Anemonia viridis, focusing in detail on the reorganization of the ECM mesoglea. In this context, both enzymatic, biometric and histological experiments reveal how this gelatinous connective layer plays a fundamental role in the correct restoration of the original structures by modifying its consistency and stiffness. Indeed, through the deposition of collagen I, it might act as a scaffold and as a guide for the reconstruction of missing tissues and parts, such as amputated tentacles.Given the anatomical simplicity and the extraordinary ability to regenerate missing parts of the body, Cnidaria represent an excellent model for the study of the mechanisms regulating regenerative processes. They possess the mesoglea, an amorphous and practically acellular extracellular matrix (ECM) located between the epidermis and the gastrodermis of the body and tentacles and consists of the same molecules present in the ECM of vertebrates, such as collagen, laminin, fibronectin and proteoglycans. This feature makes cnidarians anthozoans valid models for understanding the ECM role during regenerative processes. Indeed, it is now clear that its role in animal tissues is not just tissue support, but instead plays a key role during wound healing and tissue regeneration. This study aims to explore regenerative events after tentacle amputation in the Mediterranean anemone  The ability to restore the correct tissue architecture after injuries requires a series of closely connected cellular and molecular events. Defects in the early steps of wound healing and the tissue remodeling program can interfere with regeneration , even inAlthough in the animal kingdom different mechanisms exist, in the last decades it has become clear that the extracellular matrix (ECM) plays a pivotal role during different physiological processes, including wound healing and tissue regeneration .Since the Cnidaria, a sister group to Bilateria, possess high body regeneration capacities and can regrow missing body parts when dissected , is it iCnidarians are among the simplest living metazoans and evolved approximately 700 million years ago. They consist of two body layers derived from the endoderm (gastrodermis) and ectoderm (epidermis) of the embryo. Between these two cellular layers is an amorphous, jelly-like mesoglea connective layer. This generally contains no cellular elements other than some scattered amoebocytes and local genital cells.Nematostella vectensis [The majority and most detailed cellular and molecular regeneration studies have been carried out using Hydra ,7,8,9,10ectensis ,16,17, iectensis .Unlike the freshwater polyp Hydra, where regeneration is based on three distinct populations of multipotent stem cells, the I-cells, the ectodermal and endodermal epithelial cells ,21,22,23The ultrastructure of the mesoglea in hexacoral anthozoan polyps shares some properties with that of various Hydra species, having a basal lamina composed of a lattice of fine filaments and containing collagen IV orthologs ,29. HoweAnemonia viridis as an in vivo model for the study of the matrix/cell interaction. For this purpose, we used the Mediterranean A. viridis is a Mediterranean hexacoral anthozoan  belonging to the family Actinidae, that inhabits benthic rocky bottoms from 0 to 10 m deep. Like many anthozoans, it harbors zooxanthellae, photosynthetically active unicellular algae belonging to the genus Symbiodinium, within its gastrodermal cells. This association represents a trophic mutualistic endosymbiosis, in which zooxanthellae provide their cnidarian host with reduced organic carbon, resulting from their photosynthetic activity, while the host provides unicellular algae with inorganic carbon, inorganic nitrogen, and inorganic phosphate, as well as a refuge from herbivory [erbivory ,31. The The broader objective of the study was to analyze the mesoglea ECM, from a biochemical, biometric and morphological point of view, in unamputated and injured polyps after amputation of their oral tentacles. Specifically, we evaluated changes in mesoglea stiffness during regeneration, both in tentacles and the body’s mesenteries. The possible role of fibroblast-like cells, differentiated from hyaline amebocytes, in the remodeling of the ECM through the production of metalloproteases, the deposition and spatial organization of new collagen I fibrils in the mesoglea matrix were investigated. The anemones survived the stress of injury. The tentacles hexamerously were arranged in several cycles moderately long (to 20–30 mm length), smooth, slender, tapering distally, inner ones longer than outer ones, contractile, dark-yellow to light-brown, sometimes with whitish or light pink tips.A. viridis adult specimen the tentacles were amputated. A lateral view of the amputated tentacle is shown in In After 1 day post-amputation (dpa), the injured terminal parts of tentacles appeared lighter in color than the rest of tentacles and the body c. The blA. viridis tentacles and body extracts increased over time in amputated samples with respect to unamputated animals  for the interaction of time (T) and injury (I) factors (TxI). In the body, both time (T) and injury (I) factors resulted in significance  as well as their interaction (TxI).In both tissues, the two-way ANOVA analysis reported in The proteolytic activity was investigated in tentacles and body extracts using SDS-PAGE with gelatin and fibrinogen as substrates c,d. The p-value (<0.05) in both body and tentacle tissue extracts was revealed for the variables Time (T), Substrate (S) and their interaction (TxS).The two-way ANOVA results regarding proteolytic activity were reported in In order to evaluate biometric parameters of regenerating tissues, longitudinal sections, stained with Gomori’s Trichrome, of control (unamputated) a,b and 7Of note, in the 7 dpa tentacle, both the ectoderm and the gastrodermis layers appeared thicker than in the unamputated tentacle, while the mesoglea size remained constant during all the regenerative phases. However, a pronounced variation in the mesoglea connective tissue density was appreciated. Indeed, after 7 dpa c,d the cComparing the control tentacles with the regeneration after amputation, a significant increase in tissue thickness e and a rIn order to shed light on morphological modification occurring in the mesoglea connective tissue during tentacle regeneration, histological and ultrastructural analyses were performed on unamputated and regenerated tentacles. Control tentacle tissues revealed intact epithelia containing cells characteristic for each epithelium and separated by a loose mesoglea. Epidermis was formed by a single layer of densely packed columnar cells with the basal part mounted on the mesoglea. On the opposite side, the gastrodermis layer, with numerous endosymbiotic dinoflagellates zooxanthellae, was visible a. MesoglThe changes occurring in the ECM during the regenerative phase, both at the level of cellular and matrix reorganization, were evaluated in tentacles allowed to grow back for 1 and 7 days and subsequently cut from the base of the body. The epidermal layer of the tentacle regrown after one day from cutting, was formed by columnar cells arranged in a circular way and anchored with their basal side to the inner mesoglea e. An incAfter 7 days, the mesoglea infiltrating the newly formed epidermis increased in length together with the tentacle growth i. Bands The ECM area and the number of elongated amebocytes in both noninjured (Cnt) and amputated tentacles a were quThese data suggested that these cells might be implicated in the ECM neo-synthesis and reorganization.Based on the morphological observation, we were further interested in evaluating a possible interaction between tentacle regeneration and mesoglea expansion from the body’s mesenteries and whether the mesoglea ECM remodeling would be correlated to collagen neo-synthesis and fibroplasia events.To this aim, Masson’s trichrome staining with blue aniline, immunofluorescence and immunogold assays using anti-pro-collagen1α1 (COL1α1), to detect collagen I and bFGFR, a specific fibroblast marker in both vertebrates and invertebrates , was perIn both tentacles and mesenteries of healthy animals, the ECM had an amorphous and nonfibrotic appearance and was slightly colored by aniline blue a,b. More+ fibrils dispersed in the ECM were detected by immunofluorescence assay  activity, especially by fibroblasts localized in the connective tissue . This enA. viridis increases significantly in tentacle extracts after amputation in all three time points considered, while the enzyme amount remains at a constant level in the body extract. Here, we showed that ALP in Algae convert dissolved organic phosphorus (DOP) in the environment into dissolved inorganic phosphorus (DIP), which is used by animal organisms for their metabolic activities. The symbiotic zooxanthellae, abundant in the tentacles, play a crucial role in the absorption of phosphate dissolved in water ,38,39,40Wound healing, occurring immediately after an injury and involving cell contraction and re-adhesion, is the phase that precedes the regeneration of the lost structures. During both of these phases, the immune response facilitates protection against microbes, maintaining homeostasis and preventing infections that might compromise fitness ,42. AlonMatrix metalloproteinases (MMPs) are a family of endopeptidases collectively able to degrade the components of the extracellular matrix. They play important roles in propeptides and collagen fibril formation and normal tissue remodeling, embryogenesis, angiogenesis, and wound healing ,44.In cnidarian, several typical components of vertebrate ECM, such as fibronectin, laminin and different types of collagen are involved in cell adhesion ,48,49,50Here we found a significant increase of protease activity (both gelatinolytic and fibrinolytic) in the regenerating tissue after wound clousure from 1 to 14 dpa.Our results suggested that these metalloproteases could play an essential role in the degradation and remodeling of mesoglea connective tissue during tissue regrowth. These findings are consistent with the results obtained by tissue biometric analysis performed on sections of new formed tentacles 7 days after injury and show that during regeneration the thickness of the mesoglea did not increase, as it did for the epidermis and gastrodermis. However, the consistency of the mesoglea changed visibly and shifted from a loose matrix to a compact one. The change in stiffness was probably due to the ECM remodeling mediated by proteases, secreted by immune cells during wound healing and tissue regeneration phases . The clo+ hyaline amebocytes, acting as fibroblast-like cells. These cells, interacting with the