Although photodynamic therapy (PDT) has promising advantages in almost non‐invasion, low drug resistance, and low dark toxicity, it still suffers from limitations in the lipophilic nature of most photosensitizers (PSs), short half‐life of PS in plasma, poor tissue penetration, and low tumor specificity. To overcome these limitations and enhance PDT, liposomes, as excellent multi‐functional nano‐carriers for drug delivery, have been extensively studied in multi‐functional theranostics, including liposomal PS, targeted drug delivery, controllable drug release, image‐guided therapy, and combined therapy. This review provides researchers with a useful reference in liposome‐based drug delivery. Liposomes with high capacity for photosensitizers (PSs), other drugs and imaging agents in hydrophobic bilayer and hydrophilic aqueous core make them capable of executing multi‐functions in disease diagnosis and therapy to enhance photodynamic therapy. The flexibility of surface modification and components of liposome possess the advantages in targeted drug delivery and release. Upon absorption of light, PSs are excited to the excited singlet state (S1), and transform‐ to the triplet state (T1) by intersystem crossing (ISC). In the type I process, the excited PS at the T1 state transfers an electron to cellular substrates to form radicals and reactive oxidative species (ROS). In the type II process, there are three essential elements: PS, light, and oxygen. Singlet oxygen (1O2) is formed by transferring energy from photoexcited PSs at the T1 state to triplet oxygen (3O2), as shown in Figure high levels of ROS can disrupt the balance between ROS generation and detoxification, thus, interfering with biological systems, and causing cell damage by oxidizing biomolecules, such as lipids, amino acids, and heterocyclic bases in nucleic acids. The overall therapeutic effects of PDT include direct‐targeted cell ablation, vascular damage, and inflammatory and immune responses. Furthermore, ROS can only travel ≈10−55 nm upon excitation due to its short half‐life (≈10−320 ns) within cells. Therefore, PDT can be confined by the light‐focusing site and the distribution of PSs.Since porfimer sodium was approved as the first photosensitizer (PS) for the treatment of bladder cancer in 1993, photodynamic therapy (PDT) has been widely used in anti‐tumor and anti‐infection therapies. PDT ablates the vessel to block the vesicular delivery of PSs to the tumor interior. The tissue penetration ability of light to activate PDT greatly limits the therapeutic depth. The lipophilic/hydrophobic nature of most PSs induces low solubility and aggregation in aqueous media, which not only decreases cellular uptake, but also increases the photothermal effect and quenches the generation of ROS. Although many PSs are fluorescent dyes and fluorescent imaging (FLI) allows the detection of the delivery, distribution, and metabolites of PS in cells, surface, and shallow tissue, the poor tissue penetration of optical imaging greatly limits its application in deep tissues and organs. In addition, the tumor microenvironment (TME) contributes to the tumor resistance to therapies, including PDT. For example, oxygen‐dependent PDT suffers from hypoxia, as the median oxygen levels of most tumors are below 2%. Additionally, oxygen consumption by PDT aggravates hypoxia. Hypoxia‐activated transcription factors, such as the transcription factor activator protein 1 (AP‐1), nuclear factor E2‐related factor 2 (NRF2), and hypoxia inducible factor 1 (HIF‐1), decrease the therapeutic effects of PDT, and may even promote the tumor invasion and metastasis. Some tumor cells increase their tolerance to high oxidative stress by upregulating the expression of glutathione (GSH) to neutralize ROS. Therefore, an efficient strategy to overcome the limitations of singular PSs is urgently needed for tumor therapy.The delivery and bio‐distribution of singular PSs usually rely on their intrinsic characteristics and interactions with the physiological environment, which may greatly influence therapeutic effects. However, there remain major challenges in low efficiencies of targeted delivery, tissue penetration, and tissue accumulation of many PSs to target tissue/cell PDT. In vascular delivery, the biomolecules in the plasma and the body self‐clearance mechanism could obviously reduce the concentration of PS. activatable PS, nanoparticle delivery, and combined therapy. Among of these, liposomes have been widely applied in medicinal formulations in clinic and clinical trials for imaging, diagnosis, and therapy. Importantly, liposomes have the potential to overcome the limitations of singular PSs due to the following reasons: 1) Lipids are amphiphilic and can disperse PS to avoid aggregation and self‐quenching; 2) Liposomes are multi‐functional nano‐carriers for targeted delivery and triggered release on demand by multiple stimuli owing to the flexibility of modifications on their surface, size control, and tunable encapsulation; 3) Multi‐functional theranostics is possible by co‐loading other therapeutic agents and imaging contrasts. In addition, liposomes avoid the exposure of cargoes to normal tissue/organ and blood circulation, thus preventing the degradation of cargoes, immuno‐response, and possible toxicity, before reaching the desired lesion sites; 4) Liposomes consist of natural phospholipids, which are biocompatible and biodegradable materials, minimizes the risks induced by the formulation are minimized; and 5) Compared with free PSs, liposomal formulation of PSs improves therapeutic efficiency by prolonging tissue permeation and retention.To date, many efforts have been made to overcome these limitations, such as supramolecular PS, in 2004, Zheng et al. in 2011, Azevedo et al. in 2011, and then Hamblin et al. in 2013. Since the liposomes for PDT have experienced several stages from conventional/simple to multi‐functional liposomes, we summarize the recent progress, especially the design and applications of the multi‐functional liposomes in theranostics , nanoparticles , can be encapsulated into liposomes.The components and size of liposomes influence their stability and drug delivery. Cholesterol is often used as an additive in liposomes to induce the stability and drug release.2.2 The liposomal formulation of PSs helps to successfully overcome the limitations of the singular PS in PDT  region: 600–800 nm or longer wavelength) for better tissue penetration and high absorption efficiency to reduce the dose of PS.DT Table .2.2.1st, 2nd, and 3rd generations of PSs. Because most porphyrinoids are hydrophobic, liposomal formulations improve water solubility and cell uptake. Heger et al. reviewed liposomal 2nd generation PS for the treatment of solid cancers. Skupin‐mrugalska et al. reviewed the status of liposomal porphyrinoids as PSs and PS precursor . Recently, Duzgunes et al. reviewed the works on phorphyrinoid derivatives. Here, we focus on the application of new developments.As the most successful unit of PSs, many porphyrinoids have been investigated and have entered clinical and preclinical trials including the 1 Most porphyrinoids are derivatives of three types of structures: porphyrin, chlorin, and porphyrazin , photoprotoporphyrin IX dimethyl ester (PpIX‐DME), and 4TPPS with longer wavelengths were incorporated into liposomes for tumor therapy  for tumor therapy. Liposomal 4TPPS showed higher efficiency than free 4TPPS with ≈46% and ≈57% HeLa cell survival rate under light irradiation at 552 and 640 nm, respectively.The porphyrin derivatives photoprotoporphyrin IX , m‐tetrahydroxyphenylchlorin (mTHPC), purpurin 18 (P18), and benzoporphyrin derivative monoacid A (BPD) have been encapsulated in liposomes for tumor therapy  as cationic surfactants. Different formulations of liposomal mTHPC, such as Foscan, Foslip, and Fospeg, have been successfully employed for various tumor treatments. Bezdetnaya et al. fabricated a novel hybrid delivery system called Temoporfin‐in‐Cyclodextrin‐in‐Liposome, in which mTHPC was bound to β‐CDs located in the inner aqueous core. P18 nanoconfined liposomes (P18⊂L) were prepared to treat Taxol‐resistant tumor cells by suppressing cancer cell migration and destroying metastasis‐related extracellular matrix (ECM) in PDT.Chlorin derivatives chlorin e6  showed a high rate of cell uptake and strong phototoxicity in tumor PDT ‐tetracyanoporphyrazine  Phthalocyanine Pc1. In the liposomal formulation, Pc1 showed the highest inhibition against E. faecalis with 3.61 log reduction, in contrast to the zinc(II) analogue ZnPc‐1 (5.70 log), which is consistent with their quantum efficiencies.ZnPc was also encapsulated in DPPC liposomes for HeLa and Sk‐Cha1 cells. In a model of protein oxidation, ROS produced by liposomal ZnPc can oxidize the autofluorescent amino acid Trp on BSA, which is detected quantitatively. An analog of ZnPc, PcN, was encapsulated into the nanoliposome PcN@Lip‐FA, which significantly improved cellular uptake and phototoxicity against HeLa cells.Chelation of metal ions to porphyrinoids, such as ZnPz 1–3 as PSs activated by NIR irradiation demonstrated significantly better phototoxicity in cancer cell lines  compared with that of the solution of Pz1. Liposomal porphyrazine 5 showed better photocytotoxic effects  than its free form (IC50 = 3.135 ± 0.156 × 10−6m) to LNCaP cells under hypoxia and moderate phototoxicity at 5 × 10−5 × 10−6m for antibacterial therapy.Liposomal dendritic magnesium sulfanyl tribenzoporphyrazine ClAlPc) has been used in PDT of non‐metastatic melanoma cells and human metastatic melanoma cells  of liposomal ClAlPc was much longer than that (0.8 µs) of free ClAlPc in DMSO, resulting in a higher quantum yield (Ф△: 0.69 vs 0.29). AlPcS4 has better water solubility and poor cell permeability than ClAlPc due to the four sulfonic acid groups. Cationic liposomes enhanced the delivery efficiency of AlPcS4 and blocked its high affinity to serum albumin  exerts antitumor, antiangiogenic effects and anti‐Gram‐positive bacteria activity. Caeano et al. studied Hyp in DPPC liposomes , which greatly enhances the ISC. The flexible modification of BODIPY extends the absorption wavelength to the therapeutic window (650–800 nm) for in vivo applications. Although PSs have advantages such as good photo‐stability, high quantum yield, high molar extinction coefficient, and sharp absorption peak, hydrophobicity and lack of tumor‐specificity still limit their further application in PDT. By encapsulation in liposomes, Singh reported that a group of halogen‐BODIPY dyes  for PDT has better toxicity against human ovarian carcinoma cell line (SK‐OV‐3) with an IC50 of 0.350 × 10−6m  also showed good photosensitivity  derivative LMIC60‐2 bearing a polar cation group showed much better photodynamic activity than other analogues with pristine C60  was encapsulated in cationic liposomes for the treatment of multidrug‐resistant Gram‐negative bacteria in sewage treatment plants  based on a phenothiazinium scaffold has strong photosensitizing ability and NIR absorbance at 650–670 nm for better tissue penetration  has NIR absorbance and NIR fluorescence for optical imaging  and PDT. To overcome the drawbacks of aggregation, rapid body clearance, and short residence time in blood, liposomal ICG was applied to PDT of human triple‐negative breast cancer cells. However, compared with free ICG, some liposomal ICGs displayed negligible ROS production, which was possibly attributed to ROS absorption by nearby phospholipids and ICG molecules. More studies discussed the use of liposomally formulated ICG in optical imaging and cancer treatments including PTT and PDT in Sections Because of the long polymethine bridge, indocyanine green  in liposomes for imaging‐guided PDT  could be activated by re‐aggregation of BP following liposome disruption in the tumor  PS, AIEgen does not suffer from self‐quenching in fluorescence and photosensitivity limitations due to its special aggregation‐induced emission (AIE) enhancement phenomenon.r Figure .