Non-neoplastic Cells Research Articles

E. Coli cytotoxic necrotizing factor-1 promotes colorectal carcinogenesis by causing oxidative stress, DNA damage and intestinal permeability alteration

BackgroundBacterial toxins are emerging as promising hallmarks of colorectal cancer (CRC) pathogenesis. In particular, Cytotoxic Necrotizing Factor 1 (CNF1) from E. coli deserves special consideration due to the significantly higher prevalence of this toxin gene in CRC patients with respect to healthy subjects, and to the numerous tumor-promoting effects that have been ascribed to the toxin in vitro. Despite this evidence, a definitive causal link between CNF1 and CRC was missing. Here we investigated whether CNF1 plays an active role in CRC onset by analyzing pro-carcinogenic key effects specifically induced by the toxin in vitro and in vivo.MethodsViability assays, confocal microscopy of γH2AX and 53BP1 molecules and cytogenetic analysis were carried out to assess CNF1-induced genotoxicity on non-neoplastic intestinal epithelial cells. Caco-2 monolayers and 3D Caco-2 spheroids were used to evaluate permeability alterations specifically induced by CNF1, either in the presence or in the absence of inflammation. In vivo, an inflammatory bowel disease (IBD) model was exploited to evaluate the carcinogenic potential of CNF1. Immunohistochemistry and immunofluorescence stainings of formalin-fixed paraffin-embedded (FFPE) colon tissue were carried out as well as fecal microbiota composition analysis by 16 S rRNA gene sequencing.ResultsCNF1 induces the release of reactive oxidizing species and chromosomal instability in non-neoplastic intestinal epithelial cells. In addition, CNF1 modifies intestinal permeability by directly altering tight junctions’ distribution in 2D Caco-2 monolayers, and by hindering the differentiation of 3D Caco-2 spheroids with an irregular arrangement of these junctions. In vivo, repeated intrarectal administration of CNF1 induces the formation of dysplastic aberrant crypt foci (ACF), and produces the formation of colorectal adenomas in an IBD model. These effects are accompanied by the increased neutrophilic infiltration in colonic tissue, by a mixed pro-inflammatory and anti-inflammatory cytokine milieu, and by the pro-tumoral modulation of the fecal microbiota.ConclusionsTaken together, our results support the hypothesis that the CNF1 toxin from E. coli plays an active role in colorectal carcinogenesis. Altogether, these findings not only add new knowledge to the contribution of bacterial toxins to CRC, but also pave the way to the implementation of current screening programs and preventive strategies.

Histopathologic Comparison Among Drug Eruptions Induced by Enfortumab Vedotin, Brentuximab Vedotin, and Taxanes.

Microtubule-stabilizing agents (enfortumab vedotin and brentuximab vedotin) and microtubule-disrupting agents (docetaxel and paclitaxel) are used as anticancer agents but can also induce drug eruptions. Recently, mitotic arrest figures have been reported in various non-neoplastic cells as the histopathologic side effect of these drug eruptions. Therefore, we performed a comparative analysis of drug eruptions associated with these microtubule-targeting agents. Enfortumab vedotin-, brentuximab vedotin-, docetaxel-, and paclitaxel-associated drug eruptions were retrieved from 4 hospitals in 5, 5, 5, and 7 patients, respectively. Ring mitotic and other mitotic arrest figures were observed in the epidermis in all types of drug eruption but were most frequently (100%) observed in enfortumab vedotin-induced eruptions. Such a finding was also occasionally observed in the sweat ductoglandular units but not in the follicular epithelium. Keratinocyte multinucleation and apoptotic keratinocytes distributed predominantly in the upper part of the epidermis were also observed in these eruptions, particularly in enfortumab vedotin-induced eruptions (4/5, 80%). In conclusion, drug eruptions associated with microtubule-targeting agents, particularly enfortumab vedotin, can often exhibit mitotic arrest figures, keratinocyte multinucleation, and apoptotic keratinocytes predominantly observed in the upper part of the epidermis. These characteristic histopathologic features can be the diagnostic clues of drug eruptions induced by microtubule-targeting agents.

Detection of genome instability by 53BP1 expression as a long-lasting health effect in human epidermis surrounding radiation-induced skin cancers.

