Comprehensive clinical imaging, histopathological analysis and liquid biopsy-based surveillance of human uveal melanoma in a prolonged rabbit xenograft model.

Uveal melanoma is the most common intraocular tumor in adults. Our group has previously developed a human uveal melanoma animal model; however, adverse effects caused by the immunosuppressive agent, cyclosporine A, prevented animals from surviving more than 12 weeks. In this study, we tested multiple cyclosporine A doses over an extended disease course up to 20 weeks, providing complete clinical imaging of intraocular tumors, histopathological analysis and liquid biopsy biomarker analysis. Twenty albino rabbits were divided into four groups with different daily cyclosporine A schedules (0-10 mg/kg) and inoculated with human uveal melanoma cell lines, 92.1 or MP41, into the suprachoroidal space. Rabbits were monitored with fundoscopy, ultrasound and optical coherence tomography. Intraocular tumors (macroscopic or microscopic) were detected in all study animals. Tumor size and growth were correlated to cyclosporine A dose, with tumors regressing when cyclosporine A was arrested. All tumors expressed HMB-45 and MelanA; however, tumor size, pigmentation and cell morphology differed in 92.1 vs. MP41 tumors. Finally, across all groups, circulating tumor DNA from plasma and aqueous humor was detected earlier than tumor detection by imaging and correlated to tumor growth. In conclusion, using three clinically relevant imaging modalities (fundoscopy, ultrasonography and optical coherence tomography) and liquid biopsy, we were successfully able to monitor tumor progression in our rabbit xenograft model of human uveal melanoma.

Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions.

BACKGROUND: Although rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention. METHODS: Six UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis. RESULTS: UM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions. CONCLUSIONS: Under an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

Blood and saliva-derived ctDNA is a marker of residual disease after treatment and correlates with recurrence in human papillomavirus-associated head and neck cancer.

BACKGROUND: There is an alarming increase in human papillomavirus-associated head and neck cancer (HNC), reaching epidemic levels. While patient prognosis is generally good, off-target treatment effects are associated with decreased quality of life. Thus, non-invasive strategies to predict treatment response and risk of recurrence could help de-escalate treatment. In this study, we tested circulating tumor (ct)DNA in liquid biopsies (blood/saliva) of HPV-positive HNC patients to assess treatment response and disease progression. METHODS: A total of 235 blood and saliva samples were collected from 60 HPV-positive and 17 HPV-negative HNC patients (control group) before and/or after treatment. Samples were analyzed using ddPCR for HPV16/18/31/33/35/45 and correlated with imaging and pathological examination. RESULTS: HPV-ctDNA detection was significantly higher prior to treatment (91%) than after treatment (8.0%) (χ2 p < 0.00001), with high concordance between saliva and blood (93%). In matched samples, all patients positive for ctDNA before treatment showed significant reductions in ctDNA levels post treatment (p < 0.0001). All but one patient with persistent ctDNA after treatment showed residual tumor and subsequent recurrence. Finally, fragmentomic analysis revealed shifts in cell-free DNA fragment size after treatment, suggesting a complementary biomarker for treatment response. CONCLUSIONS: Blood and saliva were found to be good sources of HPV-ctDNA. The presence of ctDNA strongly correlated with treatment response, demonstrating clinical utility as a non-invasive biomarker to monitor tumor progression in HPV-positive HNC. Liquid biopsy based ctDNA testing could be an effective approach to predict recurrence and stratify patients for de-escalation of treatment, thereby improving quality of life.

Tumour extracellular vesicles induce neutrophil extracellular traps to promote lymph node metastasis.

Lymph nodes (LNs) are frequently the first sites of metastasis. Currently, the only prognostic LN assessment is determining metastatic status. However, there is evidence suggesting that LN metastasis is facilitated by the formation of a pre-metastatic niche induced by tumour derived extracellular vehicles (EVs). Therefore, it is important to detect and modify the LN environmental changes. Earlier work has demonstrated that neutrophil extracellular traps (NETs) can sequester and promote distant metastasis. Here, we first confirmed that LN NETs are associated with reduced patient survival. Next, we demonstrated that NETs deposition precedes LN metastasis and NETs inhibition diminishes LN metastases in animal models. Furthermore, we discovered that EVs are essential to the formation of LN NETs. Finally, we showed that lymphatic endothelial cells secrete CXCL8/2 in response to EVs inducing NETs formation and the promotion of LN metastasis. Our findings reveal the role of EV-induced NETs in LN metastasis and provide potential immunotherapeutic vulnerabilities that may occur early in the metastatic cascade.