[94]PpIX‐Ole,PL‐C17,PhLPC, PhLSM, and PC‐BPD), AIEgen‐lipid, and lipid‐ICG (iDOPE and ICG‐C18) were incorporated into liposomes by self‐assembly  resulted in mitochondrial‐related cell death in PDT. However, liposomal PC‐BPD was endocytosed to form endosomes and then lysosomes, resulting in lysosomal photodamage in PDT.To avoid PS leakage from liposomes, lipids are covalently conjugated to PS, thus improving affinity to the liposomal bilayer. Conjugation dose not significantly change the electron absorption and photosensitivity of PS. For instance, as building blocks, conjugates of lipid‐porphyrin  were incorporated into the liposomes for PDT. However, the leakage of toxic heavy metal ions (Cu2+) hinders their biomedical application. Liposomal absorber reduced graphene oxide (rGO) significantly constrained the leakage. In another case, 2d‐TiO NPs exhibited a broad absorption area from 400 to 1000 nm and photosensitivity upon irradiation with visible light (400–800 nm).2TiO has good stability and biocompatibility in physiological environments. The liposome‐TiO2 composites showed photo‐toxicity to cancer cells (HeLa and HepG2).Inorganic materials have also been successfully used in liposome‐based PDT. Normally, inorganic PS displays high satiability, high sensitizing efficiency, high photothermal effect, and activation by NIR light. In general, the amphiphilic properties of liposomes make them suitable for the delivery of all kinds of PSs and have many promising advantages, regardless of the hydrophobic or hydrophilic nature of the drugs.2.2.2 The prodrug DiBDP was released at the acidic lysosome and then activated by intracellular NTR in hypoxia only to turn on phototoxicity and fluorescence  is a typical method for PS regulation. ICG or DiR quenched the liposomal Ce6 by FRET. The photoactivity of Ce6 was recovered upon ICG or DiR was destroyed by NIR laser irradiation , and red blood cell membranes have shown promising advantages owing to their intact materials . In conventional liposomes, liposomal PSs are delivered passively to the tumor by enhanced permeability and retention (EPR) effect; however, their specificity is insufficient. In PDT, the unavoidable accumulation of PSs in normal tissues/cells may induce side effects. Fortunately, the liposome membrane provides numerous immobilized sites for recognition moieties, such as antibodies, ligands, peptides,Antibody‐conjugated liposomes: Antibodies exhibit high specific recognition ability against antigens in complicated physiological environments . Many antibodies, such as antibodies against CD19, CD20, CD74, HIF‐1α, EGFR, and HER2 have been selected for liposomal immune‐targeting  to enhance the specificity of delivery.In addition, because the size of full‐length or partial antibodies (55−150 kDa) may interfere with the preparation and biodistributions of liposomes, a single domain antibody was alternatively conjugated onto liposomes containing PS , that encapsulated upconversion nanoparticles@black phosphorus quantum dots (UCNPs@BPQDs). The aptamer‐modified liposome guided the tumor cell‐targeted combined therapies of PTT and PDT.y Figure .[113] Ligand (peptide)‐conjugated liposomes: Biomarkers, such as the folate receptor, transferrin receptor, integrin (αvβ3), and CD44, which are overexpressed on cancer cells, have been utilized for tumor‐targeting liposomal delivery.  was incorporated into the lipid‐PEG motif, which was assembled into liposomes together with PS and other drugs. Thus, the targeted RGD‐SSL‐[P]‐[S] displayed specific toxicity to human umbilical vein endothelial cells (HUVECs) with neovascularization, whereas no obvious damage to normal ARPE‐19 cells was observed. Self‐assembled liposome‐porphyrin (Lip‐p‐OH) was modified with an HA derivative to target CD44‐overexpressed cancer cells, such as MDA‐MB‐231 cells.] Figure  The receBiomimic liposomes: Compared with “artificial” methods to introduce recognition moieties on the surface of liposomes, the biomimic method to prepare “endogenous” liposomes for drug delivery has promising advantages. As its endogenous characteristics, the reassembly of natural cell membranes to liposomes as drug carriers has good biocompatibility and minimum immune‐resistance. Liposomes derived from homologous tumor cell membranes provided highly specific targeting by self‐recognition internalization in the delivery of nanoparticles into the targeted tumor  micromotors could be driven by ultrasonic waves at a speed of up to 56.5 µm s−1 in biological media, and the motion can be directed by an external magnetic field. The formulation had good biocompatibility and stability in blood.A red blood cell‐mimicking micromotor was constructed using hemoglobin (Hb) particles containing magnetic FeT Figure .[24] B2.3.2Due to their special structure, liposomes can encapsulate hydrophobic or hydrophilic materials, including dyes for imaging, chemotherapeutic drugs for chemotherapy, and genes for gene therapy. Drug delivery by conventional liposomes mainly depends on the EPR effect. However, the site specificity of drug delivery and release is not satisfactory for pursuing better therapeutic effects and fewer side effects. On the platform of liposomes, tumor‐targeting drug release has been successfully achieved by endogenous stimuli (pH and enzyme) and controllable external stimuli .FigureFor liposomes comprising natural lipids or similar components, modifications were made to the backbone of lipids to induce the destabilization of liposomes and triggered the cargo release upon changes in lipid conformation  or structure  in response to the various stimuli mentioned above Figure.Light‐sensitive liposomes: Light is a non‐invasive, site‐controllable, and safe stimulus to remotely induce the destruction of liposomes and release of cargoes. The principles are mainly dependent on the molecular mechanism of photo‐triggerable lipid and subsequent destabilization of liposomes. ROS‐sensitive liposomes are disrupted by photo‐induced oxidation of unsaturated lipids to destabilize the liposomes to release the cargoes, including chemotherapeutic agents, fluorophores, imaging contrasts, and genes. As shown in Figure  SLPC, DOPC, DOPE, DOTAP, POPC, and EPC.verteporfin, pheoA, and m‐THPP) in photo‐triggerable liposomes. Particularly, m‐THPP/DOPC was the most efficient system for DOX release and has potential in chemotherapy and PDT.The photooxidation of phospholipids and the subsequent membrane degradation was investigated on the combination of unsaturated lipids  with PSs , which induces controllable release of cargo (a fluorophore) from liposomes.DSPE‐PEG‐PheoA, DPPC, DOPE, and other components was triggered by NIR irradiation (670 nm) to release the chemotherapeutic drug gemcitabine (GEM). NIR (808 nm) irradiation also activated liposomes containing lipid egg‐yolk L‐phosphatidylcholine (EPC), PS cypate (Cy), and other drugs.The tissue penetration of cargoes released on demand depends on the efficiency of the activation of PS by light. Due to the tissue absorption, NIR has better penetration than ultraviolet and visible light. For example, a ROS‐responsive liposome GDPPL, fabricated with verteporfin and gold nanoparticles) to produce singlet oxygen, which destabilized the liposome by oxidizing unsaturated lipid DOTAP to release a gene silencing reagent (antisense oligonucleotide) or a chemotherapeutic drug . The system was also modified by triphenylphosphine (TPP) for mitochondria‐targeted delivery.Compared with visible light and NIR, X‐rays have unlimited tissue‐penetration in medical examination and therapy, such as X‐ray checking, CT, and radiotherapy. Recently, in an X‐ray‐triggerable liposome, X‐ray activated PSs  is a key factor for the construction of thermo‐sensitive liposomes. Another factor is the photothermal agent that efficiently converts light energy into heat. Thermo‐sensitive liposomes encapsulated with ICG as a photothermal agent were triggered by NIR irradiation to release cargos, where DPPC as one of the major components has a phase transition temperature of 41 °C. Thermo‐sensitive liposomes are often involved in the combined therapy of PTT with PDT, chemotherapy, and so on.pH‐responsive liposomes: In the tumor physiological environment, pH values range from 5 to 6.3, which is different from the normal physiological pH (7.0−7.4). pH is often used to activate pH‐sensitive molecules/materials, such as lipids and peptides, to induce site‐specific liposomal drug release. In a pH‐sensitive liposome encapsulated PS (DiBDP), the lipid DOPE is sensitive to pH. At a physiological pH of 7.4, DiBDP was slowly released, i.e., only 9.1% of DiBDP was released over 24 h of incubation. However, this was significantly accelerated at pH 6.0, owing to the pH‐induced degradation of DOPE in the Ab‐DiBDP NPs. The fastest release of DiBDP was observed at pH 5.0, reaching a plateau at ≈86% within 24 h. Moreover, a smart liposome was fabricated using the acid‐responsive phosphatidylethanolamine poly(L‐histidine)40(PE‐p(His)40) and temperature‐responsive FA‐conjugated PE‐p(NIPAM) (PE‐p(NIPAM)40‐FA). The swelling of the histidine block, where the imidazole was ionized under acidic conditions, was responsive to drug release. The hybrid nanocomplexes released the chemotherapeutic drug (DOX) more rapidly than bare liposomes, thus enhancing the therapeutic effects upon exposure to the acidic physiological environment in cancer cells. In other study, pH‐sensitive CaP played a similar role in drug release.Other pH‐sensitive nanoparticles also facilitate construction of hybrid nano‐platforms joined with liposomes for acid‐responsive drug release. For example, zinc oxide (ZnO) as an acid‐responsive material has been incorporated into liposomes for drug delivery in cancer therapy.,3After the illustrations of the single element of the principles of liposome‐based drug delivery, such as liposomal PSs, targeting models of liposomes and triggerable release models of liposomes, the multi‐function of liposomes were introduced to synergistically enhance PDT. To address the limitations of PDT monotherapy, co‐capsulated drugs have been developed for example, TME for therapy resistance, including hypoxia and tumor extracellular matrix. Moreover, the solutions to enhance the specificity of delivery and release may reduce the dose and side effects. Therefore, multi‐functional liposomes in response to environmental stimuli for combined diagnosis/therapy with PDT have emerged rapidly as a new tendency in applications, including image‐guided PDT, PDT/chemotherapy, PTT/PDT and etc., since Zheng et al. fabricated an excellent example of multi‐functional nano‐platform based on liposomes called “porphysomes” in 2011.3.1Tissue penetration is one of the main challenges in tumor PDT, particularly in PS delivery and light irradiation. On one hand, the challenge of PS delivery can be addressed by liposome‐guided delivery, as summarized in the previous Section  PS (ruthenium complex) was activated by the blue light transformed from light at 532 or 630 nm, respectively, through triplet‐triplet annihilation upconversion (TTA‐UC). Thereafter, Chang et al. made significant progress in a polymeric liposome co‐loaded with PS  and up‐conversion nanoparticles (UCNs) for PDT and FLI  to visible light (540 nm) to activate PS (MC 540) in depth. In another liposomal system (Hb‐NPs@liposome), in situ luminescence catalyzed by Hb in the presence of luminol and endogenous H2O2, overcome the