Overexpression of Merlin in B16F10 mouse melanoma cells reduces their metastatic activity: role of the cell surface heparan sulfate glycosaminoglycans

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.     

99mTcOAADT]-(CH2)2-NEt2: a potential small-molecule single-photon emission computed tomography probe for imaging metastatic melanoma

Evaluation of [99mTc]oxotechnetium(V) complexes of the amine-amide-dithiol (AADT) chelates containing tertiary amine substituents as small-molecule probes for the diagnostic imaging of metastatic melanoma has shown that technetium-99m-labeled AADT-(CH2)2-NEt2 (99mTc-1) has the highest tumor uptake and other favorable biological properties. We have, therefore, assessed this agent in a more realistic metastatic melanoma model in which, after i.v. tail injection, a highly invasive melanoma cell line, B16F10, forms pulmonary tumor nodules in normal C57BL6 mice. Small melanotic lesions develop in the lungs and, on histologic examination, appear as small black melanoma colonies, increasing in size and number with time after tumor cell injection. Groups of mice received tumor cell inocula of 2 x 10(5), 4 x 10(5), or 8 x 10(5) B16F10 cells; 14 days later, 2 hours after 99mTc-1 administration, lung uptake of 2.83 +/- 0.21%, 3.63 +/- 1.07%, and 4.92 +/- 1.61% injected dose per gram of tissue (% ID/g), respectively, was observed, compared with normal lung uptake of 2.13 +/- 0.2% ID/g (P < 0.05). Additionally, a higher level of 99mTc-1 accumulation was seen 17 days after tumor cell inoculation as the lung lesions grew. These in vivo studies coupled with additional in vitro and ex vivo assessment show that 99mTc-1 has high and specific uptake in melanoma metastases in lungs and can potentially follow the temporal growth of these tumors.     

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.     

Targeting foreign major histocompatibility complex molecules to tumors by tumor cell specific single chain antibody (scFv)

Down-regulation of the major histocompatibility complex (MHC) is one of the major mechanisms that tumor cells adopted to escape immunosurveillance. Therefore, specifically coating tumor cells with foreign MHC may make tumor cells a better target for immune recognition and surveillance. In this study, we designed and generated a fusion protein, H2Kd/scPSMA, consisting of a single chain antibody against human prostate specific membrane antigen (PSMA) and the extracellular domain of mouse H-2Kd. The expression of this fusion protein in B16F0 mouse melanoma cells was confirmed by RT-PCR and fluorescent activated cell sorting (FACS). Our animal study showed that the expression of H2Kd/scPSMA in B16F0/PSMA5, a B16F0 cell line expressing human PSMA, significantly inhibited tumor growth as demonstrated in the pulmonary metastasis assay and tumor growth study and improved overall survival.     

Lowered superoxide dismutase in highly metastatic B16 melanoma cells

Superoxide dismutases (SOD), which are enzymes scavenging the superoxide radical, were studied in two variant lines of the B16 melanoma: B16F1 with low metastatic potential and B16F10 with high metastatic potential. SOD activity was measured by a method utilizing reduction in the chemiluminescence of luminol. Using cell free extracts it was shown that the highly metastatic B16F10 cell line has a SOD activity lower (20.70 +/- 3.07) units/mg protein, n = 8, than that of the less metastatic B16F1 cell line (81.38 +/- 6.78) units/mg protein, n = 8. Acrylamide gel electrophoresis suggested that Mn-SOD activity is higher in B16F1 cells.     

B16-F10-luc-G5黑色素瘤细胞生物学特性的研究

目的：探讨B16-F10-luc-G5黑色素瘤细胞的生物学特性.方法：倒置荧光显微镜下连续观察B16-F10-luc-G5细胞生长动态.流式细胞术检测冻存复苏后、培养基漂浮细胞、胰酶消化损伤后的细胞死亡率以评价其对实验损伤的耐受性.1×104/孔B16-F10-luc-G5细胞按1∶2梯度稀释至0.78×102/孔,加入底物荧光素后检测其生物发光特性.Babl/C和C57 bl/6雄性小鼠各10只接种该细胞,目测肿瘤生长速度和活体成像监测Babl/C小鼠移植瘤生长动态以评价其成瘤特性.结果：B16-Fl0-luc-G5细胞生长动态符合典型B16-F10细胞特性,无自发及激发荧光.B16-F10-luc-G5细胞冻存、漂浮细胞及对数生长期细胞消化后死亡率分别为23.8％、35.8％和4.8％.B16-F10-luc-G5细胞数与实测平均光子数之间存在线性回归关系,检测光子数可反映细胞数目.两组小鼠移植成瘤率均为100％,平均肿瘤出现时间差异显著（t =9.05,P＜0.05）,移植瘤病理符合B16黑色素瘤典型生长特点;活体成像监测可灵敏监测移植瘤生长动态.结论：B16-F10-luc-G5细胞具有生长快、对各种实验操作耐受性好、标记生物发光基因易追踪等优点,且其一般特性与B16-F10细胞基本相同,是肿瘤学研究良好的实验材料.     

