Exploring Regorafenib Responsiveness and Uncovering Molecular Mechanisms in Recurrent Glioblastoma Tumors through Longitudinal In Vitro Sampling.

Glioblastoma, a deadly brain tumor, shows limited response to standard therapies like temozolomide (TMZ). Recent findings from the REGOMA trial underscore a significant survival improvement offered by Regorafenib (REGO) in recurrent glioblastoma. Our study aimed to propose a 3D ex vivo drug response precision medicine approach to investigate recurrent glioblastoma sensitivity to REGO and elucidate the underlying molecular mechanisms involved in tumor resistance or responsiveness to treatment. Three-dimensional glioblastoma organoids (GB-EXPs) obtained from 18 patients' resected recurrent glioblastoma tumors were treated with TMZ and REGO. Drug responses were evaluated using NAD(P)H FLIM, stratifying tumors as responders (Resp) or non-responders (NRs). Whole-exome sequencing was performed on 16 tissue samples, and whole-transcriptome analysis on 13 GB-EXPs treated and untreated. We found 35% (n = 9) and 77% (n = 20) of tumors responded to TMZ and REGO, respectively, with no instances of TMZ-Resp being REGO-NRs. Exome analysis revealed a unique mutational profile in REGO-Resp tumors compared to NR tumors. Transcriptome analysis identified distinct expression patterns in Resp and NR tumors, impacting Rho GTPase and NOTCH signaling, known to be involved in drug response. In conclusion, recurrent glioblastoma tumors were more responsive to REGO compared to TMZ treatment. Importantly, our approach enables a comprehensive longitudinal exploration of the molecular changes induced by treatment, unveiling promising biomarkers indicative of drug response.

Preclinical glioma models in neuro-oncology: enhancing translational research.

PURPOSE OF REVIEW: Gliomas represent approximately 25% of all primary brain and other central nervous system (CNS) tumors and 81% of malignant tumors. Unfortunately, standard treatment approaches for most CNS cancers have shown limited improvement in patient survival rates. RECENT FINDINGS: The current drug development process has been plagued by high failure rates, leading to a shift towards human disease models in biomedical research. Unfortunately, suitable preclinical models for brain tumors have been lacking, hampering our understanding of tumor initiation processes and the discovery of effective treatments. In this review, we will explore the diverse preclinical models employed in neuro-oncology research and their contributions to translational science. SUMMARY: By utilizing a combination of these preclinical models and fostering interdisciplinary collaborations, researchers can deepen their understanding of glioma brain tumors and develop novel therapeutic strategies to combat these devastating diseases. These models offer promising prospects for personalized and efficacious treatments for these challenging malignancies. Although it is unrealistic to fully replicate the complexity of the human body in vitro, the ultimate goal should be to achieve the closest possible resemblance to the clinical context.

Identification of New Potential Prognostic and Predictive Markers in High-Grade Osteosarcoma Using Whole Exome Sequencing.

Conventional high-grade osteosarcoma (OS) is the most common primary cancer of bone and it typically affects the extremities of adolescents. OS has a complex karyotype, and molecular mechanisms related to carcinogenesis, progression and resistance to therapy are still largely unknown. For this reason, the current standard of care is associated with considerable adverse effects. In this study, our aim was to identify gene alterations in OS patients using whole exome sequencing (WES) to find new potential prognostic biomarkers and therapeutic targets. We performed WES on formalin-fixed paraffin-embedded (FFPE) biopsy materials collected from 19 patients affected by conventional high-grade OS. The clinical and genetic data were analyzed according to response to therapy, presence of metastasis and disease status. By comparing good and poor responders to neoadjuvant therapy, we detected a clear prevalence of mutations in the ARID1A, CREBBP, BRCA2 and RAD50 genes in poor responders that negatively influence the progression-free survival time. Moreover, higher tumor mutational burden values correlated with worse prognosis. The identification of mutations in ARID1A, CREBBP, BRCA2 and RAD50 may support the use of a more specific therapy for tumors harboring these alterations. In particular, BRCA2 and RAD50 are involved in homologous recombination repair, and could thus be used as specific therapy targets of inhibitors of the enzyme Poly ADP Ribose Polymerase (PARP). Finally, tumor mutational burden is found to be a potential prognostic marker for OS.

