Role of SOX family of transcription factors in central nervous system tumors

SOX genes are developmental regulators with functions in the instruction of cell fate and maintenance of progenitor’s identity during embryogenesis. They play additional roles during tissue homeostasis and regeneration in adults particularly in the Central Nervous System (CNS). In the last years a growing number of evidences has shown that mutations and dysfunction of SOX factors are implicated in several human diseases, including a variety of cancers. In this review, we will summarize the current knowledge about SOX family in CNS tumors and their role in the origin and maintenance of the subpopulation of cancer stem cells in these tumors

Oncolytic adenoviruses as a therapeutic approach for osteosarcoma: A new hope

Osteosarcoma is the most common bone cancer among those with non-hematological origin and affects mainly pediatric patients. In the last 50 years, refinements in surgical procedures, as well as the introduction of aggressive neoadjuvant and adjuvant chemotherapeutic cocktails, have increased to nearly 70% the survival rate of these patients. Despite the initial therapeutic progress the fight against osteosarcoma has not substantially improved during the last three decades, and almost 30% of the patients do not respond or recur after the standard treatment. For this group there is an urgent need to implement new therapeutic approaches. Oncolytic adenoviruses are conditionally replicative viruses engineered to selectively replicate in and kill tumor cells, while remaining quiescent in healthy cells. In the last years there have been multiple preclinical and clinical studies using these viruses as therapeutic agents in the treatment of a broad range of cancers, including osteosarcoma. In this review, we summarize some of the most relevant published literature about the use of oncolytic adenoviruses to treat human osteosarcoma tumors in subcutaneous, orthotopic and metastatic mouse models. In conclusion, up to date the preclinical studies with oncolytic adenoviruses have demonstrated that are safe and efficacious against local and metastatic osteosarcoma. Knowledge arising from phase I/II clinical trials with oncolytic adenoviruses in other tumors have shown the potential of viruses to awake the patient´s own immune system generating a response against the tumor. Generating osteosarcoma immune-competent adenoviruses friendly models will allow to better understand this potential. Future clinical trials with oncolytic adenoviruses for osteosarcoma tumors are warranted

Assessment of metabolic patterns and new antitumoral treatment in osteosarcoma xenograft models by [18F]FDG and sodium [18F]fluoride PET

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and young adults that produces aberrant osteoid. The aim of this study was to assess the utility of 2-deoxy-2-[18F-] fluoro-D-glucose ([18F] FDG) and sodium [18F] Fluoride (Na [18F] F) PET scans in orthotopic murine models of osteosarcoma to describe the metabolic pattern of the tumors, to detect and diagnose tumors and to evaluate the efficacy of a new treatment based in oncolytic adenoviruses. METHODS: Orthotopic osteosarcoma murine models were created by the injection of 143B and 531MII cell lines. [18F]FDG and Na [18F] F PET scans were performed 30 days (143B) and 90 days (531MII) post-injection. The antitumor effect of two doses (107 and 108 pfu) of the oncolytic adenovirus VCN-01 was evaluated in 531 MII model by [18F] FDG PET studies. [18F] FDG uptake was quantified by SUVmax and Total Lesion Glycolysis (TLG) indexes. For Na [18F] F, the ratio tumor SUVmax/hip SUVmax was calculated. PET findings were confirmed by histopathological techniques. RESULTS: The metabolic pattern of tumors was different between both orthotopic models. All tumors showed [18F] FDG uptake, with a sensitivity and specificity of 100%. The [18F] FDG uptake was significantly higher for the 143B model (p < 0.001). Sensitivity for Na [18F] F was around 70% in both models, with a specificity of 100%. 531MII tumors showed a heterogeneous Na [18F] F uptake, significantly higher than 143B tumors (p < 0.01)

Assessment of metabolic patterns and new antitumoral treatment in osteosarcoma xenograft models by [18F]FDG and sodium [18F]fluoride PET

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and young adults that produces aberrant osteoid. The aim of this study was to assess the utility of 2-deoxy-2-[18F-] fluoro-D-glucose ([18F] FDG) and sodium [18F] Fluoride (Na [18F] F) PET scans in orthotopic murine models of osteosarcoma to describe the metabolic pattern of the tumors, to detect and diagnose tumors and to evaluate the efficacy of a new treatment based in oncolytic adenoviruses. METHODS: Orthotopic osteosarcoma murine models were created by the injection of 143B and 531MII cell lines. [18F]FDG and Na [18F] F PET scans were performed 30 days (143B) and 90 days (531MII) post-injection. The antitumor effect of two doses (107 and 108 pfu) of the oncolytic adenovirus VCN-01 was evaluated in 531 MII model by [18F] FDG PET studies. [18F] FDG uptake was quantified by SUVmax and Total Lesion Glycolysis (TLG) indexes. For Na [18F] F, the ratio tumor SUVmax/hip SUVmax was calculated. PET findings were confirmed by histopathological techniques. RESULTS: The metabolic pattern of tumors was different between both orthotopic models. All tumors showed [18F] FDG uptake, with a sensitivity and specificity of 100%. The [18F] FDG uptake was significantly higher for the 143B model (p < 0.001). Sensitivity for Na [18F] F was around 70% in both models, with a specificity of 100%. 531MII tumors showed a heterogeneous Na [18F] F uptake, significantly higher than 143B tumors (p < 0.01)

