Hair follicle-derived blood vessels vascularize tumors in skin and are inhibited by Doxorubicin

We have recently shown that the neural-stem cell marker nestin is expressed in hair follicle stem cells and the blood vessel network interconnecting hair follicles in the skin of transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). The hair follicles were shown to give rise to the nestin-expressing blood vessels in the skin. In the present study, we visualized tumor angiogenesis by dual-color fluorescence imaging in ND-GFP transgenic mice after transplantation of the murine melanoma cell line B16F10 expressing red fluorescent protein. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that the blood vessel-specific antigen CD31 was expressed in ND-GFP-expressing nascent blood vessels. ND-GFP expression was diminished in the vessels with increased blood flow. Progressive angiogenesis during tumor growth was readily visualized during tumor growth by GFP expression. Doxorubicin inhibited the nascent tumor angiogenesis as well as tumor growth in the ND-GFP mice transplanted with B16F10-RFP. This model is useful for direct visualization of tumor angiogenesis and evaluation of angiogenic inhibitors.     

Lymph node metastasis of oral cancer visualized in live tissue by green fluorescent protein expression

Here, we report the establishment of a stably transfected cell line which expresses high levels of green fluorescent protein (GFP), thus permitting the detection and visualization of developing tumors and lymph node metastases after injection into nude mice. Cells of the human oral squamous carcinoma cell line (SAS-L1) were transfected with an expression vector containing a cDNA encoding humanized GFP and the neomycin resistance gene. A clone with stable high-level expression of GFP was selected in vitro using G418. To study metastasis formation, GFP-expressing cells were injected orthotopically into the tongue of nude mice. The resultant tumor growth in the tongue and micrometastases in the lymph nodes could be visualized by GFP fluorescence. Therefore a useful model has been developed for the study of oral cancer, firstly to understand the metastatic process and secondly for the evaluation of potential treatments.     

Ferrocenes (F168, F169) and fero-sorbitol-citrate (FSC): potential anticancer drugs

We have shown earlier that the iron containing, ferric-sorbitol-citrate complex (FSC) inhibited proliferation of cultured mouse melanoma B16, GHC, KB, HeLa and CaCo2 cells and caused mouse melanoma regression in vivo. This drug did not affect the proliferation of the nonmalignant fibroblast L929 line, human bone marrow-HBS, VERO and HEF cell line. It is also known, that some metallocene derivatives posses antitumour properties resulting principally from their action on the metabolism of DNA, and subsequently, RNA and proteins. We synthesized in our laboratory some ferrocene analogs (F168 and F169) and tested their antiproliferative ability for malignant human carcinoma Hep2 and mouse melanoma F10 cell lines. As control cell lines, human HEF and mice L929 fibroblasts were used. The tested iron substances were very potent in inhibiting the growth of malignant cell lines, whereas they had no significant inhibitory effect on the viability of nonmalignant fibroblasts. The most pronounced growth inhibitory and cytotoxic effect was found in the malignant F10 cells and the most potent was ferrocene F169. Because of their selective effect on malignant cells, the ferric-sorbitol-citrate complex as well as tested ferrocenes will be further investigated and submitted as new antitumour substances.     

Clinically proven markers of metastasis predict metastatic spread of human melanoma cells engrafted in scid mice

Metastasis formation is a complex process and as such can only be modelled in vivo. As markers indicating metastatic spread in syngenic mouse models differ significantly from those in man, this study aimed to develop a human melanoma xenograft mouse model that reflects the clinical situation. Six human melanoma cell lines (LOX, MV3, FEMX-1, G361, MeWo and UISO-Mel6) were xenografted into severe combined immunodeficient mice and tumour growth, metastatic behaviour and number of lung metastases were assessed. Tumours and metastases were analysed for HPA binding and expression of CEACAM-1 and L1, all markers indicative of metastasis in clinical studies. Development of primary tumour nodules ranged from 3 weeks (MV3) to 3 months (MeWo). Whereas G361 and FEMX-1 rarely formed lung metastases, MeWo, MV3 and LOX were moderately and UISO-Mel6 was highly metastatic. Similar to clinical studies, HPA, CEACAM1 and L1 indicated metastatic spread in the xenograft melanoma model, but were not all simultaneously expressed in all cell lines. Considering the strongest expression of one marker combined with an absent or low expression of the other two markers, we conclude that LOX is the cell line of choice for analyses of the functional role of HPA-binding glycoconjugates, UISO-Mel6 is ideally suited to study CEACAM1 function in melanoma spread and L1 function can best be modelled using MeWo.     

