In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate–aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed ...on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST‐expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST‐depleted glioma. Despite their GLAST expression, GBM stem‐like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+‐ATPase. Overexpression of Na+/K+‐ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH‐101. In glioma‐bearing mice, a single intratumoral injection of UCPH‐101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.
What's new?
Increased expression of the cystine/glutamate antiporter xCT is associated with glutamate‐promoted glioblastoma (GBM) invasion into surrounding brain tissue. In this study, GLAST, an astrocyte transporter physiologically devoted to glutamate uptake, was also found to serve a role in GBM aggressiveness. GLAST was highly expressed in GBM specimens and, in association with Na+/K+/ATPase downregulation, exhibited impaired glutamate uptake, resulting in elevated extracellular glutamate levels and GMB cell protection against apoptosis. GLAST inhibition, by contrast, decreased extracellular glutamate, increased GBM cell apoptosis, and prolonged survival in glioma‐bearing mice. The findings provide preliminary background for translational research of GLAST inhibitors.
Circulating tumor cell (CTC) clusters are associated with increased metastatic potential and worse patient prognosis, but are rare, difficult to count, and poorly characterized biophysically. The ...PillarX device described here is a bimodular microfluidic device (Pillar‐device and an X‐magnetic device) to profile single CTCs and clusters from whole blood based on their size, deformability, and epithelial marker expression. Larger, less deformable clusters and large single cells are captured in the Pillar‐device and sorted according to pillar gap sizes. Smaller, deformable clusters and single cells are subsequently captured in the X‐device and separated based on epithelial marker expression using functionalized magnetic nanoparticles. Clusters of established and primary breast cancer cells with variable degrees of cohesion driven by different cell‐cell adhesion protein expression are profiled in the device. Cohesive clusters exhibit a lower deformability as they travel through the pillar array, relative to less cohesive clusters, and have greater collective invasive behavior. The ability of the PillarX device to capture clusters is validated in mouse models and patients of metastatic breast cancer. Thus, this device effectively enumerates and profiles CTC clusters based on their unique geometrical, physical, and biochemical properties, and could form the basis of a novel prognostic clinical tool.
The PillarX device sorts circulating tumor cells (CTCs) and CTC clusters according to size, epithelial state and deformability. Cohesive and compact clusters are captured between pillars and demonstrate reduced deformability; while less cohesive clusters re‐arrange shape between pillars and are captured in the X‐shaped micro‐structures according to their epithelial properties. Distinct CTC cluster populations are identified from in vivo cancer models.
ABSTRACT
Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases ...to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.
miR-145 is an important repressor of pluripotency in embryonic stem cells and a tumor suppressor in different cancers. Here, we found that miR-145 is strongly down-regulated in glioblastoma (GB) ...specimens and corresponding glioblastomaneurospheres (GB-NS, containing GB stem-like cells) compared to normal brain (NB) and to low-grade gliomas (LGG). We observed a direct correlation between miR-145 expression and the progression-free survival (PFS) in LGG patients and overall survival (OS) in GB patients. Using microarray analysis, we identified relevant differences in gene expression profiles between GB-NS over-expressing miR-145 (miRover-NS) and GB-NS Empty (Empty-NS). We focused our attention on HEF1/Cas-L/NEDD9, a scaffold protein involved in invasion in several types of cancer. We confirmed a significant down-regulation of NEDD9 in miRover-NS and we found a higher expression in GB and GB-NS compared to NB. Approximately 50% of LGG patients expressed higher levels of NEDD9 than NB, and the PFS of such patients was shorter than in patients expressing lower levels of NEDD9. We observed that intracranial injection of GB-NS over-expressing miR-145 delays significantly tumor development :deriving tumors showed a significant down-regulation of NEDD9. In addition, we demonstrated a significant inhibition of invasion in silencing experiments with GB-NS shNEDD9 (shNEDD9), and an up-regulation of miR-145 in shNEDD9, suggesting a doublenegative feedback loop between miR-145 and NEDD9. Our results demonstrate the critical role of miR-145 and NEDD9 in regulating glioblastoma invasion and suggest a potential role of NEDD9 as a biomarker for glioma progression.
Background.
Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph−) myeloproliferative disorder characterized by extramedullary haematopoiesis and abnormal neoangiogenesis in both the bone ...marrow (BM) and the spleen. We previously provided evidence that endothelial cells (ECs) from either the spleen or the splenic vein of PMF patients frequently share the JAK2V617F mutation with the hematopoietic malignant cells. More recently, we confirmed this observation also in BM-derived ECs of PMF patients. The mechanism underlying this phenomenon remains, however, not yet clarified. RUNX1 is a critical regulator of hematopoiesis, required for hematopoietic stem cell (HSC) generation and function. In human embryo, it is expressed in all emerging HSCs and progenitors and it is a necessary transcription factor for endothelial to hematopoietic transition. In the adult humans it is expressed in all blood cells, in decreasing intensity according to the maturation status, except erythrocytes. In angiogenesis, it induces endothelial differentiation and maturation as well as vascular network formation by promoting expression of VE-cadherin. Finally, it is involved in retinal aberrant neoangiogenesis.