limitation of light penetration. Radiolabeled Europium‐loaded liposomes (64Cu‐Eu/VBBO lipo) emit radioluminescence (red fluorescence) to activate PS.On the other hand, light penetration largely depends on its wavelength due to tissue absorption. For a single PS, longer wavelength of exciting light leads to better tissue penetration for activating PDT. In addition to developing new PSs with longer wavelengths of absorption toward the NIR window, in situ light‐generating strategies for PS activation have been investigated to overcome the limited penetration of visible light. For example, a flexible strategy based on the liposome system was reported by Bonnet et al. and Chang et al., respectively. In a proof‐of‐concept study by Bonnet et al.,verteporfin activation. The mitochondria‐targeted liposomes (TPP‐Lipo‐VP‐10Au) encapsulated PS verteporfin and gold nanoparticles. The results indicated that 1O2 generation was enhanced by ≈129% and 186% by gold nanoparticles with diameters of 5 and 10 nm, respectively, in the presence of X‐rays (4 Gy).Unlike the typical PDT working with three essential elements , X‐ray‐induced PDT has overcome the reliance on light, which has excellent tissue penetration ability compared with only up to 3 mm penetration of red light (620–750 nm) required by 3.2The TME is highly associated with tumor progression, intractable metastasis, and especially resistance to therapies. The TME is characterized by the typical features of tumors, such as hypoxia, oxidative stress, and acidosis, and induce changes in the components of the ECM, immune response, and angiogenesis. Therefore, new therapeutic strategies targeting the TME have become essential to overcome tumor resistance.3.2.1Figure As PDT is an oxygen‐dependent procedure, it can be suppressed by hypoxia regardless of PDT‐induced hypoxia or tumor hypoxia. In order to improve PDT efficiency under hypoxia, there are three ways to enhance the supply of cellular oxygen to PS: 1) delivery of extracellular oxygen or oxygen‐generating materials to PS, 2) transformation of other endogenous molecules to oxygen, and 3) suppression of other pathways consuming intracellular oxygen  droplets entrapping oxygen were encapsulated in a liposomal system (Lip(ASC/PFH) containing nanoparticle Au@SiO2@Cu2O (ASC) as PS for enhancing tumor PDT  for antibacterial PDT. After only 15 min, the antibacterial efficacy against Escherichia coli and Staphylococcus aureus was improved from 66.54% and 48.04%, to 99.62% and 99.63%, respectively. Hb, an oxygen‐carrying protein in red blood cells, is an ideal oxygen donor. Thus, liposomal oxygen‐saturated Hb efficiently alleviated tumor hypoxia to enhance PDT  as an oxygen‐generating material and MB were encapsulated into an oxygen‐self‐supplying liposome‐based nanoparticle, LipoMB/CaO2 , aza‐BODIPY dye (B1), and CaO2 nanoparticles were encapsulated in the hydrophobic layer and NH4HCO3 was encapsulated in the hydrophilic cavity  nanoclusters inhibited thioredoxin reductase (TrxR) to enhance tumor PDT , a pH responder anchored on the liposome membrane, was converted to MG carbocation (MG+), leading to liposome collapse and the payload release of Au nanoclusters and Ce6. Subsequently, the Au nanoparticles inhibited TrxR activity to provide more oxygen supply for the enhancement of PDT.Several strategies have been reported to suppress oxygen consumption during respiration, thus increasing the oxygen supply to PDT. Liposomal metformin which inhibits mitochondrial respiration, relieved tumor hypoxia to enhance PDT Figure .[151] T Figure .[152] 3.2.2Figure  L‐Buthionine sulfoximine (BSO), a GSH synthesis antagonist, was encapsulated in a thermally responsive liposome (PLNA) and released by NIR irradiation.In cancer cells, excessive concentration of GSH as a ROS scavenger compromises the efficiency of PDT. Therefore, NIR‐photothermal liposomal nanoantagonist amplified cancer PDT  to facilitate the penetration of Ce6 and relieve hypoxia to enhance PDT. In another study, liposomal formation of hydrophobic P18 enabled ECM destruction and facilitated the chemotherapy by Taxol.The abundant tumor ECM results in insufficient tumor retention and ineffective intra‐tumor penetration of therapeutic agents, as well as acidity and hypoxia of TME, further leading to unsatisfactory therapeutic outcomes for many therapies. Collagenase (CLG) was encapsulated into acid‐responsive nanoparticles (NCPs) and then delivered in liposomes co‐loaded with 3.3 Herein, we focused on liposome‐based imaging, which provides direct and visual evidence of the delivery, accumulation, and metabolism of the functional liposomes in vivo. These studies guide PDT protocols and those of other combined therapies, and evaluate the therapeutic efficiencies  imaging, computed tomography (CT), magnetic resonance imaging (MRI), ultrasonic imaging (USI), positron emission tomography (PET) imaging, and FLI.3.3.1 act as a PS and fluorophore in PDT, which is simultaneously serviced in PDT and FLI of tumors , a T1 magnetic MRI contrast agent, was doped into MSNs, which were encapsulated into liposomes to guide synergistic tumor therapies.] Figure ,E,F Afte3.3.44TPPS) for PDT  was encapsulated into the nanoliposome for CT together with PS  porphyrin as PS, were successfully applied in theranostic for 4T1 tumors. Interestingly, PET imaging showed that 89Zr‐Df‐MCLs efficiently accumulated in the tumor. Furthermore, another PET tracer 64Cu was chelated with PS Ce6 to give AQ4N‐64Cu‐hCe6‐liposome, which was responsible for PET, PA, and FL imaging , it clearly and quantitatively demonstrated the pharmacokinetic and time‐dependent bio‐distributions of 64Cu‐labeled liposomes in organs and tumors of mice in real‐time of up to 24 h. However, PET imaging is limited by the dosage of ionizing radiation.PET imaging has shown promising advantages in bio‐imaging and medical examinations as it has high sensitivity, high spatial resolution, excellent tissue penetration, and is capable of dynamic real‐time checking. Compared with CT and MRI, PET imaging displays metabolic activities, which can distinguish tumors from normal tissues. Isotopes with a suitable half‐life that emit positrons are key in PET imaging. Cancer cell membrane‐derived liposomes bearing g Figure –H. 3.3.7ICG as a PS and photothermal agent, as well as the disruption of photothermal‐responsive liposomes during drug release  liposomes were triggered by NIR irradiation to rapidly release DOX. the oxidations of DOPC and cholesterol by ROS were responsive to drug release. DOX/ZnPc co‐loaded MSNs@CaP@PEGylated liposomes achieved a combination of PDT and chemotherapy for tumors, where CaP was pH‐responsive for DOX release. More details on the pharmacokinetics and pharmacodynamics of liposomal chemophototherapy were studied in short drug‐light intervals. The study revealed that better penetration and accumulation resulted in an overall 7.4‐fold increase in the DOX concentration in the tumor. You et al. combined photodynamic and chemotherapy, and study DOX release from ROS‐sensitive liposomes after NIR light irradiation of PS ICG. At the same time, the fluorescent emission of DOX (Ex/Em: 498/553 nm) facilitated the study of drug delivery and distribution by optical imaging.As the first approved liposomal anticancer drug, DOX is widely used against numerous malignancieses Table , entry 1 The multikinase inhibitor cabozantinib (XL184) was released from ROS‐sensitive liposomes upon PDT, and then efficiently suppressed the resistance of tumor to PDT  signaling pathway, promoting the recovery of tumor and vessel against PDT damage. Third, XL184 inhibited the MET (a receptor tyrosine kinase) signaling transduction to suppress tumor metastasis in response to hypoxia. The clinical epidermal growth factor receptor (EGFR) inhibitor lapatinib was also applied in liposome‐based PDT  and IR 820 were combined for synergistic phototherapy and chemotherapy  for PTT and PDT Table , entry 4 Li et al. constructed a multi‐sensitive liposome (ELTSL) responsive to enzyme, light, and temperature for controllable release of PS and the chemotherapeutic agent HOC (Table  Peptide‐polyethylene glycol (PEG) corona incorporated on the surface of liposomes is cleaved specifically by MMP‐2 overexpressed on tumor to enhance tumor penetration and cellular uptake of the liposome. Under NIR laser irradiation, PPa is not only a PS for PDT, but also a fluorophore for imaging and hyperthermia‐induced drug release of the lipophilic prodrug HOC and water‐soluble DOX. The unprotected Pt (IV) prodrug HOC was reduced to OXA (II) by endogenous GSH. This prodrug strategy improved the therapeutic outcomes of platinum drugs, while suppressing the side effects.Platinum‐containing compounds, such as cisplatin (cDDP) and OXA, have been used as chemotherapeutic drugs for anti‐tumor. cDDP is a first‐line chemotherapeutic drug that inhibits DNA via DNA‐crosslinking. A dual‐effect liposome (PL‐CDDP‐Ce6) significantly improved tumor therapeutic efficacy while reducing its toxicity Table , entry 6OC Table , entry 7DVDMS) by liposomal delivery to enhance singular therapy (Table  This formulation of PTX prevented serious systemic toxicities and low selectivity.The anti‐Tublin inhibitor paclitaxel (PTX) was entrapped with PS sinoporphyrin sodium  cells contributes to the PDT‐resistance in post‐PDT survival. To overcome this resistance, HIF‐1 and topoisomerase were inhibited by liposomal acriflavine, to suppress cell survival against PDT based on liposomal Pc Table , entry 9DSPE‐PEG‐PheoA)  were transported to the tumor, PDT induced the disruption of ROS‐responsive liposomes for drug release and consumption of the endogenous oxygen to exacerbate hypoxia. Subsequently, TPZ was activated to toxic BTZ by biogradation at the hypoxic site, which induced DNA damage and subsequent cell death. Therefore, the overall therapeutic efficacy was significantly improved by cooperation with chemotherapy.Although liposome‐based combined PDT and traditional chemotherapy have shown good therapeutic effects, the risk of leakage of chemotherapeutic agents might induce potential side effects to normal tissues. A new strategy, particularly suitable for post‐PDT therapy, was developed to minimize side effects. When PDT is severely weakened by hypoxia, the toxicity of the chemotherapeutic prodrug is activated by hypoxia. The therapeutic prodrugs tirapazamine (TPZ) and TuBB‐9 against protein Ki‐67 (Table BPD) and mAb antibody (bevacizumab) was validated in a mouse model of pancreatic cancer. To enhance PDT efficacy, the antibody suppressed the VEGF‐mediated signaling pathways activated by PDT. In another study, a light‐activatable antibody conjugate, TuBB‐9‐FITC, was delivered by the liposomes to HeLa cells. After the free BPD and liposomal TuBB‐9‐FITC were absorbed by HeLa cells and assembled into the endosome, the endosome was disrupted by a NIR of 690 nm to release TuBB‐9‐FITC, which further targeted Ki‐67 in the nucleus followed by PDT activated by 490 nm light for cell elimination.In addition to chemotherapeutic agents, immunotoxic agents, such as monoclonal antibody bevacizumab against VEGFPpIX)  inhibitor, was co‐encapsulated into PpIX‐NLG@Lipo along with PS ), containing an amphiphilic and cationic D‐(KLAKLAK)2 peptide and Ce6, showed synergistic anti‐cancer activity (Table  The membrane‐lytic D‐(KLAKLAK)2 peptide disrupted the endo‐lysosomal compartment and induced mitochondria‐dependent apoptosis.Biopharmaceutical drugs are important components of chemotherapeutic drugs. Protein toxin (saprin) together with PS was delivered into MC28 fibrosarcoma cells by cell‐penetrating peptide (CPP)‐immobilized liposomes (Table N‐methyl glucamine antimoniate (SbV), respectively, to cooperate with AlClPC to enhance the PDT in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL/6 mice  co‐loaded with Ce6 and copper sulfide (CuS) as the photothermal agent, were prepared by Li et al. , gold nanofilm for PTT was coated on the surface of liposomes encapsulating ZnPc for PDT to enhance therapeutic effects on cancer cells.  