Comparative in vivo and in vitro proliferation of murine tumor cells in the bone microenvironment

To examine the effect of bone microenvironmental factors on the growth of metastatic cells, the in vivo proliferative features of three murine cell lines were determined at skeletal metastatic sites and correlated with their ability to grow in vitro in the presence of bone-derived factors. Bones, ovaries, adrenals and the brain were most affected by metastasis, following an intraarterial injection of B16/F1 and B16/F10 melanoma and FS/L10 fibrosarcoma cells into C57BL/6 mice. Melanoma cells showed a marked metastatic preference for bone, while fibrosarcoma cells developed brain metastasis in all animals. Tumor burden in bones was highest (19+/-2%) for B16/F10 cells, compared to B16/F1 (10+/-2%) or FS/L10 (3+/-1%) cells. Autoradiographic studies demonstrated organ- and cell type-specific differences in tumor cell proliferation, with B16/F10 cells displaying the lowest labelling indexes in bone (12+/-2% for B16/F10 vs 28+/-2% and 27+/-4% for B16/F1 and FS/L10 cells, respectively). To test if bone-derived factors differentially affected tumor cell growth in these three cell lines H-3-thymidine uptake by these tumor cells was assessed after in vitro incubation with bone-derived conditioned medium. Under these conditions, we observed stimulation of B16/F10 cell proliferation, but inhibition of uptake in the other two cell lines. Thus, these results demonstrate that, in this in vivo experimental model, growth properties of metastatic cells are organ- and cell type-specific. Additionally, we show that the in vitro proliferative behavior of tumor cells in the presence of bone-derived factors correlates and may predict skeletal tumor growth properties in vivo.     

Human tumor spontaneous metastasis in immunosuppressed newborn rats. I. Characterization of the bioassay

We characterize a new model of spontaneous metastasis of human tumor cells using anti-thymocyte serum (ATS) immunosuppressed newborn rats. We analyzed the intrinsic value of the bioassay of measurement of tumorigenicity and metastatic capacity using 17 human tumor cell lines, of which were 9 human malignant melanomas. Most of these cell lines revealed as tumorigenic and metastatic in lungs and/or lymph nodes 3 weeks after s.c. injection in the ventral flank of newborn rats, irrespective of their origin. All the melanoma cell lines that we injected were tumorigenic and 77% were metastatic, whereas the same cell lines grafted in nude mice showed no evidence of metastases after 6 weeks' examination. We were not able to show any relationship between tumorigenicity in nude mice and the malignant behavior of these cells in ATS-treated newborn rats. Likewise, neither chromosome abnormalities, nor antigenic marker expression were found to be related to tumor growth in nude mice or newborn rats. Two intrinsic parameters of the model were studied: number of cells injected vs. dose of ATS injected for one melanoma cell line; and role of the 3rd and 4th injections of ATS in the establishment and development of pulmonary metastases. Moreover, we show that s.c. injection in the ATS-treated newborn rat may represent a suitable method for studying melanoma cell tumor growth and spontaneous dissemination.     

