Protocol to quantify immune cell distribution from the vasculature to the glioma microenvironment on sequential immunofluorescence multiplex images.

Although myeloid-derived immune cells can be dispersed throughout the tumor microenvironment (TME), anti-tumor effector cells are confined to the perivascular space. Here, we present a protocol to quantify immune cell distribution from tumor vasculature to its glioma microenvironment on sequential immunofluorescence multiplex images. We describe steps for sequential immunofluorescence multiplex staining, image generation, and storage. We then detail the procedures for tissue, vessel, and nuclei segmentation; cell phenotyping; data extraction; and training using RStudio and Spyder.

Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context. The increasing number of biomarkers analyzed, together with whole tissue imaging, generate datasets approaching several hundreds of gigabytes in size, which are rich sources of valuable knowledge but require investments in infrastructure and resources for extracting quantitative information. The analysis of multiplex whole-tissue images requires extensive training and experience in data analysis. Here, we showcase how a set of open-source tools can allow semi-automated image data extraction to study the spatial composition of tissues with a focus on tumor microenvironment (TME). With the use of Lunaphore COMET platform, we interrogated lung cancer specimens where we examined the expression of 20 biomarkers. Subsequently, the tissue composition was interrogated using an in-house optimized nuclei detection algorithm followed by a newly developed image artifact exclusion approach. Thereafter, the data was processed using several publicly available tools, highlighting the compatibility of COMET-derived data with currently available image analysis frameworks. In summary, we showcased an innovative semi-automated workflow that highlights the ease of adoption of multiplex imaging to explore TME composition at single-cell resolution using a simple slide in, data out approach. Our workflow is easily transferrable to various cohorts of specimens to provide a toolset for spatial cellular dissection of the tissue composition.

Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

Ophthalmic applicator displacement as a method of treating large diffuse uveal melanomas.

Purpose: The presentation of results of an ophthalmic plaque displacement as a brachytherapy treatment method of large diffuse uveal melanomas. Material and methods: This was a retrospective analysis of treatment results of 9 patients with large diffuse uveal melanomas using ophthalmic plaque displacement. Patients were treated with this method in our center between 2012 and 2021 (last follow-up visit in 2023). To achieve appropriate radiation dose distribution for large tumors with a base greater than 18 mm, brachytherapy (106Ru in 7 patients and 125I in 2 patients) with applicator displacement was used as primary treatment. Median follow-up was 2.9 years, and for patients with positive primary treatment results, it was 1.7 months. Median time to local relapse was 2.3 years. Results: In 5 patients, a positive result of local treatment was obtained, out of whom, one patient underwent enucleation due to complications. In the next 4 cases, local recurrence developed. In all tumors, the use of applicator displacement method caused that planning target volume (PTV) was effectively covered with treatment isodose. Conclusions: Brachytherapy with ocular applicator displacement allows for the treatment of tumors with base measurements larger than 18 mm. The application of this method may be considered as an alternative for eye enucleation in particular cases of large diffuse tumors, such as a neoplasm of the eye with vison, or when a patient does not consent to enucleation.

Cranial imaging window implantation technique for longitudinal multimodal imaging of the brain environment in live mice.

Intravital two-photon microscopy of the mouse brain requires visual access without affecting normal cognitive functions, which is crucial for longitudinal imaging studies that may last several months. In this protocol, we describe the surgical implantation of a metal-free cranial imaging window, which can be used to perform two-photon microscopy and magnetic resonance imaging in the same animal. This multimodal imaging platform enables the investigation of dynamic processes in the central nervous system at a cellular and macroscopic level. For complete details on the use and execution of this protocol in the context of brain cancer, please refer to Zomer et al.1.

Treatment Patterns, Testing Practices, and Outcomes in Patients with EGFR Mutation-Positive Advanced Non-Small-Cell Lung Cancer in Poland: A Descriptive Analysis of National, Multicenter, Real-World Data from the REFLECT Study.

