Publications

Immunotherapies are beneficial for a considerable proportion of cancer patients, but ineffective in others. In vitro modelling of the complex interactions between cancer cells and their microenvironment could provide a path to understanding immune therapy sensitivity and resistance. Here we develop MIRO, a fully humanised in vitro platform to model the spatial organisation of the tumour/stroma interface and its interaction with immune cells. We find that stromal barriers are associated with immune exclusion and protect cancer cells from antibody-dependent cellular cytotoxicity, elicited by targeted therapy. We demonstrate that IL2-driven immunomodulation increases immune cell velocity and spreading to overcome stromal immunosuppression and restores anti-cancer response in refractory tumours. Collectively, our study underscores the translational value of MIRO as a powerful tool for exploring how the spatial organisation of the tumour microenvironment shapes the immune landscape and influences the responses to immunomodulating therapies.

JTD Keywords: Activation, Animals, Architecture, Breast-cancer, Cancer-associated fibroblasts, Cell line, tumor, Collagen, Female, Humans, Immunomodulation, Immunotherapy, Interleukin-2, Lab-on-a-chip devices, Mechanism, Mice, Microenvironment, Migration, Neoplasms, Stromal cells, T-cells, Therap, Tumor microenvironment

The tumor microenvironment (TME) comprises a heterogenous cell population within a complex threedimensional (3D) extracellular matrix (ECM). Stromal cells within this TME are altered by signaling cues from cancer cells to support uncontrolled tumor growth and invasion events. Moreover, the ECM also plays a fundamental role in tumor development through pathological remodeling, stiffening and interaction with TME cells. In healthy tissues, Hippo signaling pathway actively contributes to tissue growth, cell proliferation and apoptosis. However, in cancer, the Hippo signaling pathway is highly dysregulated, leading to nuclear translocation of the YAP/TAZ complex, which directly contributes to uncontrolled cell proliferation and tissue growth, and ECM remodeling and stiffening processes. Here, we compare the effect of increasing cell culture substrate stiffness, derived from tumor progression, upon the dysregulation of the Hippo signaling pathway in colorectal cancer-associated fibroblasts (CAFs) and normal colorectal fibroblasts (NFs). We correlate the dysregulation of Hippo pathway with the magnitude of the traction forces exerted by healthy and malignant stromal cells. We found that ECM stiffening is crucial in Hippo pathway dysregulation in CAFs, but not in normal fibroblasts.

JTD Keywords: Cancer-associated fibroblasts, Hippo pathway, Organ size control, Tissu, Tumor microenvironment, Yap-ta, Yap/taz

The diverse roles of the low-density lipoprotein receptor family (LDLR) have been associated with many processes critical to maintaining central nervous system (CNS) health and contributing to neurological diseases or cancer. In this review, we provide a comprehensive understanding of the LDLR's involvement in common brain tumors, specifically high-grade gliomas, emphasizing the receptors' critical role in the pathophysiology and progression of these tumors due to LDLR's high expression. We delve into LDLR's role in regulating cellular uptake and transport through the brain barrier. Additionally, we highlight LDLR's role in activating several signaling pathways related to tumor proliferation, migration, and invasion, engaging readers with an in-depth understanding of the molecular mechanisms at play. By synthesizing current research findings, this review underscores the significance of LDLR during tumorigenesis and explores its potential as a therapeutic target for high-grade gliomas. The collective insights presented here contribute to a deeper appreciation of LDLR's multifaceted roles and implications for physiological and pathological states, opening new avenues for tumor treatment. The role of LDLR family receptors in mediating the transport of LDL across the blood-brain barrier (BBB), facilitating processes such as survival, proliferation, and invasion in high-grade gliomas. Nanoparticles targeting LDLR can be used for drug delivery, potentially inducing cell death and reducing tumor proliferation and survival in high-grade glioma cells. image

JTD Keywords: Animals, Blood-brain-barrier, Brain neoplasms, Cancer, Delivery, Doxorubicin, Expression, Glioblastomas, Glioma, Humans, Low-density lipoprotein receptors, Low‐density lipoprotein receptors, Migratio, Nanoparticles, Neoplasm grading, Proliferation, Receptors, ldl, Signal transduction, Targeted therapie, Targeted therapies, Targeting therapy, Tumor microenvironment

Objective:To investigate the spermidine pathway capability to predict patients at risk for tumor recurrence following colorectal cancer (CRC) surgery.Background:Recurrence rates after CRC surgery remain at about 20% despite an optimal technique and adjuvant therapy when necessary. Identification of risk biomarkers of recurrence is an unmet need. The spermidine pathway is indispensable for cell proliferation and differentiation, and is suggested to accelerate tumor spread.Methods:This was a prospective cohort study of patients undergoing CRC surgery from 2015 to 2018. Plasma samples were collected before surgery and on postoperative day 4, and the spermidine pathway was assessed through mass spectrometry. Oncological outcomes were registered.Results:A total of 146 patients were included and 24 (16.4%) developed tumor recurrence. Higher levels of preoperative spermidine pathway components (spermidine, spermine, spermidine synthase enzyme, and spermine/arginine balance) were positively associated with recurrence. Surgery promoted a decrease in these pathway elements. The greater the decline was, the lower the risk of recurrence. Preoperative spermidine over the cut-off of 0.198 mu M displayed a 4.69-fold higher risk of recurrence. The spermine synthase enzyme behaved in the opposite direction.Conclusions:The spermidine pathway is associated with tumor recurrence following CRC surgery and, after confirmation in larger cohorts, could be translated as a risk biomarker of recurrence into clinical practice.

JTD Keywords: Adjuvant chemotherapy, Colon-cancer, Colorectal cancer, Energ, Metabolomics, Polyamine levels, Postoperative intraabdominal infection, Spermidine pathway, Stage, Tumor recurrenc

The performance of single-chain polymeric nanoparticles (SCPNs) in biomedical applications highly depends on their conformational stability in cellular environments. Until now, such stability studies are limited to 2D cell culture models, which do not recapitulate the 3D tumor microenvironment well. Here, a microfluidic tumor-on-a-chip model is introduced that recreates the tumor milieu and allows in-depth insights into the diffusion, cellular uptake, and stability of SCPNs. The chip contains Matrigel/collagen-hyaluronic acid as extracellular matrix (ECM) models and is seeded with cancer cell MCF7 spheroids. With this 3D platform, it is assessed how the polymer's microstructure affects the SCPN's behavior when crossing the ECM, and evaluates SCPN internalization in 3D cancer cells. A library of SCPNs varying in microstructure is prepared. All SCPNs show efficient ECM penetration but their cellular uptake/stability behavior depends on the microstructure. Glucose-based nanoparticles display the highest spheroid uptake, followed by charged nanoparticles. Charged nanoparticles possess an open conformation while nanoparticles stabilized by internal hydrogen bonding retain a folded structure inside the tumor spheroids. The 3D microfluidic tumor-on-a-chip platform is an efficient tool to elucidate the interplay between polymer microstructure and SCPN's stability, a key factor for the rational design of nanoparticles for targeted biological applications.© 2024 The Authors. Small Methods published by Wiley-VCH GmbH.

JTD Keywords: 3d cancer cell uptake, Cancer cells, Cell culture, Cell uptake, Cellular uptake, Diseases, Ecm penetration, Extracellular matrices, Extracellular matrix penetration, Functional polymers, Hydrogen bonds, Medical applications, Microfluidics, Microstructure, Nanoparticles, Polymeric nanoparticles, Scpns, Single chains, Single-chain polymeric nanoparticle, Stability, Tumor-on-a-chip, Tumors

This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.