A fluorescent orthotopic bone metastasis model of human prostate cancer

Here, we report a fluorescent spontaneous bone metastatic model of human prostate cancer developed by surgical orthotopic implantation of green fluorescent protein (GFP)-expressing prostate cancer tissue. Human prostate cancer PC-3 cells were transduced with the pLEIN expression retroviral vector containing the enhanced GFP and neomycin resistance genes. Stable GFP high-expression PC-3 clones were selected in vitro with G418, which were then combined and injected s.c. in nude mice. For metastasis studies, fragments of a single highly fluorescent s.c. growing tumor were implanted by surgical orthotopic implantation in the prostate of a series of nude mice. Subsequent micrometastases and metastases were visualized by GFP fluorescence throughout the skeleton, including the skull, rib, pelvis, femur, and tibia The central nervous system, including the brain and spinal cord, was also involved with tumor, as visualized by GFP fluorescence. Systemic organs, including the lung, plural membrane, liver, kidney, and adrenal gland, also had fluorescent metastases. The metastasis pattern in this model reflects the bone and other metastatic sites of human prostate cancer. Thus, this model should be very useful for the study and development of treatment for metastatic androgen-independent prostate cancer.     

Nude rat model for studying metastasis of human tumor cells to bone and bone marrow

Bone metastases reproducibly developed in nude rats after an injection of LOX human malignant melanoma cells into the left ventricle, with hind leg paralyses appearing in all animals within approximately 2 weeks. Manifest metastases were present exclusively in the skeletal system, predominantly in the lumbar portion of the spine, the long bones, and occasionally in the skull. Intracardially injected 125I-labeled tumor cells and monodisperse microspheres were distributed in parallel to the various tissues. Moreover, because the levels of radioactivity were significantly lower in bone than in lung, kidney, and liver, the pattern of metastases could not be explained solely by hemodynamic factors. In chemotherapy experiments, the survival time of rats given left ventricular injections of LOX cells increased in a dose-dependent manner after the animals were treated with dacarbazine. Researchers may find the model useful for studying the biology of bone metastases and for testing the sensitivity of these lesions to drugs.     

Helix pomatia agglutinin binding in human tumour cell lines: correlation with pulmonary metastases in nude mice.

The extent of lectin binding by three human melanoma (LOX, FEMX-1 and SESX) and two sarcoma lines (MHMX and OHSX) was related to their potential for experimental metastasis formation in athymic nude mice. The Helix pomatia agglutinin (HPA), which recognises the N-acetyl-D-galactosamine ligand, showed differential binding to the cell lines in a manner that correlated with their ability to give lung colonies after i.v. injection in the mice (P < 0.005). The degree of HPA binding and lung colony formation of the cell lines studied was ranked in the following order, LOX > MHMX > OHSX > SESX > FEMX-I. Similar patterns were not observed with the other lectins used in this study (WGA, Con A, PNA and UEA-I). The high HPA reacting LOX melanoma line shows extensive pulmonary metastatic formation with no extrapulmonary colonies, whereas the low HPA reacting FEMX-I cells give only extrapulmonary metastases with no detectable colonies in the lungs. Precoating of tumour cells with HPA prior to injection did not reduce the ability of cells to give pulmonary metastases, suggesting that the HPA epitope was not functionally associated with the pulmonary metastatic potential observed in nude mice. These findings support recent human studies of a correlation between HPA binding and incidence of metastasis, however, our data indicate that there is no causal relationship. Further analyses are required to identify the specific HPA-binding glycoconjugates that may be involved.     

In vitro assay demonstrates similar invasion profiles for B16F1 and B16F10 murine melanoma cells

The variants of the B16 murine melanoma cell line were assayed for their invasive characteristics in the membrane invasion culture system (MICS) and concomitantly tested for their ability to form lung metastases in vivo. Specifically, the B16F1 (low metastatic variant) and the B16F10 (high metastatic variant) murine melanoma cell lines were examined for their ability to invade human amniotic basement membranes (BMs) in vitro and simultaneously examined for lung colony formation in vivo. The B16F1 and B16F10 cell lines both demonstrated similar invasion profiles over 72 h with the total percent invasion through the BMs for both cell lines not exceeding 5.0%. In vivo observations reconfirmed the significant difference in the metastatic capabilities of the 2 variants. These data suggest that tumor cells with differing metastatic propensities can invade an amniotic BM at similar rates, but their survival and metastatic lesion forming capabilities in vivo may vary considerably.     