Innovative Approach to Isolate and Characterize Glioblastoma Circulating Tumor Cells and Correlation with Tumor Mutational Status.

Circulating tumor cells (CTCs) are one of the most important causes of tumor recurrence and distant metastases. Glioblastoma (GBM) has been considered restricted to the brain for many years. Nevertheless, in the past years, several pieces of evidence indicate that hematogenous dissemination is a reality, and this is also in the caseof GBM. Our aim was to optimize CTCs' detection in GBM and define the genetic background of single CTCs compared to the primary GBM tumor and its recurrence to demonstrate that CTCs are indeed derived from the parental tumor. We collected blood samples from a recurrent IDH wt GBM patient. We genotyped the parental recurrent tumor tissue and the respective primary GBM tissue. CTCs were analyzed using the DEPArray system. CTCs Copy Number Alterations (CNAs) and sequencing analyses were performed to compare CTCs' genetic background with the same patient's primary and recurrent GBM tissues. We identified 210 common mutations in the primary and recurrent tumors. Among these, three somatic high-frequency mutations (in PRKCB, TBX1, and COG5 genes) were selected to investigate their presence in CTCs. Almost all sorted CTCs (9/13) had at least one of the mutations tested. The presence of TERT promoter mutations was also investigated and C228T variation was found in parental tumors and CTCs (C228T heterozygous and homozygous, respectively). We were able to isolate and genotype CTCs from a patient with GBM. We found common mutations but also exclusive molecular characteristics.

Metabolic-imaging of human glioblastoma live tumors: A new precision-medicine approach to predict tumor treatment response early.

Background: Glioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy remain the primary therapeutic options for GB, and no new therapies have been introduced in recent years. This therapeutic standstill is primarily due to preclinical approaches that do not fully respect the complexity of GB cell biology and fail to test efficiently anti-cancer treatments. Therefore, better treatment screening approaches are needed. In this study, we have developed a novel functional precision medicine approach to test the response to anticancer treatments in organoids derived from the resected tumors of glioblastoma patients. Methods: GB organoids were grown for a short period of time to prevent any genetic and morphological evolution and divergence from the tumor of origin. We chose metabolic imaging by NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to predict early and non-invasively ex-vivo anti-cancer treatment responses of GB organoids. TMZ was used as the benchmark drug to validate the approach. Whole-transcriptome and whole-exome analyses were performed to characterize tumor cases stratification. Results: Our functional precision medicine approach was completed within one week after surgery and two groups of TMZ Responder and Non-Responder tumors were identified. FLIM-based metabolic tumor stratification was well reflected at the molecular level, confirming the validity of our approach, highlighting also new target genes associated with TMZ treatment and identifying a new 17-gene molecular signature associated with survival. The number of MGMT gene promoter methylated tumors was higher in the responsive group, as expected, however, some non-methylated tumor cases turned out to be nevertheless responsive to TMZ, suggesting that our procedure could be synergistic with the classical MGMT methylation biomarker. Conclusions: For the first time, FLIM-based metabolic imaging was used on live glioblastoma organoids. Unlike other approaches, ex-vivo patient-tailored drug response is performed at an early stage of tumor culturing with no animal involvement and with minimal tampering with the original tumor cytoarchitecture. This functional precision medicine approach can be exploited in a range of clinical and laboratory settings to improve the clinical management of GB patients and implemented on other cancers as well.

Sedoheptulose Kinase SHPK Expression in Glioblastoma: Emerging Role of the Nonoxidative Pentose Phosphate Pathway in Tumor Proliferation.

