Circulating Melanoma Cells Research Articles

Glutathione reductase-mediated thiol oxidative stress suppresses metastasis of murine melanoma cells

Malignant melanoma is a highly metastatic and life-threatening cancer. Reactive oxygen species (ROS) play important roles in cancer initiation and progression including metastasis. It has been reported that the oxidative stress spontaneously generated in circulating melanoma cells was able to suppress distant metastasis in vivo. However, little is known regarding the effects and mechanism of glutathione reductase (GR) inhibition-induced oxidative stress in regulation of melanoma metastasis. Here, we demonstrate that GR inhibition generates oxidative stress and suppresses lung metastasis and subcutaneous growth of melanoma in vivo. In addition, inhibitory effects by GR activity reduction were observed on cell proliferation, colony formation, cell adhesion, migration and invasion in melanoma cells in vitro. GR inhibition-induced oxidative stress was also found to block epithelial-to-mesenchymal transition (EMT) by decreasing the expression of Vimentin, ERK1/2, transcription factor Snail and increasing the expression of E-cadherin. In addition, actin rearrangement, a key element involved in cell motility, was also affected by GR-mediated oxidative stress possibly through protein S-glutathionylation on actin. In conclusion, this study identifies GR as an effective regulator of oxidative stress that affects the multistep processes of metastasis in melanoma cells, and it becomes a potential target for melanoma therapy.

PO-200 Prevention of melanoma brain colonisation by inhibiting cytokines secretion from activated astrocytes

IntroductionThe brain microenvironment consists of various cell types such as astrocytes, microglia, neurons and brain endothelial cells. It sustains the normal brain functions and the structures of the cerebral capillaries that form the blood brain barrier (BBB). During tumour progression, malignant cells release vascular endothelial growth factor and other cytokines that increase the permeability of blood vessels allowing them to intravasate into the circulation. In addition, the cells can take advantage of the patho-physiology of the tumour angiogenenic leaky blood vessels at distant organs and extravasate from them at inflamed sites. Such activated site can be the brain parenchyma, which may become a pro-inflammatory metastatic niche. Although little is known about the precise mechanisms and the factors that contribute to the brain metastatic process, there is increasing evidence that astrocytes are involved by mediating a neuroinflammation. They respond by releasing pro-inflammatory cytokines that compromise the integrity of the BBB, increasing the permeability of the capillary endothelium and preparing a metastatic niche for melanoma colonisation.Material and methodsWe established three spontaneous melanoma brain metastasis mouse models that present a pro-inflammatory microenvironment and a pro-metastatic niche preceding the colonisation of melanoma to the brain. siRNA targeting specific chemokines, neutralizating antibody, or a CRISPR/Cas9 system were used to downregulate the expression and secretion of chemokines for further in vitro studies.Results and discussionsWe identified MCP-1, a pro-inflammatory chemokine, implicated in the paracrine interaction between melanoma cells and the brain. The positive staining for GFAP and MCP-1 in the tumour area suggests a cooperation between melanoma and the tumour microenvironment, in which MCP-1 plays a pro-tumorigenic role during cell migration. Astrocytes-secreted MCP-1 expression levels increase when co-cultured with melanoma cells or their conditioned media. Downregulation of MCP-1 leads to decreased migration of melanoma cells in vitro. Moreover, neutralising antibody-targeting MCP-1 reduces melanoma invasion in matrigel towards sprouting aortas.ConclusionActivated astrocytes promote the migration of circulating melanoma cells to the brain by increased secretion of pro-inflammatory cytokines, such as MCP-1. Consequently, investigating the changes in melanoma and astrocytes gene-expression could be the key for interfering and preventing melanoma colonisation to the brain.

