The present treatments for lung cancer include surgical resection, radiation, chemotherapy, targeted therapy, and immunotherapy. Despite advances in therapies, the prognosis of lung cancer has not been substantially improved in recent years. Chimeric antigen receptor (CAR)-T cell immunotherapy has attracted growing interest in the treatment of various malignancies. Despite CAR-T cell therapy emerging as a novel potential therapeutic option with promising results in refractory and relapsed leukemia, many challenges limit its therapeutic efficacy in solid tumors including lung cancer. In this landscape, studies have identified several obstacles to the effective use of CAR-T cell therapy including antigen heterogeneity, the immunosuppressive tumor microenvironment, and tumor penetration by CAR-T cells. Here, we review CAR-T cell design; present the results of CAR-T cell therapies in preclinical and clinical studies in lung cancer; describe existing challenges and toxicities; and discuss strategies to improve therapeutic efficacy of CAR-T cells. Lung cancer is one of the most common and deadly cancer types globally . Lung caThe present main treatment strategies for lung cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy , 7. AlthCAR-T cell therapy has emerged as an innovative cancer immunotherapy for lung cancer treatment –16. AlthT cells genetically engineered to carry synthetic CAR bind specifically targeted tumor antigens and kill these targeted tumor cells. CAR are synthetic receptors composed of an antigen-binding domain/hinge motif, transmembrane domain, and intracellular signaling domain. The extracellular antigen-binding domain, composed of a single-chain variable fragment (scFv), recognizes targeted tumor-associated antigens (TAAs) and triggers downstream signaling. The hinge/spacer region provides flexibility to allow the antigen-binding domain to access the targeted antigen. The hinge/spacer region can be adjusted to its optimal length to provide a sufficient distance between CAR-T cells and targeted tumor cells. The transmembrane domain facilitates the distribution of CARs to the T cell membrane, influencing CAR expression, function, and stability. The intracellular domain or endodomain is composed of combinations of signaling domains such as the T-cell activation complex transducer CD3ζ and several costimulatory molecules  Figure, 27.in vivo  complex targeted for TAAs on cancer cells to establish tumor immunity. Similarly, for successful CAR-T therapy in solid tumors, it is important to identify specific TAAs that are highly and selectively expressed in solid tumors but weakly expressed or absent in normal tissue. Several TAAs have been proposed in CAR-T cell research in solid tumors including lung cancer. TAAs currently being investigated in clinical trials of CAR-T cells include carcinoembryonic antigen (CEA), EGFR, human epidermal growth factor receptor 2 (HER2), mesothelin (MSLN), prostate stem cell antigen (PSCA), mucin 1 (MUC1), tyrosine kinase-like orphan receptor 1 (ROR1), programmed death ligand 1 (PD-L1), and CD80/CD86 Table 1.in vivo established human lung cancer model in immune-compromised mice showed treatment with inducible IL8 (iIL8) and CEA-targeted CAR-T cells completely eliminated advanced stage of lung cancer  against EGFR-positive tumor cells  revealed anti-tumor effects of HER2-targeted CAR-T cells, including decreased tumor growth but not complete tumor elimination  family, is also a potential CAR target antigen in lung cancer . Studiesmination , 45. In in vivo subcutaneous mouse lung cancer models and ex vivo models revealed a slower tumor growth rate and inhibitory effects on cell proliferation , highly expressed in cancer-associated fibroblasts (CAFs), can modulate the tumor microenvironment by ECM remodeling. FAP overexpression on CAFs is associated with poor prognosis in many solid tumors including lung cancer. Targeting FAP is also being evaluated for CAR-T cell therapy in NSCLC. r growth , 64. Furr growth , 64.Preclinical CAR-T cell therapy studies in lung cancer by targeting several potential targets, like erythropoietin-producing hepatocellular carcinoma A2 (EphA2), lung-specific X protein (LUNX), variant domain 6 of CD44 gene (CD44V6), melanoma-associated antigen (MAGE)-A1, exhibited significant suppression of tumor growth –68. FurtAlthough there has been continuous improvement of CAR-T cell therapies and their great promise in the treatment of lung cancer and other solid tumors has been revealed, many challenges and hurdles exist. T cell intrinsic as well as tumor-driven mechanisms and treatment-related toxicities in CAR-T cell limit efficacy and safety in solid tumors including lung cancer.Following administration, CAR-T cells encounter considerable challenges in treating lung cancer, such as tumor antigen escape, TME heterogeneity, immune suppression, CAR-T cell trafficking and infiltration into the tumor, and CAR-T cell exhaustion Figure 3The challenges associated with CAR-T cell therapy in solid tumors such as lung cancer include tumor antigen escape and the emergence of multiple resistance mechanisms. Although CAR-T cell therapy can produce high initial response rate in some patients or diseases by overcoming HLA restriction and MHC I downregulation, many patients subsequently experience disease relapse because of antigen escape by cancer cells, resulting in the partial or complete loss of target antigen expression. CD19-targeted CAR-T cell therapy in ALL patients and BMCA-targeted CAR-T cell treatment in patients with multiple myeloma resulted in disease recurrence with the development of resistance and reduced target antigen expression in cancer cells after treatment , 70. SimAnother significant limiting factor in CAR-T cell therapy in solid tumors including lung cancer is tumor heterogeneity. Overall, tumor heterogeneity is a major factor in cancer treatment efficacy, resistance, and failure , 72. SpaSimilarly, as other solid tumors, CAR-T cells in lung cancer suffer the immunosuppressive effect of the TME, which hinders their effector function and impedes clinical efficacy of CAR-T cells , 76.Many infiltrating cell types contribute to an immunosuppressive TME, including myeloid-derived suppressor cells, CAFs, tumor-associated macrophages, and regulatory T cells, which secrete factors such as TGF-β, IL-10, ARG-1, inducible nitric oxide synthase (iNOS), COX2, PGE2, FAP, and PD-L1 , 78. TheUnlike observations in hematological malignancies, hurdles including effective trafficking and infiltration of CAR-T cells into the tumor site limit efficacy of CAR-T cell therapy in solid tumors such as lung cancer. T cell infiltration into lung tumors is mainly influenced by chemokines, chemokine receptors, adhesion molecules, the irregular and extensive leakage of the tumor vasculature, and a hypoxic and immunosuppressive TME –82. FurtThe success of CAR-T cell therapy is also hampered by the development of a dysfunctional state called CAR-T cell exhaustion. CAR-T cell exhaustion is one factor limiting the efficacy of CAR-T cell therapy in solid tumors including lung cancer. T cell exhaustion develops in the TME by persistent antigen stimulation, increase in expression of inhibitory receptors, and the presence of inhibitory immune cells and cytokines . In soliA major hurdle to CAR-T cell therapy is severe toxicities. The most common toxicities following infusion of CAR-T cells are CRS, neurologic toxicity, tumor lysis syndrome (TLS), on-target-off-tumor effects, anaphylaxis, and hematologic toxicities , 86, 87 CRS is the most prevalent adverse effect after CAR-T cell therapy. Fever is the most common symptom of CRS after CAR-T cell infusion can be accompanied by nausea, fatigue, hypotension, and cardiac dysfunction . After CNeurologic toxicity is the second major side effect reported in patients after CD19-specific CAR-T cell infusion . More reAnother potential adverse effect of CAR-T cell therapy is TLS. TLS describes a group of metabolic abnormalities that may occur because of the CAR-T cell-mediated lysis of malignant cells . TLS canFurther treatment-related side effects may occur if target antigens selected for CAR T cell therapy are not specific and shared healthy tissue and healthy cells. Recent single cell analysis and other studies in healthy tissues studies revealed expression of several TAAs in various non-cancerous tissues, supporting the concern for on-target – off tumor mediated side effects –102 Ta. CarboniAs the majority of currently utilized CAR-T cells carry an antigen recognition domain derived from murine monoclonal antibodies , infusioDespite the success of CAR-T cell therapy against hematologic malignancies, the effects of CAR-T cell therapies on solid tumors such as lung cancer are unsatisfactory because of antigen heterogeneity, an immunosuppressive microenvironment, and insufficient trafficking to tumor tissue. Furthermore, CAR-T therapy in the treatment of solid tumors may result in adverse cytotoxicity in healthy cells because of the presence of targeted TAA on healthy cells. Therefore, it is utmost important to develop strategies to improve safety and efficacy of CAR-T cell therapies in lung cancer and other solid tumors. To overcome these hurdles, several studies adapted genetic engineering approaches to modulate CAR-T cells to enhance their efficacy, functional activity in the immunosuppressive TME, and efficient infiltration into the tumor site.Recently, several scientists attempted to improve the efficacy and feasibility of CAR-T cell therapy in solid tumors and avoid off-tumor toxicity. To overcome antigen heterogeneity in solid tumors, several approaches have been adopted to target multiple antigens with a single CAR-T cell population. The combination of biotinylated antibodies and avidin-conjugated CAR has been used to control CAR-T cell activity and target multiple antigens , 110. OtIn vitro and in vivo studies using chemically programmed antibody fragment (ca-Fab)/CAR-system based on/off switch targeting folate binding proteins showed specific elimination of folate receptor expressing cancer cells (in vitro and in vivo using anti-CD 19 hepatitis C virus NS3 protease (HSV-NS3) between the single-chain variable fragment (scFV) demonstrated control of CAR-T activity in the presence and absence of HCV-NS3 inhibitor asunaprevir in eliminating CD19 positive tumor cells , and dastinib have been employed to develop safety switches for CAR-T cells  Switchaber cells . In addior cells .To improve efficacy of CAR -T-cell therapy, several strategies modified CAR-T cells to secrete pro-inflammatory cytokines such as IL-12  or transTo enhance the penetration of CAR-T cells into solid tumors by overcoming physical barriers in the TME, different approaches have been explored to design CAR-T cells targeting the tumor-associated stromal fibroblast protease FAP or ECM-modifying enzymes or to use distinct chemokine gradients to recruit CAR-T cells to solid tumor tissues. Several studies also reported that solid tumor-associated chemokine release characteristics can be utilized to enhance the trafficking of therapeutic T-cells using chemokine receptors –135. Oneex vivo loading of CAR-T cells with arginine , the German Center for Lung Research (DZL) and DFG, SFB 1213 (Project A10* to RS). This study also supported by the international doctoral program “i-Target: immunotargeting of cancer” (funded by the Elite Network of Bavaria), Melanoma Research Alliance (grant number 409510 to SK), Marie Sklodowaska-Curie Training Netwrok for Optimizing Adoptive T cell Therapy of Cancer (funded by the Horizon 2020 Program of the European Union: grant 955575), Else Kröner Fresenius-Stiftung, German Cancer Aid, Ernst Jung Stiftung, Institutional Strategy LMU excellent of LMU Munich (within the framework of the German Excellence Intiative), Bunderministerium für Bildung Und Forschung, European Research Council (Starting Grant 756017), Deutsche Forschungsgemeinschaft (DFG), by the SFB-TRR 338/1 2021-452881907, FritzBender Foundation, Jose´Carreras Foundation and Hector Foundation.SK has received honoraria from TCR2 Inc, Novartis, BMS and GSK. SK is an inventor of several patents in the field of immune-oncology. SK received license fees from TCR2 Inc and Carina Biotech. SK received research support from TCR2 Inc. and Arcus Bioscience for work unrelated to the manuscript.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.