Glioblastoma (GBM) is the most common form of malignant brain cancer and is considered the deadliest human cancer. Because of poor outcomes in this disease, there is an urgent need for progress in understanding the molecular mechanisms of GBM therapeutic resistance, as well as novel and innovative therapies for cancer prevention and treatment. The pentose phosphate pathway (PPP) is a metabolic pathway complementary to glycolysis, and several PPP enzymes have already been demonstrated as potential targets in cancer therapy. In this work, we aimed to evaluate the role of sedoheptulose kinase (SHPK), a key regulator of carbon flux that catalyzes the phosphorylation of sedoheptulose in the nonoxidative arm of the PPP. SHPK expression was investigated in patients with GBM using microarray data. SHPK was also overexpressed in GBM cells, and functional studies were conducted. SHPK expression in GBM shows a significant correlation with histology, prognosis, and survival. In particular, its increased expression is associated with a worse prognosis. Furthermore, its overexpression in GBM cells confirms an increase in cell proliferation. This work highlights for the first time the importance of SHPK in GBM for tumor progression and proposes this enzyme and the nonoxidative PPP as possible therapeutic targets.

Single-Cell Molecular Characterization to Partition the Human Glioblastoma Tumor Microenvironment Genetic Background.

BACKGROUND: Glioblastoma (GB) is a devastating primary brain malignancy. The recurrence of GB is inevitable despite the standard treatment of surgery, chemotherapy, and radiation, and the median survival is limited to around 15 months. The barriers to treatment include the complex interactions among the different cellular components inhabiting the tumor microenvironment. The complex heterogeneous nature of GB cells is helped by the local inflammatory tumor microenvironment, which mostly induces tumor aggressiveness and drug resistance. METHODS: By using fluorescent multiple labeling and a DEPArray cell separator, we recovered several single cells or groups of single cells from populations of different origins from IDH-WT GB samples. From each GB sample, we collected astrocytes-like (GFAP+), microglia-like (IBA1+), stem-like cells (CD133+), and endothelial-like cells (CD105+) and performed Copy Number Aberration (CNA) analysis with a low sequencing depth. The same tumors were subjected to a bulk CNA analysis. RESULTS: The tumor partition in its single components allowed single-cell molecular subtyping which revealed new aspects of the GB altered genetic background. CONCLUSIONS: Nowadays, single-cell approaches are leading to a new understanding of GB physiology and disease. Moreover, single-cell CNAs resource will permit new insights into genome heterogeneity, mutational processes, and clonal evolution in malignant tissues.

Dietary Patterns and Weight Status in Italian Preschoolers with Autism Spectrum Disorder and Typically Developing Children.

Atypical eating habits are more common in children with autism spectrum disorders (ASD) than typically developing (TD) peers. Feeding problems may lead to the double burden of specific nutrient deficiencies and excessive weight gain, with a consequent increase in obesity prevalence. The dietary intake of Italian preschoolers with ASD compared to their TD peers and the impact of their dietary choices on their weight status and relationship to food selectivity (FS) were investigated. Dietary patterns and their associations with body mass index (BMI) were evaluated in 65 children with ASD and 82 peers with TD aged 1.3-6.4 years. Eating habits were assessed with a modified version of a parent-rated semi-quantitative Food Frequency Questionnaire. Moreover, the prevalence of FS and possible links with dietary patterns and BMI were investigated in the ASD group. Children with ASD consumed significantly higher amounts of simple sugars, processed and ultra-processed carbohydrates, both low- and high-fat animal proteins, and lower amounts of vegetables and fruits compared to peers with TD. The obesity rate was 1.5% in children with TD and more than fourfold (6.2%) in children with ASD, although the difference between groups was not statistically significant. FS was significantly more frequent in children with ASD than in peers with TD. Children with ASD and FS showed significantly lower annual intakes of vegetable proteins and fiber (considered essential nutrients for a healthy diet) than children with ASD without FS. Our results showed that children with ASD showed different dietary habits than those with TD, with the higher consumption of energy-dense foods and lower amounts of food-sourced fibers, which could place them at increased risk to develop overweight, obesity, and micronutrient deficiencies later in life.

Analysis of exosome-derived microRNAs reveals insights of intercellular communication during invasion of breast, prostate and glioblastoma cancer cells.

