Mekk3 is essential for early embryonic cardiovascular development.

The early development of blood vessels consists of two phases, vasculogenesis and angiogenesis, which involve distinct and also overlapping molecular regulators, but the intracellular signal transduction pathways involved in these processes have not been well defined. We disrupted Map3k3 (also known as Mekk3), which encodes Mekk3, a member of the Mekk/Ste11 family, in mice. Map3k3-/- embryos died at approximately embryonic day (E) 11, displaying disruption of blood vessel development and the structural integrity of the yolk sac. Angiogenesis was blocked at approximately E9.5 in mutant embryos. Map3k3 disruption did not alter the expression of the genes encoding Vegf-1, angiopoietin or their receptors. The development of embryonic, but not maternal, blood vessels in the placentas of Map3k3-/- embryos was impaired, revealing an intrinsic defect in Map3k3-/- endothelial cells. Moreover, Mekk3 activated myocyte-specific enhancer factor 2C (Mef2c), a transcription factor crucial for early embryonic cardiovascular development through the p38 mitogen-activated protein kinase (Mapk) cascade. We conclude that Mekk3 is necessary for blood vessel development and may be a possible target for drugs that control angiogenesis.

CD40-CD40 ligand interactions in vivo regulate migration of antigen-bearing dendritic cells from the skin to draining lymph nodes.

Whereas CD40-CD40 ligand interactions are important for various dendritic cell (DC) functions in vitro, their in vivo relevance is unknown. We analyzed the DC status of CD40 ligand -/- mice using a contact hypersensitivity (CHS) model system that enables multiple functions of DCs to be assessed in vivo. Immunohistochemistry of skin sections revealed no differences in terms of numbers and morphology of dendritic epidermal Langerhans cells (LCs) in unsensitized CD40 ligand -/- mice as compared with wild-type C57BL/6 mice. However, after contact sensitization of CD40 ligand -/- mice, LCs failed to migrate out of the skin and substantially fewer DCs accumulated in draining lymph nodes (DLNs). Furthermore, very few antigen-bearing DCs could be detected in the paracortical region of lymph nodes draining sensitized skin. This defect in DC migration after hapten sensitization was associated with defective CHS responses and decreased cutaneous tumor necrosis factor (TNF)-alpha production and was corrected by injecting recombinant TNF-alpha or an agonistic anti-CD40 monoclonal antibody. Thus, CD40-CD40 ligand interactions in vivo regulate the migration of antigen-bearing DCs from the skin to DLNs via TNF-alpha production and play a vital role in the initiation of acquired T cell-mediated immunity.

Localization of DNA damage and its role in altered antigen-presenting cell function in ultraviolet-irradiated mice.

Prior ultraviolet (UV) irradiation of the site of application of hapten on murine skin reduces contact sensitization, impairs the ability of dendritic cells in the draining lymph nodes (DLN) to present antigen, and leads to development of hapten-specific suppressor T lymphocytes. We tested the hypothesis that UV-induced DNA damage plays a role in the impaired antigen-presenting activity of DLN cells. First, we assessed the location and persistence of cells containing DNA damage. A monoclonal antibody specific for cyclobutyl pyrimidine dimers (CPD) was used to identify UV-damaged cells in the skin and DLN of C3H mice exposed to UV radiation. Cells containing CPD were present in the epidermis, dermis, and DLN and persisted, particularly in the dermis, for at least 4 d after UV irradiation. When fluorescein isothiocyanate (FITC) was applied to UV-exposed skin, the DLN contained cells that were Ia+, FITC+, and CPD+; such cells from mice sensitized 3 d after UV irradiation exhibited reduced antigen-presenting function in vivo. We then assessed the role of DNA damage in UV-induced modulation of antigen-presenting cell (APC) function by using a novel method of increasing DNA repair in mouse skin in vivo. Liposomes containing T4 endonuclease V (T4N5) were applied to the site of UV exposure immediately after irradiation. This treatment prevented the impairment in APC function and reduced the number of CPD+ cells in the DLN of UV-irradiated mice. Treatment of unirradiated skin with T4N5 in liposomes or treatment of UV-irradiated skin with liposomes containing heat-inactivated T4N5 did not restore immune function. These studies demonstrate that cutaneous immune cells sustain DNA damage in vivo that persists for several days, and that FITC sensitization causes the migration of these to the DLN, which exhibits impaired APC function. Further, they support the hypothesis that DNA damage is an essential initiator of one or more of the steps involved in impaired APC function after UV irradiation.