neosynthesized collagen fibrils, appear to be involved in the formation and spatial organization of a compact collagen scaffold.Our morphological analyses under an optical and electron microscope, and Masson’s trichrome-specific colorimetric assays for ECM, seem to support this hypothesis, showing for the first time that during the tentacle regenerative process, mesoglea shift from a lax network to a lattice organized in bundles of collagen fibrils. Furthermore, immunolocalization assays, using polyclonal antibodies directed against the pro-collagen1α1 and bFGFR antigens, indicate that the visco-elastic behavior of mesoglea is obtained by the neo-synthesis of type I collagen from bFGFRA. viridis, the mesoglea ECM provides not only a mechanical support to maintain the shape and integrity of tissues, but also plays a fundamental role in tissue morphogenesis, cell migration and differentiation during tentacle regeneration [N. vectensis, after tentacle amputation, the change of mesoglea stiffness, obtained through metalloprotease activity, could initially play the role of a barrier against the entry of pathogens into the lesion site. Subsequently, the production of new collagen ECM by fibroblast-like cells, differentiated from amebocytes, could act as a scaffold to guide the correct regrowth of the tentacle. In conclusion, basing on the obtained evidence, we suggest that in neration . Indeed,A. viridis, the specimens have been collected between October and November, when biometric variations are less influenced by thermal stress [Considering previous studies on the seasonal changes in morphology of l stress . Adult oEach animal was amputated to the level of 25% of the total tentacles, to be then placed and fed in the tank. After 1, 7 and 14 days after the first amputation, three replicates of animals, respectively, were used for morphological, biometric, biochemical and histological analyses. Not amputated specimens were used as control.Chemicals were from Sigma-Aldrich (USA).For each experimental time, protein extraction was performed separately for body and tentacles cut in the basal zone, near the oral disk using the protocol. Tissues were homogenized in polycarbonate tubes with 10 mL TBS buffer  and centrifuged at 12,000 rpm for 30 min at 4 °C . Supernatant was collected and stored in Eppendorf tubes at −20 °C. The protein content was measured by Bradford method (1976)  using bo2 (pH 7.8). The enzymatic kinetic was evaluated [Samples were incubated in an equal volume of 4 mM p-nitrophenyl phosphate liquid in 100 mM ammonium bicarbonate containing 1 mM MgClvaluated  at regul2+  adjusted with 2% Triton X-100. Gels were left to stir overnight in TBS Ca2+ (pH 8.0) at room temperature, immersed in 50% methanol, stained in Coomassie Blue  for an hour and finally decolored . The reaction was stopped with distilled water and densitometric analysis was carried out using the open-source software Image J (http://rsbweb.nih.gov/ij/download.html) (accessed on 20 May 2021).The polyacrylamide electrophoretic run of tentacle and body extracts was performed under non-reducing conditions (7.5%) using gelatin (2 mg/mL) and fibrinogen (0.5 mg/mL) as substrates. Migration took place at 160 V for about 1 h. Gels were washed in TBS CaAnimals were placed in a plate and biometric parameters were measured  using a millimeter gauge, to select specimens of comparable size. Pictures of the part in regeneration were taken using a stereoscopic microscope . The thickness of the different tentacle layers and of the lumen area were calculated in micrometers and then the percentage increase or decrease through mathematical transformation compared to a tentacle of a control animal was also calculated. The biometric evaluation system for tentacles in regeneration was carried out using Image J software. The budding area (cutting site with regrowth tissues) of three organisms subjected to the cut of 25% of the total number of tentacles at three times point  was fixed in Bouin’s solution until complete sinking. Subsequently, samples were dehydrated in an increasing scale of ethanol  and paraffin-embedded. Sections (7 μm in thickness) have been cut in series with a rotary microtome  and processed for the Masson’s and Gomori’s and Trichrome stains.4), 0.2 g hematoxylin, 0.04 g KIO3, 40 mL glycerol) for 10 min at room temperature, washed in distilled water for 15 min, and been left in Gomori’s trichrome solution  and finally washed in acidified water (0.2% acetic acid).Sections were stained with Carazzi’s emalume  and specimens were treated as suggested by the datasheet, in order to highlight the presence of both collagen and the reticular fibers with aniline blue, while the cell cytoplasm was in red. Images were recorded with a Nikon Digital Sight DS-SM optical Microscope . A. viridis tissues, dissected from the area in regeneration, were fixed in 0.1 M cacodylate buffer (pH 7.4) containing 4% glutaraldehyde for 2 h. Specimens were then washed in the same buffer and post-fixed for 1 h with 1% osmium tetroxide in cacodylate buffer (pH 7.4). After standard serial ethanol dehydration, specimens were embedded in an Epon-Araldite 812 mixture. Sections were obtained with a Reichert Ultracut S ultratome . Data were recorded with a DS-5 M-L1 digital camera system (Nikon). Ultrathin sections (0.7 μm in thickness) were placed on copper grids (300 mesh), stained by uranyl acetate and lead citrate and observed with a Jeol 1010 EX transmission electron microscope . 2HPO4, pH 7.4), specimens were incubated with and goat anti-rabbit Cy3-conjugated secondary antibody , diluted 1:200, for 45 min. Cell nuclei were counterstained with DAPI  for 2 min and slides were mounted with Cityfluor . Negative control experiments were performed omitting primary antibodies.After etching with 3% NaOH in 100% ethanol , ultrathUltrathin sections were obtained as above and collected on gold grids (300 mesh). After etching with NaOH 3% and methanol, specimens were pre-incubated for 30 min in BSA blocking solution and then for 1 h with the anti-bFGF receptor Flg primary antibody, diluted 1:50. After two washings with PBS, the goat anti-rabbit IgG (H + L)-gold conjugate secondary antibody , diluted at 1:40, was used for 30 min. In control experiments, the primary antibody was omitted, and sections were incubated with the secondary antibody alone. After washings with PBS, samples were treated for 5 min with 0.5% glutaraldehyde in PBS solution, counterstained with uranyl acetate for 8 min and observed at TEM.p  <  0.05, ** p  <  0.01 and *** p  <  0.001. The analyses were carried out using the GraphPad Prism Version 8.0.0. for Windows (www.graphpad.com) (accessed on 20 May 2021). The mesoglea area and the number of elongated amebocytes were evaluated both in intact and injured tentacles by analyzing five different slides, using the ImageJ software. The software GraphPad Prism 7  was used to perform statistical analyses and differences between treatments were calculated by one-way ANOVA, followed by Fisher’s post-hoc test. Values of p < 0.05 were considered statistically significant.All the experiments were performed in triplicate and the values used were the mean of the three assays ± SEM. To assess multiple comparisons, a parametric two-way analysis of variance (ANOVA) was performed on the data, with a post hoc Tukey test. Differences between means were considered significant for *


All the parameters of the ADME study revealed that geraniol is an acceptable drug candidate. A docking study showed that the binding energy of geraniol  was lower than that of acetylcholine  with AChE, which exhibited around a 12.58-fold higher binding affinity of geraniol. Furthermore, molecular dynamics simulation revealed that the RMSD of AChE alone or in complex with geraniol fluctuated within acceptable limits throughout the simulation. The mean RMSF value of the complex ensures that the overall conformation of the protein remains conserved. The average values of Rg, MolSA, SASA, and PSA of the complex were 3.16 Å, 204.78, 9.13, and 51.58 Å2, respectively. We found that the total SSE of AChE in the complex was 38.84% (α-helix: 26.57% and β-sheets: 12.27%) and remained consistent throughout the simulation. These findings suggest that geraniol remained inside the binding cavity of AChE in a stable conformation. Further in vivo investigation is required to fully characterize the pharmacokinetic properties, optimization of dose administration, and efficacy of this plant-based natural compound.Acetylcholinesterase (AChE) inhibition is a key element in enhancing cholinergic transmission and subsequently relieving major symptoms of several neurological and neuromuscular disorders. Here, the inhibitory potential of geraniol and its mechanism of inhibition against AChE were elucidated in vitro and validated via an in silico study. Our in vitro enzyme inhibition kinetics results show that at increasing concentrations of geraniol and substrate, Vmax did not change significantly, but Km increased, which indicates that geraniol is a competitive inhibitor against AChE with an IC The cholinergic network affects various cellular functions through neurotransmission via acetylcholine (ACh), a neurotransmitter released from presynaptic neurons into the synaptic cleft, which binds to acetylcholine receptors (AChR) on the post-synaptic membrane, relaying the signal from the nerve or hydrolyzed by acetylcholinesterase (AChE) . A signiMoreover, cholinesterase enzymes such as AChE further decreased the concentration of ACh by hydrolyzing it . TherefoThe approved medications (cholinesterase inhibitors) for neurodegenerative disorders have several side effects such as nausea, vomiting, loss of appetite, headache, constipation, confusion, and dizziness ,12. SubsMost natural products are promising for the effective treatment of various ailments, and our team previously reported their antioxidant, anti-inflammatory, antidiabetic, hypolipidemic, and neuroprotective properties by inhibiting key regulatory enzyme activity ,20,21,2210H18O and is well known for its antioxidative , where “V” is denoted as the velocity (change in absorbance), which represents the enzyme activity, and “[S]” is denoted for substate concentration A. This lxon plot B and obtllalobos , who fouanalysis .Y-axis (Y-intercept = 1/Vmax), which showed the varying Vmax, but intersected at the same locations on the X-axis (X-intercept = −1/Km) of the plot, showing similar Km (2 = 0.22) with an increase in the concentrations of tacrine, and Vmax decreased significantly (R2 = 0.81), which indicated that tacrine is a non-competitive inhibitor of AChE and binds to the non-catalytic site of the enzyme. Our results are in agreement with a previous report where tacrine  