Non-small-cell lung cancer (NSCLC) represents 85% of new cases of lung cancer. Over the past two decades, treatment of patients with NSCLC has evolved from the empiric use of chemotherapy to more advanced targeted therapy dedicated to patients with an epidermal growth factor receptor (EGFR) mutation. The multinational REFLECT study analyzed treatment patterns, outcomes, and testing practices among patients with EGFR-mutated advanced NSCLC receiving first-line EGFR tyrosine kinase inhibitor (TKI) therapy across Europe and Israel. The aim of this study is to describe the Polish population of patients from the REFLECT study, focusing on treatment patterns and T790M mutation testing practice. A descriptive, retrospective, non-interventional, medical record-based analysis was performed on the Polish population of patients with locally advanced or metastatic NSCLC with EGFR mutations from the REFLECT study (NCT04031898). A medical chart review with data collection was conducted from May to December 2019.The study involved 110 patients. Afatinib was used as the first-line EGFR-TKI therapy in 45 (40.9%) patients, erlotinib in 41 (37.3%), and gefitinib in 24 (21.8%) patients. The first-line EGFR-TKI therapy was discontinued in 90 (81.8%) patients. The median progression-free survival (PFS) on first-line EGFR-TKI therapy was 12.9 months (95% CI 10.3-15.4). A total of 54 patients started second-line therapy, of whom osimertinib was administered to 31 (57.4%). Among 85 patients progressing on first-line EGFR-TKI therapy, 58 (68.2%) were tested for the T790M mutation. Positive results for the T790M mutation were obtained from 31 (53.4%) tested patients, all of whom received osimertinib in the next lines of therapy. The median overall survival (OS) from the start of first-line EGFR-TKI therapy was 26.2 months (95% CI 18.0-29.7). Among patients with brain metastases, the median OS from the first diagnosis of brain metastases was 15.5 months (95% CI 9.9-18.0). The results of the Polish population from the REFLECT study highlight the need for effective treatment of patients with advanced EGFR-mutated NSCLC. Nearly one-third of patients with disease progression after first-line EGFR-TKI therapy were not tested for the T790M mutation and did not have the opportunity to receive effective treatment. The presence of brain metastases was a negative prognostic factor.

Multimodal imaging of the dynamic brain tumor microenvironment during glioblastoma progression and in response to treatment.

Tumors evolve in a dynamic communication with their native tissue environment and recruited immune cells. The diverse components of the tumor microenvironment (TME) can critically regulate tumor progression and therapeutic response. In turn, anticancer treatments may alter the composition and functions of the TME. To investigate this continuous dialog in the context of primary brain cancers, we developed a multimodal longitudinal imaging strategy. We combined macroscopical magnetic resonance imaging with subcellular resolution two-photon intravital microscopy, and leveraged the power of single-cell analysis tools to gain insights into the ongoing interactions between different components of the TME and cancer cells. Our experiments revealed that the migratory behavior of tumor-associated macrophages is different in genetically distinct glioblastomas, and in response to macrophage-targeted therapy. These results underscore the importance of studying cancer longitudinally in an in vivo setting, to reveal complex and dynamic alterations in the TME during disease progression and therapeutic intervention.

Brachytherapy or enucleation in ring melanoma patients: which is better? Preliminary results of the authors' own experiences.

PURPOSE: A retrospective evaluation of effectiveness of brachytherapy or enucleation in treatment of rare form of uveal melanoma: ring melanoma. MATERIAL AND METHODS: Analysis comprised 49 patients treated from 2000 to 2019 for ring melanoma involving ciliary body. In 15 patients, primary treatment consisted of brachytherapy (106Ru or 125I), whereas in 34 patients, eyeballs were enucleated. The evaluation concerned differences between analyzed groups relating to the clinical and histopathological features as well as overall survival, cancer-specific overall survival, and disease-free survival. RESULTS: No significant differences between the analyzed groups were found with regards to clinical and histopathological features, apart from intra-ocular pressure (increased in the enucleation group). Kaplan-Meier analysis did not reveal any significant differences between the group treated with enucleation and the group undergoing brachytherapy, both with regards to overall survival (p = 0.325) and cancer-specific overall survival (p = 0.477). A significant difference was observed in disease-free survival (p = 0.009), which was significantly shorter in the group undergoing brachytherapy. In the analysis of parameters of the applied brachytherapy, no significant differences between patients with and without local recurrence were found. Mean observation period was 350.8 weeks (range, 24-996 weeks, SD = 231.6). A local recurrence occurred in 11 (22.4%) patients, including 3 (6.1%) in enucleation and 8 (16.3%) after brachytherapy groups. Metastasis developed in 11 (22.4%) cases after a mean follow-up of 133 weeks (33.25 months), range 3-655 weeks. CONCLUSIONS: Preliminary observations may suggest that brachytherapy in this rare form of uveal melanoma, such as ring melanoma involving the ciliary body, may be taken into consideration as a useful alternative to enucleation. However, the confirmation of such an approach requires a larger patients' group to be gathered, and also a longer follow-up period. This is especially important in patients with a good baseline visual acuity in the affected eye, or when the neoplasm is present in the remaining eye with vision.

Diagnostic Algorithm for Conjunctival Melanocytic Lesions.

