A Case Study of a Rare Undifferentiated Spindle Cell Sarcoma of the Penis: Establishment and Characterization of Patient-Derived Models.

Rare sarcomas present significant treatment challenges compared to more prevalent soft tissue sarcomas due to limited treatment options and a poor understanding of their biology. This study investigates a unique case of penile sarcoma, providing a comprehensive morphological and molecular analysis. Through the creation of experimental patient-derived models-including patient-derived xenograft (PDX), 3D, and monolayer primary cultures-we successfully replicated crucial molecular traits observed in the patient's tumor, such as smooth muscle actin and CD99 expression, along with specific mutations in genes like TSC2 and FGFR4. These models are helpful in assessing the potential for an in-depth exploration of this tumor's biology. This comprehensive approach holds promise in identifying potential therapeutic avenues for managing this exceedingly rare soft tissue sarcoma.

In vitro culture of mechanically isolated murine primary follicles in the presence of human platelet lysate PLTMax.

OBJECTIVE: To develop a system for the culture of murine preantral ovarian follicles using Human Serum Albumin (HSA) and Human Platelet Lysate (PLTMax). METHODS: Mechanically isolated preantral follicles (N=146) were obtained from Swiss mice and cultured in DMEM:F12 medium for ten days in a 96-well plate with conical bottom. The medium was supplemented with penicillin, streptomycin, and equine chorionic gonadotropin. Additional proteins were tested in 4 test groups: G1: human serum albumin (HSA), G2: human platelet lysate (PLTM), and G3 and G4: HSA + PLTMax at lower and higher concentrations, respectively. Cellular vitality and oocyte morphology were evaluated on day 11 of culture. RESULTS: The highest follicular growth (3.4 fold) was achieved in HSA (G1), while a significantly lower (1.8 fold) growth was achieved in the presence of PLTM (G2, G4) and even further reduced (1.2 fold) when HSA and PLTM were combined (G3). Cellular vitality was close to 70-80% among the four groups, and the highest number of intact oocytes were found in G1. CONCLUSIONS: PLTM did not improve follicular development and oocyte maturation compared to HSA but preserved cell vitality.

Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors.

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.

A novel program of infiltrative control in astrocytomas: ADAM23 depletion promotes cell invasion by activating γ-secretase complex.

Background: Infiltration is a life-threatening growth pattern in malignant astrocytomas and a significant cause of therapy resistance. It results in the tumor cell spreading deeply into the surrounding brain tissue, fostering tumor recurrence and making complete surgical resection impossible. We need to thoroughly understand the mechanisms underlying diffuse infiltration to develop effective therapies. Methods: We integrated in vitro and in vivo functional assays, RNA sequencing, clinical, and expression information from public data sets to investigate the role of ADAM23 expression coupling astrocytoma's growth and motility. Results: ADAM23 downregulation resulted in increased infiltration, reduced tumor growth, and improved overall survival in astrocytomas. Additionally, we show that ADAM23 deficiency induces γ-secretase (GS) complex activity, contributing to the production and deposition of the Amyloid-ß and release of NICD. Finally, GS ablation in ADAM23-low astrocytomas induced a significant inhibitory effect on the invasive programs. Conclusions: Our findings reveal a role for ADAM23 in regulating the balance between cell proliferation and invasiveness in astrocytoma cells, proposing GS inhibition as a therapeutic option in ADAM23 low-expressing astrocytomas.

Immune Checkpoint Blockade via PD-L1 Potentiates More CD28-Based than 4-1BB-Based Anti-Carbonic Anhydrase IX Chimeric Antigen Receptor T Cells.

The complete regression of clear cell renal cell carcinoma (ccRCC) obtained pre-clinically with anti-carbonic anhydrase IX (CAIX) G36 chimeric antigen receptor (CAR) T cells in doses equivalent to ≅108 CAR T cells/kg renewed the potential of this target to treat ccRCC and other tumors in hypoxia. The immune checkpoint blockade (ICB) brought durable clinical responses in advanced ccRCC and other tumors. Here, we tested CD8α/4-1BB compared to CD28-based anti-CAIX CAR peripheral blood mononuclear cells (PBMCs) releasing anti-programmed cell death ligand-1 (PD-L1) IgG4 for human ccRCC treatment in vitro and in an orthotopic NSG mice model in vivo. Using a ≅107 CAR PBMCs cells/kg dose, anti-CAIX CD28 CAR T cells releasing anti-PD-L1 IgG highly decrease both tumor volume and weight in vivo, avoiding the occurrence of metastasis. This antitumoral superiority of CD28-based CAR PBMCs cells compared to 4-1BB occurred under ICB via PD-L1. Furthermore, the T cell exhaustion status in peripheral CD4 T cells, additionally to CD8, was critical for CAR T cells efficiency. The lack of hepatotoxicity and nephrotoxicity upon the administration of a 107 CAR PMBCs cells/kg dose is the basis for carrying out clinical trials using anti-CAIX CD28 CAR PBMCs cells releasing anti-PD-L1 antibodies or anti-CAIX 4-1BB CAR T cells, offering exciting new prospects for the treatment of refractory ccRCC and hypoxic tumors.