was reported as a reversible non-competitive inhibitor against AChE enzyme [Moreover, we also investigated the AChE enzyme inhibition kinetics of tacrine through a Lineweaver–Burk plot and found that all the trendlines of different concentrations of tacrine at various concentrations of the substrate intersected at different locations on the milar Km A. We furmilar Km B and obtE enzyme ,59, whicE enzyme  and contE enzyme , who rep2) than tacrine (38.91 Å2) and that both can easily cross the BBB.Physicochemical properties, drug-likeness, and pharmacokinetics of geraniol and tacrine were evaluated using the SwissADME tool . Both liIn the present study, based on our in vitro results, it is well established that tacrine behaves as a non-competitive inhibitor and that geraniol showed competitive inhibition against AChE and binds to the catalytic site residues, which are mainly used for binding of the substrate (acetylcholine). Therefore, an analysis of molecular docking between AChE–geraniol and AChE–acetylcholine was performed, which revealed that both ligands were bound to the central active site cavity of AChE . AChE inThe binding pose of geraniol at the active site of AChE was further compared with the binding mode of a control ligand, that is acetylcholine . It was −1 and 1.02 × 103 M−1, respectively, for the AChE–acetylcholine interaction,—5.6 kcal mol−1 and 1.28 × 104 M−1, respectively, for the AChE–geraniol interaction at CAS cavity, and −6.8 kcal mol−1 and 9.72 × 104 M−1, respectively, for the AChE–geraniol interaction at the PAS cavity. We found that the binding affinity of geraniol for AChE was around 12.58-fold higher than that of the control ligand, acetylcholine, in the CAS cavity.Interestingly, the amino acid residues of AChE commonly engaged in the interaction with geraniol as well as acetylcholine in the CAS cavity including Trp84, Gly118, Glu199, Ser200, Phe330, His440, Gly441, and Tyr442, which show that geraniol interacted with all the specific residues of acetylcholine, except Tyr130 and Ile439 and that geraniol interacted with four more residues: Tyr121, Phe290, Phe331, and Ile444. Our molecular docking results confirmed that geraniol is a competitive inhibitor of the AChE enzyme. Moreover, the docking energy and the corresponding binding affinity were estimated to be –4.1 kcal mol−1, indicating that most of the terpenoids have high affinity for catalytic site residues, such as Asp74, Trp86, Gly120, Gly121, Ser125, Ser203, Phe295, Phe297, Tyr337, Phe338, His447, and Gly448. However, the report did not explain the residues that specifically interacted with geraniol. Moreover, we also analyzed the binding of non-hydrolysable structural analogues  in the active site of AChE at the catalytic site and at the peripheral anionic site, as previously reported by Colletier et al. [Our results are in agreement with a previous report , where gr et al. , and theThe dynamic nature of the interaction and the stability of the AChE and geraniol complex was assessed by molecular dynamics simulation under physiological conditions. The initial frame of the AChE–geraniol complex was subjected to molecular dynamics for 100 ns, and the results are presented in N- and C-terminal zones. The mean RMSF value of AChE–geraniol complex was determined to be 1.28 Å, which is much lower than the acceptable limit. It is evident that the RMSF of AChE did not deviate significantly in the presence of geraniol, assuring that the overall conformation of the protein remained conserved.The root-mean-square fluctuation (RMSF) of a protein provides insight into the local conformational changes in the side chains of a protein during simulation. 2, respectively (The dependency of radius of gyration (Rg) and surface area, such as molecular surface area (MolSA), solvent accessible surface area (SASA), and polar surface area (PSA) of a ligand on simulation time, provide information about the behavior of the ligand inside binding pocket of the enzyme. The Rg may also indicate whether the complex remains folded during MD simulation. The variation in Rg of geraniol bound to AChE as a function of simulation time is presented in ectively D. These The formation of a stable protein and ligand complex was established by determining the total number of contacts formed between them during the simulation A. It is The interaction between a ligand and protein often leads to changes in the protein’s secondary structural elements (SSE). Thus, a check on the variation in SSE during simulation is critical to provide an overview the establishment of a stable complex between geraniol and AChE. The variation in the total SSE (α-helix + β-sheet) of AChE bound with geraniol during simulation is presented in An analysis of the interaction between AChE and geraniol during the molecular dynamics simulation is presented in 50: 98.06 ± 3.9 µM) in the catalytic active site. Geraniol also exhibited the finest drug-likeness, pharmacokinetics, and physiological properties that can cross the BBB, as well as high absorption through the GI tract, suggesting that it can be a potential drug candidate for neurological disorders. The in vitro results were validated through in silico study and found that geraniol bound to most of the residues of the catalytic active site with a 12.40-fold higher binding affinity and lower binding energy  than the substrate binding energy . Moreover, molecular dynamics simulation study concluded that complex (AChE–geraniol) RMSD fluctuated within acceptable limits, and the overall conformation of the protein remained conserved and remained consistent throughout the simulation. These findings suggest that geraniol remained inside the binding cavity of AChE in a stable conformation. Further in vivo investigation is required to fully characterize the pharmacokinetic properties, optimization of dose administration, and efficacy of this plant-based natural compound.Neuromuscular and neurodegenerative disorders are generally treated by inhibiting acetylcholinesterase (AChE) enzyme to increase the level of acetylcholine in synapses. Through our in silico and in vitro findings, it has been concluded that geraniol inhibits AChE in a concentration-dependent manner and that it bound more effectively (IC


In horses, there is an increasing interest in developing long-lasting drug formulations, with biopolymers as viable carrier alternatives in addition to their use as scaffolds, suture threads, screws, pins, and plates for orthopedic surgeries. This communication focuses on the prolonged biocompatibility and biodegradation of PLA, prepared by hot pressing at 180 °C. Six samples were implanted subcutaneously on the lateral surface of the neck of one horse. The polymers remained implanted for 24 to 57 weeks. Physical examination, plasma fibrinogen, and the mechanical nociceptive threshold (MNT) were performed. After 24, 28, 34, 38, and 57 weeks, the materials were removed for histochemical analysis using hematoxylin-eosin and scanning electron microscopy (SEM). There were no essential clinical changes. MNT decreased after the implantation procedure, returning to normal after 48 h. A foreign body response was observed by histopathologic evaluation up to 38 weeks. At 57 weeks, no polymer or fibrotic capsules were identified. SEM showed surface roughness suggesting a biodegradation process, with an increase in the median pore diameter. As in the histopathological evaluation, it was not possible to detect the polymer 57 weeks after implantation. PLA showed biocompatible degradation and these findings may contribute to future research in the biomedical area. Biodegradable materials have been widely studied for medical applications during the last decades due to their numerous benefits over non-biodegradable materials. Importantly, since these materials disappear after degradation, the implants do not need to be removed. Furthermore, implants prepared using biodegradable polymers can prevent recurrences in the event of fractures, as they can be designed to degrade at a rate that will slowly transfer the load to the healing bone, thus allowing for adequate healing ,2. HowevPoly(lactic acid) (PLA) is one of the most widely used biopolymers and has the potential to be used as an alternative to high-cost, non-biodegradable biocompatible materials. PLA holds promising applications in several areas, particularly in packaging, agricultural products, disposable materials, and the biomedical industry ,5. AmongTherefore, the medical application of PLA in horses can be an exciting opportunity for the experimental development of new biomaterials, as this species undergoes a fast and excessive healing process, which tends to follow abnormal repair reactions such as the formation of exuberant granulation tissue ,11. MoreIn the equine species, therapeutic polymer implants can mimic tissues, promote cell proliferation, and tissue reconstitution, forming a conjunctive capsule. Moreover, they can be combined with drugs for systemic or local action. However, polymer implants must be biocompatible and bioabsorbable ,8. ThereOur research group recently evaluated the biocompatibility and biodegradability of PLA and a polymer blend based on PLA and poly(ε-caprolactone) (PCL) compatibilized with a copolymer derived from ε-caprolactone and tetrahydrofuran, which was implanted subcutaneously in horses. However, the materials were only implanted for 24 weeks . Herein,Clinical evaluation was carried out during the experiment to verify the health evolution of the horse and/or the possible presence of systemic changes induced by the implantation of the biomaterials. In general, there was no discomfort associated with the implantation of the materials, and no behavioral and/or appetite changes were observed. Intestinal motility, hydration status, apparent mucous color, and capillary filling time were within the normal range for the species. RR and RT values were within reference values for adult horses throughout the implantation period , Figur. HR valuPF concentration was determined to check for systemic inflammatory processes due to the implantation of the biopolymers. PF values remained within the range of reference for the equine species  throughF = 9.431; p ≤ 0.001) after implantation. Forty-eight hours after implantation MNT was reversed  (≤ 0.001) .Furthermore, over time, the polymer underwent degradation and changes in its morphology up to 57 weeks. Afterward it was no longer possible to detect the polymer within the skin fragment, thus impeding the removal of polymer fragments for analysis without the skin . We mustWe previously studied the changes related to the safe biodegradation of the PLA polymer implanted subcutaneously in horses for 24 weeks . Here weWe found that PLA