A Lipidomic Approach to Identify Potential Biomarkers in Exosomes From Melanoma Cells With Different Metastatic Potential.

Melanoma, one of the most lethal cutaneous cancers, is characterized by its ability to metastasize to other distant sites, such as the bone. Melanoma cells revealed a variable in vitro propensity to be attracted toward bone fragments, and melanoma-derived exosomes play a role in regulating the osteotropism of these cells. We have here investigated the lipid profiles of melanoma cell lines (LCP and SK-Mel28) characterized by different metastatic propensities to colonize the bone. We have purified exosomes from cell supernatants by ultracentrifugation, and their lipid composition has been compared to identify potential lipid biomarkers for different migration and invasiveness of melanoma cells. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) lipid analysis has been performed on very small amounts of intact parental cells and exosomes by skipping lipid extraction and separation steps. Statistical analysis has been applied to MALDI mass spectra in order to discover significant differences in lipid profiles. Our results clearly show more saturated and shorter fatty acid tails in poorly metastatic (LCP) cells compared with highly metastatic (SK-Mel28) cells, particularly for some species of phosphatidylinositol. Sphingomyelin, lysophosphatidylcholine, and phosphatidic acid were enriched in exosome membranes compared to parental cells. In addition, we have clearly detected a peculiar phospholipid bis(monoacylglycero)phosphate as a specific lipid marker of exosomes. MALDI-TOF/MS lipid profiles of exosomes derived from the poorly and highly metastatic cells were not significantly different.

First prospective data on breast cancer patients from the multicentre italian bone metastasis database.

Bone metastases (BM) are still the main cause of morbidity in cancer patients because of skeletal-related events (SREs) that reduce quality of life. They have also led to increased social and healthcare costs. At present, data available on BM are insufficient. This was a multicentre prospective observational study of patients with BM from breast cancer (BC) with at least 6 months' follow-up. Information on patients at the first diagnosis of BM, including demographics and characteristics of the primary tumor and BM. Data were periodically updated by participating centres and reviewed by the coordinator centre. From October 2014 to July 2019, 618 patients with BM from solid tumors were enrolled and 220 were eligible for the present study. Median age was 62 years (range 26-86). Median follow-up was 34 months (range 6-149). At the time of enrolment, 109 (50%) had only BM (BOM) and 109 (50%) had concomitant visceral lesions and BM (BVM). Median time-to-first BM was 47 months (range 0-312) in BOM and 78.6 months in BVM patients. Disease-free interval differed on the basis of BC molecular subtype and stage. Ninety-eight BM patients had at least on SRE. Zoledronate was used in 69.1% of cases and denosumab in 28.3%. First-line treatment was hormone-based (50.7%), chemotherapy-based (38.7%) or chemotherapy- + hormone therapy-based (9.7%). Median progression-free and overall survival were 15.1 months (95% CI 12.6-18.4) and 66.8 months (95% CI 52.1-79.2), respectively. Our prospective study could substantially help to better understand the natural history of BM from BC.

The Impairment in Kidney Function in the Oral Anticoagulation Era. A Pathophysiological Insight.

The need for anticoagulation in patients with atrial fibrillation (AF) is fundamental to prevent thromboembolic events. Direct oral anticoagulants (DOACs) recently demonstrated to be superior, or at least equal, to Warfarin in reducing the risk for stroke/systemic embolism and preventing major bleeding and intracranial hemorrhages. The AF population often suffers from chronic kidney disease (CKD). Indeed, the relationship between AF and renal function is bidirectional: AF can trigger kidney failure, while kidney impairment can promote alterations able to enhance AF. Therefore, there are concerns regarding prescriptions of anticoagulants to patients with AF and CKD. The worsening in kidney function can be effectively due to anticoagulants administration. Warfarin has been recognized to promote acute kidney injury in case of excessive anticoagulation levels. Nevertheless, further mechanisms can induce the chronic worsening of renal function, thus leading to terminal kidney failure as observed in post-hoc analysis from registration trials and dedicated observational studies. By contrast, DOACs seem to protect kidneys from injuries more efficiently than Warfarin, although they still continue to play a role in promoting some kidney lesions. However, the exact mechanisms remain unknown. This narrative review aimed to discuss the influence of oral anticoagulants on renal impairment as well as to overview potential pathophysiological mechanisms related to this clinical complication.