B16 melanoma cell arrest in the mouse liver induces nitric oxide release and sinusoidal cytotoxicity: a natural hepatic defense against metastasis

The formation of liver metastases involves interactions between intravascular cancer cells and the hepatic microvasculature. Here we provide evidence that the arrest of intravascular B16F1 melanoma cells in the liver induces a rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. B16F1 melanoma cells (5 x 10(5)) labeled with fluorescent microspheres were injected into the portal circulation of C57BL/6 mice. The production of NO in vivo was detected by electron paramagnetic resonance spectroscopy ex vivo using an exogenous NO-trapping agent. A burst of NO was observed in liver samples examined immediately after tumor cell injection. The relative electron paramagnetic resonance signal intensity was 667 +/- 143 units in mice injected with tumor cells versus 28 +/- 5 units after saline injection (P < 0.001). Two-thirds of cells arrested in the sinusoids compared with the terminal portal venules (TPVs). By double labeling of B16F1 cells with fluorescent microspheres and a TdT-mediated UTP end labeling assay, we determined that the melanoma cells underwent apoptosis from 4-24 h after arrest. The mean rate of apoptosis was 2-fold greater in the sinusoids than in the TPVs at 4, 8, and 24 h after injection (P < 0.05-0.01). Apoptotic cells accounted for 15.9 +/- 0.8% of tumor cells located in the sinusoids and 7.1 +/- 0.9% of tumor cells in the TPVs. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester completely blocked the NO burst (P < 0.001) and inhibited the apoptosis of B16F1 cells in the sinusoids by 77%. However, the rate of tumor cell apoptosis in the TPVs was not changed. There were 5-fold more metastatic nodules in the livers of N(G)-nitro-L-arginine methyl ester-treated mice (P < 0.05). The inactive enantiomer N(G)-nitro-D-arginine methyl ester had no effect on the initial NO burst or on apoptosis of tumor cells in vivo. Both annexin V phosphatidylserine plasma membrane labeling and DNA end labeling of apoptotic cells were demonstrated after a 5-min exposure (a time equivalent to the initial transient NO induction in vivo) of B16F1 cells to a NO donor in vitro. These results identify the existence of a natural defense mechanism against cancer metastasis whereby the arrest of tumor cells in the liver induces endogenous NO release, leading to sinusoidal tumor cell killing and reduced hepatic metastasis formation.     

Effects of cytokine-mediated modulation of nm23 expression on the invasion and metastatic behavior of B16F10 melanoma cells

The molecular mechanisms of tumor invasion and metastasis are yet to be fully elucidated. A potential tumor-metastasis-suppressor gene nm23 has been described in certain rodent and human tumors. In the present study, we examined the potential anti-invasive and anti-metastatic effect of nm23 gene in B16F10 cells, a malignant murine melanoma cell line. Transfection of nm23 gene into B16F10 melanoma ceils resulted in significant suppression of the invasiveness and metastatic ability of melanoma cells and significantly enhanced the survival of tumor-bearing mice. B16F10 melanoma cells transfected with nm23 produced significantly less soluble ICAM-I and were more susceptible to LAK-cell-mediated cytotoxicity. Co-culture of B16F10 melanoma cells with IL-2 had no effect on nm23 expression, whereas treatment with PGE₂, TNF-α and IFN-γ resulted in down-regulation of nm23 expression. Concomitantly, in vivo treatment with TNF-α or IFN-γ in experimental mice increased pulmonary metastases and lowered the overall survival period, as compared with IL-2 treatment alone. These results provide evidence that nm23, in addition to its anti-metastatic function, could also be involved in modulating tumor-target-structure expression, in down-regulating invasive potential and in production of soluble intracellular adhesion molecules. The down-regulation of nm23 by TNF-α, IFN-γ and particularly by PGE₂ warrants re-examination of current immunotherapeu-tic protocols and of the role played by PGE₂ in tumor progression. © 1995 Wiley-Liss, Inc.     

