Genomic-based precision medicine has not only improved tumour therapy but has also shown its weaknesses. Genomic profiling and mutation analysis have identified alterations that play a major role in sarcoma pathogenesis and evolution. However, they have not been sufficient in predicting tumour vulnerability and advancing treatment. The relative rarity of sarcomas and the genetic heterogeneity between subtypes also stand in the way of gaining statistically significant results from clinical trials. Personalized three-dimensional tumour models that reflect the specific histologic subtype are emerging as functional assays to test anticancer drugs, complementing genomic screening. Here, we provide an overview of current target therapy for sarcomas and discuss functional assays based on 3D models that, by recapitulating the molecular pathways and tumour microenvironment, may predict patient response to treatments. This approach opens new avenues to improve precision medicine when genomic and pathway alterations are not sufficient to guide the choice of the most promising treatment. Furthermore, we discuss the aspects of the 3D culture assays that need to be improved, such as the standardisation of growth conditions and the definition of in vitro responses that can be used as a cut-off for clinical implementation. As emphasised by original contributions in this special issue, sarcomas are rare and heterogeneous neoplasms comprising more than 70 subtypes of bone and soft tissue sarcomas (STSs) . AlthougIn the past decades, the genomic and epigenomic characteristics of tumour subtypes have been a focus of research. The hope has been to better classify sarcomas on the basis of genomic rearrangements and to identify vulnerable sites to target treatments . Drug reIn contrast to novel clinical N-1 trials , based oEWSR1:FLI1 fusion or EWSR1 fusion with other partners [SS18 at 18q11 and SSX1/SSX2 at Xp11 genes, resulting in an altered BAF complex [YAP1:TFE3-fused gene and WWTR1(TAZ)-CAMTA1 gene fusions are characteristic of haemangioendothelioma, whereas the presence of FUS:DDIT3 or EWSR1:DDIT3 genes is pathognomonic in myxoid liposarcoma (MLPS), a malignant tumour, recapitulating lipogenesis [In order to understand the aims of the clinical studies discussed below, it is first necessary to provide a short overview of the genetic drivers of sarcoma pathogenesis identified to date. Bone and soft tissue tumours are generally classified on the basis of histology and genomic alterations promoting transformation into simple karyotypes characterised by gene fusion or complex karyotypes. All fusion genes are summarised in partners . In syno complex ,16. The ogenesis ,18,19. Togenesis .Sarcomas with complex karyotypes are a heterogeneous group that include angiosarcoma, myxofibrosarcoma, pleomorphic liposarcoma, osteosarcoma, undifferentiated sarcoma, leiomyosarcoma, and dedifferentiated liposarcoma. Large-scale genomic profiling studies have identified recurrent hotspot mutations, as shown in The standard of care for localised sarcomas consists of surgery associated with neoadjuvant (preoperative) or adjuvant (postoperative) chemotherapy or radiation ,28. HoweIn this section, we review clinical trials using targeted therapy selected on the basis of actionable mutations identified by genomic profiling of patients .Mouse double minute 2 (MDM2), which mediates p53 ubiquitination and its degradation [Amplification of cyclin-dependent kinases (CDKs) is typical in sarcomas and present in over 90% of well-differentiated/dedifferentiated liposarcomas (WD/DDLPSs). Many efforts have been made to develop CDK-specific inhibitors with low toxic effects. Currently, the most effective molecules include the CDK4/6 inhibitors Palbociclib, Ribociclib, and Abemaciclib. Palbociclib, in particular, has demonstrated antitumor activity in patients with advanced or metastatic WD/DDLPS, improving survival (NCT01209598) . Palbociradation . Severalradation . The mosradation . Howeverradation . Many smradation . Recent radation ,41. TrabThe PI3K/AKT pathway and downstream signalling are activated constitutively in the majority of localised osteosarcomas and are involved in their progression . BKM120 In desmoid tumours, prolonged progression-free survival was reported following Sorafenib treatment in combination with another multi-TKI ,49, and Tumour immunotherapy can be divided according to mechanism into targeted therapies based on antibodies, checkpoint inhibitors (ICIs), cytokine therapy, adoptive T-cell transfer (ACT), and tumour vaccination. The use of monoclonal antibodies targeting receptors as monotherapy or in combination with chemotherapy for sarcomas has demonstrated limited activity or did not improve overall survival. For example, the combination of Olaratumab, a monoclonal antibody against platelet-derived growth factor receptor alpha, with doxorubicin provided no detectable clinical benefit in terms of PFS and overall survival in osteosarcoma compared to conventional chemotherapy . MonocloPotentially better outcomes compared to conventional chemotherapy were reported in some sarcoma histotypes with immune checkpoint inhibitors (ICIs). Sarcomas with a high frequency of chromosome copy number alterations, such as UPS and MFS, may be capable of eliciting an immune response and respond better to ICIs or other immunotherapies . However+ cell activation following PD-1/PD-L1 treatment [One mechanism contributing to the immunosuppressive effect of the TME is the downregulation of the HLA immunophenotype inhibiting CD8reatment . Howeverreatment . Patientreatment . In contreatment . This sureatment . The comreatment . CurrentAnother approach to immunotherapy is chimeric antigen receptor T (CAR-T) adaptive cell therapy. This involves isolating the patient’s own T-cells, modifying them to express a CAR that recognises a specific tumour antigen, and then reinjecting them into the patient . The recTrials on rare cancers are usually designed and conducted in small genetically or biomarker-defined subsets of patients. However, the restrictive criteria and the limited analyses answer only specific questions but may not yield real-world data (RWD) and real-world evidence (RWE). RWD data include information on the standard care of patients obtained from prospective and/or retrospective observational studies , and RWEThe lessons learned from these studies have influenced the design of different classes of trials. Currently, umbrella, basket, platform, and master observational trials (MOTs), as well as platform trials, put the patient’s genomic landscape at the centre of targeted therapy and attempt to speed up treatment decisions . ImportaAnother tool to study personalised treatment is NCI-MATCH-N-of-1 trials. An N-of-1 trial is a single-patient clinical trial to evaluate the efficacy and/or adverse events of one or several interventions chosen on the basis of individual molecular and clinical data . So far,Spheroids are extensively used in cancer research. They can be generated from tumour cell lines or by culturing primary tumour cells, allowing their aggregation into small 3D clusters using different methods . Many prVarious preclinical studies have utilised sarcoma spheroids of different histologies as models to study responses to chemotherapies and target therapy in osteosarcoma , chondroFUS-DDIT3 fusion gene. MLPS frequently recurs and has a poor prognosis after standard treatments, such as surgery, and is, therefore, in dire need of novel therapeutic approaches. In a preclinical study, spheroids established from two MLPS patient cell lines (NCC-MLPS2-C1 and NCC-MLPS3-C1) were successfully used for high-throughput drug screening of cytotoxic drugs. Of the 213 tested anti-cancer agents, Romidepsin was selected as the most efficient to suppress cell proliferation, even though it had the lowest IC50. These results indicated that spheroids are a useful tool for basic and preclinical studies of MLPS and probably for other rare tumours, too [Most frequently, spheroids were used as platforms to screen a panel of cytotoxic agents approved for other tumour types, and the threshold of spheroid cell death was used as a parameter of drug efficacy. For example, in one study of Ewing’s sarcoma (EwS), spheroids were used to mimic the temporal sequence of chemotherapy administration in patients, using a microfluidics droplet system. Spheroids generated inside droplets showed similar sizes and low intratumoural differences, and growth and viability could be measured by using fluorescence markers of live and dead cells. Data from an ES model showed that sequential combination treatment with etoposide 24 h before cisplatin resulted in a synergistic tumour cell death effect. The authors, thus, identified the droplet-based microfluidics approach as a valid tool for the evaluation of drug combinations . In thisurs, too .Radiation is an important treatment for sarcomas, particularly those resistant to chemotherapy. The efficacy of gadolinium oxide nanoparticles (GdoNPs) in combination with single (4 Gy) or fractionated (4 × 1 Gy) irradiation was evaluated in chondrosarcoma spheroids and in vivo in a mouse model. Gadolinium oxide treatment decreased spheroid growth and murine tumours. Based on these results, a multicentre, randomised Phase 2 trial is currently evaluating the combination of GdoNP with radiotherapy for the treatment of patients with inoperable musculoskeletal tumours . The uti+, CD4+, and CD8+), and myeloid cells (CD11b+ and/or CD11c+) [Spheroids can contain multiple cell types present in the tumour microenvironment (TME) and may, therefore, be useful for evaluating patient responses to immunotherapy. Spheroids generated from chordoma with a diameter of 40–100 μm contained immune cells, such as T lymphocytes (CD3 CD11c+) . They al CD11c+) ,95. HowePatient-derived organoid (PDO) protocols have been established for many primary and metastatic sarcomas . SeveralOrganoids are widely explored as functional assays to guide the selection of target treatments and chemotherapies. In most studies, organoids are prepared from surgery specimens in order to have sufficient material, and drug screening is performed a few days before starting patient treatment. Several reports have established correlations between