were reconstructed from the cell membrane of native glioma cells, which were specifically internalized by homogenous tumor cells. To deliver HA‐PEG‐MPLs to tumor cells site‐specifically, iron oxide magnetic nanoparticles in the aqueous core are responsive to magnetic targeting in a magnetic field, and HA‐PEG on the liposome is responsive to ligand targeting by CD44 receptors on tumors. In general, dual functional liposomal ICGs for combined PTT/PDT can efficiently suppress tumor progression.As the only FDA‐approved NIR dye, d Figure . LiposommTHPC and iron oxide nanoparticles for MHT  loaded with T Figure –E.BPD) targets mitochondria and ER, whereas the liposomal PC‐BDP targets the lysosome  regulated TME to enhance synergistic PDT and CDT , the laser source activated the cleavage reaction between ROS‐liable thioketal bond of mPEG‐TK‐Cy and PEG corona, thereby enhancing tumor cell uptake. In addition, the ROS‐sensitive lipid EPC with a double bond was oxidized to release liposomal cargoes, such as Fe3O4 and the prodrug hexadecyl‐oxaliplatin carboxylic acid (HOC). Low‐toxicity HOC and Fe3O4 were reduced to highly toxic OXA and Fe2+ by endogenous GSH, respectively. The consumption of GSH promotes the generation of H2O2, which is converted to ROS product (•OH) and oxygen. The ROS product (•OH) potentially induced liposome collapse and cell damage. Semi‐quantitative analysis of tumor oxygen saturation level  by PA imaging revealed that tumor hypoxia was relieved by RALP for PDT. NIR fluorescence imaging revealed the efficient accumulation of RPLR at the tumor sites by the EPR effect.In contrast to oxygen‐dependent PDT, CDT converts endogenous H3.5Liposomes can serve as a nano‐platform for multi‐modes of theranostics. Single imaging methods have limitations in terms of resolution, depth penetration, signal‐to‐noise ratio, and anatomy information, whereas their synergism can address these limitations.Figure  NIR irradiation induced thermo‐sensitive liposomes and phase‐change materials (PCM) to release drugs. The nano‐platform is capable of treating tumor by combined therapies  and triple‐modal imaging ‐guided therapies. Later, a similar platform loaded TPZ as a hypoxia‐activated prodrug for combined therapies.Fan et al. developed multi‐functional theranostic nano‐platforms based on thermo‐sensitive liposomes triggered by NIR irradiation  containing ICG, contrast agent Gd, and chemotherapeutic agent Dox, a thermo‐sensitive liposome was fabricated on the surface of MSNs. ICG in liposomes was responsive to release of cargoes by photothermal effect upon NIR irradiation. Folic acid‐modified liposomes improved tumor‐specific cellular uptake. Using this theranostic platform, NIRF/PA/MRI trimodal imaging successfully visualized the distribution of liposomes and guided chemotherapy and phototherapy for the depletion of tumors.Li et al. constructed a theranostic hybrid nano‐platform consisting of liposome‐conjugated mesoporous silica nanoparticles for triple‐model imaging‐guided synergistic cancer therapy , T1 MRI contrast (Gd‐DSPE‐DOTA), NIR dye (DSPE‐IRDye800CW), and EGFR antibody (DSPE‐PEG‐cetuximab). The platform integrated the good biocompatibility of liposomes and the high stability of silica. The combined therapies of PD‐L1 immunotherapy and PDT showed synergistic therapeutic effects to achieve superior therapeutic efficacy and prevent tumor recurrence.EGFR‐targeted liposomal nanohybrid cerasomes performed excellent theranostic functions and immune checkpoint inhibition in a mouse model of colorectal cancer  for gene knockdown, gene editing system (CRISPR‐Cas9) for gene knockout and plasmid DNA (pDNA) for transfection.The identification of more imaging tools and therapeutic agents that can combine with PDT for image‐guided liposomal theranostic and combined therapies is also needed. Radiolabeled liposomes can be used in theranostics, such as radiotherapy and diagnosis (SPECT/PET).Taken together, liposomes, as a multi‐functional theranostic platform for PDT, have achieved rapid progress in recent years. We believe this review will be valuable for researchers studying the drug delivery of liposome‐based PDT and that there will be further discoveries in this area in the future.The authors declare no conflict of interest.


Cord‐blood and heel‐prick TSH levels are essential in diagnosing and preventing the serious complications of congenital hypothyroidism, which mainly include intellectual disability. The study aimed to compare between cord‐blood and heel‐prick TSH sensitivity and specificity in detecting congenital hypothyroidism (CH) among newborn screened babies.The study included 21,012 newborn screened babies for congenital hypothyroidism starting from September 2013 until March 2019. Both cord‐blood and heel‐prick TSH were collected from each newborn. Heel prick and cord‐blood TSH cutoff values of >21 μU/ml and >30 mIU/L respectively were considered positive.Out of the total screened newborns, 12 were confirmed for having primary congenital hypothyroidism. Nine cases were positive for cord‐blood TSH , while 139 cases were positive for heel‐prick blood TSH .For the screening of CH, heel prick is considered a superior method, but cord blood remains a practical option due to its cost‐effectiveness, immediate action, and lower recall rate. Therefore, whenever recall is difficult and/or early discharge is the practice, cord blood is an alternative method to heel prick but not with cases of prematurity. Cord blood and heel prick TSH levels are essential in diagnosing and preventing congenital hypothyroidism (CH). Out of the total screened newborn 12 were confirmed for having primary congenital hypothyroidism. 10 cases were positive for cord blood TSH  while 139 cases were positive for heel prick blood TSH . Cord blood is an alternative method to heel‐prick but not with cases of prematurity. Therefore, abnormalities in thyroid functions might lead to severe neurological and developmental consequences, one of which is intellectual disability.In the 1970s, hypothyroidism neonatal screening programs were developed worldwide.In 1972 Dussault, Quebec‐Canada, the first congenital hypothyroidism screening was performed. Seven hypothyroid infants were detected among 47,000 newborns within 3 years. In the initial report, the method missed 10% of the cases with hypothalamic‐pituitary hypothyroidism. It is attributable to under‐developed hypothalamic‐pituitary axis in that group of newborns results in the delayed rise of TSH.In short, advancements in laboratory research have empowered clinicians to enhance the lives of newborns with congenital hypothyroidism. The development of sensitive and specific assays to measure TSH using cord blood and heel‐prick blood made it possible to initiate highly cost‐effective newborn thyroid screening programs. Thus, early diagnosis and treatment will save detected children from intellectual disability. Therefore, this study aims to compare and determine the sensitivity and specificity of cord blood and heel‐prick blood thyroid‐stimulating hormone (TSH) in detecting congenital hypothyroidism among newborn screened babies at King Abdulaziz Medical City, Jeddah, Kingdom of Saudi Arabia.22.1TM software. A non‐probability consecutive sampling technique was used.We conducted a comparative cross‐sectional study at King Abdulaziz Medical City (KAMC), Jeddah, Saudi Arabia. All the data included in the study were obtained from samples, which were delivered from the delivery unit in the hospital. All newborn screened babies from September 2013 until the end of March 2019 were included in this study. We excluded transferred patients from other hospitals, neonatal death prior to specimen collection for newborn screening, babies with incomplete screening for congenital hypothyroidism, and all lost to follow‐up patients. As shown in Figure The data of patients were collected using a data collection sheet containing the basic demographic variables (gender and date of birth), gestational age, birth weight, initial cord‐blood TSH result, initial heel‐prick TSH results, repeated cord‐blood TSH result, repeated heel‐prick TSH result, confirmed diagnosis, and patient remarks. Heel‐prick TSH blood sample levels of more than 21.0 μU/ml were considered positive and required confirmation. Cord TSH levels above 30.0 mIU/L were considered positive.The ethical committee at King Abdullah Medical City had granted ethics approval for the study (IRB# SP19‐457‐J). As data would be collected from health records, informed consent was not applicable, and all patients’ medical record numbers were anonymized during data analysis.2.2For cord blood, TSH was performed using i2000 Architect chemiluminescent immunoassay, (Abbott Diagnostics) since 2013 until now. TSH measurement in heel‐prick sample using dry blood spot filter paper was performed using Genetic Screen Processor by Perkin Elmer method.2.3t test for correlating qualitative and quantitative variables, and Spearman's correlation for correlating quantitative variables.We used proportions for categorical data, means ± SD or median (range) for quantitative data, chi‐square or Fisher exact test for correlating categorical variables, Student's 33.1Our study had a total of 149 patients who had positive heel prick or positive cord blood or both. Furthermore, it stresses that CH is a rare diagnosis with only 12 true‐positive cases. While heel‐prick positive cases were comparable in terms of birth weight , gestational age , and gender distribution with 57% females, cord‐blood‐positive cases were only term (100%) with normal birth weight (100%) and were predominantly females (70%).3.2Table 3.3Table 3.4As shown in Table 3.5While cord‐blood samples are taken immediately after delivering a child, heel‐prick samples have different collection timing depending on each center. Out of the 139‐positive heel‐prick samples, 121 samples (87%) were collected on the same day of birth. In comparison, only 18 samples (13%) were collected 24 h or more after birth.Moreover, TSH levels in those true‐positive samples were notably higher measuring around 140 uU/ml Figure . SamplesIn comparison, samples that were collected after 24 h or more of birth had lower false‐positive rates (72%) with only 5 true‐positive samples (28%) Figure .Overall, TSH levels in both heel‐prick samples collected at birthday or 24 h after birth were much higher in true‐positive cases Figure  compared4Up to date, this study is one of a few that have compared the sensitivity and specificity between cord and heel‐prick TSH neonatal screening for CH. TSH, which is the main screening test, had been measured through both cord and heel‐prick blood in order to early detect and diagnose CH.Our study had a total of 149 cases: 10 were detected by cord blood and 139 were detected by heel brick, who had positive heel prick or positive cord blood or both. Furthermore, it stresses that CH is a rare diagnosis with only 12 true‐positive cases. All the cases were true‐positive heel‐brick samples, but only 9 cases were true‐positive cord‐blood samples.Our data demonstrated that cord TSH results had fewer false‐positive samples compared with heel prick. Al‐Juraibah et al. reported that false‐positive rates were around three times higher in heel‐prick TSH compared with cord blood.Mengreli et al. found that many cases of CH were missed at 20 mU\L cutoff. About 40% of them were premature.In terms of sensitivity and specificity, 