Protocol to generate scaffold-free, multicomponent 3D melanoma spheroid models for preclinical drug testing.

Three-dimensional (3D) models play an increasingly important role in preclinical drug testing as they faithfully mimic interactions between cancer cells and the tumor microenvironment (TME). Here, we present a protocol for generating scaffold-free 3D multicomponent human melanoma spheroids. We describe steps for characterizing models using live-cell imaging and histology, followed by drug testing and assessment of cell death through various techniques such as imaging, luminescence-based assays, and flow cytometry. Finally, we demonstrate the models' adaptability for co-cultures with immune cells.

Single-cell transcriptomics of NRAS-mutated melanoma transitioning to drug resistance reveals P2RX7 as an indicator of early drug response.

Treatment options for patients with NRAS-mutant melanoma are limited and lack an efficient targeted drug combination that significantly increases overall and progression-free survival. In addition, targeted therapy success is hampered by the inevitable emergence of drug resistance. A thorough understanding of the molecular processes driving cancer cells' escape mechanisms is crucial to tailor more efficient follow-up therapies. We performed single-cell RNA sequencing of NRAS-mutant melanoma treated with MEK1/2 plus CDK4/6 inhibitors to decipher transcriptional transitions during the development of drug resistance. Cell lines resuming full proliferation (FACs [fast-adapting cells]) and cells that became senescent (SACs [slow-adapting cells]) over prolonged treatment were identified. The early drug response was characterized by transitional states involving increased ion signaling, driven by upregulation of the ATP-gated ion channel P2RX7. P2RX7 activation was associated with improved therapy responses and, in combination with targeted drugs, could contribute to the delayed onset of acquired resistance in NRAS-mutant melanoma.

MiRNAs from serum-derived extracellular vesicles as biomarkers for uveal melanoma progression.

Uveal melanoma (UM) is a rare type of malignancy that originates from melanocytes in the choroid, iris and the eye's ciliary body. Biomarkers for early detection and progression of UM, especially the molecular traits governing the development of metastasis, are still not available in clinical practice. One extensively studied components of liquid biopsies are extracellular vesicles. Due to their unique molecular cargo, they can contribute to early cancer development and at the same time carry markers for disease onset and progression. For characterisation of the miRNA profiles present in circulating serum-derived exosomes of patients with diagnosed primary and metastatic UM, we have analyzed the miRNA cargos using next-generation sequencing followed by RT-qPCR validation in a cohort of patients (control n = 20; primary n = 9; metastatic n = 11). Nine miRNAs differentiating these patient groups have been established. We show that hsa-miR-144-5p and hsa-miR-191-5p are the most promising biomarker candidates, allowing the categorization of patients into local and advanced UM. Additionally, the comparison of miRNA expression levels in exosomes derived from UM patients with those derived from healthy donors revealed that hsa-miR-191-5p, -223-3p, -483-5p, -203a has the potential to be used as an early marker for the presence of UM. This pilot study reveals that miRNAs extracted from circulating exosomes could be exploited as potential biomarkers in UM diagnosis and, more importantly, for indicating metastatic spread.

The Analysis of Inflammation-Related Proteins in a Cargo of Exosomes Derived from the Serum of Uveal Melanoma Patients Reveals Potential Biomarkers of Disease Progression.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumour in adults with a poor prognosis and extremely high mortality rate due to the development of metastatic disease. However, despite relatively good knowledge about the histological and genetic risk factors for metastasis development, there is no specific biomarker that would allow early detection of UM progression. Recently, exosomes and their molecular cargo have been widely studied in the search for potential biomarkers in several cancers. The purpose of this study was to analyze the inflammation-related protein cargo of exosomes derived from the serum of primary and metastatic UM patients and healthy donors. METHODS: The exosomes were isolated from the serum of primary and metastatic UM patients and healthy donors. Using multiplex immunoassay technology, we analyzed the concentration of 37 inflammation-related proteins in obtained exosomes. RESULTS: The analysis of protein cargo showed several molecules related to inflammation, such as interferon-gamma, interleukin 2, 22 and 12(p40), Pentraxin-3, TNFSF13B and TNFSF8 which were significantly enriched in metastatic UM exosomes. We showed a significant correlation between the disease stage and the concentration of these inflammation-related proteins from exosomal cargo. CONCLUSIONS: Based on the obtained results, we propose the panel of exosomal proteins for early detection of uveal melanoma progression into metastatic disease.

The Potential Role of Selected miRNA in Uveal Melanoma Primary Tumors as Early Biomarkers of Disease Progression.