JTD Keywords: Animals, Barrier, Cell line, tumor, Dna, Drug delivery, Drug-delivery, Enzyme catalysis, Gene delivery, Gene transfer techniques, Humans, Lactic acid, Mice, Nanobots, Nanoparticles, Pdna, Plasmids, Polyglycolic acid, Polylactic acid-polyglycolic acid copolymer, Swarming, Transfectio, Transfection, Urease, Urinary bladder neoplasms

New drugs and technologies are continuously developed to improve the efficacy and minimize the critical side effects of cancer treatments. The present investigation focuses on the development of a liposomal formulation for Idelalisib, a small-molecule kinase inhibitor approved for the treatment of lymphoid malignancies. Idelalisib is a potent and selective antitumor agent, but it is not indicated nor recommended for first-line treatment due to fatal and serious toxicities. Herein, liposomes are proposed as a delivery tool to improve the therapeutic profile of Idelalisib. Specifically, PEGylated liposomes were prepared, and their physicochemical and technological features were investigated. Light-scattering spectroscopy and cryo-transmission electron microscopy revealed nanosized unilamellar vesicles, which were proved to be stable in storage and in simulated biological fluids. The cytotoxicity of the liposome formulation was investigated in a human non-Hodgkin's lymphoma B cell line. Idelalisib was able to induce death of tumor cells if delivered by the nanocarrier system at increased efficacy. These findings suggest that combining Idelalisib and nanotechnologies may be a powerful strategy to increase the antitumor efficacy of the drug.

JTD Keywords: Antitumor drug, Apoptosis, Idelalisib, Liposomes, Lymphoma cells, Nanodeliver

Treatment-na & iuml;ve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.

JTD Keywords: Apoptosis, Death, Dinaciclib, Mcl-, Models, P-tefb, Sch 727965, Trail, Tumors

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth. The mechanical properties of heterogeneous cell populations in colorectal tumors and the relevance to cancer metastasis remain not fully understood. Here, the authors suggest that the variations in malignant phenotypes between LGR5-positive cancer stem cells and LGR5-negative cells could be due to their distinct mechanical phenotypes observed in vitro, determined by the membrane to cortex attachment proteins Ezrin/Radixin/Moesin.

JTD Keywords: , , Adhesion, Deformability, E-cadherin, Erm proteins, Expression, Metastasis, Organization, Plasticit, Stem-cells, Tumor-cells

Simple Summary Neuroblastoma (NB), a prevalent childhood cancer, presents challenges in treatment due to its cellular diversity and the presence of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs, hindering effective therapies. We developed a stiffness-based in vitro platform simulating arterial and venous conditions to address this gap. Notably, adrenergic NB cells transdifferentiated into TECs where there was an arterial-like stiffness, while mesenchymal cells did not. This platform facilitated the identification of Globotriaosylceramide (GB3) as a novel TEC biomarker. Moreover, we harnessed Shiga toxin-functionalized nanoparticles for the specific targeting of GB3-positive cells, showing promise for future therapeutic strategies. Our study provides insights into NB heterogeneity, offers a predictive tool for assessing aggressiveness, and introduces potential targets for precision therapies.Abstract Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

JTD Keywords: Alternative vasculature, Angiogenesis, Cells, Differentiation, Gb3, Neuroblastoma, Origin, Tumor-derived endothelial cells

Neuroblastoma (NB) is a highly vascularized pediatric tumor arising from undifferentiated neural crest cells early in life, exhibiting both traditional endothelial-cell-driven vasculature and an intriguing alternative vasculature. The alternative vasculature can arise from cancer cells undergoing transdifferentiation into tumor-derived endothelial cells (TEC), a trait associated with drug resistance and tumor relapse. The lack of effective treatments targeting NB vasculature primarily arises from the challenge of establishing predictive in vitro models that faithfully replicate the alternative vasculature phenomenon. In this study, we aim to recreate the intricate vascular system of NB in an in vitro context, encompassing both types of vascularization, by developing a novel neuroblastoma-on-a-chip model. We designed a collagen I/fibrin-based hydrogel closely mirroring NB's physiological composition and tumor stiffness. This biomaterial created a supportive environment for the viability of NB and endothelial cells. Implementing a physiological shear stress value, aligned with the observed range in arteries and capillaries, within the microfluidic chip facilitated the successful development of vessel-like structures and triggered transdifferentiation of NB cells into TECs. The vascularized neuroblastoma-on-a-chip model introduced here presents a promising and complementary strategy to animal-based research with a significant capacity for delving into NB tumor biology and vascular targeting therapy.

JTD Keywords: 3d tumor model, Angiogenesis, Endothelial-cells, Microfluidic device, Neuroblastoma, Origi, Transdifferentiation, Tumor, Tumor-derived endothelial cells, Tumor-on-a-chip, Vasculature

Bladder cancer treatment via intravesical drug administration achieves reasonable survival rates but suffers from low therapeutic efficacy. To address the latter, self-propelled nanoparticles or nanobots have been proposed, taking advantage of their enhanced diffusion and mixing capabilities in urine when compared with conventional drugs or passive nanoparticles. However, the translational capabilities of nanobots in treating bladder cancer are underexplored. Here, we tested radiolabelled mesoporous silica-based urease-powered nanobots in an orthotopic mouse model of bladder cancer. In vivo and ex vivo results demonstrated enhanced nanobot accumulation at the tumour site, with an eightfold increase revealed by positron emission tomography in vivo. Label-free optical contrast based on polarization-dependent scattered light-sheet microscopy of cleared bladders confirmed tumour penetration by nanobots ex vivo. Treating tumour-bearing mice with intravesically administered radio-iodinated nanobots for radionuclide therapy resulted in a tumour size reduction of about 90%, positioning nanobots as efficient delivery nanosystems for bladder cancer therapy.© 2024. The Author(s).

JTD Keywords: cell, drug-delivery, nanomotors, tissue, Bladder cancers, Cancer therapy, Diseases, Drug administration, Drug delivery, Enhanced diffusion, Enhanced mixing, Ex-vivo, In-vivo, Mammals, Nanobots, Nanoparticles, Nanosystems, Oncology, Positron emission tomography, Radioisotopes, Silica, Survival rate, Therapeutic efficacy, Tumor penetration, Tumors

Degradable polymeric micelles are promising drug delivery systems due to their hydrophobic core and responsive design. When applying micellar nanocarriers for tumor delivery, one of the bottlenecks encountered in vivo is the tumor tissue barrier: crossing the dense mesh of cells and the extracellular matrix (ECM). Sometimes overlooked, the extracellular matrix can trap nanoformulations based on charge, size, and hydrophobicity. Here, we used a simple design of a microfluidic chip with two types of ECM and MCF7 spheroids to allow high-throughput screening of the interactions between biological interfaces and polymeric micelles. To demonstrate the applicability of the chip, a small library of fluorescently labeled polymeric micelles varying in their hydrophilic shell and hydrophobic core forming blocks was studied. Three widely used hydrophilic shells were tested and compared, namely, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and poly(acrylic acid), along with two enzymatically degradable dendritic hydrophobic cores (based on hexyl or nonyl end groups). Using ratiometric imaging of unimer:micelle fluorescence and FRAP inside the chip model, we obtained the local assembly state and dynamics inside the chip. Notably, we observed different micelle behaviors in the basal lamina ECM, from avoidance of the ECM structure to binding of the poly(acrylic acid) formulations. Binding to the basal lamina correlated with higher uptake into MCF7 spheroids. Overall, we proposed a simple microfluidic chip containing dual ECM and spheroids for the assessment of the interactions of polymeric nanocarriers with biological interfaces and evaluating nanoformulations' capacity to cross the tumor tissue barrier.

JTD Keywords: Extracellular matrix, Microfluidics, Nanoparticle mobility, Polymeric micelles, Tumor-on-a-chip

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.© 2023. The Author(s).

JTD Keywords: force, migration, yap, Cancer-associated fibroblasts, Cell line, tumor, Fibroblasts, Mechanotransduction, cellular, Neoplasms, Tumor, Tumor microenvironment

Cancer progression is caused by genetic changes and associated with various alterations in cell properties, which also affect a tumor's mechanical state. While an increased stiffness has been well known for long for solid tumors, it has limited prognostic power. It is hypothesized that cancer progression is accompanied by tissue fluidization, where portions of the tissue can change position across different length scales. Supported by tabletop magnetic resonance elastography (MRE) on stroma mimicking collagen gels and microscopic analysis of live cells inside patient derived tumor explants, an overview is provided of how cancer associated mechanisms, including cellular unjamming, proliferation, microenvironment composition, and remodeling can alter a tissue's fluidity and stiffness. In vivo, state-of-the-art multifrequency MRE can distinguish tumors from their surrounding host tissue by their rheological fingerprints. Most importantly, a meta-analysis on the currently available clinical studies is conducted and universal trends are identified. The results and conclusions are condensed into a gedankenexperiment about how a tumor can grow and eventually metastasize into its environment from a physics perspective to deduce corresponding mechanical properties. Based on stiffness, fluidity, spatial heterogeneity, and texture of the tumor front a roadmap for a prognosis of a tumor's aggressiveness and metastatic potential is presented.© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.