Abstract LB-144: Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy

Abstract A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-L1 expression and mutational burden, are insufficiently precise to guide treatment selection and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma Circulating Tumor Cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC-Score) applied to microfluidically-enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (N=42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC-Score within 7 weeks of therapy correlates with marked improvement in progression-free survival (Hazard Ratio (HR): 0.17, P=0.008) and overall survival (HR: 0.12, P=0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies. Citation Format: Xin Hong, Ryan J. Sullivan, Mark Kalinich, Tanya Kwan, Shiwei Pan, Joseph A. LiCausi, John D. Milner, Linda T. Nieman, Ben S. Wittner, Uyen Ho, Tianqi Chen, Ravi Kapur, Don Lawrence, Keith T. Flaherty, Lecia V. Sequist, Sridhar Ramaswamy, David T. Miyamoto, Michael Lawrence, Anita Giobbie-Hurder, Mehmet Toner, Kurt J. Isselbacher, Shyamala Maheswaran, Daniel A. Haber. Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-144.

Abstract 2108: Detection and molecular profiling of leptomeningeal disease in melanoma

Abstract Approximately 5% of melanoma patients develop leptomeningeal disease (LMDz), a highly symptomatic complication with a dismal survival of 8-10 weeks. The aim of this study was to determine whether diagnosis and personalized treatment for melanoma-LMDz could be improved by assessing patient-derived cerebrospinal fluid (CSF) specimens. Circulating tumor cells in CSF (CSF-CTCs) were detected by Veridex CellSearch® System and the circulating melanoma cell kit, based on anti-CD146 and anti-high molecular weight melanoma associated antigen (HMW-MAA-PE (MEL-PE)). Of the 12 patients with definitive LMDz diagnosis, all but 1 patient (92%) had CSF-CTCs (range 23-3055 CTCs/ml). In contrast, only 3/8 (37%) melanoma patients without LMDz diagnosis had CSF-CTCs detected, with significantly lower CTC counts per ml CSF (range 0.13-0.6 CTCs/ml). Ex vivo studies of CSF uncovered that although patient-derived CSF does not appear to act as a chemo-attractant or a stimulant of invasion to initiate melanoma migration to the leptomeninges, it significantly reduced the ability of BRAF inhibitors to induce apoptosis in established melanoma cell lines (p<0.005). Patient-derived CSF composition is complex but includes many growth factors and mediators of adhesion and TGFβ signaling. In melanoma cells, the CSF stimulated signaling through mTOR/AKT and STAT. BRAF inhibition amplified these signals further. We show that ex vivo expansion of isolated CSF-CTCs is possible for ~25% of samples, in the presence of FBS, FGF and EGF. At this time, there is a very limited understanding of the mechanisms underlying melanoma metastasis to the leptomeninges, a truly devastating and rapidly terminal complication of melanoma. This is the first report of patient-derived CSF eliciting a protective effect in melanoma cells, suggesting melanoma cells find a protective niche in the central nervous system. Furthermore, we demonstrate that patient-derived CSF biopsies serve as a critical tool for improving diagnosis and personalized treatment for melanoma-LMDz patients. Citation Format: Inna Smalley, Brittany Evernden, Vincent Law, Rajappa Kenchappa, John Puskas, Elena Ryzhova, Nam Tran, Arnold Etame, Solmaz Sahebjam, Anthony Magliocco, Peter Forsyth, Keiran S. Smalley. Detection and molecular profiling of leptomeningeal disease in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2108.

Isolation and characterization of circulating melanoma cells by size filtration and fluorescent in-situ hybridization.

Isolation of circulating tumor cells (CTCs) from blood of melanoma patients has been difficult owing to inconsistent expression of surface antigens. Here we report on the isolation, detection, and characterization of CTCs from blood of melanoma patients using microfiltration and fluorescent in-situ hybridization (FISH). Two tubes of blood from 15 patients with advanced melanoma were collected. These two tubes subsequently underwent filtration through a membrane with pore sizes of 7.5 μm. Isolated cells from one tube were analyzed by FISH for RREB1 (6p24), MYB (6q32), SE6 (D6Z1), and CCND1 (11q13) and the other paired specimen was analyzed by immunofluorescence for HMB45, melanoma-associated antigen recognized by T cells-1, tyrosinase and melanogenesis associated transcription factor. We identified CTCs in 10 out of 13 melanoma samples by immunofluorescence (2.5-99 CTCs/3 ml of blood) and in 13 specimens by FISH (7.2-76 CTCs/3 ml of blood) with more CTCs identified by FISH in 10 out 13 samples. Two filters failed. Our results show that CTCs are detectable in the majority of patients with advanced melanoma. These tools will be useful in characterizing treatment related changes of melanoma in CTCs.

Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.

Emerging Biomarkers in Cutaneous Melanoma

Earlier identification of aggressive melanoma remains a goal in the field of melanoma research. With new targeted and immune therapies that have revolutionized the care of patients with melanoma, the ability to predict progression and monitor or predict response to therapy has become the new focus of research into biomarkers in melanoma. In this review, promising biomarkers are highlighted. These biomarkers have been used to diagnose melanoma as well as predict progression to advanced disease and response to therapy. The biomarkers take various forms, including protein expression at the level of tissue, genetic mutations of cancer cells, and detection of circulating DNA. First, a brief description is provided about the conventional tissue markers used to stage melanoma, including tumor depth. Next, protein biomarkers, which provide both diagnostic and prognostic information, are described. This is followed by a discussion of important genetic mutations, microRNA, and epigenetic modifications that can provide therapeutic and prognostic material. Finally, emerging serologic biomarkers are reviewed, including circulating melanoma cells and exosomes. Overall the goal is to identify biomarkers that aid in the earlier identification and improved treatment of aggressive melanoma.

Nuclear Receptor Nur77 Facilitates Melanoma Cell Survival under Metabolic Stress by Protecting Fatty Acid Oxidation

Fatty acid oxidation (FAO) is crucial for cells to overcome metabolic stress by providing ATP and NADPH. However, the mechanism by which FAO is regulated in tumors remains elusive. Here we show that Nur77 is required for the metabolic adaptation of melanoma cells by protecting FAO. Glucose deprivation activates ERK2 to phosphorylate and induce Nur77 translocation to the mitochondria, where Nur77 binds to TPβ, a rate-limiting enzyme in FAO. Although TPβ activity is normally inhibited by oxidation under glucose deprivation, the Nur77-TPβ association results in Nur77 self-sacrifice to protect TPβ from oxidation. FAO is therefore able to maintainNADPH and ATP levels and prevent ROS increase and cell death. The Nur77-TPβ interaction further promotes melanoma metastasis by facilitating circulating melanoma cell survival. This study demonstrates a novel regulatory function of Nur77 with linkage of the FAO-NADPH-ROS pathway during metabolic stress, suggesting Nur77 as a potential therapeutic target in melanoma.

PAX3d mRNA over 2.76 copies/µL in the bloodstream predicts cutaneous malignant melanoma relapse.

ObjectiveThe aim of this study was to evaluate if our molecular algorithm, based on tumor circulating transcripts, may predict relapse risk in cutaneous malignant melanoma (CMM).ResultsThe multi-marker panel was able to differentiate patients with CMM from HC with high diagnostic sensitivity and specificity, especially for MITF-m and TGFB2 (91–100%) whose levels decreased during follow-up of recurrence-free patients, and remained stable in the case of relapse. PAX3d higher than 2.76 copies/µL emerged as a promising biomarker [specificity = 75–93% and negative predictive value = 75–98%] to stratify subjects at high risk of CMM recurrence independently of age, gender and AJCC staging [OD = 9.5(3.2–28.0), p < 0.001]. The survival analysis confirmed PAX3d performance in relapse prediction with significant differences in recurrence risk 12 months after the basal time-point (p = 0.008).Materials and MethodsPeripheral blood was collected from 111 CMM patients and from 87 healthy controls (HC) randomly selected. Each specimen was examined by qRT-PCR analysis for the expression of 3 tumor-related transcripts (PAX3d, MITF-m and TGFB2) at diagnosis, and at the following 6 and 12 months during clinical monitoring.ConclusionsWe demonstrated the usefulness of our molecular algorithm to indirectly detect circulating melanoma cells in blood, along with PAX3d capability to assess patients’ progression and relapse prediction.