responses in PDOs and responses in patients, including disease trajectory, and have helped to characterise the landscape of drug resistance ,102,103.Rhabdomyosarcomas (RMSs) are mesenchymal-derived tumours and the most common childhood soft tissue sarcomas. Despite intense treatment, the prognosis for high-risk patients is poor. The discovery of new therapies would benefit from additional preclinical models. Recently, 19 paediatric organoids from all major RMS subtypes have been generated. Molecular, genetic, and histological characterisation showed that the models closely resembled the original tumours and were genetically stable over extended culture periods of up to 6 months. Tumours of mesenchymal origin can, therefore, be used to generate organoid models relevant to a variety of preclinical and clinical research questions . Novel tRecently, a multi-cohort proof-of-concept study (NCT04986748) involving sarcoma and melanoma patients has started. This trial will use 3D patient models as functional screening of a 14-drug panel, and the in vitro results will indicate patient treatment. Another trial of interest is Sarco PDX (NCT02910895), which will use organoids generated from preoperative biopsies and patient-derived xenografts to personalise the screening of standard chemotherapy in combination with tyrosine kinase inhibitors in advanced and metastatic sarcoma . HoweverAlthough chemotherapies remain the primary treatment for most sarcomas, immune checkpoint inhibitors (ICIs) have been increasingly used. At present, ICI treatment is recommended when there is evidence of increased PD-1 or PD-L1 expression, deficiency in mismatch repair proteins, or increased tumour mutational burden (TMB). Despite these indications, not enough evidence supports the use of ICIs as first-line monotherapy for advanced sarcomas, because ICIs often fail to induce long-lasting efficient cytotoxic responses. In contrast, the combination of chemotherapy with immunotherapy has shown promising results in unresectable or metastatic angiosarcoma and leiomyosarcoma subtypes . Ongoing+ and CD4+ cells, tumour-infiltrating lymphocytes (TILs), and stromal cells. The authors observed infiltrating CD3+ T-cells expressing PD-L1 and the reactivation of TILs following treatment with ICIs. Unfortunately, no comparisons were made between these PDOs and patients receiving the same treatments. Future prospective studies will be required to establish correlations. Of note, when organoids did not contain sufficient intertumoral immune cells, it was necessary to add peripheral blood mononuclear cells to the co-culture in order to detect immune responses. These studies constitute an important first step towards the implementation of precision immuno-oncology. Additional clinical trials are listed in The ability to predict patient-specific responses to immunotherapy with functional assays would benefit them both in terms of timely interventions and cost effectiveness. For this purpose, several organoid-based co-culture models designed to reconstitute or preserve the immune system have been developed. One of the few studies on chordoma organoids demonstrated that they may recapitulate immunosuppressive microenvironments. In particular, chordoma organoids showed the typical immune exclusion phenotype and macrophage M2 polarisation observed in patients. Studies of the culture media from organoids detected the secretion of CCL5 chemokine  and its 2) cultured ex vivo [Explant cultures consist of small tumour biopsy fragments  and colorectal carcinoma (CRC) were co-cultured with autologous plasma and peripheral blood mononuclear cells . DocetaxAnother area in which important progress has been achieved is immunotherapy. Tumour explants that preserve both inter- and intra-tumoural immune clusters are of particular interest for immunotherapy, not only as a prognostic tool but also to select predictive biomarkers of responses to anti-PD-1 antibodies . To dateIt is also conceivable that future developments to find novel tumour treatments could use 3D models generated not from individual patients but from genetically modified normal or tumour cell lines. In this case, the genetic modification is identified in a range of different tumours by the existing data from large-scale genomic or mutational studies. Using such models may provide clues about the relevance of the genetic alteration for tumour growth and indicate their susceptibility, in principle, to a broad range of novel compounds without quantitative or ethical constraints.Despite the identification of genomic profiles and aberrant mutations associated with sarcoma pathogenesis, the success rate of interventions targeting some of them has been very low. The highly variable histology of their subtypes and their rarity pose many challenges discussed in the present review. The development and implementation of different three-dimensional tumour models as functional assays to evaluate the drug vulnerability of individual patients and guide their treatment have not yet received clinical validation. Many limitations impede their use as valid tools. One of the issues is the assay time. Three-dimensional models need to be established from patient biopsy early in the disease trajectory, i.e., before any tumour-directed clinical treatment begins, to avoid alterations in tumour characteristics. Furthermore, to improve the statistical significance and predictive accuracy of the ex vivo functional assays, a great number of PDO cultures need to be generated from individual biopsies, preferentially surgical biopsies. The type of 3D model should be decided on the basis of the clinical question. Neither spheroids nor explants can survive multiple cycles of expansion, and drug tests, therefore, must be performed a short time after biopsy sampling. On the other hand, organoids grow better than spheroids and are generally preferred as a platform to model the tumour microenvironment and for personalised immunotherapy or genomic target therapy.For all these models, standardised quality criteria must be established, the conditions of cultures predefined, and algorithms created to determine the cut-off of a treatment effect in vitro, which should be supported by evidence. Furthermore, several reports have indicated a high degree of both inter-patient and intra-patient heterogeneity among different organoid platforms ,123. ThiThe results of completed and ongoing trials highlight the challenges associated with targeting genetic mutations. Optimizing treatment options on the basis of personalised preclinical assays will offer better treatment to patients and unlock the full potential of precision oncology.


Gastroesophageal reflux disease (GERD) is a highly prevalent disease. Mechanical etiology, including hiatal hernia, can be resistant to empiric proton pump inhibitor (PPI) trials; Nissen fundoplication is commonly used to treat mechanical GERD. Chylothorax is a rare complication of abdominal surgeries, including anti-reflux procedures. In this case report, a 75-year-old female presented with shortness of breath following a redo laparoscopic Nissen fundoplication. Chest CT pulmonary angiography (CTPA) showed bilateral large pleural effusions that were managed by fluid restriction, repeated thoracocentesis, and chest tube insertion; the pleural fluid analysis was significant for fluid triglycerides high at 232 mg/dL which was diagnostic for chylothorax. The patient was treated conservatively. Appropriate management of chylothorax is crucial to avoid subsequent respiratory failure, immunodeficiency, and malnutrition. Chylomicrons and triglycerides in the pleural fluid can be diagnostic for chylothorax. Treatment of chylothorax includes three main approaches: controlling the cause, conservative treatment, and surgical interventions. The prevalence of gastroesophageal reflux disease (GERD) is increasing with more than 30% of adults in the United States endorsing weekly symptoms . FurtherA 75-year-old female with hypertension, hypothyroidism, hyperlipidemia, diabetes mellitus type 2, and a history of floppy Nissen fundoplication with mesh placement 13 years earlier presented to the ED with shortness of breath. This occurred five days after undergoing a redo laparoscopic hiatal hernia repair and Nissen fundoplication with bilateral pigtail placement for recurrent symptoms of regurgitation, progressive shortness of breath, and chronic cough. She had a moderate diaphragmatic hernia on computed tomography (CT) of the abdomen and pelvis.Micrococcus luteus in one bottle, likely due to contamination. CT pulmonary angiography (CTPA) showed large bilateral pleural effusions with bilateral lower lobe atelectasis . Chest X-ray showed large bilateral pleural effusions, more on the right than on the left Figure . The patEsophageal symptom characterization of heartburn and regurgitation has 70% sensitivity and specificity for GERD, suggesting first-line therapy of PPIs with high rates of symptomatic relief without objective testing ; howeverChylothorax is characterized by the accumulation of chyle within the pleural space due to disruption or obstruction of the thoracic duct or its tributaries, the diagnosis of which is supported by pleural fluid triglyceride concentration greater than 110 mg/dL. Since the content of the chylous fluid is usually high in triglycerides, lymphocytes, and immunoglobulins, prolonged chylothorax causes dyspnea, immunodeficiency, and malnutrition . AlthougChyle is usually milky and can be mistaken for pus. In traumatic cases, it can be blood-stained. Chylomicrons are molecular complexes of proteins and lipids synthesized in the jejunum, and their presence in the pleural fluid is the gold standard diagnostic for chylothorax . ChylomiTreatment of chylothorax can be achieved through three main approaches : (i) conChylothorax is a rare complication of abdominal surgeries that should be ruled out in patients with postoperative persistent pleural effusion. Measuring the pleural fluid chylomicrons and/or triglyceride levels is essential to confirm the diagnosis of chylothorax. Management can be achieved through different approaches, including controlling the cause, conservative treatment with fluid restriction, diuresis, pleura fluid drainage, and surgical intervention.