JTD Keywords: brain, cancer, cells, collective migration, elastic energy, elastography, in vivo magnetic resonance elastography, invasion, medical imaging, solid stress, tissue fluidity, tumor mechanics, viscoelastic properties, Cancer, Collagen, Extracellular-matrix, Humans, In vivo magnetic resonance elastography, Medical imaging, Neoplasms, Prognosis, Tissue fluidity, Tumor mechanics, Tumor microenvironment

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pa-thology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE set-tings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve 1/4 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC inde-pendently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (a-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff micro -environment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities. (c) 2023 THE AUTHORS. Published by Elsevier Inc. on behalf of the United States & Canadian Academy of Pathology. This is an open access article under the CC BY-NC-ND license (http://creativecommo ns.org/licenses/by-nc-nd/4.0/).

JTD Keywords: biomarkers, collagen, ct-fire, lung cancer, mechanobiology, Adenocarcinoma, Association, Biomarkers, Collagen, Ct-fire, Differentiation, Expression, Extracellular-matrix, I collagen, Invasion, Lung cancer, Mechanobiology, Microenvironment, Signature, Survival, Tumor microenvironment

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an >80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.

JTD Keywords: cells, code, elisa, il-6, inflammation, kits, pathogenesis, procalcitonin, release, Cytokines, Humans, Immunoassay, Immunologic tests, Interleukin-6, Tumor necrosis factor-alpha

JTD Keywords: Acquisition, C-13, Detecting tumor response, Exchang, Excitation pulses, Magnetic-resonance-spectroscopy, Metabolism, Mri, Resolution, Strategies

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.© 2023 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

JTD Keywords: chemoresistance, dormancy, drives, extracellular vesicles, invasion, plasticity, premetastatic niche, triple-negative breast cancer, tumor microenvironment, Cancer cell plasticity, Cell line, tumor, Extracellular vesicles, Fibroblasts, Humans, Lung, Neoplastic stem cells, Premetastatic niche, Triple negative breast neoplasms, Triple-negative breast cancer, Tumor microenvironment

Mechanical changes in tumors have long been linked to increased malignancy and therapy resistance and attributed to mechanical changes in the tumor extracellular matrix (ECM). However, to the best of our knowledge, there have been no mechanical studies on decellularized tumors. Here, we studied the biochemical and mechanical progression of the tumor ECM in two models of lung metastases: lung carcinoma (CAR) and melanoma (MEL). We decellularized the metastatic lung sections, measured the micromechanics of the tumor ECM, and stained the sections for ECM proteins, proliferation, and cell death markers. The same methodology was applied to MEL mice treated with the clinically approved anti-fibrotic drug nintedanib. When compared to healthy ECM (~0.40 kPa), CAR and MEL lung macrometastases produced a highly dense and stiff ECM (1.79 ± 1.32 kPa, CAR and 6.39 ± 3.37 kPa, MEL). Fibronectin was overexpressed from the early stages (~118%) to developed macrometastases (~260%) in both models. Surprisingly, nintedanib caused a 4-fold increase in ECM-occupied tumor area (5.1 ± 1.6% to 18.6 ± 8.9%) and a 2-fold in-crease in ECM stiffness (6.39 ± 3.37 kPa to 12.35 ± 5.74 kPa). This increase in stiffness strongly correlated with an increase in necrosis, which reveals a potential link between tumor hypoxia and ECM deposition and stiffness. Our findings highlight fibronectin and tumor ECM mechanics as attractive targets in cancer therapy and support the need to identify new anti-fibrotic drugs to abrogate aberrant ECM mechanics in metastases.

JTD Keywords: atomic force microscopy, basement membrane, breast-cancer, decellularization, expression, extracellular matrix, extracellular-matrix, fibronectin, intermittent hypoxia, lung carcinoma, lung metastases, melanoma, metastatic niche formation, micromechanical properties, nintedanib, signature, stiffness, tumor-growth, Colorectal-cancer progression, Lung metastases, Stiffness

Nanoparticles (NPs) are commonly functionalized using targeting ligands to drive their selective uptake in cells of interest. Typical target cell types are cancer cells, which often overexpress distinct surface receptors that can be exploited for NP therapeutics. However, these targeted receptors are also moderately expressed in healthy cells, leading to unwanted off-tumor toxicities. Multivalent interactions between NP ligands and cell receptors have been investigated to increase the targeting selectivity towards cancer cells due to their non-linear response to receptor density. However, to exploit the multivalent effect, multiple variables have to be considered such as NP valency, ligand affinity, and cell receptor density. Here, we synthesize a panel of aptamer-functionalized silica-supported lipid bilayers (SSLB) to study the effect of valency, aptamer affinity, and epidermal growth factor receptor (EGFR) density on targeting specificity and selectivity. We show that there is an evident interplay among those parameters that can be tuned to increase SSLB selectivity towards high-density EGFR cells and reduce accumulation at non-tumor tissues. Specifically, the combination of high-affinity aptamers and low valency SSLBs leads to increased high-EGFR cell selectivity. These insights provide a better understanding of the multivalent interactions of NPs with cells and bring the nanomedicine field a step closer to the rational design of cancer nanotherapeutics.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: aptamer avidity and affinity, delivery, microscopy, multivalency, multivalent, nanoparticle targeting, silica -supported lipid bilayers, Aptamer avidity and affinity, Multivalency, Nanoparticle targeting, Silica-supported lipid bilayers, Supported lipid-bilayers, Tumor targeting

The involvement of the extracellular matrix (ECM) in tumor progression has motivated the development of biomaterials mimicking the tumor ECM to develop more predictive cancer models. Particularly, polypeptides based on elastin could be an interesting approach to mimic the ECM due to their tunable properties. Here, we demonstrated that elastin-like recombinamer (ELR) hydrogels can be suitable biomaterials to develop breast cancer models. This hydrogel was formed by two ELR polypeptides, one containing sequences biodegradable by matrix metalloproteinase and cyclooctyne and the other carrying arginylglycylaspartic acid and azide groups to allow cell adhesion, biodegradability, and suitable stiffness through "click-chemistry" cross-linking. Our findings show that breast cancer or nontumorigenic breast cells showed high viability and cell proliferation for up to 7 days. MCF7 and MCF10A formed spheroids whereas MDA-MB-231 formed cell networks, with the expression of ECM and high drug resistance in all cases, evidencing that ELR hydrogels are a promising biomaterial for breast cancer modeling.

JTD Keywords: clinical-trials, collagen i, discovery, mcf-7 cells, phenotype, progression, spheroids, translation, tumor microenvironment, Extracellular-matrix

The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-gamma, and bladder infiltration of selected immune cells such as ILCs and CD4(TEM). BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3(+) (CD4(+) and CD8(+)) T cells, together with high systemic IFN-gamma release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions.

JTD Keywords: bcg, innate immune response, innate-lymphoid cells, lipid, non-muscle invasive, Bcg, Calmette-guerin bcg, Glycerol, Identification, Immune-response, Innate immune response, Innate-lymphoid cells, Lipid, Lipids, Mycolic acids, Neutral-red, Non-muscle invasive, Phenolic glycolipids, Tuberculosis, Tumor microenvironment, Virulence

Mitochondrial nucleoids or mitochondrial DNA (mtDNA) encodes for a variety of enzymes and proteins that are essential for oxidative phosphorylation, mitochondrial fussion/fission and apoptotic processes. However, visulization of mtDNA dynamics in response to external stumili has not yet been achieved. Herein, we developed a fluorescent probe, named BDP, that is capable of specifically bind to mtDNA in vitro and in living cells, without interfering mitochondrial functions. Its large Stokes-Shift and red-emission tail render its suitability for stimulated emission depletion (STED) visulization of mtDNA dynamics in living cells. We sucessfully demonstrated for the first time how apoptotic induced anti-cancer drug could impact on mitochondrial nucleoids, and the morphology evolution of mtDNA from segmentation to dispersion was recorded, in a single mitochondria at nanoscale.