Chemokine receptors in advanced breast cancer: differential expression in metastatic disease sites with diagnostic and therapeutic implications.

BACKGROUND: We investigated the expression of CXCR4, CCR7, estrogen receptor (ER), progesterone receptor (PR) and HER2-neu in human metastatic breast cancers to determine whether these biological biomarkers were preferentially expressed in any organ-specific metastases. MATERIALS AND METHODS: CXCR4, CCR7, ER, PR and HER2-neu expression levels were evaluated by immunohistochemical staining using paraffin-embedded tissue sections of metastatic breast cancers (n = 41) obtained by either diagnostic biopsy or surgical resection. RESULTS: The metastatic sites included the following: bone (n = 15), brain (n = 14), lung (n = 6), liver (n = 2), and omental metastases (n = 2). CXCR4 was expressed in 41% of cases, CCR7 expression was demonstrated in 10%, and HER2-neu overexpression was present in 27%. CXCR4 was more likely to be expressed in bone metastases than visceral metastases (67% versus 26%, P = 0.020). Visceral sites demonstrated a lower rate of CXCR4 positivity (33% and 23%, respectively, for lung and brain metastases). Similarly, CCR7 was more likely to be found in bone metastases than visceral sites (27% versus 0%, P = 0.037). CONCLUSIONS: These results indicate that CXCR4 can contribute to the homing of breast cancer cells to the bone. This finding might have important clinical implications since patients with metastatic bone disease may achieve the highest benefit from a CXCR4-targeted therapy.

Innervation of the hamster Harderian gland.

Harderian glands of male and female hamsters have nerve endings associated with the secretory cells, myoepithelial cells, and the blood vessels. The nerve endings adjacent to the myoepithelial cells also have myoneural junctions.

Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C.

Members of the myocyte enhancer factor-2 (MEF2) family of MADS (MCM1, agamous, deficiens, serum response factor)-box transcription factors bind an A-T-rich DNA sequence associated with muscle-specific genes. The murine MEF2C gene is expressed in heart precursor cells before formation of the linear heart tube. In mice homozygous for a null mutation of MEF2C, the heart tube did not undergo looping morphogenesis, the future right ventricle did not form, and a subset of cardiac muscle genes was not expressed. The absence of the right ventricular region of the mutant heart correlated with down-regulation of the dHAND gene, which encodes a basic helix-loop-helix transcription factor required for cardiac morphogenesis. Thus, MEF2C is an essential regulator of cardiac myogenesis and right ventricular development.

Visualization of a guinea pig T lymphocyte surface component cross-reactive with immunoglobulin.

Thymus-derived lymphocytes (T cells) show exquisite specificity in recognition of antigens, but the nature of the cell surface receptor is controversial. Although antigen recognition mediated by immunoglobulin variable (V) regions remains the minimal hypothesis, it has been extremely difficult to definitely establish the presence of immunoglobulins on these cells. Chicken antibodies, produced against the (Fab')2fragment of mouse immunoglobulin G (IgG) and purified by binding to and elution from IgG-Sepharose 4B, bind to an endogenously synthesized surface component of guinea pig T cells. The binding occurred via a cross-reaction with murine k chain and a heavy chain determinant localized in the Fd region, and was visualized by immunofluorescence and immunoelectronmicroscopy using both transmission and scanning techniques. These data provide direct evidence for the presence of a surface component related to immunoglobulin on T lymphocytes.