We previously reported endogenous activation of the DNA damage response (DDR) in the epidermis surrounding basal cell carcinoma resected from Nagasaki atomic bomb survivors, suggesting the presence of genomic instability (GIN) in the survivors as a late effect of radiation. Dual-color immunofluorescence (IF) analysis of TP53-binding protein-1 (53BP1) and a proliferative indicator, Ki-67, to elucidate GIN in tumor tissues revealed that abnormal 53BP1 expression is closely associated with carcinogenesis in several organs. The present study aimed to confirm the presence of radiation-induced GIN in the non-neoplastic epidermis of patients with radiation-induced skin cancer. Formalin-fixed paraffin-embedded tissues were obtained from all participants between 2008 and 2019 at the Nagasaki University Hospital. 53BP1 nuclear expression was examined using dual-color IF analysis with Ki-67 expression to assess the extent and integrity of the DDR. Expressions of gamma-H2AX, p53 and p21 were also analyzed using the dual-color IF analysis for their association with 53BP1. The results of this study provide evidence for sporadic activation of the DDR in medically irradiated and ultraviolet-exposed epidermis as a long-lasting radiation effect, which is a predisposition to skin cancer. Furthermore, the incidence of abnormal 53BP1 expression in cancer cells was higher than in non-neoplastic epidermal cells surrounding cancer, suggesting a correlation between the type of 53BP1 and the malignant potential of skin tumors. This study highlights the usefulness of dual-color IF for 53BP1 (and Ki-67) as an indicator to estimate the level of GIN as a long-lasting health effect of radiation exposure.

Expression of αv Integrin in Feline Injection-Site Sarcoma (FISS): Preliminary Investigations.

Feline injection-site sarcomas (FISSs) are malignant skin tumors of mesenchymal origin arising at local post-vaccination (or injection) sites. In recent years, a fluorescence imaging technique based on probes targeting αvβ3 integrin has been effectively applied for the surgical complete resection of the tumor. In our study, we investigated the utility of a commercially available anti-αv integrin polyclonal antibody for the histopathological evaluation of FISS's surgical excision margins. We collected 10 formalin-fixed paraffin-embedded (FFPE) feline excisional biopsies with a histopathological diagnosis of FISS (7 fibrosarcomas and 3 pleomorphic sarcomas) and wide margin tissue, along with one subcutaneous injection-site granuloma and 6 osteosarcomas. Samples were processed for histology, and slides were stained for IHC with the anti-αv integrin antibody. Immunostained slides were evaluated for the cellular localization and intensity of the staining in different neoplastic and non-neoplastic cell populations. Neoplastic and non-neoplastic spindle cells had cytoplasmic positivity in all fibrosarcomas. Inflammatory cells, including macrophages of the injection-site granuloma, were negative. Multinucleated giant cells in the pleomorphic sarcomas had an intense membranous positivity. Although the anti-αv integrin antibody was ineffective for the histopathological evaluation of surgical excision margins, the membranous localization of αv integrin in multinucleated giant cells of pleomorphic sarcomas suggests that it plays a role in the oncogenesis of this FISS variant.

REST-dependent glioma progression occurs independently of the repression of the long non-coding RNA HAR1A.

The long non-coding RNA (lncRNA), HAR1A is emerging as a putative tumour suppressor. In non-neoplastic brain cells, REST suppresses HAR1A expression. In gliomas REST acts as an oncogene and is a potential therapeutic target. It is therefore conceivable that REST promotes glioma progression by down-regulating HAR1A. To test this hypothesis, glioma clinical databases were analysed to study: (I) HAR1A/REST correlation; (II) HAR1A and REST prognostic role; (III) molecular pathways associated with these genes. HAR1A expression and subcellular localization were studied in glioblastoma and paediatric glioma cells. REST function was also studied in these cells, by observing the effects of gene silencing on: (I) HAR1A expression; (II) cancer cell proliferation, apoptosis, migration; (III) expression of neural differentiation genes. The same phenotypes (and cell morphology) were studied in HAR1A overexpressing cells. Our results show that REST and HAR1A are negatively correlated in gliomas. Higher REST expression predicts worse prognosis in low-grade gliomas (the opposite is true for HAR1A). REST-silencing induces HAR1A upregulation. HAR1A is primarily detected in the nucleus. REST-silencing dramatically reduces cell proliferation and induces apoptosis, but HAR1A overexpression has no major effect on investigated cell phenotypes. We also show that REST regulates the expression of neural differentiation genes and that its oncogenic function is primarily HAR1A-independent.

Downregulated Regucalcin Expression Induces a Cancer-like Phenotype in Non-Neoplastic Prostate Cells and Augments the Aggressiveness of Prostate Cancer Cells: Interplay with the G Protein-Coupled Oestrogen Receptor?