JTD Keywords: Dna, Mitochondrial dna (mtdna), Pyridine salt derivatives, Stimulated emission depletion (sted), Tumor

Precision medicine is starting to incorporate functional assays to evaluate anticancer agents on patient-isolated tissues or cells to select for the most effective. Among these new technologies, dynamic BH3 profiling (DBP) has emerged and extensively been used to predict treatment efficacy in different types of cancer. DBP uses synthetic BH3 peptides to measure early apoptotic events ('priming') and anticipate therapy-induced cell death leading to tumor elimination. This predictive functional assay presents multiple advantages but a critical limitation: the cell number requirement, that limits drug screening on patient samples, especially in solid tumors. To solve this problem, we developed an innovative microfluidic-based DBP (µDBP) device that overcomes tissue limitations on primary samples. We used microfluidic chips to generate a gradient of BIM BH3 peptide, compared it with the standard flow cytometry based DBP, and tested different anticancer treatments. We first examined this new technology's predictive capacity using gastrointestinal stromal tumor (GIST) cell lines, by comparing imatinib sensitive and resistant cells, and we could detect differences in apoptotic priming and anticipate cytotoxicity. We then validated µDBP on a refractory GIST patient sample and identified that the combination of dactolisib and venetoclax increased apoptotic priming. In summary, this new technology could represent an important advance for precision medicine by providing a fast, easy-to-use and scalable microfluidic device to perform DBP in situ as a routine assay to identify the best treatment for cancer patients.© 2022. The Author(s).

JTD Keywords: biomarkers, cancer drugs, chemotherapy, chip, models, platform, sensitivity, strategy, tumor-cells, Precision medicine

Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.© 2022. The Author(s), under exclusive licence to Springer Nature Limited.

JTD Keywords: colonization, defines, human colon, mutations, plasticity, retrieval, stem-cells, subtypes, underlie, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Article, Cancer, Cancer growth, Cancer immunotherapy, Cancer inhibition, Cancer recurrence, Cancer staging, Cell, Cell adhesion, Cell migration, Cell population, Cell surface receptor, Cohort analysis, Colorectal cancer, Colorectal neoplasms, Colorectal tumor, Comprehensive molecular characterization, Controlled study, Crispr-cas9 system, Cytoskeleton, Disease exacerbation, Disease progression, Dynamics, Emp1 gene, Epithelial membrane protein-1, Extracellular matrix, Flow cytometry, Fluorescence intensity, Gene expression, Genetics, Human, Human cell, Humans, Immune response, Immunofluorescence, In situ hybridization, Marker gene, Metastasis potential, Mice, Minimal residual disease, Mouse, Neoplasm proteins, Neoplasm recurrence, local, Neoplasm, residual, Nonhuman, Pathology, Phenotype, Prevention and control, Protein, Receptors, cell surface, Single cell rna seq, Tumor, Tumor protein, Tumor recurrence

Great advances in cancer treatment have been undertaken in the last years as a consequence of the development of new antitumoral drugs able to target cancer cells with decreasing side effects and a better understanding of the behavior of neoplastic cells during invasion and metastasis. Specifically, drug delivery systems (DDS) based on the use of hydroxyapatite nanoparticles (HAp NPs) are gaining attention and merit a comprehensive review focused on their potential applications. These are derived from the intrinsic properties of HAp (e.g., biocompatibility and biodegradability), together with the easy functionalization and easy control of porosity, crystallinity and morphology of HAp NPs. The capacity to tailor the properties of DLS based on HAp NPs has well-recognized advantages for the control of both drug loading and release. Furthermore, the functionalization of NPs allows a targeted uptake in tumoral cells while their rapid elimination by the reticuloendothelial system (RES) can be avoided. Advances in HAp NPs involve not only their use as drug nanocarriers but also their employment as nanosystems for magnetic hyperthermia therapy, gene delivery systems, adjuvants for cancer immunotherapy and nanoparticles for cell imaging.

JTD Keywords: antitumoral, cancer, cell imaging, controlled-release, drug-carrier, efficient drug-delivery, fatty-acid-metabolism, fe3o4 nanoparticles, gene delivery, hydroxyapatite, hyperthermia, immunotherapy, in-vitro, magnetic hydroxyapatite, nano-hydroxyapatite, protein adsorption, tumor-growth, Calcium-phosphate nanoparticles, Cancer, Immunotherapy

Nature has inspired the creation of artificial micro- and nanomotors that self-propel converting chemical energy into mechanical action. These tiny machines have appeared as promising biomedical tools for treatment and diagnosis and have also been used for environmental, antimicrobial or sensing applications. Among the possible catalytic engines, enzymes have emerged as an alternative to inorganic catalysts due to their biocompatibility and the variety and bioavailability of fuels. Although the field of enzyme-powered micro- and nano-motors has a trajectory of more than a decade, a comprehensive framework on how to rationally design, control and optimize their motion is still missing. With this purpose, herein we performed a thorough bibliographic study on the key parameters governing the propulsion of these enzyme-powered devices, namely the chassis shape, the material composition, the motor size, the enzyme type, the method used to incorporate enzymes, the distribution of the product released, the motion mechanism, the motion media and the technique used for motion detection. In conclusion, from the library of options that each parameter offers there needs to be a rational selection and intelligent design of enzymatic motors based on the specific application envisioned.

JTD Keywords: Catalase, Hydrogen-peroxide, Micro/nanomotors, Micromotors, Movement, Nanomotors, Propulsion, Surfactants, Therapy, Tumor microenvironment

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metal-loproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor micro-environment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarci-noma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interac-tion. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-61/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-61-activated ADC-TAFs is both nec-essary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenu-ated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer. (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

JTD Keywords: cancer-associated fibroblast, cd63, fibrosis, smad3, tgf-β1, timp-1, Angiogenesis, Cancer cells, Cancer-associated fibroblast, Cd63, Expression, Fibrosis, Hepatocellular-carcinoma, Metalloproteinases, Nintedanib, Prognostic-significance, Protein, Smad3, Squamous-cell carcinoma, Tgf-? 1, Tgf-β1, Timp-1, Tissue inhibitor, Tumor microenvironment

Targeted agents have emerged as promising molecules for cancer treatment, but most of them fail to achieve complete tumor regression or attain durable remissions due to tumor adaptations. We used dynamic BH3 profiling to identify targeted agents effectiveness and anti-apoptotic adaptations upon targeted treatment in rhabdomyosarcoma. We focused on studying the use of BH3 mimetics to specifically inhibit pro-survival BCL-2 family proteins, overwhelm resistance to therapy and prevent relapse. We observed that the MEK1/2 inhibitor trametinib rapidly depleted the pro-apoptotic protein NOXA, thus increasing MCL-1 availability. Indeed, we found that the MCL-1 inhibitor S63845 synergistically enhanced trametinib cytotoxicity in rhabdomyosarcoma cells in vitro and in vivo. In conclusion, our findings indicate that the combination of a BH3 mimetic targeting MCL-1 with trametinib improves efficiency on rhabdomyosarcoma by blocking tumor adaptation to treatment.

JTD Keywords: apoptosis, bcl-2, combination, expression, pathway, resistance, survival, therapy, tumors, Histone deacetylase inhibitor

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cyto-kines IFN? and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. © Gawish et al.