implantation did not induce systemic inflammatory responses in the horse studied. HR values increased up to 144 h after implantation. This may have occurred because there was more frequent handling of the animal in this evaluation period, which probably triggered a mild anticipatory sympathetic response to general management. Indeed, HR decreased from the seventh day of implantation, remaining within normal values for equine species. Likewise, the concentrations of plasma fibrinogen, an acute-phase protein commonly used to diagnose and monitor various inflammatory conditions in equine medicine , revealeOn the other hand, MNT evaluation revealed the presence of a local inflammatory response, characterized by a decrease in nociception. Von Frey Filaments, used to assess the skin MNT, measures cutaneous hyperalgesia and allodynia, and usefully mimics clinical conditions that present increased cutaneous sensitivity . In a stHistopathological analysis revealed the formation of a fibrotic capsule delimiting the biomaterial from the initial stage of the inflammatory response, remaining present until 38 weeks post-implantation. Encapsulation of biomaterials occurs due to a chronic foreign body reaction . In compInflammation around the material, characterized by the presence mainly of lymphocytes and macrophages, accompanied the cell growth of the fibrous tissue amidst the biopolymers, which represented the local chronic inflammation and organization of this tissue. The presence of macrophages in the capsule and at the material’s interface was observed in other studies, being related to phagocytosis and implant clearance ,30. InteThe proliferation of fibrous tissue around polymer fragments may be associated with the presence of pores and cracks in the materials, as observed by SEM. These pores promote cell growth as they provide a greater surface area . Fifty-sIn addition to macrophages and multinucleated giant cells, lymphocytes were often present in the infiltrates around the implants. These findings are probably associated with a chronic inflammatory response at the injury site, which is usually characterized by the presence of macrophages, multinucleated giant cells, monocytes, and lymphocytes, in addition to fibrosis and angioplasia . FurtherSEM data, obtained from the sample removed 24 weeks following implantation, revealed signs of biodegradation of the PLA. However, few pores were present with an median diameter of 0.477 μm. As post-implantation time progressed, we observed a gradual increase in the number of surface pores and a large variation in pore size. In vivo biodegradation of PLA depends on the intrinsic characteristics of the material, such as the D-PLA content, crystallinity, and molar mass and the recipient’s conditions such as temperature, pH, and presence of cellular infiltrates . The PLA−1). The main in vivo biodegradation mechanism of PLA is the hydrolysis of ester groups, which leads to a slow reduction in chain size by chain scission to small oligomers or unitary lactic acid molecules. Once reduced to small lactic acid oligomers, they can be phagocytosed and take part in cellular metabolism [Another important factor is the size of the PLA chains since only small molecules can enter cells and be used for cell metabolism. The average molar mass of the PLA used in this study is high  under protocol n° 006548/17.−1, 1.4% of D-PLA content, 48 MPa tensile strength, 2.5% strain at break, and 16 J.m−1 Izod impact resistance. Polymer implants (1 cm2 and 1 mm thick) were produced via hot pressing at 180 °C. Sterilization was performed with ethylene oxide .The polymer was prepared at the Department of Materials Engineering at the São Carlos School of Engineering, University of São Paulo, Brazil, according to Dias e Chinelatto (2019) . The matWe used one adult horse, gelding, crossbreed, weighing 350 kg, 15 years old, from the didactic herd of the Laboratory of Equine Exercise Physiology and Pharmacology (LAFEQ/DMFA/FCAV), UNESP. The animal was kept in a paddock, fed with corn silage, water, and mineral salt ad libitum, in addition to 0.2% of body weight of a mash feed once a day. Before study commencement, the horse underwent a complete physical examination, in addition to hematological and biochemical tests, to determine its health status. The horse was previously treated with anthelmintic (repeated every four months) and vaccinated against rabies, tetanus toxoid, east and west equine encephalomyelitis, and equine influenza types A1 and A2.An overview of the implantation sites is depicted in −1). Afterward, local anesthetic infiltration was performed around each incision site with 2.0 mL of 2% lidocaine hydrochloride. A 2 cm horizontal incision was made using a #15 scalpel blade in the determined area on the LS. A space between the skin and cutaneous muscle was obtained by blunt divulsion and the polymer implanted. It is important to highlight that the divulsion was performed ventrally to the incision so that the skin suture did not interfere in the evaluations. Then, skin suture was performed in a simple interrupted pattern with nylon 0. Postoperative procedures consisted of cleaning the area using gauze and 0.9% saline solution and fly repellent ointment around the surgical wound once a day for 10 days. No analgesic and/or anti-inflammatory medication was provided during the experimental period. The stitches were removed on the 7th postoperative day. The same surgeon performed all surgical procedures.Before implanting the biomaterial, six areas of approximately 8 cm2 on the LS (three on the left and three on the right) were shaved, and antisepsis was performed with 2% chlorhexidine digluconate and 70% ethyl alcohol. Subsequently, the animal was sedated with intravenous administration of xylazine hydrochloride , respiratory rate (RR), rectal temperature (RT) as well as the intestinal motility, hydration status, apparent mucous color, and capillary filling time. Physical examination was performed before the commencement of the study and 6, 12, 24, 48, 72, 96, 120, 144, 168 h after implantation and then weekly up to 8 weeks, fortnightly up to 38 weeks, and 57 weeks. The same evaluator obtained all the measurements.−1). PF was determined before the commencement of the study and 6, 12, 24, 48, 72, 96, 120, 144, 168 h after implantation and again at 2 and 3 weeks.To determine PF concentration, the chronometric technique described by Clauss (1957)  was usedA commercial set of Von Frey Filaments (VFF) were used to assess the skin MNT . Six filaments of sizes 5.07 to 6.65 were used, which represent applied forces ranging from 11.8 to 446.7 g, respectively. The filaments were applied perpendicularly to the horse’s skin until the nylon thread started to bend. Four applications, separated approximately 1 cm from each other, were performed around the implantation site, at intervals of 3 s. Initially, the thinnest filament was used and, when no aversive response was observed, the next filament was used until the animal showed an aversive reaction or the largest filament was used. An aversive reaction was defined as a movement of the tail, ears, or head, kicking, or stepping to the side. Simple motion reflexes upon first touching the filament on the skin were not accepted as an aversion response, and in these cases, the test was repeated after 10 s. Evaluations were performed before the commencement of the study and 6, 12, 24, 48, 72, 96, 120, 144, 168 h after implantation, then weekly until 8 weeks, and fortnightly until 38 weeks. The same operator performed all measurements, and these were made with the horse in a quadrupedal position in an area without movement restrictions. The values obtained were converted into force (g) according to the table provided by the manufacturer.The implants were removed along with surrounding tissue fragments. Two implants were removed after 24 weeks of implantation (PLA24 and PLA24F), one implant was removed at 28 weeks (PLA28), one implant at 34 weeks (PLA34), one at 38 weeks (PLA38), and the final implant was removed at 57 weeks (PLA57). As in the implantation procedure, the animal was properly sedated, and local infiltrative anesthesia was performed before the surgical removal of the fragment. One of the fragments removed after 24 weeks of implantation (PLA24F) was fixed in 10% buffered formalin for 24 h. The other implants and surrounding tissue fragments were fixed in 3.5% glutaraldehyde solution with PBS (pH 7.2) for 24–48 h and subsequently subjected to three washes using 5% glucose in 0.1 m sodium cacodylate buffer solution for 5 min. As we did not know whether fixation in glutaraldehyde would interfere with the histological evaluation and because this horse was used in the pilot study by Carvalho et al., 2020 , at 24 wFor histopathologic evaluation, tissue samples were processed for paraffin embedding, and 5 µm sections were obtained and stained with hematoxylin and eosin. Tissue samples were evaluated in a light optic microscope by a pathologist with no knowledge about the implant’s removal time, thus eliminating bias. Lesions were classified semiquantitatively using the scores described by De Jong et al. (2005) , with soMorphological characterization of the materials was performed by SEM. Implant samples with surrounding tissue fragments underwent a dehydration process in an increasing alcohol solution and dried at a critical point. Samples that had the polymer removed from the obtained fragments were dried in an oven at 35 °C for 12 h. All samples were mounted on supports and sputtered with gold in a vacuum. The material surfaces were analyzed in a scanning electron microscope  operated at 10–15 kV. Images were examined with Fiji ImageJ 1.50i software, and the average pore diameter was determined manually from at least 300 pores. For non-circular pores, the greatest distance was considered.n = 6) for the same animal. MNT was subjected to analysis of variance (ANOVA) for repeated measurements in unilateral time, followed by Tukey’s post-hoc test. Pore diameter evaluated by SEM images were submitted to the Kruskal–Wallis H-test for one-way analysis of variance (ANOVA), followed by the Dunn’s post-hoc test. All statistical analyzes were performed using Sigma-Plot software, version 12.0, with a significance level of p < 0.05.All variables were checked for normal distribution using the Shapiro-Wilk test. To evaluate the MNT, the six implants were considered as repetitions (Over time the polymer changed its morphology, indicating biocompatible degradation. The kinetics of PLA biodegradation revealed herein reinforce PLA’s potential for clinical use and may contribute to future research efforts to enable its use in the biomedical industry, including equine medicine.