Angiogenesis has been associated with numbers of solid tumours. Anti-angiogenesis drugs starve tumours of nutrients and oxygen but also make it difficult for a chemo reagent to distribute into a tumour, leading to aggressive tumour growth. Anti-angiogenesis drugs do not appear to improve the overall survival rate of pancreatic cancer. Vessel normalisation is merging as one of the new approaches for halting tumour progression by facilitating the tumour infiltration of immune cells and the delivery of chemo reagents. Targeting p21-activated kinases (PAKs) in cancer has been shown to inhibit cancer cell growth and improve the efficacy of chemotherapy. Inhibition of PAK enhances anti-tumour immunity and stimulates the efficacy of immune checkpoint blockades. Inhibition of PAK also improves Car-T immunotherapy by reprogramming the vascular microenvironment. This review summarizes current research on PAK’s role in tumour vasculature and therapeutical response, with a focus on pancreatic cancer. The global incidence of pancreatic ductal adenocarcinoma (PDA) is increasing. Pancreatic cancer now ranks among the top ten most prevalent cancers . The pooOne of the main barriers to developing effective treatments for PDA is its unique characteristic of the tumour microenvironment (TME). The TME of PDA exhibits a dense stromal environment, containing cancer-associated fibroblasts and immune cells that interact with different subsets of cancerous cells, including cancer stem cells (CSCs) . In addiInhibiting blood vessels may limit the expansion of the tumour and enhance the survival of cancer patients ,8. In 20Over 90% of PDA carry KRas mutations . KRas isTumour blood vessels exhibit many defects, such as abnormal vessel growth, leaky structure with an absence of proper pericyte support, and loss of the normal structures found in arterioles, capillaries, and venules . These dIn the TME of pancreatic cancer, the accumulation of fibrotic stroma attributes to a robust desmoplastic response which increases the interstitial fluid pressure . The elePreclinical studies have shown that tumour vasculature normalisation decreased hypoxia and thus metastasis while increasing drug delivery to the tumour site, enhancing the anti-tumour effect of the drug . Tumour Histidine-rich glycoprotein (HRG), a highly prevalent and extensively characterised protein in the plasma , facilitThe TME of pancreatic cancer contains an inefficient and leaky vasculature associated with poor tumour perfusion, leading to hypoxia and tumour metastasis. The failure of anti-angiogenesis drugs in the treatment of pancreatic cancer has urged the approach of tumour vasculature normalisation. PAKs have played important roles in endothelial cell proliferation, migration, and permeability, as well as in angiogenesis in cancer settings ,49. In tPDA is dominated by mutations in the oncogene of KRas, which activates downstream signalling pathways including other GTPases such as CDC42 and Rac , which iThe most extensively studied members among the PAK proteins are PAK1 and PAK4, which have been found to be overexpressed in many types of cancers ,56,57. TPAK4 stimulates cell proliferation and survival through AKT and ERK pathways and plays a key role in regulating the proliferation of vascular smooth muscle cells (VSMCs) through the AKT pathway . PAK4 acThe unique dense stromal which exists in the TME of PDA compresses vasculatures and reduces blood vessel function, preventing the effective delivery of drugs into tumour sites and inhibiting the immune cell penetration to the tumour site. The regulation of tumour vasculature will have a significant impact on the efficacy of chemo- and immune-therapies. PAKs have recently gained attention in angiogenesis and the modulation of vasculature in cancer settings, including PDA ,67,68,69Rho, Rac, and Cdc42, members of the Rho family of small GTPases, are responsible for the rearrangement of actin cytoskeleton in cells and regulate integrin adhesion complexes located at the cell surface in response to various signals . They alPAK1 has a vital role in the process of tumour angiogenesis. PAK1 amplification has been associated with advanced tumour stages, higher MVD, and notable colony stimulating factor 2 expression (CSF2) in myxofibrosarcoma. CSF2 is a cytokine that regulates the differentiation of macrophages and promotes angiogenesis, which is downregulated in PAK1 knockdown groups . In breaIn the TME of pancreatic cancer, there is a positive correlation between PAK1 expression and the expression of α-SMA and Desmin, suggesting the potential involvement of cancer-associated fibroblasts (CAFs), which are known contributors to angiogenesis . PAK1 prPAK4 plays a critical role in coordinating the abnormal development of blood vessels and the exacerbation of low-oxygen (hypoxic) conditions within a tumour. In a kinome-wide screening of mesenchymal-like transcriptional activation in human glioblastoma-derived endothelial cells, PAK4 was identified as a selective regulator of genetic reprogramming in tumour vasculature, leading to aberrant vascularisation . PAK4 knIn prostate cancer, inhibition of PAK4 increased the width of blood vessels in the tumour context . FurtherThe roles of PAKs in tumour vasculature are summarised in In pancreatic cancer, the extensive desmoplastic reaction leads to the formation of a dense stroma, causing inadequate vascularisation, inefficient drug delivery, and ineffective immune cell infiltration into the tumour sites, which eventually leads to therapeutical resistance and cancer progression . PAKs haPAKs are activated and play a crucial role in facilitating the resistance of cancer cells ,95. ModuTumour vasculature normalisation would potentially facilitate immune cell infiltration and increase the efficacy of immunotherapy. Immune checkpoint inhibitors have been found to enhance vessel normalisation by engaging CD4+ T lymphocytes . PD-L1 ePAK4 expression is negatively correlated with immune cell infiltration in other cancers. High PAK4 expression is associated with limited infiltration of T cells and dendritic cells in immune checkpoint inhibitor-resistant melanoma patients. However, genetic deletion of PAK4 reversed PD-1 blockade resistance by increasing CD8 T cell infiltration in mice carrying flank tumours of melanoma. A combination of anti-PD-1 treatment and a PAK4 inhibitor enhanced anti-tumour responses compared to anti-PD-1 treatment alone. The effects of PAK4 KO were mediated via inhibition of the WNT pathway . Wnt/β-cInhibition of PAK4 enhanced the infiltration of CD103+ dendritic cells and CD8 T cells, and its expression correlates with CCL21 levels in melanoma biopsies. Notably, PAK4 KO cells exhibited increased expression of genes associated with blood vessel formation, including Cercam, Enpep, Itga, and Lgals. CD31, a primary blood vessel marker, was increased in tumours treated with anti-PD-1 in PAK4 KO mice . In glioTumour vasculature plays a significant role in regulating the supply of nutrients, oxygen, and immune cell infiltration in the TME. Efforts in anti-angiogenesis treatments, designed to deprive cancer cells of nourishment by diminishing tumour blood vessel formation, have yielded outcomes below optimistic projection. The reduction in tumour vascularisation results in increased tumour hypoxia, leading to increased therapeutical resistance and cancer metastasis. The vasculature normalisation approach aims to reinstate proper blood flow, thereby improving the distribution of therapeutic drugs, enhancing the tumour infiltration of immune cells, and decreasing the spread of metastasis , which hIn view of the importance of the immune system in cancer, it is crucial to understand the molecular mechanisms involved in immune cell infiltration and vasculature changes. The interaction between vasculature, tumour immune response, and PAKs in cancer biology presents a complicated and dynamic landscape. As simplified in


Enzyme replacement therapy (ERT) with cerliponase alfa (Brineura) has been shown to slow progression of milestone deterioration in Neuronal Ceroid Lipofuscinosis Type 2 (CLN2), an inherited neurodegenerative lysosomal storage disorder. Brineura must be administered intraventricularly every 2 weeks, necessitating the placement of ventricular reservoirs and requiring frequent access. Traditionally, positive cerebrospinal fluid (CSF) cultures are managed with device replacement and antibiotic treatment. We sought to establish which circumstances might allow for careful observation without device removal. In this study, we report our single-institution experience with clinical outcomes of positive CSF cultures in 16 CLN2 patients over 6 years.We retrospectively reviewed a cohort of 16 patients with CLN2 disease who had ventricular reservoirs placed for ERT administration. At each ERT infusion, CSF was collected by sterile technique and cultured by both thioglycolate broth and agar plate. Epidemiologic and microbiologic data, symptomatology, total antibiotic days, removal and replacement of ventricular reservoir, hospital length of stay, and mortality were analyzed for each patient.Cutibacterium acnes, which typically grew in only broth culture (82%), suggesting low bacterial burden . The other 7 patients with positive cultures remained asymptomatic with no intervention required.In our cohort, 11 of 16 patients (69%) had at least one positive CSF culture . Of the 11 with positive cultures, only 3 had their device removed and replaced for a positive culture with concurrent antibiotic treatment at our center, and 2 patients subsequently have been treated with prophylactic antibiotics at infusions . Out of 1401 total CSF cultures, 64 (4.56%) were positive. The most common organism grown was At our institution, we have found that in asymptomatic patients with frequently accessed ventricular reservoirs, positive CSF culture with low virulence skin flora was common, and rarely required intervention. This strategy reduced the risks of invasive surgery, prolonged antibiotic courses, and missed infusions. In the future, routine screening cultures in asymptomatic patients can be avoided.Raymond Wang, MD, Biomarin Pharmaceutical: Advisor/Consultant|Biomarin Pharmaceutical: Grant/Research Support|Biomarin Pharmaceutical: equity interest Antonio C. Arrieta, MD, FIDSA, FPIDS, Astellas Pharma Global Development, Inc.: Advisor/Consultant|Astellas Pharma Global Development, Inc.: Grant/Research Support|Astellas Pharma Global Development, Inc.: Honoraria|Cumberland Pharmaceutical: Grant/Research Support|IDbyDNA: Advisor/Consultant|IDbyDNA: Grant/Research Support|Melinta: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Nabriva: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support|Pfizer, Inc: Advisor/Consultant|Pfizer, Inc: Grant/Research Support|Roche/Genentech: Grant/Research Support|The Medicine Company: Grant/Research Support


The aim of present work is to apply the Caputo–Fabrizio fractional derivative in the constitutive equations of heat transfer. Natural convection flow of an unsteady second grade fluid over a vertical plate with exponential heating is discussed. The generalized Fourier law is substituted in temperature profile. A portion of the dimensionless factors are utilized to make the governing equations into dimensionless structures. The solutions for temperature and velocity profiles of Caputo–Fabrizio model are acquired through the Laplace transform method. These solutions are greatly affected through the variation of different dimensionless variables like Prandtl number, Grashof number, and second-grade fluid parameter. Finally, the influence of embedded parameters is shown by plotting graphs through Mathcad. From the graphical results it is concluded that, the temperature of the fluid decreases with the increasing values of the Prandtl number and Second grade fluid parameter and increases with the passage of time. The velocity of the fluid increases with increasing values of the Grashof number, second grade parameter and time while decreases with increasing values of fractional parameter and Prandtl number. Samiulhaq et al.2 studied the fractional second-grade fluid of MHD flow considering modified Darcy’s law and exponential heating. The LT is utilized for the solution of CF model. The influence of fractional order derivative having non-singular kernel on second-grade fluid was analyzed by Fetecau et al.3. The impact of second-grade fluid in vertical and circular cylinder was considered by Refs.5. The exact solution is acquired via Laplace transform (LT). References7 investigated of fractional immiscible multi-layer flow of second-grade fluid in rectangular channel. The remedy is acquired via finite Fourier sine and LT. Siddique et al.8 studied the heat transfer analysis of fractional second-grade fluid with Newtonian heating. CF and ABC fractional operators are introduced in the fluid model. Elnaqeeb et al.9 analyzed the natural convection flow of second-grade nanofluid using Prabhakar fractional operator for the solution. Abro10 investigated the second-grade MHD fluid with permeable medium utilizing CF fractional operator. References12 studied the fractional  rotational second-grade fluid with thermal stratification. The remedy is got via Laplace transform. Importance of magnetic field of second-grade fluid on the dynamical analysis was given by Riaz et al.13. The CF and ABC fractionalized model is solving through LT method. Sene14 investigated the generalized second-grade fluid using Caputo–Liouvile fractional operator. Nisa et al.15 studied the second-grade fluid with damped thermal flux and radiation among vertical channel. Generalized Fourier law has been utilized in energy equation. The solution is acquired from the most powerful method called Laplace transform. Haq et al.16 analyzed the fractional second-grade fluid with ramped wall heating and slippage effects on heat and mass transfer model. The CF time derivative is utilized in temperature profile. The solution is acquired via Laplace transform.The study of non-Newtonian fluids in fluid mechanics is more useful than Newtonian fluids because of the intricate interaction between stress and strain in non-Newtonian fluids and its industrial and engineering applications. The extraction of energy from geothermal regions, the flow of oil through porous rocks, the filtering of particles from fluids, and medication penetration through human skin are all examples of non-Newtonian fluid applications. Second-grade fluids are non-Newtonian fluids that have up to a second derivative in the strain tensor relation, whereas Newtonian fluids have just a first derivative. Many researchers and engineers were interested in the flow of second-grade fluid. Nehad and KhanThe above literature motivates us to work on natural convection flow of fractional second grade fluid with generalized Fourier’s law. The main novelty of this article is the analysis of heat transfer through natural convection under the effects of exponential heating with generalized Fourier thermal flux. The solution for velocity and temperature field is acquired via Laplace transform technique. The effects of different embedded parameters were finally visually and conceptually demonstrated.17. Fluid flow problem is represented geometrically by Fig. Consider the second grade fluid of unsteady flow closer to an infinite upright plate with exponential heating the straight upward direction of the plate. Consider as x-axis direction and y-axis is put up with perpendicular to the flat established by the plate when t = 0 both the fluid and the plate are remainder to the constant temperature T infinity. After some time, the plate begins to move at the specified speed. The governing equation is provided by, according to the standard Boussinesq’s approximationFollowing are the IC’s and BC’s;Substituting the bellow dimensionless factors,Introducing the above unit less factor in –3) and  and 3) a18 and Hristov20, which addresses the thermal heat flux.The fractional generalization of Fourier law is given by Henry et al.21Caputo–Fabrizio fractional derivative and its Laplace transform is defined asUsing the above CF definition in Eq.  in \docuThe dimensionless initial and boundary condition are follow;Put Eq.  in Eq.  and takiThe transform IC and BC are following,After solving Eq.  and usinEquation  inverse Taking LT on Eq.  and theiAfter solving Eq.  and intrThe inverse LT of Eq.  and util(i)Considering 22.The result given in  is unifoIn this section, we examine physical comprehension. The physical sketch of the problem is given in Fig. The effects of time on the temperature profile are depicted in Fig. Figure The effect of the second-grade fluid component on velocity is seen in Fig. The temperature of the fluid decreases with the increasing values of the Prandtl number and fractional parameter.The temperature of the fluid increases with the increasing values of the time.The velocity of the fluid decreases with the increasing values of the fractional parameter.The velocity of the fluid increases with the increasing values of the Grashof number and time.The velocity of the fluid decreases with the increasing values of the second grade parameter and Prandtl number.We looked into the generalized Fourier law, thermal heat flux, and natural convection flow of a second-grade fluid in this paper’s for closed-form solution. Using the Caputo–Fabrizio fractional derivative, the well-known model is converted to a fractional model. The Laplace transform is used to obtain the solutions to the momentum and energy equation. Plots are made for a number of embedded parameters that are part of the momentum and energy equation. The following list includes the thesis’ principal conclusions:This problem can be extended by solving this problem for Heat transfer analysis of generalized second-grade fluid with exponential heating and non-Fourier heat flux.This problem can be extended by solving this problem by adding heat generation and radiation source to the same problem.This problem can be extended by solving this problem by adding concentration class to the same problem.