Chronic low-level JUUL aerosol exposure causes pulmonary immunologic, transcriptomic, and proteomic changes.

E-cigarettes currently divide public opinion, with some considering them a useful tool for smoking cessation and while others are concerned with potentially adverse health consequences. However, it may take decades to fully understand the effects of e-cigarette use in humans given their relative newness on the market. This highlights the need for comprehensive preclinical studies investigating the effects of e-cigarette exposure on health outcomes. Here, we investigated the impact of chronic, low-level JUUL aerosol exposure on multiple lung outcomes. JUUL is a brand of e-cigarettes popular with youth and young adults. To replicate human exposures, 8- to 12-week-old male and female C57BL/6J mice were exposed to commercially available JUUL products (containing 59 mg/ml nicotine). Mice were exposed to room air, PG/VG, or JUUL daily for 4 weeks. After the exposure period, inflammatory markers were assessed via qRT-PCR, multiplex cytokine assays, and differential cell count. Proteomic and transcriptomic analyses were also performed on samples isolated from the lavage of the lungs; this included unbiased analysis of proteins contained within extracellular vesicles (EVs). Mice exposed to JUUL aerosols for 4 weeks had significantly increased neutrophil and lymphocyte populations in the BAL and some changes in cytokine mRNA expression. However, BAL cytokines did not change. Proteomic and transcriptomic analysis revealed significant changes in numerous biological pathways including neutrophil degranulation, PPAR signaling, and xenobiotic metabolism. Thus, e-cigarettes are not inert and can cause significant cellular and molecular changes in the lungs.

EV-ADD, a database for EV-associated DNA in human liquid biopsy samples.

Extracellular vesicles (EVs) play a key role in cellular communication both in physiological conditions and in pathologies such as cancer. Emerging evidence has shown that EVs are active carriers of molecular cargo (e.g. protein and nucleic acids) and a powerful source of biomarkers and targets. While recent studies on EV-associated DNA (EV-DNA) in human biofluids have generated a large amount of data, there is currently no database that catalogues information on EV-DNA. To fill this gap, we have manually curated a database of EV-DNA data derived from human biofluids (liquid biopsy) and in-vitro studies, called the Extracellular Vesicle-Associated DNA Database (EV-ADD). This database contains validated experimental details and data extracted from peer-reviewed published literature. It can be easily queried to search for EV isolation methods and characterization, EV-DNA isolation techniques, quality validation, DNA fragment size, volume of starting material, gene names and disease context. Currently, our database contains samples representing 23 diseases, with 13 different types of EV isolation techniques applied on eight different human biofluids (e.g. blood, saliva). In addition, EV-ADD encompasses EV-DNA data both representing the whole genome and specifically including oncogenes, such as KRAS, EGFR, BRAF, MYC, and mitochondrial DNA (mtDNA). An EV-ADD data metric system was also integrated to assign a compliancy score to the MISEV guidelines based on experimental parameters reported in each study. While currently available databases document the presence of proteins, lipids, RNA and metabolites in EVs (e.g. Vesiclepedia, ExoCarta, ExoBCD, EVpedia, and EV-TRACK), to the best of our knowledge, EV-ADD is the first of its kind to compile all available EV-DNA datasets derived from human biofluid samples. We believe that this database provides an important reference resource on EV-DNA-based liquid biopsy research, serving as a learning tool and to showcase the latest developments in the EV-DNA field. EV-ADD will be updated yearly as newly published EV-DNA data becomes available and it is freely available at www.evdnadatabase.com.