Cellular therapies have transformed the treatment of relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL), which typically does not respond well to salvage chemotherapy. Recently, approximately 40% of r/r DLBCL patients across three different trials achieved a complete remission at 1 year after receiving treatment with autologous chimeric antigen receptor (CAR) T cells (auto-CARs). These successes have prompted studies of auto-CARs in second-line settings, in which axicabtagene ciloleucel and lisocabtagene maraleucel both showed improved event-free survival over autologous hematopoietic cell transplantation (AHCT). While encouraging, this data also highlights that 60% of patients relapse or progress following treatment with auto-CARs. Individual disease characteristics and logistical challenges of cell engineering also limit patients’ eligibility for auto-CARs. Allogeneic CAR T cells  may address some of these limitations as they may mitigate delays associated with auto-CARs, thereby reducing the need for bridging chemotherapies and increasing availability of cellular products for patients with aggressive lymphomas. By being sourced from healthy donors who have never been exposed to cytotoxic chemotherapy, allo-CARs can be created from T cells with better fitness. Allo-CARs made from specific cellular subsets  may also have increased efficacy and long-term persistence. Additionally, allo-CARs have been successfully created from other cell types, including natural killer cells, gamma-delta T-cells and induced pluripotent stem cells. These cell types can be engineered to target viral antigens, enabling precision targeting of virally driven DLBCL. As allogeneic donor cells can be banked and cryopreserved in batches, they can be made more readily available, potentially reducing logistical hurdles and costs compared to engineering auto-CARs. This may ultimately create a more sustainable platform for cell therapies. Challenges with allo-CARs that will need to be addressed include graft versus host disease, alloimmunization, potentially decreased persistence relative to auto-CARs, and antigen escape. In short, the adaptability of allo-CARs makes them ideal for treating patients with r/r DLBCL who have progressed through standard chemotherapy, AHCT, or auto-CARs. Here, we review the published literature on patients with r/r DLBCL treated with allogeneic CAR products manufactured from various cell types as well as forthcoming allogeneic CAR technologies. The treatment of r/r DLBCL is rapidly evolving as adoptive cellular technologies advance to the forefront. The impetus for their development was partially driven by the poor treatment options for this disease, demonstrated by the SCHOLAR-1 study in which only 26% of patients with r/r DLBCL treated with salvage chemotherapy achieved an objective response (OR), with a median survival of 6.3 months . Axi-celnd-line auto-CAR studies in which bridging was permitted  have the potential to address many of the above limitations. Advantages inherent to allo-CARs include the ability to optimize cell source, donor type, and manufacturing such that allo-CARs can be generated relatively rapidly, banked, and made readily available for patients who need urgent therapy. These qualities also should theoretically decrease production costs, thereby improving the long-term sustainability of adoptive cell therapy. Graft versus host disease (GVHD), alloimmunization, decreased long-term persistence, and antigen escape are major challenges to overcome for allo-CARs to become mainstays in the treatment of r/r DLBCL.Allo-CARs have thus far largely been used as donor lymphocyte infusions after allogeneic hematopoietic cell transplantation  to either treat or mitigate relapse. Smith et al. previously reviewed clinical outcomes in patients who received either donor-derived allo-CARs or recipient-derived “pseudo-allo” CARs in the post-transplant setting . Among tvs-lymphoma effect induced by the donor-derived allo-CARs  cells have recently shown promise as another platform for CAR engineering. These relatively rare cells co-express T and NK cell markers with a semi-invariant TCR that recognizes antigens presented on CD1d, an MHC class I-like molecule . CAR-modLike iNKT cells, γδ T cells participate in both innate and adaptive immunity and have cytotoxic mechanisms independent of the TCR-MHC interaction. γδ T cells recognize peptide antigens in an MHC-unrestricted fashion and can express ligands such as NK receptors and toll-like receptors to identify and destroy target cells damaged by infection, malignancy, or other stressors . Their mnd-generation CAR, and directing the differentiation of the CAR-iPSC clone towards T-lymphoid lineages  phenotype, and a decreased frequency of these cells relative to tumor burden has been associated with increased CAR-T doubling times and failure to achieve durable responses in patients with DLBCL  was associated with treatment resistance . Prior cmulation . Studiesmulation . Anothermulation . CCR7+CDth DLBCL . Enrichiesponses . These ced state . A Phaseed state . CAR proed state  and haveed state .As alluded to in prior sections, the key obstacles that allo-CARs will need to overcome to become more broadly used include GVHD, allo-CAR rejection , and potentially decreased long-term persistence. Though not specific to allo-CARs, resistance mechanisms such as antigen escape will also need to be addressed to maximize allo-CAR effectiveness.GVHD and alloimmunization both reflect the challenge of addressing HLA-incompatibility between the allo-CAR donors and patients. Overall, early clinical data with allo-CARs revealed encouragingly low rates of GVHD , 16, 18,Alloimmunization is expected to be a significant issue with allo-CARs. Though lymphodepletion is necessary for expansion of infused cells, immune recovery with time is expected to result in rejection of HLA-mismatched cells. Preexisting donor-specific antibodies  may also mediate rejection . AlemtuzCAR T-cell persistence is a multifactorial issue that has yet to be fully explained. A prior review of auto-CAR resistance mechanisms  identifiAntigen escape is a common CAR resistance mechanism observed with leukemias , though in vitro compared to their counterparts with 4–1BB endodomains  have been shown to induce durable remissions in CD19-low or negative disease previously treated with anti-CD19 auto-CARs . Studiesodomains . Alteratodomains . These fThis review attempts to illustrate the features that make allo-CARs appealing alternatives to auto-CARs. Considering that many auto-CAR patients currently receive 3 or more lines of therapy prior to cell collection, it is likely that their CARs are made from less fit T cells at baseline. Less-differentiated T-cells are also relatively less abundant in circulation  and naïvNJ and MS together developed the conceptual ideas behind this project. NJ wrote the manuscript with assistance and editorial support by MS. All authors contributed to the article and approved the submitted version.MS is supported by the following funding sources: Burroughs Wellcome Fund Postdoctoral Enrichment Program, American Society of Hematology-Robert Wood Johnson Foundation Harold Amos Medical Faculty Development Program, NHLBI K08 HL156082-01A1 and V Scholar Grant for Black/African American Cancer Researchers.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.