Photoacoustic detection of circulating melanoma cells as a predictor of metastasis in stage III patients.

e21053 Background: Circulating tumor cells have been correlated with disease state and distant metastatic spread in cancer patients. We postulated that enumerating circulating melanoma cells (CMCs) may predict the onset of distant metastasis in Stage III patients. We detected CMCs using our photoacoustic flow cytometer, in which we irradiated enriched blood samples with nanosecond pulsed laser light. While there is no effect on non-optically active leukocytes, absorption of laser light by pigmented melanoma cells resulted in robust ultrasonic waves that indicated CMCs in the sample. Methods: We tested 32 archived samples from 9 Stage III melanoma patients using our photoacoustic flow cytometer. Each patient had between 2 and 6 serial blood samples. We used a pulsed Nd:YAG laser to irradiate mononuclear cells in suspension and under flow. The number of CMCs detected after testing was recorded, indicating the time sequence of circulating tumor cell activity. Results: The numbers of CTCs for each sample is shown in the table below. The ultimate disease state, whether the patient became metastatic or not, was blinded to the investigators who performed the photoacoustic tests. One sample for patient 3 indicated 63 CMCs, though this test was known to be contaminated and had an unknown number of false detections. Conclusions: We found that patients who had a series of more than 4 CMCs were more likely to become metastatic than those patients who tested for 4 CMCs for fewer, indicating that a sequence of CMC detections in serial blood draws provides a potentially strong predictor of metastasis in Stage III melanoma patients warranting further investigation at this and lower stages of melanoma. We are developing a more rigorous model based on time series analysis of CMCs for prediction of metastasis. [Table: see text]

Photoacoustic tomography: Breathtaking whole-body imaging

High-frame-rate, high-resolution photoacoustic computed tomography reveals, for small live animals, the brain's functional connectivity and the dynamics of breathing, blood oxygenation and circulating melanoma cells.

Single-impulse Panoramic Photoacoustic Computed Tomography of Small-animal Whole-body Dynamics at High Spatiotemporal Resolution.

Imaging of small animals has played an indispensable role in preclinical research by providing high dimensional physiological, pathological, and phenotypic insights with clinical relevance. Yet pure optical imaging suffers from either shallow penetration (up to ~1–2 mm) or a poor depth-to-resolution ratio (~1/3), and non-optical techniques for whole-body imaging of small animals lack either spatiotemporal resolution or functional contrast. Here, we demonstrate that standalone single-impulse photoacoustic computed tomography (SIP-PACT) mitigates these limitations by combining high spatiotemporal resolution (125-µm in-plane resolution, 50 µs / frame data acquisition and 50-Hz frame rate), deep penetration (48-mm cross-sectional width in vivo), anatomical, dynamical and functional contrasts, and full-view fidelity. By using SIP-PACT, we imaged in vivo whole-body dynamics of small animals in real time and obtained clear sub-organ anatomical and functional details. We tracked unlabeled circulating melanoma cells and imaged the vasculature and functional connectivity of whole rat brains. SIP-PACT holds great potential for both pre-clinical imaging and clinical translation.

Potenziale und Limitationen der Detektion von zirkulierenden Melanomzellen

The detection and characterisation of circulating melanoma cells (CMC) is a non-invasive procedure with a low complication rate and can potentially distinguish choroidal nevi from uveal melanomas and help to initiate early adequate therapy. It has the perspective to select and monitor treatment. This technique may lead to new insights into the metastasis process itself and help to detect specific targets for future therapies. This article introduces the different techniques for CMC detection; their potentials and limitations are discussed.

Targeting cholesterol transport in circulating melanoma cells toinhibit metastasis.

Despite recent breakthroughs in targeted- and immune-based therapies, rapid development of drug resistance remains a hurdle for the long-term treatment of patients with melanoma. Targeting metastatically spreading circulating tumor cells (CTCs) may provide an additional approach to manage melanoma. This study investigates whether targeting cholesterol transport in melanoma CTCs can retard metastasis development. Nanolipolee-007, the liposomal form of leelamine, reduced melanoma metastasis in both a novel in vitro flow system mimicking the circulating system and in experimental as well as spontaneous animal metastasis models, irrespective of the BRAF mutational status of the CTCs. Leelamine led to cholesterol trapping in lysosomes, which subsequently shut down receptor-mediated endocytosis, endosome trafficking, and inhibited the major oncogenic signaling cascades important for survival such as the AKT pathway. As pAKT is important in CTC survival, inhibition by targeting cholesterol metabolism led to apoptosis, suggesting this approach might be particularly effective for those CTCs having high levels of pAKT to aid survival in the circulation system.