Regucalcin (RGN) is a calcium-binding protein and an oestrogen target gene, which has been shown to play essential roles beyond calcium homeostasis. Decreased RGN expression was identified in several cancers, including prostate cancer (PCa). However, it is unknown if the loss of RGN is a cause or a consequence of malignancy. Also, it needs confirmation if RGN oestrogenic regulation occurs through the G-protein-coupled oestrogen receptor (GPER). This study investigates how RGN knockdown affects prostate cell fate and metabolism and highlights the GPER/RGN interplay in PCa. Bioinformatic analysis assessed the relationship between RGN expression levels and patients' outcomes. RGN knockdown (siRNA) was performed in non-neoplastic prostate and castration-resistant PCa. Wild-type and RGN knockdown PCa cells were treated with the GPER agonist G1. Viability (MTT), proliferation (Ki-67 immunocytochemistry), apoptosis (caspase-3-like activity) and migration (Transwell assays) were evaluated. Spectrophotometric analysis was used to determine glucose consumption, lactate production and lactate dehydrogenase activity. Lipid content was assessed using the Oil Red assay. Bioinformatic analysis showed that the loss of RGN correlates with the development of metastatic PCa and poor survival outcomes. RGN knockdown induced a cancer-like phenotype in PNT1A cells, indicated by increased cell viability and proliferation and reduced apoptosis. In DU145 PCa cells, RGN knockdown augmented migration and enhanced the glycolytic profile, which indicates increased aggressiveness, in line with patients' data. GPER activation modulated RGN expression in PCa cells and RGN knockdown in DU145 cells influenced GPER actions, which highlighted an interplay between these molecular players with relevance for their potential use as biomarkers or therapeutic targets.

EPCO-51. A MULTI-OMIC ANALYSIS OF REGIONAL HETEROGENEITY IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults. A hallmark of GBM is its intratumoral heterogeneity as well as infiltration into the surrounding brain. The growing understanding of the cellular diversity and cellular state diversity within GBM necessitates a need for a more granular evaluation of the molecular landscape of this disease. This study aimed to investigate this using single cell RNA sequencing combined with single-cell ATAC sequencing and spatial transcriptomics in order to delineate cellular states and enriched pathways between malignant cells in different regions of GBM. The study cohort consisted of 15 patients with primary GBM. Tumor samples were taken at the time of surgical resection using intraoperative stereotactic navigation from three anatomically distinct locations: periphery – the cortex or white matter in the peri-tumoral region that is beyond contrast-enhancing tumor, border – the contrast-enhancing border of the tumor on T1-weighted MRI, and core - the hypointense core region on T1-weighted MRI. Samples underwent combined snRNA and ATAC sequencing (10x Multiome) as well as spatial transcriptomics (10x Visium). Single-nucleus RNA + ATAC sequencing captured a total of 37,547 neoplastic cells and 18,498 non-neoplastic cells within the tumor microenvironment. The majority of non-neoplastic cells were identified within the periphery alongside a distinct group of neoplastic cells that exhibited unique a unique distribution of malignant cellular state and differentially regulated genetic pathways compared to malignant cells within the tumor border and core. Gene regulatory analysis of the malignant cells in the periphery identified a previously undefined set of enriched transcription factors that are periphery-specific regulon drivers. Overall, this analysis sheds light on the genetic and epigenetic differences between malignant GBM cells based on their location and warrant further investigation into the biology and targetable aspects of the malignant cells that exist beyond the contrast-enhancing border of the tumor.

CNSC-43. NEUROTRANSMITTER DEPENDENCY UNDERLIES TUMOR GROWTH IN HUMANIZED PEDIATRIC LOW-GRADE GLIOMA MODELS

Abstract Brain tumors arise in close association with neurons, suggesting that these non-neoplastic cells may be critical stromal drivers of brain tumor initiation and growth. Previously, we have shown that murine low-grade optic glioma formation and progression in the setting of Neurofibromatosis type 1 (NF1) is dictated by neurons and neuronal activity. In these studies, these neuronal dependencies reflected neuronal activity-driven enzymatic cleavage of a growth factor (neuroligin-3) from oligodendrocyte precursors cells (tumor initiation) or neuronal production of a paracrine factor to stimulate T cell support of optic glioma growth (tumor progression). Since neurons typically communicate with other neurons through neurotransmitters, we sought to explore the possibility that neurotransmitters operate to modulate low-grade glioma growth using humanized mouse models of pilocytic astrocytoma (PA). Leveraging single cell RNA sequencing of three independent sets of pediatric PAs, we identified neurotransmitter pathway enrichment in the tumor cells. This neurotransmitter pathway enrichment reflected aberrant expression of specific neurotransmitter receptors, which we confirmed in three independently generated PA tissue microarrays and in five distinct primary PA cell lines grown in vitro. Moreover, this aberrant neurotransmitter receptor expression established differential neurotransmitter PA growth dependencies in vitro. Importantly, interruption of neurotransmitter signaling in human PA xenografts attenuated tumor growth and ERK activation in Rag1-/- mice in vivo. Finally, we discovered crosstalk between neurotransmitter receptor and receptor tyrosine kinase signaling that revealed another target for therapeutic inhibition. Taken together, these findings elucidate a previously unknown neurotransmitter PA growth dependency amenable to therapeutic targeting.