Delta-24-RGD combined with radiotherapy exerts a potent antitumor effect in diffuse intrinsic pontine glioma and pediatric high grade glioma models

Pediatric high grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPGs), are aggressive tumors with a dismal outcome. Radiotherapy (RT) is part of the standard of care of these tumors; however, radiotherapy only leads to a transient clinical improvement. Delta-24-RGD is a genetically engineered tumor-selective adenovirus that has shown safety and clinical efficacy in adults with recurrent gliomas. In this work, we evaluated the feasibility, safety and therapeutic efficacy of Delta-24-RGD in combination with radiotherapy in pHGGs and DIPGs models. Our results showed that the combination of Delta-24-RGD with radiotherapy was feasible and resulted in a synergistic anti-glioma effect in vitro and in vivo in pHGG and DIPG models. Interestingly, Delta-24-RGD treatment led to the downregulation of relevant DNA damage repair proteins, further sensitizing tumors cells to the effect of radiotherapy. Additionally, Delta-24-RGD/radiotherapy treatment significantly increased the trafficking of immune cells (CD3, CD4+ and CD8+) to the tumor niche compared with single treatments. In summary, administration of the Delta-24-RGD/radiotherapy combination to pHGG and DIPG models is safe and significantly increases the overall survival of mice bearing these tumors. Our data offer a rationale for the combination Delta-24-RGD/radiotherapy as a therapeutic option for children with these tumors. SIGNIFICANCE: Delta-24-RGD/radiotherapy administration is safe and significantly increases the survival of treated mice. These positive data underscore the urge to translate this approach to the clinical treatment of children with pHGG and DIPGs

Involvement of miRNAs in the differentiation of human glioblastoma multiforme stem-like cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process

Development of a DIPG Orthotopic Model in Mice Using an Implantable Guide-Screw System

Objective In this work we set to develop and to validate a new in vivo frameless orthotopic Diffuse Intrinsic Pontine Glioma (DIPG) model based in the implantation of a guide-screw system. Methods It consisted of a guide-screw also called bolt, a Hamilton syringe with a 26-gauge needle and an insulin-like 15-gauge needle. The guide screw is 2.6 mm in length and harbors a 0.5 mm central hole which accepts the needle of the Hamilton syringe avoiding a theoretical displacement during insertion. The guide-screw is fixed on the mouse skull according to the coordinates: 1mm right to and 0.8 mm posterior to lambda. To reach the pons the Hamilton syringe is adjusted to a 6.5 mm depth using a cuff that serves as a stopper. This system allows delivering not only cells but also any kind of intratumoral chemotherapy, antibodies or gene/viral therapies. Results The guide-screw was successfully implanted in 10 immunodeficient mice and the animals were inoculated with DIPG human cell lines during the same anesthetic period. All the mice developed severe neurologic symptoms and had a median overall survival of 95 days ranging the time of death from 81 to 116 days. Histopathological analysis confirmed tumor into the pons in all animals confirming the validity of this model. Conclusion Here we presented a reproducible and frameless DIPG model that allows for rapid evaluation of tumorigenicity and efficacy of chemotherapeutic or gene therapy products delivered intratumorally to the pons

Oncolytic adenovirus Delta-24- RGD engineered to express 4-1BBL or OX40L as therapeutic approach for diffuse intrinsic pontine gliomas

Pediatric tumors appear during childhood and adolescence and are diagnosed from 0 to 19 years old.1 The WHO estimates that 400,000 cases are diagnosed every year globally, which account for about 1% of all cancer diagnoses. 2 Pediatric tumors differ substantially from tumors occurring in adults. While adult cancer occurs due to the accumulation of genetic mutations and cell damage with age, pediatric tumors are usually caused by a blockage in the maturation of immature developing cell types. Therefore, the latter show much lower genetic aberrations. 3,4 The most frequently diagnosed tumors during childhood are hematological malignancies (including leukemia and lymphoma), followed by tumors of the central nervous system (CNS). Specifically, leukemia (24.7%), tumors of the central nervous system (17.2%), non-Hodgking lymphoma (7.5%), Hodgking lymphoma (6.5%), soft-tissue sarcoma (5.9%) and bone tumors (4-8%) are the most common groups5 (Figure 1). In this work, we will focus on brain tumors, and specifically on diffuse midline gliomas (DMGs)