Interleukin-6 Signaling in Triple Negative Breast Cancer Cells Elicits the Annexin A1/Formyl Peptide Receptor 1 Axis and Affects the Tumor Microenvironment.

Annexin A1 (AnxA1) is a pleiotropic protein that exerts essential roles in breast cancer (BC) growth and aggressiveness. In our previous work, we described the autocrine signaling of AnxA1 through formyl peptide receptor 1 (FPR1) in the triple-negative (TN) BC cell line, MDA-MB-231. Here, we aimed to describe the interaction between the AnxA1/FPR1 and the Interleukin-6 (IL-6) signaling pathways and their role in the tumor microenvironment (TME). First, we demonstrated that AnxA1 and IL-6 expression levels are correlated in BC tissue samples. In three TNBC cell lines, overexpression of both AnxA1 and IL-6 was also identified. Next, we inhibited FPR1, the IL-6 receptor and STAT3 in both MDA-MB-231 and MDA-MB-157 cells. The FPR1 inhibition led to increased levels of IL-6 and secreted AnxA1 in both cell lines. On the other side, inhibition of the IL-6 receptor or STAT3 led to the impairment of AnxA1 secretion, suggesting the essential role of the IL-6 signaling cascade in the activation of the AnxA1/FPR1 autocrine axis. Finally, we described the interaction between IL-6 and the AnxA1/FPR1 pathways and their role on the TME by analyzing the effect of supernatants derived from MDA-MB-231 and MDA-MB-157 cells under the inhibition of FPR1 or IL-6 signaling on fibroblast cell motility.

Prion protein is associated with a worse prognosis of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSC) etiopathogenesis remains unclear, and the biological changes with the activation of heat shock proteins (HSPs) and prion protein (PRNP) promoted by hypoxia in HNSC are undetermined. This study investigates hypoxia's effect in lymph node metastasis by PRNP expression changes and its main partners. METHODS: The study combined a theoretical/cell culture study with a case-control study. First, bioinformatics and cell culture were performed. A case-control study was performed in a second step by comparing HNSC patients with and without lymph node metastasis. ANALYSES: The Cancer Genome Atlas (TCGA) data source validates the theory in the global population study. RESULTS: Bioinformatics analysis suggests that hypoxia-inducible factor-1α (HIF1A) is associated with HSPA4, HSP90AA1 and PRNP expression. TCGA data validate the hypothesis that higher HSP90AA1, HSPA4 and PRNP are related to metastases and low survival. Herein, the cell study demonstrated that muted PRNP did not respond to hypoxia. DISCUSSION: Our results collectively provide the first evidence that PRNP promotes HNSC lymph node metastasis progression through the upregulation of HSPA4, HSP90AA1 and HIF1A. Our findings may provide a molecular basis for the promoting Role of PRNP in HNSC progression.

Desmoplastic Small Round Cell Tumor: A Review of Main Molecular Abnormalities and Emerging Therapy.

Desmoplastic small round cell tumor (DSRCT) is an extremely rare, aggressive sarcoma affecting adolescents and young adults with male predominance. Generally, it originates from the serosal surface of the abdominal cavity. The hallmark characteristic of DSRCT is the EWSR1-WT1 gene fusion. This translocation up-regulates the expression of PDGFRα, VEGF and other proteins related to tumor and vascular cell proliferation. Current management of DSRCT includes a combination of chemotherapy, radiation and aggressive cytoreductive surgery plus intra-peritoneal hyperthermic chemotherapy (HIPEC). Despite advances in multimodal therapy, outcomes remain poor since the majority of patients present disease recurrence and die within three years. The dismal survival makes DSRCT an orphan disease with an urgent need for new drugs. The treatment of advanced and recurrent disease with tyrosine kinase inhibitors, such as pazopanib, sunitinib, and mTOR inhibitors was evaluated by small trials. Recent studies using comprehensive molecular profiling of DSRCT identified potential therapeutic targets. In this review, we aim to describe the current studies conducted to better understand DSRCT biology and to explore the new therapeutic strategies under investigation in preclinical models and in early phase clinical trials.

Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity.

Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs.

Unconventional Secretion of Heat Shock Proteins in Cancer.

Heat shock proteins (HSPs) are abundant cellular proteins involved with protein homeostasis. They have both constitutive and inducible isoforms, whose expression levels are further increased by stress conditions, such as temperature elevation, reduced oxygen levels, infection, inflammation and exposure to toxic substances. In these situations, HSPs exert a pivotal role in offering protection, preventing cell death and promoting cell recovery. Although the majority of HSPs functions are exerted in the cytoplasm and organelles, several lines of evidence reveal that HSPs are able to induce cell responses in the extracellular milieu. HSPs do not possess secretion signal peptides, and their secretion was subject to widespread skepticism until the demonstration of the role of unconventional secretion forms such as exosomes. Secretion of HSPs may confer immune system modulation and be a cell-to-cell mediated form of increasing stress resistance. Thus, there is a wide potential for secreted HSPs in resistance of cancer therapy and in the development new therapeutic strategies.

Engagement of cellular prion protein with the co-chaperone Hsp70/90 organizing protein regulates the proliferation of glioblastoma stem-like cells.

BACKGROUND: Glioblastoma (GBM), a highly aggressive brain tumor, contains a subpopulation of glioblastoma stem-like cells (GSCs) that play roles in tumor maintenance, invasion, and therapeutic resistance. GSCs are therefore a promising target for GBM treatment. Our group identified the cellular prion protein (PrPC) and its partner, the co-chaperone Hsp70/90 organizing protein (HOP), as potential target candidates due to their role in GBM tumorigenesis and in neural stem cell maintenance. METHODS: GSCs expressing different levels of PrPC were cultured as neurospheres with growth factors, and characterized with stem cells markers and adhesion molecules markers through immunofluorescence and flow cytometry. We than evaluated GSC self-renewal and proliferation by clonal density assays and BrdU incorporation, respectively, in front of recombinant HOP treatment, combined or not with a HOP peptide which mimics the PrPC binding site. Stable silencing of HOP was also performed in parental and/or PrPC-depleted cell populations, and proliferation in vitro and tumor growth in vivo were evaluated. Migration assays were performed on laminin-1 pre-coated glass. RESULTS: We observed that, when GBM cells are cultured as neurospheres, they express specific stemness markers such as CD133, CD15, Oct4, and SOX2; PrPC is upregulated compared to monolayer culture and co-localizes with CD133. PrPC silencing downregulates the expression of molecules associated with cancer stem cells, upregulates markers of cell differentiation and affects GSC self-renewal, pointing to a pivotal role for PrPC in the maintenance of GSCs. Exogenous HOP treatment increases proliferation and self-renewal of GSCs in a PrPC-dependent manner while HOP knockdown disturbs the proliferation process. In vivo, PrPC and/or HOP knockdown potently inhibits the growth of subcutaneously implanted glioblastoma cells. In addition, disruption of the PrPC-HOP complex by a HOP peptide, which mimics the PrPC binding site, affects GSC self-renewal and proliferation indicating that the HOP-PrPC complex is required for GSC stemness. Furthermore, PrPC-depleted GSCs downregulate cell adhesion-related proteins and impair cell migration indicating a putative role for PrPC in the cell surface stability of cell adhesion molecules and GBM cell invasiveness, respectively. CONCLUSIONS: In conclusion, our results show that the modulation of HOP-PrPC engagement or the decrease of PrPC and HOP expression may represent a potential therapeutic intervention in GBM, regulating glioblastoma stem-like cell self-renewal, proliferation, and migration.

Prion protein binding to HOP modulates the migration and invasion of colorectal cancer cells.

Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies. The generation of conventional treatments has improved, but approximately 50 % of patients with CRC who undergo potentially curative surgery ultimately relapse and die, usually as a consequence of metastatic disease. Our previous findings showed that engagement of the cellular prion protein (PrP(C)) to its ligand HSP70/90 heat shock organizing protein (HOP) induces proliferation of glioblastomas. In addition, PrP(C) has been described as an important modulator of colorectal tumor growth. Here, we investigated the biological relevance of the PrP(C)-HOP interaction in CRC cells. We demonstrate that HOP induced the migration and invasion of CRC cell lines in a PrP(C)-dependent manner and that phosphorylation of the ERK1/2 pathway is a downstream mediator of these effects. Additionally, we show that a HOP peptide with the ability to bind PrP(C) and abolish the PrP(C)-HOP interaction inhibited the migration and invasion of CRC cells. Together, these data indicate that the disruption of the PrP(C)-HOP complex could be a potential therapeutic target for modulating the migratory and invasive cellular properties that lead to metastatic CRC.