TOP2A expression predicts responsiveness to carfilzomib in myeloma and informs novel combinatorial strategies for enhanced proteasome inhibitor cell killing.

Microarray was utilized to determine if a genetic signature associated with resistance to carfilzomib (CFZ) could be identified. Twelve human myeloma (MM) cell lines (HMCLs) were treated with CFZ and a cell-viability profile was assessed categorizing HMCLs as sensitive or resistant to CFZ. The gene expression profiles (GEP) of untreated resistant versus sensitive HMCLs revealed 29 differentially expressed genes. TOP2A, an enzyme involved in cell cycle and proliferation, was overexpressed in carfilzomib-resistant HMCLs. TOP2A protein expression levels, evaluated utilizing trephine biopsy specimens acquired prior to treatment with proteasome inhibitors, were higher in patients failing to achieve a response when compared to responding patients. Logistic-regression analysis confirmed that TOP2A protein expression was a highly significant predictor of response to PIs (AUC 0.738). Further, the combination of CFZ with TOP2A inhibitors, demonstrated synergistic cytotoxic effects in vitro, providing a rationale for combining topoisomerase inhibitors with CFZ to overcome resistance in MM.

Immune System Evasion as Hallmark of Melanoma Progression: The Role of Dendritic Cells.

Melanoma is an immunogenic tumor whose relationship with immune cells resident in the microenvironment significantly influences cancer cell proliferation, progression, and metastasis. During melanomagenesis, both immune and melanoma cells undergo the immunoediting process that includes interconnected phases as elimination, equilibrium, and escape or immune evasion. In this context, dendritic cells (DCs) are active players that indirectly counteract the proliferation of melanoma cells. Moreover, DC maturation, migration, and cross-priming as well as their functional interplay with cytotoxic T-cells through ligands of immune checkpoint receptors result impaired. A number of signals propagated by highly proliferating melanoma cells and accessory cells as T-cells, natural killer cells (NKs), tumor-associated macrophages (TAMs), T-regulatory cells (T-regs), myeloid-derived suppressor cells (MDSCs), and endothelial cells participate to create an immunosuppressive milieu that results engulfed of tolerogenic factors and interleukins (IL) as IL-6 and IL-10. To underline the role of the immune infiltrate in blocking the melanoma progression, it has been described that the composition, density, and distribution of cytotoxic T-cells in the surrounding stroma is predictive of responsiveness to immunotherapy. Here, we review the major mechanisms implicated in melanoma progression, focusing on the role of DCs.

Revisiting the Role of Exosomes in Colorectal Cancer: Where Are We Now?

Exosomes (Exos) are nano-sized extracellular vesicles constitutively released by both prokaryotic and eukaryotic cells. Their role as inter-cellular messengers involved in both physiological and pathological processes has overwhelmingly come to light in the last decade, and their contribution to cancerogenesis and tumor metastasis is under intensive investigation. Here we review the most recent information concerning Exos in colorectal cancer (CRC) and focus on their effects on tumor microenvironment and the immune system, as well as unravel their role in the formation of the pre-metastatic niche and in drug resistance. Such a recent knowledge on Exos depicts their potential translations into the clinical arena, either as an alternative tool of "liquid biopsy" or novel therapeutic approaches for CRC. However, due to the limited data available from clinical trials, they need further validations before addressing their putative application in oncology.

Tumor-derived exosomes promote the in vitro osteotropism of melanoma cells by activating the SDF-1/CXCR4/CXCR7 axis.