Systemic lupus erythematosus (SLE) is an autoantibody-related disease that affects multiple organs. Stercoral colitis (SC) is a rare type of inflammatory colitis with a high mortality rate. Here, we report the first case of pediatric-onset lupus in a case complicated by stercoral colitis. We also conducted a literature review of patients with SC under 30 years old to provide useful clues for rapid diagnosis at a young age. A 28-year-old female with a history of lupus and neuropsychiatric SLE was admitted with severe abdominal pain. She was found to have stercoral colitis during surgery. Two years later, the patient underwent Hartman's operation due to ischemia of the colon. In addition, 10 patients younger than 30 years old with a diagnosis of SC were analyzed based on clinical presentation, physical examination, laboratory exam, imaging and treatment. All cases had a favorable outcome without mortality. Stercoral colitis is a rare but lethal complication, emphasizing the importance of a multidisciplinary approach. Differential diagnosis should include stercoral colitis for patients with SLE developing unexplained sharp abdominal pain. Systemic lupus erythematosus (SLE) is an autoantibody-related disease that affects multiple organs. In addition, some SLE patients have gastrointestinal presentations or complications, including 6% of those with chronic constipation . Such inStercoral colitis is a rare type of inflammatory colitis with a high mortality rate ranging from 32 to 57%; it occurs mainly in elderly individuals with a history of chronic constipation, and it presents in a moribund state .To our knowledge, this is the first reported pediatric-onset lupus case complicated by stercoral colitis. The importance of the possibility of the occurrence of this disease in patients with SLE should be highlighted, even in young adults and especially in those with early childhood-onset disease. Moreover, due to the high mortality rate of stercoral colitis, awareness is promptly required for immediate diagnosis and treatment.2 intravenous cyclophosphamide pulse therapy. Furthermore, she experienced a brain abscess at the age of 25 years. After the above presentations, she regained clear consciousness without cognitive dysfunction or neurologic deficits. Daily medication was maintained with 5 mg prednisolone  and 100 mg azathioprine daily, with regular follow-up at our pediatric rheumatology clinic.A 28-year-old female was diagnosed with pediatric-onset lupus at the age of 8, with initial presentations of fever, malar rash, arthritis, positive antinuclear antibodies (ANAs), and elevated anti-dsDNA antibodies. In addition, she had episodes of neuropsychiatric SLE (NPSLE) at the ages of 19, 22, and 24, with seizures, cranial neuropathies of the facial nerve and transverse myelitis with neurogenic bladder, respectively, and treated with one dose of 0.5 g/mLater, she presented to our emergency department with severe abdominal pain and fever for 1 day. She had a history of chronic constipation and urinary tract infection for 2 weeks before admission, and her last bowel movement was 3 days prior. On arrival, she showed tachycardia (123 beats per min) and a body temperature of 38°C but no hypertension or hypotension 121/70 mmHg). Physical examination revealed a distended abdomen, severe tenderness in the lower abdomen, positive peritoneal signs, rebounding pain and decreased bowel sounds; rectal examination was not performed. Blood laboratory tests showed elevated inflammation markers, a C-reactive protein (CRP) level of 77.35 mg/L, a white blood cell count of 7,900/μL, and normal platelet count, serum creatinine, electrolyte and liver function test results. There were no obvious symptoms of lupus attacks, such as discoid rash, oral ulcer or arthralgia. SLE activity remained stable. C3 and C4 levels were 63.4 and 19.1 mg/dL, respectively, and her anti-dsDNA antibody level was 382.0 U/mL, lower than those over the previous 6 months. Disease activity, as quantified by the SLEDAI-2K score . Informed consent was obtained from the patient. We report this case according to CARE (for Case Reports) guidelines ,” “stercoral colitis,” and “stercoral perforation” to search for studies in PubMed published between 1965 and August 2020 in English. Relevant articles and additional references were found by checking the citations in the articles retrieved.To date, fewer than 200 cases of stercoral colitis or stercoral perforation have been reported, and only 10 patients [3 males –11 and 7Stercoral perforation is a rare but fatal complication of constipation and fecal impaction. Before our patient, fewer than 200 cases have been reported since the first report in 1894 . Maurer By exploring the published English literature (PubMed search from 1965 through August 2020), we found only two cases of stercoral colitis in patients with SLE , 23. In Various causes of abdominal pain in lupus may indicate misdiagnosis, such as lupus enteritis, primary peritonitis and chronic lupus peritonitis , 28. LupAccording to Maurer et al.'s definition , there aEarly diagnosis of stercoral colitis remains a dilemma. According to a previous review, not all cases involve fever, peritoneal signs, elevation of inflammatory markers, metabolic acidosis, or electrolyte imbalance , 29. In Although previous experts have mentioned that pain during an upright abdominal X-ray examination can provide clues about stercoral colitis or perforation, including colonic swelling at the impaction site, calcified fecaloma, or free air, fecal matter may obscure these findings , 33. TheAccording to Wu et al.'s research, 52% of patients can be treated non-operatively through an intestinal regimen . HoweverIt is important to identify SLE patients with severe abdominal pain who may be at risk of stercoral colitis or perforation. Stercoral colitis is a rare yet lethal complication that can occur in patients as young as 28 years old, and differential diagnosis should highlight the importance of a multidisciplinary approach. In addition to appendicitis, peritonitis, or vasculitis, differential diagnosis should include stercoral colitis for patients with SLE who develop unexplained sharp abdominal pain.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee on Human Studies at Chang Gung Memorial Hospital in Taiwan, R.O.C. (IRB 201800989A3). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.C-CG and L-LL carried out the case analysis, participated in the sequence alignment, and drafted the manuscript. C-YW and J-LH participated in the design of the study and performed the statistical analysis. J-LH conceived of the study and participated in its design. All authors contributed to the article and approved the submitted version.This work was supported by the Research Fund of Chang Gung Memorial Hospital CMRPG3H1201-2, CMRPVVH0023, the National Science Council NMRPVVK6021-3 and the Ministry of Science and Technology .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The efficacy and safety of the combination of endocrine therapy (ET) and CDK4/6 inhibitors for patients with hormone receptor (HR)-positive HER2-negative metastatic breast cancer (BC) presenting with visceral crisis or life-threatening conditions represent a challenge for daily clinical practice. Indeed, the peculiarity of this clinical presentation (signs and symptoms of rapidly progressive disease) does not allow to include such patients in a trial aiming for drug approval. On the basis of the scientific evidence available so far, chemotherapy represents the standard of care according to guidelines, on the basis of the more rapid activity in comparison with ET alone. Besides, the combination of ET and CDK4/6 inhibitors have demonstrated in clinical trials to have clinically impactful activity in a short time, thus suggesting a potential role in advanced tumors that require rapid response. Herein, we report the clinical history of a young woman with HR-positive HER2-negative metastatic BC and a pancytopenia due to carcinomatosis of the bone marrow receiving letrozole and leuprorelin plus the CDK4/6 inhibitor palbociclib, who significantly derived clinical benefit from treatment. Considering that these peculiar cases are excluded from clinical trials, the estimation of the magnitude of the benefit of the newer ET combination may potentially represent a practical question for large case series and real-world studies. Breast cancer (BC) is the most common cancer in women  and the de novo metastatic or recurrence after neoadjuvant/adjuvant treatment), on the type of neoadjuvant/adjuvant ET, if any, and whether relapse occurs while under adjuvant ET or after the end of adjuvant ET. Patients are defined as endocrine sensitive if they have never been exposed to ET or relapsed more than 1 year after completion of adjuvant ET (de novo metastatic disease or presenting more than 12 months after completion of adjuvant aromatase inhibitor (AI), first-line ET standard of care includes AI plus a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor or fulvestrant plus a CDK4/6 inhibitor, while tamoxifen, AI alone, or fulvestrant alone are reserved for selected cases  includes endocrine treatment (ET) alone or in combination with a targeted therapy for patients without visceral crisis, defined as severe organ dysfunction, as assessed by signs and symptoms, laboratory studies, and rapid progression of disease . The chouvant ET . For pated cases . For pated cases . With AICDK4/6 inhibitors are the most recently introduced class of agents for the treatment of advanced LBC . The dysp = 0.00973) (p = 0.09)  . In the 0.00973) –24. Whil = 0.09) , in the  = 0.09) , 25. HowIn patients with advanced LBC, chemotherapy is indicated for the treatment of visceral crisis or significant functional organ impairment and when it is no longer possible to consider the endocrine-responsive disease , 26. ImpA 46-year-old premenopausal woman had a past medical history of 1-cm papillary thyroid cancer (pT1a) diagnosed at the age of 40 and treated with total thyroidectomy, uterine fibromatosis treated with hysterectomy and bilateral breast augmentation. She was diagnosed in August 2018 with metastatic bilateral infiltrating lobular carcinoma of the breast. In June 2018, she presented with general fatigue. Blood tests performed in August 2018 revealed severe anemia , which required red blood cell transfusions, and a reduction in white blood cell and platelet counts . Other laboratory studies showed elevated alkaline phosphatase. A bone marrow biopsy was performed; pathology revealed that poorly differentiated lobular carcinoma with strong immunohistochemical (IHC) expression of ER (80%) had infiltrated the bone marrow, with reduced numbers of the three hemopoietic cell lines . An 18-fluorodeoxyglucose-positron emission tomography computed tomography (FDG-PET/CT) showed uptake with activation of the bone marrow in all bone segments, a nuanced uptake in bilateral breast and a pelvic ascitic effusion B. MammoThe patient had been suggested to start chemotherapy. However, she was concerned about alopecia and chemotherapy-related side effects in general. Thus, the patient