Bullous pemphigoid (BP) is an autoimmune blistering disease that targets the haemidesmosomal proteins, mainly BP180. Extracellular vesicles (EVs) have been demonstrated to carry tissue-specific autoantigens in the setting of autoimmune diseases and transplant organ rejection; this phenomenon was demonstrated to have pathogenic implications in autoimmune diseases and to correlate with transplant rejection severity. The purpose of this study was to identify the presence of BP targeted autoantigens in blister fluid derived EVs. We isolated, by size exclusion chromatography, EVs derived from blisters of BP-patients and from suction blisters of healthy donors. EV characterization was performed by flow cytometry and nanoparticle tracking analysis. Western blot analysis was used to investigate the presence of autoantigens. A suspension enriched in EVs was efficiently obtained from blister fluid from patients and healthy donors. EV-enriched fractions were enriched in particles with a size distribution characterizing small-EVs (main peak was present at 94.5 nm). BP180 was found, by western blot analysis, in EVs derived from blister fluid of 3 out 6 BP patients and in none of EVs isolated from suction blister fluid of healthy donors. BP230 and Dsg1 were not detectable in EVs of any of the samples. No specific clinical characteristics seemed to correlate to the presence of BP180 in EVs. The discovery of BP180 in EVs derived from blister fluid might help understanding BP pathogenesis.The online version contains supplementary material available at 10.1007/s00403-023-02560-2. Bullous pemphigoid (BP) is an organ-specific autoimmune blistering disease, mediated by autoantibodies directed against haemidesmosomal proteins, mainly BP180 [Extracellular vesicles (EVs) are small membranous structures that encapsulate a variegate cargo  and mediate intercellular communication . Based oThe aim of our study was to identify the presence of BP targeted autoantigens in blister fluid derived EVs (BF-EVs) in BP patients.All patients had recently diagnosed active forms of BP, confirmed by characteristic histological and immunological findings. Study population included 9 BP patients with idiopathic BP and 2 patients with drug-induced BP and 6 healthy controls . Suction blisters on the volar surface of forearms were obtained by Hijama cups . BlisterEVs were isolated from BF by size-exclusion chromatography (SEC) with qEV70 columns , that contain a resin with approximately 70 nm pore size . Prior SEV characterization by flow cytometry was performed as previously described . For eacFor western blot analysis EVs were resuspended in RIPA buffer  and protein content was quantified by Bicinchoninic acid (BCA) assay . Two μg of proteins for each sample, were loaded on 4%–12% NuPAGE Bis–Tris gel . Electrophoresis was performed at 150 V and proteins were blotted on a polyvinylidene fluoride membrane . After blocking nonspecific sites with 5% non-fat dry milk  in Tris Buffered Saline with Tween 20 , blot membrane was incubated overnight at 4 °C with specific primary antibodies for: rabbit anti-human BP180 , goat anti-human BP230  goat anti-human DSG1 , and mouse anti-human CD63 , prepared in 2.5% non-fat dry milk/TTBS. After three washes with TTBS, membranes were incubated with a specific HRP-conjugated secondary antibody . Positivity was highlighted by providing the substrates for the chemiluminescence reaction of HRP  and impressing a photographic sheet by autoradiography . Images were scanned using the Epson perfection 1260 scanner. Gel running was performed under not denatured conditions for the detection of CD63 and under denatured conditions for other markers.Demographic and clinical characteristics of patients and controls are summarized in Table 10 particles/ml . At flowcytometry analysis, CFDA-SE stained particles under the level of the dimensional gate in the FL1 intensity channel expressed high levels of both CD81 and CD9 , while the expression of CD63 was lower (5.6%), indicating that the suspension collected from the SEC column was mainly composed by EVs .A suspension enriched in EVs was efficiently obtained from blister fluid of patients and healthy controls. From an average amount of 677 ml of patient-derived blister fluid and 532 ml of healthy control-blister fluid we were able to purify 25 mg and 19 mg of EVs, respectively, as measured by BCA assay. Nanoparticle tracking analysis indicated that the suspension was enriched in particles with a size distribution characterizing small-EVs (exosomes). The main peak was present at 94.5 nm, and a median size distribution of 123.9 nm, while the concentration of vesicles retrieved from 250 µl was 6.5 × 10For what concerns the idiopathic BP cases, a band at 180 kDa was detectable at variable intensity in 5 out of 9 patients . A band at 120KDa was clearly visible in 3 cases  and a faint band was detectable in 3 cases . Bands at a molecular weight of approximately 150KDa were visible in four cases   Representative size distribution for blister fluid-EVs, analysed by Zetaview NTA. (b) Representative bidimensional dot plots  for CFDA-SE specificity: EVs stained with CFDA-SE at 4°C (left panel) and EVs stained with CFDA-SE at room temperature (RT) (right panel). (C) Flow cytometry analysis of blister fluid-EVs. Areas under the black lines identify vesicles reacting with CD81 (left panel), CD63 (middle panel), and CD9 (right panel). Areas under the gray lines indicate the interactions of vesicles with corresponding non-reactive immunoglobulin of the same isotype (JPG 7 KB)Supplementary file2 Western Blot results of Drug-induced BP (PNG 142 KB)Below is the link to the electronic supplementary material.