Dynamic change in phosphorylated platelet-derived growth factor receptor in peripheral blood leukocytes following docetaxel therapy predicts progression-free and overall survival in prostate cancer.

In a placebo-controlled randomised study of the platelet-derived growth factor receptor (PDGFR) inhibitor imatinib mesylate and docetaxel in metastatic prostate cancer with bone metastases (n=116), no significant differences in progression-free and overall survival were observed. To evaluate pharmacodynamic correlates of outcomes, we assessed the association of plasma platelet-derived growth factor (PDGF) isoform kinetics and PDGFR inhibition with progression-free and overall survival by individual treatment arm. We found that in the docetaxel-placebo arm alone, the probability of decrease in PDGFR phosphorylation (Pr-Decr-pPDGFR) above 0.5 (vs 30 months (HR 3.1; P=0.04 in log-rank test). By contrast, in the docetaxel plus imatinib arm, the association of Pr-Decr-pPDGFR >0.5 with a rise in plasma PDGF isoform concentrations and inferior survival was not observed. The data suggest that dynamic changes in PDGFR phosphorylation in peripheral blood leukocytes predict docetaxel efficacy. Rising plasma PDGF concentrations may explain and/or mark docetaxel resistance. Validation and mechanistic studies addressing these unexpected findings should anticipate a confounding influence of concurrent PDGFR inhibitor therapy.

Correlation of tumor growth inhibitory activity of macrophages exposed to adriamycin and adriamycin sensitivity of the target tumor cells.

Variant cell lines of the murine fibrosarcoma UV-2237, selected for doxorubicin [adriamycin (ADM)] resistance, were used to study the tumoricidal activity of macrophages that had been exposed to ADM. Free ADM (0.01-1 microgram/ml) was cytotoxic to the sensitive UV-2237M parent and to the partially sensitive UV-2237M-revertant cell lines, whereas the ADM-resistant UV-2237M ADMR line was unaffected by these levels of ADM. Macrophages harvested from the peritoneal cavities of mice given ip injections of 10 mg ADM/kg body weight 1 day or 4 days previously inhibited proliferation of the 3 cell lines, an effect that was directly correlated to the degree of ADM sensitivity of each cell line. Macrophages exposed in vitro to ADM (from 0.01 to 1 microgram/ml) inhibited the growth of, and eventually were cytolytic to, the parent and the revertant cell lines; these ADM-exposed macrophages did not affect the UV-2237M-ADMR cell line. The correlation between the antitumor effects of macrophages exposed to ADM and the ADM susceptibility of tumor cells suggests that ADM-exposed macrophages exert their effect by the release or transfer of the stored drug.

Influence of organ environment on extracellular matrix degradative activity and metastasis of human colon carcinoma cells.

Orthotopic implantation of human colon carcinoma cells is useful for studying the behavior of metastatic subpopulations. We observed that the parental line and variants of human colon carcinoma KM12 cells were all tumorigenic following implantation into the subcutis or cecal wall of BALB/c nude mice. Their ability to metastasize to distant organ sites varied, however, with the site of growth. Subcutaneous (SC) tumors did not produce visceral metastases, whereas cecal tumors metastasized to the regional mesenteric lymph nodes and to the liver. To examine the influence of organ environment on the extracellular matrix-degrading activity of the tumors, we inoculated human colon carcinoma cells into the subcutis or cecal wall and after 7 weeks isolated and cultured the tumors in serum-free medium. The conditioned media of SC tumors contained very low levels of type IV collagenase (gelatinase) and heparanase (heparan sulfate-specific endo-beta-D-glucuronidase), whereas the media of the cecal wall tumors contained high levels of both. Zymograms of the media revealed that the intracecal human colon carcinomas secreted more than three times the amount of latent and active forms of 92-kd type IV collagenase than did the SC tumors. Moreover, only the conditioned media of intracecal tumors contained latent and active forms of 64-kd type IV collagenase. Histochemical analysis using rabbit antiserum raised against the synthetic peptides of 72-kd procollagenase type IV showed type IV collagenase in the intracecal tumors; human colon carcinoma growing SC, however, were not stained significantly. These results suggest that factors in the organ environment may affect production and secretion of tumor extracellular matrix-degrading enzymes, and these factors may modify the metastatic behavior of human colon carcinoma cells in nude mice.

