In recent decades, increasing interest in the use of natural products in anticancer therapy field has been observed, mainly due to unsolved drug-resistance problems. The antitumoral effect of natural compounds involving different signaling pathways and cellular mechanisms has been largely demonstrated in in vitro and in vivo studies. The encapsulation of natural products into different delivery systems may lead to a significant enhancement of their anticancer efficacy by increasing in vivo stability and bioavailability, reducing side adverse effects and improving target-specific activity. This review will focus on research studies related to nanostructured systems containing natural compounds for new drug delivery tools in anticancer therapies. Cancer is among the leading causes of death worldwide. The number of new cancer cases per year is expected to rise to 23.6 million by 2030 . In EuroIn recent years a better understanding of the mechanisms underlying tumor biology led to a significant progress in the prevention, detection and treatment of cancer. Traditional approaches for cancer treatment include surgery, radiation therapy, chemotherapy, immunotherapy and targeted and hormone therapy . UnfortuDrug resistance is still a great problem in cancer therapy. Resistance can be intrinsic or acquired when there is low or no response to anticancer therapy from the beginning or during the course of therapy, respectively. One-drug resistance refers to resistance to one specific drug; when patients show resistance to one drug and become resistant to other unrelated drugs (with different structures and mechanisms of action), the term multidrug resistance (MDR) is employed .A number of factors can contribute to clinical MDR in cancer: factors related to the host as well as factors strictly associated to tumor mass and its interaction with surrounding tissues  6]..6].Among host factors, genetic variants, interactions between different drugs and alteration in the pharmacokinetics are the most important. The host genetic variants are mainly related to genes encoding drug-metabolizing enzymes, drug uptake/efflux transporters, drug targets, DNA repair and cell cycle control mechanisms . When diAmong tumor factors, alterations in the intracellular drug concentration is one of the most important. The amount of a drug accumulated in drug-resistant cells is usually reduced with respect to sensitive cells. This event can be due to a reduced drug diffusion/permeability or to the activity of transmembrane proteins belonging to the ATP-binding cassette (ABC) transporter superfamily. These proteins are often overexpressed and are able to extrude structurally different compounds across plasma and intracellular membranes, through an active mechanism coupled to ATP hydrolysis . In diffOther tumor factors that can exert a pivotal role in inducing clinical MDR are: (1) deregulation of cell death mechanisms ; (2) alteration of DNA damage response and repair mechanisms; (3) epigenetic modifications ; (4) tumor microenvironment (TME) .TME is a dynamic network of cancer cells, extracellular matrix components and stromal cells . TME is characterized by a reduced supply of nutrients and oxygen and low pH. Hypoxic conditions contribute to the reduced development of vessels within the tumor mass, thus causing a decreased drug amount in target sites and, consequently, drug efficacy. All these elements strongly suggest that TME plays a crucial role in cell response to chemo, radio and targeted therapies . AnotherCancer stem cells (CSCs), initially identified in AML and later in solid tumors , represent a very small subpopulation in a tumor and possess the ability of self-renewal and to form new tumors in nude mice . CSC bioIn addition to stemness and tumor heterogeneity, epithelial-to-mesenchymal transition (EMT) has been associated with resistance to anticancer therapies, although a direct correlation has been demonstrated mostly in cellular and preclinical models and, to a lesser extent, in clinical samples. During tumor progression, EMT is responsible for the coexistence of different cellular phenotypes showing both epithelial and mesenchymal features: cancer cells tend to lose intercellular contacts, as well as apical-basal polarity, detach from extracellular matrix components and acquire mesenchymal features . Thus, these hybrid cells can also show invasive and stem cell-like features, be adaptable to microenvironment changes and contribute to the onset of tumor resistance to anticancer therapy and immune escape .Despite the positive outcomes in the field of cancer research, considering all the above factors responsible for limited chemotherapy success, a lot of studies are continuously concerned to develop more efficient therapeutic strategies, in order to selectively target cancer cells, avoid a MDR response, overcome biological barriers and achieve a spatial, temporal and dose control of drug release.Natural products, either in their naturally occurring forms or their synthetically modified forms, have been demonstrated over 40 years to be an important source for cancer preventive and chemotherapeutic agents. Indeed, considering the period between the 1940s and the end of 2014, almost 50% of all small molecules approved for cancer therapy is represented by natural products or compounds that are directly developed from them . UnfortuNanomedicine  has receNanoparticles (NPs) or nanocarries (NCs) used in medicines are increasingly considered as potential candidates to safely carry therapeutic agents into targeted compartments in an organ, particular tissue or cell. NPs are a broad class of materials intended for a broad spectrum of clinical applications  . A numbeThe base knowledge of NPs and the assessment of their safety and efficacy are still expanding. A wide variety of classes are being investigated as nanocarriers for cancer treatment, including lipid-based, polymer-based, inorganic nanoparticles and drug-conjugated nanoparticles .Some of these tools have also been approved for use in clinical protocols . In this®) [®, recently approved by U.S. Food and Drug Administration [Liposomes and lipid nanocapsules (LNCs) are two nanodelivery systems based on lipids. Liposomes, constituted by a hydrophilic “core” surrounded by lipid bilayers, are able to incorporate hydrophobic drugs within the lipid bilayer or hydrophilic drugs within the aqueous core . Since t®) . A triggstration ,35.Another type of lipid-mediated delivery system is represented by lipid nanocapsules (LNCs) that are surrounded by a membrane composed of PEGylated surfactants . The encIn multicellular organisms, cells communicate via extracellular molecules such as nucleotides, lipids, short peptides and proteins. Released by cells, these molecules can bind to receptors of other cells, inducing intracellular signaling and physiological modification of the recipient cells. In addition to these single molecules, eukaryotic cells also release biological micro- and nano-structures in the extracellular environment, called membrane vesicles, containing lipids, proteins and even nucleic acids, which affect the encounter cells in a complex way . ExosomeExosomes are secreted by most cell types  in normal and pathological conditions. They have been found in cell culture media, in different biological ,42,43,44The fundamental characteristics of a system for an efficient administration of drugs are the control and precision of the process. The fast release of drugs would result in their incomplete absorption, gastrointestinal disorders and other side effects. It is also necessary that the drugs do not decompose during administration, as some components may be toxic on their own. Therefore, the drugs should be encapsulated within carriers made of compatible materials, in which they maintain their biological and chemical properties. Furthermore, carriers should decompose by dissolving in the solution or by excretory routes at the end of transport.Carbon nanomaterials are mainly composed of carbon, usually in the form of hollow spheres, ellipsoids or tubes. Studies aimed at carbon nanomaterials have led to the discovery and development of carbon nanotubes (CNTs), which are made of graphite sheets  rolled up to form a cylindrical structure. The diameter of a nanotube is between 0.7 and 30 nm . CNTs are very interesting due to their mechanical strength and electrical properties . In addiRecent discoveries on graphene, a two-dimensional, crystalline allotrope of carbon, stimulated research on related structures, such as pristine Graphite NanoPlatelets (GNPs), a 1–15 nm thick flake, constituted of 3–48 layers of graphene, obtained from Intercalated Graphite Compounds (GIC) via thermochemical exfoliation . These nThe use of graphene in therapeutics delivery is based on its high surface-area to volume ratio; polyaromatic structure can be functionalized, improving targeting to tissues. Moreover, the possibility to combine hydrophobic and hydrophilic regions on the surface of GNPs supports their interaction with lipids in cell membranes . RecentlOne of the earliest reports of graphene-based cancer drug delivery was by Yang et al. They obtained a graphene oxide-doxorubicin hydrochloride nanohybrid (GO-DXR) through a simple noncovalent method. Results showed an efficient high loading and pH-dependent release of the drug on GO, opening these novel GO-based nanohybrids for applications as drug carriers and biosensors . Jokar ePolymeric nanoparticles (NPs) are systems composed by natural, synthetic or semisynthetic polymers. The main features related to polymeric nanoparticles employment are the limited shape and wide size distribution. Polymeric NP are typically spherical, although their final size can be modulated during the synthesis process. Moreover, polymeric NP properties, as well as the amount, rate and pathway used for cellular uptake of the encapsulated drug molecule, may be modified in order to improve the final product . As promPolymeric nanoparticles of natural origins are mainly made by polysaccharides, such as cellulose and lisozima . NaturalPolymers utilized for nanoparticles of synthetic origin production are totally artificial, such as PLGA, PLA (Poly-lactic acid) and PMA (thiolated poly (methacrylic acid) ,75. AmonVarious forms of inorganic nanoparticles, including superparamagnetic iron oxides, gold nanoparticles and other metallic and non-metallic nanoparticles or nanoclusters, enhance the efficiency of radiotherapy and improve tumor imaging. Several of these inorganic nanoparticles  are sufficiently small (10–100 nm) to penetrate in distinct tissues through capillaries ,84. Othe3O4) nanoparticles are under development as possible new strategies to deliver conjugated drugs using a local hyperthermia or oscillation strategies to targeted area. Moreover, magnetic fields can also be used to guide the drug within the body [Such devices can contain the drug and also specific receptor-targeting agents, as antibodies or ligands, as well as magnetic resonance imaging contrast agents . In earlthe body .The application of nanotechnology to drug delivery has an enormous potential concerning the improvement of selectivity in targeting neoplastic cells. Thanks to the enhanced permeability and retention (EPR) effect in tumor and inflamed tissue vasculature, chemotherapeutic drugs can be selectively delivered in specific regions . ScarcelThe EPR effect is based on two factors: the first exploits the fact that the endothelium around the blood vessels in the tumor is often very discontinuous and thus allows the passage of large particles; the second factor relies on the lack of lymphatic drainage in tumors that would normally remove these particles. Consequently, they can accumulate and remain in diseased tissues for a longer time than healthy ones.For the EPR effect to occur, however, nanoparticles must remain long enough in the bloodstream to reach the tumor without being removed first by macrophages, as they are recognized as foreign agents by the immune system. It is therefore necessary to inhibit the phenomenon of opsonization, i.e., the recognition by the body the reticuloendothelial system of the foreign body; it occurs through the covering of this foreign body by specific components of the blood, such as opsonins, with subsequent elimination by macrophages. To avoid opsonization, many studies have shown that it is possible to coat nanosystems with polyethylene glycol (PEG) at a concentration of at least 0.1% ,95,96.In addition to the EPR effect, also known as passive target (the NP is administered with no modifications), the active target allows nanoparticles to reach and selectively bind cancer cells . To obtaRelating to the delivery modalities (ability or not to target specific cells), nanocarriers can be divided into three generations of compounds. “First generation” nanocarriers are able to accumulate by passive mechanisms through EPR effect . Among the first-generation vectors, liposomes-based drug delivery is the most successfully used in the clinic, as demonstrated by liposomal doxorubicin for breast, ovarian carcinoma and Kaposi’s sarcoma. The “second generation” of therapeutic nanovectors are characterized by additional functions; the surface modification with ligands allows their binding to specific molecules on tumor cells (active target mechanism) . MoreoveThe above-mentioned strategies allowed the developing of more than 200 products that have been approved or are under clinical investigation . In contNatural products represent a large family of different chemical entities with a wide variety of activities and pharmacological effects . They orIn recent years, biomedical research has focused its attention at searching substances of natural origin as possible chemosensitizing and chemopreventive agents ,112,113.Actinomyces antibioticus [®), collected from the bark of Taxus brevifolia [In 1940, the first antitumor antibiotic, actinomycin D, was isolated from the fungus bioticus  Since thevifolia , is a seevifolia ,119. Dr.evifolia .There are many in vitro and in vivo studies focusing on drug delivery systems of natural compounds in the field of oncology aimed at improve their solubility, bioavailability and selectivity ,122,123.Liposomes represent one of the most employed nanoparticle systems for cancer therapy. They are able to encapsulate both lipophilic and hydrophilic compounds within their phospholipid bilayer and the inner core. Many studies have shown that encapsulating natural substances in liposomes can improve their biological activity compared to non-encapsulation . One exaTo date, many nanodelivery systems have been developed and reported to effectively bypass MDR both in vitro and in vivo. The encapsulation of chemotherapeutics can avoid their direct interaction with ABC transporters both at plasma and intracellular membranes, thus modifying the intracellular drug concentration and localization. In addition to bypassing drug transporter activity, nanoparticle delivery systems can also be effective in inducing apoptotic cell death .Cheng et al.  reportedNano-based systems are increasingly employed to simultaneously deliver multiple natural compounds in order to overcome MDR-associated side effects. In a recent in vitro and in vivo study, resveratrol and doxorubicin were co-encapsulated in poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles. Resveratrol is a natural stilbene and a non-flavonoid polyphenol, present in grapes, peanuts and red wine. This phytoestrogen possesses anti-oxidant, anti-inflammatory, cardioprotective and anti-cancer properties . The PLGPescheria fuchsiaefolia tree, has been demonstrated to enhance doxorubicin cytotoxicity and induce chemosensitizing effect on cultured multidrug-resistant U-2 OS-DX osteosarcoma and melanoma cell line Me30966 when used at noncytotoxic concentrations [The plant alkaloid voacamine, isolated from the bark of the trations . Voacamitrations .In a study by Gupta et al., synthesized chitosan-based nanoparticles loaded with PTX were employed for the treatment of different kinds of cancer. Nanoparticle-loaded drug exhibited better activity with sustained release, higher cell uptake and reduced hemolytic toxicity in MDA-MB-231 breast cancer cell lines compared with pure PTX .®. The in vivo studies showed a decrease of tumor volume compared with Doxil® alone, while folate aided and enhanced specific targeting. These results indicate that this nano-drug is a promising candidate for treating liver cancer and monitoring the progress of the cancer using magnetic resonance imaging [Maeng et al. demonstrated that folate-functionalized superparamagnetic iron oxide nanoparticles, developed previously for liver cancer cure, are also been used for the delivery of Doxil imaging .Curcumin is a natural polyphenolic compound extracted from the plant turmeric; a lot of in vitro and some in vivo studies demonstrated its anticancer properties in breast, prostate, bone, cervices, lung and liver cancer cell lines. Unfortunately, free curcumin is not soluble in water and not very bioavailable, features that limit its application in the clinics; the encapsulation in nanoparticles such as liposomes has solved these problems ,140,141.Cheng et al. have developed a method based on the self-assembly behavior of phospholipid in water and the diffusion of curcumin into liposomal membranes driven by hydrophobic forces. This method has allowed a good encapsulation and bio-accessibility of curcumin for its applications in cancer treatment .TM) can reduce the tumor growth of pancreatic and colorectal cancer. In a phase I dose escalation study the safety, pharmacokinetics, tolerability and activity of intravenously administered liposomal curcumin were evaluated in patients with locally advanced or metastatic cancer. The results showed that 300 mg/m2 liposomal curcumin over 6 h was the maximum tolerated dose in these heavily pretreated patients, and is the recommended starting dose for anti-cancer trials [Liposomal curcumin  plus curcumin. These results highlight the potential use of such nano-carriers in the treatment of patients with pancreatic cancer .Plasmodium falciparum that causes malaria infection. Artemisinin is a sesquiterpene lactone obtained from sweet wormwood, Artemisia annua [Artemisinin was discovered by Youyou Tu in 1972 and is used as an antimalarial for the treatment of multi-drug resistant strains of ia annua . This suia annua ,148.Leto et al. investigated the antitumor properties of Artemisinin encapsulated in a PEGylated nanoliposome decorated with transferrin receptors on HCT-8 cell line. The results confirmed the enhanced delivery of Artemisinin loaded in liposomes actively targeted with transferrin in comparison with the other Artemisinin-loaded liposomes and an improved cytotoxicity .Many in vitro studies on resveratrol have reported its involvement in different cellular responses, such as cell cycle arrest, induction of differentiation, apoptosis and growth inhibition in several types of cancer, principally prostate and colon cancers . UnfortuOther natural substances with promising anticancer properties are essential oils (EOs). They are a complex mixture of hydrophobic and volatile compounds synthesized from aromatic plants. They are constituted of terpenoids, phenol-derived aromatic components and aliphatic components. EOs have been widely used for their antimicrobial, antioxidant, anti-inflammatory, immunomodulatory and anticancer properties in vitro . The chaBoswellia and Commiphora. Frankincense and myrrh oil exhibit many biological activities such as antimicrobial, anti-inflammatory and antitumor. The study of Shi et al. focused on the preparation of SLNs, which are capable of improving the stability and antitumor efficacy of FMO. The results demonstrated that SLNs can be used to increase the stability and/or to improve the in vivo antitumor efficacy of FMO [An in vivo study on mice investigated the oral delivery of frankincense and myrrh oil (FMO) loaded in Solid lipid nanoparticles (SLNs), a new nanoparticle-based drug-delivery system with particles that range in diameter from 10 to 1000 nm (FMO-SLNs). Frankincense and myrrh are gum resins obtained from the genera y of FMO .Carvacrol is a monoterpene present in the essential oil of oregano. It has been studied for its therapeutic properties, especially in the control of painful conditions and inflammation during cancer condition. The encapsulation of carvacrol in a complex of β-cyclodextrin and administered  in mice with sarcoma tumor reduced hyperalgesia. However, pure carvacrol did not cause significant changes in nociceptive responses. These results produced evidence that the encapsulation of carvacrol in β-cyclodextrin can be useful for the development of new options for pain management .Aloe emodin  is one of the constituents of Aloe vera gel. It shows multiple properties  [tective) . A lot otective) ,161,162 tective) . Unfortutective) .Li et al. investigated the effect of transfection of r-caspase-3 with nanoliposomes loaded with AE and photodynamic therapy in human gastric cancer cells . Nano-AECurrently, many nanomedicines that are used for cancer therapy and approved by the U.S. Food and Drug Administration (FDA), are extracted from natural compounds, such as Doxorubicin, Paclitaxel, Vincristin, Kaempferol, Silamarin, Resveratrol and Curcumin ,170,171.