BACKGROUND: Bone metastases occur rarely in patients suffering from malignant melanoma, although their onset severely worsens both prognosis and quality of life. Extracellular vesicles (EVs) including exosomes (Exos) are active players in melanoma progression involved in the formation of the pre-metastatic niche. METHODS: Trans-well assays explored the basal migratory and invasive potential of four melanoma cell lines and investigated their different propensity to be attracted toward the bone. Exosomes were purified from cell supernatants by ultracentrifugation and explored in their ability to influence the bone tropism of melanoma cells. The molecular machinery activated during this process was investigated by RT-PCR, droplet digital-PCR, flow-cytometry and Western blot, while loss of function studies with dedicated siRNAs defined the single contribute of CXCR4 and CXCR7 molecules. RESULTS: Melanoma cells revealed a variable propensity to be attracted toward bone fragments. Gene profiling of both osteotropic and not-osteotropic cells did not show a different expression of those genes notoriously correlated to chemotaxis and bone metastasis. However, bone conditioned medium significantly increased CXCR4, CXCR7 and PTHrP expression solely to osteotropic cells, while their Exos were able to revert the original poor bone tropism of not-osteotropic cells through CXCR7 up-regulation. Silencing experiments also demonstrated that membrane expression of CXCR7 is required by melanoma cells to promote their chemotaxis toward SDF-1 gradients. CONCLUSIONS: Our data correlated the osteotropism of melanoma cells to the activation of the SDF-1/CXCR4/CXCR7 axis following the exposition of tumor cells to bone-derived soluble factors. Also, we demonstrated in vitro that tumor-derived Exos can reprogram the innate osteotropism of melanoma cells by up-regulating membrane CXCR7. These results may have a potential translation to future identification of druggable targets for the treatment of skeletal metastases from malignant melanoma.

Metastatic bone disease: Pathogenesis and therapeutic options: Up-date on bone metastasis management.

Bone metastases (BM) are a common complication of cancer, whose management often requires a multidisciplinary approach. Despite the recent therapeutic advances, patients with BM may still experience skeletal-related events and symptomatic skeletal events, with detrimental impact on quality of life and survival. A deeper knowledge of the mechanisms underlying the onset of lytic and sclerotic BM has been acquired in the last decades, leading to the development of bone-targeting agents (BTA), mainly represented by anti-resorptive drugs and bone-seeking radiopharmaceuticals. Recent pre-clinical and clinical studies have showed promising effects of novel agents, whose safety and efficacy need to be confirmed by prospective clinical trials. Among BTA, adjuvant bisphosphonates have also been shown to reduce the risk of BM in selected breast cancer patients, but failed to reduce the incidence of BM from lung and prostate cancer. Moreover, adjuvant denosumab did not improve BM free survival in patients with breast cancer, suggesting the need for further investigation to clarify BTA role in early-stage malignancies. The aim of this review is to describe BM pathogenesis and current treatment options in different clinical settings, as well as to explore the mechanism of action of novel potential therapeutic agents for which further investigation is needed.

The metabolic milieu in melanoma: Role of immune suppression by CD73/adenosine.

The mechanisms leading to immune escape of melanoma have been largely investigated in relation to its tumour immunogenicity and features of inflamed microenvironment that promote the immune suppression during the disease progression. These findings have recently led to advantages in terms of immunotherapy-based approaches as rationale for overcoming the immune escape. However, besides immune checkpoints, other mechanisms including the adenosine produced by ectonucleotidases CD39 and CD73 contribute to the melanoma progression due to the immunosuppression induced by the tumour milieu. On the other hand, CD73 has recently emerged as both promising therapeutic target and unfavourable prognostic biomarker. Here, we review the major mechanisms of immune escape activated by the CD39/CD73/adenosine pathway in melanoma and focus potential therapeutic strategies based on the control of CD39/CD73 downstream adenosine receptor signalling. These evidences provide the basis for translational strategies of immune combination, while CD73 would serve as potential prognostic biomarker in metastatic melanoma.

Vitamin D in melanoma: Controversies and potential role in combination with immune check-point inhibitors.

