The activity of a PI3K δ-sparing inhibitor, MEN1611, in non-small cell lung cancer cells with constitutive activation of the PI3K/AKT/mTOR pathway.

Background: Lung cancer remains the leading cause of cancer-related death worldwide. Targeted therapies with tyrosine kinase inhibitors (TKIs) result in improvement in survival for non-small cell lung cancer (NSCLC) with activating mutations of the epidermal growth factor receptor (EGFR). Unfortunately, most patients who initially respond to EGFR-TKI ultimately develop resistance to therapy, resulting in cancer progression and relapse. Combination therapy is today a common strategy for the treatment of tumors to increase the success rate, improve the outcome and survival of patients, and avoid the selection of resistant cancer cells through the activation of compensatory pathways. In NSCLC, the phosphoinositide-3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway has been heavily implicated in both tumorigenesis and the progression of disease. Objectives: In this study, we investigated the efficacy of a PI3K δ-sparing inhibitor, MEN1611, in models of NSCLC sensitive and resistant to EGFR inhibitors (erlotinib and gefitinib) with a wild-type PIK3CA gene. Methods: We performed functional, biochemical, and immunohistochemistry studies. Results: We demonstrated good efficacy of MEN1611 in NSCLC devoid of PIK3CA gene mutations but with constitutive activation of the PI3K/AKT pathway and its synergistic effect with gefitinib both in vitro and in vivo. Conclusions: Overall, this preclinical study indicates that the inhibitor could be a candidate for the treatment of NSCLC with an erlotinib/gefitinib-resistant phenotype and constitutive activation of the PI3K/AKT pathway, a phenotype mimicked by our model system.

Combining rotary wet-spinning biofabrication and electro-mechanical stimulation for thein vitroproduction of functional myo-substitutes.

In this work, we present an innovative, high-throughput rotary wet-spinning biofabrication method for manufacturing cellularized constructs composed of highly-aligned hydrogel fibers. The platform is supported by an innovative microfluidic printing head (MPH) bearing a crosslinking bath microtank with a co-axial nozzle placed at the bottom of it for the immediate gelation of extruded core/shell fibers. After a thorough characterization and optimization of the new MPH and the fiber deposition parameters, we demonstrate the suitability of the proposed system for thein vitroengineering of functional myo-substitutes. The samples produced through the described approach were first characterizedin vitroand then used as a substrate to ascertain the effects of electro-mechanical stimulation on myogenic maturation. Of note, we found a characteristic gene expression modulation of fast (MyH1), intermediate (MyH2), and slow (MyH7) twitching myosin heavy chain isoforms, depending on the applied stimulation protocol. This feature should be further investigated in the future to biofabricate engineered myo-substitutes with specific functionalities.

In Vitro and In Vivo Effects of the Urokinase Plasminogen Activator Inhibitor WX-340 on Anaplastic Thyroid Cancer Cell Lines.

Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.

Transition Metal Dichalcogenides (TMDC)-Based Nanozymes for Biosensing and Therapeutic Applications.

Nanozymes, a type of nanomaterial with enzyme-like properties, are a promising alternative to natural enzymes. In particular, transition metal dichalcogenides (TMDCs, with the general formula MX2, where M represents a transition metal and X is a chalcogen element)-based nanozymes have demonstrated exceptional potential in the healthcare and diagnostic sectors. TMDCs have different enzymatic properties due to their unique nano-architecture, high surface area, and semiconducting properties with tunable band gaps. Furthermore, the compatibility of TMDCs with various chemical or physical modification strategies provide a simple and scalable way to engineer and control their enzymatic activity. Here, we discuss recent advances made with TMDC-based nanozymes for biosensing and therapeutic applications. We also discuss their synthesis strategies, various enzymatic properties, current challenges, and the outlook for future developments in this field.

Tackling Current Biomedical Challenges With Frontier Biofabrication and Organ-On-A-Chip Technologies.

In the last decades, biomedical research has significantly boomed in the academia and industrial sectors, and it is expected to continue to grow at a rapid pace in the future. An in-depth analysis of such growth is not trivial, given the intrinsic multidisciplinary nature of biomedical research. Nevertheless, technological advances are among the main factors which have enabled such progress. In this review, we discuss the contribution of two state-of-the-art technologies-namely biofabrication and organ-on-a-chip-in a selection of biomedical research areas. We start by providing an overview of these technologies and their capacities in fabricating advanced in vitro tissue/organ models. We then analyze their impact on addressing a range of current biomedical challenges. Ultimately, we speculate about their future developments by integrating these technologies with other cutting-edge research fields such as artificial intelligence and big data analysis.