139 neonates had positive heel‐prick TSH samples with 100% sensitivity and 99.3% specificity. Similarly, Al‐Juraibah et al. had 305 positive heel‐prick TSH samples with 100% sensitivity, 98.3% specificity but higher recall rate of 1.68% compared to 0.6% and less positive predictive value of 2.3 compared to 8.63% in our study.Our study had a lower recall rate than different studies.N = 139), our study showed a more of female predominance with male to female ratio of (1:3 in TSH Heel Prick and 1:3.5 in TSH cord blood). These are similar outcomes to what was shown in other studies such as Al‐Maghmasi et al. and Henry et al. (1:3 and 1:2 respectively).Our study included 21,012 babies who were screened for CH. Out of this number, only 12 babies were confirmed for having CH with an incidence rate of 1:1751. All confirmed cases were positive for heel prick, cord blood, or both. Al‐Juraibah et al. study screened 17,729 babies; 7 cases were confirmed for the diagnosis of CH, which reflects that it is a rare diagnosis to be found.Our study suggested that the prevalence of CH is comparable to figures such as Bisha province in Saudi Arabia, which showed the highest prevalence in the region, 1:1173 using cord‐blood TSH screening. On the contrary, the province of both Al Baha and Hail represented the lowest prevalence in the country, 1:7709 and 1:6550, respectively.5Cord‐blood TSH appears to be more practical option as a screening method for CH disorders due to low cost, low recall rate, and immediate action. Especially in countries where the earliest possible discharge is their current practice, it is challenging to get the newborn back to do the test in the hospital. Healthcare centers that are looking for high sensitivity regardless of the recall rate can use the heel prick as screening method for CH. Heel‐prick test is the superior screening choice for premature/low birth weight babies.The authors declare no competing interests.SA researched the literature and conceived the idea of the study. EA and AB carried out data retrieval. SA, MT, FB, and MM carried out analytical. AA done statistical analysis. SA and AB finally reviewed the article. All authors participated in reviewing and writing the article.


To analyze the risk factors for progression of urolith associated with obstructive urosepsis to severe sepsis or septic shock, we had done the retrospective cross-sectional study, which would facilitate the early identification of high-risk patients.Datas were retrospectively reviewed from 160 patients, suffering from obstructive urosepsis associated with urolith between December 2013 and December 2019. There were 49 patients complicating by severe sepsis (severe sepsis group), 12 patients complicating by septic shock (septic shock group), and 99 patients without progressing to severe sepsis or septic shock (sepsis group). The data covered age, gender, BMI (body mass index), time interval from ED (emergency department) to admission, WBC count (white blood cell count), NLR (neutrophil/lymphocyte ratio), HGB (hemoglobin), etc. Datas were analyzed by univariate analyses and multivariate logistic regression analysis. The corresponding nomogram prediction model was drawn according to the regression coefficients.Univariate analysis showed that the differences of age, the time interval from ED to admission, history of diabetes mellitus, history of CKI (chronic kidney disease), NLR, HGB, platelet count, TBil , SCr (serum creatinine), ALB , PT (prothrombin time), APTT , INR , PCT , and positive rate of pathogens in blood culture were statistically significant (P < 0.05). Multivariatelogistic regression analysis showed that age, SCr, and history of CKI were independent risk factors for progression to severe sepsis, or septic shock (P < 0.05).Aged ≥ 65 years, SCr ≥ 248 mol/L, and history of CKI were independent risk factors for progression of urolith associated with obstructive urosepsis to severe sepsis or septic shock. We need to pay more attention to these aspects, when coming across the patients with urolithic sepsis.The online version contains supplementary material available at 10.1186/s12894-022-00988-8. Urosepsis refers to the systemic inflammatory response caused by urinary tract infection, whose onset and progression is rapid. People pay more and more attention to urosepsis, because the fatality rate is as high as 28.3–41.1% once it develops into severe sepsis and septic shock , whereas9 /L or leukopenia < 4 × 109 /L, or the ratio of immature white blood cells are ≥ 10%). 160 patientswere divided into sepsis group (99 cases), severe sepsis group (49 cases), and septic shock group (12 cases). For the definition of severe sepsis and septic shock, refer to the Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016 (2016 SCC Guidelines). Severe sepsis is sepsis with consequent organ dysfunction and/or tissue insufficiency, one of the following: (1) Hypotension caused by sepsis; (2) Lactic acid was greater than normal; (3) Even with adequate fluid resuscitation, urine volume remains < 0.5 ml/kg/h at least 2 h; (4) Non-pneumonia-induced acute lung injury with PaO2/FiO2 < 250 mmHg. (5) Acute lung injury caused by pneumonia with PaO2/FiO2 < 200 mmHg. (6) SCr > 176.8 μmol/L (2.0 mg/ dL); (7) TBil > 34.2 μmol/L (2 mg/dL); (8) PLT < 100 000 mu l; (9) Coagulation disorders (INR > 1.5). Septic shock was defifined as a systolic arterial pressure below 90 mmHg, a mean arterial pressure < 60 mmHg, or a reduction in systolic blood pressure > 40 mmHg from baseline, despite adequate fluid replacement or using vasopressors for at least 1 h.A retrospective cross-sectional study was made to analyse the data collected from 160 patients suffering from urolithic sepsis between December 2013 and December 2019 in three branches of Guangdong Provincial Hospital of Traditional Chinese Medicine. The diagnostic criteria for urosepsis ). Gender distribution was 41 male and 119 female, aged 20‒92 years (median 63 years). General underlyingdiseases included 34 diabetes, 41 CKIs, 30 anemias, 69 hypertensions, 11 heart diseases, 7 cancers and 46 urolithiasis surgeries. The average time interval from ED to admission was 9.66 h (range 1–48 h).A total of 160 patients met the inclusion criteria. The average BMI was 24.29 kg/mThere were significant differences among the sepsis group, the severe sepsis group and the septic shock group in terms of NLR (P = 0.005), platelet count (P = 0.000), HBG P = 0.101), ALB (P = 0.000), SCr (P = 0.000), Tbil (P = 0.004), PT (P = 0.000), APTT (P = 0.000), INR (P = 0.000), PCT (P = 0.000), and positive rate of pathogens in blood culture (P = 0.001). The complete set of results includingthe highest temperature level, WBC count, CRP etc., is listed below  and gram-negative bacteria occurred in the the majority  of our cases and blood culture (85/160) were both low. Sixty-three (44.06%) cases (143 bacteria) were positive for bacteria in urine culture, and twenty-five (29.41%) cases (85 bacteria) were positive for bacteria in blood culture.As to the causative bacteria in bacteremia, /160 and Based on the logistic multivariate regression analysis, the three independent risk factors  were brought into in predicting the progression of urolith associated with obstructive urosepsis to severe sepsis or septic shock by ROC curve. We establish an individualized nomogram prediction model of pre-hospital patients with urolith associated with obstructive urosepsis Fig. .Fig. 1AgUrolith associated with obstructive urosepsis is one of the most common emergencies encountered in urology, which is characterized by its severity and rapid progression. Of the 160 pre-hospital patients with urolith associated with obstructive urosepsis in this study, 61 patients developed severe sepsis, or septic shock, whose incidence reaches to 38.13 percents. So it is particularly important and necessary for clinicians to identify highly risk factors for its progression to severe sepsis, or septic shock.Urolith associated with obstructive urosepsis occurs predominantly in female patients,  which alEarly and multidisciplinary comprehensive intervention of sepsis are essential to improve the prognosis and reduce the mortality , 11. TheThe pathogenesis of sepsis is still unclear, which may be closely related to the secretion of inflammatory mediators, immune dysfunction, endotoxin translocation, and other factors. When sepsis occurs, neutrophils are released from the bone marrow into the peripheral circulation and apoptosis is delayed . Tulzo eCRP and PCT are widely used in clinical practice as important indicators to reflect the degree of inflammation in the body. Fukashi Yamamichi et al.  analysedSepsis and septic shock are the main causes of AKI (acute kidney injury), and more than 50% of ICU patients with AKI are associated with sepsis . The incThe massive release of inflammatory mediators in septic patients activates the coagulation system, which leads to coagulation disturbance and thrombocytopenia , 24. KamPathogen culture and drug sensitivity identification are important clinically for the treatment and prognosis. In this study, the collection rates of urine culture 143/160) and blood culture (85/160) were both low, which raises the concern of standard of care and should be improved in the future. In this study, 63 strains of pathogens were isolated in urine culture and a total of 27 strains of pathogens were isolated in blood culture . Older age is one of the independent risk factors for the progression of sepsis. According to our study, if the patients are older than 65 years, their systemic inflammatory responses are more intense. Scr level has been used to evaluate the degree of AKI in patients with urosepsis.Patients with urosepsis associated with urolith obstructive may have rapid deterioration of renal function in a short period of time, especially when SCr ≥ 248 mol/L, which leads to increased serum concentrations of systemic inflammatory mediators and further aggravates the risk of infection. This phenomenon is more obvious in patients with CKI.For septic patients with chronic renal insufficiency, early intervention to protect renal function is helpful to improve the prognosis and prevent disease progr ession. If the patients who progressed to severe sepsis group and septic shock group were grouped into one group, that is the progressive group ; this study, the datas were simply divided into the sepsis group (99 cases) and the progressive group. By comparing the differences in age, Scr, and history of CKI between the two groups, the ROC curve was used to compare the value of these three risk factors in predicting the progression of urolith associated with obstructive urosepsis to severe sepsis or septic shock, respectively. The results showed that age, Scr and history of CKI all had good predictive functions. The area under ROC curve was 0.718, 0.923 and 0.611, respectively, and the prediction accuracy of Scr was higher than the other two datas (Fig. The main limitation of this study lies in that this is a retrospective cross-sectional study, with deficiencies such as incomplete test panels, ambiguous definition of indicators, and inconsistent treatment regimens, which need to be addressed by a better designed prospective study.To summarize our findings from this multicenter retrospective cross-sectional study, patients aged ≥ 65 years, Scr ≥ 248 mol/L, and history of CKI were independent risk factors for progression of urolith associated with obstructive urosepsis to severe sepsis or septic shock. It is essential to halt the progression of urosepsis by identifying its risk factors and to treat it as early as possible.Additional file 1. Author information.Additional file 2. Table legend.Additional file 3. Original data.