The role of vitamin D in melanoma is still controversial. Although several Authors described a correlation between vitamin D deficiency and poor survival in metastatic melanoma patients, clinical trials exploring the effects of vitamin D supplementation in this clinical setting were mostly inconclusive. However, recent evidence suggests that vitamin D exerts both anti-proliferative effects on tumor cells and immune-modulating activities, that have been widely explored in auto-immune disorders. On the one hand, vitamin D has been shown to inhibit T-helper17 lymphocytes, notoriously involved in the pathogenesis of immune-related adverse events (iAEs) which complicate immune-checkpoint inhibitor (ICI) treatment. On the other hand, vitamin D up-regulates PDL-1 expression on both epithelial and immune cells, suggesting a synergic effect in combination with ICIs, for which further investigation is needed.

Uric Acid, Metabolic Syndrome and Atherosclerosis: The Chicken or the Egg, Which Comes First?

BACKGROUND: A great debate in literature exists nowadays on the role of uric acid as a marker of cardiovascular and metabolic organ damage or a risk factor for cardiovascular and metabolic disease. METHODS: The study aimed to determine the relationship among serum uric acid and metabolic syndrome and atherosclerosis, by means of carotid intima media-thickness, in a cohort of 811 otherwise healthy overweight/obese subjects, without overt atherosclerosis not using any kind of drug. RESULTS: Uric acid levels were positively related to male gender, waist circumference, BMI, systolic and diastolic pressure levels, fasting insulin, fasting glucose, HOMA-IR, triglycerides, total cholesterol, LDL cholesterol, the presence of metabolic syndrome and the number of the components of metabolic syndrome and negatively related to HDL cholesterol levels. No correlation was found between uric acid and carotid intima media thickness. At the multiple regression analysis, only waist circumference and triglycerides (positively) and HDL-cholesterol (negatively) maintained an independent association with uric acid as dependent variable, while age, female gender and uric acid showed a significant independent association with metabolic syndrome as dependent variable. Moreover, the analysis of the odd ratios showed that the risk of developing metabolic syndrome was consistent with uric acid levels ranging from 3 mg/dl to 8 mg/dl. CONCLUSION: The presence of metabolic syndrome does not seem to provide hyperuricemia. By contrast, higher serum uric acid level may predict the risk of metabolic syndrome. Moreover, our results suggest that uric acid cannot be considered a risk factor for early atherosclerosis, at least when assessed using carotid ultrasound.

Serum exosomes as predictors of clinical response to ipilimumab in metastatic melanoma.

Immunotherapy is effective in metastatic melanoma (MM) but most studies failed in discovering a biomarker predictive of clinical response. Exosomes (Exo) from melanoma cells are detectable in sera of MM patients similarly to those produced by immune cells that control the tumor progression. Here, we investigated by flow-cytometry the levels of Exo from both T-cells and dendritic cells (DCs) in 59 patients with MM treated with IPI and the relative expression of PD-1, CD28 and ICOS as well as CD80 and CD86. We found a significant increment of PD-1 and CD28 expression in patients achieving a clinical response reflected by improvement of both PFS and OS. Furthermore, MM patients receiving IPI who showed extended PFS underwent increased expression of CD80 and CD86 on DC-derived Exo at the end of treatment. These results suggest a possible association of both PD-1 and CD28 up-regulation on immune cell-derived Exo in patients with better clinical response to IPI.

Immune system and melanoma biology: a balance between immunosurveillance and immune escape.

Melanoma is one of the most immunogenic tumors and its relationship with host immune system is currently under investigation. Many immunomodulatory mechanisms, favoring melanomagenesis and progression, have been described to interfere with the disablement of melanoma recognition and attack by immune cells resulting in immune resistance and immunosuppression. This knowledge produced therapeutic advantages, such as immunotherapy, aiming to overcome the immune evasion. Here, we review the current advances in cancer immunoediting and focus on melanoma immunology, which involves a dynamic interplay between melanoma and immune system, as well as on effects of "targeted therapies" on tumor microenvironment for combination strategies.

pIL6-TRAIL-engineered umbilical cord mesenchymal/stromal stem cells are highly cytotoxic for myeloma cells both in vitro and in vivo.