Nanotechnology-Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID-19 Vaccines.

In recent years, the main quest of science has been the pioneering of the groundbreaking biomedical strategies needed for achieving a personalized medicine. Ribonucleic acids (RNAs) are outstanding bioactive macromolecules identified as pivotal actors in regulating a wide range of biochemical pathways. The ability to intimately control the cell fate and tissue activities makes RNA-based drugs the most fascinating family of bioactive agents. However, achieving a widespread application of RNA therapeutics in humans is still a challenging feat, due to both the instability of naked RNA and the presence of biological barriers aimed at hindering the entrance of RNA into cells. Recently, material scientists' enormous efforts have led to the development of various classes of nanostructured carriers customized to overcome these limitations. This work systematically reviews the current advances in developing the next generation of drugs based on nanotechnology-assisted RNA delivery. The features of the most used RNA molecules are presented, together with the development strategies and properties of nanostructured vehicles. Also provided is an in-depth overview of various therapeutic applications of the presented systems, including coronavirus disease vaccines and the newest trends in the field. Lastly, emerging challenges and future perspectives for nanotechnology-mediated RNA therapies are discussed.

Tumor Extracellular Matrix Stiffness Promptly Modulates the Phenotype and Gene Expression of Infiltrating T Lymphocytes.

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4+ and CD8+ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8+ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4+ and CD8+ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.

Recent advances in bioprinting technologies for engineering cardiac tissue.

Annually increasing incidence of cardiac-related disorders and cardiac tissue's minimal regenerative capacity have motivated the researchers to explore effective therapeutic strategies. In the recent years, bioprinting technologies have witnessed a great wave of enthusiasm and have undergone steady advancements over a short period, opening the possibilities for recreating engineered functional cardiac tissue models for regenerative and diagnostic applications. With this perspective, the current review delineates recent developments in the sphere of engineered cardiac tissue fabrication, using traditional and advanced bioprinting strategies. The review also highlights different printing ink formulations, available cellular opportunities, and aspects of personalized medicines in the context of cardiac tissue engineering and bioprinting. On a concluding note, current challenges and prospects for further advancements are also discussed.

Recent advances in bioprinting technologies for engineering different cartilage-based tissues.

Inadequate self-repair and regenerative efficiency of the cartilage tissues has motivated the researchers to devise advanced and effective strategies to resolve this issue. Introduction of bioprinting to tissue engineering has paved the way for fabricating complex biomimetic engineered constructs. In this context, the current review gears off with the discussion of standard and advanced 3D/4D printing technologies and their implications for the repair of different cartilage tissues, namely, articular, meniscal, nasoseptal, auricular, costal, and tracheal cartilage. The review is then directed towards highlighting the current stem cell opportunities. On a concluding note, associated critical issues and prospects for future developments, particularly in this sphere of personalized medicines have been discussed.

In vivoorganized neovascularization induced by 3D bioprinted endothelial-derived extracellular vesicles.

Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability to mediate intercellular communication. This feature has been explored and is under constant investigation by researchers, who have demonstrated the important role of EVs in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limitations to overcome before clinical application, including the inability to confine the EVs to strategically defined sites of interest to avoid side effects. In this study, for the first time, EV application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of human umbilical vein endothelial cell-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bioadditives for the formulation of advanced bioinks. Afterin vivosubcutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculaturein situ, consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favour the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischaemic tissues, which represent the fundamental substrate for advanced regenerative medicine applications.

Extracellular Vesicles from Skeletal Muscle Cells Efficiently Promote Myogenesis in Induced Pluripotent Stem Cells.

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological "shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

CASP4 gene silencing in epithelial cancer cells leads to impairment of cell migration, cell-matrix adhesion and tissue invasion.

Inflammatory caspases, including human caspase-4 (CASP4), play key roles in innate immune responses to promote fusion of phagosomes harboring pathogenic bacteria with lysosomes, halt intracellular replication of pathogens, maturation and secretion of pro-inflammatory cytokines. The role of inflammatory caspases in cancer cells remains poorly investigated. Here, we explored the consequences of modulating CASP4 expression levels on the migratory behavior of epithelial cancer cell lines. By a gene silencing approach and in vitro and in vivo studies we show that down-regulation of CASP4 leads to impaired cell migration and cell-matrix adhesion. This phenotype is accompanied by an increased actin cytoskeleton polymerization, changes in the overall organization of adherens junctions (AJs) and number and size of focal adhesions. Interestingly, the cell migration deficit could be reversed by epithelial growth factor treatment, and depletion of calcium ions unveiled a role of CASP4 in the novo assembly of AJs, suggesting that the role of CASP4 is not cell-autonomous. Finally, CASP4-silenced A431 cells exhibited a severe reduction in their ability to invade lung tissue, when injected into nude mice. Overall, our data support the emerging evidence that inflammatory caspases can regulate cell migration through actin remodeling and uncover a novel role of CASP4 in cancer cell behavior.