Polymethine cyanine dyes, as the most important class of organic near-infrared-II (NIR-II) fluorophores, recently received increasing attention due to their high molar extinction coefficients, intensive fluorescence brightness, and flexible wavelength tunability for fluorescent bioimaging applications. Very recently, remarkable advances have been made in the development of NIR-II polymethine fluorophores with improved optical performance, mainly including tunable fluorescence, improved brightness, improved water solubility and stability. In this review, we summarize the recent research advances in molecular tailoring design strategies of NIR-II polymethine fluorophores, and then emphasize the representative bioimaging and biosensing applications. The potential challenges and perspectives of NIR-II polymethine fluorophores in this emerging field are also discussed. This review may provide guidance and reference for further development of high-performance NIR-II polymethine fluorophores to boost their clinical translation in the future. in vivo applications.Overview of historical development for polymethine fluorophores with NIR-II emission and high brightness for  Over the past few decades, NIR-I fluorescence imaging has undergone explosive growth, but NIR-I technique still suffers from the roadblock of low photon tissue penetration depth which severely restricts its further in vivo biological applications. Hence, it remains a great challenge to meet the multilevel and diverse requirements for high-definition imaging results through NIR-I fluorescence imaging. To this end, researchers never stop to pursue the more high-performance fluorophores to expand their in vivo biological applications.Fluorescence imaging, as a non-invasive or minimally invasive diagnostic imaging modality, holds great promise for improving disease detection and image-guided surgery in preclinical studies and clinical trials.9–11 The NIR-II fluorescence imaging technique can markedly improve the sensitivity and specificity of disease detection and diagnosis in biomedical research and clinical practice. More recently, remarkable achievements have been made in vivo fluorescence imaging with NIR-II emission.12–14 And a range of NIR-II molecular fluorophores have been successfully developed and employed for diverse biosensing and bioimaging applications. Though the research on NIR-II fluorescent imaging is continuously flourishing, currently available high-performance NIR-II fluorophores remain extremely scarce which largely limits the further development of NIR-II imaging technology. Currently, most of the reported fluorescence quantum yields of NIR-II fluorophores remain far lower than that of NIR-I fluorophores on account of the small HOMO–LUMO gap and poor structural rigidity, which generally leads to weak fluorescent brightness in a living system.15 During the development of molecular and structural engineering, most NIR-II fluorophores suffer from the pending dilemma that wavelength and brightness can't be firmly balanced, namely long enough wavelength with low brightness or high brightness with short wavelength.16 Furthermore, poor water solubility and low stability also have limited their further applications and clinical transformation for NIR-II imaging techniques. Therefore, intensive effort should be continually made to develop NIR-II fluorophores with a longer wavelength in the NIR-II window, high brightness, favorable stability, and good biocompatibility. Until now, small-molecular NIR-II fluorophores primarily include these three structural types: donor–acceptor–donor (D–A–D), polymethine (D–π–A), and BODIPY scaffolds.17–19 Among them, polymethine fluorophores, as a class of indispensable NIR-II fluorophores for chemical and biological research, received remarkable attention due to their high molar extinction coefficients, intensive brightness, and flexile wavelength tunability for versatile biomedical applications.5,20,21 And polymethine fluorophores have been successfully explored to enable the acquisition of high-fidelity imaging at wavelengths beyond 1000 nm, including commercially available IR-1048 (ref. 23 flavylium polymethine dye (Flav7),24 Flav7 analog dyes,25 and FD-1080,26etc. However, there are still some unresolved critical issues in the development of NIR-II polymethine fluorophores for further applications and clinical trials due to unsatisfactory fluorescent brightness, poor molecular stability, and bad biocompatibility.Since 2009, fluorescence imaging in the second near-infrared  window, as a cutting-edge imaging technique, has received considerable attention worldwide due to its low photon scattering, minimal autofluorescence, and deep tissue penetration depth.048 ref.  and IR-1In this review, we systemically summarize recent research advances in the development of NIR-II polymethine fluorophores for biosensing and bioimaging in living systems. We emphatically introduce their rational productive manipulation strategy, spectroscopic properties, and highlight some selected biological applications. Meanwhile, we also discuss the challenges and prospects in the future development of polymethine fluorophores in this newly emerging field.227 Generally, the classic cyanine dyes are composed of two nitrogen-containing heterocycles, one of which is positively charged and is covalently linked by a tunable conjugated chain of an odd number of carbon atoms to the other nitrogen center.28 This unique molecular structure imparts cyanine dyes with multiple intrinsically inherent advantages, including flexible synthesis and modification, and wavelength-tunable absorption and emission. In the past few decades ago, there were already many polymethine fluorophores with a long-wavelength absorption or emission, but not for further applications due to the limitation of supporting imaging instruments and systems.29 With the development of NIR-II fluorescence imaging system, polymethine cyanine dyes received increasing attention for wide-ranging biomedical applications due to their above-mentioned multiple inherent advantages. Polymethine dye scaffolds are popular and promising candidates for constructing NIR-II fluorescent contrast agents. The length of the polymethine chain, structures of heterocycle, nature and position of substituents, electronic asymmetry, interactions of fluorophores, and structures of ion-pairs can influence the spectroscopic properties of polymethine cyanine dyes.30 Among them, the polymethine chain is the major structural factor determining the spectroscopic properties of polymethine fluorophores. Lengthening the polymethine chain can effectively extend the π-conjugate system promoting the emission wavelength bathochromic shift of polymethine fluorophores, even beyond 1000 nm. But polymethine cyanine dyes with more than seven methines, namely beyond Cy7, are well-known to suffer from the bottleneck of “cyanine limit” and undergo a symmetry-breaking Peierls transition process which results in an unsymmetric distribution of π-electron density in the ground state, localized positive charge, and substantial bond length transformation along with the π-conjugation chain.31 Compared with symmetrical counterparts, the polar unsymmetric cyanine fluorophores exhibit a broader charge-transfer type absorption and lower fluorescence quantum yields. Since the Peierls transition is vigorously favored in aqueous solution, the “cyanine limit” problem is a non-negligible main holdback for small-molecular polymethine dyes design that largely limits the preparation of high-performance NIR-II polymethine fluorophores for diverse biosensing and bioimaging applications. Otherwise, the different structures of heterocycle also have an extremely significant influence on the spectroscopic properties of polymethine fluorophores. In this way, it is possible to obtain long-wavelength polymethine fluorophores through rationally modifying heterocycle, but not need extending the polymethine chain length. Such an effective strategy allows to obtain more stable polymethine fluorophores than cyanine dyes with long polymethine chain. Introducing substituents with various electronic characteristics into different positions of the heteroradicals can also regulate the extent of vibrational interactions in polymethine dye molecules and then influence their spectroscopic properties. It is also a good option to fine-tune the optical performance of polymethine fluorophores by rationally introducing selective substituents. To enable real-time and high-contrast bioimaging and biosensing, an ideal NIR-II polymethine fluorophore should comply with three crucial principles: (1) long absorption/emission wavelengths in the NIR-II window; (2) high NIR-II fluorescent brightness in biological system; (3) enhanced fluorophore chemostability and photostability.Since 1856, cyanine dyes have almost dominated the research field and applications of photography and other relevant biological and medicinal research fields.In the past decade, considering the significant competitive advantages of NIR-II imaging, intensive efforts have been devoted to designing and synthesizing NIR-II polymethine fluorophores with improved optical properties, mainly including longer wavelength, higher brightness, better biocompatibility, and superior optical/physiological stability. In this section, we will detailly discuss the molecular tailoring strategies for NIR-II polymethine fluorophores based on these above-mentioned pivotal factors to avoid the “cyanine limit” problem. The typical chemical structures of NIR-II polymethine dyes recently applied for living system applications are summarized in 2.116Polymethine fluorophores have conjugated π-electrons and a push–pull structural element. And the intramolecular charge transfer (ICT) usually occurs in push–pull molecular π-conjugated systems containing an electron donor (D) and an electron acceptor (A). Generally, NIR-II fluorophores display a relatively smaller HOMO–LUMO gap than NIR-I chromophores. Consequently, through reasonably regulating the charge transfer effect between different electron donors and acceptors to achieve a smaller HOMO–LUMO gap, the fluorescence emission of polymethine fluorophores can be extended to the NIR-II spectrum range. The rational and effective design strategies have been explored to develop polymethine fluorophores with NIR-II emission wavelength, mainly including these three types: (a) extending conjugation of π-electrons, (b) increasing the electron density of the donor or/and decreasing the electron density of the acceptor, (c) constructing fluorescent J-aggregates for larger red-shifted absorption and emission .1635,40 Surprisingly, dyes C5 and 5H5, as Cy5 and Cy3 derivatives, respectively, can effectively absorb light and emit fluorescence in the NIR-II region whose absorption and emission wavelengths realize a more obvious bathochromic shift than Cy7 systems. In addition, Martin groups also reported two new classes of persulfonated indocyanine dyes FIN-872 and FNIR-1072 through catechol-ring