Melanoma Cell-conditioned Medium Research Articles

Abstract PR07: The role of keratinocyte desmoglein 1 in shaping the melanoma tumor niche

Abstract Melanoma is an aggressive cancer arising from transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. Keratinocytes and melanocytes communicate through both contact and paracrine signaling, a niche called the pigmentary unit. Ultraviolet (UV) radiation-induced melanocyte mutations drive melanoma formation and progression, but the role of keratinocytes and an altered microenvironment in melanomagenesis is understudied. We showed that the keratinocyte-specific desmosomal cadherin, desmoglein 1 (Dsg1), is reduced in response to acute UV exposure. Here, we show that Dsg1 is progressively reduced in regions surrounding human dysplastic nevi, melanoma in situ, and stage I/II melanomas, preceding loss of E-cadherin that occurs in later stages. Since we previously showed that Dsg1 suppresses EGFR/Erk1/2 signaling and regulates keratinocyte cytokine profiles, we hypothesized that altered keratinocyte Dsg1 expression governs melanocyte behavior and transformation through paracrine signaling. Dsg1 knockdown via shRNA elevated phospho-EGFR, phospho-Stat3, phospho-ERK and nuclear NFκB, accompanied by increases in IL-1α, IL-1β, IL-6, and IL-8, and CXCL1 proinflammatory cytokine mRNAs and secretion of IL6, IL8, and CXCL1. Melanocytes grown in conditioned media from Dsg1-deficient keratinocytes increased MITF transcription, while AXL transcript levels remained unchanged, a signature associated with early-stage melanoma. Consistent with increased MITF, melanocytes cultured in conditioned media from Dsg1-depleted keratinocytes increased pigment secretion, reversible by inhibiting the melanocortin 1 receptor. Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents mis-localized to suprabasal layers, similar to pagetoid behavior in early melanoma. This, combined with the loss of keratinocyte Dsg1 surrounding human melanoma lesions, led us to examine mechanisms of sustained Dsg1 loss in the tumor niche. Keratinocytes cultured in melanoma cell-conditioned media reduced Dsg1 expression, suggesting that Dsg1 downregulation is maintained by melanoma cells post-transformation. We postulate that keratinocyte Dsg1 downregulation may initially be part of the skin’s protective response against UV exposure through its role in increasing pigmentation. However, chronic Dsg1 loss may contribute to melanoma initiation and be important for the maintenance of the promelanoma tumor niche. This abstract is also being presented as Poster A02. Note: This abstract was not presented at the conference. Citation Format: Hope E. Burks, Christopher R. Arnette, Jennifer L. Koetsier, Joshua A. Broussard, Valarie Ogwo, Quinn R. Roth-Carter, Pedram Gerami, Jodi L. Johnson, Kathleen J. Green. The role of keratinocyte desmoglein 1 in shaping the melanoma tumor niche [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR07.

Abstract 1164: sHER3 inhibits the proliferation and migration of melanoma-derived cells in a tenascin-dependent manner

Abstract The expression of HER3 receptor tyrosine kinase, a member of the EGFR/HER family of cellular oncogenes, is common in both primary and metastatic melanoma, and high levels of HER3 expression are correlated with poor patient survival. However, no one has demonstrated the mechanistic basis for HER3 as a poor prognostic determinant in melanoma patients, even though these tumors poorly express other members of the EGFR/HER receptor family. In this study we present evidence demonstrating that 13 of 14 melanoma cell lines tested express HER3, supporting previous reports regarding the frequency of expression of this gene product in melanoma samples. We further report that a soluble isoform of HER3 (sHER3) that we previously have shown to be a potent inhibitor of neuregulin mediated, Akt-dependent breast cancer cell growth strongly inhibits the growth of HER3 expressing melanoma cells. Interestingly, however, the phosphorylation of Akt, a known mediator of ligand-dependent HER3 signaling, is not reduced in melanoma cells growth inhibited by sHER3. To explore alternative mechanisms of HER3 signaling in melanoma, we identified sHER3 interacting partners using a mass spectrometry proteomic approach. Only one prominent sHER3 binding partner was identified in melanoma cell conditioned media: tenascin. This was an unexpected finding since ligand-dependent HER3 signaling has not previously been linked to tenascin. Interestingly, tenascin expression has independently been identified as another negative prognostic indicator in melanoma. Our initial studies to test the functional relationship between HER3 and tenascin suggest that tenascin expression is correlated with both HER3 expression and migration in all of the melanoma lines tested. Moreover, sHER3 inhibition of melanoma migration was dependent on the level of tenascin present. Treatment of melanoma cells with purified sHER3 inhibits the migration only in the presence of high levels of tenascin. These results suggest the existence of an unanticipated tenascin- and HER3-dependent signaling pathway in melanoma cells, and suggest that this pathway is functionally correlated with melanoma cell migratory potential and poor patient survival. (This work was supported by the Harry J. Lloyd Charitable Trust Foundation.) Citation Format: Chunlin Cai, Nita J. Maihle. sHER3 inhibits the proliferation and migration of melanoma-derived cells in a tenascin-dependent manner. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1164. doi:10.1158/1538-7445.AM2014-1164

Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling

In many cancers hyaluronan content is increased, either by tumor cells or the surrounding stromal cells and this increased hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of melanoma cell (aggressive melanoma cell line C8161)-derived factors on fibroblast hyaluronan synthesis, intracellular signaling, MMP expression and invasion. Treatment of the fibroblast cultures with melanoma cell conditioned medium (CM) caused accumulation of hyaluronan in the culture medium and formation of thick pericellular hyaluronan coat and hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161 melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with siRNA showed that Has2 was responsible for the increased hyaluronan synthesis induced by the melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46 kinases were screened with phosphokinase array kit. Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the melanoma cell CM-induced hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition, siRNA for PDGFRα/β inhibited Has2 upregulation in melanoma cell CM-treated fibroblasts. In parallel with the increased hyaluronan synthesis the melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced MMP expression and increased invasion into collagen-Cultrex matrix. siRNA blocking of Has2 or PDGFRα/β expression reversed the stimulatory effect of melanoma cell CM on fibroblast invasion. PDGF secreted by melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor tumor growth.

4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors

There is a close correlation between tumour progression and hyaluronan production, either by tumour cells or by stromal cells that are stimulated by tumour-derived factors. Inhibition of tumour stimulation of fibroblast hyaluronan may suppress tumour growth and invasion. To examine the effect of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) on the growth of and hyaluronan synthesis by fibroblasts and C8161 and MV3 melanoma cell lines, invasion, and inhibition of tumour cell-derived factor activation of fibroblasts. Effects of 4-MU on growth and hyaluronan synthesis by fibroblasts and melanoma cells were examined in monolayer culture and fibroblast-contracted collagen lattices, and their effects on the growth and invasion of tumour cells into collagen lattices were also studied. 4-MU caused a dose-dependent growth inhibition of fibroblast and melanoma cells with maximum inhibition at 0·5 mmol L(-1) 4-MU. At this dose, 4-MU inhibited (3) H-glucosamine incorporation into fibroblast glycosaminoglycans by 52%, and hyaluronan synthesis by 64%. The relative inhibition was more pronounced when fibroblasts were stimulated with C8161 melanoma cell-conditioned medium. 4-MU reduced the level of hyaluronan in fibroblast-contracted collagen lattices, and inhibited both the growth on and invasion into the lattices by melanoma cells. This growth inhibition appears to be predominantly independent of inhibition of hyaluronan synthesis. The effect on growth inhibition was reversible, and 4-MU had no effect on apoptosis. 4-MU is a potent inhibitor of hyaluronan synthesis, induction of stromal hyaluronan accumulation by tumour cells, and fibroblast and melanoma cell proliferation, and results suggest that 4-MU may have potential as a tumour cell anti-invasive and antiproliferative agent.

Carba Analogs of Cyclic Phosphatidic Acid Are Selective Inhibitors of Autotaxin and Cancer Cell Invasion and Metastasis

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.

Carba Analogs of Cyclic Phosphatidic Acid are Selective Inhibitors of Autotaxin and Cancer Cell Invasion and Metastasis

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase 2, NPP2) is an autocrine motility factor that was initially isolated from melanoma cell conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). LPA is also known to be an important mediator of cancer cell survival, growth and invasion. Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring, stable analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect has remained unresolved. Here we show that carba analogs of cPA lack agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo. This work was supported by USPHS & UT Cancer Institute Pilot Grant.

Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion

Hyaluronan, a high molecular weight glycosaminoglycan is associated with cellular proliferation and migration. In a number of different tumour types, there is a close correlation between tumour progression and hyaluronan production, either by the tumour cells or the surrounding stromal cells. We have examined the ability of an aggressive melanoma cell line (C8161) to stimulate the synthesis of fibroblast hyaluronan, and the association of cell-surface CD44 receptors and hyaluronan with invasion. Melanoma cell-conditioned medium (CM) prepared in low glucose medium (1 mg/ml) stimulated the synthesis of fibroblast glycosaminoglycan as measured by [3H] glucosamine incorporation, and the synthesis of hyaluronan as measured using a specific hyaluronan-binding plate assay, while tumour cell-CM prepared in high glucose medium (4.5 mg/ml) inhibited the synthesis of fibroblast glycosaminoglycan. High glucose tumour cell-CM contained large amounts of lactate that appeared to inhibit the tumour-derived factor stimulation of fibroblast glycosaminoglycan synthesis, as removal of the lactate restored the stimulating activity. Melanoma cells seeded on contracted collagen lattices and incubated at the air/liquid interface rapidly formed a multilayered cell mass on the surface, with significant invasion of the gel. Hyaluronan staining was apparent within the collagen gel, and strong staining was seen around the invading tumour cells, but not around those cell layers near the surface. CD44 expression on the tumour cells was confined to those invading cells and corresponded to cellular hyaluronan staining. Hyaluronan staining was also apparent around and between tumour cells invading fibroblast-free collagen lattices. Monolayer cultures of C8161 cells stained strongly for CD44, but few cells stained for hyaluronan, while no detectable hyaluronan was released into the medium. In summary, the C8161 melanoma cells stimulated the synthesis of fibroblast hyaluronan, and in collagen lattices, only the invasive tumour cells expressed CD44 and hyaluronan, either in the presence or absence of fibroblasts.

Fibroblasts enhance the invasive capacity of melanoma cells in vitro.

In previous experiments we have shown an enhanced expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts obtained from the border of invasive melanoma in comparison to fibroblasts more distant from the tumour. In the study reported here we sought to determine whether melanoma-derived soluble factors are responsible for the stimulation of MMP-1 expression in fibroblasts. By real-time PCR and enzyme-linked immunosorbent assays, we demonstrated that the stimulation of fibroblasts with melanoma cell conditioned medium led to an increased expression of MMP-1 mRNA as well as MMP-1 protein, whereas melanoma cells themselves did not produce detectable amounts of MMP-1 protein. Basic fibroblast growth factor (bFGF) was detected as an important factor responsible for the enhanced expression of MMP-1 by fibroblasts after stimulation with melanoma cell conditioned medium. In a three-dimensional in vitro invasion assay, we demonstrated that fibroblasts are essential for melanoma cell invasion into a collagen I matrix. These findings support the hypothesis that stromal fibroblasts assist the invasion of melanoma cells through the extracellular matrix by producing elevated amounts of proteolytic enzymes after interaction with soluble factors (e.g. bFGF).

Melanoma cells stimulate osteoclastogenesis, c-Src expression and osteoblast cytokines

Malignant melanomas metastasise to the bone and enhance osteoclast bone resorption. We demonstrated that a 48-h-B16 melanoma cell conditioned media (B16CM) induced osteoclastogenesis in mouse bone marrow cultures, without the requirement of B16 cell-bone marrow cell co-culture. B16 cells transcriptionally expressed detectable levels of TGFβ1, IL-6, M-CSF, GM-CSF and TNFα mRNAs, albeit to a lower extent compared with levels in osteoblasts, and failed to express PTHrP, OPGL, OPG and IL-1β. Interestingly, B16CM greatly upregulated IL-1β, IL-6 and GM-CSF, and modestly enhanced TNFα and OPGL mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. B16CM barely upregulated c-Fos, but strongly and time-dependently enhanced c-Src expression in the total bone marrow cultures during osteoclast differentiation. Moreover, c-Src expression was enhanced in differentiated and purified osteoclast preparations to higher levels than in stromal cells. In conclusion, melanoma induces osteoclast generation with a paracrine mechanism independent of cell-cell contact, specifically upregulating c-Src in osteoclasts and cytokine expression in osteoblasts.