®/Caelyx® (1995) [As known, the first nano-drug FDA-approved was a PEGylated liposomal formulation of doxorubicin, Doxil® (1995) , which s® (1995) .TM is a non-pegylated liposomal formulation loaded with doxorubicin. It is used to treat women with metastatic breast cancer usually in combination with cyclophosphamide. It has received a marketing authorization valid throughout the EU on 13 July 2000. MyocetTM shows reduced cardiotoxicity and more prolonged intratumor accumulation, as compared to free drug, thanking to the citrate complex and the loading method through a pH gradient [Myocetgradient .® is another natural nanodrug currently used in clinical mostly for advanced HIV-associated Kaposi’s sarcoma. It is an aqueous solution of the citrate salt of daunorubicin encapsulated into liposomes. Daunorubicin is an anthracycline antibiotic with antineoplastic activity, obtained from Streptomyces peucetius and Streptomyces coeruleorubidus. Free daunorubicin was employed for acute lymphocytic leukemia and acute myeloid leukemia and induces many adverse effects such as cardiovascular, dermatologic , gastrointestinal and genitourinary. If administered as DaunoXome®, daunorubicin displays substantially altered pharmacokinetics with reduced side effects [DaunoXome effects .® uses lysolipid thermally sensitive liposome to encapsulate doxorubicin. Heat-sensitive liposomes rapidly change structure when heated to 40–45 °C, creating openings in the liposome, thus allowing doxorubicin delivery directly into and around the targeted tumor. ThermoDox® is used for primary liver cancer and it is being evaluated in a two-arm, double-blinded, placebo-controlled, randomized phase 3 clinical study, designed to assess ThermoDox® in combination with standardized radiofrequency thermal ablation (RFA ≥ 45). The goal of this study is to increase the effective treatment zone in order to capture micrometastases [ThermoDoxtastases .®, employed for treatment of hematologic malignancies and solid tumors. This nano-drug is a sphingomyelin and cholesterol-based nanoparticle formulation of Vincristine that is able to overcome the dosing and pharmacokinetic limitations, increase the circulation time, optimize delivery to target tissues and facilitate dose intensification without increasing toxicity compared to drug free [Another nano-drug FDA-approved is the vincristine sulfate liposome called Marqiborug free .A clinical/phase III study of another nano-drug is based on nanoparticle drug delivery formulation that encapsulates PTX in polymeric micelles. It is an open-label phase III non-inferiority trial to compare the efficacy and safety of the nano-drug called NK105 and PTX in metastatic or recurrent breast cancer. The primary endpoint was not met, but NK105 had a better peripheral sensory neuropathy toxicity profile than free PTX .An Australian clinical/Phase I open-label single-ascending study is still ongoing to measure the safety, tolerability and pharmacokinetics of OP-101 (Dendrimer N-acetyl-cysteine), normally used for the treatment of cell or squamous cell skin cancer, after subcutaneous administration in healthy volunteers. The trial includes two outcomes: primary outcome evaluates the safety and tolerability of OP-101 after single subcutaneous doses in healthy subjects (adverse events), which include laboratory test variables; the secondary outcome is to determine the pharmacokinetic profile of OP-101 after single doses in healthy subjects (plasma and urine concentrations). Recently, the US FDA approved to initiate phase II trial in COVID-19 infections .® is the clinically approved albumin nanoparticle formulation of PTX, in the form of nanospheres (130 nm in diameter). This formulation has increased the bioavailability of paclitaxel, resulting in higher intratumor concentrations facilitated by albumin receptor (gp60)-mediated endothelial transcytosis. This represents a very successful drug delivery system of PTX, approved in different countries for the treatment of metastatic breast cancer, advanced pancreatic cancer and for advanced non-small cell lung cancer in combination with carboplatin for patients who are not candidates for curative surgery or radiation therapy [Abraxane therapy .The FDA approved nanodevices described in this section and the corresponding reference are summarized in Traditional approaches for cancer treatment are sometimes limited because of their low specificity, responsible for severe side effects and toxicity, and the possible induction of the MDR phenotype. As a consequence, the searching for more effective anticancer therapeutic strategies is increasingly driven by the need to selectively kill cancer cells, defeat the MDR phenomenon and increase the specificity of a drug through the spatial, temporal and dose control of its release.Natural products represent an important source for the discovery of novel anticancer drugs to be used both at a preventive and therapeutic level. Unfortunately, some of the natural compounds’ features restrict their application in anticancer therapy. A lot of studies have already demonstrated that strategies based on nanomedicine and nanodelivery systems allow to improve the efficacy of natural compounds with anticancer properties, increasing their solubility, bioavailability and selectivity, reducing their systemic toxicity and in some cases circumventing a drug resistance response. Nowadays, there are different types of drug carriers with unique and versatile properties that make them appropriate for oncology applications. Increasingly advanced research allows to design multifunctional nanocarriers, characterized by a gradual and selective release of multiple compounds at specific tumor sites. NPs carrying and delivering both compounds already used in chemotherapy and natural products are demonstrated in in vitro and preclinical studies to be very effective for both therapeutic and chemosensitizing purposes.


The comprehensive analysis of biological and clinical aspects of circulating tumor cells (CTCs) has attracted interest as a means of enabling non-invasive, real-time monitoring of cancer patients and enhancing our fundamental understanding of tumor metastasis. However, CTC populations are extremely small when compared to other cell populations in the blood, limiting our comprehension of CTC biology and their clinical utility. Recently developed proteomic and genomic techniques that require only a small amount of sample have attracted much interest and expanded the potential utility of CTCs. Cancer heterogeneity, including specific mutations, greatly impacts disease diagnosis and the choice of available therapeutic strategies. The CTC population consists primarily of cancer stem cells, and CTC subpopulations are thought to undergo epithelial–mesenchymal transition during dissemination. To better characterize tumor cell populations, we demonstrated that changes in genomic profiles identified via next-generation sequencing of liquid biopsy samples could be expanded upon to increase sensitivity without decreasing specificity by using a combination of assays with CTCs and circulating tumor DNA. To enhance our understanding of CTC biology, we developed a metabolome analysis method applicable to single CTCs. Here, we review―omics studies related to CTC analysis and discuss various clinical and biological issues related to CTCs. Cancer is the second leading cause of death in the United States . ComprehA drawback to the use of CTCs is their rarity. The median CTC count in various cancers is only 1 to 84 per 7.5 mL of peripheral blood . Indeed,The heterogeneity of cancers, including specific mutations, greatly impacts disease diagnosis and the choice of available therapeutic strategies. The advent of genomic medicine and single-cell analysis techniques has increased research focused on intratumoral heterogeneity in individual patients. Intratumoral heterogeneity includes the uneven distribution of genetically diverse tumor subpopulations across different lesions as well as dynamic variations in the genetic diversity of individual tumors over time . SubstanThe evidence clearly indicates that tumors exhibit heterogeneity and may differ before and after cancer treatment. This heterogeneity can lead to different responses to therapy. Single CTCs have been used to elucidate the complex tumor genomic profiles of colon cancer tumors . MutatioTraditionally, CTCs are thought to induce epithelial–mesenchymal transition (EMT) during dissemination. Although normal epithelial cells are immobile, some cancerous epithelial cells may begin expressing proteins associated with motility and cease expressing proteins involved in forming connections between cells during embryonic development. This transformation produces mobile ‘mesenchymal’ cells that can migrate and form other lesions. Cancer cells that have an EMT phenotype can penetrate and pass through blood vessel walls and enter the bloodstream. These CTCs eventually attach to endothelial cells of blood vessels and exit the bloodstream, forming new metastatic lesions in other organs ,24,25. TMoreover, EMT is associated with the properties of cancer stem cells (CSCs). The metastatic potential of a tumor depends upon the appearance of