JTD Keywords: covid-19 mouse model, covid-19 therapy, cytokine storm, immunology, inflammation, mavie16, mouse, mouse-adapted sars-cov-2, program, recombinant soluble ace2, tmprss2, Adaptive immunity, Angiotensin converting enzyme 2, Angiotensin-converting enzyme 2, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Apoptosis, Article, Bagg albino mouse, Breathing rate, Bronchoalveolar lavage fluid, C57bl mouse, Cell composition, Cell infiltration, Controlled study, Coronavirus disease 2019, Coronavirus spike glycoprotein, Covid-19, Cytokeratin 18, Cytokine production, Dipeptidyl carboxypeptidase, Disease model, Disease models, animal, Disease severity, Drosophila-melanogaster, Enzyme linked immunosorbent assay, Expression vector, Flow cytometry, Gamma interferon, Gene editing, Gene expression, Gene mutation, Genetic engineering, Genetics, Glycosylation, High mobility group b1 protein, Histology, Histopathology, Immune response, Immunocompetent cell, Immunology, Immunopathology, Interferon-gamma, Interleukin 2, Metabolism, Mice, inbred balb c, Mice, inbred c57bl, Mouse-adapted sars-cov-2, Myeloperoxidase, Neuropilin 1, Nonhuman, Nucleocapsid protein, Pathogenicity, Peptidyl-dipeptidase a, Pyroptosis, Recombinant soluble ace2, Renin angiotensin aldosterone system, Rna extraction, Rna isolation, Sars-cov-2, Severe acute respiratory syndrome coronavirus 2, Spike glycoprotein, coronavirus, T lymphocyte activation, Trabecular meshwork, Tumor necrosis factor, Virology, Virus load, Virus replication, Virus transmission, Virus virulence

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

JTD Keywords: cancer therapy, mirnas delivery, nanocarriers, nanovesicles, neuroblastoma, pediatric cancer, quatsomes, Biodistribution, Cancer therapy, Cell engineering, Cells, Cholesterol, Controlled drug delivery, Diseases, Dna, Dysregulated ph, Lipoplex, Microrna delivery, Mirnas delivery, Nanocarriers, Nanoparticles, Nanovesicle, Nanovesicles, Neuroblastoma, Neuroblastomas, Pediatric cancer, Ph sensitive, Ph sensors, Quatsome, Quatsomes, Rna, Sirna, Sirna delivery, Sirnas delivery, Small interfering rna, Small rna, Targeted drug delivery, Tumors, Vesicles

Nowadays, liquid biopsy represents one of the most promising techniques for early diagnosis, monitoring, and therapy screening of cancer. This novel methodology includes, among other techniques, the isolation, capture, and analysis of circulating tumor cells (CTCs). Nonetheless, the identification of CTC from whole blood is challenging due to their extremely low concentration (1-100 per ml of whole blood), and traditional methods result insufficient in terms of purity, recovery, throughput and/or viability of the processed sample. In this context, the development of microfluidic devices for detecting and isolating CTCs offers a wide range of new opportunities due to their excellent properties for cell manipulation and the advantages to integrate and bring different laboratory processes into the microscale improving the sensitivity, portability, reducing cost and time. This chapter explores current and recent microfluidic approaches that have been developed for the analysis and detection of CTCs, which involve cell capture methods based on affinity binding and label-free methods and detection based on electrical, chemical, and optical sensors. All the exposed technologies seek to overcome the limitations of commercial systems for the analysis and isolation of CTCs, as well as to provide extended analysis that will allow the development of novel and more efficient diagnostic tools.

JTD Keywords: Cancer detection, Cancer diagnosis, Cancer-cells, Capture, Chip, Circulating tumor cells, Enrichment, Label-free isolation, Liquid biopsy, Microchannel, Microfluidics, Separation, Ultra-fast

Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in estrogen receptor-alpha (ER alpha)-dependent breast cancers and off-target indications such as infections, although the immune activity of TAM and its active metabolite, 4-OH tamoxifen (4HT), is poorly characterized. Here, we aimed at investigating the endocrine and immune activity of these SERMs in macrophages. Using primary cultures of female mouse macrophages, we analyzed the expression of immune mediators and activation of effector functions in competition experiments with SERMs and 17 beta-estradiol (E2) or the bacterial endotoxin LPS. We observed that 4HT and TAM induce estrogen antagonist effects when used at nanomolar concentrations, while pharmacological concentrations that are reached by TAM in clinical settings regulate the expression of VEGF alpha and other immune activation genes by ER alpha- and G protein-coupled receptor 1 (GPER1)-independent mechanisms that involve NRF2 through PI3K/Akt-dependent mechanisms. Importantly, we observed that SERMs potentiate cell phagocytosis and modify the effects of LPS on the expression of inflammatory cytokines, such as TNF alpha and IL1 beta, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1 beta secretion through caspase-1 activation.

JTD Keywords: drug repurposing, inflammation, macrophage, nrf2, Afimoxifene, Animals, Apoptosis, Breast-cancer, Cells, cultured, Drug repurposing, Esr1 protein, mouse, Estrogen receptor alpha, Expression, Female, Gper1 protein, mouse, Immunomodulating agents, Inflammation, Inflammation mediators, Lipopolysaccharide, escherichia coli o111 b4, Lipopolysaccharides, Macrophage, Macrophages, peritoneal, Mice, Mice, inbred c57bl, Mice, knockout, Nf-e2-related factor 2, Nfe2l2 protein, mouse, Nrf2, Phagocytosis, Phenotype, Receptors, estrogen, Receptors, g-protein-coupled, Resistance, Selective estrogen receptor modulators, Sex-differences, Signal transduction, Tamoxifen, Tumor-associated macrophages

Neuroblastoma (NB) is the most common extracranial pediatric solid cancer originating from undifferentiated neural crest cells. NB cells express EZH2 and GLI1 genes that are known to maintain the undifferentiated phenotype of cancer stem cells (CSC) in NB. Recent studies suggest that tumor-derived extracellular vesicles (EVs) can regulate the transformation of surrounding cells into CSC by transferring tumor-specific molecules they contain. However, the horizontal transfer of EVs molecules in NB remains largely unknown. We report the analysis of NB-derived EVs in bioengineered models of NB that are based on a collagen 1/hyaluronic acid scaffold designed to mimic the native tumor niche. Using these models, we observed an enrichment of GLI1 and EZH2 mRNAs in NB-derived EVs. As a consequence of the uptake of NB-derived EVs, the host cells increased the expression levels of GLI1 and EZH2. These results suggest the alteration of the expression profile of stromal cells through an EV-based mechanism, and point the GLI1 and EZH2 mRNAs in the EV cargo as diagnostic biomarkers in NB.

JTD Keywords: exosomes, genes, lines, maintenance, pathway, proliferation, rna, stemness, tumor, Cancer

[Figure: see text].

JTD Keywords: activation, cell extrusion, contraction, drives, homeostasis, interface, junctions, kinase, tension, Adhesion, Article, Cell membranes, Chemical activation, Cytology, E-cadherin, Epithelial monolayers, Epithelium, Homoeostasis, Human, Mechanical instabilities, Monolayers, Morphological transformations, Morphology, Normal tissue, Oncogenics, Soft substrates, Substrates, Tissue, Tissue mechanics, Tissue morphology, Tumor development

Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.

JTD Keywords: 3d cell-derived matrices, adipose mesenchymal stem cells, collagen matrix, colorectal adenocarcinoma, cytotoxicity assay, deposition, expansion, extracellular microenvironment, extracellular-matrix, fibronectin, growth, macromolecular crowders, microcarriers, scaffolds, tissue, 3d cell-derived matrices, Adipose mesenchymal stem cells, Antineoplastic agents, Cell culture techniques, three dimensional, Cell line, tumor, Cell survival, Cytotoxicity assay, Decellularized extracellular matrix, Doxorubicin, Drug resistance, neoplasm, Extracellular microenvironment, Humans, Macromolecular crowders, Mesenchymal stem cells, Mesenchymal stem-cells, Microcarriers, Models, biological, Proof of concept study, Tissue scaffolds, Tumor microenvironment

Multiple targeted therapies are currently explored for pediatric and young adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment. However, this new armamentarium of therapies faces an old problem: choosing the right treatment for each patient. The lack of predictive biomarkers is particularly worrying for pediatric patients since it impairs the implementation of new treatments in the clinic. In this study, we used the functional assay dynamic BH3 profiling (DBP) to evaluate two new treatments for BCP-ALL that could improve clinical outcome, especially for relapsed patients. We found that the MEK inhibitor trametinib and the multi-target tyrosine kinase inhibitor sunitinib exquisitely increased apoptotic priming in an NRAS-mutant and in a KMT2A-rearranged cell line presenting a high expression of FLT3, respectively. Following these observations, we sought to study potential adaptations to these treatments. Indeed, we identified with DBP anti-apoptotic changes in the BCL-2 family after treatment, particularly involving MCL-1 – a pro-survival strategy previously observed in adult cancers. To overcome this adaptation, we employed the BH3 mimetic S63845, a specific MCL-1 inhibitor, and evaluated its sequential addition to both kinase inhibitors to overcome resistance. We observed that the metronomic combination of both drugs with S63845 was synergistic and showed an increased efficacy compared to single agents. Similar observations were made in BCP-ALL KMT2A-rearranged PDX cells in response to sunitinib, showing an analogous DBP profile to the SEM cell line. These findings demonstrate that rational sequences of targeted agents with BH3 mimetics, now extensively explored in clinical trials, may improve treatment effectiveness by overcoming anti-apoptotic adaptations in BCP-ALL.