Broth microdilution at high inoculum is the gold standard for the detection of the CzIE, but this method is time and labor-intensive. We developed a nitrocefin-based rapid test that can identify the CzIE among MSSA. However, the original protocol for this rapid test includes the use of ampicillin in solution, which is difficult to prepare and unstable once reconstituted. In this study, we aimed to show that simple elution from ampicillin disks available in the typical clinical microbiology lab performs as well as an ampicillin powder-based assay.The cefazolin (Cz) inoculum effect, defined as a Cz MIC ≥16 µg/mL at 10Original Protocol for the performance of the Nitrocefin-based rapid test as published by Rincon et at JCM, 2022). The CzIE was determined using gold standard broth microdilution at high inoculum (107 CFU/mL). The published protocol of the nitrocefin test  was modified as follows : instead of adding ampicillin from powder to BHI broth (150 ug/mL), ampicillin disks (10 ug) of 2 different brands (Oxoid and BD) were added to 1 mL of BHI broth of 2 different brands  and incubated for 2 hours at room temperature with frequent shaking . The remaining steps of the protocol were not modified. The original protocol was used as control. Performance metrics were calculated for the complete data set.We included 107 unique MSSA bloodstream isolates which had been previously characterized  were compared to the use of ampicillin from powder to a final concentration of 150 ug/mL51.4%(55/107) of the isolates were CzIE+ by reference broth microdilution at high inoculum. When using Oxoid BHI broth, the sensitivity and specificity of the test were 96.3% and 90.3% regardless of the ampicillin form (disk vs powder) or disk brand (Table 1). When using BD BHI broth, the sensitivity and specificity of the test were 69.1% and 94.2%, regardless of the ampicillin form or disk brand.The modification of the nitrocefin-based rapid to use ampicillin disks instead of ampicillin powder did not impact the accuracy of the tests as it was able to correctly detect the CzIE in isolates, regardless of the brand of ampicillin disk. Implementation of this test may contribute to therapeutic decisions in deep-seated MSSA infections.Jennifer Dien Bard, PhD, Abbott Molecular: Grant/Research Support|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|BioMerieux: Honoraria|Genetic Signature: Advisor/Consultant|Genetic Signature: Grant/Research Support|Luminex: Grant/Research Support|Salve: Stocks/Bonds|Thermo Fisher: Honoraria Lars Westblade, PhD, Accelerate Diagnostics, Inc.: Grant/Research Support|bioMerieux, Inc.: Grant/Research Support|Hardy Diagnostics: Grant/Research Support|Roche Molecular Systems, Inc.: Advisor/Consultant|Roche Molecular Systems, Inc.: Grant/Research Support|Selux Diagnostics, Inc.: Grant/Research Support|Shionogi, Inc.: Advisor/Consultant|Talis Biomedical: Advisor/Consultant William R. Miller, M.D., Merck: Grant/Research Support|UpToDate: Honoraria


Tumours do not exist in isolation from the organism; their growth, proliferation, motility, and immunosuppressive response are intricately connected to the tumour’s microenvironment. As tumour cells and the microenvironment coevolve, an inflammatory microenvironment ensues, propelling the phenomenon of inflammation-cancer transformation—an idea proposed by modern medicine. This review aims to encapsulate the array of representative factors within the tumour’s inflammatory microenvironment, such as interleukins , transforming growth factor-beta (TGF-β), interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Moreover, drawing upon research in traditional Chinese medicine (TCM) and pharmacology, we explore the delicate interplay between these factors and tumour-associated inflammatory cells: tumour-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumour-associated neutrophils (TANs) and dendritic cells (DCs). By analyzing the tumour-promoting effects of these entities, we delve into the connotations of Academician Tong Xiao-lin’s novel model of “state-target differentiation” and its application in the diagnosis and treatment of tumours. Our aim is to enhance the precision and targeting of tumour treatment in clinical practice. Delving deeper into our understanding of tumour pathogenesis through the lens of modern medicine, we discern the key etiology and pathogenesis throughout the entire developmental stage of tumours, unveiling the evolutionary patterns of Chinese Medicine (CM) states: heat state → phlegm state → stagnation state → deficiency state. Building upon this foundation, we devised a state-regulating formula. Simultaneously, drawing on pharmacological research in traditional Chinese medicine (TCM), we meticulously identified a range of targeted drugs that effectively modulate the aforementioned tumour-related mediators. This comprehensive strategy—a harmonious integration of state identification, target recognition, and simultaneous regulation—aims to elevate clinical efficacy. The fusion of TCM with Western medicine in tumour treatment introduces novel dimensions to the precise and refined application of TCM in clinical practice. In the mid-1800s, Rudolf Virchow was the first who correlated inflammation to long-term ailments, like cancer. Inflammatory factors released by the tumours trigger malignant growth, while infections in the body lead to the transformation of healthy cells or obscure tumours into cancer. Since then, numerous researches are on-going to explore the association between inflammation and cancer transformation . Within The concept of “State-target differentiation” was proposed by the esteemed academician Tong Xiaolin, with the intention of drawing inspiration from the physiological and pathological changes in diseases within modern medicine. By incorporating CM perspectives, this concept aims to unravel the disease’s evolutionary trends and comprehensively comprehend its core pathogenesis, consequently establishing corresponding state-regulating prescriptions and medications. Simultaneously, the integration of contemporary pharmacological research facilitates the administration of targeted drugs to amplify disease-specific treatments, thereby achieving a comprehensive therapeutic effect targeting both symptoms and underlying causes. In this context, the term “state” pertains to the dynamic nature, state of affairs, and overall picture of a human body’s internal environment, serving as a comprehensive summary of the core pathogenic mechanisms at various stages of disease development—falling under the macro category. On the other hand, the term “target” refers to the framework of Western medicine, where disease-specific prescriptions and medications are identified based on the aforementioned “state,” thereby targeting the disease itself, its typical symptoms, and relevant clinical physicochemical indicators—falling under the micro category .In our quest to delve into the utilization of the “state-target-cause-effect” therapeutic approach in diagnosing and treating the intricacies of the tumour inflammatory microenvironment, we aim to synergistically integrate the comprehensive insights offered by Chinese and Western medicine regarding the entire trajectory of tumour inflammatory microenvironment development. Furthermore, we shall draw upon the profound wisdom contained within the classifications expounded in the “12th Five-Year” textbook of “Internal Medicine of Traditional Chinese Medicine” , cancer Tumour microenvironment is a dynamic entity that acts like a home for tumour cells development and progression . Tumour The inflammatory microenvironment serves as a catalyst to transform the normal cells into tumour cells, thus promoting the multiplication and spread. The prolonged release of inflammatory components from these mutated tumour cells keeps the balance of inflammation, which in turns fosters the growth of tumour cells and increases the risk of malignancy. Tumour inflammation exerts a significant impact on the growth and spread of the cancerous cells, hence acts as an essential factor for cancer disease. Figure Cancer can be exacerbated due to the extrinsic factors such as infection, and intrinsic factors like genetic mutations that induce the activation of oncogenes and suppression of tumour suppressor genes, forms a relationship between inflammation and cancer. The dynamic interactions between the extrinsic and intrinsic factors create a convoluted and ever-evolving inflammatory atmosphere, which is regulated by various inflammatory factors released by the infiltrating cells. The inflammatory cancer transformation and the generation development of a cancer-associated microenvironment is shown in Fig. The inflammatory tumour microenvironment, which is low in oxygen, pH and high in tissue pressure allows tumour cells to thrive. Hypoxia has been proven to suppress the killing capacity of NK cells , reduce Tumour cells stimulate a sustained inflammatory reaction and cause the tumour cells to hold under a prolonged chronic inflammatory state, which is a significant component of tumour inflammation. The chronic inflammatory state is essentially unmanageable due to they are influenced by multiple mechanisms and factors within the inflammatory environment. Besides, the irregulated process also causes the accumulation of pro-inflammatory molecules in the inflamed area, hence acts in reciprocation to keep the inflammation continuing, resulting in tumour cells destruction .Several inflammatory factors related to tumours, including interleukins , TGF-β, IFN-γ, TNF-α, VEGF, and MMPs, are also the cancer-associated genes. The release of these inflammatory substances summons and energizes the inflammatory cells such as TAMs, MDSCs, TANs, DCs, NKs, B-cells, and T-cells. The collaboration between these cytokines and inflammatory cells promotes the development and metastasis of cancer cells, genetic mutations, and angiogenesis in the tumour inflammatory microenvironment.Studies proved that IL-6, a cytokine with various roles in inflammatory condition, can initiate epithelial–mesenchymal transition (EMT) in tumour cells, and this is evidenced by the decrease of E-cadherin and increase of N-cadherin and vimentin expression which boosting the movement and infiltration of tumour cells . IL-6 trThe level of IL-10 expression is closely related to tumour immunosuppression. In the ovarian cancer TME, studies have shown that IL-10 impairs the ability of DCs to stimulate T-cell responses. This is achieved by blocking the expression of IFN-γ, TNF-α, and IL-12, as well as down-regulating MHC class II molecules. Consequently, T cell immunotherapy against tumour growth is inhibited .IL-17 is a pro-inflammatory cytokine produced by cells expressing RAR-associated orphan receptor γt (RORγt), including T cells  and non-T cells. Research has demonstrated that IL-17 plays a significant role in mediating tumour drug resistance, promoting tumour proliferation, and facilitating metastasis. Its specific mechanisms involve mediating resistance to tumour therapy by enriching bone marrow-derived cells (BMDCs) in the TME, as well as up-regulating the activity of stromal cells . AdditioIL-1β is abundant in the TME and plays a crucial role in promoting tumour growth and inducing tumour immunosuppression. Sequential treatment of wild-type (WT) mice with anti-IL-1β antibodies and anti-PD-1 antibodies has been shown to completely inhibit tumour progression . In non-Evidence shows that TGF-β pathway is a suppressor of tumour growth in the context of the tumour environment . DespiteIn 1965, Wheelock made a pioneer discovery on the antiviral substance IFN-γ, which is the part of the type II interferon family and is a cytokine that stimulates an inflammatory response initiated by T cells and NK cells. Despite the initial evidence suggesting that IFN-γ could promote tumour immunity and antiviral activity, results of clinical trials reported in contrary. IFN-γ was reported to induce tumour immunosuppression and pro-tumour growth when used in combination with carboplatin/paclitaxel, thus resulting in shorter survival times for ovarian cancer patients . IFN-γ aIt has been proven that TNF-α fosters tumour proliferation . The priNon-small cell lung cancer (NSCLC) is a prevalent and serious type of lung cancer. In NSCLC, the role of VEGF and its receptor is significant in promoting an immunosuppressive tumour microenvironment. This occurs through direct effects on both innate and adaptive immune cells, as well as indirect effects on endothelial cells . ConsequMatrix metalloproteinases (MMPs) are a class of proteases closely associated with tumour cell infiltration and metastasis. They play a key role in degrading and altering the extracellular matrix structure, allowing cancer cells to invade surrounding tissues. Research has shown that the key substances regulating the process of matrix degradation are the endogenous