Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream TM that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 Â 10 6 peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% 6 3.1% (n ¼ 12) and 71.2% 6 1.6% (n ¼ 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R

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DNA derived from NSCLC tumors can be detected with high sensitivity in urine and plasma, enabling diagnostic detection and monitoring of therapeutic response from these noninvasive "liquid biopsy" samples.

The thrombin receptor [protease-activated receptor-1 (PAR-1)] is overexpressed in highly metastatic melanoma cell lines and in patients with metastatic lesions. Activation of PAR-1 leads to cell signaling and up-regulation of genes involved in adhesion, invasion, and angiogenesis. Herein, we stably silence PAR-1 through the use of lentiviral short hairpin RNA and found significant decreases in both tumor growth (P < 0.01) and metastasis (P < 0.001) of highly metastatic melanoma cell lines in vivo. The use of viruses for therapy is not ideal as it can induce toxic immune responses and possible gene alterations following viral integration. Therefore, we also used systemic delivery of PAR-1 small interfering RNA (siRNA) incorporated into neutral liposomes [1,2-dioleoyl-sn -glycero-3-phosphatidylcholine (DOPC)] to decrease melanoma growth and metastasis in vivo. Significant decreases in tumor growth, weight, and metastatic lung colonies (P < 0.001 for all) were found in mice treated with PAR-1 siRNA-DOPC. The in vivo effects of PAR-1 on invasion and angiogenesis were analyzed via immunohistochemistry. Concomitant decreases in vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 expression levels, as well as decreased blood vessel density (CD31), were found in tumor samples from PAR-1 siRNA-treated mice, suggesting that PAR-1 is a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. We propose that siRNA incorporated into DOPC nanoparticles could be delivered systemically and used as a new modality for melanoma treatment. [Cancer Res 2008;68(21):9078-86]

Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/ CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/ CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/ CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis.Cutaneous melanoma is the most aggressive type of skin cancer, and it can metastasize very rapidly (1). An estimated 62,480 new cases of melanoma were diagnosed in the United States during 2008, 8,420 of which resulted in death (2). The transition of melanoma from the radial growth phase to the vertical growth phase to metastasis is accompanied by multiple molecular changes (3-8). We and others have shown that two transcription factors, activating transcription factor-1 (ATF-1) 2 and cAMP-response element-binding protein (CREB), are activated and overexpressed in melanoma during its progression toward the malignant phenotype (9 -13).CREB and ATF-1 belong to the leucine zipper class of transcription factors. Stimuli such as growth factors, neurotransmitters, inflammatory biolipids, stress signals, or other factors that elevate intracellular cAMP or Ca 2ϩ levels can activate CREB/ATF-1 through phosphorylation at Ser 133 by protein kinase A or mitogen-activated protein kinases (MAPK) (14 -17). Following activation, CREB/ATF-1 regulates the expression of genes that suppress apoptosis, induce cell proliferation, and mediate inflammation and tumor metastasis by binding to cAMP-response elements (CREs) within the promoter and enhancer regions of these genes (15, 18 -20).A number of reports have suggested that CREB is involved in melanoma progression We have demonstrated previously that quenching CREB activity in metastatic melanoma cells by means of a dominant-negative form of CREB (KCREB) leads to a decrease in their tumorigenicity a...

Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFRmediated signal transduction mechanism that required pertussis toxin-insensitive G␣ q protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAFinduced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment. cAMP-response element (CRE)2 -binding protein (CREB), activating transcription factor (ATF) 1, and CRE modulators are members of the large basic region leucine zipper superfamily of transcription factors that regulate gene expression in response to cAMP, intracellular Ca 2ϩ mobilization, and cell stimulation with growth factors (1). The ubiquitously expressed CREB and ATF-1 and the preferentially expressed (in neuroendocrine tissues) CRE modulator bind to complete and partial palindromic CRE DNA consensus sites 5Ј-TGACGTCA-3Ј and 5Ј-CGTCA-3Ј. These transcription factors induce genes involved in cellular metabolism and respiration, gene transcription, cell cycle regulation and cell survival, as well as growth factor and cytokine genes (reviewed in Ref. 1). The transcriptional activity of the DNA-bound CREB protein dimers is regulated by phosphorylation of the serine 133 site in the kinase-inducible domain, wh...

Melanocytes accumulate galectin-3 with tumor progression, particularly in the nucleus. The strong association of cytoplasmic and nuclear expression in lesions of sun-exposed areas suggests an involvement of UV light in activation of galectin-3.

Galectin-3 (Gal-3) is a ␤-galactoside-binding protein that is involved in cancer progression and metastasis. Using a progressive human melanoma tissue microarray, we previously demonstrated that melanocytes accumulate Gal-3 during the progression from benign to dysplastic nevi to melanoma and further to metastatic melanoma. Herein, we show that silencing of Gal-3 expression with small hairpin RNA results in a loss of tumorigenic and metastatic potential of melanoma cells. In vitro , Gal-3 silencing resulted in loss of tumor cell invasiveness and capacity to form tube-like structures on collagen ("vasculogenic mimicry"). cDNA microarray analysis after Gal-3 silencing revealed that Gal-3 regulates the expression of multiple genes , including endothelial cell markers that appear to be aberrantly expressed in highly aggressive melanoma cells , causing melanoma cell plasticity. These genes included vascular endothelial-cadherin , which plays a pivotal role in vasculogenic mimicry , as well as interleukin-8 , fibronectin-1 , endothelial differentiation sphingolipid G-protein receptor-1 , and matrix metalloproteinase-2. Chromatin immunoprecipitation assays and promoter analyses revealed that Gal-3 silencing resulted in a decrease of vascular endothelial-cadherin and interleukin-8 promoter activities due to enhanced recruitment of transcription factor early growth response-1. Moreover , transient overexpression of early growth response-1 in C8161-c9 cells resulted in a loss of vascular endothelial-cadherin and interleukin-8 promoter activities and protein expression. Thus , Gal-3 plays an essential role during the acquisition of vasculogenic mimicry and angiogenic properties associated with melanoma progression.