JTD Keywords: apoptosis, bh3 mimetics, cancer, dependence, increases, kinase inhibition, pediatric leukemia, precision medicine, resistance, sensitivity, targeted therapies, tumor-cells, venetoclax, Apoptosis, Bcl-2 family proteins, Bh3 mimetics, Pediatric leukemia, Resistance, Targeted therapies

Lung cancer is the leading cause of cancer-related death worldwide. The desmoplastic stroma of lung cancer and other solid tumors is rich in tumor-associated fibroblasts (TAFs) exhibiting an activated/myofibroblast-like phenotype. There is growing awareness that TAFs support key steps of tumor progression and are epigenetically reprogrammed compared to healthy fibroblasts. Although the mechanisms underlying such epigenetic reprogramming are incompletely understood, there is increasing evidence that they involve interactions with either cancer cells, pro-fibrotic cytokines such as TGF-β, the stiffening of the surrounding extracellular matrix, smoking cigarette particles and other environmental cues. These aberrant interactions elicit a global DNA hypomethylation and a selective transcriptional repression through hypermethylation of the TGF-β transcription factor SMAD3 in lung TAFs. Likewise, similar DNA methylation changes have been reported in TAFs from other cancer types, as well as histone core modifications and altered microRNA expression. In this review we summarize the evidence of the epigenetic reprogramming of TAFs, how this reprogramming contributes to the acquisition and maintenance of a tumor-promoting phenotype, and how it provides novel venues for therapeutic intervention, with a special focus on lung TAFs.

JTD Keywords: cancer-associated fibroblasts, desmoplasia, dna methylation, epigenetics, expression, genomic dna, lung cancer, mechanical memory, myofibroblast differentiation, pulmonary fibroblasts, smoking, stromal fibroblasts, tgf-?, tgf-beta, tgf-β, transforming growth-factor-beta-1, tumor stroma, Cancer-associated fibroblasts, Carcinoma-associated fibroblasts, Desmoplasia, Epigenetics, Lung cancer, Smoking, Tgf-β, Tumor stroma

For decades, fetal bovine serum (FBS) has been used routinely for culturing many cell types, based on its empirically demonstrated effects on cell growth, and the lack of suitable non-xenogeneic alternatives. The FBS-based culture media do not represent the human physiological conditions, and can compromise biomimicry of preclinical models. To recapitulate in vitro the features of human bone and bone cancer, we investigated the effects of human serum and human platelet lysate on modeling osteogenesis, osteoclastogenesis, and bone cancer in two-dimensional (2D) and three-dimensional (3D) settings. For monitoring tumor growth within tissue-engineered bone in a non-destructive fashion, we generated cancer cell lines expressing and secreting luciferase. Culture media containing human serum enhanced osteogenesis and osteoclasts differentiation, and provided a more realistic in vitro mimic of human cancer cell proliferation. When human serum was used for building 3D engineered bone, the tissue recapitulated bone homeostasis and response to bisphosphonates observed in native bone. We found disparities in cell behavior and drug responses between the metastatic and primary cancer cells cultured in the bone niche, with the effectiveness of bisphosphonates observed only in metastatic models. Overall, these data support the utility of human serum for bioengineering of bone and bone cancers.

JTD Keywords: 3d cancer models, 3rs, alpha tnf-alpha, culture, cypridina luciferase, ewings-sarcoma, ewing’s sarcoma, human platelet lysate, human serum, human tumor, in-vitro, osteogenic differentiation, stem-cells, zoledronic acid, 3d cancer models, 3rs, Cypridina luciferase, Ewing's sarcoma, Ewing’s sarcoma, Fetal bovine serum, Human serum

A new methodology for the pH-triggered degradation of polymers or for the release of drugs under visible light irradiation based on the cyclization of ortho-hydroxy-cinnamates (oHC) to coumarins is described. The key oHC structural motif can be readily incorporated into the rational design of novel photocleavable polymers via click chemistry. This main-chain moiety undergoes a fast photocleavage when irradiated with 455 nm light provided that a suitable base is added. A series of polyethylene glycol-alt-ortho-hydroxy cinnamate (polyethylene glycol (PEG)(n)-alt-oHC)-based polymers are synthesized and the time-dependent visible-light initiated cleavage of the photoactive monomer and polymer is investigated in solution by a variety of spectroscopic and chromatographic techniques. The photo-degradation behavior of the water-soluble poly(PEG(2000)-alt-oHC) is investigated within a broad pH range (pH = 2.1-11.8), demonstrating fast degradation at pH 11.8, while the stability of the polymer is greatly enhanced at pH 2.1. Moreover, the neat polymer shows long-term stability under daylight conditions, thus allowing its storage without special precautions. In addition, two water-soluble PEG-based drug-carrier molecules (mPEG(2000)-oHC-benzhydrol/phenol) are synthesized and used for drug delivery studies, monitoring the process by UV-vis spectroscopy in an ON/OFF intermittent manner.

JTD Keywords: coumarins, drug delivery, e/z-double bond isomerization, o-hydroxy cinnamates, polymer degradation, Aliphatic compounds, Antioxidant activity, Antitumor, Chromatographic techniques, Chromatography, Cis/trans isomerization, Controlled drug delivery, Coumarin derivatives, Coumarins, Drug delivery, Drug delivery system, E/z-double bond isomerization, Films, Hydrogels, Image enhancement, Light, Long term stability, O-hydroxy cinnamates, Particles, Photoactive monomers, Photodegradation, Polyethylene glycols, Polyethylenes, Polymer degradation, Responsive polymers, Salts, Structural motifs, Synthesis (chemical), Targeted drug delivery, Visible light photocatalysis, Visible-light irradiation

Tumor vessel-on-a-chip systems have attracted the interest of the cancer research community due to their ability to accurately recapitulate the multiple dynamic events of the metastatic cascade. Vessel-on-a-chip microfluidic platforms have been less utilized for investigating the distinctive features and functional heterogeneities of tumor-derived vascular networks. In particular, vascular tumors are characterized by the massive formation of thrombi and severe bleeding, a rare and life-threatening situation for which there are yet no clear therapeutic guidelines. This is mainly due to the lack of technological platforms capable of reproducing these characteristic traits of the pathology in a simple and well-controlled manner. Herein, we report the fabrication of a versatile tumor vessel-on-a-chip platform to reproduce, investigate, and characterize the massive formation of thrombi and hemorrhage on-chip in a fast and easy manner. Despite its simplicity, this method offers multiple advantages to recapitulate the pathophysiological events of vascular tumors, and therefore, may find useful applications in the field of vascular-related diseases, while at the same time being an alternative to more complex approaches. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: in vitro model, microfluidics, organ-on-chip, vascular tumor, vessel, In vitro model, Microfluidics, Organ-on-chip, Vascular tumor, Vessel

Cold atmospheric plasma (CAP) is a potential anticancer therapy. CAP has cytotoxic effects when applied either directly to cancer cell cultures or indirectly through plasma-conditioned liquids. This protocol describes how to treat adherent cultures of human cancer cell lines with CAP or plasma-conditioned medium and determine cell viability following treatment. The protocol also includes details on how to quantify the reactive oxygen and nitrogen species present in medium following CAP treatment, using chemical probes using UV-visible or fluorescence spectroscopy. CAP treatment takes ~30 min, and 3 h are required to complete quantification of reactive oxygen and nitrogen species. By providing a standardized protocol for evaluation of the effects of CAP and plasma-conditioned medium, we hope to facilitate the comparison and interpretation of results seen across different laboratories. © 2021, The Author(s), under exclusive licence to Springer Nature Limited.