Quantitative fluorescent microassay for identification of antiproliferative compounds.

A reproducible and convenient microassay was developed by us to measure the antiproliferative activity of biological materials and anticancer drugs. The assay involved the labeling of target cells after their incubation with anticancer agents with the fluorescent vital dye hydroethidine at a dose of 28 micrograms/ml for 30 minutes. Once internalized into live cells, hydroethidine is dehydrogenated to ethidium, which then intercalates into DNA. Hydroethidine labeled the cells uniformly. The cells were lysed with detergent, and the relative fluorescence of this dye was monitored in a microfluorimeter at the speed of 30 seconds per 96-well plate. Because this assay is accurate and reproducible, does not present problems of disposal of labeling agents, and is extremely rapid, it should prove very useful for the screening of many potential anticancer agents with antiproliferative activity.

Development of a model of chronic lymphocytic leukemia in inbred strain 2 guinea pigs.

A new transplantable leukemia (KSL) of unknown etiology that arose in a female Sewall-Wright strain 2 guinea pig is described. KSL cells morphologically resembled medium to large lymphocytes, displayed surface Ia antigen and receptors for complement and for the Fc portion of immunoglobulin, and synthesized surface immunoglobulin (IgM). These characteristics suggest a leukemia of B-lymphocyte origin. KSL cells were shown to be sensitive in vivo to both cyclophosphamide and 1,3-bis(2-chloroethyl)-1-nitrosourea; however, neither drug effectively prevented eventual recurrence of the disease. KSL leukemia was also shown to be distinct from another guinea pig lymphatic leukemia (L2C) with respect to cell morphology, antigenicity, and in vivo growth rate. In this last respect, KSL appeared more closely related to the chronic lymphocytic leukemias. Thus KSL is the first chronic lymphocytic leukemia in the guinea pig to be characterized; it is also the only guinea pig model of lymphatic leukemia that is distinct from L2C leukemia currently available for study.

Demonstration of multiple phenotypic diversity in a murine melanoma of recent origin.

The purpose of these studies was to examine whether the metastatic heterogeneity that is frequently found in serially transplanted neoplasms could be observed in a murine melanoma of recent origin. The primary K-1735 melanoma that arose in an inbred C3H/HeN murine mammary tumor virus-negative (C3H-) mouse was transplanted once into an immunosuppressed recipient and then established in culture. Cells from the fifth in vitro passage were used to produce clones. The parent K-1735 and 22 cloned lines were tumorigenic in syngeneic and outbred N:NIH(S) nude mice. Metastatic properties were assessed by observing the ability of the cells to produce pulmonary and extrapulmonary lesions after they were injected iv into 6-week-old C3H- mice. The number of metastases produced, their relative size, and pigmentation varied dramatically among the clones. Only 2 of 22 clones were indistinguishable from the parent tumor. Most of the nonmetastatic (but tumorigenic clones) were also nonmetastatic in 3-week-old nude mice, which suggests that the absence of metastasis formation was not merely due to their immunologic rejection by the normal C3H- mouse. Control subcloning experiments demonstrated that the procedure of cloning in vitro was not responsible for the variability among the clones. The clones did not differ in their karyotype or cell size, but they did differ in their growth rate in vitro. These phenotypes, however, did not correlate with metastatic propensity. In conclusion, the K-1735, a murine melanoma of most recent origin, is heterogeneous and contains subpopulations of cells with diverse biologic behavior.

Organ-specific modulation of steady-state mdr gene expression and drug resistance in murine colon cancer cells.