TMIC-69. HUMAN ORGANOID TUMOR TRANSPLANTATION IDENTIFIES FUNCTIONAL GLIOBLASTOMA MICROENVIRONMENTAL COMMUNICATION MEDIATED BY PTPRZ1

Abstract Glioblastoma, the most aggressive form of primary brain cancer, is driven by both intrinsic cellular properties and extrinsic factors from the tumor microenvironment (TME). Here, we leverage our novel human organoid tumor transplantation (HOTT) system to explore how external cues modulate glioblastoma cell type specification, heterogeneity, and migration. Comparing the single-cell transcriptomes of HOTT and matched primary tumors, we found that HOTT recapitulates not only core features of major patient tumor cell types but also those of peritumor non-neoplastic cell types. This enables us to explore tumor-TME interactions and identify PTPRZ1, a receptor tyrosine phosphatase implicated in brain tumor migration, as a key mediator in intercellular communication. This finding was further confirmed by interrogation of published datasets from patients containing non-neoplastic microenvironmental cells. PTPRZ1 activation, resulting from the reactivation of developmental programs in both the tumor and its TME, highlights the relevance of the organoid system for modeling the GBM microenvironment. Moreover, HOTT uniquely allows for perturbations in either patient tumors or their microenvironments, or both, to verify PTPRZ1 function. Tumor knockdown of PTPRZ1 confirmed its role in promoting tumor migration and maintaining progenitor identity. Surprisingly, environmental PTPRZ1 knockdown in human cortical organoids before tumor transplantation inhibited tumor migration and promoted differentiation, even without direct tumor manipulation. Overall, this fundamental result demonstrates that a single treatment delivered to the tumor and its TME can exert opposite effects on the tumor. Consequently, it underscores the necessity of studying human glioblastoma within a human microenvironment context, such as HOTT, especially when evaluating therapeutic efficacy in GBM, as effective treatments must address both the tumor and its complex TME, rather than the tumor alone.

EPCO-07. INTEGRATING SPATIALLY RESOLVED MULTI-OMICS DATA TO UNCOVER DYSFUNCTIONAL METABOLISM DRIVEN NETWORKS THAT ENHANCE INFILTRATION OF DIFFUSE GLIOMAS

Abstract BACKGROUND Diffuse infiltration is an aggressive feature of high-grade gliomas with survival implications. The contribution of crosstalk between non-neoplastic and neoplastic cells to tumor infiltration remains largely understudied due to the lack of profiling techniques that retain spatial information. Spatial multi-omic profiling is a promising approach to comprehensively analyze transcript-omics, prote-omics and metabol-omics on the same tissue section while preserving information about the spatial organization of cells. Integration of these spatial studies allows for inferring the consequences of complex cell-cell communication underlying tumor infiltration. We hypothesize that the glioma edge is enriched with pro-infiltration ligands-receptors driving tumor infiltration. Strategies that disrupt these ligand-receptor networks may suppress glioma infiltration and improve clinical outcomes. METHODS We utilized a glioma tissue micro-array (TMA) to establish the neoplastic and non-neoplastic heterogeneity in the GBM infiltration edge. Each TMA slide consists of pathologist annotated tumor core and edge samples of Glioblastoma (IDH Wildtype, WHO Grade 4; n=10), Diffuse Astrocytoma (IDH Mutant, WHO Grade 3; n=3), Oligodendroglioma (IDH mutant, WHO Grade 2; n=5) and 2 non-brain control samples. We performed spatial multi-omics profiling on adjacent sections of the TMA using 10x Xenium spatial transcriptomics, imaging mass cytometry (IMC) and mass spectrometry imaging (MSI). Integration, visualization, and quantification of the spatial data was done on VisioPharm. RESULTS In GBM, we identified candidate mRNA transcripts and proteins for ligands enriched at the infiltrating edge (compared to tumor core; p<0.0001) that correlated with a poor progression free survival (PFS; r2=0.22). These candidate ligands were also significantly enriched at the edge of oligodendroglioma WHO Grade 2 and astrocytoma WHO Grade 3. CONCLUSIONS Spatial multi-omics profiling on a TMA consisting of Glioma WHO grades 2-4 identified differentially expressed and targetable pro-tumor infiltration ligands associated with lower PFS in GBM. Functional studies to uncover the role of metabolites enriched at the glioma edge for the expression of the identified pro-infiltration ligands are ongoing.

