Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy.

BACKGROUND: Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. METHODS: NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. RESULTS: We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. CONCLUSIONS: These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.

Spatial Transcriptomics: Emerging Technologies in Tissue Gene Expression Profiling.

In this Perspective, we discuss the current status and advances in spatial transcriptomics technologies, which allow high-resolution mapping of gene expression in intact cell and tissue samples. Spatial transcriptomics enables the creation of high-resolution maps of gene expression patterns within their native spatial context, adding an extra layer of information to the bulk sequencing data. Spatial transcriptomics has expanded significantly in recent years and is making a notable impact on a range of fields, including tissue architecture, developmental biology, cancer, and neurodegenerative and infectious diseases. The latest advancements in spatial transcriptomics have resulted in the development of highly multiplexed methods, transcriptomic-wide analysis, and single-cell resolution utilizing diverse technological approaches. In this Perspective, we provide a detailed analysis of the molecular foundations behind the main spatial transcriptomics technologies, including methods based on microdissection, in situ sequencing, single-molecule FISH, spatial capturing, selection of regions of interest, and single-cell or nuclei dissociation. We contextualize the detection and capturing efficiency, strengths, limitations, tissue compatibility, and applications of these techniques as well as provide information on data analysis. In addition, this Perspective discusses future directions and potential applications of spatial transcriptomics, highlighting the importance of the continued development to promote widespread adoption of these techniques within the research community.

N-aryltetrahydroisoquinoline derivatives as HA-CD44 interaction inhibitors: Design, synthesis, computational studies, and antitumor effect.

Hyaluronic acid (HA) plays a crucial role in tumor growth and invasion through its interaction with cluster of differentiation 44 (CD44), a non-kinase transmembrane glycoprotein, among other hyaladherins. CD44 expression is elevated in many solid tumors, and its interaction with HA is associated with cancer and angiogenesis. Despite efforts to inhibit HA-CD44 interaction, there has been limited progress in the development of small molecule inhibitors. As a contribution to this endeavour, we designed and synthesized a series of N-aryltetrahydroisoquinoline derivatives based on existing crystallographic data available for CD44 and HA. Hit 2e was identified within these structures for its antiproliferative effect against two CD44+ cancer cell lines, and two new analogs (5 and 6) were then synthesized and evaluated as CD44-HA inhibitors by applying computational and cell-based CD44 binding studies. Compound 2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-5-ol (5) has an EC50 value of 0.59 µM against MDA-MB-231 cells and is effective to disrupt the integrity of cancer spheroids and reduce the viability of MDA-MB-231 cells in a dose-dependent manner. These results suggest lead 5 as a promising candidate for further investigation in cancer treatment.

Direct detection of alpha satellite DNA with single-base resolution by using abasic Peptide Nucleic Acids and Fluorescent in situ Hybridization.

The detection of repetitive sequences with single-base resolution is becoming increasingly important aiming to understand the biological implications of genomic variation in these sequences. However, there is a lack of techniques to experimentally validate sequencing data from repetitive sequences obtained by Next-Generation Sequencing methods, especially in the case of Single-Nucleotide Variations (SNVs). That is one of the reasons why repetitive sequences have been poorly studied and excluded from most genomic studies. Therefore, in addition to sequencing data, there is an urgent need for efficient validation methods of genomic variation in these sequences. Herein we report the development of chemFISH, an alternative method for the detection of SNVs in repetitive sequences. ChemFISH is an innovative method based on dynamic chemistry labelling and abasic Peptide Nucleic Acid (PNA) probes to detect in situ the α-satellite DNA, organized in tandem repeats, with single-base resolution in a direct and rapid reaction. With this approach, we detected by microscopy the α-satellite DNA in a variety of human cell lines, we quantified the detection showing a low coefficient of variation among samples (13.16%-25.33%) and we detected single-base specificity with high sensitivity (82.41%-88.82%). These results indicate that chemFISH can serve as a rapid method to validate previously detected SNVs in sequencing data, as well as to find novel SNVs in repetitive sequences. Furthermore, the versatile chemistry behind chemFISH can lead to develop novel molecular assays for the in situ detection of nucleic acids.

Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells.

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis-the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.

Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding.

Barcoding and pooling cells for processing as a composite sample are critical to minimize technical variability in multiplex technologies. Fluorescent cell barcoding has been established as a standard method for multiplexing in flow cytometry analysis. In parallel, mass-tag barcoding is routinely used to label cells for mass cytometry. Barcode reagents currently used label intracellular proteins in fixed and permeabilized cells and, therefore, are not suitable for studies with live cells in long-term culture prior to analysis. In this study, we report the development of fluorescent palladium-based hybrid-tag nanotrackers to barcode live cells for flow and mass cytometry dual-modal readout. We describe the preparation, physicochemical characterization, efficiency of cell internalization, and durability of these nanotrackers in live cells cultured over time. In addition, we demonstrate their compatibility with standardized cytometry reagents and protocols. Finally, we validated these nanotrackers for drug response assays during a long-term coculture experiment with two barcoded cell lines. This method represents a new and widely applicable advance for fluorescent and mass-tag barcoding that is independent of protein expression levels and can be used to label cells before long-term drug studies.

Selective Anticancer Therapy Based on a HA-CD44 Interaction Inhibitor Loaded on Polymeric Nanoparticles.

Hyaluronic acid (HA), through its interactions with the cluster of differentiation 44 (CD44), acts as a potent modulator of the tumor microenvironment, creating a wide range of extracellular stimuli for tumor growth, angiogenesis, invasion, and metastasis. An innovative antitumor treatment strategy based on the development of a nanodevice for selective release of an inhibitor of the HA-CD44 interaction is presented. Computational analysis was performed to evaluate the interaction of the designed tetrahydroisoquinoline-ketone derivative (JE22) with CD44 binding site. Cell viability, efficiency, and selectivity of drug release under acidic conditions together with CD44 binding capacity, effect on cell migration, and apoptotic activity were successfully evaluated. Remarkably, the conjugation of this CD44 inhibitor to the nanodevice generated a reduction of the dosis required to achieve a significant therapeutic effect.

Nanomedicine as a Promising Tool to Overcome Immune Escape in Breast Cancer.

Breast cancer is the most common type of malignancy and leading cause of cancer death among women worldwide. Despite the current revolutionary advances in the field of cancer immunotherapy, clinical response in breast cancer is frequently below expectations, in part due to various mechanisms of cancer immune escape that produce tumor variants that are resistant to treatment. Thus, a further understanding of the molecular events underlying immune evasion in breast cancer may guarantee a significant improvement in the clinical success of immunotherapy. Furthermore, nanomedicine provides a promising opportunity to enhance the efficacy of cancer immunotherapy by improving the delivery, retention and release of immunostimulatory agents in targeted cells and tumor tissues. Hence, it can be used to overcome tumor immune escape and increase tumor rejection in numerous malignancies, including breast cancer. In this review, we summarize the current status and emerging trends in nanomedicine-based strategies targeting cancer immune evasion and modulating the immunosuppressive tumor microenvironment, including the inhibition of immunosuppressive cells in the tumor area, the activation of dendritic cells and the stimulation of the specific antitumor T-cell response.

Development of a nanotechnology-based approach for capturing and detecting nucleic acids by using flow cytometry.

Nucleic acid-based molecular diagnosis has gained special importance for the detection and early diagnosis of genetic diseases as well as for the control of infectious disease outbreaks. The development of systems that allow for the detection and analysis of nucleic acids in a low-cost and easy-to-use way is of great importance. In this context, we present a combination of a nanotechnology-based approach with the already validated dynamic chemical labeling (DCL) technology, capable of reading nucleic acids with single-base resolution. This system allows for the detection of biotinylated molecular products followed by simple detection using a standard flow cytometer, a widely used platform in clinical and molecular laboratories, and therefore, is easy to implement. This proof-of-concept assay has been developed to detect mutations in KRAS codon 12, as these mutations are highly important in cancer development and cancer treatments.