BACKGROUND: Mesenchymal/stromal stem cells (MSCs) are favorably regarded in anti-cancer cytotherapies for their spontaneous chemotaxis toward inflammatory and tumor environments associated with an intrinsic cytotoxicity against tumor cells. Placenta-derived or TRAIL-engineered adipose MSCs have been shown to exert anti-tumor activity in both in-vitro and in-vivo models of multiple myeloma (MM) while TRAIL-transduced umbilical cord (UC)-MSCs appear efficient inducers of apoptosis in a few solid tumors. However, apoptosis is not selective for cancer cells since specific TRAIL receptors are also expressed by a number of normal cells. To overcome this drawback, we propose to transduce UC-MSCs with a bicistronic vector including the TRAIL sequence under the control of IL-6 promoter (pIL6) whose transcriptional activation is promoted by the MM milieu. METHODS: UC-MSCs were transduced with a bicistronic retroviral vector (pMIGR1) encoding for green fluorescent protein (GFP) and modified to include the pIL6 sequence upstream of the full-length human TRAIL cDNA. TRAIL expression after stimulation with U-266 cell conditioned medium, or IL-1α/IL-1ß, was evaluated by flow cytometry, confocal microscopy, real-time PCR, western blot analysis, and ELISA. Apoptosis in MM cells was assayed by Annexin V staining and by caspase-8 activation. The cytotoxic effect of pIL6-TRAIL + -GFP + -UC-MSCs on MM growth was evaluated in SCID mice by bioluminescence and ex vivo by caspase-3 activation and X-ray imaging. Statistical analyses were performed by Student's t test, ANOVA, and logrank test for survival curves. RESULTS: pIL6-TRAIL + -GFP + -UC-MSCs significantly expressed TRAIL after stimulation by either conditioned medium or by IL-1α/IL-1ß, and induced apoptosis in U-266 cells. Moreover, when systemically injected in SCID mice intratibially xenografted with U-266, those cells underwent within MM tibia lesions and significantly reduced the tumor burden by specific induction of apoptosis in MM cells as revealed by caspase-3 activation. CONCLUSIONS: Our tumor microenvironment-sensitive model of anti-MM cytotherapy is regulated by the axis pIL6/IL-1α/IL-1ß and appears suitable for further preclinical investigation not only in myeloma bone disease in which UC-MSCs would even participate to bone healing as described, but also in other osteotropic tumors whose milieu is enriched of cytokines triggering the pIL6.

1,25(OH)2 vitamin D(3) contributes to osteoclast-like trans-differentiation of malignant plasma cells.

1,25-dihydroxyvitamin D (1,25(OH)2D) exerts pleiotropic effects including bone turnover and immune system regulation. It inhibits both T and B cell proliferation while decreasing the secretion of inflammatory cytokines and immunoglobulins. 1,25(OH)2D also modulates monocyte-macrophage and osteoclast (OC) maturation. Since we have previously described that malignant plasma cells may trans-differentiate towards the myeloid lineage participating to skeletal devastation in multiple myeloma (MM), we here evaluated in vitro the role of 1,25(OH)2D in this lineage switch. We investigated the gene and protein expression of vitamin D receptor (VDR) in MM cell lines. Thus, after cell treatment with 1,25(OH)2D, we analyzed their morphology and the expression of myeloid and OC markers. Finally, we assessed their bone resorption property on calcium phosphate slices. All MM cells expressed VDR in nuclear and perinuclear sites. Treatment with 1,25(OH)2D altered their morphology from round to fusiform, while inducing paxillin focalization. 1,25(OH)2D administration also up-regulated myeloid and OC genes, including C/EBPα, RANK, M-CSFR and V-ATPase, whose promoters contain potential 1,25(OH)2D responsive elements. Finally, 1,25(OH)2D increased MM cell capability to generate pits of erosion on calcium phosphate discs. This data suggest that myeloma cells may undergo a functional trans-differentiation into OCs and, under appropriate experimental conditions, 1,25(OH)2D triggers this lineage switch.