and aryl-ring fusion, respectively.36 Compared to the catechol fusion, the different manner of aryl rings on the nonamethine scaffold can bring about the intriguing results of the maximum absorption wavelength red-shifted from 872 to 1072 nm through extending the conjugation system.Lengthening the polymethine chain is a classic and valid strategy to push the wavelength redshift, but this strategy can simultaneously induce the negative result of compromised fluorescence quantum yield, decreased stability, and the loss of symmetric electron delocalization due to the “cyanine limit” problem. Different from extending conjugation by increasing the length of the polymethine bridge, Delcamp and Hong developed a new strategy to force all the orbitals of the indolizine and thiopyrylium moieties to participate in the resonance enlarging the conjugation system .35,40 Su41,42 For example, the oxygen atoms of IR-27 were substituted by sulfur atoms, leading to an about 100 nm red-shifted absorption from 988 to 1080 nm  and created a clear relation between structure and optical performance for this kind of bright NIR-II fluorescent imaging reagents.25 Similarly, BTC1070 bearing two electron-donating diethylamino moieties has an approximately 100 nm emission wavelength bathochromic shift compared to BTC980, which could be attributed to the strong intramolecular charge transfer effect of nitrogen atoms.32 Based on extending heterocycle conjugation of IR1048, Yang groups successfully designed and developed a novel heptamethine cyanine dye Cy-PA with the maximal fluorescence emission at approximately 1120 nm by introducing indolium-derived polycyclic aromatic hydrocarbons on the terminal ends of the conjugated polyene backbone.37Heterocycle modification is an alternative and useful approach to construct NIR-II emissive stable polymethine fluorophores. Recent studies show that replacing the heteroatom from oxygen with a carbon or chalcogen atom can lead to a bathochromic shift. 1080 nm . But thilavylium .24 To ob28 Specially, the π-extension on polymethine chain can inevitably accelerate the self-aggregation of fluorophore molecules. Every coin has two sides, as the old saying goes. The fluorophores with a planar conjugation structure in water solution may readily orient in a head-to-tail fashion to form J-aggregates. Compared with the monomer molecules, J-aggregates, as highly ordered assembled organic dyes via the self-assembly processes, display the spectroscopic properties of bathochromically-shifted absorption and emission, small Stokes shift, and increased absorption coefficients.45,46 The unique optical properties of J-aggregates are desired and valuable for in vivo bioimaging applications. Therefore, it is also an attractive and efficient strategy to achieve longer wavelength fluorescent contrast agents by constructing fluorescent J-aggregates. For example, the self-assembly of amphiphilic cyanine dye FD-1080 and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) presents a stabilized FD-1080 J-aggregate with remarkably bathochromically-shifted absorption at 1360 nm and emission at 1370 nm  × quantum yield (ΦF)]. An ideal fluorophore should have intense absorption and emission properties upon light excitation. Most small-molecular NIR-II polymethine fluorophores have the common characteristic of a highly bulky conjugated system with a small HOMO/LUMO energy gap which leads to the low quantum yield. In general, the fluorescent brightness of most NIR-II fluorophores (below 103 M−1 cm−1) is almost two orders of magnitude lower than that of visible and NIR-I fluorophores (around 105 M−1 cm−1).49 The weak brightness of NIR-II fluorophores in an aqueous solution is mainly attributed to their interactions with surrounding water molecules, which causes the energy loss of the excited fluorophore.50 To increase the quantum yield of polymethine fluorophores, some enlightening and attractive strategies have been proposed to reduce nonradiative processes by interactions with biomolecules, introduce conformational restraint on the polymethine skeleton structure, or decrease intersystem crossing by changing heavy atoms.51,52 Furthermore, intensive efforts to improve the brightness of polymethine dyes can take steps to reduce aggregation effects as far as possible to keep them in a molecular monomer form for effective absorbing and emitting capacity  dyes with tunable maximal absorption/emission ranging from 1100 to 1400 nm through the molecular engineering strategy  borate (F5-TPB) reference to their parent forms. This work provides a successful paradigm to overcome the problem of fluorophore self-aggregation and fluorescence self-quenching and reserve the NIR-II multiplexed imaging capacity for high-contrast dual-color bioimaging. Besides, supermolecule is also a good strategy to introduce steric hindrance. To mitigate the “cyanine limit” and improve fluorescent brightness, Bradley groups encapsulated the newly developed cationic cyanine dye 3 with cucurbit[7]uril (CB7) through the host/guest self-assembly strategy, transforming the π-electron distribution of the cyanine fluorophore , and enhanced fluorescence brightness up to 2778-fold. Similarly, FD-1080 containing two sulfonic acid groups and cyclohexene group has favorable water solubility and stability with both maximal absorption and emission in the NIR-II window.26 Through combining with fetal bovine serum (FBS), the quantum yield of FD-1080-FBS complex can effectively increase from 0.31% to 5.94%  in situ to decompose the polymethine chains which is the leading cause of fluorophore photobleaching.58,59 Hence, avoiding the reactive species attack as far as possible may be beneficial for the improvement of chemostability. For example, introducing large steric hindrance substitutes or electron-withdrawing groups into polymethine dyes is an available approach to prevent small-molecular fluorophores from attacking by various reactive species. Moreover, combining the fluorophores with protein to present a stable protein-fluorophore complex not only can improve the fluorescence quantum yield, but also enhance the NIR-II fluorophore stability. In addition, intensive efforts should be made to largely maximize the rigidity and effectively reduce the isomerization behavior for more stable fluorophores. Therefore, there is no doubt that the performance of polymethine fluorophores can be remarkably strengthened by improving the rigidity of conjugated backbone structures. Considering the rigid of xanthene structure, CX-2 and CX-3 were synthesized by covalently linking two xanthene units with an odd number of methine moieties.38 They are partially rigidified fluorophores and stable in aqueous solution due to partial restriction of the polymethine chain. Similarly, 5H5 is also a partially rigidified polymethine cyanine dye with excellent optical properties for NIR-II fluorescence bioimaging.35 Otherwise, the asymmetric Rh1029 fluorophore composed of xanthene and benzo[cd]indolium moieties also performed very well in NIR-II bioimaging.33The chemostability and photostability of fluorophores in a complex microenvironment is also regarded as a matter worth profoundly considering in the development of NIR-II polymethine fluorophores which is of great importance for high-quality and high-contrast bioimaging. Unfortunately, the chemostability of NIR polymethine fluorophores is inherently poor on account of the small HOMO–LUMO gap. A too low LUMO orbital is easy to be attacked by nucleophiles, such as ubiquitous intracellular cysteine, homocysteine, glutathione, and so on. On the contrary, a too high HOMO orbital is susceptible to oxidation or electrophilic attack.2.412,60 For example, the frequently-used sulfonic acid groups were chemically modified on the molecular structure of polymethine fluorophores to improve their water solubility, such as FD-1080,26 LZ-1105,34 and FINR1072,36etc. PEGylation is also an effective strategy to improve water solubility and biocompatibility functionality.61 5H5 with a terminal alkyne group was covalently modified with cRGD-PEG8via Cu(i)-catalyzed azide–alkyne click reaction for improving tumor targeted ability and water solubility.35 Additionally, hydrophobic fluorophores can also be imparted water solubility via a non-covalent hydrophilic encapsulation method. Polyethylene glycol conjugated phospholipids (DSPE-mPEG) is one of the most commonly used hydrophilic encapsulation materials which shows enormous potential for clinical biomedical application. The hydrophobic fluorophores were changed into hydrophilic nanoparticles for further biological imaging applications through the nano-precipitation method, such as CXs,38 BTC1070,32 Flav7,24 Rh1029,33 IR1061,23 and HCs,39etc.The water solubility of fluorophores is also an important factor for biological applications in the biological environment. So far, there are two commonly used strategies to increase the water solubility of NIR-II polymethine fluorophores: one is to embed hydrophilic groups on the structure of fluorophores; the other is to package hydrophobic fluorophores with micelle or other carriers.362 The past decade has witnessed tremendous progress in the development of NIR-II polymethine cyanine dyes for versatile bioimaging applications. Herein, we introduce some representative application examples of polymethine-based NIR-II fluorescent imaging research, such as tumor targeted imaging, dynamic vessel imaging, and intravital multicolour imaging.NIR-II fluorescent imaging allows detailed imaging of diverse biological processes with deep tissue penetration depth, high spatial and temporal resolution, and high signal-to-noise ratio benefiting from the longer wavelength with negligible autofluorescence and tissue scattering. NIR-II small-molecular polymethine fluorophores have various intrinsically inherent advantages, such as clear molecular structure, good biocompatibility, superior metabolic properties, and minimal long-term toxicity, which exhibit great potential for NIR-II bioimaging applications.3.1e.g., tumor, vasculature, lymph nodes, and skeleton) and monitor the dynamic physiological processes  owing to its high temporal and high spatial resolution and deep tissue penetration.58,63 NIR-II polymethine fluorophores have been successfully applied in tumor imaging in vivo, showing great penetration depth, outstanding spatial resolution, and high signal-to-noise ratio. For example, the polymethine thiopyrylium salt 5H5 has both absorption and emission in the NIR-II window with a maximum absorption at 1069 nm and maximum emission at 1125 nm.35 To improve the tumor targeting ability and water solubility, the small-molecular fluorophore 5H5 with terminal alkyne function was covalently conjugated with an integrin-targeted peptide c(RGD)fk via copper-mediated azide–alkyne cycloaddition. In vivo NIR-II or NIR-IIa targeted imaging of human U87MG glioma, the highest tumor contrast was achieved at 8 h with a tumor tissue/nontumor tissue (T/NT) value of 4.0 upon the 808 nm laser irradiation, whereas