MiRNAs represent a mechanism that regulates gene expression in many pathological conditions. Exosomes are known to be secreted from all types of cells, and the exosomes-released molecules are crucial messengers that can regulate cellular processes. We investigated the miRNAs content of exosomes released by cancer cells during the invasion . An invasion stimulus has been generated through scratches created on the confluent cells of cancer cell lines: glioblastoma, breast and prostate cancers.Several miRNAs were found to be significantly differentially abundant during the cell invasion , both in common among different cell lines and exclusive. Understanding the language codes among cells involved in invasion can lead to the development of therapies that can inhibit cellular communication, slowing or eventually stopping their activity.

Multiregional Sequencing of IDH-WT Glioblastoma Reveals High Genetic Heterogeneity and a Dynamic Evolutionary History.

Glioblastoma is one of the most common and lethal primary neoplasms of the brain. Patient survival has not improved significantly over the past three decades and the patient median survival is just over one year. Tumor heterogeneity is thought to be a major determinant of therapeutic failure and a major reason for poor overall survival. This work aims to comprehensively define intra- and inter-tumor heterogeneity by mapping the genomic and mutational landscape of multiple areas of three primary IDH wild-type (IDH-WT) glioblastomas. Using whole exome sequencing, we explored how copy number variation, chromosomal and single loci amplifications/deletions, and mutational burden are spatially distributed across nine different tumor regions. The results show that all tumors exhibit a different signature despite the same diagnosis. Above all, a high inter-tumor heterogeneity emerges. The evolutionary dynamics of all identified mutations within each region underline the questionable value of a single biopsy and thus the therapeutic approach for the patient. Multiregional collection and subsequent sequencing are essential to try to address the clinical challenge of precision medicine. Especially in glioblastoma, this approach could provide powerful support to pathologists and oncologists in evaluating the diagnosis and defining the best treatment option.

Gastrointestinal symptoms and behavioral problems in preschoolers with Autism Spectrum Disorder.

BACKGROUND: Gastrointestinal (GI) symptoms are frequently reported in children with Autism Spectrum Disorder (ASD), and an impact of GI comorbidity on ASD behavioral problems has been hypothesized. AIMS: To explore the type and the prevalence of GI symptoms in ASD patients and typical development (TD) controls, and to investigate their possible association with behavioral problems. METHODS: A total of 230 preschoolers were included in this study. Specifically, four groups of children were evaluated: ASD individuals suffering from GI symptoms (ASD/GI+), ASD subjects without GI symptoms (ASD/GI-), TD peers with (TD/GI+) and without (TD/GI-) GI symptoms. Parental report of behavioral problems and GI symptoms were assessed through the Child Behavior Check List 1½-5. RESULTS: A significant higher percentage of ASD (37.4%) versus TD (14.8%) with GI symptoms was observed. 'Constipated' and 'Not-Eat' were the most frequent GI symptoms both in ASD and in TD groups, but they were evaluated as more severe in ASD patients. ASD/GI+ children had more anxiety problems, somatic complaints, externalizing and total problems than ASD/GI- individuals. TD/GI+ did not show more behavioral problems than TD/GI-. CONCLUSION: Development of evidence-based guidelines for identification of GI problems in ASD preschoolers is warranted. GI symptomatology should be accurately assessed, especially in ASD children with anxiety and/or externalizing behavioral problems.

Ectopic fat: the true culprit linking obesity and cardiovascular disease?

Obesity is a major risk factor for cardiovascular disease and its complications. However, not all fat depots share the same characteristics. Recent studies have found that ectopic rather than subcutaneous fat accumulation is associated with increased cardiometabolic risk. However, ectopic fat accumulation can be seen initially as a protective mechanism against lipotoxicity. Subsequently the adipose tissue becomes dysfunctional, thus inducing systemic metabolic alterations (through release of cytokines) or specific organ dysfunctions. The purpose of this review is to summarise the current available data on the impact of excess adiposity vs ectopic fat in the development of cardio-metabolic diseases.

Non-alcoholic fatty liver disease (NAFLD) and its connection with insulin resistance, dyslipidemia, atherosclerosis and coronary heart disease.