An embryo-specific expressing TGF-β family protein,growth-differentiation factor 3 (GDF3), augments progression of B16 melanoma

Malignant tumor cells often express embryonic antigens which share the expression with embryonic stem (ES) cells. The embryonic antigens are usually encoded by ES cell-specific genes, a number of which are associated with tumorigenesis and/or tumor progression. We examined the expression of ES cell-specific genes in the mouse B16 melanoma cell line to identify the factors promoting tumorigenesis. We found that endogenous growth-differentiation factor 3 (GDF3) expression was induced in implant B16 tumor during tumor progression in syngenic C57BL/6 mice. B16 F10, a subline with a high metastatic potential, continuously expressed GDF3 while low metastatic B16 F1 expressed comparatively decreased levels of GDF3. Overexpression of GDF3 promoted growth of implanted melanoma B16 F1 and F10 in syngenic mice. Ectopic expression of GDF3 was accompanied by an increased level of production of CD24/CD44. Such a profile was reported to be characteristic of melanoma stem cell-like cells. GDF3 expression was observed in embryonal carcinomas, primary testicular germ cell tumors, seminomas and breast carcinomas. However, the role of GDF3 in these cancers remains undetermined. Overexpression of GDF3 did not affect the growth of mouse hepatoma high or low metastatic sublines G5 or G1, both of which do not express GDF3. Since GDF3-driven CD24 acts as a receptor for endogenous innate immune ligands that modulate cell proliferation, CD24 is an effective determinant of tumorigenesis in malignant cell transformation. Finally, our results support the view that GDF3 has the ability to induce progression of CD24-inducible melanoma in mice.     

Effect of an anti-CD54 (ICAM-1) monoclonal antibody (UV3) on the growth of human uveal melanoma cells transplanted heterotopically and orthotopically in SCID mice

We have shown that administration of a novel anti-CD54 monoclonal antibody (UV3) results in long-term survival of SCID mice bearing human myeloma xenografts. Previous studies have demonstrated a link between the expression of CD54 and the progression of uveal melanoma. Our study assessed the expression of CD54 on 7 human uveal melanoma cell lines and 3 cell lines established from uveal melanoma metastases. In vivo studies examined the efficacy of systemic and local administration of UV3 antibody on the progression of uveal melanoma cells transplanted either heterotopically or orthotopically into SCID mice. Five of the 7 primary uveal melanoma cell lines and all 3 of the metastases cell lines expressed CD54. Intraperitoneal injection of either IgG or F(ab')2 fragments of UV3 significantly inhibited the growth of subcutaneous and intraocular melanomas. Subconjunctival injection of either IgG or F(ab')2 fragments of UV3 produced a significant reduction in the growth of intraocular melanomas, even if the antibody was administered after the appearance of intraocular tumors. The results indicate that both primary and metastatic human uveal melanoma cells express CD54. The marked inhibition of intraocular and subcutaneous uveal melanoma progression suggests that UV3 antibody is a promising therapeutic agent for further evaluation in patients with uveal melanoma. This is especially noteworthy, as no existing therapeutic modality prevents metastasis of uveal melanoma or prolongs the survival of patients with uveal melanoma.     

Differentiation and the tumorigenic and metastatic phenotype of murine melanoma cells