STEM-19. TARGETINGGIHCG LNCRNA IN GLIOBLASTOMA STEM CELLS: A POTENTIAL THERAPEUTIC STRATEGY

Abstract Glioblastoma multiforme (GBM) remains the most lethal and prevalent primary brain tumor worldwide. Despite advances in understanding its biology and multimodal therapy options, the overall prognosis for GBM patients remains bleak, primarily due to the high recurrence rate, which is intimately linked to tumor resistance against standard therapeutic interventions. Glioblastoma stem cells (GSCs) contribute to therapeutic resistance and cellular heterogeneity through their self-renewal properties and ability to adapt. Thus, identifying new molecular targets driving GBM progression is crucial for developing effective therapies. Recent advancements in genome biology have revealed that long non-coding RNAs (lncRNAs) play roles in various biological functions, and the dysregulation of numerous lncRNAs has been shown to be associated with specific cancers. To identify GSC-associated lncRNAs, a comprehensive analysis was performed using single-cell RNA sequencing datasets from GBM tumors, GBM organoids, GSC-enriched GBM tumors, as well as normal human brain samples. A chromatin-enriched lncRNA, GIHCG, was identified as being highly expressed in GSCs compared to non-neoplastic neural stem cells. The GIHCG gene, located on human chromosome 12q14.1, is frequently amplified in glioblastoma and impacts overall survival. Bioinformatic and functional analyses revealed that GIHCG is amplified in 13% of GBM patient samples, and high expression of GIHCG is a negative prognostic marker for GBM patients, with significantly reduced overall survival compared to the low expression group. In addition, the depletion of GIHCG significantly reduced GSC proliferation and migration. Further, the knockdown of GIHCG decreased both mRNA and protein expression levels of critical GSC stemness factors, including SOX2, NESTIN, and OLIG2. Moreover, GIHCG depletion reduced the sphere formation capacity of GSCs, demonstrating the functional importance of GIHCG in the maintenance of GSC stemness. These results indicate that GIHCG is essential for GSC proliferation, migration, and stemness, underscoring its potential therapeutic value in RNA-based therapies for combating glioblastoma malignancy.

TMIC-06. TUMOR ASSOCIATED MACROPHAGES DRIVE TUMOR PROGRESSION AND ARE TRANSCRIPTIONALLY SHAPED BY HISTONE MUTATIONS IN DIFFUSE MIDLINE GLIOMA

Abstract Little progress has been made to delineate the inflammatory microenvironment in pediatric high-grade glioma (pHGG) and diffuse midline glioma (DMG). We utilize molecularly defined human samples and immunocompetent mouse models to study how tumor location and genetic driver mutations influence the tumor microenvironment (TME). We first induced tumors of each pHGG/DMG molecular subtype (H3WT, H3G34R, H3.1K27M, H3.3K27M) into both the cortical hemisphere and midline of separate mice and performed survival studies and flow cytometry. The main driving factor shaping the immune landscape and survival is the driver mutation, not tumor location. Next, we performed single cell RNA sequencing on murine H3WT cortical pHGG, H3WT DMG, H3.1 and H3.3K27M DMGs, paired with multiplex flow cytometry. H3K27M DMGs have sparse T cell infiltration and limited T cell recruiting chemokine production. The main non-neoplastic cell type in all tumors was infiltrating myeloid cells and microglia. H3WT DMGs had the greatest myeloid cell and monocyte-derived macrophage (MDM) infiltration, while H3.3K27M DMGs were enriched for microglia. Disease associated gene signatures were found in tumor-associated macrophages (TAMs) that are found in other neurodegenerative diseases, marked by down-regulation of inflammatory signaling, interferon responses, and up-regulation of metabolism-related pathways. H3.3K27M DMGs were enriched for disease-associated TAMs. These gene signatures were then identified in human pHGG/DMG scRNA sequencing data. CD16 monocytes and microglia were enriched for diseased signatures, while CD14 monocytes were pro-inflammatory. We demonstrate that reprogramming this diseased signature in mice results in increased T cell infiltration and extension of survival in H3K27M DMG. Last, through pharmacologic inhibition of CCR1 and CCR5, we demonstrate preventing TAM infiltration in DMG results in enhanced survival, comparable to radiation therapy. Together, this work provides the foundation for developing immunotherapies targeted at specific subgroups of pHGG and DMGs, such as CAR-T-cell, oncolytic viral therapy, and checkpoint blockade.