BACKGROUND: The major cause of death from cancer is metastases that are resistant to conventional therapies. The resistance of metastatic tumor cells to chemotherapy can be caused by their intrinsic properties, such as increased expression of the mdr genes. PURPOSE: The purpose of our present study was to determine some of the mechanisms by which the organ microenvironment influences the response of tumor cells to chemotherapy. METHODS: Murine CT-26 colon cancer cells growing in continuous culture (parental cells) were harvested and injected subcutaneously into the lateral flank (to produce subcutaneous tumors) or the lateral tail vein (to produce experimental lung metastases) of 10 8-week-old syngeneic male BALB/c mice. Seven days after tumor-cell injection, the mice were given intravenous injections of either doxorubicin (10 mg/kg) or 0.9% NaCl (controls). This in vivo injection was repeated 7 days later. Mice with subcutaneous tumors and lung metastases were killed by cervical dislocation on day 21, and tumor samples from control mice were harvested and adapted to culture. The sensitivity of the cultured cells to doxorubicin and fluorouracil (5-FU) was determined at multiple time points. Levels of mdr-1 DNA were measured by slot-blot and Southern-blot analyses. mdr mRNA expression levels were measured by Northern-blot analysis using mdr-1- and mdr-3-specific hybridization probes, and P-glycoprotein level was determined by fluorescence-activated cell sorting using different monoclonal antibodies. RESULTS: Treatment with doxorubicin produced 80% growth inhibition of CT-26 subcutaneous tumors but had little effect on the number (and size) of experimental lung metastases. Collectively, the results suggest that the multidrug-resistant phenotype developed in CT-26 cells growing in the lung environment. Cultures established from lung metastases were initially resistant to doxorubicin (but not to 5-FU) and showed elevated expression of mdr-1 mRNA transcripts and P-glycoprotein. This resistance could be overcome by verapamil and disappeared after 21 days in culture. No mdr gene amplification was detected. The expression level of mdr-specific mRNA (predominance of mdr-1) and P-glycoprotein was directly associated with resistance to doxorubicin. CONCLUSIONS: Results of this study have demonstrated that the in vivo sensitivity of murine CT-26 colon carcinoma cells to doxorubicin depends on the organ environment. The organ environment can influence the P-glycoprotein-mediated multidrug-resistant phenotype in tumor cells, and the increased expression of P-glycoprotein is transient; once removed from the environment (lung), the cell's resistance reverts to that of the sensitive parent cells.

Augmentation of antiproliferative activity of interferon alfa against human bladder tumor cell lines by encapsulation of interferon alfa within liposomes.

Present therapy for human bladder cancer includes the intravesical administration of antiproliferative agents, such as recombinant human interferon alfa (IFN-alpha). The administration of cytotoxic molecules encapsulated in liposomes could provide a more efficient method for such therapy. Therefore, we determined whether encapsulation of the recombinant human IFN-alpha hybrid BBDD within liposomes will produce antitumor effects against the human bladder cancer cell line 253J superior to those observed with free IFN-alpha. Adherent cells were cultured in medium alone, in medium containing different concentrations of IFN-alpha, or in medium containing multilamellar liposomes (phosphatidylcholine-phosphatidylserine at a molar ratio of 7:3) that encapsulated saline or IFN-alpha. Cell growth was determined 96-120 hours later. Additional control groups consisted of target cells cultured with free IFN-alpha or with IFN-alpha plus liposomes containing saline. Cytostasis mediated by free IFN-alpha alone or IFN-alpha in the presence of liposome-saline was identical and ranged from 0%-30% (10 IU/mL) to 45%-70% (1,000 IU/mL). Liposomes containing saline produced no effects. Liposome-encapsulated IFN-alpha produced significantly greater growth inhibition than free IFN-alpha: 40%-70% (10 IU/mL) and 80%-90% (1,000 IU/mL), respectively. Moreover, a 253J variant subline selected for resistance to free IFN-alpha was sensitive to IFN-alpha presented in liposomes. These data suggest that the encapsulation of antiproliferative agents such as IFN-alpha in liposomes can improve therapeutic results.

Correlation of growth capacity of human tumor cells in hard agarose with their in vivo proliferative capacity at specific metastatic sites.