inhibitors of MMPs, known as tissue inhibitors of metalloproteinases (TIMPs). In normal cells, MMPs are involved in the degradation and remodeling of the extracellular matrix, maintaining a balance. However, in cancer, the activity and regulation mechanisms of MMPs are often imbalanced, thereby promoting tumour infiltration and metastasis . It is wTAMs can be classified into M1-type, which exhibits the anti-tumour effect, and M2-type, which exhibits the pro-tumour effect . The tral-arginine is deficient, MDSC will inhibit t cell production and thus inhibit the immune response [MDSCs derived from immature and undifferentiated cells which can be categorized into G-MDSC and M-MDSC . MDSC inresponse –63. M-MDresponse . MDSC caresponse . MDSC inresponse . MDSC caNeutrophils have both anti-tumour N1 type and pro-tumour N2 type behaviours. Fridlender et al. found their pro-tumourigenic effects were associated with inhibition of the N1 phenotype and activation of the N2 phenotype by TGF-β . and TGFDendritic cells (DCs) play a key role in anti-tumour immune responses . It was Academician Xiaolin Tong developed the “Target Identification and Treatment” strategy through the combination of CM and modern medical techniques. CM holds the holistic notion in high regard, and viewing a healthy body must be in a constant equilibrium. If this equilibrium is being disrupted, the body’s internal environment will be imbalanced, and potentially develop various diseases. Malignant tumour is a long-term disease that disrupts the body’s equilibrium, and swelling plays a pivotal role in their expansion. Tumour inflammation is responsible for the disruption of the microenvironment.CM treatment focuses on rectifying any imbalances in the body and restoring its internal equilibrium to combat diseases.The “state-target differentiation” strategy is a medical innovation system that combines the theories of CM and modern medicine proposed by Academician Tong Xiaolin. CM emphasizes the concept of wholeness and believes that a healthy human body is in a state of dynamic balance. When the balance is broken, the internal environment of the body is disturbed, and it is easy to be attacked by diseases, and various “distortions” appear. Malignant tumour is a chronic disease with imbalance of homeostasis, and inflammation is one of the important factors for tumour development. Tumour inflammation microenvironment is a manifestation of microenvironmental imbalance caused by tumour inflammation.According to the theory of “state-target differentiation,“ the treatment of malignant tumours can be divided into four stages. In the early stage, evil qi invades. In the middle stage, there is an interplay between good and evil. In the late stage, cancerous toxins gradually penetrate into the body, resulting in a deficiency of positive qi. Scholars who start from the perspective of inflammation have discovered a close relationship between tumours and abnormal secretion of inflammatory factors as well as immune imbalance. They propose the theory of inflammation-cancer transformation, which suggests that the formation of cancerous toxins is closely related to heat, phlegm, stasis, and deficiency. The process includes the transformation of heat into phlegm, phlegm into stagnation, and finally into positive deficiency. Under the influence of dampness and heat, they interact and intensify each other. Dampness becomes hotter due to heat, leading to the formation of Damp-Heat Proof. Over time, damp-heat evil qi further disrupts the body’s fluid balance, causing damp-heat to transform into phlegm. The hot dampness burns the fluids and forms phlegm, obstructing the flow of qi and blood. Eventually, the combination of phlegm, dampness, and heat gradually results in stagnation. Blood stagnation hinders the normal circulation of qi and exacerbates the disease’s progression. Ultimately, the prolonged effects of damp-heat evil and stagnation weaken the positive qi, damaging it and causing a progressive decline. This weakened positive qi state accelerates tumour development and worsens the disease. At this stage, toxins have deeply penetrated the bloodstream, significantly escalating the severity of the disease. Phlegm evil disrupts normal blood flow and combines with dampness and heat evils, gradually manifesting as palpable lumps.Hedyotis diffusa and Asarum sagittarioides can be employed to alleviate the body’s inflammatory response. To specifically address tumor-related physiological and chemical indicators such as IL-6, TGF-β, IFN-γ, TNF-α, MMPs, and others, Chinese herbs and representative formulas that demonstrate specific effects on these indicators can be sought as interventions for tumors at both macro and micro levels (Fig. To maintain the balance of yin and yang in the body, various treatments can be utilized to address the aforementioned imbalances. These may include heat-clearing and detoxifying therapies, phlegm elimination and stagnation dispersal techniques, blood circulation promotion and blood stagnation elimination methods, as well as positive support and dispersal of evil spirits. Representative formulas, such as prescription of Qingrejiedu , Xiaotanels Fig. .CM is unique in providing a comprehensive assessment on the progression of the disease based on its staging system. Compared to the Western approaches, CM emphasizes on the maintenance of the equilibrium between human body and environment, relying on the holistic notion of harmony between Heaven and Earth, and yin and yang. The Treatments are thus approached to a more macroscopic viewpoint . By takiAmong the CM professionals, cancer development is widely perceived as a result of an imbalance between good and evil forces . The “TrCM believes that abundance of negative energy in the body over an extended period can cause tumours. Heat damages the body, causing an increase in fluids and mucus, a decrease in Qi and blood flow, and blockages of organs and channels due to the heat and toxins released by the blood clot, mucus, and impurities. The blood clots, mucus and impurities can cause sores and lesions in the body. “The Golden Mirror of Medicine” states that carbuncles and gangrene are caused by the toxic heat that blocked the meridians, which results in the stagnation of both blood and Qi. The literatures of both antiquity and modernity indicate that heat toxicity causes tumours . PatientThe text “Jing Yue Quan Shu” warns that if one’s diet remains stagnant, it may impede progress and will not yield the desired outcome . In the Patients with tumour typically display a hypercoagulable shape due to the increased blood viscosity. However, CM views it as a symptom of the body’s fluids depletion due to the excessive evil heat generated by inflammation, thereby causing the blood to become stagnant and slow down the circulation. This concept is now widely accepted by modern medicine, whereby the generation of an inflammatory reaction activates the emission of cancer-related substances and triggers the rise of inflammatory mediators, which trigger the external clotting system to amplify the thrombin level . TherefoThe “Jing Yue Quan Shu” states that the key of treating the tumour’s progression is to recognize the appropriate timing of intervention and supplement energy. If the accumulation is persisted for a prolonged period, the vital energy will become increasingly feeble, rendering it unwise to launch an attack, as the stomach is near to the gas and would be the first to suffer from the harm. The more the patient strikes, the weaker the patient . The lacWhen dealing with the cancerous growths, addressing the underlying cause is paramount, and in the case where the tumours are caused by inflammation, the source of the inflammation must be identified. As inflammation is the primary target of the disease, CM believes anti-inflammatory treatments and immediate improving the microenvironment of tumour inflammation will be an effective strategy. By using herbs with anti-inflammatory effects, inflammation conditions in the cancer patients can be reduced, the inflammatory environment can be regulated, the pain can be reduced, and eventually immunity can be enhanced. The goal of controlling blood pressure in the patient is to address the four primary pathogenic conditions of heat, phlegm, stasis, and deficiency for malignant tumour progression by employing a combination of medications and formulas. Western medicine focuses on treating the symptoms of blood pressure by specifically targeting the blood pressure. To optimize the treatment of malignant tumours using Chinese herbs, it is essential to determine the most efficient medications for this purpose. This paper takes into the account of the recent pharmacological findings in Chinese medicine, whereby herbs that are targeting to the release of cytokines like IL-6, TGF-β, IFN-γ, and TNF-α are focused, then employs in combination with the evaluation methods of Western medicine and CM to achieve precise disease management.Various inflammatory molecules like IL-6, TGF-β, IFN-γ, and TNF-α are associated with tumour development, while tumour recurrence is associated with its spread . AdditioHerb used in Traditional Chinese Medicine, such as scutellarin barbate herba, are proven to effectively control the body’s heat state, which is a significant factor that causes tumours. Controlling the body’s heat state can reduce abdominal mass, bloating, and pain in the patients with colorectal cancer, and lower AFP levels in the liver cancer patients. The active ingredient, galbanum polysaccharide, is proven to effectively hinder the immunosuppression caused by the tumour inflammatory microenvironment. The study conducted by Ye Hua revealed that galbanum polysaccharide disrupts the immunosuppression of the tumour by altering the ratio of cytokines in the TH1/TH2 subgroup of the tumour microenvironment when it was administered to the serum of C26 tumour-bearing mice . FurtherIn addition, herbs like Coicis Semen can be utilized to reduce VEGF expression, hence preventing the growth of tumours, and treating dampness accumulation and phlegm congestion in cancer patients. Cervical cancer patients have reported an improvement in stool hardening. The Weakened spleen yang, laxity, and loose stools after prescription of this herb. In addition, this herb also reduced abdominal distension in liver cancer patients and caused a marked decrease in the abnormal CEA index in gastric cancer patients. Furthermore, Xu et al. discovered that Coix lachrymal oil efficiently reduced the generation of VEGF and bFGF, a basic fibroblast growth factor, in the human pancreatic cancer on site BxPC-3 cells, which affected the cell growth cycle and prevent the growth of new blood vessels for the survival of tumours . In spitSalvia miltiorrhiza which contains Tanshinone, has been found to effectively impede the progression of hepatocellular carcinoma in mice by suppressing the release of TGF-β [Studies have shown that herbs that stimulate blood flow and disperse stagnation can reduce tumour growth. When taken together with the herbs that invigorate qi, the efficacy of the herbs in preventing tumour spread and augmenting immune response are improved . Zhou etof TGF-β , increasHerbs which are known to nurture and sustain are widely used in CM to bring up the beneficial energy and maintain the equilibrium of yin and yang, to improve the body’s immunity, and mitigate the insufficiency of the cancer patients. Taking Ginseng Radix et Rhizoma as an example, Wang Lei et al. reported that ginsenoside Rb1 could counteract the TGF-β1 cytokines released by the liver cancer cells, thereby preventing the NK cells from being immunosuppressed . Du et aCM has a long history of research in tumor prevention and treatment, and clinical trials in recent decades have shown that CM is one of the most important tools for tumor treatment. Therefore, treatment by combining with the tumor inflammatory microenvironment may provide new ideas for cancer treatment.In this paper, we summarize the tumor inflammatory microenvironment regulated by inflammatory mediators and cells, and the role of in regulating tumor inflammation. According to the theory of CM, the pathogenesis of malignant tumors lies in the invasion of external evil, dysfunction of qi and deficiency of positive qi, accompanied by states of heat, phlegm, stagnation and deficiency. Therefore, in the CM treatment of tumors, TCM is first used to regulate the imbalanced environment inside the organism, and the treatment plan is adjusted according to the actual situation of the patient to achieve the therapeutic purpose. However, the improvement of clinical physical and chemical indices of tumors by TCM treatment is still unclear.Table This article presents some research results and possible mechanisms of TCM in tumor therapy, in order to provide a reference for further research and development of TCM in cancer therapy.


