin suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated NF-κB-inducing kinase (NIK), inhibitor of kappa B kinase (IKK), and inhibitor of kappa B (IκB) and increases the expression of IκB protein in vivo. In addition, we found that mangiferin inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs) in vivo. Mangiferin treatment also increased the expression of cleaved caspase-3, cleaved Poly ADP ribose polymerase-1 (PARP-1), p53 upregulated modulator of apoptosis (PUMA), p53, and phosphorylated p53 proteins, and decreased the expression of Survivin and Bcl-associated X (Bcl-xL) proteins in vivo. These results indicate that mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation, thereby inhibiting metastasis and tumor growth. Importantly, the number of reported NIK selective inhibitors is limited. Taken together, our data suggest that mangiferin may be a potential therapeutic agent with a new mechanism of targeting NIK for the treatment of metastatic melanoma.

Abstract A29: MIF controls human myeloid-derived immunosuppressive phenotype/function in melanoma educated CD14+ monocytes

Abstract Tumors suppress the host immune system by employing a variety of cellular immune modulators such as regulatory T cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). Recently, MDSCs have been recognized as critically important cellular immune modulators that serve to suppress both innate and adaptive immune responses in a wide variety of human malignancies. Macrophage migration inhibitory factor (MIF) is one the oldest cytokine activities described and is an important determinant of both innate and adaptive immune responses. Recent studies by our group revealed that MDSCs isolated from melanoma-bearing MIF-deficient mice are substantially less immunosuppressive than those isolated from MIF wildtype mice and these phenotypes correspond to significantly reduced primary and metastatic melanoma growth and progression. Importantly, 4-iodo-6-phenylpyrimidine (4-IPP – our previously discovered small MIF inhibitor) fully recapitulates MIF-deficiency in vitro and in vivo and serves to attenuate MDSC immunosuppression and melanoma disease progression in mice. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive functions, an in vitro MDSC induction model was established using normal, primary human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist, 4-IPP. Our studies suggest that the induction of CD14+/CD33+/HLADRlo/- myeloid-suppressive cells recently shown to represent the bulk of immunosuppressive activity in late stage melanoma patients is largely dependent on MIF. Importantly, small molecule inhibition of MIF in these MDSC-like cells almost completely abolishes their ability to suppress proliferation and IFN-gamma production by autologous T cells. Further, MIF-dependent suppressive activity of MDSCs is regulated via an NADPH oxidase induced ROS-mediated signaling mechanism. Finally, we demonstrate that small molecule inhibitors of MIF attenuate the suppressive properties of circulating CD14+/HLADRlo/- MDSCs isolated from stage III/IV melanoma patients confirming MIF as a critical mediator of MDSC-dependent immunosuppression in patients with advanced stage melanoma. These studies using human, clinically relevant experimental melanoma models lend strong support to the rationale that therapeutic inhibition of MIF in human melanoma MDSCs is a novel and clinically viable approach to enhancing anti-tumor T cell immunity in advanced melanoma patients. Citation Format: Kavitha Yaddanapudi, Beatriz E. Rendon, Gwyneth J. Lamont, John W. Eaton, Robert A. Mitchell. MIF controls human myeloid-derived immunosuppressive phenotype/function in melanoma educated CD14+ monocytes. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A29. doi:10.1158/1538-7445.CHTME14-A29

Anti-metastatic immunotherapy based on mucosal administration of flagellin and immunomodulatory P10.

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system

OBJECTIVE:Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines.METHODS:We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed.RESULTS:FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma.CONCLUSION:Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished.

Abstract 1833: A large-scale synthetic lethal RNAi screening identifies unique sensitizing targets for anti-inflammatory CDDO-Me in metastatic melanoma

Abstract Metastatic melanoma is the most lethal form of skin cancer strongly associated with the inflammatory process by secreting high levels of proinflammatory cytokines and producing reactive oxygen and nitrogen species. Chronic inflammation is considered as a major factor to promote the tumor microenvironment in advantage of resistance to radiation, chemotherapy, and biotherapy for metastatic melanoma. CDDO-Me, a novel synthetic triterpenoid, has been shown to exert potent anti-inflammatory and antitumor activity by decreasing oxidative stress and the production of pro-inflammatory molecules, such as Nrf2 and iNOS, in several tumor models. CDDO-Me is presently under evaluation in clinical studies for chronic kidney disease and pancreatic cancer. Here, we used CDDO-Me as a primary agent to inhibit metastatic melanoma growth. In order to improve CDDO-Me response in melanoma cells, we utilized a large-scale synthetic lethal RNAi screen with 24,000 siRNAs targeting ∼6,000 human druggable genes to identify targets that when silenced would sensitize melanoma cells to CDDO-Me. Results from the screens identified twenty of siRNA pools that significantly potentiated the growth inhibitory effects of CDDO-Me in A375 cells. We selected five unique genes, GNPAT, SUMO1, SPUNT2, FLI1, and SSX1, which have not been addressed as drug targets in melanoma before, for target validation and functional analysis in four metastatic melanoma and control cell lines. Specific siRNA against each of these five genes could substantially sensitize four tested melanoma cells to CDDO-me by increasing the growth inhibition and induction of apoptosis, while treatment with nontarget siRNA resulted in no change compared to the treatment with CDDO-Me only. Further studies of downstream signaling revealed that these five genes involved in regulating melanoma cell proliferation and cell death. We identified for the first time that the levels of phosphorylated Erk1/2, Akt, GSK-2, and PRAS40, were dramatically decreased by individual siRNA against GNPAT, SUMO1, SPUNT2, FLI1, and SSX1. Our findings indicate that GNPAT, SUMO1, SPUNT2, FLI1, and SSX1 may play critical roles in synergy with inflammation pathways in modulating melanoma cell survival, which could serve as sensitizing targets to enhance CDDO-Me efficacy in melanoma. Supported by MDACC SPORE in Melanoma P50CA093459. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1833. doi:1538-7445.AM2012-1833