The purpose of this study was to determine whether the degree of anchorage-independent growth of human tumor cells in increasing concentrations of agarose correlated with the capacity of the cells to produce experimental metastases in nude mice. Human melanoma, breast carcinoma, and colon carcinoma cells from parental lines and variants selected in vivo for metastasis and in vitro cloned lines were plated into medium containing 0.3%, 0.6%, 0.9%, or 1.2% of agarose. These cells were also injected into nude mice: intravenously for melanoma, into the mammary fat pad for breast carcinoma, and into the spleen for colon carcinoma. Production of tumor cell colonies in dense agarose (greater than 0.6%) correlated with production of experimental metastases in the lung (melanoma, breast carcinoma) or liver (colon carcinoma). We conclude that the degree of anchorage-independent growth of tumor cells can predict their biological behavior and metastatic potential in vivo. Thus, this technique may be useful for the isolation of metastatic cells from heterogeneous human neoplasms.

Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice.

BACKGROUND: By the time patients are diagnosed with ovarian carcinoma, peritoneal dissemination of the tumor often has occurred. The progressive growth and spread of ovarian carcinoma depend, in part, on the formation of an adequate blood supply. We determined whether the expression of genes that regulate distinct steps in angiogenesis (i.e., the formation of new blood vessels) was associated with the pattern and progressive growth of human ovarian carcinomas implanted in the peritoneal cavity of nude mice. METHODS: Five different human ovarian carcinomas were injected individually into the peritoneal cavity of female NCr-nu/nu nude mice. The expression of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), interleukin 8 (IL-8), and collagenase type IV (MMP-2 [matrix metalloproteinase-2] and MMP-9) was determined by northern blot analysis, in situ hybridization of messenger RNA, and immunohistochemical analysis. Blood vessel distribution and density, macrophage infiltration pattern, and stromal reaction were determined by immunohistochemical analysis with specific antibodies. RESULTS: Three of the carcinomas produced both solid lesions and ascitic tumors, whereas the remaining two produced only solid lesions. Two of the carcinomas produced rapidly progressive disease, two produced slow disease, and one produced intermediate disease. The formation of ascites was directly associated with expression of VEGF/ VPF, and survival was inversely associated with expression of IL-8. In rapidly growing tumors, the number of blood vessels was high throughout the lesion; in contrast, in slow-growing tumors, most vessels (and infiltrating macrophages) were located at the periphery. CONCLUSIONS: The expression of various genes that regulate angiogenesis in human ovarian carcinomas is associated with the pattern of the disease and its progression. Therefore, targeting specific genes that regulate angiogenesis could offer new approaches to the treatment of ovarian cancer.

Platelet-derived endothelial cell growth factor in human colon cancer angiogenesis: role of infiltrating cells.