the T/NT value was increased up to 6.8 under the 1064 nm excitation  were clearly visualized by means of the NIR-IIa fluorescence of LZ1105  detection in the NIR-II window with high spatial resolution; (2) establishing multiple orthogonal excitations and/or emission detection wavelengths without the problem of crosstalk between different color channel; (3) rapidly detecting each channel on the millisecond timescale.25 NIR-II polymethine fluorophores have high molar extinction coefficients, intense fluorescent brightness, and particularly flexible wavelength tunability, which exhibits tremendous potential for multiplexed fluorescence imaging. For example, HC1222/F5-TPB and HC1342/F5-TPB have orthogonal excitation wavelengths (1120 and 1319 nm) and emission detection channels  which is beneficial for multiplexed imaging.39 The standard dual-color imaging of the circulatory system and metabolic organs was carried out in living mice through successively performing oral or intravenous injection of HC1222/F5-TPB nanoparticles and intravenous administration of HC1342/F5-TPB nanoparticles , JuloChrom5 (λex = 892 nm), Chrom7 (λex = 968 nm), and JuloFlav7 (λex = 1065 nm), Sletten's group successfully realizes the four-color video-rate imaging in mice which is the first time to be reported , excited-state intramolecular charge transfer, and Förster resonance energy transfer (FRET), 4.170,71 NIR-II polymethine fluorophores were chemically tailored to construct off–on probes for biosensing due to their merits of flexible modification and wavelength tunability. A nitro group, exerted as a common fluorescence quencher, is generally adopted to develop activatable fluorescent probes. For example, Cai groups developed a hypoxia activatable NIR-II fluorescent probe IR1048-MZ through covalently conjugating the nitroreductase (NTR) enzyme-responsive nitro imidazole group with the commercially available NIR-II fluorophore IR-1048  for pH, adenosine triphosphate (ATP), and metal-ion detection to verify the universality of these NIR-II platforms.74ICT is an effective and popular sensing mechanism which is usually exerted in off–on probes. For example, Zhang and Lei groups developed a NIR-II fluorescent probe PN910 toward H in vivo .72 In th enzymes .73 This et al. designed and developed a pH or viscosity activatable fluorescent probe LET-1052 for acidic tumor microenvironment (TME) triggered NIR-II PTT, along with viscosity triggered the assessment of therapeutic efficacy by itself.75 In acidic TME, the maximal absorption of LET-1052 at 1052 nm displays a 3.4-fold enhancement from pH 7.4 to 5.0 on account of the protonation on the nitrogen atoms and conjugated π-electron rearrangement, which activates NIR-II PTT and tumor-specific fluorescence imaging upon 1064 nm light irradiation. Afterward, PTT-induced cellular death brings about the elevated intracellular viscosity, along with the emerging NIR-I fluorescence signal for real-time evaluation of therapeutic efficacy due to the restricted intramolecular rotation of LET-1052. This work provides an advanced therapeutic modality with instant self-evaluation functionality for modern precise personalized medicine.The real-time evaluation of tumor therapeutic efficacy during the treatment process is extremely important for successful tumor treatment. To achieve this goal, Lin 4.276,77 ICT is a frequently used sensing mechanism to develop NIR-II ratiometric fluorescent probes. For example, BTC1070 decorated with two amino groups possesses the absorption/emission beyond 1000 nm and has been calculated pKa values at 0.29 and 3.81.32 BTC1070 exhibits a ratiometric fluorescence emission signal transformation from 1065 nm (BTC1070) to 980 nm  between pH 1–4 which is attributed to the intramolecular charge transfer process of the amino groups switching from on to off  adjustable sensors, namely pTAS, were established by encapsulating the donor aza-BODIPY (NAB) with NIR-II emission and pH sensitive polymethine based pre-acceptor (NRh) to form the FRET system through simply regulating the component ratio.78 Combining the pH response regions of pTAS, a two-fold widened pH detection range (6.11–7.22) is obtained compared to the pHt settled sensor (6.38–6.94). With the help of the adjustable pHt, the real-time dynamic variation processes of tumor pH in vivo could be quickly captured through dual-channel ratiometric bioimaging in the NIR-II window which displays an ideal coefficient of variation under 1% reference to the standard pH meter.Constructing the FRET system is also an alternative and powerful avenue to obtain a ratiometric fluorescence probe. Based on the FRET strategy, the fluorescence probe PN1100 composed of CX-1 and CX-3 dyes in micelles can be exerted as a ratiometric nanoprobe to monitor drug-induced hepatotoxicity by responding to corresponding marker analyte OONO− ref.   can obtain higher-quality images with almost “zero” background due to their minimized photon scattering, negligible autofluorescence, and deeper tissue penetration.58 However, the number of polymethine fluorophores with the absorption and/or emission wavelengths beyond 1200 nm remains rare. At the same time, the “cyanine limit” problem can never be ignored since fluorophores with the flexible long-wavelength polymethine chain usually encounter this dilemma. Crossing the “cyanine limit” generally leads to broader charge-transfer type absorption bands and lower fluorescence brightness of polymethine fluorophores which tremendously restricts their further biological imaging applications. One feasible manner to overcome this issue is not to lengthen the conjugation chain length, but rationally regulate and enhance the heterocyclic terminal unit, such as fluorophores Fav7 and its derivatives, C5, and 5H5. Conformational restraint and heterocycle exchange may also be exerted as alternative and feasible ways to obtain NIR-II emission while not crossing the cyanine limit, such as CX dyes, FNIR1072, HC dyes, and Flav7 analog dye. Therefore, it is futile to blindly chase the expansion of absorption or emission wavelengths in the NIR-IIa/IIb window. What even more important is that increasing wavelength to profoundly consider the fluorescent brightness in the biological aqueous environment for feasible imaging applications.As discussed above, an ideal polymethine fluorophore should have high enough fluorescent brightness in the NIR-II spectral region with favorable stability and biocompatibility in a biological system. In the past decade, various strategies have been employed to push the absorption/emission peaks of polymethine fluorophores to more bathochromic shifts, mainly including lengthening the polymethine chain, modifying the heterocycle, and constructing stable fluorophore-protein J-aggregates, in vivo applications. This matter may be prevented by introducing large steric hindrance via covalent or non-covalent manners on the fluorophore skeleton. At the same time, the hindrance may also avoid the self-aggregation of fluorophores in aqueous solutions. Strengthening the rigidity of the conjugated backbone structures may also significantly improve the performance of polymethine fluorophores. In short, an ideal polymethine dye should not solely have a single outstanding advantage, but possess excellent comprehensive performance, especially high fluorescent brightness with a long wavelength in an aqueous solution.The fluorescent brightness of fluorophores in aqueous solutions directly determines the success or failure of bioimaging applications. Most polymethine fluorophores have enough long emission wavelength in the NIR-II window, but it is regrettable that they generally encounter the bottleneck of extremely low fluorescent brightness. In the development of pursuing more long-wavelength fluorophores in the NIR-II spectral region, it is challenging but valuable to reserve the fluorescent brightness of polymethine fluorophores in aqueous solutions. There are three common strategies to improve the fluorescent brightness, including introducing large steric hindrance, constructing a stable protein-fluorophore complex, and increasing the rigidity of polymethine skeleton structure. Besides, improving the stability and biocompatibility of polymethine fluorophores is also an unavoidable issue for successful application practice. Most of the NIR-II polymethine cyanine dyes in aqueous solution are not stable enough and vulnerable to reactive species which largely limits their further etc. But it remains challenging to simultaneously track or even quantify multiple relevant analytes in vivo through one single probe. However, the biological microenvironment is a dynamic and complex network. Exploring the physiological processes or distinctly elucidating pathogenesis and realizing the multiple factors sensed simultaneously in living systems is of great importance for modern biochemistry and biomedicine. Hence, future efforts should be devoted to developing NIR-II fluorescent probes for multi-analyte detection, especially those probes that can respond to different analytes with orthogonal fluorescent signals or multimodal signals.In addition, more intensive efforts should also be devoted to the development of polymethine-based NIR-II multiple-detection probes for biosensing. Up to now, some NIR-II polymethine-based fluorescent probes have been successfully developed and used for biological detection, such as NIR-II HD 5 series, PN1100, PN910, and LET1052, in vivo bio-applications. For example, bioluminescence and chemiluminescence imaging, as autofluorescence-free optical imaging which do not require extrinsic light excitation, can remarkably improve the signal-to-noise ratio.79 Combining bioluminescence and chemiluminescence imaging with NIR-II polymethine fluorophores to bathochromically shift their emission wavelength reaching the NIR-II window is still a great challenge. To achieve this, it requires highly efficient energy transfer systems between the donor  and the acceptor (NIR-II polymethine fluorophores) which is envisioned to bring about significant improvement in the spatial resolution. Besides, exploring the NIR-II polymethine fluorophores based on the biorthogonal chemistry would extend theirs in vivo imaging applications. The labeling strategy based on metabolic engineering and bioorthogonal chemistry is an ideal artificial targeting and labeling technology without interfering with native biochemical processes. One straightforward method to introduce this labeling technology into NIR-II polymethine fluorophores is to modify them with bioorthogonal tags . Of course, combining the NIR-II polymethine fluorophores with tagged proteins also can significantly improve fluorescent labeling efficiency in biological applications.Rationally combining NIR-II polymethine fluorophores with other optical imaging or advanced biotechnology tactics is challenging but intriguing to further improve the imaging performance and extend their The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.There are no conflicts to declare.