Decreased NK cell cytotoxicity and increased T regulatory cells facilitate progression of metastatic murine melanoma

Malignant melanoma is the most aggressive form of skin cancer. Metastatic dissemination in distant organs is one of the hallmarks of melanoma progression. Immunosuppression and tumour escape from immune surveillance are thought to be the major factors responsible for the establishment and progression of melanoma; however, the exact mechanisms leading to decreased anti-tumour immunity are not completely understood. We aimed to analyse the anti-tumour immune response during hematogenous metastasis using a B16-F1 metastatic melanoma model in C57BL/6 mice. At 21 days after tumour cell inoculation, rapid metastatic melanoma growth was observed, reflected through the increased incidence, number and size of metastatic colonies in the lungs (B16-F1). Phenotypic analyses of splenocytes revealed an increased percentage of CD3+T cells, a markedly reduced percentage of CD19+ B cells and an increased percentage and absolute number of CD4+Foxp3+T regulatory cells. The cytotoxic activities of total splenocytes and isolated NK cells were significantly decreased in tumour-bearing mice. Thus, the metastatic progression of melanoma in this model is associated with diminished NK cytotoxicity, which may be due to an increased expansion of suppressive CD4+Foxp3+ T regulatory cells in the spleen.

Association between Pterostilbene and Quercetin Inhibits Metastatic Activity of B16 Melanoma

Inhibition of cancer growth by resveratrol (trans-3,5,4'trihydroxystilbene; RESV), a phytoalexin present in many plant species, is limited by its low bioavailability. Pterostilbene (3,5-dimethoxy-4'-hydroxystilbene; PTER) and quercetin (3,3',4',5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols, show longer half-life in vivo. In vitro growth of highly malignant B16 melanoma F10 cells (B16M-F10) is inhibited (56%) by short-time exposure (60 min/day) to PTER (40 μM) and QUER (20 μM) (approximate mean values of plasma concentrations measured within the first hour after intravenous administration of 20 mg/kg of each polyphenol). Intravenous administration of PTER and QUER (20 mg/kg per day) to mice inhibits (73%) metastatic growth of B16M-F10 cells in the liver, a common site for metastasis development. The antimetastatic mechanism involves: 1) a PTER-induced inhibition of vascular adhesion molecule 1 expression in the hepatic sinusoidal endothelium, which consequently decreases B16M-F10 cell adhesion to the endothelium through very late activation antigen 4; and 2) a QUER- and PTER-induced inhibition of Bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. Our findings demonstrate that the association of PTER and QUER inhibits metastatic melanoma growth and extends host survival.

Melanoma antigens and their recognition by T cells.

During the last 10 years many melanoma antigens recognized by T cells have been molecularly characterized. This review summarizes the main features of these antigens, including both classes I and II HLA-restricted peptides, and describes their classification into diverse groups according to the tissue distribution of the antigens. The different in vitro and in vivo immunogenicity of such antigens is then discussed leading to the conclusion that Melan-A/MART-1 is the strongest among those tested being frequently recognized by patients' T cells both in vitro and in vivo. However, no correlation was found between T-cell response of melanoma patients to Melan-A/MART-1 and clinical response when it was used for vaccination. Data are also presented that suggest, through an ex vivo analysis carried out with tetramers staining of melanoma-specific T cells, that only in a limited number of advanced patients does a specific immune response develop. This response, however, appears unable to effectively counteract metastatic melanoma growth.

Effect of Bacillus calmette guerin on local and metastatic melanoma growth

Recent interest in the effect of Bacillus calmette Guerin (B.C.G.) on tumor growth was created by the demonstration that B. C.G. could inhibit growth of different cancers in man [12, 141. Variable results, ranging from stimulation or no effect to inhibition of the growth of different tumors by inoculating B.C.G., have been obtained using animal models. Most reports studied the effect of B.C.G. on local tumor growth, but this may have little relevance clinically, since current reports suggest that immunotherapy will be most useful for treating small metastases which are present at the time of primary tumor excision [ 141. A carcinogen-induced hepatoma in guinea pigs has been extensively studied [5, 91 and a number of important observations relating to dosage, time and mode of inoculation, and the effect of different strains of B.C.G. have been recorded. However, this tumor is immunogenic [ 161 and theoretically would be more sensitive to B.C.G. therapy [l]. When a weakly immunogenic, transplantable neoplasm of spontaneous origin, the B16 melanoma, was used, variable responses to B.C.G. therapy were found [7, 111. Since it is considered that weakly immunogenic, spontaneously metastasizing tumors most likely resemble the common human cancers [4, lo], the B16 melanoma was further studied to determine whether more consis-