Patients with advanced clear cell renal cell carcinoma (ccRCC) have a poor prognosis and lack effective prognostic biomarkers. N6‐methyladenosine‐related lncRNAs (m6A‐related long noncoding RNAs [lncRNAs]) have been confirmed to be associated with the development of multiple tumors, but its role in ccRCC is not clear.Gene expression data and clinical information of ccRCC patients were extracted from The Cancer Genome Atlas Database. The prognostic m6A‐related lncRNAs were obtained by Pearson's correlation analysis and univariate Cox regression analysis. Afterward, the cluster classification and its correlation with prognosis, clinical characteristics, and immunity were analyzed. LASSO regression was used to establish the prognostic risk model. The predictive performance of the prognostic model was evaluated and validated by survival analysis and receiver operating characteristic curve analysis, et al. The expression of immune checkpoints and immune cell infiltration in patients with different risks were systematically analyzed.PD‐1, PD‐L1, CTLA‐4, LAG‐3, TIM‐3, and TIGIT were significantly increased in the high‐risk patients, and there was a correlation between the risk score and immune cell infiltration.A total of 27 prognostic m6A‐related lncRNAs were identified. These m6A‐related lncRNAs were differentially expressed between tumor and normal tissues. Among them, 24 high‐risk m6A‐related lncRNAs were overexpressed in Cluster 2 and correlated with poor prognosis, low stromal score, high expression of immune checkpoints, and immunosuppressive cells infiltration. Based upon, a prognostic risk model composed of seven m6A‐related lncRNAs was constructed. After a series of analyses, it was proved that this model had good sensitivity and specificity, and could predict the prognosis of patients with different clinical stratification. The expression of The seven m6A‐related lncRNAs prognostic risk signature showed reliable prognostic predictive power for ccRCC and was associated with the expression of immune checkpoints and immune cell infiltration. This seven m6A‐related lncRNAs signature will be helpful in managing ccRCC and guiding individualized immunotherapy. M6A‐related long noncoding RNAs can well predict the prognosis of clear cell renal cell carcinoma of different ages, genders, pathological characteristics and stages and disease progression, and are associated with the expression of immune checkpoints and immune cell infiltration. Its incidence is increasing in recent years, and it occurs more in males than in females.small nucleolar RNA host gene 14 (SNHG14) is significantly increased in ovarian cancer tissues, and the inhibition of SNHG14 significantly inhibits the migration and invasion of cells.gastric cancer metastasis‐associated long noncoding RNA in gastric cancer tissues is associated with metastasis in patients.urothelial cancer‐associated 1 (UCA1) is significantly upregulated in RCCs and is positively correlated with tumor differentiation and tumor node metastasis (TNM) staging. UCA1 promotes the malignant phenotype of RCCs by regulating of the miR‐182‐5p/DLL4 axis.Long noncoding RNAs (lncRNAs) are a group of noncoding RNAs with a length greater than 200 nucleotides.METTL3, METTL14, and RBM15); removed by demethylases ; and recognized by binding proteins .N6‐methyladenosine (m6A) methylation occurs in the N6‐position of adenosine.In this study, we used bioinformatics to screen for m6A‐related lncRNAs that are associated with the prognosis of ccRCC and constructed a prognostic model. This prognostic model will help to assess the prognosis of ccRCC patients and guide clinical treatment. Our results will provide a basis for further exploration of the potential mechanism of m6A modification of lncRNAs in ccRCC.22.1https://portal.gdc.cancer.gov/, until February 1, 2021). The Genome Reference Consortium Human Build 38 (GRCh38) was downloaded from GENCODE (https://www.gencodegenes.org/human), and the expression data of lncRNAs in the transcriptome were extracted based on the gene biotype. These data were obtained from a public database, and therefore, ethics approval was not required.Transcriptomic sequencing (RNA‐seq) data, fragments per kilobase of transcript per million mapped reads, and related clinical information of ccRCC patients were downloaded from The Cancer Genome Atlas (TCGA) database .According to previously published studies, we found 23 m6A‐related genes, namely, methyltransferases 2.3To investigate the clinical significance of m6A‐related lncRNAs that are associated with prognosis, we used the Consensus Clustering algorithm and the ConsensusClusterPlus packagep value  < .05 were used to determine the difference between different genotypes.To further explore the biological processes mediated by m6A‐related lncRNAs, we used GSEA software (version 4.1.0) to determine gene expression enrichment for different subtypes in the Molecular Signatures Database Collection  to analyze the difference of Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. A normalized enrichment score (NES) > 1 and nominal 2.4p value of deconvolution to represent the accuracy of the results. p < .05 indicated that the inferred cell composition was reliable. Leukocyte signature matrix (LM22) contains 547 genes that distinguish 22 human immune cell phenotypes, including seven T‐cell types, naive and memory B cells, plasma cells, natural killer (NK) cells, and myeloid subsets. We used the CIBERSORT algorithm to analyze the RNA‐seq expression profile based on the LM22 classification. We evaluated the abundance of immune cell subsets in samples and visualized the differences in immune cell infiltration between different types.LncRNAs are involved in the regulation of the tumor microenvironment (TME) and signaling transduction in tumor cells. To understand the correlation between m6A‐related lncRNAs and TME, we used the ESTIMATE algorithm in the R estimate package2.5(PD‐1), programmed cell death‐ligand 1 (PD‐L1), cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), lymphocyte activation gene‐3 (LAG‐3), T‐cell immunoglobulin, and mucin domain‐containing protein 3 (TIM‐3) and T‐cell immunoglobulin and ITIM domain (TIGIT), that are the key genes associated with currently used tumor immune checkpoint inhibitors, and we used the R corrplot packagep values. The differential expression of the above genes between tumor and normal samples and different clusters was analyzed using the limma packagep < .05).To explore the correlation between m6A‐related lncRNAs and immune‐related genes, we selected programmed cell death 1 2.6∑i=1n(Coefi×βi), where Coef represents the regression coefficient, and β represents the m6A‐related lncRNA expression value. The risk score for each patient was calculated using this formula, and then, the training set and test set were divided into a high‐risk group and a low‐risk group based on the median score. The R survival package was used for Kaplan–Meier analysis. The log‐rank test was used to compare the overall survival rates for the high‐ and low‐risk groups. A time‐dependent receiver operating characteristic (ROC) curve was plotted. The area under the curve (AUC) > 0.60 was considered an acceptable prediction. In addition, we also used univariate and multivariate Cox regression analyses to assess whether the risk score could be an independent prognostic factor for ccRCC. To evaluate the predictive ability of the model for different populations, Kaplan–Meier analysis was performed for age, sex, grade, stage, and TNM staging. Finally, we analyzed the correlation between risk scores and clinicopathological features to assess the ability of this model to predict ccRCC progression.First, the R caret package2.7To understand the significance of this risk model in the assessment of the immune microenvironment and immunotherapeutic efficacy in ccRCC, we analyzed the differences in the expression of immune checkpoints between the high‐ and low‐risk patients and the correlation between risk scores and tumor‐infiltrating immune cells.33.1R | > .7; p < .001) that were positively correlated with RBM15 and METTL3  downloaded from TCGA were combined with GRCh38 downloaded from GENCODE to obtain 14086 lncRNAs. The workflow was shown in Figure 3 Figure . Throughp < .05). Three low‐risk lncRNAs, AC018752.1, RPL34‐AS1, and COL18A1‐AS1, had higher expression in normal tissue than in tumor tissue, and the remaining 24 high‐risk lncRNAs  were significantly overexpressed in tumor tissues  and Custer 2 , ATP‐binding cassette (ABC) transporter , Notch signaling pathway , and vascular endothelial growth factor (VEGF) signaling pathway  were significantly enriched in Cluster 2 , regulation of microtubule‐based movement  and phospholipid acid metabolism  were significantly enriched in Cluster 2  signaling pathway ; the higher the score, the lower the tumor purity. It was found that the stromal score of Cluster 1 was higher than that of Cluster 2  and the expression of PD‐1, PD‐L1, and CTLA‐4, LAG‐3, and TIGIT in Cluster 2 was significantly higher than those in Cluster 1  and a test set (n = 264). There was no significant difference in the clinical baseline characteristics between the training set and the test set  − (AC018752.1 × 0.1872) − (RPL34‐AS1 × 4.5020) + (AF117829.1 × 0.3137) + (AC009948.2 × 0.9556) + (SNHG10 × 0.0863) + (AL133243.3 × 0.3944).The ccRCC patients in the TCGA database were randomly divided into a training set  were upregulated, and two low‐risk lncRNAs (AC018752.1 and RPL34‐AS1) were downregulated. In contrast, the expression of these lncRNAs in low‐risk patients were opposite.The risk score curve, survival status, and heat map of gene expression of patients in the high‐risk and low‐risk groups are shown in Figure Next, we performed univariate and multivariate Cox regression analyses to evaluate the prognostic value of risk scores and other clinical features of the prognostic model. Univariate Cox regression analysis indicated that age, grade, stage, and risk score were correlated with the prognosis of ccRCC patients Figure . Multiva3.6We further performed Kaplan–Meier survival analysis on different ages, sex, grades, stages, and TNM stages and compared the survival rates for high‐ and low‐risk patients in all stratifications to explore whether our prognostic model is suitable for patients with different clinical stratifications. The results are shown in Figure Finally, we analyzed the correlation of risk scores with different clinicopathological characteristics and found that risk scores were significantly different among different clusters, grades, immune scores, TNM staging, and stage. Risk scores were significantly higher for patients in Cluster 2, G3–4, low‐immune scores, T3–4, N1, and M1 staging, and Stage III–IV Figure , suggest3.7PD‐1, PD‐L1, CTLA‐4, LAG‐3, TIM‐3, and TIGIT, were significantly increased in the high‐risk group  and liver cancer tissues and cells and closely associated with the poor prognosis of patients.PD‐1, PD‐L1, CTLA‐4, LAG‐3, and TIGIT were highly expressed and positively correlated with m6A‐related lncRNAs. These results suggest that the prognostic m6A‐related lncRNAs could be a signature for the assessment of the immune cell infiltrating TME and immunotherapeutic response.In this study, 27 prognostic m6A‐related lncRNAs were identified by analyzing gene expression data of 530 ccRCC patients in the TCGA database. Among those 27 lncRNAs, 24 high‐risk lncRNAs were overexpressed in tumor tissue, suggesting that these lncRNAs might have an oncogenic effect in the occurrence and development of ccRCC. The expression of these lncRNAs was positively correlated with the m6A “writers” To understand the regulatory mechanism of m6A‐related