Uveal melanoma (UM) is the most common primary tumor of the eye diagnosed in adults, associated with a high risk of metastasis and thereby, poor prognosis. Among known risk factors for the development of metastatic disease is the loss of BAP1 expression and chromosome 3 monosomy in the primary tumor. However, the expression levels of specific micro RNAs (miRNA) in tumor tissue may also serve as a valuable marker for determining the risk of metastatic disease in patients with primary uveal melanoma. In our study, we analyzed the miRNA expression data of cases selected from The Cancer Genome Atlas study on uveal melanoma, and determined a panel of 15 miRNAs differentially expressed between patients with primary and metastatic disease. Next, 6 miRNAs were validated on a group of 46 tumor samples from primary and metastatic patients. We have shown, that expression of hsa-miR-592, hsa-miR-346, and hsa-miR-1247 was significantly increased, while hsa-miR-506 and hsa-miR-513c were decreased in the tumors of patients with metastatic disease. Hsa-miR-196b expression did not differ between the two subgroups, however, we showed significant correlation with BAP1 expression. Moreover, hsa-miR-592 also showed correlation with monosomy 3 tumors. Gene ontology analysis revealed involvement of those miRNAs with cellular processes mediating the metastatic process. Our results showed that miRNAs play an important role in the deregulation of several oncogenic pathways in UM and can, thereby, promote metastatic spread to distant organs. Moreover, differentially expressed miRNAs may be used as an interesting biomarker for the assessment of metastatic risk in uveal melanoma patients.

Surgical Wound Fluids from Patients with Breast Cancer Reveal Similarities in the Biological Response Induced by Intraoperative Radiation Therapy and the Radiation-Induced Bystander Effect-Transcriptomic Approach.

In patients with breast cancer who undergo breast-conserving surgery (BCS), more than 90% of local recurrences occur in the same quadrant as the primary cancer. Surgical wound fluids (SWF) are believed to play a role in this process by inducing an inflammatory process in the scar tissue area. Despite strong clinical data demonstrating the benefits of intraoperative radiotherapy (IORT), the biological basis underlying this process remains poorly understood. Ionizing radiation (IR) directly affects cells by damaging DNA, thereby altering the cell phenotype. IR directly affects cancer cells and also influences unirradiated cells located nearby, a phenomenon known as the radiation-induced bystander effect (RIBE), significantly modifying the tumor microenvironment. We hypothesized that SWF obtained from patients after BCS and IORT would induce a radiobiological response (due to RIBE) in unirradiated cells, thereby modifying their phenotype. To confirm this hypothesis, breast cancer cells were incubated with SWF collected from patients after BCS: (1) without IORT (wound fluid (WF) group), (2) with IORT (radiotherapy wound fluid (RT-WF) group), and (3) WF with conditioned medium from irradiated cells (WF+RIBE group) and then subjected to microarray analysis. We performed gene set enrichment analysis to determine the biological processes present in these cells. This analysis showed that the RT-WF and WF+RIBE groups shared common biological processes, including the enhancement of processes involved in cell-cycle regulation, DNA repair, and oxidative phosphorylation. The WF group was characterized by overrepresentation of pathways involved in the INF-α and INF-γ response, inflammatory response, and the IL6 JAK/STAT3 signaling pathway. These findings show that MDA-MB-468 cells stimulated with surgical wound fluids obtained from patients who underwent BCS plus IORT and from cells stimulated with SWF plus RIBE share common biological processes. This confirms the role of the radiation-induced bystander effect in altering the biological properties of wound fluids.

The Composition of Surgical Wound Fluids from Breast Cancer Patients is Affected by Intraoperative Radiotherapy Treatment and Depends on the Molecular Subtype of Breast Cancer.

Invasive oncological procedures affect the remaining tumor cells by increasing their survival, proliferation, and migration through the induction of wound healing response. The phenomena of local relapse after breast-conserving surgery (BCS) has resulted in a series of research and clinical trials with the aim of assessing whether localized intraoperative radiotherapy (IORT), may be beneficial in inhibiting local recurrences. Therefore, it is essential to assess the impact of intraoperative radiotherapy in modulating the immunological response and wound healing process. Thus, we decided to perform a quantitative analysis of the composition of surgical wound fluids (SWF) in two groups of breast cancer (BC) patients: those treated with BCS followed by IORT, and those who underwent BCS alone. We found that several cytokines, which are believed to have anti-tumor properties, were highly expressed in the luminal A breast cancer subtype in the IORT treatment group. Interestingly, we also found significant differences between IORT patients with tumors of different molecular subtypes. Based on these findings, we hypothesized that IORT treatment might be beneficial in changing the tumor bed microenvironment, making it less favorable for tumor recurrence due to decreased concentration of tumor-facilitating cytokines, especially in the luminal A subtype of BC.