CSCs in the primary tumor tissue. These CSCs exhibit two characteristics: self-renewal and the ability to efficiently regenerate phenotypic heterogeneity in the parental tumor ,28,29,30Various CTC enrichment technologies have been developed , includiMicrofluidic technologies are well suited for label-free analyses. As such, microfluidic methods for size-based isolation and concentration of cells (positive test rate using cell line of 84%) as well as automated size-based selection (positive test rate using cell line between 42% and 70%) have been developed ,53. In aThe sensitivity of genomic profiling using liquid biopsy specimens could be increased without decreasing specificity by employing a combination of assays for CTCs, circulating tumor DNA (ctDNA), and cell-free DNA (cfDNA).KRAS, NRAS, and PIK3CA genes in patients exhibiting resistance to anti-EGFR therapy via combined NGS analysis of CTCs and ctDNA. Moreover, mutations in codon 61 in KRAS and NRAS were detected more frequently in colorectal cancer patients with acquired resistance to anti-EGFR therapy than before initiation of anti-EGFR therapy.Recently, we investigated whether CTCs can be used as a tool for selecting clinical strategies by observing molecular changes in real time in patients with advanced stages of cancer . We enriIn another study of 28 patients with multiple myeloma , discordIn our recent study, we used the microfluidics flow method to enrich CTCs and found an average of 14.5 CTCs/mL of blood  in one patient, and CTCs were observed in 27 of 31 patients enrolled in our study . These rm/z peak was detected, we identified a trend in the frequency of peaks distributed along the m/z scale. This trend was noticed following the comparison of histograms of the average spectra of CTCs from gastric cancer patients to those from colorectal cancer patients, as shown in n = 9 and n = 13, respectively) to identify possible metabolites or lipids that are unique to these specific cancers. Among the statistically significant peaks identified, acyl carnitine metabolites and sterol lipids levels were more elevated in colorectal cancer than gastric cancer. Eicosanoids were also more abundant in the CTCs of colorectal cancer patients, a finding that was corroborated by other studies examining this cancer type [To enhance our understanding of CTC biology, we developed a metabolomic analysis method that can be performed with a single CTC . Althougcer type .Although robust analysis of MS signals from single cells remains a challenging task, our method has the potential to be utilized as a novel biomarker on the single-cell level.Culturing CTCs represents a future challenge and goal. Several reports of attempts to culture CTCs have been published. CTC culture is very challenging due to the extremely low number of CTCs when compared to other cell populations in the blood and because the behavior of CTCs is unclear. The formation of heterotypic cell clusters between CTCs and white blood cells (WBCs) has been previously reported ,72,73, aTo date, there are reports of successful culture of CTCs from colon cancer , head anThe heterogeneity of cancers, including specific mutations, greatly impacts disease diagnosis and the choice of available therapeutic strategies. Tumors are heterogeneous, and they may also exhibit differences based on the time of assessment . Heterogeneity is also related to differences in response to therapy. As noted above, cancer heterogeneity may lead to discordance between the results of assays for primary and metastatic lesions, as well as for CTCs, ctDNA, and cfDNA. A number of studies on the biological behavior of tumor cells have been reported. Rapid autopsies of seven individuals who died of advanced pancreatic cancer were reported . To deteThese results suggest that there is slight concordance between the genetic alteration profiles of primary/metastatic sites and CTCs. Moreover, the biological behavior of tumor cells can change on a moment-to-moment basis, and clonal evolution can occur in primary tumors or in response to selective pressures associated with cancer therapies. We should not focus only on concordance between the primary tumor site and CTCs as a surrogate biomarker for monitoring the primary sites; instead, we should focus on the clinical association between CTCs and clinical findings, such as metastatic potential, the efficacy of targeted therapies, and long-term prognosis.Liquid biopsy techniques allow for repeated sampling, which is not usually possible with tissue biopsies. Because they are cancer cells, CTCs could be used to determine the biological characteristics of cancers in individual patients. Indeed, melanoma patients with detectable programmed cell-death ligand 1 (PD-L1)-positive CTCs exhibited significantly longer progression-free survival (PFS) following treatment with an immune checkpoint inhibitor than patients with PD-L1-negative CTCs  . The 12-Subpopulations of CTCs are thought to undergo EMT during dissemination, and as such, the CTC population consists primarily of CSCs. CSCs are associated with both initiation of primary tumors and the establishment of metastatic lesions. EMT and the acquisition of stem cell properties are related . A recent study reported the use of CD44, CD133, and vimentin as expression markers . It is tThe development of novel CTC culturing technologies may enable the evaluation of drug response by taking advantage of liquid biopsy, a minimally invasive real-time monitoring approach, thus guiding drug discovery and development as well as therapeutic decision-making for personalized treatment.Various CTC enrichment technologies have been developed, including density gradient centrifugation, microfiltration in two and three dimensions, inertial microfluidics, DEP, acoustophoresis, direct imaging modalities, functional assays, and immunoaffinity techniques. Therefore, CTCs can now be used as a liquid biopsy marker that reflects the biological and clinical aspects of cancers and enables the non-invasive real-time monitoring of cancer patients. Tumors are heterogeneous and may differ before and after cancer treatment. This heterogeneity can lead to different responses to therapy. Subpopulations of CTCs are thought to undergo EMT during dissemination, and as such, the CTC population consists primarily of CSCs. To better understand tumor cell populations, we demonstrated that information regarding alterations in genomic profiles as determined by NGS analysis of liquid biopsy samples could be expanded upon by employing a combination of assays involving CTCs and ctDNA, thus increasing sensitivity without decreasing specificity. Moreover, to enhance our understanding of CTC biology, we developed a metabolomic analysis method that can be performed with a single CTC. Each cancer type has a specific metabolic profile that could provide new biomarkers that could be monitored on the single-cell level. Among future challenges and goals is the culturing of CTCs. The development of techniques for culturing CTCs may lead to the development of novel omics  analyses and new drug screening techniques.


Osteoarthritis, rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis, all have one clear common denominator; an altered turnover of bone. However, this may be more complex than a simple change in bone matrix and mineral turnover. While these diseases share a common tissue axis, their manifestations in the area of pathology are highly diverse, ranging from sclerosis to erosion of bone in different regions. The management of these diseases will benefit from a deeper understanding of the local versus systemic effects, the relation to the equilibrium of the bone balance , and the physiological and pathophysiological phenotypes of the cells involved . For example, the process of endochondral bone formation in chondrocytes occurs exists during skeletal development and healthy conditions, but also in pathological conditions. This review focuses on the complex molecular and cellular taxonomy of bone in the context of rheumatological diseases that alter bone matrix composition and maintenance, giving rise to different bone turnover phenotypes, and how biomarkers  can be applied to potentially describe specific bone phenotypic tissue profiles. Maintenance of bone homeostasis is essential for proper function of the skeleton. Mechanical stress, trauma, autoimmunity, ageing, menopause and other hormonal changes, genetics, diet and lifestyle lead to alterations in bone and joint structures and can result in pathological changes. Aberrant bone turnover is a feature shared by many rheumatic diseases, including osteoarthritis (OA) , rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) –4. The bThe aim of this review is to review the relevant literature in order to provide an understanding of the bone manifestations underlying joint diseases in rheumatology, and how these may be characterized using biomarkers to identify potential bone specific endotypes within and across rheumatic diseases.Endochondral ossification, or endochondral bone formation, is the process underlying longitudinal bone growth during skeletal maturation. A significant reason for the interest in this process is the close similarity between endochondral bone formation and the bone pathology of for example the SpA joint , 9–11. IMononuclear osteoclast precursors and endothelial cells invade the osteoid resulting in capillary invasion and formation of bone marrow cavity. Chondrocytes in the growth plate proliferate in columns towards the diaphysis, becoming hypertrophied as they deposit cartilage matrix and finally undergoing apoptosis . LongituInflammatory signaling such as tumor necrosis factor α (TNFα) and interleukin 23 (IL-23) or altered expression of growth factors such as bone morphogenic proteins (BMPs) and Wnts may disturb or misdirect these processes depending anatomical site and/or external stimuli such as mechanical changes as seen in various arthritic diseases including RA and SpA , 7.Bone remodeling is required for maintenance of calcium homeostasis and strength of the bones and thus for the sustained health and functionality of the skeleton . Bone reIn healthy adults bone resorption is