had approached our center for a second opinion. We kept in mind the risks that chemotherapy carried due to the low blood cell count. On the other hand, we were not confident enough to start endocrine monotherapy. We considered the encouraging survival results, together with the not relevant hematologic toxicity, of the combination of ET with the CDK4/6 inhibitor palbociclib. We shared with the patient the possibility of undertaking enhanced ET, adding a new drug recently available in clinical practice, palbociclib, specifying that no data were available on the efficacy and safety of the proposed combination in similar clinical situations. Therefore, we agreed with the patient to start ET with a combination of an AI, letrozole, and a CDK4/6 inhibitor, palbociclib, switching to chemotherapy in case of worsened signs and symptoms of the metastatic disease. We also added ovarian suppression with a luteinizing hormone-releasing hormone agonist (LH-RHa) as the patient was premenopausal.In September 2018, she started palbociclib 125 mg orally daily 3 weeks on and 1 week off, letrozole 2.5 mg orally daily continuously and leuprorelin 3.75 mg intramuscular injections every 28 days. The patient tolerated treatment well, except for grade 1 fatigue, arthralgia, and hot flashes. After the first two weeks of therapy with palbociclib, the patient also had grade 4 neutropenia without fever, which resolved with discontinuation of palbociclib for 3 weeks. Then, the CDK4/6 inhibitor was restarted at the reduced dose of 100 mg daily, with subsequent persistent asymptomatic grade 2 neutropenia.After 4 months of treatment, her red blood cells and platelet count was restored to within the normal range  with grade 2 neutropenia being treated with palbociclib. Serum levels of Ca15.3 decreased to 18 U/ml. In February 2019, follow-up PET/CT scan showed the resolution of the hypermetabolic breast tumors and bone metastatic foci with the disappearance of the ascitic effusion    is currently being conducted. The objective of the trial is to demonstrate an improvement in time to treatment failure with endocrine combination therapy compared to chemotherapy, in order to provide real-world evidence that palbociclib plus ET is the better first-line choice not only compared to ET, but also compared to chemotherapy. Noteworthy, the combination of ET plus a CDK4/6 inhibitor is also tested in comparison with chemotherapy in the setting of significant visceral impairment. The RIGHT Choice study (NCT03839823) is a phase II randomized trial that is exploring ET plus ribociclib compared with chemotherapy in terms of PFS in non-pretreated premenopausal LBC patients with symptomatic visceral metastases, or rapid progression of disease or impending visceral compromise.According to international guidelines, the initial treatment of HR-positive, HER2-negative advanced BC involves the administration of ET, even in the presence of visceral disease, excluding those patients with life-threatening conditions or with visceral crisis as presentations of the disease , 26. Pal 0.0235) . A simil= 0.537) . OngoingStudies regarding the efficacy and safety of ET, with or without CDK4/6 inhibitors, in LBC with DCBM are extremely limited because patients with visceral crisis or life-threatening conditions, such as bone marrow infiltration, are excluded from clinical randomized trials. In relation to our clinical case, chemotherapy, due to its rapid time to response, represents an important therapeutic resource, even as a less toxic and better tolerated weekly administration, and it is generally recommended for the treatment of BC with DCBM , 29, 30.However, in the current case, due to its cytotoxic effect, chemotherapy may initially worsen hematological disorders due to pre-existing marrow infiltration by cancer cells. The hematological chemotherapy toxicity sometimes requires supportive care, such as transfusion of red blood cells and platelets and the administration of granulocyte growth factors. In contrast, ET, although less rapid than chemotherapy in obtaining a disease response, is better tolerated and has no hematological toxicity. Therefore, endocrine agents, from the point of view of toxicity, can be used safely in the treatment of BC with DCBM .There are few published reports in the literature on the efficacy of ET in the treatment of LBC with DCBM. The case of a 64-year-old woman who presented at the diagnosis with diffuse bone marrow metastasis from an occult HR-positive, HER2-negative BC was described; tamoxifen treatment achieved a sustained response, with normalization of hematological parameters . At our vs. 0%–1% of patients treated with ET alone  because we were aware that the hematological toxicity of CDK4/6 inhibitors is due to their cytostatic but non-cytotoxic effect on the blood marrow cells and therefore the hematological adverse events are generally low grade; while neutropenia is frequent, febrile neutropenia occurs only in 1%–1.8% among patients treated with CDK4/6 inhibitors ET alone , 36–39, p < 0.001)  , then co< 0.001) . Further< 0.001) . Given tIn conclusion, the combination of ET and CDK4/6 inhibitor may provide longer clinical benefit than chemotherapy in advanced LBC, with better response rates and time to response similar to chemotherapy. On the other hand, the combination of ET and a CDK4/6 inhibitor is less toxic and leads to better quality-of-life outcomes than chemotherapy. However, the population treated in daily clinical practice often differs from that included in randomized controlled trials, such as the patient in our clinical case. Indeed, there are no prospective data on the use of CDK4/6 inhibitors in patients with visceral crisis or rapidly developing life-threatening clinical manifestations. For this reason, it is difficult to conclude on any specific treatment recommendation in these patients. Therefore, the publication of case reports, case series, or real-word studies should be encouraged, because they help to clarify the efficacy and safety of innovative drugs, saving or delaying the use of chemotherapy, even in borderline clinical situations.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.Written informed consent was obtained from the relevant individual(s), and/or minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.GG and EB conceived the idea of the case report. GG drafted the manuscript, and LC and EB critically reviewed it. GG, AO, AP, and EB were involved in the patient’s clinical management. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Corynebacterium glutamicum is a promising host for surface display due to its natural advantages, while single screening methods and fewer anchor proteins restrict its application. In this study, the subcellular localization (SCL) predictor LocateP and tied-mixture hidden Markov models were used to analyze all five known endogenous anchor proteins of C. glutamicum and test the accuracy of the predictions. Using these two tools, the SCLs of all proteins encoded by the genome of C. glutamicum 13032 were predicted, and 14 potential anchor proteins were screened. Compared with the positive controls NCgl1221 and NCgl1337, three anchoring proteins—NCgl1307, NCgl2775, and NCgl0717—performed better. This study also discussed the applicability of the anchor protein screening method used in this experiment to other bacteria.The display of recombinant proteins on the surfaces of bacteria is a research topic with many possible biotechnology applications—among which, the choice of host cell and anchoring motif is the key for efficient display.  Corynebacterium glutamicum possesses many useful characteristics, and some successful platforms have been reported. Firstly, C. glutamicum exhibits low extracellular protease activity, which is conducive to protein display on its surface , mycoloylated proteins , and membrane protein   . These r, MscCG) , glucana, MscCG) , glucosi, MscCG) , cellula, MscCG)  etc., whC. glutamicum has been based mainly on partial screening rather than direct genome-wide screening of the characteristics of anchor proteins—for example, the process of screening Ncgl1337 as the anchor protein was to identify and analyze the mycolic acid layer protein by SignalP and tied-mixture hidden Markov models (TMHMM) and then display the reporter for further analysis and verification  and mCherry as reporters to verify the potential anchor proteins and then compared the performance of these two reporters. Then, through the display of α-amylase, the practicality of displaying three highly effective anchor proteins on the surface of C. glutamicum was examined. Finally, we discussed the general applicability of this screening method for use with Gram-positive bacteria.In this study, we reported a novel method for screening anchor proteins of Escherichia coli Top10 was used as cloning host for plasmid construction, and C. glutamicum ATCC 13032 was used for gene editing. E. coli was cultured in LB medium  at 37°C, and C. glutamicum ATCC 13032 was cultured in BHISG medium  at 30°C. For resistant strains, antibiotics were added as follows: final concentration of 50 μg L–1 kanamycin for E. coli and 25 μg L–1 kanamycin for C. glutamicum. In addition, the concentration of isopropyl-βd-thiogalactoside (IPTG) used to induce the expression of α-amylase was 0.5 mM.The reagents and primers used in this study are listed in C. glutamicum have been described, namely, NCgl1337, NCgl1221, PorB, PorC, and PorH. In this study, we downloaded the protein sequences of these known endogenous anchor proteins of C. glutamicum in FASTA format from the National Center for Biotechnology Information website1 and subjected them to LocateP and TMHMM for SCL prediction analysis and transmembrane structure prediction  genome in FASTA format2 and then subjected the protein sequence to LocateP and TMHMM for prediction and analysis. Then, based on the characteristics of the anchor proteins, the parameter standards for anchor protein screening were formulated  and Taq DNA polymerase . The Seamless Assembly Cloning Kit  was used to assemble the plasmids using the principle of homologous recombination. In all expression vectors, the ribosome binding site (AAAGGAGGCCCTTCAG) is added before the open reading frame of the fusion protein. The constructed plasmids were verified by DNA sequencing . All primers were synthesized by Shanghai Generay Biotech Co., Ltd. .C. glutamicum surface display system used pEC-XK99e as the carrier and EGFP, mCherry, and α-amylase as the reporters, respectively. The gene (amyE) of α-amylase (BSU_03040) was derived from B. subtilis 168. The N-terminal fusion or C-terminal fusion was determined according to the characteristics of the anchor proteins screened. Among these 25 anchor proteins, NCgl0550, NCgl0633, and NCgl2291 are C-terminal fusions, and the rest are N-terminal fusions . The N-terminal/C-terminal of the anchor protein has a FLAG  tag.The construction of the C. glutamicum ATCC13032 as a template and using 5am F and 5am R as primers, the Ncgl1307 gene sequence fragment 1 was amplified by PCR; using the EGFP gene as the template and using EGFP F-N site and EGFP R-N site as