The hypoxic tumour microenvironment (hTME), arising from inadequate and chaotic vascularity, can present a major obstacle for the treatment of solid tumours. Hypoxic tumour cells compromise responses to treatment since they can generate resistance to radiotherapy, chemotherapy and immunotherapy. The hTME impairs the delivery of a range of anti‐cancer drugs, creates routes for metastasis and exerts selection pressures for aggressive phenotypes; these changes potentially occur within an immunosuppressed environment. Therapeutic strategies aimed at the hTME include targeting the molecular changes associated with hypoxia. An alternative approach is to exploit the prevailing lack of oxygen as a principle for the selective activation of prodrugs to target cellular components within the hTME. This review focuses on the design concepts and rationale for the use of unidirectional Hypoxia‐Activated Prodrugs (uHAPs) to target the hTME as exemplified by the uHAPs AQ4N and OCT1002. These agents undergo irreversible reduction in a hypoxic environment to active forms that target DNA topoisomerase IIα (TOP2A). This nuclear enzyme is essential for cell division and is a recognised chemotherapeutic target. An activated uHAP interacts with the enzyme‐DNA complex to induce DNA damage, cell cycle arrest and tumour cell death. uHAPs are designed to overcome the shortcomings of conventional HAPs and offer unique pharmacodynamic properties for effective targeting of TOP2A in the hTME. uHAP therapy in combination with standard of care treatments has the potential to enhance outcomes by co‐addressing the therapeutic challenge presented by the hTME. AQ4NBanoxantrone dihydrochlorideBBBblood brain barrierCA9carbonic anhydrase IXCTCscirculating tumour cellsCYP450cytochrome P450DSBDNA double strand breakECMextracellular matrixGLUT1glucose transporter 1 also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)GBMglioblastoma multiformeHAPhypoxia‐activated prodrugHIFhypoxia‐inducible factorhTMEhypoxic tumour microenvironmentIQRinterquartile rangeMTXmitoxantronePDACpancreatic ductal adenocarcinomaPO2partial pressure of oxygenPTENphosphatase and tensin homologRUNX2Runt domain transcription factorTAMtumour‐associated macrophagesTIFPtumour interstitial fluid pressureTOP2DNA topoisomerase II (EC 5.99.1.3)TOP2ADNA topoisomerase IIaTOP2BDNA topoisomerase IIbuHAPunidirectional hypoxia‐activated prodrugVEGFvascular endothelial growth factor1The tumour microenvironment (TME) comprises regions with very low levels of oxygen (<1%). Such hypoxic TMEs (hTMEs) arise as a result of continued tumour cell proliferation and accompanying vascular insufficiency across a wide range of cancer types.In this review, we focus on the unidirectional HAPs (uHAPs) AQ4N (banoxantrone) and OCT1002. These uHAPs are both di‐N‐oxide analogues of the anti‐cancer TOP2A poison mitoxantrone.22.12 level of about 42.6 mmHg —defined as physoxia. This is much less than inspired air  which is frequently referred to as 'normoxia'. Although it is difficult to be exact, tumour hypoxia can be considered to occur below 3% oxygen, and in many situations, these levels are significantly lower (<1%). The ability to target the hTME via prodrug activation is a key design feature of HAPs and uHAPs; activation in the hypoxic range thereby protects normal tissues from exposure to the reduction product. In part, HAPs have been compromised during their clinical evaluation by an inability or failure to assess the degree of hypoxia in individual tumours as a means of effective patient selection.In many studies, the level of oxygenation and extent of hypoxia in tumours is poorly understood. It is crucial to recognise that normal tissues have a median pOin vivo tissue physoxic environments. This caveat also applies to preparatory protocols for cell‐based therapiesDifferentiation between physoxia and tumour hypoxia2.2Tumour hypoxia drives a plethora of changes that re‐shape cellular behaviour in the hTME, resulting in functional immunosuppression, enhanced tumour aggressiveness, therapeutic resistance and opportunities for metastasis Figure . These cThe genetic instabilities of tumour cells in the hTME are subject to Darwinian selection under varying hypoxic stress conditions with downstream impact on DNA repair pathways.Gene expression and mutation profiles of tumours have recently provided evidence that hypoxia can direct the evolutionary trajectory of mutations presented in a given tumour.Given that the hypoxia‐induced cellular recruitment cascade involves components of immune response pathways  comprises multiple components including proteoglycans and fibrous proteins such as the collagens, elastins, fibronectins and laminins.These factors can also affect HAP behaviour within the TME. Prodrug design reviews61: atte3A prodrug is ideally a pharmacologically inert compound that undergoes biotransformation to facilitate its therapeutic action. Prodrug structures carry non‐toxic functionalities that serve to modify or reduce unwanted properties of the activated compound. This aspect is important for HAP performance to minimise normal tissue toxicity and the dose restrictions typically imposed in combination regimens. An ideal anti‐cancer HAP would undergo intracellular conversion selectively in the hTME3.1Since 1960s, a concept was pursued of harnessing nitroaryl compounds to substitute for reactive oxygen in the radiotherapy of cancers whose treatment was compromised by refractory hypoxia. This gave way to the development of prodrugs bioreductively activated in low oxygen. The view that HAPs undergoing 1e‐bioreduction will generate tumour cell DNA damage via nitro radical anion formation selectively under hypoxia was compelling. Ensuing clinical trials of nitroaryl HAPs to date have largely explored agents with nitroimidazole (TH‐302), nitroaniline (PR104A) or aryl‐di‐N‐oxides (Tirapazamine) bioreduction triggers. TH‐302 has provided the most recent clinical experience for hTME targeting. However, TH‐302 failed to improve overall survival compared with doxorubicin alone in a phase III study of soft‐tissue sarcoma, although several explanations have been proffered following retrospective analyses.Collectively reversible HAPs rely on a 1e‐bioreduction step, most often catalysed by flavoprotein reductases, a process that can be inhibited even in the presence of moderate hypoxia  forcing the HAP to undergo redox cycling ; a type I DNA topoisomerase targeting HAP involving C‐10 substituted derivatives  of a camptothecin analogue (SN‐38).A more recent approach for HAP design is to incorporate a standard bioreductive trigger to release a pharmacological agent that interferes with a specific cellular process, and these include: the experimental agent CH‐01 that releases a Chk1/Aurora A inhibitor following nitroimidazole bioreduction3.2irreversible formation of a stable targeted agent and water as a by‐product.HAP design employing aliphatic N‐oxides, that can be activated through an obligate 2e‐bioreduction process, addresses the disadvantages of reversible HAPs. A key characteristic of the activation process is that the intrinsic oxygen atom transfer under hypoxic conditions is a unidirectional reaction resulting in 3.3The first compound of the uHAP type to be developed was AQ4N.Insight into the action of the activated form of AQ4N can be found in its structural similarity to mitoxantrone (MTX), an anti‐cancer DNA intercalator and TOP2A poison  under 1% oxygen.Cells in the hTME undertaking cell cycle progression face late cell cycle checkpoints activated by failed DNA decatenation or an accumulation of unresolved DNA damage.In summary, the key properties of an activated uHAP that targets the hTME are as follows: (i) capacity to be delivered to the hTME,5There is clearly a de facto case for the continued pursuit of overcoming the adverse impact of tumour hypoxia on treatment options and outcomes. This is further highlighted by the growing need to address underlying reasons for the failure of promising approaches to immunotherapy. This review has attempted to bring a focus on the importance of uHAP design in targeting the hTME and address the restrictions and limited therapeutic usefulness of the reversible HAPs. It is increasingly clear that the impact of the hTME is far‐reaching and not just confined to tumour cells in situ. Such niches recruit local stromal cell cohorts and multiple cell types that both infiltrate and transit the hTME—whether at primary or metastatic sites. Neoplastic cells escaping from primary niches present a tipping point for treatment options and the anti‐metastatic effects of uHAPs are clearly relevant in this scenario.In vitro experimental evaluations should at least attempt to mimic these low oxygen levels and deploy new generations of hypoxia reporters for 3D and organoid culture models. This underlines the problem when trying to re‐create the hTME experimentally and indicates the importance of an existing clinical proof‐of‐concept already gained for AQ4N.The review has set uHAP development against a backdrop of the profiles of hypoxia and physoxia found in preclinical tumour models and in a range of the most clinically prevalent cancers , SRM (director) and LHP are shareholders of OncoTherics Limited. PJS and LHP are directors and shareholders of BioStatus Limited. BioStatus created OncoTherics Limited to commercially exploit intellectual property concerning a new series of anti‐cancer drugs that can be activated under hypoxia. PJS, SRM and LHP are actively involved in commercialising this technology through OncoTherics Poland S.A.Writing, review and/or revision of the manuscript: all authors.