always followed by a balanced amount of bone formation in a process termed coupling . CouplinPathologies arising from alterations in how bone is formed and resorbed are numerous, and include different forms of osteoporosis, calcium metabolic disorders and severe genetic malignancies; Such diseases have been instrumental in understanding bone processes, cellular communication of the bone, and identifying potential markers describing bone compartment changes. Here, we have selected three non-rheumatic situations underlining extremes of osteoclast and osteoblast function and bone modeling and remodeling gone wrong, namely hypogonadal , osteopetrosis and fibrodysplasia ossificans progressive (FOP).Hypogonadal osteoporosis arises from a decrease of sex steroid production . As sex Most therapies for osteoporosis target the accelerated osteoclastogenesis and/or function. Bisphosphonates, denosumab (anti-RANKL antibody), hormone replacement therapy and Selective Estrogen Receptor Modulators (SERMs) focus on reducing the number of osteoclasts and, thereby, bone resorption . Due to OPT is a rare, inherited high bone mass disease . The bonAn interesting difference between the two types of OPT lies within the overall bone turnover. Both types show reduced bone resorption and the osteoclast-poor type shows reduced bone formation as expected from the coupling between these processes . ImportaFOP is a rare genetic disease characterized by abnormal heterotopic ossification of the muscles, tendons, aponeurons, and ligaments. With progressive ossification affecting peri-articular and soft tissues the disease will lead to skeletal deformities growth impairment, joint ankylosis and chronic pain affecting gait, posture and respiration. The median life expectancy of individuals with FOP is approximately 40 years . The molOA is the most common form of chronic arthritis , 54. A hStudies of the periarticular bone in knee and hip OA patients show abnormal subchondral bone, with sclerosis, thinning of the vertical trabecular structure, and under-mineralization, thought to be result from accelerated bone remodeling Fig.  66, 67], 6766, 6OA development is associated with changes in chondrocyte behavior, including matrix calcification and expression of hypertrophic markers such as MMP13 and type X collagen, which are both signs of hypertrophy , 77. TheWhile not occuring in the subcondral region one of the cardinal bone alterations in OA is osteophytes Fig. . These bIn summary, changes in the subchondral region are associated with bone marrow changes, BMLs, microfractures and vascularization, leading to significant increases in bone remodeling and tissue turnover causing perturbed bone structure in the joint . Not surThe heterogenous nature of OA pathology has driven the field towards defining OA phenotypes and developing and applying biomarkers to describe disease state and to predict prognosis and treatment response. The extracellular matrix (ECM) centralized pathology of OA has centered efforts around proteins and peptides arising from the remodeling of the different joint matrices, including bone and cartilage Table . Here thAlthough not a neo-epitope, the cartilage residing cartilage oligomeric protein (COMP) is increased in serum and synovial fluid of OA patients , 103, anThe successful application of biomarkers in clinical trials in the osteoporosis field has further ensured access to already validated markers such as urinary and serum CTX-I  and its While an established definition of endotypes is still lacking in OA, studies are beginning to utilize biomarkers to explore and better understand the underlying changes in OA and how to separate these into categories that can be used to optimize treatment –114. FutRA is a complex autoimmune inflammatory disease that primarily affects peripheral joints, presenting with or without systemic, extra-articular involvement. RA is histologically characterized by synovial inflammation (synovitis) and hyperplasia leading to the formation of invasive pannus tissue. The pannus invades and erodes the adjacent cartilage and bone tissue leading to cartilage destruction and bone loss, characteristic of RA Fig.  115, 11. Both trFocal bone loss in RA appears in the form of bone erosions and subchondral osteolysis at the articular surface. Several lines of evidence indicate that the erosive process is carried out by osteoclasts that populate the interface between pannus and articular bone , 119–122The second form of focal bone loss characterizing RA is periarticular osteopenia, the loss of trabecular size and number in the metaphyseal regions adjacent to the inflamed joints. Periarticular osteopenia is also believed to be mediated by osteoclasts, albeit through different mechanisms . As periThe last decade has seen an increasing number of novel treatments targeting a variety of different immunological pathways involved in the RA pathology, including TNFα, IL-1 and IL-6 production and intracellular pathways such Janus kinase (JAK) inhibitors. Treatment effects are determined by the degree of disease activity, patient-reported quality of life, and acute phase responses, such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). While markers of inflammation can clinically be relevant for determining treatment response, markers that could accurately describe tissue damage in the different joint compartments would likely be better suitable for guiding targeted treatment.Several serological markers which are based on measuring fragments released from the matrix in both bone and cartilage, as a result of increased tissue destruction or turnover, exist in the RA arena Table . The CatBone biomarkers have also shown promise in predicting response to treatment. A recent study found that RA patients who had a high bone turnover levels measured as the ratio between serum CTX-I and osteocalcin and who simultaneously had low serum C2M levels, indicating slow cartilage degradation, were more likely to respond to anti-IL-6R treatment .Cartilage turnover markers have also proven useful in RA. High levels of CTX-II predict an increased risk of radiographic progression . CTX-II Type IV collagen is an abundant protein of basement membranes, but it is also part of the interstitial matrix of the synovial lining layer. The MMP-generated neo-epitope fragment of type IV collagen, C4M, was found to be associated with treatment efficacy in two phase III clinical studies with tocilizumab. Furthermore, higher baseline levels were associated with structural progression in the form of joint space narrowing (JSN) and Sharp score after 52 weeks. Tocilizumab treatment decreased C4M levels in a dose dependent manner and early reduction was associated with a better treatment response . In summPsA is a form of inflammatory arthritis associated with psoriasis . A signiAlthough the bone resorptive phenotype in both RA and PsA appear similar, at least early in disease, there are significant differences between the two. RA typically involves phalangeal joints in a symmetric pattern with bone erosions and inadequate repair mechanisms resulting in local and systemic bone loss , 161. PsBoth RANK and RANKL expressing cells have been shown to be highly elevated in PsA compared to OA and the upregulated RANKL expression was highly localized to the synovial lining layer . MeasuriTNFα, like in RA, also plays a central role in the pathophysiology of PsA. The levels of TNFα and its soluble receptor (TNF-R55) are elevated in serum and synovial fluid of PsA patients . Furtherh17 cells is significantly elevated compared to healthy controls [+CD11b+Gr1lowRANK+CSF-1R+ osteoclast precursors with increased differentiation capacity compared to precursors from control mice even before arthritis onset, suggesting that IL-17A expression may exacerbate bone destruction even in the absence of inflammation [IL-17 is another prominent cytokine that has been implicated in inflammatory joint diseases – including RA, PsA and AS. In both PsA and psoriasis patients, IL-17 secretion by T helper 17 (Tcontrols . IL-17 hcontrols . IL-17 ccontrols . Furtherammation . In lineammation .IL-23 is involved in bone remodeling by inducing osteoclastogenesis but also modulates differentiation of IL-17 and IL-22 secreting immune cells. The secretion of IL-17, − 22 and − 23 has also been associated with arthritis and enthesitis in a mouse model of SpA . PerhapsWhile the mechanisms underlying bone destruction in PsA are relatively well described, the mechanisms underlying the bone formation typically observed in PsA are still not well understood. The excessive bone formation can occur both in the axial skeleton, presenting as syndesmophytes, and in the peripheral joints, as joint ankylosis, enthesophytes or periosteal bone formation . AlthougThe bone formation in PsA arises from anabolic factors that stimulate the formation of hypertrophic chondrocytes and osteoblasts. However, the link between inflammatory cytokines and this bone formation is still elusive. Nonetheless, the importance of Wnt/β-catenin signaling in directing osteoblast differentiation and function in PsA is well established. In contrast to RA, decreased serum levels of Wnt pathway antagonist DKK1 have been reported in PsA patients as compared to healthy controls which is in line with an increase in bone formation . MoreoveEvidence of the involvement of BMP signaling has been suggested by studies in DBA/1 mice that spontaneously develop PsA-like arthritis, but also ankylosing enthesitis , 184. ThAssessment of biomarkers reflecting bone turnover in human PsA patients has shown variable results depending on the stage and activity of the disease Table . There aIn contrast to RA, increased levels of bone formation markers, osteocalcin and bone specific ALP, have been reported for PsA patients in some studies , 188 butBoth the cartilage formation markers PRO-C2 , CPII 1 and PIIATogether with bone and cartilage biomarkers in PsA, skin biomarkers may be useful tools to identify the psoriatic component of PsA. For psoriasis the ideal biomarker has not yet been identified, however serum measurements of different S100 proteins are associated with disease activity , 203, VEAS is part of a group