Using B16 F10 murine melanoma cells and sublines generated from the JB/MS melanoma which exhibit various degrees of melanogenesis, the relationships among differentiation, tumorigenicity, and metastatic potential were examined. The effect of melanocyte-stimulating hormone (MSH), which specifically stimulates differentiation of melanocytes, was also studied. All melanoma lines tested were capable of growing as experimental pulmonary metastases but, surprisingly, the undifferentiated and amelanotic JB/MS-w cells failed to grow as primary subcutaneous tumors. JB/MS-w cells, which had few surface MSH receptors, did not respond to MSH with an increase in melanin production, unlike the other cell lines. Although in vitro treatment with MSH did not change the rates of growth of primary tumors by these cell lines, such treatment decreased the number of pulmonary metastases from B16 F10, JB/MS cells, JB/MS-b1 cells and JB/MS-w cells. Conversely, MSH treatment significantly increased the rates of pulmonary metastases from JB/MS-p cells. The expression of surface melanoma antigens, urokinase-type plasminogen activity and susceptibility to natural killer cells were examined. MSH did not significantly alter surface melanoma antigen expression, but increased the natural killer cell susceptibility of B16 F10, JB/MS and JB/MS-b1 cells, cells which possess abundant surface MSH receptors. There was an inverse correlation between differentiation (pigmentation) and proliferation in vitro, and the more pigmented melanoma cells (B16 F10, JB/MS and JB/MS-b1) expressed relatively lower levels of class-I MHC, relatively higher levels of class-II MHC and the highest metastatic capacity. These results demonstrate that MSH possesses the capacity to regulate not only melanogenesis, but also other factors critical to the metastatic growth of the cells.     

How Do Bisphosphonates Inhibit Bone Metastasis In Vivo?

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and have demonstrated clinical utility in the treatment of patients with osteolytic bone metastases. They also exhibit direct antitumor activity in vitro and can reduce skeletal tumor burden and inhibit the formation of bone metastases in vivo. However, whether such effects are caused by a direct action of bisphosphonates on tumor cells or indirectly through inhibition of bone resorption remains unclear. To address this question, we used here a structural analog of the bisphosphonate risedronate, NE-58051, which has a bone mineral affinity similar to that of risedronate, but a 3000-fold lower bone antiresorptive activity. In vitro, risedronate and NE-58051 inhibited proliferation of breast cancer and melanoma cell lines. In vivo, risedronate and NE-58051 did not inhibit the growth of subcutaneous B02 breast tumor xenografts or the formation of B16F10 melanoma lung metastasis. In contrast to NE-58051, risedronate did inhibit B02 breast cancer bone metastasis formation by reducing both bone destruction and skeletal tumor burden, indicating that the antitumor effect of bisphosphonates is achieved mainly through inhibition of osteoclast-mediated bone resorption.     

The bisphosphonate olpadronate inhibits skeletal prostate cancer progression in a green fluorescent protein nude mouse model.

PURPOSE: Metastatic bone disease is one of the major causes of morbidity and mortality in prostate cancer patients. Bisphosphonates are currently used to inhibit bone resorption and reduce tumor-induced skeletal complications. More effective bisphosphonates would enhance their clinical value. EXPERIMENTAL DESIGN: We tested several bisphosphonates in a green fluorescent protein (GFP)-expressing human prostate cancer nude mouse model. The in vivo effects of four bisphosphonates, including pamidronate, etidronic acid, and olpadronate, on bone tumor burden in mice intratibially inoculated with PC-3-GFP human prostate cancer cells were visualized by whole-body fluorescence imaging and X-ray. RESULTS: The PC-3-GFP cells produced extensive bone lesions when injected into the tibia of immunocompromised mice. The skeletal progression of the PC-3-GFP cell growth was monitored by GFP fluorescence and the bone destruction was evaluated by X-ray. We showed that 3,3-dimethylaminopropane-1-hydroxy-1,1-diphosphonic acid (olpadronate) was the most effective bisphosphonate treatment in reducing tumor burden as assessed by GFP imaging and radiography. The GFP tumor area and X-ray score significantly correlated. Reduced tumor growth in the bone was accompanied by reduced serum calcium, parathyroid hormone-related protein, and osteoprotegerin. CONCLUSIONS: The serum calcium, parathyroid hormone-related protein, and osteoprotegerin levels were significantly correlated with GFP area and X-ray scores. Treatment with olpadronate reduced tumor growth in the bone measured by GFP and X-ray imaging procedures. Imaging of GFP expression enables monitoring of tumor growth in the bone and the GFP results complement the X-ray assessment of bone disease. The data in this report suggest that olpadronate has potential as an effective inhibitor of the skeletal progression of clinical prostate cancer.     