BACKGROUND: Development of new blood vessels is essential for tumor growth and metastasis and depends on the production of angiogenic factors by tumor and/or infiltrating cells. We previously showed that vascular endothelial growth factor (VEGF) expression and vessel count correlate with metastasis in human colon cancer. Although most tumors with high vessel counts express high levels of VEGF, some do not. Recently, platelet-derived endothelial cell growth factor (PD-ECGF), another potent angiogenic factor, has been reported to be expressed in colon cancer. PURPOSE: In this study, we examined the role of PD-ECGF in colon cancer angiogenesis and whether PD-ECGF is derived from the tumor or infiltrating cells. METHODS: Immunostaining for PD-ECGF was performed on 96 colon cancer specimens, some of which were previously stained for VEGF and factor VIII, a marker that is specific for endothelial cells. Double staining was done by using antibodies to PD-ECGF and to CD68 (macrophage specific) or CD3 (lymphocyte specific) to confirm which infiltrating cells produce PD-ECGF. Northern blot analysis for PD-ECGF messenger RNA (mRNA) was performed on four colon cancer specimens and corresponding normal colon mucosae (same patients) and four human colon cancer cell lines (KM12SM, SW620, HT29, and NCI-H508) to determine whether colon cancer epithelium expresses PD-ECGF. RESULTS: Immunohistochemical analysis demonstrated that PD-ECGF was expressed in infiltrating cells in most of the colon cancer specimens (80 [83%] of 96) but rarely in tumor epithelium (five [5%] of 96). Double staining demonstrated that infiltrating cells staining positive for both PD-ECGF and CD68 were more predominant than those staining positive for both PD-ECGF and CD3. The intensity of staining for PD-ECGF in infiltrating cells correlated with vessel counts (Spearman's rank correlation coefficient (R) = .29; P = .004), but did not correlate with the intensity of VEGF staining (R = .176, P = .086) or metastasis (Mann-Whitney U test, P = .253). PD-ECGF staining intensity was higher in specimens with a high vessel count (> 50 at high magnification) and low VEGF-staining intensity (< or = 2+) than in specimens with a high vessel count (again, > 50) and high VEGF-staining intensity (3+). Northern blot analysis revealed that colon cancer specimens and normal mucosae expressed relatively high levels of PD-ECGF mRNA, whereas PD-ECGF mRNA transcripts were not detectable in colon cancer cell lines. CONCLUSIONS AND IMPLICATIONS: PD-ECGF expression in human colon cancer specimens is associated with vessel count and may be responsible for tumor vascularity in those tumors with low VEGF expression. Infiltrating cells expressing PD-ECGF may contribute to angiogenesis, thus providing an additional mechanism for tumor neovascularization.

Isolation of tumoricidal macrophages from lung melanoma metastases of mice treated systemically with liposomes containing a lipophilic derivative of muramyl dipeptide.

The systemic administration of multilamellar liposomes--composed of phosphatidylcholine and phosphatidylserine (7:3 mol ratio), containing the immunomodulator, muramyl tripeptide phosphatidylethanolamine (MTP-PE)--into C57BL/6N mice bearing the syngeneic B16-BL6 melanoma was associated with the eradication of spontaneous lung and lymph node metastases. Immunofluorescence and electron microscopic analyses revealed that 24 hours after the tumor-bearing mice were given iv injections of liposomes, 15% of the alveolar macrophages and 5% of the metastasis-associated macrophages contained phagocytosed liposomes. However, only macrophages isolated from lungs or metastases of mice given injections of liposomes containing MTP-PE (treatment success), but not macrophages from mice treated with empty liposomes (treatment failure), were tumoricidal against the target cells in vitro. These data provide direct evidence that the regression of established metastases, after treatment of tumor-bearing mice with liposomes containing MTP-PE, was associated with tumoricidal macrophages residing within the metastases.

Mechanism of tumor cell resistance to lysis by syngeneic lymphocytes.

B16 melanoma variant lines, which resisted lysis by syngeneic lymphocytes, were selected in vitro by repeated exposure of the tumor cells to purified cytotoxic lymphocytes. The resistance of the tumor cells to lysis mediated by syngeneic lymphocytes was not accompanied by a loss or masking of major histocompatibility antigens. Neither the lymphocyte-susceptible B16 (F10) nor the lymphocyte-resistant B16 (F10Lr) cells grew in allogeneic recipients, and both were destroyed in vitro by allogeneic lymphocytes. The resistance to lysis by syngeneic lymphocytes was not accompanied by loss or masking of receptors for macrophage recognition and destruction. F10Lr cells did not protect F10 cells from lymphocyte-mediated lysis in cocultivation experiments. Immunization of syngeneic mice in vivo with B16-F10 cells successfully protected mice against challenge with B16-F10 cells. However, mice immunized with B16-F10Lr cells were not protected against challenge with B16-F10 cells. B16-F10Lr cells were not immunogenic when tested under this condition. Light, scanning, and transmission electron microscopic studies of tumor cell-lymphocyte interaction suggested that the resistance of B16-F10Lr cells to destruction by syngeneic lymphocytes in vitro was due to the masking or absence of tumor-specific antigen(s) present on the lymphocyte-susceptible F10 cells.