of diseases termed spondyloarthritides, characterized by chronic inflammation affecting the spine and sacroiliac joints. It typically presents in young adults and imposes a significant burden. AS is diagnosed by symptom, a physical exam, inflammation markers and by radiography or MRI. AS ss characterized by the presence of structural changes to the skeleton, mainly manifesting as progressive ankylosis of the spine and the sacroiliac joints, but also by inflammation associated trabecular bone loss resulting in increased fracture risk Fig. . Both tyThe bone phenotype in AS is a complex mix of different processes where both pathological bone loss and excessive bone formation shape the clinical manifestation of the disease . The losThe mechanisms responsible for the characteristic alterations to bone formation leading to ankylosis between adjacent vertebrae or other bones is still a feature of the disease which is not fully understood. It is proposed that the process is initiated around entheses, the connection or transition zone between tendons or ligaments and bone , 213. ThPolymorphisms in BMP6 have been associated with radiologic severity in Korean AS patients . HoweverLike in PsA, Wnt signaling has been implicated in the bone formation associated with AS. Preclinical evidence comes from studies in a human TNFα transgenic mouse model where inhibition of the wnt signaling antagonist DKK1 resulted in a switch from RA-like erosive phenotype to bone formation phenotype with progressive ankylosis of both limb and sacroiliac joints, suggesting that Wnts may promote bone formation associated with pathological development of AS , 223. AlSeveral studies evaluated the serum levels of bone turnover markers in AS, with inconsistent results. One explanation for this, could be the challenge of relating changes occurring in a single joint or enthesis, and compare it too systemic levels of low systemic inflammation. The systemic measurement may therefore disturb the serological measurement in terms of bone formation. In contrast to the observed pathological changes, most studies found no difference in serum levels of bone formation markers, like osteocalcin, PINP and bone specific ALP, in patients with AS compared with their control groups , 229–232CTX-I is elevated in the serum  and urinThe serological patterns of cartilage turnover biomarkers in AS are very similar to PsA, whereby both formation markers, like PIIANP , CPII 2 and PRO-The bone manifestations listed here, while still arising from different mechanism and arising in different tissue areas, share some degree of similarity in their manifestation; namely the aberrant remodelling leading to alterations in bone density and architecture, and the osteochondral ossification like alterations leading to undesired calcification and bone growth or pannus formation. We hypothesize that endotypes across joint diseases exist which share similarities that can be exploited both when trying to treat the right patient with the right drug or potentially when repurposing drugs from other indications. The challenge of course lies in defining such endotype. Efforts are gaining traction in the OA field, where a number of endotypes have been suggested, among these, a bone driven endotype. For other rheumatic diseases involving bone alterations such as RA, the focus has rather been in trying to understand the underlying inflammatory patterns in order to better target anti-inflammatory treatment, and less attention have been given to structural aspects. Specific and sensitive biomarkers such as imaging and biochemical markers, that can accurately describe structural changes, combined with clinical assessment, seems crucial to defining common underlying endotypes. Longitudinal clinical studies including early to late stage disease patients with careful clinical examinations and state of the art imaging combined with continuous and consistent body fluid sampling would further enable us to study the link between systemic biomarkers and disease specific pathological bone turnover.It is apparent that unique molecular pathways underlie the manifestation of typical bone phenotypes in rheumatic disease. Common to all rheumatic diseases is aberrant bone homeostasis, leading to both excessive destruction and deposition of bone matrix in bones, joints and the spine, causing pain and disability. Despite the differing nature of the molecular processes in bone between different rheumatic diseases, biomarker signatures and platforms provide a valuable opportunity to objectively define the underlying pathobiology, which largely recapitulate the phenotypic changes arising with disease onset, progression and treatment. With the varying phenotypes, underlying aetiologies, and in some rheumatic diseases a lack of effective responses to treatment in bone, joint and spine disease, the field is becoming increasingly focused on application of biomarkers to accurately identify patient profiles and ensure more appropriate and targeted treatment, ultimately working towards more personalized healthcare approaches. In the coming years, it will be increasingly important to test markers in both well characterized, longitudinal observational cohorts to better understand the natural courses of the diseases and intervention studies in order to identify which patients are more likely to respond to treatment, for the benefit of patients, healthcare systems, and pharmaceutical companies.


Lupus is experienced as an “aggressive and expansive” disease affecting patients’ activities of daily living, sense of self, and social functioning . CurrentLike many autoimmune diseases, one of the most notable features of systemic lupus erythematosus is its greater prevalence in females than males. Therefore, it has been hypothesized that differences in gene expression and regulation between may underlie sex-biased autoimmunity including lupus ,3.Indeed, a female-biased gene network has been described in human skin which associates significantly with susceptibility to autoimmunity. This network is downstream of a putative transcription factor, VGLL3 , which is also female biased . Genome-Building on this discovery, a recent study examines the immunological function of VGLL3 in mice and describes a novel mouse model that recapitulates many aspects of human cutaneous and systemic lupus . Mouse aThe transgenic mouse model was generated by introducing a construct that produced murine VGLL3 under the control of the bovine keratin K5 promoter, which has previously been shown to drive expression primarily in the basal compartment and epithelia of the skin and secondarily in other stratified epithelia . OverexpThe mouse model exhibited gross and microscopic similarities to cutaneous human lupus. Lesions, skin thickening, and scaling developed at 6–12 weeks of age. These features were preferentially located towards the ears and face, sites that are commonly associated with human cutaneous lupus. Microscopic examination of mouse lesional skin using simple hematoxylin-eosin and periodic acid-Schiff techniques revealed more similarities to human lupus lesions. These microscopic features included neutrophil infiltration and exocytosis in the dermis, thickening of the squamous epidermis, accumulation of melanin in the upper dermis, formation of vacuoles in basal cells, apoptotic keratinocytes, and basement membrane thickening.Immunological analysis of transgenic inflamed skin tissue was in concordance with that observed in lupus patients and accepted mouse models of lupus. Immune complex deposition was observed by immunofluorescence in skin tissue. Immunohistochemistry revealed an inflammatory infiltrate that was enriched with markers for T cells; B cells dendritic cells, neutrophils, and macrophages. Flow cytometry analysis of skin associated cells showed B cell, plasmacytoid dendritic cell, and neutrophil expansion.Gene expression in the transgenic and control skin was analyzed revealing similarities to expression patterns seen in lupus patients. Significantly increased mRNA expression of eleven inflammatory and lupus-related markers was observed in the skin of VGLL3 overexpressing mice compared to wild type mice. Several of these were confirmed by immunofluorescence. A broader RNA-Seq approach analyzing 811 genes in the same skin sites revealed a shared pattern in differential gene expression compared to that observed in orthologous genes dysregulated in the skin of discoid lupus and subacute cutaneous lupus patients.Overexpression of VGLL3 in skin led to the development of systemic inflammation in the mouse model. Skin-draining lymph nodes and spleen mean mass was swollen by 4-fold and 2-fold, respectively. Mass cytometry analysis of the skin-draining lymph nodes and spleen showed significantly expanded B-cell populations. Significant increases in autoantibody production were detected in the serum by indirect immunofluorescence and enzyme-linked immunosorbent assay. Peripheral blood mononuclear cells in the mice displayed altered gene expression which, across 477 genes, was significantly associated with the pattern of orthologous genes dysregulated in patients with systemic lupus.The transgenic mice’s skin symptoms were frequently severe enough to warrant euthanasia at 4–5 months of age, rendering certain types of studies, particularly regarding kidney damage, impracticable. Despite this, subtle inflammation of the glomeruli could be observed through histology. Immune complexes, an early marker of lupus nephritis, were also detected in the glomeruli by immunofluorescence.The lupus-like phenotypes in the Vgll3-transgenic mice raise the question whether VGLL3 also sensitizes skin to UV radiation. In lupus-related photoprovocation an above threshold dose of UV irradiation, which only penetrates the deeper dermis, can lead to systemic autoimmune symptoms over a period of days or even weeks after an event ,8. It isIn conclusion, the findings from the new mouse model together with the previous report supports the continued focus on the skin-immune system interface in lupus. Taken together with previous findings, VGLL3 represents a female-biased factor that can drive development of cutaneous and systemic lupus.