Expression and function of CD9 in melanoma cells

CD9, a member of the tetraspanin family, functions as an organizer in “tetraspanin webs,” through interacting with other cell adhesion molecules. It plays a role in differentiation, fertilization, and cell migration. We investigated the expression and function of CD9 in melanoma. CD9 protein expression in B16 mouse melanoma and six human melanoma cell lines was decreased compared to normal melanocytes. B16F1 clones stably overexpressing CD9 had reduced ability to form colonies in soft agar; however, paradoxically these overexpressing clones had increased ability to invade Matrigel. Similarly, transient overexpression of CD9 in the human metastatic melanoma cell line WM9 dramatically decreased anchorage‐independent growth, while transient overexpression of CD9 in the radial growth phase cell line SbCl2 resulted in the gain of Matrigel invasion activity. DNA sequencing of CD9 cDNA from all six human melanoma cell lines did not show deletions, insertions, or mutations. Treatment of all six human melanoma cell lines with the histone deacetylase inhibitor trichostatin A increased CD9 levels. The DNA methylation inhibitor 5‐aza‐cytidine also increased CD9 protein levels with greater increases seen in cell lines derived from more malignant melanomas. © 2009 Wiley‐Liss, Inc.     

Influence of nitric oxide synthase II gene disruption on tumor growth and metastasis

The relationship between nitric oxide (NO) synthase II (NOS II) expression and the metastatic ability of tumor cells is inconclusive. We determined the role of host NOS II expression in the growth and metastasis of the B16-BL6 murine melanoma and M5076 murine ovarian sarcoma cell lines. The cells were either s.c. or i.v. injected into syngeneic wild-type (NOS H+/+) and NOS II-null (NOS H-/-) C57BL/6 mice. Both cell lines produced slightly larger s.c. tumors in NOS H-/- mice than in NOS II+/+ mice. However, B16- BL6 cells produced more and larger experimental lung metastases in NOS II+/+ mice than in NOS II-/- mice, whereas M5076 cells produced fewer and smaller experimental lung metastases in NOS II+/+ mice than in NOS II-/- mice. After activation with IFN-gamma and lipopolysaccharide, macrophages isolated from NOS II+/+ C57BL/6 mice produced NO-dependent cytotoxicity in sarcoma cells, whereas macrophages from NOS II-/- C57BL/6 mice did not. In contrast, activated macrophages produced little to no NO-mediated cytotoxicity in melanoma cells. Immunostaining analyses indicated that NOS II expression was apparent in the metastases growing in NOS H+/+ mice and correlated with increased cell proliferation in B16-BL6 lung metastases but with decreased cell proliferation in M5076 liver metastases. Our data suggest that disruption of host NOS II expression enhanced the growth and metastasis of NO-sensitive tumor cells but suppressed the metastasis of NO-resistant tumor cells, proposing that host-derived NO may differentially modulate tumor progression.     

A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis

Hematogenous dissemination of melanoma is a life-threatening complication of this malignant tumor. Here, we identified junctional adhesion molecule-C (JAM-C) as a novel player in melanoma metastasis to the lung. JAM-C expression was identified in human and murine melanoma cell lines, in human malignant melanoma, as well as in metastatic melanoma including melanoma lung metastasis. JAM-C expressed on both murine B16 melanoma cells as well as on endothelial cells promoted the transendothelial migration of the melanoma cells. We generated mice with inactivation of JAM-C. JAM-C(-/-) mice as well as endothelial-specific JAM-C-deficient mice displayed significantly decreased B16 melanoma cell metastasis to the lung, whereas treatment of mice with soluble JAM-C prevented melanoma lung metastasis. Together, JAM-C represents a novel therapeutic target for melanoma metastasis.