An effective polymeric nanocarrier that allows for active targeting and selective drug delivery in cell coculture systems.

In this manuscript, we report the development of a versatile, robust, and stable targeting nanocarrier for active delivery. This nanocarrier is based on bifunctionalized polymeric nanoparticles conjugated to a monoclonal antibody that allows for active targeting of either (i) a fluorophore for tracking or (ii) a drug for monitoring specific cell responses. This nanodevice can efficiently discriminate between cells in coculture based on the expression levels of cell surface receptors. As a proof of concept, we have demonstrated efficient delivery using a broadly established cell surface receptor as the target, the epidermal growth factor receptor (EGFR), which is overexpressed in several types of cancers. Additionally, a second validation of this nanodevice was successfully carried out using another cell surface receptor as the target, the cluster of differentiation 147 (CD147). Our results suggest that this versatile nanocarrier can be expanded to other cell receptors and bioactive cargoes, offering remarkable discrimination efficiency between cells with different expression levels of a specific marker. This work supports the ability of nanoplatforms to boost and improve the progress towards personalized medicine.

Mass Cytometry Tags: Where Chemistry Meets Single-Cell Analysis.

Mass cytometry is a highly multiparametric proteomic technology that allows the measurement and quantification of nearly 50 markers with single-cell resolution. Mass cytometry reagents are probes tagged with metal isotopes of defined mass and act as reporters. Metals are detected using inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS). Many different types of mass-tag reagents have been developed to afford myriad applications. We have classified these compounds into polymer-based mass-tag reagents, nonpolymer-based mass-tag reagents, and inorganic nanoparticles. Metal-chelating polymers (MCPs) are widely used to profile and quantify cellular biomarkers; however, both the range of metals that can be detected and the metal signals have to be improved. Several strategies such as the inclusion of chelating agents or highly branched polymers may overcome these issues. Biocompatible materials such as polystyrene and inorganic nanoparticles are also of profound interest in mass cytometry. While polystyrene allows the inclusion of a wide variety of metals, the high metal content of inorganic nanoparticles offers an excellent opportunity to increase the signal from the metals to detect low-abundance biomarkers. Nonpolymer-based mass-tag reagents offer multiple applications: cell detection, cell cycle property determination, biomarker detection, and mass-tag cellular barcoding (MCB). Recent developments have been achieved in live cell barcoding by targeting proteins (CD45, b2m, and CD298), by using small and nonpolar probes or by ratiometric barcoding. From this perspective, the principal applications, strengths, and shortcomings of mass-tag reagents are highlighted, summarized, and discussed, with special emphasis on mass-tag reagents for MCB. Thereafter, the future perspectives of mass-tag reagents are discussed considering the current state-of-the-art technologies.

Amplification-free profiling of microRNA-122 biomarker in DILI patient serums, using the luminex MAGPIX system.

Dynamic chemical labelling is a single-base specific method to enable detection and quantification of micro-Ribonucleic Acids in biological fluids without extraction and pre-amplification. In this study, dynamic chemical labelling was combined with the Luminex MAGPIX system to profile levels of microRNA-122 biomarker in serum from patients with Drug-Induced Liver Injury.

A versatile theranostic nanodevice based on an orthogonal bioconjugation strategy for efficient targeted treatment and monitoring of triple negative breast cancer.

A novel chemical-based orthogonal bioconjugation strategy to produce tri-functionalized nanoparticles (NPs) an chemotherapy drug, doxorubicin (DOX), a near-infrared cyanine dye (Cy7) and CRGDK homing peptide, a peptide specifically binds to neuropilin-1 (Nrp-1) overexpressed on triple negative breast cancer (TNBC) cells, has been validated. These theranostic NPs have been evaluated in vitro and in vivo using an orthotopic xenotransplant mouse model using TNBC cells. In vitro assays show that theranostic NPs improve the therapeutic index in comparison with free DOX. Remarkably, in vivo studies showed preferred location of theranostic NPs in the tumor area reducing the volume at the same level than free DOX while presenting lower side effects. This multifunctionalized theranostic nanodevice based on orthogonal conjugation strategies could be a good candidate for the treatment and monitoring of Nrp-1 overexpressing tumors. Moreover, this versatile nanodevice can be easily adapted to treat and monitor different cancer types by adapting the conjugation strategy.