Alterations of sensory retinal explants exposed to choroidal melanoma cells ex vivo.

Cultures of retinal explants have been established as a useful tool to investigate effects of pathogenic agents in vitro. We used such cultures as a model to study the effects of choroidal melanoma on retinal organisation and function. Rabbit retinal explants were co-cultured with human choroidal melanoma cells, or exposed to supernatants from choroidal melanoma cell cultures, for various periods from 1 day to 10 days. The retinal explants were then studied by histology and immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin. The release of the pro-inflammatory interleukins IL-6 and IL-8 into the media was measured by enzyme-linked immunosorbent assay. Both in the co-cultures and after treatment with choroidal melanoma cell supernatants for more than 1 week, the layered structure of the retinae became disorganised. Retinal glial (Müller) cells displayed gliosis as indicated by increased GFAP immunoreactivity and decreased immunoreactivity for vimentin. Additionally, the secretion of cytokines, particularly of IL-8, was significantly modulated. The retinal explants produced much less IL-8 than the melanoma cells in separate cultures but increased their IL-8 release significantly after a few days' exposure to melanoma cell-conditioned medium. The results show that in cases of choroidal melanoma, the well-known morphological and inflammatory alterations of the retina are accompanied by glial cell reactivity and up-regulated retinal cytokine secretion, and may be caused by soluble factors secreted and induced by the melanoma.

Fibroblasts surrounding melanoma express elevated levels of matrix metalloproteinase-1 (MMP-1) and intercellular adhesion molecule-1 (ICAM-1) in vitro.

Tumour growth and metastasis involve the degradation of extracellular matrix components by matrix degrading enzymes produced by tumour cells and stromal fibroblasts. In this study, fibroblasts were obtained from biopsies on the border (TB) and 1 cm distant from the melanoma (TD) and cultured separately. Similar studies were performed with fibroblasts surrounding melanocytic nevi as control. The expression of matrix metalloproteinase-1 (MMP-1) mRNA and tissue matrix metalloproteinase inhibitor 1 (TIMP-1) were studied by Northern blot analysis. The activation antigen intercellular adhesion molecule-1 (ICAM-1) in TB-and TD-fibroblasts was investigated by flow cytometry. In melanoma, TB-fibroblasts showed an increased expression of MMP-1 mRNA mainly in fibroblasts obtained from tumours with extended invasive growth demonstrated by Clark level whereas the expression of the major specific inhibitor TIMP-1 was unaltered. In contrast, fibroblasts surrounding benign melanocytic nevi did not express elevated levels of MMP-1. The upregulation of MMP-1 in TB-fibroblasts compared to TD-fibroblasts was maintained during cultivation. Furthermore, MMP-1 mRNA expression and MMP-1 total protein amount in normal fibroblasts were increased by melanoma cell conditioned medium. We demonstrated an increased expression of ICAM-1 in TB-fibroblasts compared to TD-fibroblasts in vitro depending on the amount of inflammatory infiltrate in situ. The differences of ICAM expression disappeared during continued cell culture. These results support the idea that fibroblasts surrounding melanoma are activated and are possibly involved in the degradation of matrix proteins surrounding the tumour.

Melanoma cell-derived factor stimulation of fibroblast glycosaminoglycan synthesis—The role of platelet-derived growth factor