Transcriptional networks controlled by SOX2 in glioblastoma stem cells

Glioblastoma is the most common and aggressive primary malignant brain tumor in adults. It constitutes 45,2% of all malignant central nervous system (CNS) tumors and 80% of all primary malignant CNS tumors. The current standard of care for glioblastoma includes maximal surgical resection, radiation and chemotherapy. Despite steady advances in new treatments to improve survival rates, the overall survival of patients with glioblastoma has not improved over the last decades, remaining at 12 to 18 months from diagnosis. At the cellular level glioblastoma is composed by heterogeneous cell populations, among which the glioma stem-like cells (GSCs) exhibits self-renewal potential and the ability to reconstitute the original tumor upon orthotopic implantation. GSCs are the culprit of glioma chemo- and radio-resistance ultimately leading to relapse. The elucidation of the transcriptome and the molecular pathways involved in the generation and maintenance of GSCs is critical to understand the molecular underpinnings of glioblastoma malignancy and could allow the identification of relevant and novel therapeutic targets. SOX2, a critical transcription regulator of embryonic and neural stem cell function, is deregulated in GSCs and is highly expressed in glioblastoma. However the precise molecular pathways regulated by SOX2 in GSCs remain poorly understood. We hypothesized that SOX2 plays a prominent role in driving the growth, treatment resistance and recurrence of glioblastoma cells, through the orchestration of different transcriptional pathways. In this work we performed a genome-wide analysis of SOX2-regulated coding and non-coding transcripts in GSCs. We identified 2048 differentially expressed coding transcripts regulated by SOX2. These genes are involved in different biological process, such as cell adhesion and cell-cell signaling, and in canonical pathways related with intracellular signaling cascades and amino-acid metabolism pathways associated with GSC propagation. SOX2 regulates 261 non-coding transcripts differentially expressed in GSCs, including miRNAs and lncRNAs amongst other. We identified 2 interesting miRNAs regulated by SOX2. miR-301a-3p is over-expressed in glioblastoma tissues, positively correlates with SOX2 expression and participates in the invasive properties of GSCs, acting as an onco-miR. miR-425-5p is significantly overexpressed in glioblastoma tissue and also correlates with SOX2 expression. Moreover, SOX2 activates its expression by directly binding to its promoter. Inhibition of miR-425-5p expression results in inhibition of neurosphere formation, cell proliferation and finally apoptotic cell death. Inhibition of mir-425-5p in vivo leads to a significant increase in overall median survival time of mice bearing orthotopic glioma xenografts. In summary this work integrates for the first time the coding and non-coding transcriptome controlled by SOX2 in GSCs, defining miR-301 and miR-425 as novel onco-miRs in GSCs and gaining new insights about the molecular circuitries governing glioblastoma biology

TIM-3 blockade as a therapeutic approach for diffuse intrinsic pontine glioma

Diffuse intrinsic pontine gliomas (DIPG) is an aggressive brain tumor and the leading cause of pediatric death caused by cancer. Despite great strides in understanding this disease, prognosis is dismal, with over 90% of patients dying within two years of diagnosis and a median overall survival time of 9-12 months. These grim statistics underscore that DIPG is an unmet clinical need. In this doctoral thesis, I have evaluated the local administration of an anti-TIM-3 antibody in anti-TIM-3 antibody in syngeneic orthotopic models of DIPG as a therapeutic approach for this disease. Our work uncovered, through in silico studies in patient datasets (whole RNAseq and scRNAseq) and samples (by multiplex IF) that TIM-3 was robustly expressed in tumor cells and tumor microenvironment, mainly in microglia and macrophages, suggesting this molecule as a potential therapeutic target in DIPGs. Mechanistic studies showed that TIM-3 provided intrinsic survival cues to the tumor cell while modulating the tumor microenvironment when expressed in the myeloid compartment. In vivo studies showed that TIM-3 blockade significantly increased the overall survival of DIPG immunocompetent orthotopic models, led to long-term survivors, and showed immune memory. TIM-3 inhibition resulted in an increase in the number and proliferative state of microglia, NK cells, and CD8+ T cells and higher levels of IFN, GrzB, and TNF&945; corresponding to NK and T-cell activate phenotypes. Interestingly, there was a decrease in the Treg population, which caused an increase in the pro-inflammatory CD8/Treg ratio. Chemokine studies demonstrated an augmentation of CCL5, CCL2 chemotactic chemokines, and CXCL10, IL-1;, and IFN- pro-inflammatory cytokines in the tumor microenvironment of treated mice. Additionally, DCs, CD4+, and CD8+ T cells were increased in treated draining lymph nodes and of functional significance, expressed higher amounts of pro-inflammatory cytokines than in control mice. Interestingly, the depletion of NK cells, CD4+, and CD8+ T cells immune populations did not completely abrogate the treatment efficacy. However, microglia and macrophages depletion with an anti-CSF1R resulted in a total loss due to a loss of microglia and CD8 T cells pro-inflammatory populations, chemokines, and cytokines indicating a critical role of these populations in the therapeutic effect of TIM-3 blockade. This study provides a new and previously unstudied view of DIPG treatment. TIM-3 blockade emerges as an exciting alternative to classical immune checkpoints, such as PD-1, that did not obtain the desired results in DIPG clinical trials. Moreover, the lack of other effective therapies for these devastating pediatric brain tumors makes these pre-clinical results especially promising and offers strong support for initiating a clinical trial with an anti-TIM-3 antibody for the treatment of DIPG