Filtering blue light mitigates the deleterious effects induced by the oxidative stress in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a major cause of blindness in elderly. It is characterized by the loss of central vision due to damaged retinal pigment epithelial (RPE) cells and photoreceptors. Blue Light (BL) exposure was proposed as a risk factor for AMD progression. We undertook this study to determine the effects of BL on the behaviour of RPE cells and their potential mitigation by BL-filtering intraocular lenses (IOL). Human RPE cells were exposed or not to BL, with the absence or presence of either a clear ultraviolet (UV)-filtering IOL (CIOL), or a yellow UV- and BL-filtering IOL (YIOL). Cells were analyzed for their oxidative stress by measuring the levels of reactive oxygen species (ROS), and their viability. BL exposure significantly increased the levels of both total cellular and mitochondrial ROS. While this increase was not affected by placing the CIOL in the BL beam, YIOL decreased the levels of both ROS reservoirs. Increased ROS production was accompanied by increased cell death which was similarly decreased when cells were protected with the YIOL. Pre-treatment of cells with N-acetylcycteine (NAC) abolished the increased cell death, suggesting that the effects of BL on cell viability were mainly due to increased levels of ROS. BL is deleterious to RPE cells due to increased oxidative stress and cell death. These effects were mitigated by filtering these radiations. The use of BL-filtering devices may represent a strategy to reduce these effects on RPE cells and delay the onset of AMD.

Anticancer effects of mifepristone on human uveal melanoma cells.

BACKGROUND: Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. METHODS: The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 µM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. RESULTS: MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. CONCLUSION: This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Numerical and Experimental Validation of Mixing Efficiency in Periodic Disturbance Mixers.

The shape and dimensions of a micromixer are key elements in the mixing process. Accurately quantifying the mixing efficiency enables the evaluation of the performance of a micromixer and the selection of the most suitable one for specific applications. In this paper, two methods are investigated to evaluate the mixing efficiency: a numerical model and an experimental model with a software image processing technique. Using two methods to calculate the mixing efficiency, in addition to corroborating the results and increasing their reliability, creates various possible approaches that can be selected depending on the circumstances, resources, amount of data to be processed and processing time. Image processing is an easy-to-implement tool, is applicable to different programming languages, is flexible, and provides a quick response that allows the calculation of the mixing efficiency using a process of filtering of images and quantifying the intensity of the color, which is associated with the percentage of mixing. The results showed high similarity between the two methods, with a difference ranging between 0 and 6% in all the evaluated points.

Parametric Study of the Factors Influencing Liposome Physicochemical Characteristics in a Periodic Disturbance Mixer.

Liposomes encapsulate different substances ranging from drugs to genes. Control over the average size and size distribution of these nanoparticles is vital for biomedical applications since these characteristics determine to a high degree where liposomes will accumulate in the human body. Micromixers enable the continuous flow synthesis of liposomes, improving size control and reproducibility. Recently, Dean flow dynamics-based micromixers, such as the periodic disturbance mixer (PDM), have been shown to produce controlled-size liposomes in a scalable and reproducible way. However, contrary to micromixers based on molecular diffusion or chaotic advection, their production factors and their influence over liposome properties have not yet been addressed thoroughly. In this work, we present a comprehensive parametric study of the effects of flow conditions and molecular changing factors such as concentration, lipid type, and temperature on the physicochemical characteristics of liposomes. Numerical models and confocal images are used to quantitatively and qualitatively evaluate mixing performance under different liposome production conditions and their relationship with vesicle properties. The total flow rate (TFR) and, to a lesser extent, the flow rate ratio (FRR) control the liposome size and size distribution. Effects on liposome size are also observed by changing the molecular factors. Moreover, the liposome ζ potential is independent of the factors studied here. The micromixer presented in this work enables the production of liposomes as small as 24 nm, with monodispersed to low or close to low polydispersed liposome populations as well as a production rate as high as 41 mg/h.

Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. METHODS: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCß4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). RESULTS: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. CONCLUSIONS: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Liquid Biopsy in Uveal Melanoma: Are We There Yet?

In the era of precision oncology, major strides are being made to use individual tumor information for clinical decision-making. Differing from traditional biopsy methods, the emerging practice of liquid biopsy provides a minimally invasive way of obtaining tumor cells and derived molecules. Liquid biopsy provides a means to detect and monitor disease progression, recurrence, and treatment response in a noninvasive way, and to potentially complement classical biopsy. Uveal melanoma (UM) is a unique malignancy, with diagnosis heavily reliant on imaging, few repeat biopsies, and a high rate of metastasis, which occurs hematogenously and often many years after diagnosis. In this disease setting, a noninvasive biomarker to detect, monitor, and study the disease in real time could lead to better disease understanding and patient care. While advances have been made in the detection of tumor-disseminated components, sensitivity and specificity remain important challenges. Ambiguity remains in how to interpret current findings and in how liquid biopsy can have a place in clinical practice. Related publications in UM are few compared to other cancers, but with further studies we may be able to uncover more about the biology of disseminated molecules and the mechanisms involved in the progression to metastasis.