抑制整合素α9基因的表达对黑色素瘤细胞B16F1的生长和肺转移的影响

目的 观察shRNA干扰整合素α9（ITGA9）的表达对黑色素瘤细胞B16F1的生长和肺转移的影响。方法 用RNA干扰技术下调B16F1中ITGA9的表达,建立小鼠皮下成瘤和肺转移模型,观察肿瘤生长情况,计数肺转移灶数量。结果 ITGA9-shRNA转染组的肿瘤生长速度减慢（P＜0.05）,实验终点,该组肿瘤平均体积与scramble-shRNA组相比下降36%;肺转移灶数量显著减少（P＜0.05）。结论 下调ITGA9的表达可抑制黑色素瘤细胞B16F1在小鼠体内的生长和肺转移。ITGA9可能成为黑色素瘤的治疗靶点。     

The environmental stressor ultraviolet B radiation inhibits murine antitumor immunity through its ability to generate platelet-activating factor agonists

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr -/- mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3(EGFP) transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.     

TAP expression reduces IL-10 expressing tumor infiltrating lymphocytes and restores immunosurveillance against melanoma

Many immune therapeutic strategies are under development for melanoma to treat metastatic disease and prevent disease reoccurrence. However, human melanoma cells are often deficient in antigen processing and this appears to play a role in their expansion and escape from immunosurveillance. For example, expression of the transporters associated with antigen processing (TAP1 and TAP2) is down-regulated in the mouse melanoma cell line B16F10. This results in a lack of tumor-associated antigen processing, low surface expression of MHC Class I molecules and low immunogenicity. We observe that restoration of TAP1 expression by transfection resurrects the processing and presentation of viral antigens, and the melanoma-associated antigen, TRP-2. Immunization with irradiated B16F10/rTAP1 transfected cells generates CTLs that are capable of killing B16F10/rTAP1 transfected targets and B16F10 targets deficient in TAP1. Furthermore, B16F10/rTAP1 transfectants grow at a significantly slower rate in mice than B16F10 cells. In an experimental model that closely recapitulates the clinical situation, treatment of B16F10 tumors in mice with a vaccinia virus vector expressing TAP1 also significantly decreases tumor growth in vivo. Furthermore, tumors treated with vaccinia TAP1 had significantly reduced numbers of immunosuppressive, CD3(+)/IL-10 positive, tumor infiltrating lymphocytes. Therefore, TAP1 expression restores both antigen presentation and immunogenicity in B16F10 melanoma cells and concomitantly reduces immunosuppressive IL-10 production at the local tumor site, thereby increasing immunosurveillance mechanisms against tumors.     

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.     

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.     

Pentoxifylline modulates cell surface integrin expression and integrin mediated adhesion of B16F10 cells to extracellular matrix components

Our previous studies demonstrated that Pentoxifylline (PTX), a phosphodiesterase inhibitor, could inhibit the lung homing of B16-F10 melanoma cells in C57BL/6 mice. In this study we have looked at the effect of PTX on cell surface integrin expression and integrin mediated adhesion of B16-F10 melanoma cells. B16-F10 cells treated with PTX when injected through the tail vein of mice showed a 75% reduction in pulmonary nodules as compared to control untreated cells. PTX brought about a significant reduction in the integrin mediated adhesion of F10 cells to Fibronectin and Vitronectin (58.75% +/- 3.4 S.E and 60% +/- 1.7 S.E respectively if control was considered as 100%). This inhibition in adhesion was evident up to four hours only and treatment for 24 hours brought about an increase in adhesion (135.5% +/- 0.5 S.E). Flow cytometric analysis showed higher surface expressions of alphav, alpha5 and alphaIIb integrin subunits in B16-F10 as compared to the low metastatic cell line B16-F1 suggesting a role for these integrins in determining the metastatic potential. PTX brought about a significant decrease in the cell surface expression of alpha5, alphaIIb and beta1 integrin subunits but not that of the alphav subunit on B16-F10 cells. PTX also brought about a reduction in the total cellular protein levels of beta1 and alphav integrin subunits. Various isoforms of Protein Kinase C (PKC) has been shown to regulate integrin expression, localization and activity. Hence we looked at the effect of PTX on total cellular PKC activity. PTX brought about a significant reduction in total cellular PKC activity (82.66 +/- 0.593). Collectively our results indicate that the antimetastatic action of PTX is mediated, at least in part through its effects on adhesion and the surface expression of specific integrin receptors.     