lncRNAs in ccRCC, we performed pathway and functional enrichment analysis for Clusters 1 and 2. The results showed that the signaling pathways associated with tumor occurrence and development, drug resistance, and angiogenesis, such as the mTOR signaling pathway, Notch signaling pathway, VEGF signaling pathway, and ABC transporters, were enriched to varying degrees in Cluster 2 patients with a poor prognosis. mTOR is regulated by a variety of cell signals, mainly through the phosphatidylinositol 3‐kinase/protein kinase B/mTOR signaling pathway, to regulate cell proliferation, autophagy, and apoptosis, among other regulatory functions. In many cancer types, the mTOR signaling pathway is abnormally activated and is involved in tumor formation, the regulation of immune cell differentiation, and tumor metabolism.LINC00342, AC018752.1, RPL34‐AS1, AF117829.1, AC009948.2, SNHG10, and AL133243.3) that were significantly correlated with prognosis and constructed a prognosis risk model. Studies have found that the long intergenic nonprotein coding RNA 00342 (LINC00342) can regulate the growth, invasion, and metastasis of colorectal cancer and NSCLC cells and is closely associated with the poor prognosis of patients.RPL34‐AS1) is localized on human chromosome 4q25 and has antitumor effects in esophageal carcinoma and papillary thyroid carcinoma. RPL34‐AS1 overexpression can inhibit tumor cell proliferation and invasion and promote apoptosis.Based on the above results, we suggested that the m6A‐related lncRNAs are closely associated with the prognosis and biological process of ccRCC patients. According to the LASSO regression analyses, we ultimately obtained seven m6A‐related lncRNAs (Survival analysis, ROC curves, and risk curves were used to analyze the accuracy and stability of the model. The results indicated that the model can accurately distinguish high‐ and low‐risk patients and accurately predict the prognostic risk of ccRCC. The stratification of different clinical traits showed that this model could effectively predict the prognosis of patients of different ages, sex, and stages. Univariate and multivariate Cox regression analyses showed that this risk model could be used as an independent prognostic indicator for ccRCC patients. In addition, we also analyzed the relationship between the expression of the prognostic m6A‐related lncRNAs in the model and different clinicopathological characteristics. We found that the high‐risk score was associated with the progression of ccRCC. Taken together, our prognostic model is reliable and can be used to identify the risk and prognosis of ccRCC patients, information that is conducive to early intervention and treatment.NKILA alters the balance between immune activating and immunosuppressive T‐cell subsets in the TME by regulating the sensitivity of apoptosis of T‐cell subsets, resulting in tumor immune escape. Wang et al.MALAT1 promotes the immune escape of diffuse large B‐cell lymphoma by targeting miR‐195. MALAT1 gene knockout promotes the proliferation of CD8+ T cells and inhibits the epithelial–mesenchymal transition‐like signal transduction process through Ras‐extracellular signal‐regulated kinase signaling pathways. The lncRNA SNHG15 promotes PD‐L1 expression through the inhibition of miR‐141 and participates in the immune escape of gastric cancer.PD‐1, PD‐L1, CTLA‐4, LAG‐3, TIM‐3, and TIGIT was upregulated in high‐risk patients, who are more likely to benefit from immunotherapy. The risk score was positively correlated with the infiltration of memory B cells, CD8 T cells, follicular helper T cells, and Tregs. The risk score was negatively correlated with the infiltration of naive B cells, activated dendritic cells, resting dendritic cells, resting mast cells, monocytes, and resting memory CD4 T cells. These results suggest that m6A‐related lncRNAs are involved in the regulation of the immune microenvironment. Pan et al.In recent years, immunotherapy targeting immune checkpoints has made substantial breakthroughs, bringing new hope to ccRCC patients. However, the complex microenvironment of tumors can mediate immune escape, leading to the failure of immunotherapy.This study has some limitations. The risk model constructed in this study was based on a public clinical database TCGA. The results were confirmed in a TCGA cohort but lacked external validation. Therefore, further validation in larger multicenter clinical patient cohorts is needed in the future. In addition, the specific regulatory mechanism of the m6A‐related lncRNAs in ccRCC requires further study.5This study systematically analyzed the prognostic value of m6A‐related lncRNAs in ccRCC patients, as well as their significance in the assessment of immune microenvironment and immunotherapeutic response. A prognostic risk model based on seven m6A‐related lncRNAs was constructed and validated. The model can predict the prognosis of patients with different clinical stratifications and the progression of ccRCC. The risk score can be used as an independent prognostic indicator for ccRCC. In addition, the m6A‐related lncRNAs were related to the expression of immune checkpoints and immune cell infiltration. Our study provides a method for the individualized risk stratification of ccRCC patients, provides a basis for further exploring the mechanisms underlying the occurrence and development of ccRCC. These m6A‐related lncRNAs could be potential targets for improving the response to immunotherapy in patients with ccRCC.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Jinchang Huang and Xin Jiang conceived and designed the study; Zhenjia Fan and Shanhui Zhan downloaded and organized The Cancer Genome Atlas data; Yuqin Qiu and Xiaogang Wang performed data analysis and wrote the paper; Jinchang Huang and Xin Jiang critically revised the article for essential intellectual content and administrative support. All authors read and approved the final manuscript.The data of the patients in this study were obtained from the public database datasets.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


ERCC1, ERCC5, and BRCA1) and did not evaluate several other DDR‐related genes that could have a relevant role for trabectedin efficacy. In this retrospective translational study, 118 genes involved in DDR were evaluated to determine, by transcriptomics, a predictive gene signature of trabectedin efficacy. A six‐gene predictive signature of trabectedin efficacy was built in a series of 139 tumor samples from patients with advanced STS. Patients in the high‐risk gene signature group showed a significantly worse progression‐free survival compared with patients in the low‐risk group . Differential gene expression analysis defined new potential predictive biomarkers of trabectedin sensitivity (PARP3 and CCNH) or resistance (DNAJB11 and PARP1). Our study identified a new gene signature that significantly predicts patients with higher probability to respond to treatment with trabectedin. Targeting some genes of this signature emerges as a potential strategy to enhance trabectedin efficacy.Predictive biomarkers of trabectedin represent an unmet need in advanced soft‐tissue sarcomas (STS). DNA damage repair (DDR) genes, involved in homologous recombination or nucleotide excision repair, had been previously described as biomarkers of trabectedin resistance or sensitivity, respectively. The majority of these studies only focused on specific factors . Patients in the high‐risk gene signature group showed a significantly worse progression‐free survival compared to patients in the low‐risk group. These findings may help in identifying new therapeutic targets that could enhance the efficacy of trabectedin in STS. APapurinic/apyrimidinicAUCarea under the ROC curveCRcomplete responseDDRDNA damage repairDSBdouble‐strand breakFCfold changeFDRfalse discovery rateFFPEformalin‐fixed paraffin‐embeddedGMIgrowth modulation indexHRhomologous recombinationNERnucleotide excision repairNHEJnon‐homologous end joiningOSoverall survivalPDprogression diseasePFSprogression‐free survivalPRpartial responseSDstable diseaseSTSsoft‐tissue sarcomaTMMtrimmed mean of M‐valuesTPMtranscripts per millionTTPtime‐to‐progression1Sarcomas are a heterogeneous group of rare malignant tumors with mesenchymal origin that can affect soft, bone, and visceral tissue. Sarcoma incidence is around 50–60 new cases per year for every million people, 20% being metastatic at diagnosis. Around 30–40% of localized sarcomas will eventually develop metastasis. In other words, a half of sarcoma population will suffer, sooner or later, metastatic spread. For locally advanced or metastatic soft‐tissue sarcoma (STS), the therapeutic options are scarce and the Response Evaluation Criteria in Solid Tumors (RECIST) responses to chemotherapy are infrequent, while the median of survival ranges between 18 and 22 months  and worse OS , and diagnosis of L‐sarcoma subtypes was correlated with a better PFS  and better OS . Somatic location was associated with a better PFS in our series . Patients receiving trabectedin in first or second line had better PFS  (Table P = 0.002] and non‐L‐sarcoma  were independent factors associated with a worse PFS of trabectedin in the multivariate analysis.In the univariate analysis, grade 3 tumors were associated with a worse PFS ; however, neither the expression of ERCC1  nor of ERCC5  showed a significant association with PFS of trabectedin , XPC , DNAJB14 , ATM , DNAJC7 , and NEIL2 , compared with those with a GMI < 1.33 . Patients with a GMI ≥ 1.33 showed significantly higher expression of PARP3 , POLL , RAD52 , TOP3B , XRCC5 , DNAJB14 , XPC , ATM , MSH5 , and NEIL2  were associated with clinical benefit of trabectedin, whereas overexpression of RAD21 , MSH6 (logFC = −0.369: FDR = 0.009), DNAJB11 , TDG , XRCC4 , PARP1 , ERCC3 , PRKDC , RAD23B , and TOP3A  were associated with progressive disease after trabectedin. By contrast, no different gene expression was significantly detected, by FDR, when the grouping was responders (CR+PR) vs non‐responders patients  Table . High exts Table .ATM, DNAJB14, and XPC , cyclin H (CCNH), xeroderma pigmentosum group A‐complementing protein (XPA), poly(ADP‐ribose) polymerase 1 (PARP1), BRCA1, and apurinic/apyrimidinic endodeoxyribonuclease 2 (APEX2).Univariate Cox regression model on a cohort of 139 patients identified eight genes whose expression was significantly associated with PFS of trabectedin. Further selection of genes whose Kaplan–Meier curves displayed significant differences  yielded six final genes. Lasso‐penalized Cox regression analysis was performed to construct a predictive gene signature for trabectedin. The six genes identified were as follows: exonuclease 1  showed a significantly worse PFS compared with the patients in the low‐risk group , [2.1 months (95% CI 1.8–2.4) vs 6.0 months (95% CI 4.5–7.5), P < 0.001  was determined. An additional univariate Cox regression analysis was performed using risk scores, resulting in a significant association with PFS Table . Time‐de.146 was ue Table .In the patients treated with either doxorubicin or gemcitabine, the DDR‐related gene signature was also correlated with the PFS of these drugs. The six‐gene‐based signature failed to significantly predict gemcitabine or doxorubicin activity, in terms of PFS, in our series Table .4ERCC1 and ERCC5, did not show any relevant prognostic role in our series. Rather, other DDR‐related genes showed more consistent prediction of trabectedin activity, either in terms of PFS, GMI, and/or clinical benefit. For this study, we decided to focus only in the relevance of DDR‐related genes, since the DNA DSBs induced by trabectedin along with the involvement of NER machinery and HR repair genes were