JTD Keywords: bacteria, decontamination, jet, skin, surface, Cell line, tumor, Humans, Neoplasms, Physical plasma, Plasma gases

© 2020 Wiley-VCH GmbH Cytosolic protein delivery remains elusive. The inability of most proteins to cross the cellular membrane is a huge hurdle. Here we explore the unique photothermal properties of gold nanorods (AuNRs) to trigger cytosolic delivery of proteins. Both partners, protein and AuNRs, are modified with a protease-resistant cell-penetrating peptide with nuclear targeting properties to induce internalization. Once internalized, spatiotemporal control of protein release is achieved by near-infrared laser irradiation in the safe second biological window. Importantly, catalytic amounts of AuNRs are sufficient to trigger cytosolic protein delivery. To the best of our knowledge, this is the first time that AuNRs with their maximum of absorption in the second biological window are used to deliver proteins into the intracellular space. This strategy represents a powerful tool for the cytosolic delivery of virtually any class of protein.

JTD Keywords: cell-penetrating peptide, cytosolic delivery, gold nanorod, near-infrared irradiation, Cell line, tumor, Cell-penetrating peptide, Cytosolic delivery, Gold, Gold nanorod, Metal nanoparticles, Nanotubes, Near-infrared irradiation, Phototherapy

© 2020 The Author(s) The use of oxidative stress generated by Cold Atmospheric Plasma (CAP) in oncology is being recently studied as a novel potential anti-cancer therapy. However, the beneficial effects of CAP for treating osteosarcoma have mostly been demonstrated in 2-dimensional cultures of cells, which do not mimic the complexity of the 3-dimensional (3D) bone microenvironment. In order to evaluate the effects of CAP in a relevant context of the human disease, we developed a 3D tissue-engineered model of osteosarcoma using a bone-like scaffold made of collagen type I and hydroxyapatite nanoparticles. Human osteosarcoma cells cultured within the scaffold showed a high capacity to infiltrate and proliferate and to exhibit osteomimicry in vitro. As expected, we observed significantly different functional behaviors between monolayer and 3D cultures when treated with Cold Plasma-Activated Ringer's Solution (PAR). Our data reveal that the 3D environment not only protects cells from PAR-induced lethality by scavenging and diminishing the amount of reactive oxygen and nitrogen species generated by CAP, but also favours the stemness phenotype of osteosarcoma cells. This is the first study that demonstrates the negative effect of PAR on cancer stem-like cell subpopulations in a 3D biomimetic model of cancer. These findings will allow to suitably re-focus research on plasma-based therapies in future.

JTD Keywords: 3d tumor model, cancer stem-like cells, cold atmospheric plasma, osteosarcoma, oxidative stress, plasma activated liquids, reactive oxygen and nitrogen species, 3d tumor model, Bone neoplasms, Cancer stem-like cells, Cell line, tumor, Cold atmospheric plasma, Humans, Neoplastic stem cells, Osteosarcoma, Oxidative stress, Plasma activated liquids, Plasma gases, Reactive oxygen and nitrogen species, Tumor microenvironment

© 2020 The Authors Although the concept of selective delivery has been postulated over 100 years ago, no targeted nanomedicine has been clinically approved so far. Nanoparticles modified with targeting ligands to promote the selective delivery of therapeutics towards a specific cell population have been extensively reported. However, the rational design of selective particles is still challenging. One of the main reasons for this is the lack of quantitative theoretical and experimental understanding of the interactions involved in cell targeting. In this review, we discuss new theoretical models and experimental methods that provide a quantitative view of targeting. We show the new advancements in multivalency theory enabling the rational design of super-selective nanoparticles. Furthermore, we present the innovative approaches to obtain key targeting parameters at the single-cell and single molecule level and their role in the design of targeting nanoparticles. We believe that the combination of new theoretical multivalent design and experimental methods to quantify receptors and ligands aids in the rational design and clinical translation of targeted nanomedicines.

JTD Keywords: binding-kinetics, biological identity, biomolecular corona, blood-brain-barrier, drug-delivery, gold nanoparticles, multivalency, nanotechnology, protein corona, quantitative characterization, rational design, super-selectivity, superresolution microscopy, tumor heterogeneity, Ligand-receptor interactions, Multivalency, Nanotechnology, Quantitative characterization, Rational design, Super-selectivity

Osteosarcoma is the most common primary bone tumor, and its first line of treatment presents a high failure rate. The 5-year survival for children and teenagers with osteosarcoma is 70% (if diagnosed before it has metastasized) or 20% (if spread at the time of diagnosis), stressing the need for novel therapies. Recently, cold atmospheric plasmas (ionized gases consisting of UV-Vis radiation, electromagnetic fields and a great variety of reactive species) and plasma-treated liquids have been shown to have the potential to selectively eliminate cancer cells in different tumors through an oxidative stress-dependent mechanism. In this work, we review the current state of the art in cold plasma therapy for osteosarcoma. Specifically, we emphasize the mechanisms unveiled thus far regarding the action of plasmas on osteosarcoma. Finally, we review current and potential future approaches, emphasizing the most critical challenges for the development of osteosarcoma therapies based on this emerging technique.

JTD Keywords: cancer stem cells, cold atmospheric plasma, osteosarcoma, oxidative stress, plasma treated liquids, reactive oxygen and nitrogen species, Antineoplastic activity, Antineoplastic agent, Cancer chemotherapy, Cancer stem cell, Cancer stem cells, Cancer surgery, Cancer survival, Cell therapy, Cold atmospheric plasma, Cold atmospheric plasma therapy, Electromagnetism, Human, In vitro study, Intracellular signaling, Oncogene, Osteosarcoma, Oxidative stress, Plasma treated liquids, Reactive nitrogen species, Reactive oxygen and nitrogen species, Reactive oxygen metabolite, Review, Tumor microenvironment

© 2020 Elsevier B.V. Resveratrol and artemisinin, two naturally occurring compounds with a wide range of biological activities, have been reported to exert antitumor effects against several types of cancer. In this work, Eudragit-coated liposomes were developed to safely transport resveratrol and artemisinin through the gastrointestinal tract and target the intestine. The physico-chemical properties of the Eudragit-coated liposomes were assessed by light scattering and cryogenic transmission electron microscopy. Nanosized (around 100 nm), spherical or elongated, unilamellar vesicles were produced. The protective effect of the Eudragit coating was confirmed by assessing the physical stability of the vesicles in fluids mimicking the gastrointestinal environment. Furthermore, the vesicles were found to exert a pro-oxidant activity in intestinal adenocarcinoma cells, which resulted in a marked mortality due to the generation of reactive oxygen species (ROS). A time- and dose-dependent cell growth inhibitory effect was detected, with elevated ROS levels when resveratrol and artemisinin were combined. Therefore, the proposed formulations may represent a valuable means to counteract intestinal tumor growth.

JTD Keywords: antitumor, artemisinin, eudragit, intestinal delivery, liposomes, Antitumor, Artemisinin, Eudragit, Intestinal delivery, Liposomes, Resveratrol

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell–cell interactions and cell–matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.

JTD Keywords: 3d spheroid invasion, cancer associated fibroblasts, collective migration, dissemination, epithelial cancer cells, leader/follower cells, 3d spheroid invasion, Cancer associated fibroblasts, Cancer-associated fibroblasts, Cell culture techniques, Cell line, tumor, Cell movement, Cell tracking, Collective invasion, Collective migration, Epithelial cancer cells, Extracellular matrix, Humans, Imaging, three-dimensional, Leader/follower cells, Microscopy, fluorescence, Spheroids, cellular, Tumor cells, cultured

Goel and colleagues show that CDK4/6 inhibition induces global chromatin changes mediated by AP-1 factors, which mediate key biological and clinical effects in breast cancer. Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that are enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several activator protein-1 transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.