Stem cell-based therapy has been considered as a promising option in the treatment of ischemic heart disease. Although stem cell administration resulted in the temporary improvement of myocardial contractility in the majority of studies, the formation of new cardiomyocytes within the injured myocardium has not been conclusively demonstrated. Consequently, the focus of research in the field has since shifted to stem cell-derived paracrine factors, including cytokines, growth factors, mRNA, and miRNA. Notably, both mRNA and miRNA can enter into the extracellular space either in soluble form or packed into membrane vesicles. Stem cell-derived paracrine factors have been shown to suppress inflammation and apoptosis, stimulate angiogenesis, and amplify the proliferation and differentiation of resident cardiac stem cells (CSCs). Such features have led to exosomes being considered as potential drug candidates affording myocardial regeneration. The search for chemical signals capable of stimulating cardiomyogenesis is ongoing despite continuous debates regarding the ability of mature cardiomyocytes to divide or dedifferentiate, transdifferentiation of other cells into cardiomyocytes, and the ability of CSCs to differentiate into cardiomyocytes. Future research is aimed at identifying novel cell candidates capable of differentiating into cardiomyocytes. The observation that CSCs can undergo intracellular development with the formation of “cell-in-cell structure” and subsequent release of transitory amplifying cells with the capacity to differentiate into cardiomyocytes may provide clues for stimulating regenerative cardiomyogenesis. The idea of extending the lifetime of the human heart has been fuelled by a series of major advances in transplantation and drug therapies. Nevertheless, myocardial infarction is characterized by the irreversible loss of cardiomyocytes because of their ischemic necrosis. Therefore, the need to re-establish the structural and functional features of native heart tissue represents a major challenge for the field of cardiac tissue engineering.Over the past decade, stem cell (SC) transplantation has become a promising therapeutic strategy for treating acute and chronic myocardial ischemia. Both preclinical and clinical studies involving ES cells , inducedUnderstanding how multipotent progenitors generate specific immature heart-cell lineages and form different heart-tissue components serves as a biotemplate for SC engineering to target cardiovascular regeneration. Accordingly, by utilising a primitive cell-fate map for the generation of such progenitors, the possibility also exists of regenerating the heart by transplanting specific progenitors derived from human SCs into patients with or at risk for cardiovascular disease. Towards this end, a clinically useful tissue-engineered graft needs to be designed to perform multiple different tasks including (i) reestablishment of the normal structure and function of injured myocardium across different size scales; (ii) functional integration with the host tissue; and (iii) remodelling in response to, e.g., environmental factors, growth, and aging. A “perfect” graft would balance these requirements to provide robust functionality on a long-term basis along with the capacity for vascularization.In this review, our primary focus is on bioinspired strategies along with the knowledge gained to date regarding the challenges that remain to be addressed for engineered heart regeneration to become a clinical reality, within the framework of preventive and treatment strategies rooted in personalized and precision healthcare-based and translational resources. However, this field still lacks sufficiently conclusive results to support full-scale implementation of such approaches, as exemplified by the poor survival and long-term engraftment of transplanted cells. To address these issues, new strategies are being subjected to intense evaluation with regard to such applications, with the results suggesting that these strategies may improve the efficiency of next-generation SC-based therapies.+ CSCs and even denial of the very existence of c-kit+ CSCs in adult mammals [The lack of convincing clinical evidence to support the efficacy of CSC-based therapy of ischemic heart disease has questioned the initial hypothesis that new CMs are derived from CSCs and has led to the suggestion that myocardial renewal and regeneration in adult mammals are provided by dedifferentiation and subsequent division of mature CMs . Althoug mammals .via the phenomenon of intracellular development of CSCs accompanied by formation of cell-in-cell structures (CICSs) and the subsequent release of transitory amplifying cells (TACs) capable of differentiating into mature CMs. Such approaches open up new perspectives for promoting SC-induced cardiogenesis as a paradigm for regenerative medicine in the near future.In this review, we highlight the emerging approaches to afford regeneration of heart muscle +, Sca-1+, Isl-1+, side-population (SP), cardiospheres, ALDH+ cells) in mammalian hearts and their availability in mature myocardium [+ CSCs, CD90+/CD105+ MSCs, and CD133+ endothelial progenitor cells, which led to adhesion and cluster formation [Comprehensive success has been accomplished in identifying several populations of resident CSCs  [Numerous laboratory and clinical studies have shown that after performing cell therapy for congestive heart failure, improvement in left ventricular function occurred , 27. In  effect) , 29. Thi effect) . The hos effect) ; notably effect) .via an array of immunosuppressive factors, cytokines, growth factors, and differentiation factors [β, prostaglandin E2, HGF, EGF, FGF, PDGF, VEGF, IGF, stromal cell-derived factor 1, and, as discussed in more detail below, the tryptophan-catabolic enzyme IDO and NO, a product of iNOS. Cumulatively, these secreted factors may inhibit inflammatory responses, promote endothelial and fibroblast activities, and facilitate the proliferation and differentiation of progenitor cells in tissues in situ [It has been shown that intravenous infusion of human MSCs reduces infarct size in mice via the anti-inflammatory protein TSG6; moreover, TSG6 alone was sufficient to provide a therapeutic effect . This su factors . These i in situ .SC secretome components can be secreted from the cell in different ways. Some utilise the classical secretory pathway involving a signal sequence that mediates individual protein secretion at the molecular level. Others are released through the process of exocytosis. Finally, many bioactive molecules are packed into membrane vesicles termed exosomes. At present, exosomes are considered as a potential alternative to stem cell therapy in mediating cardiac regeneration . For examiRNA sequencing indicated that MSC-derived exosomes and MSCs themselves utilise similar mechanisms to enhance cardiac repair. In addition, it revealed that MSC-derived exosomes alone could be used to promote cardiac repair of the injured myocardium as a cardioprotective approach, along with the therapeutic potential of cardiac regeneration strategies –40. The To understand the biological role of extracellular vesicles (EVs), such as exosomes and microvesicles, and their involvement in myocardial regeneration, it is important to recognise that cardiac EV production increases in stress conditions, such as hypoxia, inflammation, or injury. In hypoxic conditions, cardiac EVs contain angiogenic and prosurvival factors. In acute myocardial infarction (AMI), damaged CMs produce EVs with increased content of angiogenic, anti-apoptotic, mitogenic, and growth factors . These pin vitro), (iii) transdifferentiation of one of the various CM types, and (iv) activation of endogenous CSC differentiation into other myocardial cell types [The concept of innate heart regeneration is based on the assumption that myocardium in principle is capable of rebuilding damaged cardiac muscle. However, this capability is highly variable and dependent on numerous as-yet-unidentified factors , 48. Curll types . To datell types  whereas ll types . Specifill types . These mll types .The use of unique combinations of cardiospecific transcription factors, miRNAs, and/or chemical molecules is currently being considered for transdifferentiation of fibroblasts into CMs . MoreoveNotably, studies of cardiomyogenesis-related stimuli are being performed concomitant with ongoing debates regarding the possibility of dedifferentiation and division of mature CMs and the ability of resident CSCs to differentiate into mature CMs in the adult damaged heart . AlthougAccordingly, recent studies explored the potential of different approaches capable of stimulating cardiomyogenesis from mature CMs. For example, Ma et al.  performeμm, which is much smaller than that of mature CMs (L > 50 μm). However, the issue of CM proliferation is also controversial. In particular, it was established that systemic hypoxemia could alleviate oxidative DNA damage, thereby inducing CM proliferation in adult mammals [In this regard, it is worth noting that hypoxia , ischemi mammals , 76. Con mammals  previousex vivo) myocardial cell suspensions from rats of varying ages, mature specific pathogen free (SPF) mice, a one-year-old bull, and a human [L = 10–12 μm reserve their proliferative potential and differentiate into mature CMs upon exiting the CICSs. The imitation of ischemia  in in vitro experiments led to 5- to 10-fold increase in CICS amount and suppressed colony cell development [As the existing concepts fail to provide clear understanding of the observed phenomena, we suggested a new model based on the results obtained in our laboratory. By studying the behaviour of mammalian CSCs, we showed that the formation of new CMs from resident CSCs occurs through colony formation  and cons a human . In bothelopment .2) imposed 1 week after MI has resulted in a burst of myocardial cell proliferation. These data point to an important conclusion that high myocardial regenerative capacity observed in adult zebrafishes and newts, as well as in mammalian embryos and newborn mammals, is explained by the intensive proliferation of their CMs in the hypoxic environment [The comparison of our data with those obtained by other groups shows that hypoxia exerts dual effects on cardiomyogenesis. First, it prompts intracellular development of CSCs, which is evident from the increased number of CICSs. Upon maturation and rupture, CICSs release significant amounts of proliferating TACs , 80; we ironment , 76.It is known that hypoxia inhibits the proliferation of cardiac cells necessary to ensure embryonic and neonatal cardiomyogenesis . TherefoDespite recent skepticism about the role of CSCs in myocardial self-renewal and regeneration in adult mammals –16, 18, μm), which were named by the authors as “endogenous cardioblasts,” are activated, and their amount increases more than 10-fold following infarction. Moreover, they showed that genetically labelled cardioblasts expressed heart transcription factors and sarcomere proteins, exhibited spontaneous contractions, and formed mature CMs (in vivo) following injection into the recipient heart.By comparing our results with others, we concluded that all previously described small cells likely actually constitute TACs that “go through cardiac school (education)” inside mature CMs. It is important to highlight that intracellular CSC development is associated not only with partial cardiomyogenic differentiation of their progeny but also by the decreased level of their stemness. This fact hampers the identification of daughter cells for TACs and also ignores that the small size of TACs may lead to their erroneous interpretation as proliferating CMs. In addition, free CSCs are almost nondetectable in the adult mammalian myocardium, which strongly contributed to the negation of their role in adult cardiomyogenesis. However, the identification and in-depth exploration of the phenomenon of intracellular CSC development have encouraged us to claim that CSCs are not just innocent bystanders but rather working tools in an adult heart, which form TACs that afford myocardium renewal during their lifespan. The results from Malliaras et al.  confirmeTherefore, we consider that the existence of CSC-derived TACs has been conclusively demonstrated and should be considered in models of cardiomyogenesis. The schemes shown in Our data indicate that cardiomyogenic stimuli should be focused on TACs, rather than on CSCs or mature CMs. It is also clear that the proliferative activity of TACs is enhanced following ischemia and hypoxia and that their cardiomyogenic differentiation ability renders them as top candidates for therapeutic cardiomyogenesis in the ailing heart.Nevertheless, extensive work is still required to generate powerful off-the-shelf SC therapeutics as the data from human studies are contradictory, overall showing modest to no therapeutic SC effects , 81, 82.via clinical studies and translational applications as new consolidated entities will enhance the efficacy of cardiac cell therapy and further contribute to cardiac regenerative medicine.The heart is an extremely complex organ, and the techniques influencing its regeneration depend on many variables of nontrivial character. Physician scientists and practitioners are working hand-in-hand with SC scientists, biodesigners, and bioengineers and are likely to play a key role in developing the new paradigms for heart regeneration that appear imminent. In this review and discussion, we aim to establish a “case study” for the field of regenerative medicine using cardiovascular regeneration as a model suitable for further utilisation. We expect that advanced modalities that integrate cellular, bioengineering, and information (IT) technologies