previously reported as a potential relevant mechanism of trabectedin activity in STS patients [A six‐gene predictive signature of trabectedin efficacy was built based on the transcriptomic analysis of 118 DDR‐related genes and PFS, in a retrospective series of 139 advanced STS patients. Previously described predictive genes for trabectedin efficacy, as those related to NER machinery, such as patients , 21. HowBRCA1 (expression levels or haplotype status) had been previously associated with the clinical benefit of trabectedin [XPA gene, another component of the signature and a NER‐associated gene, was in line with previous reports that showed a direct correlation between higher expression of NER machinery‐related genes and better trabectedin efficacy [EXO1, CCNH, and APEX2 had been associated with trabectedin efficacy. EXO1 is an XPG family nuclease that participates in several DDR pathways, such as DNA mismatch repair, HR, and non‐homologous end joining (NHEJ) [CCNH gene, is a protein that forms a cyclin‐activating kinase‐subcomplex with CDK7 and MAT1. This subcomplex is part of the transcription factor II human (TFIIH) protein complex and it is involved in NER, by regulating cell cycling progression during DNA damage [APEX2 is a weak apurinic/apyrimidinic (AP) endonuclease in the DNA base excision repair pathway and functions in the context of DNA damage by oxidative and alkylating agents. APEX2 also exhibits strong activities of 3′–5′ exonuclease and 3′‐phosphodiesterase and that has been described to be necessary for the recovery of B‐cell lymphocyte progenitors after chemotherapy [XPA and CCNH) and the low expression of HR genes  would help to identify the STS patients that benefit from trabectedin treatment.Even when bectedin , 21, itsefficacy , 23. Besg (NHEJ) . Of noteg (NHEJ) , 25. CycA damage . As expeotherapy , 28. Morotherapy . APEX2 potherapy . Of noteotherapy . Accordiotherapy , 15, 16,PARP3 overexpression significantly associated with a better PFS and clinical benefit following trabectedin. PARP3, initially described in 1999 [PARP3 is involved in transcriptional silencing and in the maintenance of genomic integrity [PARP1 expression, a gene also included in our potential predictive molecular signature, was significantly associated with shorter PFS and lack of clinical benefit after trabectedin treatment. The distinct behavior or PARP1 and PARP3 genes in our study may indicate that these proteins may participate differently in some cellular mechanisms, which could affect the response to trabectedin. Both proteins are relevant for DNA genomic integrity [PARP3 seems to be overexpressed in tumors that benefit from trabectedin treatment , while PARP1 expression is higher in sarcomas with lower activity of trabectedin . Therefore, the expression of these genes may be important biomarkers of trabectedin efficacy or resistance, in specific clinical contexts that depend on the histologic subtype or the location of the primary tumor. Besides, the association of PARP1 high expression with worse trabectedin outcome supports the combination of PARP1 inhibitors with this chemotherapeutic agent. In line with these observations, the combination of olaparib with trabectedin has been tested in a phase 1b clinical trial in patients with advanced and non‐resectable bone and STS . Of 50 patients recruited, 7 had a PR, suggesting that this combination has antitumor activity [Furthermore, several DDR‐related genes were significantly associated with trabectedin activity, in terms of PFS, GMI, and/or clinical benefit. Among these genes,  in 1999 , is a po in 1999 . PARP3 integrity , being pntegrity . In turnntegrity ; howeverntegrity , 36, 37,ntegrity . Of notentegrity . Accordiactivity ; howeverBRCA1 high expression was significantly associated with shorter PFS and OS, while ERCC1 and ERCC5 expression, which were previously associated with good prognosis to trabectedin [ERCC1 and ERCC5 are not robust and reliable biomarkers of trabectedin activity. Their predictive value should accordingly be considered with caution; however, a further study with a larger cohort of cases should validate our data.In the study presented here, only bectedin , 15, 16,bectedin , 42. AccAmong the study limitations, it is important to mention that the tumor samples used for this retrospective analysis were collected at the diagnostic time, and gene expression levels could have been altered due to the several lines of previous treatment or during the natural progression of the disease. Moreover, another important limitation is related with its initial hypothesis; for this analysis, only genes related with DDR were selected and studied. This selection excluded, from the molecular signature, genes involved in other cellular processes, which may also predict trabectedin activity. Since we cannot exclude the relevance of other genes beyond DDR, further analysis, considering all the genes included in the OBP assay, is currently ongoing. The absence of an independent validation cohort, or a control cohort treated with any other drug approved for advanced STS, is other limitation of this study. The validation of the predictive value of the gene‐based signature in a larger series of cases could also help improve the performance of ROC curves.5In summary, this translational study identified a new molecular signature that could significantly predict patients with higher probability for trabectedin efficacy. This signature was based on wide transcriptomic analysis of DDR‐related genes and provides new insights for potential targets that could enhance the trabectedin efficacy. Validation of this signature is in process with an independent series of advanced STS treated with trabectedin. Finally, the potential value of specific biomarkers  is currently being tested in the preclinical setting, in order to describe the mechanisms in which they participate under drug treatment.DSM reports institutional research grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work and travel support from PharmaMar, Eisai, Celgene, Bayer, and Pfizer. NH declares Honoraria from PharmaMar and Lilly; Travel Grants from PharmaMar; Research Grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work. Research funding for her Institution  from PharmaMar, Eli Lilly and Company, AROG, Bayer, Eisai, Lixte, Karyopharm, Deciphera, GSK, Novartis, Blueprint, Nektar, Forma, Amgen, Adaptimmune, and Daichii‐Sankyo. JM‐B: Honoraria from PharmaMar, Lilly, Bayer, and Eisai; travel grants from PharmaMar; research grants from PharmaMar, Eisai, Immix BioPharma, and Novartis outside the submitted work. Research funding for his institution  from PharmaMar, Eli Lilly and Company, AROG, Bayer, Eisai, Lixte, Karyopharm, Deciphera, GSK, Novartis, Blueprint, Nektar, Forma, Amgen, Adaptimmune, and Daichii‐Sankyo.DSM and JM‐B designed the experiments and analysis. All the authors participated in data acquisition. DSM, MP‐C, JACV, and JM‐B performed data analysis and interpretation. DSM, MP‐C, JACV, and JM‐B drafted the manuscript. All the authors reviewed the manuscript and approved its final version.Fig. S1. Volcano plot of genes with prognostic relevance in progression‐free survival (PFS) of trabectedin.Click here for additional data file.Fig. S2. Venn diagram comparing the unique and shared genes between progression‐free survival, growth modulation index and clinical benefit.Click here for additional data file.Fig. S3. Time‐dependent ROC curve to assess the predictive value of the gene signature.Click here for additional data file.Table S1. Expression values of DNA damage repair‐related genes.Click here for additional data file.Table S2. Gene significantly expressed between L‐sarcoma and non‐L‐sarcomas and between grade 3 tumors vs grade 1 + 2 tumors.Click here for additional data file.Table S3. Univariate analysis of DNA damage repair‐related genes with impact in progression‐free survival to trabectedin line.Click here for additional data file.Table S4. Differential gene expression according to objective response.Click here for additional data file.Table S5. Differential gene expression according to histologic subtype grouping.Click here for additional data file.Table S6. Differential gene expression according to tumor location.Click here for additional data file.Table S7. Univariate Cox regression analysis for the values of the gene signature (risk scores).Click here for additional data file.Table S8. Multivariate analysis.Click here for additional data file.Table S9. Correlation between risk groups and progression‐free survival of doxorubicin or gemcitabine.Click here for additional data file.


Cannabis Sativa L. (cannabis) has attracted an enormous amount of attention and become one of the most popularly cultivated plants worldwide , a medicinal marijuana strain with an average THC level at 20% and a non‐detect level of cannabidiol (CBD) (van Bakel https://gdb.supercann.net), including 5.84 Gb genome sequences of 8 cultivated cannabis plants downloaded from NCBI Assembly database. Additionally, a total of 195.7 Gb RNA sequencing (RNA‐seq) data cover 16 varieties downloaded from the NCBI SRA database and one sequenced by this study. Furthermore, 7 liquid chromatography mass spectral (LC‐MS) projects quantifying major cannabinoids were collected from literatures. We also collected 5 independent data sets of MS‐based proteomics in the literatures covering 4 tissues. After masking the repeat sequences, the genomic sequences were used to predict genes based on homology prediction of related species , transcriptional evidence together with ab initio gene prediction programs. In addition, we comprehensively annotated genes from different perspectives including Gene Ontology (GO), KEGG orthology (KO), non‐redundant (Nr) peptide database, UniProt protein database and PFAM domain database  varieties, (2) gene loci, (3) metabolites and (4) proteins Figure . The ‘vaCannbiasGDB provides some popular bioinformatics tools for browsing, searching, analysing and downloading located in the navigation bar Figure . The ‘Seterpenes synthase (CsTPS) gene family. There are 22 TPS proteins of cannabis in Uniprot annotated with two Pfam keywords, ‘PF1397 Terpene_synth’ and ‘PF03936 Terpene_synth_C’. Then, we searched CsTPSs in CannabisGDB with these two Pfam keywords. A total of 217 CsTPSs from 8 varieties was found, including 26 from CsCBD, 32 from CsFN, 58 from CsJLD, 48 from CsPK and 6 from CsLAC, 16 from CsPBB, 5 from CsCD91 and 26 from CsCAN. The phylogenetic tree using maximum likelihood method with 1000 bootstrap was constructed based on the protein sequences of the high‐quality genome assemblies  together with Arabidopsis thaliana TPSs defined as outgroups using MEGAX program. The TPS genes have been divided into three classes: Class I consists of TPS‐c, TPS‐e/f and TPS‐h (Selaginella specific); Class II consists of TPS‐d (Gymnosperm specific) and Class III consists of TPS‐a, TPS‐b and TPS‐g (Chen et al., CsTPSs among nine different cannabis varieties and presented as a heatmap (Figure A typical user case picked from our web tests is present in Figure p Figure .By integrating the genomic, transcriptomics, proteomics and metabolomics data, CannabisGDB can be used to assemble valuable information to facilitate basic, translational and applied research in cannabis. CannabisGDB is the first part of the SuperCann series database and will become a central gateway for the global cannabis community to better understand cannabis biology, thus benefitting the whole cannabis industry.The authors declare no conflict of interest.SC, YJZ, WT, SY and YL conceived and designed this research. SC, ZZ, SH, XB, ZH and LH performed the experiments. GH, SY and YL prepared the article.