In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells

Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma. Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis. Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism. We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice. Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage. A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature.

Melanoma antigens are biomarkers for ipilimumab response.

Novel immunotherapy modalities significantly improve survival of patients with metastatic melanoma. However, CTLA-4-blocking monoclonal antibody ipilimumab is effective only in a small proportion of patients. Biomarkers for prediction of treatment response are indispensably needed. To determine the utility of multimarker detection of circulating melanoma cells as prognostic and pharmacodynamic biomarker in patients with metastatic melanoma treated with ipilimumab. Patients (n = 62) with metastatic melanoma in unresectable stage III or metastatic stage IV treated with ipilimumab were recruited prospectively. The values of four melanoma markers on circulating cells Melan-A, gp100, MAGE-3 and melanoma inhibitory antigen prior to the treatment and within the therapy were compared to the data collected at baseline - after the melanoma surgery. The immunotherapy pretreatment marker level was found to be prognostic of overall survival; lower levels were linked to longer survival time. Moreover, longitudinal follow-up of melanoma markers in patients treated with ipilimumab correlates with therapy response. A decline of marker levels by >30% at week 6 (in 83% of the responding subjects) to week 9 (in all responders) of ipilimumab administration was associated with response to therapy. Elevation of the tumour markers during the treatment precedes clinical progression and gives an early warning of treatment failure. Melanoma circulating cells hold potential as predictive and pharmacodynamic biomarker of immunotherapy.

Analysis of monosomy‐3 in immunomagnetically isolated circulating melanoma cells in uveal melanoma patients

Monosomy-3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno-FISH assay to detect chromosome-3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n=44) with primary non-metastatic UM, of which 58% were positive for monosomy-3. The monosomy-3 status of CMC corresponded to the monosomy-3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy-3 in the CMC was associated with an advanced tumor stage (P=0.046) and was detected in all four patients who developed metastasis within the follow-up period of 4yr. This non-invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.

Abstract 471: Detection of circulating tumor cells in high-risk primary uveal melanoma by the photoacoustic method

Abstract Circulating tumor cells (CTCs) have been shown to be a prognostic marker in breast cancer1. We hypothesize that circulating melanoma cell (CMC) detection could be utilized in the management of uveal melanoma, including early intervention. Prior methodologies for circulating uveal melanoma cell (CUMC) detection have been fraught with poor sensitivity, limiting their clinical utility2. Development of an improved method is necessary to establish the clinical utility of CUMC monitoring. Photoacoustics, also referred to as laser-induced ultrasound, is a novel platform for the detection and capture of CMCs. Photoacoustics uses short duration pulsed light to create ultrasonic acoustic waves in an optically absorbing medium, in this case melanin within melanoma3. As light is absorbed by irradiated chromophores, the optical energy gets converted into kinetic thermal energy trapped within the chromophore and subsequent thermal expansion ensues. Transient thermoelastic expansion of the absorbent cell results in the propagation of ultrasonic acoustic waves which can be detected and analyzed using a piezoelectric response mechanism. In addition, detected CMCs can be isolated by a two-phase flow cell separation technique4. Due to the low cost and melanoma specific capabilities of photoacoustics, we evaluated this technology for the purpose of CUMC detection. Methods: Cells from uveal melanoma cell line UM002B, established at Thomas Jefferson, were titrated to various cell concentrations and analyzed in a neutral density solution utilizing the photoacoustic method. Uveal melanoma cells of differing concentrations were spiked into isolated healthy donor peripheral blood mononuclear cells (PBMCs) and healthy whole blood samples. PBMC isolates were analyzed for CUMCs. Results: CUMCs were successfully quantified by the photoacoustic method including single cell detection. Recovery rates of cultured cells in a neutral density solution approached 25%. Recovery rates for CUMCs in whole blood averaged 10% of expected cell yield (56/540 cells detected) with a higher detection rate at lower cell concentrations. Photoacoustics offers a viable method for the detection of CUMCs with an accuracy that meets or exceeds previously reported CUMC yields. Studies analyzing CUMCs from patients with metastatic disease are ongoing. 1. Cristofanilli, M, et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med, 2004. 351(8): 781-91. 2. Bidard FC, et al., Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma. Int J Cancer. 2014 Mar 1;134(5):1207-13. 3. Weight RM, et al. Photoacoustic detection of metastatic melanoma cells in the human circulatory system. Opt Lett. 2006 Oct 15;31(20):2998-3000. 4. O’Brien CM, et al. Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow. J Biomed Opt. 2012 Jun;17(6):061221. Citation Format: Ryan M. Weight, Shingo Sato, Masahiro Ohara, Mizue Terai, Michael Mastrangelo, Marlana Orloff, Benjamin Goldschmidt, John Viator, Takami Sato. Detection of circulating tumor cells in high-risk primary uveal melanoma by the photoacoustic method. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 471.