BACKGROUND/AIM: This study aimed to develop an algorithm allowing the differentiation between conjunctival melanoma and other melanocytic infiltrations of the conjunctiva, on the basis of a dermatoscopic examination. PATIENTS AND METHODS: A total of 160 conjunctival pigmented lesions were studied (40 melanomas and 120 non-melanoma conjunctival infiltrations). The clinical characteristics of the tumours were assessed with the use of dermatoscopic characteristics as described by Kittler, and with taking into consideration the typical characteristics of conjunctival lesions. RESULTS: On the basis of modified dermatoscopic criteria, an algorithm was generated consisting of an assessment of the presence of 9 suspicious characteristics, e.g. more than two colours, colour asymmetry, pattern asymmetry, vascular polymorphism, presence of short vessels, linear vascular pattern, the presence of a peripheral structureless area, the presence of a grey structureless area and black dots in any part of the lesion. The presence of any of these characteristics scores 1 point. If a melanocytic lesion scores ≥3 points, the probability of diagnosing melanoma is on the level of p>0.001. CONCLUSION: The use of the proposed algorithm, based on modified dermatoscopic characteristics, may be a valuable tool for the diagnosis of conjunctival melanoma.

Choroidal metastases of thymic carcinoma in ultrasonographic imaging and optical coherence tomography.

Not required for Clinical Vignette.

The Evaluation of the Malignant Characteristics of Conjunctival Lesions Based on the Dermatoscopic Algorithm.

BACKGROUND/AIM: This study analysed the prevalence of the characteristics evaluated in dermatoscopy for melanocytic infiltrations of the conjunctiva with various degrees of malignancy. PATIENTS AND METHODS: A total of 160 conjunctival pigmented lesions were studied. Each lesion was scored using dermatoscopic patterns and the characteristics of malignancy described by Kittler. Also, the Authors' own clues were added to the evaluation. RESULTS: In melanomas, the following characteristics were identified: asymmetry of the pattern and colour, larger average number of colours, the presence of grey colour, structureless area, polymorphic vessels and feeder vessels. A pattern of black dots and a black colour was typical of malignant lesions and pre-cancerous (premalignant) lesions - primary acquired melanosis (PAM) with atypia. Cysts were observed only in the group of naevi. CONCLUSION: The patterns evaluated with dermatoscopy are present in pigmented lesions of the conjunctiva. There are, however, some characteristics which allow differentiation between melanoma and pigmented naevus and melanosis and also between PAM.

Dendritic cell extracellular vesicles.

In addition to direct cell-to-cell contact, dendritic cells (DCs) can regulate the onset of adaptive immunity through the secretion of nano-sized membrane structures, called extracellular vesicles (EVs). This novel mode of communication between cells has added a new layer of complexity to the regulation of immune responses. DCs secrete into their environment different types of EVs containing immunomodulatory molecules that have distinct structural and biochemical properties depending on their intracellular site of origin. Exosomes are generated inside multivesicular bodies and are secreted when these compartments fuse with the plasma membrane, whereas microvesicles are formed and released by budding from the cells' plasma membrane. Once outside the cell of origin, these vesicles can reach target cells through membrane receptor-ligand interactions, modifying their physiological state by the transfer of the EV content or by triggering cell signaling at the cells' surface. Particularly, EVs released by DCs contain major histocompatibility complex (MHC) class I and class II molecules able to activate cognate T cells and promote humoral responses. These activities motivated the use of DC-derived EVs in the treatment of cancer, infectious diseases and autoimmune disorders. The therapeutic potential of these vesicles led to the use of EVs from tumor antigen-loaded DCs in cancer clinical trials, although with limited clinical effects. In this review we will focus on the different EVs released by DCs, their composition and biogenesis, together with their proposed functions as immune regulators.

Re-education of macrophages as a therapeutic strategy in cancer.

Tumor-associated macrophages (TAMs) can be educated within the tumor microenvironment to promote cancer development and progression. While TAM-targeted agents have largely focused on macrophage depletion as an anticancer strategy, it is becoming increasingly evident that TAM re-education may represent a more effective approach. In this perspective, we discuss different means to achieve TAM re-education, and review the beneficial effects of these strategies, particularly when combined with immune checkpoint inhibitors.

Quality of Life in Patients with Advanced Non-Small-Cell Lung Cancer Receiving Palliative Chemotherapy.

The goal of this study was to explore quality of life in patients with advanced non-small-cell lung cancer (NSCLC) in an attempt to single out features that could help predict the possibility of non-completion of chemotherapy. The survey tool was the Quality of Life Questionnaire Core-30 (QLQ-C30) with the module Lung Cancer 13 (LC-13) developed by the European Organization for Research and Treatment of Cancer. The assessment of quality of life (QoL) was performed in 58 patients with advanced NSCLC before palliative chemotherapy and it was repeated in 43 patients who completed at least three cycles of chemotherapy. We found that the patients who failed to complete the chemotherapy course distinctly showed, in contradistinction to those who completed it, poor physical functioning in (67.6 ± 16.3 vs. 78.3 ± 21.3 points, respectively, p < 0.05) and the lack of appetite (27.1 ± 38.0 vs. 48.9 ± 37.5 points, respectively p < 0.05). At the end of palliative chemotherapy alopecia, sore throat, and constipation significantly worsened QoL, but global health status remained unchanged. In conclusion, poor physical functioning and loss of appetite seem to harbinger a risk of non-completion of chemotherapy in advanced NSCLC.