primers, the target protein EGFP gene fragment 2 was amplified; using pEC-XK99e as a template and pEC F and pEC R as primers, the gene sequence linearized vector of pEC-XK99e was amplified. Finally, these three fragments were assembled into a plasmid by Seamless Assembly Cloning Kit. For the expression vector where the passenger protein is mCherry, here is the construction of Ncgl1307-mCherry as an example  for 24 h, centrifuged at 6,000 rpm for 10 min, and then resuspended in 1 ml phosphate-buffered saline (PBS), and the optical density at 600 nm (OD600nm) was adjusted to 1. The cells were centrifuged at 6,000 rpm for 10 min and resuspended in 0.2 ml PBS containing 1% bovine serum albumin (BSA). Then, 1 μl of anti-FLAG monoclonal antibodies  was added, and the mixture was incubated at room temperature for 2 h, with gentle shaking to avoid precipitation. Subsequently, the cells were washed twice with PBS and resuspended with 0.2 ml PBS containing 1% BSA. For the strain whose reporter was EGFP, 1 μl of 2 μg/μl Alexa Fluor 647-labeled goat anti-mouse IgG antibody  was added, incubated at room temperature for 1 h, and then shaken continuously to suspend it. Finally, a BD Accuri™ C6 Plus Flow Cytometer  was used to measure the fluorescence intensity. A total of 50,000 cells per sample was collected. Subsequently, the sample was centrifuged and resuspended, then coated on a microscope slide, and finally observed through a confocal microscope . The image was acquired at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. For strains whose reporter was mCherry, an Alexa Fluor 488-labeled goat anti-mouse IgG antibody  was used. A confocal microscope was used at the excitation wavelength of 667 nm and the emission wavelength of 651 nm to acquire the images. For flow cytometry analysis, Comp-FL1-A:APC-A was used to detect AF647, and Comp-FL1-A:FITC-A was used to detect AF 488. The data of flow cytometry and confocal microscopy were analyzed and processed by GraphPad Prism 8 and LAS X software, respectively.The recombinant Corynebacterium glutamicum harboring NCgl1307-Amy or NCgl2775-Amy or NCgl0717-Amy was selected and induced by 0.5 mM IPTG. The induced cells grew at 30°C with continuous shaking for 24 h before the cells were harvested. The cell surface fraction was prepared as described by ® Ultra Amylase Assay Kit , and the specific steps used relate to the method used by The amylase activity is determined by using the EnzChek600nm of cell density was 0.1–0.3, and the strain was cultured at 30°C and 280 rpm in 12-well plates. The OD600 optical density was measured every 4 h for 32 h. During the cultivation process, final concentrations of 25 μg/ml kanamycin and 0.5 mM IPTG were added to the resistant strains of C. glutamicum. The data was analyzed by GraphPad Prism 8 software. Each incubation was conducted in triplicate, and the results are presented as mean ± standard error from at least three independent experiments.The strains picked from the plate were inoculated into BHISG and cultured overnight at 30°C and 280 rpm. CGXII medium  was usedC. glutamicum have been described, including NCgl1337, NCgl1221, PorB, PorC, and PorH. All except NCgl1221 are located in the mycolic acid layer. Of these five proteins, PorB, PorC, and PorH are porins, and they function as channels for transporting small molecules in the mycolic acid layer. NCgl1221 is a mechanosensitive channel and the main glutamate exporter of C. glutamicum, which is located on the cell membrane  sequence of the predicted proteins of C. glutamicum ATCC13032 were subjected in the FASTA format to LocateP and TMHMM. Then, based on the characteristics of these subcellular compartments and the requirements for screening anchor proteins in this experiment, we developed several screening criteria , secretory proteins are usually synthesized in the form of a precursor protein, carrying a split N-terminal signal peptide, which is used to locate the protein in a membrane-embedded export device . C. glutproteins . Fluores listeri  and E. c and Ncgl1337 (PC2–EGFP) were used as the two positive controls. In order to evaluate the fluorescence localization, we used FACS and confocal fluorescence microscopy based on the immunofluorescence reaction. In FACS analysis, the 14 cells harboring FLAG-tagged fusion-anchored proteins showed a significant drift compared to the negative control (cells harboring pEC-EGFP) (–EGFP and PC2–EGFP)  . In addiC2–EGFP) .C. glutamicum.In order to further confirm the localization of fluorescence, we observed these 25 recombinant strains with immunofluorescence under a confocal microscope. The results showed that these 14 recombinant strains mentioned above all showed the red fluorescent signal of the secondary antibody under a laser confocal microscope, including cells harboring NCgl1307, NCgl2775, and NCgl0717 , which pThe active fluorescent EGFP needs to be secreted through the Tat pathway, while the monomeric red fluorescent protein mCherry is functional regardless of the translocation pathway . Almost –mCherry) and Ncgl1337 (PC2–mCherry) were used as the two positive controls of anchor protein. In order to evaluate the fluorescence localization, we used FACS and confocal fluorescence microscopy. In FACS analysis, the protein-anchored cells showed a more significant drift than the negative control   . In addiC-XK99e) ; these rC-XK99e) , the peaC-XK99e) .B. subtilis 168 as the passenger protein. The expression of the anchor protein—Amy fusion proteins—in C. glutamicum was analyzed by SDS-PAGE and Western blot analysis of the cell wall fraction. The molecular size of the individual NCgl1307, NCgl2775, NCgl0717, and Amy proteins is approximately 38, 40  of y 38, 40 , 27, andy 38, 40 . HoweverNext, the amylase activity and cell growth of display platforms with NCgl1307-Amy, NCgl2775-Amy, and NCgl0717-Amy were analyzed . Compare600 value in the stable phase  used in this experiment includes signal peptide (1--27 AA) and propeptide (28--41 AA)3. The signal peptide and propeptide sequences will be cleaved off after guiding the transport of the amylase protein across the membrane. This should be the main reason why most amylases are present in the supernatant in this experiment. In subsequent experiments, we plan to remove the gene sequences of the signal peptide and propeptide and further explore the application of the 14 anchored proteins to the display of this α-amylase or other functional proteins.However, the protein sequence of the α-amylase gene  and protein functional region analysis, it was found that, among the three anchor proteins screened, NCgl2775 is a membrane protein and possesses comparable esterase and thioesterase activities in vitro , which mC. glutamicum genome. A total of 14 potential anchor proteins were screened. Among them, three had a higher rivet efficiency than the previously mined NCgl1221 and NCgl1337 and showed enzyme activities with amylase. These results confirmed the effectiveness of the screening method. At the same time, any protein that can rivet the protein of interest to the cell surface could be used as a potential anchor protein. Therefore, our screening method is likely to be applicable to other Gram-positive bacteria. Moreover, the operations during the screening by LocateP and TMHMM can be carried out according to specific parameters. LocateP has completed the prediction of SCLs of all of the proteins encoded by the Gram-positive bacterial genome (In this study, we used the subcellular localization and parameter prediction of LocateP and the transmembrane structure prediction and visualization of TMHMM to screen the anchor proteins in the protein sequence of the l genome . Those lOn the other hand, in the screening process of anchor proteins, the reporters EGFP and mCherry have their own characteristics. The different preferences in secretion pathway of these two reporters may have few effects on secretion and anchoring. Meanwhile, the secretion pathway of the signal peptide may play a major role in the secretion and transport of the anchor protein–reporter. It is worth mentioning that, no matter which fluorescent protein is selected, the ideal anchoring effect cannot be directly observed under the confocal laser microscope without immunofluorescence reactions because of the interference from intracellular fluorescence. In summary, functional proteins with FLAG tags against secondary antibodies AF488 might be a good choice in the screening process for anchor proteins. This combination can achieve better results in both FACS and confocal microscopy and directly verify the practicality of anchor proteins.The original contributions presented in the study are included in the article/KL and SZ designed and carried out the main studies. SH, NZ, YC, and YL have made a lot of contributions in the experiment design, experiment execution, and manuscript revision. All authors read and approved the final manuscript.YC was employed by Star Lake Bioscience Co. Inc. The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Due to the strong heterogeneity of bladder cancer (BC), there is often substantial variation in the prognosis and efficiency of immunotherapy among BC patients. For the precision treatment and assessment of prognosis, the subtyping of BC plays a critical role. Despite various subtyping methods proposed previously, most of them are based on a limited number of molecules, and none of them is developed on the basis of cell states. In this study, we construct a single-cell atlas by integrating single cell RNA-seq, RNA microarray, and bulk RNA-seq data to identify the absolute proportion of 22 different cell states in BC, including immune and nonimmune cell states derived from tumor tissues. To explore the heterogeneity of BC, BC was identified into four different subtypes in multiple cohorts using an improved consensus clustering algorithm based on cell states. Among the four subtypes, C1 had median prognosis and best overall response rate (ORR), which characterized an immunosuppressive tumor microenvironment. C2 was enriched in epithelial-mesenchymal transition/invasion, angiogenesis, immunosuppression, and immune exhaustion. Surely, C2 performed the worst in prognosis and ORR. C3 with worse ORR than C2 was enriched in angiogenesis and almost nonimmune exhaustion. Displaying an immune effective environment, C4 performed the best in prognosis and ORR. We found that patients with just an immunosuppressive environment are suitable for immunotherapy, but patients with an immunosuppressive environment accompanied by immune exhaustion or angiogenesis may resist immunotherapy. Furthermore, we conducted exploration into the heterogeneity of the transcriptome, mutational profiles, and somatic copy-number alterations in four subtypes, which could explain the significant differences related to cell states in prognosis and ORR. We also found that PD-1 in immune and tumor cells could both influence ORR in BC. The level of TGFβ in a cell state can be opposite to the overall level in the tissues, and the level in a specific cell state could predict ORR more accurately. Thus, our work furthers the understanding of