Adverse drug reactions after intravenous rituximab infusion are more common in hematologic malignancies than in autoimmune disorders and can be predicted by the combination of few clinical and laboratory parameters: results from a retrospective, multicenter study of 374 patients.

Rituximab is an effective treatment for CD20 + B-cell malignancies and autoimmune disorders. However, adverse drug reactions (ADRs) may occur after rituximab infusion, causing, in rare cases, its discontinuation. In this multicenter, retrospective study, among 374 patients treated with rituximab i.v., 23.5% experienced ADRs. Mean follow-up was 20.6 months (range 8-135). Overall, ADRs were significantly more frequent in non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemias (25-35.9%), than in autoimmune diseases (9.4-17.5%) (p < .0001). Grade 3-4 toxicity was observed in eight patients (2.1%), and in four of them (1% of all patients) definitive drug discontinuation was necessary. Interestingly, three groups of patients with different risk of developing ADR were identified, according to a predictive heat-map developed combining four parameters (splenomegaly, history of allergy, hemoglobin levels and gender) selected by multivariate analysis. This model may be useful in identifying patients at higher risk of ADRs, needing appropriate preventing therapies.

PTHrP produced by myeloma plasma cells regulates their survival and pro-osteoclast activity for bone disease progression.

To promote their survival and progression in the skeleton, osteotropic malignancies of breast, lung, and prostate produce parathyroid hormone-related protein (PTHrP), which induces hypercalcemia. PTHrP serum elevations have also been described in multiple myeloma (MM), although their role is not well defined. When we investigated MM cells from patients and cell lines, we found that PTHrP and its receptor (PTH-R1) are highly expressed, and that PTHrP is secreted both as a full-length molecule and as small subunits. Among these subunits, the mid-region, including the nuclear localization sequence (NLS), exerted a proliferative effect because it was accumulated in nuclei of MM cells surviving in starvation conditions. This was confirmed by increased transcription of several genes enrolled in proliferation and apoptosis control. PTHrP was also found to stimulate PTH-R1 in MM cells. PTH-R1's selective activation by the full-length PTHrP molecule or the NH2 -terminal fragment resulted in a significant increase of intracellular Ca(2+) influx, cyclic adenosine monophosphate (cAMP) content, and expression of receptor activator of NF-κB ligand (RANKL) and monocyte chemoattractant protein-1 (MCP-1). Our data definitely clarify the role of PTHrP in MM. The PTHrP peptide is functionally secreted by malignant plasma cells and contributes to MM tumor biology and progression, both by intracrine maintenance of cell proliferation in stress conditions and by autocrine or paracrine stimulation of PTH-R1, which in turn reinforces the production of osteoclastogenic factors. © 2014 American Society for Bone and Mineral Research.

Cytokine overproduction, T-cell activation, and defective T-regulatory functions promote nephritis in systemic lupus erythematosus.

Lupus nephritis (LN) occurs in more than one-third of patients with systemic lupus erythematosus. Its pathogenesis is mostly attributable to the glomerular deposition of immune complexes and overproduction of T helper- (Th-) 1 cytokines. In this context, the high glomerular expression of IL-12 and IL-18 exerts a major pathogenetic role. These cytokines are locally produced by both macrophages and dendritic cells (DCs) which attract other inflammatory cells leading to maintenance of the kidney inflammation. However, other populations including T-cells and B-cells are integral for the development and worsening of renal damage. T-cells include many pathogenetic subsets, and the activation of Th-17 in keeping with defective T-regulatory (Treg) cell function regards as further event contributing to the glomerular damage. These populations also activate B-cells to produce nephritogenic auto-antibodies. Thus, LN includes a complex pathogenetic mechanism that involves different players and the evaluation of their activity may provide an effective tool for monitoring the onset of the disease.