Tis21-gene therapy inhibits medulloblastoma growth in a murine allograft model.

Medulloblastoma (MB), the tumor of the cerebellum, is the most frequent brain cancer in childhood and a major cause of pediatric mortality. Based on gene profiling, four MB subgroups have been identified, i.e., Wnt or Sonic Hedgehog (Shh) types, and subgroup 3 or 4. The Shh-type MB has been shown to arise from the cerebellar precursors of granule neurons (GCPs), where a hyperactivation of the Shh pathway leads to their neoplastic transformation. We have previously shown that the gene Tis21 (PC3/Btg2) inhibits the proliferation and promotes the differentiation and migration of GCPs. Moreover, the overexpression or the deletion of Tis21 in Patched1 heterozygous mice, a model of spontaneous Shh-type MB, highly reduces or increases, respectively, the frequency of MB. Here we tested whether Tis21 can inhibit MB allografts. Athymic nude mice were subcutaneously grafted with MB cells explanted from Patched1 heterozygous mice. MB allografts were then injected with adeno-associated viruses either carrying Tis21 (AAV-Tis21) or empty (AAV-CBA). We observed that the treatment with AAV-Tis21 significantly inhibited the growth of tumor nodules, as judged by their volume, and reduced the number of proliferating tumor cells (labeled with Ki67 or BrdU), relative to AAV-CBA-treated control mice. In parallel, AAV-Tis21 increased significantly tumor cells labeled with early and late neural differentiation markers. Overall the results suggest that Tis21-gene therapy slows down MB tumor growth through inhibition of proliferation and enhancement of neural differentiation. These results validate Tis21 as a relevant target for MB therapy.

Characterization of epithelial-mesenchymal transition intermediate/hybrid phenotypes associated to resistance to EGFR inhibitors in non-small cell lung cancer cell lines.

Increasing evidence points to a key role played by epithelial-mesenchymal transition (EMT) in cancer progression and drug resistance. In this study, we used wet and in silico approaches to investigate whether EMT phenotypes are associated to resistance to target therapy in a non-small cell lung cancer model system harboring activating mutations of the epidermal growth factor receptor. The combination of different analysis techniques allowed us to describe intermediate/hybrid and complete EMT phenotypes respectively in HCC827- and HCC4006-derived drug-resistant human cancer cell lines. Interestingly, intermediate/hybrid EMT phenotypes, a collective cell migration and increased stem-like ability associate to resistance to the epidermal growth factor receptor inhibitor, erlotinib, in HCC827 derived cell lines. Moreover, the use of three complementary approaches for gene expression analysis supported the identification of a small EMT-related gene list, which may have otherwise been overlooked by standard stand-alone methods for gene expression analysis.

ΔNp63-mediated regulation of hyaluronic acid metabolism and signaling supports HNSCC tumorigenesis.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and several molecular pathways that underlie the molecular tumorigenesis of HNSCC have been identified. Among them, amplification or overexpression of ΔNp63 isoforms is observed in the majority of HNSCCs. Here, we unveiled a ΔNp63-dependent transcriptional program able to regulate the metabolism and the signaling of hyaluronic acid (HA), the major component of the extracellular matrix (ECM). We found that ∆Np63 is capable of sustaining the production of HA levels in cell culture and in vivo by regulating the expression of the HA synthase HAS3 and two hyaluronidase genes, HYAL-1 and HYAL-3. In addition, ∆Np63 directly regulates the expression of CD44, the major HA cell membrane receptor. By controlling this transcriptional program, ∆Np63 sustains the epithelial growth factor receptor (EGF-R) activation and the expression of ABCC1 multidrug transporter gene, thus contributing to tumor cell proliferation and chemoresistance. Importantly, p63 expression is positively correlated with CD44, HAS3, and ABCC1 expression in squamous cell carcinoma datasets and p63-HA pathway is a negative prognostic factor of HNSCC patient survival. Altogether, our data shed light on a ∆Np63-dependent pathway functionally important to the regulation of HNSCC progression.

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells.

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30-40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.