Given their hematopoiesis supporting capacity, it has been reasonable to investigate MSCs’ potential involvement in MDS. This review discusses this issue by summarizing existing findings obtained by in vitro studies and murine disease models of MDS. Furthermore, the theoretical background of targeting the BM-MSCs in MDS is outlined and available therapeutic modalities are described.Myelodysplastic syndromes include a broad spectrum of malignant myeloid disorders that are characterized by dysplastic ineffective hematopoiesis, reduced peripheral blood cells counts and a high risk of progression to acute myeloid leukemia. The disease arises primarily because of accumulating chromosomal, genetic and epigenetic changes as well as immune-mediated alterations of the hematopoietic stem cells (HSCs). However, mounting evidence suggests that aberrations within the bone marrow microenvironment critically contribute to myelodysplastic syndrome (MDS) initiation and evolution by providing permissive cues that enable the abnormal HSCs to grow and eventually establish and propagate the disease. Mesenchymal stromal cells (MSCs) are crucial elements of the bone marrow microenvironment that play a key role in the regulation of HSCs by providing appropriate signals  Within the adult Bone Marrow (BM) hematopoiesis take place in specialized microenvironments called niches that regulate the balance between quiescence, proliferation, differentiation and self-renewal of hematopoietic stem and progenitor cells (HSPCs) . The BM + cells, nestin+ cells and neural–glial antigen 2 (NG2)+ cells . U. U5). Us)+ cells –8 .Due to the low frequency of MSCs within the BM, data regarding their impact in regulating the homeostasis of HSCs is mainly derived from studies using ex vivo-expanded cells. The latter are defined based on the three minimal criteria established by the International Society for Cellular Therapy (ISCT)  i.e. (a)As the crucial role of BM-MSCs and their progeny in the control of hematopoiesis is increasingly being acknowledged , 13, it Myelodysplastic syndromes consist of an heterogeneous group of clonal hematological disorders characterized by ineffective dysplastic hematopoiesis, peripheral blood cytopenias and an increased risk of transformation into acute myeloid leukemia (AML) . Patientin vitro studies and mouse models supporting the notion of an impaired BM-MSC compartment in MDS and discuss its contribution in the pathogenesis of the disorder. Furthermore, the theoretical background of BM-MSCs’ therapeutic targeting in MDS will be outlined and an overview of relevant in vitro data will be provided  and increase their clonogenic potential and viability. Whether BM-MSC derived exosomes similarly support clonal hematopoiesis in the MDS setting remains to be seen. On the other hand, many studies have demonstrated that MDS-derived BM-MSCs have an impaired potential to support normal HSPCs (HGF), C-X-C motif chemokine ligand 12 (CXCL12), insulin like growth factor-1 (IGF1), insulin growth factor binding protein 2 (IGFBP2), thrombopoietin (TPO) (+ BM-MSCs from LR-MDS the up-regulation of inflammatory factors and inhibitors of hematopoiesis such as interleukin 6 (IL-6), IL-8 and C-C motif chemokine ligand (CCL3)  , 19, 29. (CCL3) , 36, 47,ic cells . This waic cells , in patiic cells .in vitro T cell activation and proliferation . A largferation . Anotherferation . More spferation . Moreoveferation . Taken tferation  BM-MSCs de novo MDS, data regarding the presence of genetic aberrations in MDS-derived BM MSCs have been contradictory. While several studies  gene  gene . Mutatiods) gene . Of noteds) gene . Sbds deme cells . Consistpatients . On the leukemia . Thus, tignaling . Activatignaling .NUP98 fusion genes have been identified in various hematologic malignancies, including MDS. The NUP98-HOXD13 fusion gene has been detected in patients with MDS  lackingse model . These mML cells . Collectdel/+Apc MDS mouse model is a conditional knock-out mouse based on the Mx1-Cre system the most commonly used system to delete the gene of interest in experimental hematology  mice significantly increased engraftment of hematopoietic cells as compared to transplantation in the absence of BM-MSCs or with BM-MSCs derived from healthy donors. Furthermore, ex vivo expanded MDS-derived BM-MSCs were shown to differ from their normal counterparts regarding expression of genes related with osteogenesis, adipogenesis, fibrosis, inflammation, cell adhesion, extracellular matrix remodeling and cytokine signaling. Of note, normal MSCs acquired similar properties upon co-culture with patient-derived bone marrow cells, substantiating the issue of hematopoietic MDS cells inducing niche alterations, favoring malignant cell expansion and disease evolution  and the ROS-associated Wnt/b-catenin pathway  is a common finding in MDS patients that occurs as a result of ineffective hematopoiesis as well as red blood cell transfusions , 77. Asid TGFβ1 . In addi pathway . IO has patients . The afoOn the other hand Hu et al.  have repex vivo expanded MDS-derived BM-MSCs have significantly different DNA methylation patterns from their normal counterparts  (PDL1) (Decitabine is another hypomethylating agent which has been approved by the Food and Drug Administration (FDA) for the treatment of patients with MDS . IncubatCDKN1A) . Moreove (PDL1) . Taken tex vivo expanded lower risk MDS-derived BM-MSCs with lenalidomide has been reported to improve their capacity to support normal clonogenic HSPCs  aberrant BM-MSCs promote the development of the myeloid malignancy and b) malignant HSPCs alter BM-MSCs causing them to facilitate disease propagation and/or evolution. Based on these notions a matter of utmost importance that has not been investigated thus far is the delineation of the potential impact of BM-MSCs in the setting of clonal hematopoiesis detected in individuals with otherwise normal complete blood cell counts and without any overt underlying hematological malignancy (We have summarized herein the current views on the implication of BM-MSCs in the MDS pathogenesis. Collectively, there is large number of studies that support the notion that BM-MSCs play a major part in the hematopoietic failure that characterizes MDS and demonstrate their involvement in the initiation and progression of the disease. The interactions between BM-MSCs and HSCs have been historically assessed lignancy . Such cllignancy  may be llignancy  and is clignancy , 87. Notlignancy , includiAt present, we have only gained a first insight on the dependence of MDS cells on the BM-microenvironment and lots of issues still remain obscure. The emergence of studies using humanized bone marrow like structures , 90 thatConceptualization, Writing-Review and Editing, CP. Writing-Original Draft, AM. Writing-Review and Editing, HP. All authors contributed to the article and approved the submitted version.


A 25-year-old female with diabetes and hypertension presented with progressive painless blurred vision in her left eye ten days after she received her third dose of the SARS-CoV-2 mRNA vaccine BNT162b2 (Pfizer-BioNTech). The clinical examination confirmed the diagnosis of Central Retinal Vein Occlusion (CRVO) complicated with macular edema. Three doses of anti-vascular endothelial growth factor (VEGF) were injected intravitreally. Visual acuity was improved from 20/100 to 20/30, but recurrence was noted at 6 months. Several cases of retinal vein occlusion (RVO) after COVID-19 vaccination have been reported. However, the present case is the youngest female individual documented to have CRVO after SARS-CoV-2 vaccination. This case demonstrates that the macular edema might be recurrent in patients with risk factors for CRVO who receive SARS-CoV-2 vaccination, suggesting the need for careful consideration of the treatment strategy and close follow-up. Although the definite pathogenesis still needs to be carefully determined, this report highlights the possible association between RVO and mRNA-based COVID-19 vaccination, even in young individuals. Given the threat of the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection worldwide, there has been vigorous research for effective treatment strategies and vaccine development to contain the pandemic. Vaccines help protect against certain diseases by providing individual and herd immunity, serving an important role in global public health. The four categories of SARS-CoV-2 vaccines include mRNA vaccines, which deliver genetic code through lipid nanoparticles , vectorThe reported side effects of COVID-19 vaccination include thrombosis with thrombocytopenia, Guillain–Barre syndrome and myocarditis. In addition, several ocular adverse effects after COVID-19 vaccination have been reported . Here, wA 25-year-old obese female with a history of hypertension managed by regular oral medications presented with progressive, painless blurred vision in her left eye for several days. Ten days before symptom onset, she received her third dose of the SARS-CoV-2 mRNA vaccine BNT162b2 (Pfizer-BioNTech). She did not have a history of taking oral contraceptive pills or a history of smoking. She had no history of allergies. At presentation, the best corrected visual acuity was 20/100 in the left eye and 20/20 in the right eye. Intraocular pressure was 13 mmHg in the right eye and 18 mmHg in the left eye. No relative afferent pupil defect was found. Slit lamp anterior segment examination showed no abnormal findings. In the left eye, fundus examination revealed a diffuse flame shape hemorrhage, disc edema and severe macula edema A,B. FluoAlthough rare, several ocular manifestations after SARS-CoV-2 vaccination have been documented. Graves’ disease activation, corneal graft rejection, herpes zoster ophthalmicus (HZO), uveitis, Vogt–Koyanagi–Harada (VKH) disease, acute abducens nerve palsy, Bell’s palsy, arteritic anterior ischemic optic neuropathy, central serous chorioretinopathy, RVO and cerebral venous sinus thrombosis have been observed in patients who received the Pfizer-BioN-Tech vaccine ,8,9,10. Retinal vein occlusion, commonly seen in the ophthalmologic clinical practice, is the second leading cause of retinal vascular blindness after diabetic retinopathy. It is classified into branch RVO (BRVO), hemiretinal vein occlusion (HRVO) and CRVO based on the location of the obstruction. The typical fundus findings associated with CRVO include tortuosity and dilatation of all branches of the central retinal vein, dot/blot and flame-shaped hemorrhages, optic disc and macula edema and cotton-wool spots. CRVO is further categorized into the ischemic and non-ischemic types based on the perfusion status. Ischemic CRVO tends to present with poor visual acuity, the presence of relative afferent pupillary defects, non-perfusion of more than 10 disc areas on FA and a higher probability of negative ERG patterns. Venous compression at arteriovenous crossings, degenerative venous change including venous stasis and endothelial damage along with hypercoagulable states may be involved in the pathogenesis. The likelihood of developing RVO increases with age . A totalA total of 22 papers with 51 cases that present with RVO after COVID-19 vaccination are summarized in Recurrent retinal hemorrhage and macular edema occurred in the present case after three doses of intravitreal injection in a six-month follow-up period while the patient’s systemic diseases were well-managed. Most published cases have relatively short periods of follow-up time and lack presentation of the patients’ final visual acuities. There are three cases of CRVO after BNT162b2 with initial VA of 20/30, 20/40 and 20/40, who had VA of 20/20 after IVI anti-VEGF without detailed follow-up information ,34. Our Based on the Naranjo scoresheet for assessing the treatment-adverse reaction association , the subThe early onset of CRVO at 25 years of age indicates that other mechanisms associated with hypercoagulability and inflammatory processes need to be considered. For this rare ocular complication associated with vaccination, it is difficult to confirm a causal relationship. Findings from previous case reports, and the young age of the patient, allow for the supposition that vaccination may have played a role in CRVO development. Vaccines for COVID-19 were designed to produce high levels of neutralizing antibodies targeting spike proteins and to activate T-helper responses. Several possible mechanisms of adverse reactions to vaccination have been described, including autoimmune/inflammatory syndrome induced by adjuvants (ASIA), altered metabolism of RNA (spike protein) in susceptible individuals, stimulation of innate immunity through nucleic acid receptors , molecular mimicry between vaccine peptide fragments and human-derived proteins and delayed hypersensitivity with deposition of immune complexes ,8. When To date, the present case is the youngest (25 years old) female individual who presented with CRVO after COVID-19 vaccination. However, definite causality needs to be established carefully. Physicians should closely monitor patients for ocular adverse effects following COVID-19 vaccination, and this report provides information on potential treatments for approaching similar complications. Patients with risk factors for RVO, even those who are young, should be closely followed for complications after vaccination. Overall, the incidence of CRVO following COVID-19 vaccination appears to be low. The benefits of vaccinations still outweigh the potential risks, but patients should receive prompt ocular examination when they notice minor visual impairments after vaccination.