Papel da proteína prion celular e seus ligantes no desenvolvimento do sistema nervoso / Gene expression characterization of WNT signaling pathway and epithelial-mesenchymal transition during kidney embryogenesis and their implications in Wilms tumors

A proteína prion celular (PrPC) é altamente expressa no sistema nervoso e sua. modificação estrutural está relacionada as encefalopatias espongiformes transmissíveis.. PrPC associa-se com proteínas de matriz extracelular, como a laminina (Ln) e. vitronectina (Vn) e também com a co-chaperonina stress inducible protein 1 (STI1).. Estes complexos estão envolvidos em diferentes processos relacionados ao. desenvolvimento do sistema nervoso. No desenvolvimento embrionário de camundongo. a distribuição de PrPC, Vn e STI1 é espaço-temporalmente relacionada, iniciando-se a. partir do oitavo dia embrionário para STI1 e Vn e no décimo para PrPC. As três proteínas. apresentam um padrão de expressão na medula espinhal em forma de gradiente, com. maior expressão na região ventral do tubo neural e notocorda e diminuindo na porção. dorsal, o que sugere o seu envolvimento na organização do sistema nervoso. A STI1. assim como o peptídeo da cadeia γ1 laminina (Ln-γ1), correspondente ao domínio de. ligação de PrPC, são capazes de promover axonogênese em neurônios de gânglios da raiz. dorsal. Quando combinados, Ln-γ1 e STI1 apresentam um efeito sinérgico sobre a. axonogênese através da mobilização intracelular de cálcio e pela ativação de Erk1/2. O. aumento na concentração intracelular de cálcio pela ligação de STI1 a PrPC é mediado. pela abertura de canais presentes na membrana. Por outro lado, o complexo PrPC-Ln-γ1. mobiliza cálcio a partir de estoques intracelulares. A interação PrPC-STI1 também é. importante na biologia de células tronco neurais. Precursores neurais (neuroesferas). derivados de animais deficientes para PrPC apresentam comprometimento de autorenovação. quando comparados aqueles provenientes animais tipo-selvagem. A ligação. de STI1 a PrPC promove um aumento na proliferação de precursores neurais,. desempenhado um papel chave nos mecanismos de auto-renovação destas. Portanto,. PrPC é capaz de se associar a diferentes ligantes desempenhando um papel relevante no desenvolvimento do sistema nervoso central e periférico.

The cellular prion protein (PrPC) is highly expressed in thenervous system and its structural modification is associated to transmissible espongiform encephalopathies. PrPC associates with extracellular matrix proteins, such as laminin (Ln) and vitronectin (Vn) and also to the co-chaperonin stress inducible protein 1 (STI1).These PrPC complexes are involved in to the development of the nervous system. During the mouse embryo development, PrPC , Vn and STI1 distribution is spatiotemporally related. STI1 and Vn expression became evident at embrionary day 8 (E8), while PrPC is initialy detected at E10. These three proteins present a gradient of expression in spinal cord, more abundant in the notochord and floor plate, suggesting that they can have a role in brain patterning. STI1 as well as the peptide from laminin γ1 chain (Ln-γ1), which corresponds to PrPC binding site, are able to promote axonogenesis in dorsal root ganglia neurons. When combined, STI1 and Ln-γ1 shown a synergic effect upon axonogenesis through intracellular Ca2+ mobilization and Erk1/2 activation. The increment of intracellular Ca2+ promoted by STI1 binding to PrPC is mediated by Ca2+ channels at the plasma membrane. On the other hand, the complex Ln-γ1-PrPC mobilizes Ca2+ from intracellular stores. The interaction between PrPC and STI1 is also important in neural stem cells biology. Neural precursors (neurospheres) derived from PrPC -null mice present impairment of selfrenewal compared to wild-type neuronal precursors. In addition, STI1 binding to PrPC promotes the proliferation of neural precursors, performing a key role in the self-renewal of stem cells. Thus, PrPC is able to associate with different ligands and presents a relevant role in the development of central and peripheric nervous system.