Evolutionary medicine argues that disease can arise because modern conditions do not match those in which we evolved. For example, a decline in exposure to commensal microbes and gastrointestinal helminths in developed countries has been linked to increased prevalence of allergic and autoimmune inflammatory disorders (the hygiene hypothesis). Accordingly, probiotic therapies that restore ‘old friend’ microbes and helminths have been explored as Darwinian treatments for these disorders. A further possibility is that loss of old friend commensals also increases the sterile, aging-associated inflammation known as inflammaging, which contributes to a range of age-related diseases, including cardiovascular disease, dementia, and cancer. Interestingly, Crowe et al., 2020 recently reported that treatment with a secreted glycoprotein from a parasitic nematode can protect against murine aging by induction of anti-inflammatory mechanisms. Here, we explore the hypothesis that restorative helminth therapy would have anti-inflammaging effects. Could worm infections provide broad-spectrum protection against age-related disease? The aging process is the main cause of senescent multimorbidity, the co-occurrence of multiple chronic pathologies that includes the major diseases of late life. The geroscience approach views preventative intervention in the aging process as a means to simultaneously pre-empt the development of multiple age-related diseases . How doeOne cause of inflammaging is gut dysbiosis, an imbalance in the composition of the intestinal microbiome. Such imbalance can manifest as loss of immunomodulatory microbial species and is exacerbated by pro-dysbiotic aspects of the modern lifestyle, including antibiotic usage and the so-called Western diet . One intWuchereria bancrofti , rheumatoid arthritis, and type 1 diabetes . For exaThe Lancet, which describes how maintaining an infection with the intestinal hookworm Necator americanus alleviated his allergies . Interestingly, reduced levels of circulating pro-inflammatory cytokines and CRP have been seen in people infected by strongylid and filarial helminths in a number of studies  though . FurtherA. viteae with anti-inflammatory properties. In the first study of its kind, ES-62 is a 62 kDa glycoprotein found in the excretory secretory products of the filarial nematode parasite Bacteroidetes:Firmicutes ratio and increases in Proteobacteria in the gut  in cadavers was found to be negatively correlated with the severity of aortic atherosclerosis  epidemiclerosis . In athes by 50% ; similarque size , and ES-clerosis .Taenia crassiceps and its antigens have been found to attenuate and prevent colon tumorigenesis in mice through anti-inflammatory mechanisms . Regarding ES-62 in particular, it would be worthwhile to test its effects on aging in mice maintained on a normal diet rather than on a life-shortening high-calorie diet.Brugia malayi microfilariae (The possibility of anti-aging helminth therapy raises various questions. How do responses to such therapy change with age? Would higher or lower parasite (or antigen) levels be needed to take account of immunosenescence? To what extent do risks of helminth therapy increase in old age? What are optimal ages to apply such therapy to reduce inflammaging? Would helminth therapy act only in a preventative fashion , delaying the genesis or worsening of inflammaging-linked pathologies, or could it reverse existing disease symptoms? At least for murine rheumatoid arthritis, there is some evidence that helminth therapy could be both prophylactic and therapeutic . Thus, ifilariae .Effective application of helminth therapy would also require a clear understanding of its specific anti-inflammaging mechanisms. Theoretically, helminths could counter inflammaging in several ways. For example, they could inhibit sources of inflammaging by preventing gut barrier permeabilization and obesity, neutralize existing inflammaging by increasing the proportion of anti-inflammatory to pro-inflammatory cytokines, or repair inflammaging-afflicted tissue damage, for example through promotion of IL-22 secretion .It goes without saying that improvements in hygiene and elimination of helminth parasites have been of incalculable benefit to humanity. But a cost coupled with this benefit is abnormalities of immune function, specifically inflammatory hyperfunction. Available evidence suggests that restorative helminth therapies are effective against not only allergic and autoimmune inflammatory disorders, but also age-associated inflammation in later life, at least to some extent. Should this be confirmed, helminth therapy could provide protection against the wide spectrum of age-related diseases promoted by inflammaging. In the wake of successes during the last century in eliminating the evils of helminth infections, the time now seems propitious to explore further their possible benefits, particularly for our aging population – strange though this may sound.


Collaborative monitoring over broad scales and levels of ecological organization can inform conservation efforts necessary to address the contemporary biodiversity crisis. An important challenge to collaborative monitoring is motivating local engagement with enough buy-in from stakeholders while providing adequate top-down direction for scientific rigor, quality control, and coordination. Collaborative monitoring must reconcile this inherent tension between top-down control and bottom-up engagement. Highly mobile and cryptic taxa, such as bats, present a particularly acute challenge. Given their scale of movement, complex life histories, and rapidly expanding threats, understanding population trends of bats requires coordinated broad-scale collaborative monitoring. The North American Bat Monitoring Program (NABat) reconciles top-down, bottom-up tension with a hierarchical master sample survey design, integrated data analysis, dynamic data curation, regional monitoring hubs, and knowledge delivery through web-based infrastructure. NABat supports collaborative monitoring across spatial and organizational scales and the full annual lifecycle of bats. Preserving the natural heritage of Earth requires informed conservation action operating over multiple, often hierarchical, jurisdictions among a wide array of constituencies  How does the monitoring program remain relevant for satisfying information needs at a variety of spatial scales? (2) How can participation and collaboration be motivated and sustained over the spatial extent of the domain of interest? (3) How is scientific rigor assured without the rigor mortis imposed by overly prescriptive control? Here, we present the collaborative monitoring framework of the North American Bat Monitoring Program (NABat) as a model approach for addressing these challenges.Bats play important roles in maintaining healthy ecosystems . Other important contributors include environmental consultants, local naturalist groups, private industry , and trained citizen scientists. These partners have utilized the NABat’s probabilistic sample design, monitoring guidance, and data management tools to contribute standardized bat population data , monitoring efforts have been coordinated and/or conducted by state, provincial, federal, and tribal land resource management and conservation agencies, universities, and non-governmental conservation organizations through a growing number of regional monitoring hubs    . NABat partners are expected to survey the same grid cells annually following national guidance . This entity coordinates members of the hub to ensure that data collection efforts throughout the region follow the predetermined GRTS sampling order of the NABat Master Sample. Coordinated efforts through the hub help prevent redundancies, facilitate equipment sharing, provide training, and streamline data submission and analysis  is enhanced by sampling in both the summer and winter seasons. Rapid declines in numbers of observed hibernating northern long-eared bats at winter roosting sites after the emergence of WNS . Thus, NABat offered a collaborative sampling framework for gathering reliable information to meet each partner’s needs.Local information needs are not always met through top-down, standardized monitoring, sometimes leading to the demise of such efforts  in other parts of the world. National monitoring programs for bats are not common outside of Europe, yet share some similarities to NABat in that they often incorporate and use different data types, such as acoustic detections during summer and winter colony counts, and have some elements of a multi-scale approach by adhering to general guidance provided by the Eurobats Secretariat . One hurdle to building broader engagement of community scientists in the NABat effort is in providing timely results to potential community participants, which is currently limited by the cost and time-delay required for processing acoustic data. Recording and classifying the echolocation calls to a species requires specialized and often expensive equipment and software , and information needs. Collaborating partners can apportion the NABat master sample across their land management jurisdiction to meet local needs while displaying “selected cells” to inform regional partners of extant survey effort. Gaps in regional-scale survey effort can then be targeted to increase coverage of high-priority units and increase statistical power. A probabilistic master sample design with an accompanying centralized database for both the sampling design and resulting data ensures defensible data integration to inform range-wide species assessments.As technological tools for monitoring bats become more widely available and easily deployable across all seasons, the increasing data volumes and acquisition rates will require new collection, integration, data transfer and storage, and synthesis strategies. Scaling and integration of multiple data types that are seasonally misaligned will require new statistical approaches. The NABat program foresees these challenges and is building the infrastructure to meet them.Given limited resources, aligning local conservation and resource management objectives with important continental-scale priorities is essential for a successful long-term conservation program. NABat provides an important and efficient bridge between local, regional, and continental scales. When objectives differ across conservation and management scales, NABat’s design can satisfy local information needs while providing data to species status and trend assessments at regional and continental scales, thus achieving an economy of scale for conservation monitoring.