Uveal melanoma pathobiology: Metastasis to the liver.

Uveal melanoma (UM) is a type of intraocular tumor with a propensity to disseminate to the liver. Despite the identification of the early driver mutations during the development of the pathology, the process of UM metastasis is still not fully comprehended. A better understanding of the genetic, molecular, and environmental factors participating to its spread and metastatic outgrowth could provide additional approaches for UM treatment. In this review, we will discuss the advances made towards the understanding of the pathogenesis of metastatic UM, summarize the current and prospective treatments, and introduce some of the ongoing research in this field.

Nelfinavir Induces Cytotoxicity towards High-Grade Serous Ovarian Cancer Cells, Involving Induction of the Unfolded Protein Response, Modulation of Protein Synthesis, DNA Damage, Lysosomal Impairment, and Potentiation of Toxicity Caused by Proteasome Inhibition.

High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction in the HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction in clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho-eIF2α, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile.

The effect of different organic solvents in liposome properties produced in a periodic disturbance mixer: Transcutol®, a potential organic solvent replacement.

Liposomes are versatile particles used in the biomedical field as drug delivery systems (DDS). Liposome production using micromixers have shown to yield nanoparticles for DDS in a single step with a controllable size by changing flow conditions. Nonetheless, other factors such as the organic solvent, play a crucial role in the liposome formation process. Furthermore, drug solubility and toxicity are pivotal when deciding which organic solvent to choose. In this work, liposomes were produced in a periodic disturbance mixer (PDM). We investigated three conventional organic solvents: ethanol, methanol, and isopropanol as well as Transcutol®. We assessed the organic solvent influence on liposome characteristics (size, size distribution and zeta potential). Among the four organic solvents, Transcutol® yielded the smallest liposomes, which ranged from 80 nm to 160 nm. Moreover, a more in-depth investigation showed that Transcutol® produced smaller or similar-sized particles under different temperature and lipid concentration conditions, compared with ethanol. Furthermore, we proved that particles zeta potential was not influenced by the organic solvent, production temperature, or lipid concentration. This work results show that Transcutol® could replace the conventional alcohol-based solvents and can potentially avoid filtration steps due to its low toxicity. Therefore, the present approach is appealing for DDS development.

Novel Methylation Patterns Predict Outcome in Uveal Melanoma.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite effective local treatments, 50% of patients develop metastasis. Better ways to determine prognosis are needed as well as new therapeutic targets. Epigenetic changes are important events driving cancer progression; however, few studies exist on methylation changes in UM. Our aim was to identify methylation events associated with UM prognosis. Matched clinical, genetic, and methylation data for 80 UM cases were obtained from The Cancer Genome Atlas (TCGA). Top differentially methylated loci were sorted through hierarchical clustering based on methylation patterns, and these patterns were compared to tumor characteristics, genomic aberrations, and patient outcome. Hierarchical clustering revealed two distinct groups. These classifications effectively separated high and low-risk cases, with significant differences between groups in patient survival (p < 0.0001) and correlation with known prognostic factors. Major differences in methylation of specific genes, notably NFIA, HDAC4, and IL12RB2, were also seen. The methylation patterns identified in this study indicate potential novel prognostic indicators of UM and highlight the power of methylation changes in predicting outcome. The methylation events enriched in the high-risk group suggest that epigenetic modulating drugs may be useful in reducing metastatic potential, and that specific differentially methylated loci could act as biomarkers of therapeutic response.

Uveal Melanoma-Derived Extracellular Vesicles Display Transforming Potential and Carry Protein Cargo Involved in Metastatic Niche Preparation.