Effect of Diosgenin in Suppressing Viability and Promoting Apoptosis of Human Prostate Cancer Cells: An Interplay with the G Protein-Coupled Oestrogen Receptor?

Diosgenin is a phytosteroid sapogenin with reported antitumoral activity. Despite the evidence indicating a lower incidence of prostate cancer (PCa) associated with a higher consumption of phytosteroids and the beneficial role of these compounds, only a few studies have investigated the effects of diosgenin in PCa, and its mechanisms of action remain to be disclosed. The present study investigated the effect of diosgenin in modulating PCa cell fate and glycolytic metabolism and explored its potential interplay with G protein-coupled oestrogen receptor (GPER). Non-neoplastic (PNT1A) and neoplastic (LNCaP, DU145, and PC3) human prostate cell lines were stimulated with diosgenin in the presence or absence of the GPER agonist G1 and upon GPER knockdown. Diosgenin decreased the cell viability, as indicated by the MTT assay results, which also demonstrated that castrate-resistant PCa cells were the most sensitive to treatment (PC3 > DU145 > LNCaP > PNT1A; IC50 values of 14.02, 23.21, 56.12, and 66.10 µM, respectively). Apoptosis was enhanced in diosgenin-treated cells, based on the increased caspase-3-like activity, underpinned by the altered expression of apoptosis regulators evaluated by Western blot analysis, which indicated the activation of the extrinsic pathway. Exposure to diosgenin also altered glucose metabolism. Overall, the effects of diosgenin were potentiated in the presence of G1. Moreover, diosgenin treatment augmented GPER expression, and the knockdown of the GPER gene suppressed the proapoptotic effects of diosgenin in PC3 cells. Our results support the antitumorigenic role of diosgenin and its interest in PCa therapy, alone or in combination with G1, mainly targeting the more aggressive stages of the disease.

Concordance in the estimation of tumor percentage in non-small cell lung cancer using digital pathology

The incorporation of digital pathology in clinical practice will require the training of pathologists in digital skills. Our study aimed to assess the reliability among pathologists in determining tumor percentage in whole slide images (WSI) of non-small cell lung cancer (NSCLC) using digital image analysis, and study how the results correlate with the molecular findings. Pathologists from nine centers were trained to quantify epithelial tumor cells, tumor-associated stromal cells, and non-neoplastic cells from NSCLC WSI using QuPath. Then, we conducted two consecutive ring trials. In the first trial, analyzing four WSI, reliability between pathologists in the assessment of tumor cell percentage was poor (intraclass correlation coefficient (ICC) 0.09). After performing the first ring trial pathologists received feedback. The second trial, comprising 10 WSI with paired next-generation sequencing results, also showed poor reliability (ICC 0.24). Cases near the recommended 20% visual threshold for molecular techniques exhibited higher values with digital analysis. In the second ring trial reliability slightly improved and human errors were reduced from 5.6% to 1.25%. Most discrepancies arose from subjective tasks, such as the annotation process, suggesting potential improvement with future artificial intelligence solutions.

Microglia and monocyte-derived macrophages drive progression of pediatric high-grade gliomas and are transcriptionally shaped by histone mutations

Pediatric high-grade gliomas (pHGGs), including hemispheric pHGGs and diffuse midline gliomas (DMGs), harbor mutually exclusive tumor location-specific histone mutations. Using immunocompetent de novo mouse models of pHGGs, we demonstrated that myeloid cells were the predominant infiltrating non-neoplastic cell population. Single-cell RNA sequencing (scRNA-seq), flow cytometry, and immunohistochemistry illustrated the presence of heterogeneous myeloid cell populations shaped by histone mutations and tumor location. Disease-associated myeloid (DAM) cell phenotypes demonstrating immune permissive characteristics were identified in murine and human pHGG samples. H3.3K27M DMGs, the most aggressive DMG, demonstrated enrichment of DAMs. Genetic ablation of chemokines Ccl8 and Ccl12 resulted in a reduction of DAMs and an increase in lymphocyte infiltration, leading to increased survival of tumor-bearing mice. Pharmacologic inhibition of chemokine receptors CCR1 and CCR5 resulted in extended survival and decreased myeloid cell infiltration. This work establishes the tumor-promoting role of myeloid cells in DMG and the potential therapeutic opportunities for targeting them.