heterogeneity and immunotherapy resistance in BC, which is expected to assist clinical practice and serve as a supplement to the current subtyping method from a novel perspective of cell states. Around the world, there were a total of 5,73,278 new cases and 2,12,536 new deaths linked to bladder cancer (BC) in 2020 . In the However, the emergence of immunotherapy has made immune checkpoint blockades applicable to BC patients, especially those with intolerance to platinum-based chemotherapy. Currently, there have been five immune checkpoint inhibitors approved for metastatic BC and adopted as the standard second-line treatment for BC after the failure of platinum-based chemotherapy. In spite of this, the proportion of BC patients responding to immunotherapy reaches as low as about 20%, which is probably attributed to the heterogeneity of the tumor microenvironment (TME). However, discovering the relationship between the heterogeneity of TME and immunotherapy resistance against BC and identifying those patients fit for immunotherapy remain arduous tasks. To solve this problem and obtain a deeper understanding of heterogeneity in BC, there are various subtyping methods proposed. In histology, BC can be subtyped into NMIBC and MIBC, depending on the pathological features and molecular characteristics. Based on DNA, somatic copy-number alterations (SCNAs), and methylation profiles, subtypes are classified and some of them associated with prognosis . TCGA-BLDespite these previously proposed subtyping methods, most of them are based on a limited number of molecules, which leads to the lack of a subtyping method based on cell states. As a part of TME, peritumor cells are closely related to tumor initiation, progression, recurrence, and drug response, and cell states are more comprehensive than molecules. Since then, the subtypes based on cell states may be more effective in revealing the relationship between the heterogeneity of TME and immunotherapy resistance, identifying therapeutic targets, and assisting in the selection of treatment and assessment of prognosis in BC. To be specific, single cell RNA-seq makes it possible to provide detailed information about cell states. Therefore, BC can be divided into subtypes with distinct heterogeneity based on cell states through the combination of single cell RNA-seq, RNA microarray, and bulk RNA-Seq.In our work, a single-cell atlas consisted of 22 immune and nonimmune cell states in BC was constructed by means of reanalyzing single-cell RNA-seq data. Then, the CIBERSORTx algorithm was adopted to identify the cell states of BC in multiple cohorts, and BC was divided into four subtypes according to the levels of 22 cell states. To verify that our subtypes classified by cell states can be relied on to distinguish the heterogeneity of BC and guide the treatment, the molecular, genomic, and clinical characteristics of the four subtypes were evaluated thoroughly. Besides this, our cell-states subtyping method was compared with other subtyping methods. According to these results, the cell-states subtyping method proposed in this study can effectively identify heterogeneity in BC TME and discover the relationship between the heterogeneity of TME and immunotherapy resistance. Our work is expected to assist in the selection of appropriate therapy for BC patients and contribute a supplement to the existing subtyping methods.A total of seven BC cohorts were retrieved from the GEO and SRA databases, including tumor samples from the TCGA BC project (TCGA-BLCA). Among these cohorts, a single cell RNA-seq cohort was used to obtain cell states in BC, three cohorts  were utilized for BC cell-state subtype construction, and three cohorts  were used for BC classification with the cell-state subtyping method. Besides this, immunotherapy response among subtypes was explored in an immunotherapy cohort (IMvigor210). The single-cell RNA-seq data of BC was downloaded from SRAp < .05 were retained for the next analysis.The raw data of eight BC samples were processed and mapped reads to the transcriptome by using Cell Ranger 6.1. After getting a raw unique molecular identifier (UMI) count matrix, SeuratV4.0  was emplTo identify BC subtypes, the R package CrossICC  was applp < .05 noted as *, p < .01 noted as **, and p < .001 noted as ***. The line graph was used to present the difference of the mean value of six checkpoints among four subtypes. Hierarchical bar charts were finally plotted to exhibit the PD-1 level in tumor cells and immune cells.First, counts or FPKM data downloaded from TCGA-BLCA, UROMOL, and IMvigor210 were transformed to TPM data. Next, the function “calculate_sig_score” of IOBR  was usedThe package maftools was used to analyze the mutation profiles of the TCGA-BLCA cohort, and the mutation rate of genes was presented by the function “oncoplot,” followed by identification of driver mutations in each subtype using the function “oncodrive” and exhibition with the function “plotOncodrive.” The Kaplan–Meier plot was utilized for analyzing some driver mutations in cBioPortal for Cancer GenomicsThe analysis of our study was done under R software version 3.6.3 or 4.03 according to the requirements of the R package. The ANOVA and Kruskal–Wallis tests were used to compare normally and nonnormally distributed data for each of the four subtypes. Contingency tables were analyzed by using the Fisher’s exact or Chi-square test, and FDR was assessed by using the Benjamini–Hochberg method for multiple hypothesis testing.The single cell RNA-seq data of eight BC samples were downloaded and reanalyzed with CellRanger, followed by quality control and removal of batch effects of the single-cell RNA-seq data with Seurat as described in Materials and Methods. After that, a total of 70,387 cells were included in this study. Signacx, a framework using neural networks, which was said to be good at identifying cell states, was used to initially identify the cells into 16 cell states, including immune cells (dendritic cell (DC), classical monocyte , non-classical monocyte , macrophage, CD4T_ naive, CD4T_memory, CD8T_naive, exhausted memory CD8T (CD8T_exm), central memory CD8T (CD8T_cm), regulatory T cells (Tregs), B_naive, B_memory, plasma_cell, natural killer cells (NK)), and nonimmune cells , and some other nonimmune cells had not been yet identified . SubsequBased on the absolute proportion of 22 cell states, the CrossICC package was employed to cluster BC in UROMOL, GSE13507, and GSE48276 cohorts jointly. The cohorts from multiple platforms were intergrated using CrossICC with an iterative algorithm, and the collection of three training cohorts could be clustered into four subtypes, namely, C1, C2, C3, and C4. The results of clustering by cell states were consistent among three cohorts, which were validated in TCGA-BLCA, IMvigor210, and GSE31684 and presented with a heatmap in TCGA-BLCA, UROMOL cohorts  and GSE1p < .05). Moreover, overall survival (OS) and relapse-free survival (RFS) in GSE31684 and OS and PFS in TCGA-BLCA also showed remarkable differences (p < .05). Strikingly, in all these cohorts, C4 had the best survival rate, whereas C2 had the worst survival rate (p < .05)  of the UROMOL cohort and in PFS, and disease-specific survival (DSS) of the GSE13507 cohort  . In concTo investigate whether the four subtypes are heterogeneous in TME, immunotherapy, and inflammation, the signature score of three cohorts (TCGA-BLCA cohort (MIBC), UROMOL cohort (NMIBC), and IMvigor210 cohort (immunotherapy)) were calculated by using the IOBR package and displayed in Somatic mutations are reported to play an extremely important role in the development of cancer, which may be related to immunophenotypes , and BC p < .05). C4 had the highest SCNAs level, and C2 had the lowest SCNAs level; although the mutation frequency of C2 was the highest, its mutation amplitude was the lowest . Patients with basal subtype always had basal and squamous differentiation markers, and prognosis was poor (p < .001). Both miRNA and lncRNA S3 subtypes were positively correlated with the good prognosis and low EMT. In contrast, C1 and C2 were mainly included in S4 subtype, whereas the S4 subtype was associated with relatively high EMT with the worst prognosis, which was consistent with the characterization of C1 and C2. Compared with the RPPA subtyping method, C4 were enriched with the most S1 subtype and the least S4 subtypes , which suggests good prognosis, whereas C2 was exactly the opposite . To further understand the heterogeneity of BC TME and the relationship between immunotherapy resistance and TME and find suitable patients for immune checkpoint blockade, a subtyping method based on cell states in BC is proposed for the first time in this study.In the present study, BC was classified into four subtypes based on 22 cell states in multiple cohorts. C1 was characterized by high Tregs and M2_macrophage, indicating that C1 had a suppressive environment. C1 responded to immune checkpoint blockade well and had a relatively better prognosis than C2. C2 was characterized by high CAFs, endothelial, M2_macrophage, Tregs, CD8T_exm, and low CD8T_cm, which suggests that C2 was enriched in EMT, angiogenesis, immune suppressive, and immune exhausted. C2 had the lowest immune response rate and the worst prognosis. C3 was characterized by the highest endothelial cells and lower CD8T_exm, suggesting that C3 was an angiogenesis-enriched subtype. The prognosis of C3 was better than that of C2, but the immune response rate was as low as that of C2. C4 was characterized by high CD8T_cm and moderate immunosuppression, which means that C4 was an immune effective subtype with the best prognosis and highest immune response rate.In general, patients with high PD1 expression had a strong response to immune checkpoint blockade therapy, whereas C2 with high expression in all six immune checkpoints unexpectedly had the lowest immune response rate. A prior study reported that tumor cells can express PD-1 and bind to their own PD-L1, contributing to failure of immune checkpoint blockade therapy . We founPrevious studies identify many subtypes of BC, such as subtypes identified by mRNA, miRNA, lncRNA, and RPPA in TCGA-BLCA. Compared with these subtyping methods, our cell-state subtyping method offers some advantages. First, previous TME subtyping methods were mostly based on a specific marker panel . These mIn summary, from a novel perspective of cell states, single cell RNA-seq combined with RNA microarray and bulk RNA-seq was conducted to classify BC into four subtypes, which could distinguish the heterogeneity of transcriptome, genome, clinical characteristics, and TME in BC. Our subtyping method explored the relationship between TME and immunotherapy resistance and indicated that cell states could be biomarkers to predict prognosis and immunotherapy response, which is helpful to develop flow cytometry for aiding diagnosis and further guide treatment using postoperative cancer tissues. Our work is expected to attract more attention to study cancer from comprehensive cell states, not just molecules, because TME is composed not only of molecules, but also of cells.