Abstract 2806: Oxidative stress limits metastasis of human melanoma cells

Abstract Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. We studied the complex and poorly understood process of metastasis by using a clinically relevant model of melanoma metastasis: patient-derived xenografts (PDX) that differ in their capacity to metastasize in NOD-SCID-Il2rg−/− (NSG) mice, mirroring their metastasis histories in the patients from which they were derived. We systematically dissected distinct steps of the metastatic cascade in vivo to show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Using Liquid Chromatography-Mass Spectrometry (LC-MS) and flow cytometry, we discovered that melanoma cells in the blood and visceral organs experienced high levels of oxidative stress not observed in subcutaneous tumours. This work demonstrates that successfully metastasizing melanomas undergo reversible functional and metabolic changes during metastasis that increases their capacity to withstand oxidative stress through an increased dependence on NADPH-generating enzymes in the folate pathway. Chronic antioxidant treatment promotes circulating melanoma cell survival and distant metastases in NSG mice. Folate pathway inhibition, using low-dose methotrexate or depletion of several NADPH-regenerating enzymes, inhibits distant metastases without significantly affecting the growth of subcutaneous tumours in the same mice. We are currently investigating the role of other NADPH-regenerating pathways such as the pentose phosphate pathway in metastasis of human melanoma cells. Finally, we hypothesize that other stress types may also play a role in limiting metastasis in vivo and we are looking for additional metabolic adaptations that may increase stress-resistance of metastasizing melanoma cells and represent novel therapeutic targets in metastatic disease. Citation Format: Elena Piskounova, Michail Agathocleous, Malea Murphy, Zeping Hu, Ralph DeBerardinis, Sean Morrison. Oxidative stress limits metastasis of human melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2806.

Circulating melanoma cells as a potential biomarker to detect metastasis and evaluate prognosis.

TNM staging is mainly used to evaluate the prognosis of melanoma patients. Serum biomarkers such as 5-S-cysteinyldopa (5-S-CD) have occasionally been used but most do not respond until the tumour burden becomes high. Recently, circulating melanoma cells (CMC) have been reported as a possible new biomarker to detect metastasis, monitor treatment response and predict prognosis. The object of this exploratory study was to evaluate the efficacy of CMC to detect metastasis and predict prognosis by cross-sectional and prospective observational analyses, respectively. Altogether 15 patients with stages II-IV melanoma were enrolled and CMC were enumerated by CellSearch system with cut-off values of two cells/7.5 mL. Serum 5-S-CD and lactate dehydrogenase (LDH) were also measured. The sensitivity of CMC and 5-S-CD for the detection of metastasis was 33 and 50%, respectively. The combination of CMC and 5-S-CD showed a sensitivity of 67%, the best performance among CMC, 5-S-CD, LDH and any combination of two of the markers. Additionally, a 30-month prospective observation showed that CMC could segregate patients with poorer prognosis. The median survival time for the patients with <2 CMC and those with ≥2 CMC was 19.5 and 4.5 months, respectively. The limitation of this study is the small sample size. These preliminary results indicate CMC may complement the efficacy of 5-S-CD to detect metastasis and can be a prognostic marker. Although there is still room for improvement to maximise the sensitivity, the CellSearch system is reproducible, standardised and suitable for multi-centre studies.