Extracellular vesicles (EVs) carry molecules derived from donor cells and are able to alter the properties of recipient cells. They are important players during the genesis and progression of tumors. Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is associated with a high rate of metastasis, primarily to the liver. However, the mechanisms underlying this process are poorly understood. In the present study, we analyzed the oncogenic potential of UM-derived EVs and their protein signature. We isolated and characterized EVs from five UM cell lines and from normal choroidal melanocytes (NCMs). BRCA1-deficient fibroblasts (Fibro-BKO) were exposed to the EVs and analyzed for their growth in vitro and their reprograming potential in vivo following inoculation into NOD-SCID mice. Mass spectrometry of proteins from UM-EVs and NCM-EVs was performed to determine a protein signature that could elucidate potential key players in UM progression. In-depth analyses showed the presence of exosomal markers, and proteins involved in cell-cell and focal adhesion, endocytosis, and PI3K-Akt signaling pathway. Notably, we observed high expression levels of HSP90, HSP70 and integrin V in UM-EVs. Our data bring new evidence on the involvement of UM-EVs in cancer progression and metastasis.

Phase II Trial of Cabozantinib in Recurrent/Metastatic Endometrial Cancer: A Study of the Princess Margaret, Chicago, and California Consortia (NCI9322/PHL86).

PURPOSE: The relevance of the MET/hepatocyte growth factor pathway in endometrial cancer tumor biology supports the clinical evaluation of cabozantinib in this disease. PATIENTS AND METHODS: PHL86/NCI#9322 (NCT01935934) is a single arm study that evaluated cabozantinib (60 mg once daily) in women with endometrial cancer with progression after chemotherapy. Coprimary endpoints were response rate and 12-week progression-free-survival (PFS). Patients with uncommon histology endometrial cancer (eg, carcinosarcoma and clear cell) were enrolled in a parallel exploratory cohort. RESULTS: A total of 102 patients were accrued. Among 36 endometrioid histology patients, response rate was 14%, 12-week PFS rate was 67%, and median PFS was 4.8 months. In serous cohort of 34 patients, response rate was 12%, 12-week PFS was 56%, and median PFS was 4.0 months. In a separate cohort of 32 patients with uncommon histology endometrial cancer (including carcinosarcoma), response rate was 6% and 12-week PFS was 47%. Six patients were on treatment for >12 months, including two for >30 months. Common cabozantinib-related toxicities (>30% patients) included hypertension, fatigue, diarrhea, nausea, and hand-foot syndrome. Gastrointestinal fistula/perforation occurred in four of 70 (6%) patients with serous/endometrioid cancer and five of 32 (16%) patients in exploratory cohort. We observed increased frequency of responses with somatic CTNNB1 mutation [four partial responses (PRs) in 10 patients, median PFS 7.6 months] and concurrent KRAS and PTEN/PIK3CA mutations (three PRs in 12 patients, median PFS 5.9 months). CONCLUSIONS: Cabozantinib has activity in serous and endometrioid histology endometrial cancer. These results support further evaluation in genomically characterized patient cohorts.

Acetylsalicylic Acid Exerts Potent Antitumor and Antiangiogenic Effects in Cutaneous and Uveal Melanoma Cell Lines.

INTRODUCTION: Acetylsalicylic acid (ASA) has been investigated for a potential anticancer role in several cancers, such as colorectal, ovarian, and endometrial cancer. Moreover, ASA has been shown to abrogate various processes that contribute to tumor growth and progression. OBJECTIVE: The aim of this study was to evaluate the effects of ASA on cutaneous melanoma (CM) and uveal melanoma (UM). METHODS: Human CM and UM cells were treated with 5 mM ASA and assessed for changes in cellular functions. Antiangiogenic effects of ASA were determined using an ELISA-based assay for 10 proangiogenic cytokines, and then validated by Western blot. Finally, proteomic analysis of ASA-treated cells was performed to elucidate the changes that may be responsible for ASA-mediated effects in melanoma cells. RESULTS: Treatment with ASA significantly inhibited the proliferation, invasion, and migration capabilities, and caused a significant decrease in angiogenin and PIGF secretion in both CM and UM. Mass spectrometry revealed 179 protein changes associated with ASA in the CM and UM cell lines. CONCLUSIONS: These results suggest that ASA may be effective as an adjuvant therapy in metastatic CM and UM. Future studies are needed to determine the regulating targets that are responsible for the antitumor effects of ASA.