JTD Keywords: Abemaciclib, Androgen receptor, Animal experiment, Animal model, Animal tissue, Apoptosis, Article, Breast cancer, C-jun, Cancer cell, Carcinoembryonic antigen related cell adhesion molecule 1, Caspase 3, Cell cycle arrest, Cells, Chromatin, Chromatin immunoprecipitation, Controlled study, Cyclin dependent kinase 4, Cyclin dependent kinase 6, Dna damage, Epidermal growth factor receptor 2, Estrogen receptor, Female, Flow cytometry, Fulvestrant, Hla drb1 antigen, Human, Human cell, Immunoblotting, Immunogenicity, Immunoprecipitation, Interferon, Luciferase assay, Mcf-7 cell line, Mda-mb-231 cell line, Microarray analysis, Morphogenesis, Mouse, Nonhuman, Palbociclib, Protein, Protein expression, Rb, Resistance, Rna polymerase ii, Rna sequence, Selective-inhibition, Senescence, Short tandem repeat, Signal transduction, Tamoxifen, Transcription elongation, Transcription factor, Transcription factor ap 1, Transcriptome, Tumor biopsy, Tumor differentiation, Tumor spheroid, Tumor xenograft, Vinculin, Whole exome sequencing

Intratumour heterogeneity due to cancer cell clonal evolution and microenvironment composition and tumor differences due to genetic variations between patients suffering of the same cancer pathology play a crucial role in patient response to therapies. This study is oriented to show that matrix-assisted laser-desorption ionization-Mass spectrometry imaging (MALDI-MSI), combined with an advanced multivariate data processing pipeline can be used to discriminate subtle variations between highly similar colorectal tumors. To this aim, experimental tumors reproducing the emergence of drug-resistant clones were generated in athymic mice using subcutaneous injection of different mixes of two isogenic cell lines, the irinotecan-resistant HCT116-SN50 (R) and its sibling human colon adenocarcinoma sensitive cell line HCT116 (S). Because irinotecan-resistant and irinotecan-sensitive are derived from the same original parental HCT116 cell line, their genetic characteristics and molecular compositions are closely related. The multivariate data processing pipeline proposed relies on three steps: (a) multiset multivariate curve resolution (MCR) to separate biological contributions from background; (b) multiset K-means segmentation using MCR scores of the biological contributions to separate between tumor and necrotic parts of the tissues; and (c) partial-least squares discriminant analysis (PLS-DA) applied to tumor pixel spectra to discriminate between R and S tumor populations. High levels of correct classification rates (0.85), sensitivity (0.92) and specificity (0.77) for the PLS-DA classification model were obtained. If previously labelled tissue is available, the multistep modeling strategy proposed constitutes a good approach for the identification and characterization of highly similar phenotypic tumor subpopulations that could be potentially applicable to any kind of cancer tissue that exhibits substantial heterogeneity. © 2019 Elsevier B.V.

JTD Keywords: Chemometrics, Colorectal cancer, MALDI imaging, Multivariate analysis, Tumor heterogeneity

Cancer represents one of the conditions with the most causes of death worldwide. Common methods for its diagnosis are based on tissue biopsies—the extraction of tissue from the primary tumor, which is used for its histological analysis. However, this technique represents a risk for the patient, along with being expensive and time-consuming and so it cannot be frequently used to follow the progress of the disease. Liquid biopsy is a new cancer diagnostic alternative, which allows the analysis of the molecular information of the solid tumors via a body fluid draw. This fluid-based diagnostic method displays relevant advantages, including its minimal invasiveness, lower risk, use as often as required, it can be analyzed with the use of microfluidic-based platforms with low consumption of reagent, and it does not require specialized personnel and expensive equipment for the diagnosis. In recent years, the integration of sensors in microfluidics lab-on-a-chip devices was performed for liquid biopsies applications, granting significant advantages in the separation and detection of circulating tumor nucleic acids (ctNAs), circulating tumor cells (CTCs) and exosomes. The improvements in isolation and detection technologies offer increasingly sensitive and selective equipment’s, and the integration in microfluidic devices provides a better characterization and analysis of these biomarkers. These fully integrated systems will facilitate the generation of fully automatized platforms at low-cost for compact cancer diagnosis systems at an early stage and for the prediction and prognosis of cancer treatment through the biomarkers for personalized tumor analysis.

JTD Keywords: Cancer, Circulant tumor cells (CTC), Circulant tumor DNA (ctDNA), Exosomes, Liquid biopsy, Microfluidic, Sensors

Neuroblastoma (NB) is a malignant tumor that affects the peripheral nervous system and represents one of the most frequent cancers in infants. Its prognosis is poor in older patients and the presence of genetic abnormalities. Metastasis is often present at the time of diagnosis, making treatment more intensive and unsuccessful. Poor prognosis and variable treatment efficacy require a better understanding of the underlying biology. Evidence has shown that the tumor microenvironment is the characteristic of tumor malignancy and progression. A more highly differentiated tissue phenotype represents a positive prognostic marker, while the tumoral tissue is characterized by a distinct composition and morphology of the extracellular matrix (ECM). In this chapter, we discuss the application of decellularized cell-derived matrices (CDMs) to model in vitro the morphology of the ECM encountered in histological hallmarks of NB patients. This technique allows for the in vitro reproduction of the fine structure and composition of native microenvironments. Because of recent advances in culture systems and decellularization techniques, it is possible to engineer CDM composition and microarchitecture to produce differentiated models of tissue niches. The final goal is to repopulate the “scaffold” with malignant NB cells for drug screening and target discovery applications, studying the impact of patient-inspired tissues on signaling, migration, and tissue remodeling.

JTD Keywords: Neuroblastoma, Cancer, Bioengineering, Tumor microenvironment, Cell-derived matrices, Decellularization

Hypoxia is a common characteristic of many solid tumors that has been associated with tumor aggressiveness. Limited diffusion of oxygen generates a gradient of oxygen availability from the blood vessel to the interstitial space and may underlie the recruitment of macrophages fostering cancer progression. However, the available data based on the recruitment of circulating cells to the tumor microenvironment has been so far carried out by conventional co-culture systems which ignore the hypoxic gradient between the vessel to the tumor interstitium. Here, we have designed a novel easy-to-build cell culture device that enables evaluation of cellular cross-talk and cell migration while they are being simultaneously exposed to different oxygenation environments. As a proof-of-concept of the potential role of differential oxygenation among interacting cells we have evaluated the activation and recruitment of macrophages in response to hypoxic melanoma, breast, and kidney cancer cells. We found that hypoxic melanoma and breast cancer cells co-cultured with normoxic macrophages enhanced their directional migration. By contrast, hypoxic kidney cells were not able to increase their recruitment. We also identified well-described hypoxia-induced pathways which could contribute in the immune cell recruitment (VEGFA and PTGS2 genes). Moreover, melanoma and breast cancer increased their proliferation. However, oxygenation levels affected neither kidney cancer cell proliferation nor gene expression, which in turn resulted in no significant changes in macrophage migration and polarization. Therefore, the cell culture device presented here provides an excellent opportunity for researchers to reproduce the in vivo hypoxic gradients in solid tumors and to study their role in recruiting circulating cells to the tumor in specific types of cancer.

JTD Keywords: Hypoxia gradient, Macrophage motility, Models of host-tumor interactions, Novel assay technology, Tumor progression

Cancer is one of the greatest threats facing our society, being the second leading cause of death globally. Currents strategies for cancer diagnosis consist of the extraction of a solid tissue from the affected area. This sample enables the study of specific biomarkers and the genetic nature of the tumor. However, the tissue extraction is risky and painful for the patient and in some cases is unavailable in inaccessible tumors. Moreover, a solid biopsy is expensive and time consuming and cannot be applied repeatedly. New alternatives that overcome these drawbacks are rising up nowadays, such as liquid biopsy. A liquid biopsy is the analysis of biomarkers in a non-solid biological tissue, mainly blood, which has remarkable advantages over the traditional method; it has no risk, it is non-invasive and painless, it does not require surgery and reduces cost and diagnosis time. The most studied cancer non-invasive biomarkers are circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes. These circulating biomarkers play a key role in the understanding of metastasis and tumorigenesis, which could provide a better insight into the evolution of the tumor dynamics during treatment and disease progression. Improvements in isolation technologies, based on a higher grade of purification of CTCs, exosomes, and ctDNA, will provide a better characterization of biomarkers and give rise to a wide range of clinical applications, such as early detection of diseases, and the prediction of treatment responses due to the discovery of personalized tumor-related biomarkers

JTD Keywords: Liquid biopsy, Cancer, Biomarkers, Non-invasive, Circulant tumor DNA (ctDNA), Circulant tumor cells (CTC)