To learn about the history and development of en bloc resection of bladder tumour (ERBT), and to discuss its future directions in managing bladder cancer.In this narrative review, we summarised the history and early development of ERBT, previous attempts in overcoming the tumour size limitation, consolidative effort in standardising the ERBT procedure, emerging evidence in ERBT, evolving concepts in treating large bladder tumours, and the future directions of ERBT.Since the first report on ERBT in 1980, there has been tremendous advancement in terms of its technique, energy modalities and tumour retrieval methods. In 2020, the international consensus statement on ERBT has been developed and it serves as a standard reference for urologists to practise ERBT. Recently, high-quality evidence on ERBT has been emerging. Of note, the EB-StaR study showed that ERBT led to a reduction in 1-year recurrence rate from 38.1 to 28.5%. An individual patient data meta-analysis is currently underway, and it will be instrumental in defining the true value of ERBT in treating non-muscle-invasive bladder cancer. For large bladder tumours, modified approaches of ERBT should be accepted, as the quality of resection is more important than a mere removal of tumour in one piece. The global ERBT registry has been launched to study the value of ERBT in a real-world setting.ERBT is a promising surgical technique in treating bladder cancer and it has gained increasing interest globally. It is about time for us to embrace this technique in our clinical practice. The development of transurethral resection of bladder tumour (TURBT) dates back to 1806, when Bozzini invented the Lichtleiter, a speculum with a candle and a mirror, which allowed visualisation of internal body cavities . Apart fTURBT is no doubt a revolutionary invention in treating bladder cancer. However, there are two main limitations with the procedure. First, bladder tumour is actively fragmented during TURBT. It results in floating tumour cells which may re-implant to the bladder wall and lead to early disease recurrence , 7. SecoThe birth and early development of ERBT was largely pioneered by urologists from Japan. In 1980, Kitamura et al. published the first report on ERBT using a polypectomy snare through a transurethral resectoscope Fig.  [9]. In Removal of large bladder tumour has always been a challenge in ERBT. Teoh and Mostafid et al. published their experiences on routine implementation of ERBT in their clinical practices . The oveSince the introduction of ERBT back in 1980, there has been a gradual realisation of the critical steps of ERBT, and the energy modalities that can be used to perform a proper ERBT. Kramer and Herrmann et al. initiated the en bloc resection of urothelium carcinoma of the bladder (EBRUC) project, which is the first attempt ever to systematically research ERBT in a multi-centre setting . In 2015In 2020, Teoh et al. developed an international collaborative consensus statement of ERBT incorporating two systematic reviews, a two-round modified Delphi survey and a consensus meeting . The “efERBT should always be considered for treating non–muscle-invasive bladder cancer (NMIBC).ERBT should be considered feasible even for bladder tumours larger than 3 cm.Number and location of bladder tumours are not major limitations in performing ERBT.The planned circumferential margin should be at least 5 mm from any visible bladder tumour.After ERBT, additional biopsy of the tumour edge or tumour base should not be performed routinely.For the ERBT specimen, T1 substage, and circumferential and deep resection margins must be assessed.It is safe to give a single dose of immediate intravesical chemotherapy, perform second-look transurethral resection, and give intravesical bacillus Calmette–Guérin (BCG) therapy after ERBT.In studies of ERBT, both per-patient and -tumour analysis should be performed for different outcomes as appropriate.Important outcomes for future ERBT studies were also identified. Specifically for ERBT, successful en bloc resection rate and resection margins should be reported. For the oncological outcomes, 3-month recurrence rate, 1-year recurrence and progression rates, and 5-year recurrence and progression rates are important outcomes to measure.The key messages are as follows:With a solid effort from global experts in ERBT, the consensus statement serves as a standard reference for urologists to practise ERBT and for researchers to conduct ERBT-related studies in the future.p < 0.001) [ p < 0.001) [p = 0.86) and 24-month recurrence  were similar between ERBT and TURBT [Over the past few decades, there has been an increasing interest in ERBT globally , 27. In < 0.001) . Detruso< 0.001) . Howevernd TURBT .p = 0.8). T1 substaging was more feasible in the ERBT group . Peri-operative outcomes including complications rates, catheterisation time and hospital stay were similar between the two groups, and the recurrence rate at median follow-up of 15 months was 18% for TURBT and 13% for ERBT (p = 0.16). In the multi-centre randomised trial by D’Andrea and Shariat et al. [p = 0.01). Operative time was similar between the two groups, but the ERBT group had a lower rate of bladder perforation (5.6% vs 12%) than the TURBT group. With a median follow-up of 13 months, recurrence rates were 18.4% in the ERBT group and 16.7% in the TURBT group (p = 0.6). In the multi-centre randomised trial by Teoh et al. [p = 0.007) [p = 0.065) in the TURBT group [p < 0.001), but detrusor muscle sampling rates, hospital stay and 30-day complications were similar between the two groups [Subsequently, results from three other randomised trials comparing ERBT with TURBT have been reported. In the single-centre randomised trial by Gallioli and Breda et al. , a totalt et al. , a totalh et al. , a total= 0.007) . The 1-yBT group . Operatio groups . To dateo groups , and theMost urologists would agree that ERBT has two main goals and potential benefits, (1) to ensure complete resection of bladder tumour, and (2) to minimise the risk of tumour seeding by extracting the bladder tumour in one piece. While the term ‘en bloc’ sounds appealing, its literal definition of ‘removal in one piece’ does not reflect the potential benefits of the procedure completely. For large NMIBC, there is a higher chance of residual disease following TURBT, and being able to ensure complete resection of bladder tumour is probably more important than a mere removal of bladder tumour in one piece. In case of muscle-invasive bladder cancer (MIBC), ensuring proper local staging of the disease is most important, and whether you remove the bladder tumour in one piece or multiple pieces has minimal implications in the subsequent management of the disease. In addition, ERBT can possibly achieve maximal transurethral resection, which may be helpful in optimising subsequent treatment such as radical cystectomy and trimodality therapy –34. TherThe general principle of modified ERBT is to resect large bladder tumours as en bloc as reasonably achievable. In the most ideal case, the whole bladder tumour can be resected and removed in one piece Fig. A. HoweveAs high-quality evidence on ERBT is emerging, it is also important to assess its feasibility and generalizability in everyday clinical practice from a global perspective. The global ERBT registry has been launched and it aims to recruit 2000 patients in total . This reSurgical training is paramount to a proper and successful dissemination of the ERBT technique. A porcine bladder TURBT model has been developed and validated, and it is useful for ERBT training . CurrentThis review paper summarises the history, development and future directions of ERBT, which represents the hard work of several general generations of urologists from different parts of the world Fig. . Through


Extracellular matrix (ECM) interacts with solid tumour cells and cells from the tumour microenvironment (ΤΜΕ). Components of the ECM and various types of mechanical forces which are generated from ECM stiffening modulate properties of solid tumour cells, thereby affecting tumour proliferation, migration, angiogenesis, immune evasion, stemness and resistance to treatment. Clarification of the role of ECM‐associated protein molecules during solid tumour progression offers new therapeutic strategies aiming at overcoming ECM‐mediated oncogenic signalling and drug resistance.A well‐investigated signalling pathway linked to alterations of ECM rigidity is the Hippo signal transduction pathway. It is an evolutionary conserved kinase cassette that controls cell proliferation, apoptosis, self‐renewal of stem cells and organ size, and is regulated by mechanical cues, the status of cellular energy and multiple receptor signalling.2+ channel, and this evokes tumour cell proliferation.Changes in ECM stiffness elicit mechano‐stimulated tumorigenic mechanisms through activation of YAP/TAZ Figure . ActivatIt has been documented that YAP/TAZ activation mediates resistance to pharmacological intervention as a result of changes in ECM stiffness.As most solid tumour cells depend on their mechanical properties to promote their malignant features, small‐molecule compounds  targeting YAP/TAZ, either directly or by disrupting vital interactions with transcription factors/complexes, have been developed and have already progressed to clinical trials with promising results.YAP is also implicated in the angiogenic process of diverse solid tumours and small‐molecule inhibitors targeting that aspect of YAP function have been developed. Verteporfin (VP) is a low‐molecular mass compound that suppresses YAP signalling by hampering YAP/TEAD interactions and can restrain proliferation, migration, tube formation and facilitate apoptosis of human umbilical vein endothelial cells (HUVECs).There are also selective inhibitors of TEAD. K‐975 is a newly identified highly selective TEAD inhibitor that impairs YAP/TAZ‐TEAD protein–protein interactions. It exerts antitumor effects by suppressing YAP/TAZ‐TEAD signalling in pleural mesothelioma.Recent findings further implicate YAP/TAZ in signalling circuitries of the ΤΜΕ as candidate targets to improve chemotherapy efficacy. Nonetheless, as shown in breast tumours, stage‐specific YAP/TAZ expression profiles and/or crosstalk with co‐operating signalling molecules should also be taken into account in predicting response to combinatorial YAP/TAZ/TEAD‐targeted treatment approaches.Conclusively, accumulating evidence suggests that anomalous YAP/TAZ signalling contributes significantly to solid tumour development and progression and is a critical factor of intrinsic and acquired resistance to a variety of targeted regimens and chemotherapies. Further research is required to elucidate the underpinning mechanisms of YAP/TAZ operational aberrations in order to formulate more efficacious treatments for a broad spectrum of solid tumours and their clinical complications.Antonios N. Gargalionis: Conceptualization ; data curation ; writing – original draft (lead). Kostas A. Papavassiliou: Conceptualization ; data curation ; writing – original draft . Athanasios G. Papavassiliou: Conceptualization (lead); data curation (lead); supervision (lead); writing – review and editing (lead).The authors declare no competing financial interests.


In many observational studies and clinical trials, this 0.9% NS composition has been associated with multiple microcirculation and immune response complications, acute kidney injury, and worse clinical outcomes. Ringer’s lactate has less sodium than plasma, as well as other electrolytes which can cause problems in patients with traumatic brain injury. This review provides a brief summary of the most important historical aspects of the origin of the most frequently used intravenous crystalloids today.Fluid resuscitation with crystalloids has been used in humans for more than 100 years. In patients with trauma, sepsis or shock of any etiology, they can help modify the clinical course of the illness. However, these solutions are medications which are not side-effect free. Recently, they have been questioned in terms of quantity (fluid overload) and their composition. The most frequently used crystalloids, both in high and low-income countries, are 0.9% normal saline (NS) and Ringer's lactate. The first descriptions of the use of sodium and water solutions in humans date from the cholera epidemic which spread throughout Europe in 1831. The composition of the fluids used by medical pioneers at that time differs greatly from the 0.9% NS used routinely today. The term “ This practice has led to early recognition of critically ill patients and changes in mortality in high and low-income countries  and calcium. This solution had a higher pH than 0.9% NS and less chloride , 35. SubOther balanced solutions have been developed recently. The use of intravenous fluids for maintenance therapy or fluid resuscitation in children in critical care is a universal practice. Multiple electrolytes solutions that are more similar to plasma in all its components are increasingly wanted. This is the case of Plasma-Lyte 148® (PL-148). This is a crystalloid which was patented in 1982 by Baxter International Inc® . They wein vivo osmolality (mOsm/kg) of the solution   and PL-148 has an osmolality of 270–290 mOsmol/kg. To avoid confusion between tonicity and composition, it is preferable to classify crystalloids as balanced or unbalanced ( of Cl−) . Howeverbalanced , 48, 49.The Pragmatic Pediatric Trial of Balanced vs. Normal Saline Fluid in Sepsis (PRoMPT BOLUS) is currently being conducted . This clThe indiscriminate use of crystalloid boluses is being questioned in terms of their quantity and quality of composition. Intravenous fluids were established in clinical practice and licensed for use without robust investigation of their efficacy or safety.in vitro experiments rather than use in humans. The term “physiological solution” should be considered inappropriate for this solution due to its composition. Balanced solutions have a composition more similar to plasma, and recent evidence suggests that they may have fewer side effects. The decision to perform fluid resuscitation should include an appropriate choice of the type of crystalloid to be used, with an individualized approach, considering the potential risks and complications associated with their use. The precision medicine based strategy for fluid resuscitation should be the fundamental principle of treatment for all patients, considering the best crystalloid  according to each particular case.0.9% NS has a historical basis which suggests that its development was aimed at