We examine whether inclusion of artificial intelligence (AI)-enabled retinal vasculometry (RV) improves existing risk algorithms for incident stroke, myocardial infarction (MI) and circulatory mortality.2 statistics. Performance of Framingham risk scores (FRS) for incident stroke and incident MI, with addition of RV to FRS, were compared with a simpler model based on RV, age, smoking status and medical history .AI-enabled retinal vessel image analysis processed images from 88 052 UK Biobank (UKB) participants (aged 40–69 years at image capture) and 7411 European Prospective Investigation into Cancer (EPIC)-Norfolk participants (aged 48–92). Retinal arteriolar and venular width, tortuosity and area were extracted. Prediction models were developed in UKB using multivariable Cox proportional hazards regression for circulatory mortality, incident stroke and MI, and externally validated in EPIC-Norfolk. Model performance was assessed using optimism adjusted calibration, C-statistics and R2 statistics between 0.75–0.77 and 0.33–0.44, respectively. For incident stroke and MI, addition of RV to FRS did not improve model performance in either cohort. However, the simpler RV model performed equally or better than FRS.UKB prognostic models were developed on 65 144 participants  and validated in 5862 EPIC-Norfolk participants . Prediction models for circulatory mortality in men and women had optimism adjusted C-statistics and RRV offers an alternative predictive biomarker to traditional risk-scores for vascular health, without the need for blood sampling or blood pressure measurement. Further work is needed to examine RV in population screening to triage individuals at high-risk. Population screening for myocardial infarction (MI) and stroke using risk prediction tools exist but have limited uptake; risk scores for circulator mortality do not exist.Risk models developed in UK Biobank  using artificial intelligence (AI)-enabled retinal vasculometry (RV), age, history of cardiovascular disease, use of hypertensive medication and smoking yielded high predictive test performance for circulatory mortality.Risk scores for MI and stroke performed similarly to established risk scores.AI-enabled RV extraction offers a non-invasive prognostic biomarker of vascular health that does not require blood sampling or blood pressure measurement, and potentially has greater community reach to identify individuals at medium-high risk requiring further clinical assessment.Circulatory mortality, including cardiovascular disease (CVD), coronary heart disease (CHD), heart failure and stroke, is a major cause of morbidity and mortality worldwide.Examination of retinal blood vessels (arterioles and venules) may offer a microvascular phenotype more indicative of the presence of early circulatory related disease processes, providing a non-invasive window on the circulatory system. Narrow retinal arterioles show a clear association with higher blood pressure (BP), hypertension and with incident CVD.We developed a fully automated artificial intelligence(AI)-enabled system ) for examining the retinal vascular tree, which overcomes many of the difficulties of earlier approaches, allowing detailed vasculometry quantification in large population studies.UKB is a prospective cohort study for which baseline biomedical and physical assessments were carried out 2006–2013, in 502 682 adults aged 40–69 years recruited from 22 UK centres.EPIC-Norfolk was the UK component of the European Prospective Investigation into Cancer (EPIC) study.10.1136/bjo-2022-321842.supp1Supplementary datahttps://www.ukbiobank.ac.uk/enable-your-research/about-our-data/health-related-outcomes-data).The primary outcome was circulatory mortality as defined using International Classification of Diseases (ICD) (ICD-10 codes I00-I99 and ICD-9 390-459) coded death registry data from the Office for National Statistics and the Health and Social Care Information Centre  for England and Wales, and the Information Services Department for Scotland, provided information on date and cause(s) of death to 31 January 2018 for UKB and 31 March 2018 for EPIC-Norfolk. Incident MI and stroke events after retinal image capture were based on medical records linkage with hospital diagnoses of non-fatal events, supplemented with participant health and lifestyle questionnaire data from repeat surveys in UKB and EPIC-Norfolk (2012–2018). ICD-10 codes I21-I25  were used for fatal and non-fatal MI; and ICD-10 codes I60, 61, 63, 64  for ischaemic and haemorrhagic stroke , tortuosity (arbitrary units)A validated, fully automated AI-enabled system (QUARTZ)Statistical analyses were carried out using STATA software . Retinal vessel widths and area showed normal distributions, tortuosity required log-transformation and within-vessel-width-variance required inverse square-root transformation to normalise distributions. Models were developed in UKB for men and women separately throughout, and externally validated in EPIC-Norfolk. We hypothesised that retinal vessel characteristics in relation to circulatory mortality might be modified by age, smoking status, presence of CVD/diabetes and use of BP lowering medications. Hence, two-way interactions between RV and age, smoking status and self-reported use of BP medication, prevalent diabetes and CVD were first examined in mutually adjusted Cox proportional hazard2 statistic (representing a measure of explained variation)2 and calibration slopes and baseline hazard. Model performance was graphically assessed from plots of the observed probability of event at 5 years by deciles of predicted risk at 5 years in UKB and by octiles in EPIC-Norfolk.Bootstrapping with 100 replications was used for internal validation to adjust model performance measures for optimism, including Harrel’s C-statistic for discrimination, RFRS for incident fatal and non-fatal stroke use age, systolic BP, treatment of hypertension, presence of diabetes and smoking statusSensitivity analyses restricted model development and validation to white ethnicity. Using EPIC-Norfolk, external validation was extended to a broader spectrum of incident cerebrovascular disease  and incident ischaemic heart disease . We followed Transparent Reporting of a multivariable prediction model for Individual Prognosis or Diagnosis guidelines for reporting of model development and validation.2 (0.33–0.44) statistics in UKB and EPIC-Norfolk, were reasonably high  and Rbly high . UKB wombly high .2 (less so in EPIC-Norfolk men). Observed risks were more aligned with predicted risks in men than in women  of retinal images captured by non-experts providing ‘vasculomic’ indices of vascular health. External validation in an older higher risk cohort (EPIC-Norfolk) replicated the findings, and models were also robust to inclusion of a wider spectrum of cerebrovascular and ischaemic heart disease events.UKB and EPIC-Norfolk are ‘healthy’ cohorts with relatively low event rates compared with other geographically similar middle-aged cohorts.Retinal imaging is established within clinic and hospital eye care and in optometric practices in the US and UK. AI-enabled vasculometry risk prediction is fully automated, low cost, non-invasive and has the potential for reaching a higher proportion of the population in the community because of ‘high street’ availability and because blood sampling or sphygmomanometry are not needed. RV is a microvascular marker, hence offers better prediction for circulatory mortality and stroke compared with MI which is more macrovascular, except perhaps in women. In the general population it could be used as a non-contact form of systemic vascular health check, to triage those at medium-high risk of circulatory mortality for further clinical risk assessment and appropriate intervention. In 2017–2018 in the UK, 41% of 40–74 years old attended their primary care NHS Health Check, which includes QRISK based screening for CVD.