Analysis of local recurrence causes in uveal melanoma patients treated with 125I brachytherapy - a single institution study.

PURPOSE: To perform a retrospective analysis of factors which might affect the occurrence of a relapse of uveal melanoma after 125I brachytherapy. MATERIAL AND METHODS: The analysis concerned 343 patients treated in the years 2001-2012. The effect on local recurrence of such factors as patient's sex, age, tumour size, shape, pigmentation, location, presence of orange pigment or petechiae on tumour surface, retinal detachment, and blood or dispersed pigment in vitreous body were studied. Additional analysis concerned physical properties of brachytherapy (total dose, irradiation dose applied to tumour apex and base and irradiation time). Two groups of patients were distinguished: with and without a relapse. The diagnostic criterion for the relapse was growth of the tumour base or height by 0.5 mm. RESULTS: Local recurrence of the uveal melanoma was observed in 29 patients (8.5%). Recurrences occurred with significantly higher frequency (p < 0.001), when the anterior tumour edge involved the ciliary body. Patients' survival in relation to the moment the occurrence of the relapse was statistically significant for application time (p = 0.004) and tumour pigmentation (p = 0.010). The deaths of patients with a local relapse were most rare when brachytherapy lasted from 72 to 95.9 hours and most frequent in cases of brownish tumour pigmentation. Patient sex, tumour shape and size, presence of orange pigment, retinal detachment, petechiae and bleeding to vitreous body as well as the dose of irradiation to tumour top and base did not have any significant effect on relapse occurrence. CONCLUSIONS: Treatment of uveal melanomas with 125I applicators allows for a high rate of positive local results. Nonetheless, the recurrence probability always exists. The involvement of the ciliary body could influence this. The survival depending on the time of relapse could be statistically significant for application time and dark-brown tumour pigmentation.

Why the need and how to approach the functional diversity of extracellular vesicles.

In the past decade, cell-to-cell communication mediated by exosomes has attracted growing attention from biomedical scientists and physicians, leading to several recent publications in top-tier journals. Exosomes are generally defined as secreted membrane vesicles, or extracellular vesicles (EVs), corresponding to the intraluminal vesicles of late endosomal compartments, which are secreted upon fusion of multi-vesicular endosomes with the cell's plasma membrane. Cells, however, were shown to release other types of EVs, for instance, by direct budding off their plasma membrane. Some of these EVs share with exosomes major biophysical and biochemical characteristics, such as size, density and membrane orientation, which impose difficulties in their efficient separation. Despite frequent claims in the literature, whether exosomes really display more important patho/physiological functions, or are endowed with higher potential as diagnostic or therapeutic tools than other EVs, is not yet convincingly demonstrated. In this opinion article, we describe reasons for this lack of precision knowledge in the current stage of the EV field, we review recently described approaches to overcome these caveats, and we propose ways to improve our knowledge on the respective functions of distinct EVs, which will be crucial for future development of well-designed EV-based clinical applications.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

Outcomes of I-125 brachytherapy for uveal melanomas depending on irradiation dose applied to the tumor apex - a single institution study.

PURPOSE: The aim of the study was an evaluation of I-125 brachytherapy patients with uveal melanoma with special consideration for the relationship of the treatment results and the irradiation dose applied to the tumor apex. MATERIAL AND METHODS: Medical records of 344 adults with uveal melanoma treated with I-125 brachytherapy in the Department of Ophthalmology and Ocular Oncology of the Jagiellonian University, Medical College in Cracow, Poland were retrospectively analyzed. The study was conducted between 2003 and 2012, and the study group was divided into two subgroups depending on the irradiation dose applied to the top of the tumor: 80 Gy to 100 Gy (n = 177) and 100 Gy to 120 Gy (n = 167). RESULTS: It was found that the height of the tumor and the largest diameter of the tumor base decreased with every consecutive follow-up measurement and differed significantly in all comparisons (p < 0.0001). No significant correlation between frequency of complications was found between both study groups (χ2 = 0.27; p = 0.6067). The correlation between survival and the irradiation dose as applied to the tumor top was statistically irrelevant (χ2 = 0.44; p = 0.5084). A logistic regression model showed that patient survival depended on the largest diameter of the base and the height of tumor (p = 0.0216), and the risk of death was larger as these dimensions increased (IR, 1.17). An increase of the largest diameter of the base by 1 mm meant a 17% increase in chances of death. In 13.4% of cases, an enucleation was necessary. CONCLUSIONS: The treatment of choroidal melanomas with I-125 iodine isotope brachytherapy is an efficient and recommended method of treatment and in many cases, an alternative to the enucleation of an eyeball.

Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes.

Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles.

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.