Platelet-activating factor induces up-regulation of antiapoptotic factors in a melanoma cell line through nuclear factor-kappaB activation

In this study, we investigated the influence of platelet-activating factor (PAF) on the induction of apoptosis-regulating factors in B16F10 melanoma cells. PAF increased the expression of mRNA and the protein synthesis of antiapoptotic factors, such as Bcl-2 and Bcl-xL, but did not increase the expression of the proapoptotic factor, Bax. A selective nuclear factor-kappaB (NF-kappaB) inhibitor, parthenolide, inhibited the effects of PAF. Furthermore, PAF inhibited etoposide-induced increases in caspase-3, caspase-8, and caspase-9 activities, as well as cell death. p50/p65 heterodimer increased the mRNA expression of Bcl-2 and Bcl-xL and decreased etoposide-induced caspase activities and cell death. In an in vivo model in which Matrigel was injected s.c., PAF augmented the growth of B16F10 cells and attenuated etoposide-induced inhibition of B16F10 cells growth. These data indicate that PAF induces up-regulation of antiapoptotic factors in a NF-kappaB-dependent manner in a melanoma cell line, therefore suggesting that PAF may diminish the cytotoxic effect of chemotherapeutic agents.     

Chronic alcohol consumption stimulates VEGF expression, tumor angiogenesis and progression of melanoma in mice

The mechanisms of alcohol-induced cancer in humans are unclear. We used the immunocompetent mice implanted with B16F10 cells to evaluate the effects of physiologically relevant EtOH intake on tumor growth and angiogenesis of melanoma. Six-wk-old male mice (C57BL/6J) were given 1% EtOH in drinking water for 12-hrs during the night which was then replaced with regular water during the remaining 12-hrs each day for 4 wks (n = 10). The control mice received regular drinking water only. In the second week, all mice were inoculated subcutaneously on the right proximal dorsal with 5 x 10(5) B16F10 cells. In the end, the tumors were isolated for measuring tumor size, average microvascular density (AMVD) using CD31 immunohistochemistry, and the expression of VEGF and its receptor (Flt-1) using Northern blot, ELISA, and immunohistochemistry. EtOH intake caused a 2.16-fold increase in tumor weight over the control (4.81 +/- 0.39 vs. 2.23 +/- 0.48 g; n = 10; p = 0.003), a 2.02-fold increase in AMVD (60.63 +/- 5.56 vs. 30.01 +/- 7.41/mm(2); p = 0.0014), and a significant increase in VEGF mRNA and protein expression plus Flt-1 protein levels in melanoma compared to the control group (p < 0.01). These results suggest that progression of melanoma growth and angiogenesis may be mediated by upregulation of VEGF and Flt-1, especially under the influence of EtOH.     

Genomic alterations in spontaneous and carcinogen-induced murine melanoma cell lines

We have conducted an analysis of genetic alterations in spontaneous murine melanoma cell line B16F0 and its two metastatic clones, B16F1 and B16F10 and the carcinogen-induced murine melanoma cell lines CM519, CM3205, and K1735. We found that unlike human melanomas, the murine melanoma cell lines did not have activating mutations in the Braf oncogene at exon 11 or 15. However, there were distinct patterns of alterations in the ras, Ink4a/Arf, and p53 genes in the two melanoma groups. In the spontaneous B16 melanoma cell lines, expression of p16Ink4a and p19Arf tumor suppressor proteins was lost as a consequence of a large deletion spanning Ink4a/Arf exons 1alpha, 1beta, and 2. In contrast, the carcinogen-induced melanoma cell lines expressed p16Ink4a but had inactivating mutations in either p19Arf (K1735) or p53 (CM519 and CM3205). Inactivation of p19Arf or p53 in carcinogen-induced melanomas was accompanied by constitutive activation of mitogen-activated protein kinases (MAPKs) and/or mutation-associated activation of N-ras. These results indicate that genetic alterations in p16Ink4a/p19Arf, p53 and ras-MAPK pathways can cooperate in the development of murine melanoma.     

Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo.

Seven different tumor cell lines (human melanoma SK MEL 28; hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma HCT8; murine colon carcinoma CT26; and murine Lewis lung carcinoma) were treated with thrombin at 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; HM29 was studied for its ability to bind to fibronectin and von Willebrand factor; CT26, B16F1, B16F10, and B16a were studied for their ability to form pulmonary metastasis after i.v. injection of thrombin-treated tumor cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin-treated tumor cell lines increased their adhesion to adherent platelets 2-to 3-fold. HM29 increased its adherence to fibronectin and von Willebrand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated CT26 cells demonstrated 2-fold greater growth in vivo after s.c. injection. The mechanism of enhanced adhesion of thrombin-treated tumor cells to platelets required the platelet integrin GPIIb-GPIIIa since it could be inhibited by agents known to block adhesion of ligands to GPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could be inhibited by treatment of thrombin-treated tumor cells with 10E5 and RGDS. The thrombin effect on tumor cells was optimum at 1 h of incubation with thrombin, did not require active thrombin on the tumor cell surface, and did not require protein synthesis (not inhibited by cycloheximide). Thus, thrombin-treated tumor cells markedly enhance pulmonary metastasis. It is suggested that this may be secondary to thrombin-induced enhanced adhesion as well as growth of tumor cells.     

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.