Non-alcoholic fatty liver disease is marked by hepatic fat accumulation not due to alcohol abuse. Several studies have demonstrated that NAFLD is associated with insulin resistance leading to a resistance in the antilipolytic effect of insulin in the adipose tissue with an increase of free fatty acids (FFAs). The increase of FFAs induces mitochondrial dysfunction and development of lipotoxicity. Moreover, in subjects with NAFLD, ectopic fat also accumulates as cardiac and pancreatic fat. In this review we analyzed the mechanisms that relate NAFLD with metabolic syndrome and dyslipidemia and its association with the development and progression of cardiovascular disease.

Loss of heterozygosity status of D9S105 marker is associated with downregulation of Krüppel-like factor 4 expression in pancreatic ductal adenocarcinoma and pancreatic intraepithelial lesions.

The transcription factor Krüppel-like factor 4 (KLF4) may act both as an oncogene and a tumor suppressor in a tissue-dependent manner, and further studies on its role in pancreatic ductal adenocarcinoma (PDAC) progression and clinical outcome are warranted. Therefore, we investigated the loss of heterozygosity (LOH) in the 9q22.3-32 region and loss of KFL4 gene expression in epithelial cells from 35 PDAC, 6 pancreatic intraductal neoplasias (PanINs) and 6 normal ducts, isolated by laser microdissection, as well as their correlation with overall survival (OS) in patients treated with gemcitabine in the adjuvant setting. LOH was evaluated with 4 microsatellite markers and in situ hybridization, while KLF4 expression was studied by reverse transcription-PCR and immunohistochemistry. LOH in at least 1 locus was observed in 25 of 35 PDAC cases and in 5 of 6 PanINs, respectively. In particular, the loss of the D9S105 marker was present in 46.9% of PDAC and 83.3% of PanINs, becoming the most deleted marker, while no LOH in D9S105 was observed in normal Wirsung pancreatic duct. Lack of KLF4 mRNA expression was significantly associated with: (1) genomic deletion flanking KLF4 in PDAC and in PanINs (with LOH of D9S105), (2) low-grade PDAC-associated PanIN, (3) lack of KLF4 protein expression, and (4) shorter OS. These results strongly suggest a relationship between D9S105 deletion and downregulation of KLF4 gene expression as an early event in PDAC progression, as well as a possible role of KLF4 as a prognostic biomarker in gemcitabine-treated patients. and IAP.

KLF4 is a novel candidate tumor suppressor gene in pancreatic ductal carcinoma.

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.

Establishment and characterization of 4 new human pancreatic cancer cell lines: evidences of different tumor phenotypes.

OBJECTIVES: Pancreatic cancer still remains a challenge for its biological complexity and lack of effective therapeutic strategies. Establishing new pancreatic cancer cell lines is therefore of paramount importance to clarify its biology. METHODS: We established and characterized 4 new pancreatic cancer cell lines (PP78, PP109, PP117, and PP161) according to their genetic (K-Ras, TP53, CDKN2A, and MADH4; DNA fingerprinting; karyotype), cytostructural (cytokeratins 7, 8, 18, and 19 vimentin, and ezrin), and functional profiles (doubling time; migration assay). RESULTS: K-Ras, TP53, and CDKN2A gene alterations were detected in all 4 of them. Each cell line had a unique DNA profile revealed by DNA fingerprinting. A complex karyotype with numerous structural and numeric chromosomal abnormalities was present in each cell line. All 4 cell lines showed positivity for cytokeratins 7, 8, and 18. All but PP78 expressed cytokeratin 19, whereas vimentin was expressed only in PP117 and PP78 cells. A different ezrin cellular distribution was noticed in PP78 and PP117, being mostly located at membrane ruffles. This peculiar distribution was associated with the strongest migratory capability. CONCLUSIONS: Our results seem to confirm the pancreatic ductal adenocarcinoma heterogeneity; in fact, the same genetic abnormalities (K-Ras, TP53, and CDKN2A) may have different effects on tumor biology depending on cellular differentiation.