Although single-cytokine inhibitors can be considered in treating severe or refractory Behçet disease (BD), these biologic agents are associated with potential therapeutic failure due to the multi-cytokine pathogenesis involving Th1- and Th17-type cytokines with activated Janus kinase/signal transducer and activator of transcription signaling pathways. Notably, there is an increasing trend toward the use of small-molecule targeted drug tofacitinib (TOF), a pan-Janus kinase inhibitor, with immediate-release formulations for treating patients with severe or refractory systemic vasculitis involving different vessel sizes. Despite no reported efficacy of extended-release formulations in refractory BD yet, such a dosage form has pharmacokinetic parameters that are comparable to those of conventional immediate-release formulations.We report the case of a 27-year-old local woman with recurrent manifestations of arthritis, orogential ulcerations, papulopustular lesions, and anterior uveitis. She was diagnosed with BD for more than 3 years, and received long-term corticosteroids plus immunosuppressants therapy with the complication of opportunistic candidiasis infection.Under extended-release TOF 11 mg once-daily therapy, the patient achieved disease remission while sparing the use of corticosteroids during follow-up.Our clinical observations implicate the oral convenience and therapeutic efficacy of extended-release TOF formulations in controlling the disease activity of BD. Biologic agents and small molecule-targeted drugs such as anti-tumor necrosis factor (TNF) monoclonal antibodies (mAbs) and apremilast have been recommended by the European League Against Rheumatism to treat severe or refractory disease. Tofacitinib (TOF), the first small-molecule pan-Janus kinase (JAK) inhibitor, was approved by the Federal Food and Drug Administration (FDA) in November 2012 for the treatment of rheumatoid arthritis (RA) with moderate to high activity and an inadequate response to methotrexate. Since individual cytokine receptors can recruit their own combinations of JAKs to activate distinct processes in targeted cells, antagonizing a specific JAK can inhibit more than 1 cytokine pathway, thereby expanding the therapeutic efficacy. Based on the distinct mechanisms of action, JAK inhibitors are at a focus of research in treating miscellaneous autoimmune and inflammatory disorders, and have the potential to provide positive outcomes by sparing the use or minimizing the dosages of corticosteroids (CS) and nonspecific immunosuppressants.Behçet disease (BD), first described by Hulusi Behçet from Istanbul in 1937, is an autoimmune systemic vasculitis affecting small and large vessels with widespread presentations including recurrent orogenital ulcerations and articular, cutaneous, gastrointestinal, neurological, ophthalmological, and vascular involvement.–9 Notably, extended-release formulations have pharmacokinetic parameters, tolerability and safety that are comparable to those of conventional immediate-release formulations. Herein, we report a case of refractory BD with articular, mucocutaneous and ocular manifestations under long-term CS plus immunosuppressants therapy complicated by opportunistic candidiasis infection. She received extended-release TOF 11 mg once-daily therapy, leading to disease remission while sparing the use of CS.Despite not an indicated use, there is an increasing trend toward using TOF with immediate-release formulations in difficult-to-treat systemic vasculitis involving different vessel sizes, including antineutrophil cytoplasmic antibody-associated vasculitis, BD, polyarteritis nodosa, and Takayasu arteritis (TAK).2 Furthermore, despite under a long-term prescription of CS plus colchicine and immunosuppressants such as methotrexate, there were recurrent articular, mucocutaneous and ocular activities. Except for BD, the patient had no other co-morbidities. At admission, physical examination revealed a body temperature of 37.5°C, injected eyes and multiple irregular-shaped ulcers over the tongue, gingiva, palate, and lips as well as whitish patches over the tongue, palate and throat  and oral fluconazole/nystatin were prescribed for disease activity and oropharyngeal candidiasis, respectively, with clinical improvements. After discharge, she underwent regular follow-up at the outpatient rheumatology clinic with an additional prescription of azathioprine (2 mg/kg/d). Nevertheless, a relapse of disease activity occurred with articular, mucocutaneous, and ocular manifestations during tapering down the daily CS dosages. Ophthalmological consultation revealed iritis without chorioretinitis, which was suggestive of anterior uveitis. Owing to the reported efficacy of TOF for refractory BD and patient's preference for once-daily therapy, we initiated extended-release TOF 11 mg once-daily treatment in June 2021. During follow-up, she had disease remission with normalized CRP levels, ESR and leukocyte counts. The daily use of prednisolone was discontinued with TOF monotherapy for more than 6 months.A 27-year-old local woman was admitted to the Internal Medicine Department of the National Cheng Kung University Hospital with a 2-week history of orogenital ulcers, polyarthralgia and swallowing pain. Before her visit, she was diagnosed with BD at other hospitals for more than 3 years, based on the manifestations of arthritis, orogential ulcerations, papulopustular skin lesions, and uveitis.oat Fig. . There w3 It has been demonstrated that both Th1 and Th17 activities are upregulated in BD,,14 similar to the T-cell responses in RA, a systemic rheumatic disease with the therapeutic indication of JAK inhibitors. The activation of JAK/STAT signaling pathways in BD is mediated through the interaction of Th1- and Th17-type cytokines with their individual surface receptors on the targeted cells.,17 Although single-cytokine inhibitors including anti-TNF mAbs and interleukin (IL)-6 blockers can be considered in severe or refractory BD, these biologic agents are associated with potential therapeutic failure due to its multi-cytokine pathogenesis. Notably, TOF has been shown to treat autoimmune diseases by suppressing the differentiation of pathogenic Th1 and Th17 cells through the inhibition of T-bet and STAT1 activation. Interestingly, owing to the multi-cytokine mechanism involving Th1 and Th17 pathways, therapeutic failure under anti-TNF or anti-IL-6 therapy has been observed in TAK patients resistant to conventional disease-modifying anti-rheumatic drugs. In 13 patients with refractory TAK including 11 treated earlier with biologic agents, 10 had clinical benefits after receiving TOF therapy for 2 to 18 months.Cytokines produced by T helper (Th) cells, Th1 and Th17, can bind to types I and II cytokine receptors associated with JAKs, causing their phosphorylation and further recruitment of signal transducer and activator of transcription (STAT) proteins to modulate gene transcription in pathogenic cells involved in autoimmune diseases. Among them, 5 cases had ever received anti-TNF mAbs and 1 was treated earlier with an IL-6 blocker. Their disease activities improved after a follow-up period of 5 to 21 months, with remission in articular and cardiovascular manifestations. The lack of effects on their gastrointestinal involvement might be attributed to the resemblance of such a manifestation in BD to Crohn disease for which TOF is known to be ineffective. Moreover, another report investigated 13 patients with severe posterior uveitis including 6 refractory to anti-TNF therapy. They received 30 mg daily doses of prednisone and 5 mg TOF 2 times a day, resulting in a rapid and sustained improvement in visual acuity. Despite no cardiovascular, gastrointestinal involvement and posterior uveitis in our reported patient, extended-release TOF 11 mg once-daily therapy led to disease remission with weaning off the daily use of CS.A recent study in BD examined the efficacy of immediate-release TOF 5 mg twice-daily therapy under preserved background medications in 13 patients with articular, cardiovascular, and/or gastrointestinal manifestations refractory to conventional disease modifying anti-rheumatic drugs.,22 On the basis of the equivalence in the area under the plasma concentration-time curve between the 2 formulations with evidence from the exposure-response relationship that area under the plasma concentration-time curve is the relevant parameter for clinical responses, extended-release TOF formulations with osmotic drug delivery were granted regulatory approval by the FDA for RA therapy in February 2016 without the need for advanced clinical trials. Later on, the FDA approved new indications for such formulations in treating patients with active diseases of psoriatic arthritis, ulcerative colitis and ankylosing spondylitis in December 2017, December 2019 and December 2021, respectively. Despite not an indicated use, extended-release TOF has been shown to have therapeutic effects that are not inferior to immediate-release formulations in refractory TAK. In addition, extended-release formulations enable less frequent dosing than immediate-release formulations. Collectively, these findings suggest that extended-release TOF can provide the less frequent oral dosing convenience with similar efficacy in comparison with immediate-release formulations in treating refractory systemic rheumatic diseases.Although there are no available data demonstrating the superiority of TOF over biologic agents in treating refractory BD patients, using this small-molecule targeted drug is associated with oral convenience and therapeutic efficacy as demonstrated in previous studies and this reported case.4There is an increasing trend toward the use of TOF with immediate-release formulations in refractory systemic vasculitis involving different vessel sizes. We report the case of 27-year-old local woman with BD. She had recurrent articular, mucocutaneous and ocular manifestations, and received long-term CS plus immunosuppressants therapy complicated by the opportunistic candidiasis infection. Under extended-release TOF therapy, the patient achieved disease remission with sparing the use of CS. Our clinical observations implicate the oral convenience and therapeutic efficacy of extended-release TOF formulations in controlling the disease activity of BD.The authors are indebted to the doctors and nurses involved in the management of the reported case at the National Cheng Kung University Hospital.Conceptualization: Chrong-Reen Wang, Tak-Wah Wong.Data curation: Chrong-Reen Wang, Tak-Wah Wong.Formal analysis: Chrong-Reen Wang, Tak-Wah Wong, Sheng-Min Hsu.Investigation: Chrong-Reen Wang, Tak-Wah Wong, Sheng-Min Hsu.Supervision: Chrong-Reen Wang.Validation: Chrong-Reen Wang, Tak-Wah Wong, Sheng-Min Hsu.Writing – original draft: Chrong-Reen Wang.