Aim. To assess if neoangiogenetic activity observed in spleen and BM of PMF patients is associated with RUNX1 expression in ECs.
Patients and Methods. Paraffin-embedded BM sections from patients with MF (n=3), and from patients with lymphomas (named as CTRLs), who underwent BM biopsy for disease staging (n=2), were used for immunostaining analysis. Spleens samples were collected from 3 patients with PMF, who received splenectomy for clinical reasons, and from 2 healthy subjects (HS), who underwent surgery after traumatic damage. Fresh spleen samples were embedded in OCT, and then snap-frozen and stored in liquid nitrogen. Endothelial colony forming cells (ECFCs) were obtained from peripheral blood (n=1 PMF, n=1 HS), PMF spleen tissue (n=1) and cord blood (n=1), according to Ingram et al (Blood 2004;104:2752) and cytospun on slides at confluence. ECFCs, BM and spleen sections were stained with antibodies directed against RUNX1 and VE-cadherin. The images were obtained by confocal laser scanning microscopy (Olympus Fluoview FV10i, 60x objective) and processed by IMAGEJ software. We evaluated 10 fields for each BM and spleen section and measured the number of RUNX1-positive vessels/total vessels. We considered positive a vessel that contained at least one RUNX1-positive cell. The results are shown as mean ± SD.
Results. Immunofluorescence staining of spleen and BM sections confirmed the presence of increased neoangiogenetic processes in samples from PMF patients with respect to CTRLs. The staining of BM and spleen sections obtained from PMF patients with antibodies anti-RUNX1 and anti-VE-cadherin (endothelial marker) allowed the detection of RUNX1+VE-cadherin- cells in the parenchima of BM and spleen (Figure 1A, 1C), occasionally in perivascular position (Figure 1A, arrowhead). Interestingly, in all patients analyzed, we could detect RUNX1+VE-cadherin+ cells in both the parenchima and in the vessels both in the BM and the spleen (Figure 1A, 1C, arrow). In BM, 22 ± 12% of the vessels had at least one double positive cell in the microvessel wall; in spleen tissue the percentage of RUNX1+ ECs increased to 60 ± 13%.
We detected RUNX1+VE-cadherin- cells only in the BM parenchima of CTRLs but not in the spleen of HS, whereas RUNX1+VE-cadherin+ cells were never observed in the vessels of neither the spleen nor the BM (Figure 1B, 1D) of HS and CTRLs, respectively.
The circulating ECFCs obtained from both PMF and HS were, as expected, VE-cadherin+ but did not express RUNX1; however, 30% of spleen-derived-ECFCs of the PMF patient were RUNX1+. In cord blood-derived ECFCs a small percentage (3%) of RUNX1+ cells was observed.
Conclusions.
Our data show a selective expression of RUNX1 in splenic- and BM-ECs of PMF patients, suggesting that activation of RUNX1 expression could be associated with the neoangiogenetic processes that characterize the disease. The expression of RUNX1 in ECFCs from spleen but not in their circulating counterpart suggests a role for the splenic microenvironment in determining RUNX1 expression in ECs. Further studies are ongoing to assess the genotype of RUNX1+ ECs of PMF patients.
No relevant conflicts of interest to declare.
MYC deregulation is common in human cancer and has a role in sustaining the aggressive cancer stem cell populations. MYC mediates a broad transcriptional response controlling normal biological ...programmes, but its activity is not clearly understood. We address MYC function in cancer stem cells through the inducible expression of Omomyc—a MYC‐derived polypeptide interfering with MYC activity—taking as model the most lethal brain tumour, glioblastoma. Omomyc bridles the key cancer stemlike cell features and affects the tumour microenvironment, inhibiting angiogenesis. This occurs because Omomyc interferes with proper MYC localization and itself associates with the genome, with a preference for sites occupied by MYC. This is accompanied by selective repression of master transcription factors for glioblastoma stemlike cell identity such as OLIG2, POU3F2, SOX2, upregulation of effectors of tumour suppression and differentiation such as ID4, MIAT, PTEN, and modulation of the expression of microRNAs that target molecules implicated in glioblastoma growth and invasion such as EGFR and ZEB1. Data support a novel view of MYC as a network stabilizer that strengthens the regulatory nodes of gene expression networks controlling cell phenotype and highlight Omomyc as model molecule for targeting cancer stem cells.
Synopsis
Cancer stemlike cells are key to cancer development and therapy. The MYC‐interfering polypeptide, Omomyc, impairs the carcinogenic potential of human glioblastoma stemlike cells (GSCs) and affects proper MYC genomic localization. This indicates that the gene regulatory nodes determining GSC identity are MYC dependent.