Comparison of the effects of photodynamic exposure with the use of chlorine E6 on glioblastoma cells of the U251 line and human embryonic kidney cells of the HEK293 line in vitro

Malignant gliomas of the brain are a global medical and social problem with a trend toward a steady increase in morbidity and mortality rates. A method that enables the visual identification of tumor tissue and simultaneously selectively destroys it is photodynamic therapy, which involves the introduction of a photosensitizer (PS) followed by its activation at a certain wavelength of light. The selectivity of the accumulation of PS in the tumor tissue of the malignant gliomas is one of the key issues in the problem of increasing the effectiveness of photodynamic therapy. Objective: to compare the effects of photodynamic exposure using PS chlorin E6 on human glioblastoma (GB) cells of the U251 line and non-malignant human embryonic kidney cells of the HEK293 line. Material and methods. Groups of cell cultures were formed depending on the conditions of cultivation and exogenous influence: 1) control - cultivated in a standard nutrient medium (Modified Eagle's Medium (MEM)) with L-glutamine, 1 mmol of sodium pyruvate, 10% fetal bovine serum) and experimental: 2) cultivated under the conditions of adding chlorin E6 (concentrations 1.0 and 2.0 μg/ml); 3) cultivated on a nutrient medium without the addition of PS and exposed to laser irradiation (LI) (λ=660 nm, power in the range 0.4-0.6 W, dose in the range 10-75 J/cm2, continuous or pulse mode); 4) cultured under conditions of chlorin E6 addition and subsequent exposure to LI (power in the range 0.4-0.6 W, dose in the range 10-75 J/cm2, continuous or pulse mode). After exposure to the specified experimental factors, dynamic observation with microphotographic registration was performed for 24 h, followed by microscopic and micrometric studies (number of viable cells, total number of cells, mitotic index (MI,%)). Results. PS chlorin E6 is incorporated into the cytoplasm of cells of U251 and HEK293 cell lines, the intensity of fluorescence is comparable. Upon exposure to chlorin E6 (1.0 and 2.0 μg/ml), cytodestructive and antimitotic effects are increased in a dose-dependent manner in the culture of human GB cells of the U251 line. The cytodestructive effect of chlorin E6 on cell cultures of the HEK293 line is less pronounced, but the antimitotic effect is comparable in both types of cell cultures. Under the influence of LI, cytodestructive and antimitotic effects increase in a dose-dependent manner in the culture of human GB cells of the U251 line. The level of cytodestructive and antimitotic effects is significantly lower in the cultures of non-neoplastic HEK293 cells. The most significant drop in the mitotic activity of GB U251 cells (~100%) was recorded at the lowest LI dose of 25 J/cm2, power of 0.6 W in pulse mode. For HEK293 cells, the most significant decrease in mitotic activity (~80%) was recorded at LI with a power of 0.6 W and dose of 75 J/cm2 in continuous mode. Under the combined effect of chlorin E6 (1 and 2 μg/ml, pre-incubation of 4 h) and LI in different modes, the viability of tumor cells in U251 culture decreases in a dose-dependent manner; the smallest dose of LI to achieve the maximum cytotoxic effect is 25 J/cm2, with a power of 0.6 W in pulse mode when using chlorin E6 at a concentration of 2 μg/ml. The specified characteristics of photodynamic exposure do not cause irreversible effects in HEK293 cultures (reference cells). Conclusions. An effective mode of photodynamic exposure to achieve a cytodestructive and antimitotic effect in the culture of human GB cells of the U251 line, which is relatively safe for non-malignant cells, has been established: the combined application of a laser irradiation dose of 25 J/cm2, with a power of 0.6 W in pulse mode during the preliminary incubation of the cell culture with chlorin E6 at a concentration of 2 μg/ml for 4 h.

Unveiling the Inflammatory Landscape of Recurrent Glioblastoma through Histological-Based Assessments.

The glioblastoma (GBM) tumor microenvironment consists of a heterogeneous mixture of neoplastic and non-neoplastic cells, including immune cells. Tumor recurrence following standard-of-care therapy results in a rich landscape of inflammatory cells throughout the glioma-infiltrated cortex. Immune cells consisting of glioma-associated macrophages and microglia (GAMMs) overwhelmingly constitute the bulk of the recurrent glioblastoma (rGBM) microenvironment, in comparison to the highly cellular and proliferative tumor microenvironment characteristic of primary GBM. These immune cells dynamically interact within the tumor microenvironment and can contribute to disease progression and therapy resistance while also providing novel targets for emerging immunotherapies. Within these varying contexts, histological-based assessments of immune cells in rGBM, including immunohistochemistry (IHC) and immunofluorescence (IF), offer a critical way to visualize and examine the inflammatory landscape. Here, we exhaustively review the available body of literature on the inflammatory landscape in rGBM as identified through histological-based assessments. We highlight the heterogeneity of immune cells throughout the glioma-infiltrated cortex with a focus on microglia and macrophages, drawing insights from canonical and novel immune-cell histological markers to estimate cell phenotypes and function. Lastly, we discuss opportunities for immunomodulatory treatments aiming to harness the inflammatory landscape in rGBM.

Somatic MED12 Mutations in Myometrial Cells.

Over 70% of leiomyoma (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131 (GG). Myometrial cells are the cell origin of leiomyoma, but the MED12 mutation status in non-neoplastic myometrial cells is unknown. In this study, we investigated the mutation burden of MED12 in myometrium. As traditional Sanger or even NGS sequencing may not be able to detect MED12 mutations that are lower than 0.1% in the testing sample, we used duplex deep sequencing analysis (DDS) to overcome this limitation. Tumor-free myometria (confirmed by pathology evaluation) were dissected, and genomic DNA from MED12 exon 2 (test) and TP53 exon 5 (control) were captured by customer-designed probe sets, followed by DDS. Notably, DDS demonstrated that myometrial cells harbored a high frequency of mutations in MED12 exon 2 and predominantly in code c.130-131. In contrast, the baseline mutations in other coding sequences of MED12 exon 2 as well as in the TP53 mutation hotspot, c.477-488 were comparably low in myometrial cells. This is the first report demonstrating a non-random accumulation of MED12 mutations at c.130-131 sites in non-neoplastic myometrial cells which provide molecular evidence of early somatic mutation events in myometrial cells. This early mutation may contribute to the cell origin for uterine LM development in women of reproductive age.

Sodium diethyldithiocarbamate trihydrate: an effective and selective compound for hematological malignancies.

Myeloid leukemias and lymphomas are among the most common and well-studied hematological malignancies. However, due to the aggressiveness and rapid progression of certain subtypes, treating these diseases remains a challenge. Considering the promising results of diethyldithiocarbamates in preclinical and clinical oncology trials, this study aimed to investigate the potential of sodium diethyldithiocarbamate trihydrate (DETC) as a prototype for developing new drugs to treat hematological malignancies. In silico analysis using SwissADME was conducted to evaluate the physicochemical characteristics and pharmacokinetic properties of DETC. An in vitro investigation utilizing the MTT assay assessed the cytotoxic effects of DETC on neoplastic and non-neoplastic cell lines. Selectivity was determined using a selectivity index and a hemolysis assay, while the mechanism of cell death in neoplastic cell lines was examined through flow cytometry analysis of pro-apoptotic and anti-apoptotic protein levels. The results demonstrated that the physicochemical characteristics of DETC are suitable for oral administration. Furthermore, the compound showed promising cytotoxic activity against human myeloid leukemia (K562) and Burkitt's lymphoma (Daudi) cell lines, with high selectivity for neoplastic cells over non-neoplastic cells of the bone marrow microenvironment (HS-5 cell line). Moreover, hemolysis was observed only at very high concentrations. The cytotoxicity mechanism of DETC against both neoplastic cell lines involved cell cycle arrest and the production of reactive oxygen species. In K562 cells, cell death was induced via apoptosis. Additional experiments are needed to confirm the exact mechanism of cell death in Daudi Burkitt's lymphoma cells.

Anticancer Activity of Benzo[a]phenoxazine Compounds Promoting Lysosomal Dysfunction.

Specific cancer therapy remains a problem to be solved. Breast and colorectal cancer are among the cancers with the highest prevalence and mortality rates. Although there are some therapeutic options, there are still few effective agents for those cancers, which constitutes a clinical problem that requires further research efforts. Lysosomes play an important role in cancer cells' survival, and targeting lysosomes has gained increased interest. In recent years, our team has been synthetizing and testing novel benzo[a]phenoxazine derivatives, as they have been shown to possess potent pharmacological activities. Here, we investigated the anticancer activity of three of the most potent derivatives from our library, C9, A36, and A42, on colorectal- and breast-cancer-derived cell lines, and compared this with the effect on non-neoplastic cell lines. We observed that the three compounds were selective for the cancer cells, namely the RKO colorectal cancer cell line and the MCF7 breast cancer cell line. In both models, the compounds reduced cell proliferation, cell survival, and cell migration, accumulated on the lysosome, and induced cell death accompanied by lysosomal membrane permeabilization (LMP), increasing the intracellular pH and ROS accumulation. Our results demonstrated that these compounds specifically target lysosomes from cancer cells, making them promising candidates as LMP inducers for cancer therapy.