PCR-free and chemistry-based technology for miR-21 rapid detection directly from tumour cells.

miRNAs are well known for being implicated in a myriad of biological situations, including those related to serious diseases. Amongst miRNAs, miRNA-21 has the spotlight as it is reported to be up-regulated in multiple severe pathological conditions, being its quantification a key point in medicine. To date, most of the techniques for miRNA quantification have shown to be less effective than expected; thus, we herein present a novel, rapid, cost-effective, robust and PCR-free approach, based on dynamic chemistry, for the identification and quantification of miRNA directly from tumour cells using both FACS and a fluorescent microplate. This dynamic chemistry novel application involves bead based reagents and allows quantifying the number of miR-21 molecules presented in MDA-MB-468 and H1975 tumour cells.

A PCR-free technology to detect and quantify microRNAs directly from human plasma.

A novel sensitive, specific and rapid method for the detection and quantification of microRNAs without requiring extraction from their biological sources is now available using a novel chemical based, PCR-free technology for nucleic acid testing. In this study, we both demonstrate how this method can be used to profile miR-451a, an important miRNA in erythropoiesis, and compare with the gold standard RT-qPCR.

Drug "Clicking" on Cell-Penetrating Fluorescent Nanoparticles for In Cellulo Chemical Proteomics.

Chemical proteomics approaches are widely used to identify molecular targets of existing or novel drugs. This manuscript describes the development of a straightforward approach to conjugate azide-labeled drugs via click chemistry to alkyne-tagged cell-penetrating fluorescent nanoparticles as a novel tool to study target engagement and/or identification inside living cells. A modification of the Baeyer test for alkynes allows monitoring the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, guaranteeing the presence of the drug on the solid support. As a proof of concept, the conjugation of the promiscuous kinase inhibitor dasatinib to Cy5-labeled nanoparticles is presented. Dasatinib-decorated fluorescent nanoparticles efficiently inhibited its protein target SRC in vitro, entered cancer cells, and colocalized with SRC in cellulo.

Metallofluorescent Nanoparticles for Multimodal Applications.

Herein, we describe the synthesis and application of cross-linked polystyrene-based dual-function nano- and microparticles containing both fluorescent tags and metals. Despite containing a single dye, these particles exhibit a characteristic dual-band fluorescence emission. Moreover, these particles can be combined with different metal ions to obtain hybrid metallofluorescent particles. We demonstrate that these particles are easily nanofected into living cells, allowing them to be used for effective fingerprinting in multimodal fluorescence-based and mass spectrometry-based flow cytometry experiments. Likewise, the in situ reductions of the metal ions enable other potential uses of the particles as heterogeneous catalysts.

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial-scale recombinant production, purification and full characterization, including X-ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross-infection.

Number of Nanoparticles per Cell through a Spectrophotometric Method - A key parameter to Assess Nanoparticle-based Cellular Assays.

Engineered nanoparticles (eNPs) for biological and biomedical applications are produced from functionalised nanoparticles (NPs) after undergoing multiple handling steps, giving rise to an inevitable loss of NPs. Herein we present a practical method to quantify nanoparticles (NPs) number per volume in an aqueous suspension using standard spectrophotometers and minute amounts of the suspensions (up to 1 µL). This method allows, for the first time, to analyse cellular uptake by reporting NPs number added per cell, as opposed to current methods which are related to solid content (w/V) of NPs. In analogy to the parameter used in viral infective assays (multiplicity of infection), we propose to name this novel parameter as multiplicity of nanofection.

miRNA in situ hybridization in circulating tumor cells--MishCTC.

Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.