Omomyc occupies DNA E‐boxes targeted by MYC network complexes, weakening the gene expression programme control nodes and facilitating phenotype changes in the presence of appropriate stimuli.
Expression of Omomyc in GSCs—in vitro and in xenografts—rebalances their transcriptome towards differentiation and tumour suppression by affecting the transcript levels of master transcription factors and key non‐coding RNAs.
Blunting MYC activity by Omomyc restrains GSC tumorigenic features—self‐renewal, proliferation, differentiation, migration and tumour vascularization—in vitro and in vivo by both cell‐autonomous and non‐cell‐autonomous mechanisms.
Cancer stemlike cells are key to cancer development and therapy. The MYC‐interfering polypeptide, Omomyc, impairs the carcinogenic potential of human glioblastoma stemlike cells (GSCs) and affects proper MYC genomic localization. This indicates that the gene regulatory nodes determining GSC identity are MYC dependent.
Abstract The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like ...state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS–STING (cyclic GMP-AMP synthase–signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.
Abstract The immune system orchestra suffer a deficit of function during aging, and the incidence of the large majority of cancer types is significantly increased in elderly humans. Although BC can ...occur in young women, it is way more common in middle-age and elderly women. There is evidence that a tonic immune system is pivotal for the success of therapies aimed at awakening a dormant or exhausted immunological response against cancer cells by mean of checkpoint inhibitors (CI). However, rodent models of cancers are currently using very young mice of a few weeks of age, which have a fully functional and tonic immune system and thymus and thus might be poorly representative of adult human cancer patients. We have recently reported that that a triple therapy (TT) involving antigen-presenting cell activation by vinca alkaloids and the generation of new TCF1+ stem cell-like T cell (scT) clones by an alkylating agent can significantly improve the efficacy of the CI anti-PD-1 in models of cancer known to be otherwise CI-resistant such as TNBC and lymphoma (Falvo et al, Cancer Res 2021; Orecchioni et al, JCM 2023). TT effect was due to T cells, as it was abrogated by their in vivo depletion. In the present study we investigated differences in TNBC growth kinetics, TT preclinical activity and tumor microenvironment (TME, including intratumoral immune and stroma cells) in young (6-8w, representative of a 6y old human) versus adult (12m, representative of 40y humans) mice. Models included 4T1 and EMT6 TNBC cells, the former generating a mostly lymphoid TME and the latter generating a mostly myeloid TME. In both models, TT efficacy was similar in young and in adult mice, as the treatment abrogated TNBC local and metastatic growth. CD8+ (but not CD4+) scTs, likely crucial players in TT efficacy, were only slightly reduced in adult mice in spite of age-related thymus involution. This notwithstanding, single cell RNA (10x), IHC and flow cytometry analyses of immune (CD45+EPCAM-) and stroma (CD45-EPCAM-) cell populations indicated major differences in the TME of young vs adult mice. Adults had significantly less CD4+ scTs, B naïve and NK cells and significantly increased memory B cells. TME matrix CAF were skewed in young, while pro-inflammatory stromal populations and myofibroblasts were skewed in adults. Matrix CAFs down regulated a signature involved in different ECM-remodeling abilities, and up regulated metabolic and hypoxic pathways. Expression of genes encoding for glycoproteins, basement membrane components, and collagens were also upregulated. Our data indicate profound differences between young and adult mice immune and stromal TMEs that should be taken into consideration when selecting the age of mice to be used in preclinical efficacy studies in orthotopic immune competent mice. Citation Format: Paolo Falvo, Stephan Gruener, Stefania Orecchioni, Giovanna Talarico, Giulia Bravetti, Giulia Mitola, Federica Pisati, Iros Barozzi, Francesco Bertolini. Middle-aged murine models of triple negative breast cancer (TNBC) show significant differences in tumor immune and stromal environments when compared to classic 6-8 week old models; Challenges and opportunities for preclinical efficacy studies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 288.
Vascular lumen formation is a fundamental step during angiogenesis; yet, the molecular mechanisms underlying this process are poorly understood. Recent studies have shown that neural and vascular ...systems share common anatomical, functional and molecular similarities. Here we show that the organization of endothelial lumen is controlled at the post-transcriptional level by the alternative splicing (AS) regulator Nova2, which was previously considered to be neural cell-specific. Nova2 is expressed during angiogenesis and its depletion disrupts vascular lumen formation in vivo. Similarly, Nova2 depletion in cultured endothelial cells (ECs) impairs the apical distribution and the downstream signalling of the Par polarity complex, resulting in altered EC polarity, a process required for vascular lumen formation. These defects are linked to AS changes of Nova2 target exons affecting the Par complex and its regulators. Collectively, our results reveal that Nova2 functions as an AS regulator in angiogenesis and is a novel member of the 'angioneurins' family.
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