The hyaluronan-rich matrix surrounding many tumours may facilitate tumour growth, invasion and angiogenesis, with the majority of this hyaluronan apparently being synthesised by normal fibroblasts, stimulated to do so by tumour cell-derived factors. Melanoma cell-conditioned medium (CM) stimulates up to a 6-fold increase in fibroblast glycosaminoglycan (GAG) synthesis, with the active factors being present in tumour CM ultrafiltration fractions >30 kDa and <1 kDa. These fractions are poorly active individually, but when recombined, the activity is substantially greater than the additive effect. The objective of this study was to identify the factors present in the ultrafiltration fraction >30 kDa that produce a greater than additive effect with the fraction <1 kDa in stimulating the incorporation of 3H glucosamine into fibroblast GAGs. A number of factors including basic fibroblast growth factor (bFGF), interleukin (IL)-1β, pleiotrophin, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), tumour necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) failed to stimulate any significant increase in GAG synthesis, but when added to the <1 kDa tumour CM fraction, both PDGF and to a lesser extent, bFGF, exhibited potent stimulating activities. Neutralising antibodies to PDGF and bFGF added to the melanoma CM decreased the fibroblast GAG-stimulating activity by 29% and 40%, respectively, in C8161 melanoma CM and by 47% and 45%, respectively, in Hs294T melanoma CM. The activities of PDGF-AA and PDGF-BB isoforms were indistinguishable, suggesting the PDGF-α receptor plays a role in the GAG-stimulatory response. Western analysis following treatment with PDGF, bFGF or melanoma CM revealed banding patterns for PDGF and melanoma CM that were similar. Immunoprecipitation of the PDGF-α receptor revealed it to be phosphorylated in fibroblasts treated with PDGF and melanoma CM, but not with control fibroblast CM. These studies suggest that PDGF plays an important role in the GAG-stimulating activity of the melanoma CM, but requires the presence of an as yet unidentified novel low molecular weight factor for full activity.

Interleukin-1-mediated H2O2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion

We have examined H2O2 production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin-1 beta (IL-1 beta) stimulation and B16 melanoma cell adhesion. Production of H2O2 was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellular 2', 7'-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1 beta treatment there was a 6-fold increase in endothelial cells producing H2O2 (67%) and a 4-fold augmentation in the Kupffer cell population (86%). The average H2O2 content per cell size unit significantly (P < 0.01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7-fold). In contrast to the homogeneity of Kupffer cells, H2O2 production intensity was largely heterogeneous in IL-1 beta-activated HSE. Enhancement of H2O2 production by IL-beta-treated HSE started at the 4th h and peaked 2-3 h later. The addition of increasing concentration of IL-1 beta to HSE for 4 h caused the progressive activation of H2O2 production by treated cells. The addition of 80 M excess of IL-1 receptor antagonist (IL-1 Ra) 10 min before IL-1 beta treatment abrogated IL-1 beta-mediated enhancement of H2O2. From the 2nd h of B16 melanoma adhesion to HSE there was significantly (P < 0.05) enhancement of H2O2 content in HSE. This activation increased 2.25-fold by the 3rd h of coculture and had reduced again by the 5th h. IL-1 Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H2O2 production response, indicating that HSE-activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 micrograms/ml anti-mouse IL-1 beta neutralizing antibody reduced the melanoma cell-induced HSE production of H2O2 by 80%. On the contrary, B16 melanoma cell-conditioned medium did not vary HSE production of H2O2 compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL-1 beta (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H2O2. HSE activation was IL-1-dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells.

Alterations in endothelial cell proteinase and inhibitor polarized secretion following treatment with interleukin-1, phorbol ester, and human melanoma cell conditioned medium.

Polarized secretion of matrix metalloproteinases and plasminogen activators by monkey aortic endothelial cells was studied in vitro, using transwell inserts. The endothelial cells constitutively expressed matrix metalloproteinase-2, tissue inhibitors of metalloproteinases 1 and 2, urokinase, and tissue plasminogen activator, all with basal preference. Matrix metalloproteinase-9 activity was induced by phorbol 12-myristate 13-acetate (apical), interleukin-1 alpha (basal), and by conditioned medium from DX3 human melanoma cells (basal). The DX3 melanoma conditioned medium also stimulated basal secretion of matrix metalloproteinase-2, urokinase, tissue plasminogen activator, and tissue inhibitors of metalloproteinases. The rise in proteolytic activity in the basal direction was reflected by increased capacity to degrade subendothelial basement membrane type IV collagen, shown immunohistologically, using monkey kidney tissue sections and basement membrane deposited by endothelial cells into the transwell membrane. Thus, IL-1 alpha and DX3 melanoma conditioned medium can stimulate endothelial cells in vitro to concentrate secretion of proteinases spatially onto the underlying basement membrane. We suggest that the stimulation of endothelial cell proteinase activity by tumor cells may facilitate tumor cell extravasation.

Endothelial cell Ca2+ increases upon tumor cell contact and modulates cell-cell adhesion.

The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion.