A new approach by investigating the intra-tumoral microbiome raised great interest because they may influence the host immune response and natural history of the disease. However, previous studies on the intra-tumoral microbiome of pancreatic ductal adenocarcinoma (PDAC) were mostly based on examining the formalin-fixed paraffin-embedded tumor specimens. This study aims to investigate the feasibility of using endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) as a complementary procedure of surgical biopsy to obtain adequate fresh pancreatic cancer tissue for intra-tumoral microbial research. This was a prospective pilot study performed at a single tertiary referral center. We obtained pancreatic cancer tissue by EUS-FNB and surgical biopsy, respectively. We amplified the V3-V4 hyper-variable region of bacterial 16S ribosomal ribonucleic acid (rRNA) genes, constructed a pair-end library, and performed high-throughput sequencing. From August 2020 to November 2020, nine eligible patients with PDAC were enrolled in this study. The intra-tumoral microbiome profile was successfully generated from the PDAC cancer tissue obtained by EUS-FNB as well as by surgical biopsy. There was no significant difference in intra-tumoral alpha-diversity or bacterial taxonomic composition between tissues obtained by EUS-FNB and by surgical biopsy. EUS-FNB can collect sufficient fresh cancer tissue for microbiome analyses without complication. The intra-tumoral microbiome profile in tissues obtained by EUS-FNB had similar alpha-diversity and taxonomic profiles with those obtained by surgical biopsy. It implicated, except for surgical biopsy, EUS-FNB can be another valid and valuable tool for studying intra-tumoral microbiome in patients with resectable and unresectable PDAC. Moreover, cancer patients seem to harbor a specific microbiome composition in the tumor niche which differs from healthy controls7. Nejman et al. found that intra-tumoral microbiome composition is diverse and cancer type-specific4. Riquelme et al. disclosed the intra-tumoral microbiome composition of pancreatic ductal adenocarcinoma (PDAC) patients. They identified a specific intra-tumoral microbiome signature predicting the long-term survivorship of PDAC6. However, studies on the intra-tumoral microbiome of PDAC were mostly based on examining the formalin-fixed paraffin-embedded (FFPE) tumor specimens obtained from patients who underwent surgical resection6. The risk of contamination with the environmental microbiota can hardly be avoided when handling the FFPE tumor specimens in a retrospective way. Ensuring that fresh tumor tissues are obtained in a sterile way is the basis of microbial research of PDAC.In recent years, there was a remarkable increase in the number of studies investigating the gut microbiome and cancer. It ranged from oncogenesis, cancer progression, outcome prediction to resistance to anticancer therapies8. Recently, EUS-FNB has assumed a growing role in the diagnosis and management of PDAC10. It can usually provide sufficient materials for cytological and histological examination of cancerous tissue in patients with unresectable PDAC13.PDAC is usually diagnosed late or detected until with metastases, and only a small proportion of PDAC patients can receive curative surgery. Therefore, most PDAC patients cannot provide fresh and sufficient cancer tissue for microbial studies. Endoscopic ultrasound-guided tissue acquisition become the irreplaceable tool in the diagnostic algorithm of solid pancreatic lesions. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) have the same safety profile, but EUS-FNB bring the better diagnostic accuracy than EUS-FNAWhether EUS-FNB can be used as a complementary procedure to obtain adequate fresh pancreatic cancer tissue to investigate the intra-tumoral microbiome remained unclear. Therefore, we conducted a prospective study to investigate the intra-tumoral microbiome profile of pancreatic cancerous tissue obtained by EUS-FNB and surgical biopsy. We compared the yield rates, adverse events, and complications between these two procedures.This was a prospective pilot study performed at a single tertiary referral center . All EUS-FNB procedures and surgical operations were performed at China Medical University Hospital in accordance with the guideline of European Society of Gastrointestinal Endoscopy and operative regulations, respectively. The patient considered eligible for this study included patients with suspected pancreatic cancer by computed tomography or magnetic resonance imaging. Patients were excluded if they were unable to provide informed consent or were using antibiotics or probiotics before the procedure. Written informed consent was obtained from each patient or family. This study was approved by the Institutional Review Boards, China Medical University Hospital. (CMUH109-REC3-026).2O, and were immediately sent for microbial analysis.During the surgical operation, we performed an ultrasound-guided core-needle biopsy for the pancreatic cancerous tissue with a 14-gauge needle. All surgical specimens were sterilely immersed in the lysis buffer containing 2.5% tris-HCI, 2.0% EDTA, 0.5% sodium dodecyl sulfate, and 95% distilled HEUS-FNB was performed by an experienced endoscopist using a linear array echoendoscope  while the patient was conscious sedated. The pancreatic lesion was carefully examined to assure no major vessels within the needle pathway before puncture in color Doppler mode. A 22-gauge needle  was used for tissue sampling. The pancreatic lesion was identified and then punctured under EUS guidance. When the needle was inserted into the lesion, the stylet was slowly withdrawn. The FNB specimens were collected without negative pressure after 20 to 40 back-and-forth movements by fanning technique. In the first pass, the tissue specimens were immediately immersed in the lysis buffer, and it was delivered immediately for microbial analysis. In the second and third passes, the tissue specimens were collected and fixed with 10% neutral buffered formalin for histological examination.14, with a minimum overlap of read pair set at 8 base pairs (bp). Merged reads were quality-filtered with Mothur (v1.34.1)15 to remove reads shorter than 400 bp or longer than 550 bp, as well as reads with a minimum average quality score lower than 27. In addition, reads containing an ambiguous base or homopolymer exceeding 8 bp were excluded. Chimera detection was performed using USEARCH (reference mode and 3% minimum divergence) and removed from further analysis. Quality-filtered and non-chimeric reads were analyzed (UPARSE pipeline)16 to generate operational taxonomic units (OTUs) per sample (at 97% identity level). The OTU representative sequences were searched against the Greengenes 13_5 database by using USEARCH global alignment to identify the corresponding taxonomy of the best hit. OTUs without a hit or with only a weak hit, that is, the function “/2” less than 9317, was excluded from the following analysis. Diversity indices  were estimated with the R package phylosea18.Tumor samples were kept on ice and transferred to a laboratory for DNA extraction in accordance with manufacturer’s protocol, which was done by using Real Genomics DNA Extraction Kit YGE100R . The isolated DNA aliquot was stored at − 80 °C before 16 s ribosomal ribonucleic acid (rRNA) gene sequencing. DNA concentration and quality were evaluated by NanoDrop ND-1000 . The hypervariable region V3-V4 of bacterial 16S rRNA genes was amplified by polymerase chain reaction using bar-coded universal primers 341F  and 805R . Library construction and sequencing of amplicon DNA samples were committed to Germark Biotechnology . A pair-end (2 × 300) library (insert size of 465 base pairs for each sample) was constructed with TruSeq Nano DNA Library Prep kit , and high-throughput sequencing was performed on an Illumina MiSeq 2000 sequencer with MiSeq Reagent Kit v3 (Illumina). The bioinformatics analysis of 16S rRNA amplicon was conducted by Germark Biotechnology . Briefly, on a per-sample basis, paired-end reads were merged using USEARCH (v8.0.1623)19. A p-value less than 0.05 was considered statistically significant.The alpha-diversity in the tissues obtained by EUS-FNB and surgical biopsy was compared using the Mann-Whitney test. Hierarchical clustering  of microbiomes was conducted using the Bray-Curtis distance of OTU-level relative abundance profile, based on which principal coordinates analysis (PCoA) was also performed using the R package ade4From August 2020 to November 2020, nine eligible patients with PDAC were enrolled in this study. They were six men and three women, with a mean age of 61.8 (47–76) years. Five patients had PDAC located in the head of pancreas, one in the body, and three in the tail. Five PDAC patients were diagnosed with stage III and four with stage IV. We used EUS-FNB to obtain pancreatic cancer tissues from six patients. We obtained tissue samples from four patients during surgery. One patient underwent both EUS-FNB and surgery. This patient underwent EUS-FNB, and the result revealed atypical glands initially; the patient was later confirmed as adenocarcinoma by a subsequent surgical operation. Five patients  who underwent EUS-FNB were confirmed PDAC, while all four patients  in the surgical group were diagnosed as PDAC. There was no internal bleeding, pancreatitis, and other adverse events after EUS-FNB. No internal bleeding nor other complications occurred in subjects who underwent surgery.The intra-tumoral microbiome profile was successfully generated from the PDAC cancer tissue obtained by EUS-FNB as well as by surgical biopsy. For microbial profiling, a total of 1.2 million pair-end reads were generated, of which 962 thousand reads passed quality filtering and were not chimera. To determine microbial diversity and composition, reads were aligned to the Greengenes database, and non-bacterial sequences were removed. The number of observed OTUs and the intra-tumoral bacterial diversity  were not significantly different between the two groups  which was predictive of long-term survivor in PDAC. They demonstrated that PDAC microbiome composition, which cross-talked to the gut microbiome, could influence the host immune response and natural history of the disease6. Chakladar et al. outlined the intra-tumoral microbiome of 187 PDAC samples through large-scale sequencing data from The Cancer Genome Atlas (TCGA), and they found potentially cancer-promoting or immune-inhibiting microbes—most of them belonged to Proteobacteria phylum30. Nejman et al. reported that intra-tumoral microbiome composition is diverse and cancer type-specific. They analyzed the intra-tumoral microbiome of 1,526 samples from seven human tumor types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. Bacteria belonging to the Firmicutes and Bacteroidetes phyla were the most abundant species in colorectal tumors, while Proteobacteria dominated the microbiome of PDAC4.Recently, a new approach by investigating the intra-tumoral microbiome raise great interest. Geller et al. showed the presence of 6. The majority of PDAC patients were diagnosed at advanced stages and thus precluded surgical resection. Masi et al. compared the results of microbiome profile in FFPE specimens obtained by surgical biopsy and EUS-FNB using Decontam31 (http://github.com/benjjneb/decontam), an open-source R package, to remove contaminant DNA sequences. They found there was no significant difference in alpha-diversity, beta-diversity, or taxonomic profiles between EUS-FNB and surgical biopsy in three patients with matched samples32. They claimed that EUS-FNB could substitute surgical biopsy in the PDAC tissue sampling for microbial research. This prospective study confirmed that both two methods can collect sufficient cancer tissue for microbiome analyses.However, previous studies on the intra-tumoral microbiome of PDAC were mostly based on the FFPE tumor specimens obtained during surgical resection8, and the EUS-FNB with the newest generation of needles has better histological procurement yield than older ones while performing solid pancreatic lesion biopsy34. In our study, we collected fresh PDAC tissue via EUS-FNB with newest generation of EUS-FNB needles and surgical biopsy. Both EUS-FNB and surgical biopsy can collect sufficient fresh cancer tissue for microbiome analyses without major complication. Furthermore, the intra-tumoral microbiome profile from EUS-FNB had similar alpha-diversity and taxonomic profiles with surgical biopsy. There are some limitations of our study including the small number of patients and the possible rare bias from 16S rRNA amplicon sequencing. Thus, further larger studies with whole genome sequencing are warranted.Since the EUS-FNB has the better diagnostic accuracy than EUS-FNAIn conclusion, except for surgical biopsy, EUS-FNB can be another valid and valuable tool for studying intra-tumoral microbiome in patients with resectable and unresectable PDAC.


Stimulator of interferon genes (STING) is currently recognized as a driver for anti-tumor immunity against various malignancies and is expected to enhance the anti-tumor effects. In this review, we summarized recent knowledges gained from epigenetics-mediated skin cancer development and discussed the clinical application of STING agonists in the treatment of skin cancer.Recent developments in immunotherapy against malignancies overcome the disadvantages of traditional systemic treatments; however, this immune checkpoint treatment is not perfect and cannot obtain a satisfactory clinical outcome in all cases. Therefore, an additional therapeutic option for malignancy is needed in oncology. Stimulator of interferon genes (STING) has recently been highlighted as a strong type I interferon driver and shows anti-tumor immunity against various malignancies. STING-targeted anti-tumor immunotherapy is expected to enhance the anti-tumor effects and clinical outcomes of immunotherapy against malignancies. In this review, we focus on recent advancements in the knowledge gained from research on STING signaling in skin cancers. In addition to the limitations of STING-targeted immunotherapy, we also discuss the clinical application of STING agonists in the treatment of skin cancer. Immune cells circulate in the human body for protection against external stimuli, such as antigens and microorganisms, by innate immunity and antigen-specific acquired immune responses ,2. TumorType I interferon (IFN) is a representative inflammatory cytokine that activates the acquired immune response mediated by antigen-presenting cells and subsequently drives cytotoxic cell expansion and activation ,9. Type Among various type I IFN-inducible candidates, stimulator of IFN genes (STING) has been recently recognized as a strong type I IFN driver and it has shown anti-tumor immunity against various malignancies ,13. STINIn the present review, we focus on recent advancements in the knowledge gained from research regarding STING signaling in skin cancers. In addition to the limitations of STING-targeted immunotherapy, we also discuss the clinical application of STING agonists in skin cancer immunotherapy in the future.STING is a crucial positive immune driver that induces the production of type I IFN by triggering intracellular pathogens, such as viruses . STING-mAs an activation mechanism, cytosolic DNA becomes a trigger for the activation of cGAS-cGAMP-STING signaling ,16,17 F. CytoplaIn addition, the STING signaling pathway plays an important role in various skin diseases. Keratinocytes are major constitutive cells in the epidermis and regulate inflammatory responses against external environmental stimuli involved in various inflammatory skin diseases ,26,27,28There are recent updates in the research on STING in skin cancers. In this section, we refer to skin malignancies in the detailed molecular mechanism of STING-mediated anti-tumor immunity, such as melanoma, squamous cell carcinoma, Merkel cell carcinoma, and adult T-cell leukemia/lymphoma.Melanoma is a malignancy derived from melanocytes that shows unfavorable clinical outcomes following current treatment . Recent STING enhances anti-tumor immunity against melanoma mediated by IFN ,39,40,41Several studies suggest a potent additional therapeutic effect of STING signaling in combination with immunotherapy. cGAS-deficient mice showed impaired growth of B16 melanomas in response to PD-L1 antibody treatment . DepletiSTING is also responsible for the efficacy of chemotherapy in melanoma. 5-fluorouracil (5-FU) is a representative chemotherapeutic agent, and its responsiveness depends on intrinsic STING signaling in tumor cells and subsequent type I IFN production. Consistently, the deficiency of STING in tumor cells is related to the requirement of a higher dose of 5-FU to exert anti-tumor effects .Radiation is one of the therapeutic options for melanoma treatment, and combination therapy with an immune checkpoint inhibitor is expected to show abscopal effects against melanoma . ConsistCutaneous squamous cell carcinoma is a skin cancer derived from epithelial keratinocytes, and its advanced form has an unfavorable clinical course because of the limited number of therapeutic options in the current treatment ,57,58. ISTING has been identified as an intrinsic regulator of squamous cell carcinoma survival ,60. STINIn contrast, cytosolic DNA triggers chromosomal instability in tumor cells and enhances tumor metastasis. This depends on the activation of cGAS-STING signaling by cytosolic DNA-sensing stimulation .Activated-STING increases IFN production and enhances the expression of PD-1 pathway in vivo . STING iMerkel cell carcinoma is a rare cutaneous malignancy derived from neuroendocrine cells, and Merkel cells act as mechanoreceptors. Merkel cell carcinoma exhibits aggressive clinical behavior with a high mortality rate. Elderly and immunocompromised host conditions contribute to the development of Merkel cell carcinoma. Although there are a limited number of therapeutic options for Merkel cell carcinoma with distant metastasis and immune checkpoint inhibitor treatment, avelumab shows favorable clinical outcomes. Therefore, anti-tumor immunity against Merkel cell carcinoma is needed as an additional therapeutic option.The Merkel cell polyoma virus plays an important role in the oncogenesis of Merkel cell carcinoma, and its replication and/or transcription drive an innate immune response via cGAS-STING . FurtherAdult T-cell leukemia/lymphoma is a malignancy associated with human T-cell lymphotropic virus type I (HTLV-1)-infected mature CD4+ T-cells ,69. AdulIFN is a key therapeutic target for the innate immune response to viruses. IFN-α is a standard therapeutic option for adult T-cell leukemia/lymphoma with a combination of the nucleoside reverse transcriptase inhibitor zidovudine. Tax expression, which is responsible for the development of adult T-cell leukemia/lymphoma, suppresses the induction of IFN production by cGAMP synthase plus STING stimulation . STING eAlthough there have been no reports regarding the relationship between STING and its downstream cytokines, these are involved in the suppression of basal cell carcinoma ,80,81, dBasal cell carcinoma is a malignancy arising from the epidermal basal cells . AlthougIFN-α injection into the tumor is effective in basal cell carcinoma . IFN-α iDiffuse large B-cell lymphoma is a malignancy of B-cells and it is a common type of non-Hodgkin cutaneous lymphoma . CutaneoAlthough there have been no reports regarding STING and diffuse large B-cell lymphoma, IFNs have been used for treatment. Among IFNs, IFN-β shows more potent direct suppressive effects on tumor cell growth by inducing apoptotic cell death following DNA damage, caspase-3 activation, and the annexin V binding effect . TherefoMycosis fungoides are malignant tumors of T lymphocytes with epidermotropism in the skin ,100,101.Several studies have shown the therapeutic effects of IFN-α on mycosis fungoides . IFN-α eSézary syndrome is a T lymphocyte-derived malignancy characterized by erythroderma as a clinical manifestation ,99,102. Other effects of IFNs are those against tumor and anti-tumor immune responses. IFN-α/β enhances immunoglobulin production by B-cells. IFN-γ enhances NK cell activity, suppresses tumor growth, and reduces IL-4 production in patients with mycosis fungoides. Therefore, STING-targeted treatment is expected to show the effect of these downstream cytokines on mycosis fungoides.To obtain a more potent anti-tumor effect mediated by the STING signal, some modifications of the STING stimulator have been investigated. Cationic liposomes with varying surface polyethylene glycol (PEG) are used to encapsulate cGAMP to facilitate its cytosolic delivery, and antigen-presenting cells improve the cellular uptake of cGAMP and pro-inflammatory gene induction . BiodegrIn addition, several novel STING agonists have been developed for melanoma treatment. Manganese potentiates the anti-tumor immune response as a STING agonist . DimericCombination therapy with STING has been proposed for the treatment of melanoma. The phagocytosis checkpoint of signal regulatory protein α (SIRPα) and STING in antigen presentation cells enhance the anti-tumor immune response . STING sThere are several reports on STING-targeted therapeutic options. SOX2 acts as a STING inhibitor. SOX2 enhances STING degradation in an autophagy-dependent manner and subsequently suppresses the production of type I IFN. Consistently, SOX2 supports squamous cell carcinoma cell growth by suppressing anti-tumor immunity. A combination of a STING agonist with anti-PD-L1 antibody treatment enhances the tumor-specific cytotoxic lymphocyte reaction, thereby improving survival . NLRX1 iEpigenetic changes are chemical modifications of DNA and DNA-binding histones, which can modulate chromatin structure and gene transcription by exposure to environmental stimuli without changing DNA sequence information .STING signaling is not suitable for melanoma cells in terms of long-term tumor development because it triggers anti-tumor immunity. As one of the strategies for melanoma development, STING is suppressed in melanoma cells by epigenetic silencing of STING and cyclic GMP-AMP synthase , possiblIn the STING modification of epigenetic changes in squamous cell carcinoma, KDM4A inhibition activates immune responses to squamous cell carcinoma and enhances the therapeutic potential of anti-PD-1 antibody treatment for squamous cell carcinoma. KDM4A is a histone demethylase that targets histone H3 lysine 9 trimethylation (H3K9me3) and is associated with the development of squamous cell carcinoma growth and metastasis. KDM4A inhibition activates cGAS-STING signaling in tumor cells. Consistently, KDM4A inhibition with the combination of anti-PD-1 antibody treatment suppresses tumor growth and metastasis , suggestAlthough STING is anticipated as an additional immunotherapy treatment against skin cancers, downstream cytokines of IFNs are needed to boost immunotherapy against cancers. However, IFN-treated patients did not exhibit the dramatic therapeutic potential of IFN-β during the treatment of skin cancers. Therefore, it seems that STING might not show satisfactory clinical outcomes in all cases as a driver of the anti-tumor immune reaction to skin cancers.The current administration of IFN-β transiently increases its concentration in the human body. Therefore, sustainable IFN-β production from the host might have more potent anti-tumor effects against skin cancers. As so, STING agonist administration can drive IFNs; however, the treatment itself might limit the transient production of IFNs.Because various cells can react with IFNs, it is important for IFNs to react to anti-tumor immune cell-specific or tumor sites to obtain the maximum effects of STING. Therefore, systemic STING agonists might result in the same pitfall as systemic IFN treatment or subcutaneous injection of IFNs. A recent study identified that IFN-independent STING signaling enhances autophagy , which cIn addition, STING signaling activation might result in the enhancement of autoimmune reactions, as seen in immune checkpoint inhibitor treatment. STING activation has been observed in various autoimmune diseases, such as rheumatoid arthritis  and dermSTING is anticipated as a therapeutic candidate for the treatment of an advanced form of skin cancer; however, it remains unclear whether STING signaling is involved in the regulation of tumor development. On the other hand, STING-targeted treatment might have disadvantages, possibly autoimmune-mediated systemic reactions, indicating that clinicians might keep in mind this advantage of STING-targeted treatment for skin cancers. Furthermore, STING-mediated inflammation may also contribute to the development of another malignancy . Given t


We present a case of an 86-year-old woman who presented with a progressive quadriparesis two days after her second dose of Moderna SARS-CoV-2 vaccine, with cerebrospinal fluid (CSF) evidence of cytoalbuminocytological dissociation and electromyogram/nerve conduction studies (EMG/NCS) findings suggestive of acute axonal motor neuropathy. Her clinical symptoms did not improve with plasmapheresis.There appears to be a potential temporal association between the inoculation of mRNA-based SARS-CoV-2 vaccines and the development of Guillain-Barre Syndrome (GBS). Despite this possible association, infection prevention using highly effective mRNA-based vaccines remains highly recommended. Large epidemiological studies of SARS-CoV-2 vaccine-related adverse events are needed. Physicians should be aware of this possible temporal association since the prompt diagnosis and treatment of GBS can drastically improve outcomes.The aim is to report a case of axonal-variant GBS that was temporally associated with an mRNA-based SARS-CoV-2 vaccine. Guillain-Barre syndrome (GBS) is an immune-mediated polyradiculoneuropathy that often presents as an acute paralyzing illness preceded by an infection, or less commonly, immunizations . VariousAn 86-year-old woman with atrial fibrillation, diabetes mellitus type 2, aortic stenosis, and ischemic stroke with no residual deficits presented with lower extremity weakness and pain. Her neurological examination progressed from a subtle paraparesis to a dense lower-extremity predominant quadriparesis within one week. She had received the second dose of the Moderna SARS-CoV-2 vaccine two days prior to her onset of symptoms. Cerebrospinal fluid (CSF) analysis demonstrated albuminocytological dissociation (WBC < 1 cells/µL and protein of 129 mg/dL) consistent with GBS. The remainder of her CSF immunological studies was negative including protein electrophoresis, gram stain, glucose, viral PCR panel, and paraneoplastic antibody evaluation. Immunoserologies were negative including thyroid function tests and anti-ganglioside antibodies. Confirmatory electromyography and nerve conduction studies (NCS) were performed which demonstrated low amplitude right peroneal, tibial, and ulnar motor evoked responses with normal distal latencies, segmental conduction velocities, and sensory responses, consistent with acute axonal motor neuropathy. She was treated with plasmapheresis for five sessions without notable improvement in her strength.This case highlights a patient who developed a progressive quadriparesis two days after receiving the second dose of the Moderna mRNA-based SARS-CoV-2 vaccine, in line with the time course of previously reported cases of post-vaccine associated GBS . The rapOverall, there were very few neuroinflammatory events reported across all of the major SARS-CoV-2 vaccine trials, which used various platforms. Two cases of GBS occurred ten days after injection with both placebo and vaccine in the clinical trial for Johnson & Johnson’s vector-based vaccine . There wThere have been only a few reports of GBS after the administration of SARS-CoV-2 vaccines in clinical practice. One report involved a patient that developed symptoms two weeks after the administration of the Pfizer-BioNtech vaccine , and a sWhile causality between vaccine administration and the development of GBS in our patient cannot be proved, the onset of symptoms is consistent with previously reported cases of post-vaccination GBS. Overall, there have been very few cases of GBS reported after the administration of SARS-CoV-2 vaccines in all the safety trials, and only two reported cases in clinical practice to date. In the absence of large epidemiological studies of SARS-CoV-2 vaccine-related adverse events, the incidence of GBS after vaccine administration remains uncertain, although the paucity of cases suggest that it is likely rare. Since infections in general, including SARS-CoV-2, may trigger GBS, infection prevention using the highly effective mRNA-based SARS-CoV-2 vaccines remain highly recommended. Physicians should be aware of the possible temporal association between the SARS-CoV-2 vaccine and GBS since the prompt diagnosis and treatment of GBS can drastically improve outcomes.


Beta-catenin is linked with colorectal cancer (CRC). Therefore, it is of interest to design and develop novel compounds to combat CRC. Hence, we document compounds  from Lycopersiconesculentum with optimal binding features for further consideration. Colon and rectum carcinoma is a significant health concern globally, with more than 700,000 deaths each year [1 - see PDF]. Nearly 30% of CRC patients develop recurrences despite curative surgery .InfectiThe three-dimensional structure of Beta-catenin from Homo sapiens PDB: ID 1JDH downloaded from the protein databank (PDB) was used in this study .10 known compounds from the tomato plant have been selected from the literature for this Study (Table 1 - see PDF). The structures of these compounds were obtained from the PubChem Compound Database in the Spatial Data File (.SDF) format and converted to thePDB file format using the Online Smile Translator. Energy minimization of ligands was completed with ChemBio 3D Ultra 12.0 using standard procedure.Molecular docking analysis of beta-catenin with compounds derived from Lycopersicon esculentum was completed using the Hex 8.0.0. Docking software .The HEX docking results of selected compounds with beta catenin showed that they would have a great docking score  as shown in Table 2 (see PDF). The more negative the value, the more stable the complex is and the more binding affinity. Accordingto energy funnel theory, less energy represents extremely reliable conformation. More negative Etotal value indicates that there must be a substantial relationship between ligand and receptor, leading to the activation of receptor activity .The molThe resultant docked complexes were further examined with pymol to show the H-bond interactions between the selected compounds and the beta-catenin protein. The findings showed that many of the compounds displayed significant H-bonding interactions with beta-cateninthrough the amino acids SER-318, THR-330, GLY-397, MET-398, ASN-434, GLU-399, LYS-433, LYS-435, SER-473, ASN-516 and MET-437 . Higher We document compounds  from Lycopersicon esculentum with optimal binding features for further consideration to combat CRC.


Testing for SARS-CoV-2 antibodies is commonly used to determine prior COVID-19 infections and to gauge levels of infection- or vaccine-induced immunity. Michigan Medicine, a primary regional health center, provided an ideal setting to understand serologic testing patterns over time. Between 27 April 2020 and 3 May 2021, characteristics for 10,416 individuals presenting for SARS-CoV-2 antibody tests  were collected. Relative to the COVID-19 vaccine roll-out date, 14 December 2020, the data were split into a pre-  and post-vaccine launch  period and contrasted with untested individuals to identify factors associated with tested individuals and seropositivity. Exploratory analysis of vaccine-mediated seropositivity was performed in 347 fully vaccinated individuals. Predictors of tested individuals included age, sex, smoking, neighborhood variables, and pre-existing conditions. Seropositivity in the pre-vaccine launch period was 9.2% and increased to 46.7% in the post-vaccine launch period. In the pre-vaccine launch period, seropositivity was significantly associated with age ), ever-smoker status ), respiratory disease ), circulatory disease ), liver disease ), non-Hispanic Black race/ethnicity ), and population density ). Except for the latter two, these associations remained statistically significant in the post-vaccine launch period. The positivity rate of fully vaccinated individual was 296/347). Testing for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) broadly falls into two categories: (1) reverse transcription polymerase chain reaction (RT-PCR) testing for active infection  and (2) immunoglobulin G or M (IgG or IgM) antibody testing for SARS-CoV-2 antibodies. While the former is crucial for identifying acute, likely contagious cases, the latter is important for determining prior infection status. In addition, these tests can help to estimate the duration of infection-induced antibody protection/immunity against COVID-19 and to understand the extent of community spread  ,4. With Recent works have examined COVID-19 outcomes  and testTo consider the influence of vaccinations on serology testing, we split the testing data into a pre-vaccine launch period, before 14 December 2020—the date when the first approved COVID-19 vaccines became publicly available to members of the University of Michigan Health System—and a post-vaccine launch period, after 14 December 2020. Specifically, this study seeks to: (1) describe the frequency and pattern of serologic testing at Michigan Medicine over the course of the pandemic; (2) identify characteristics associated with having an antibody test or seropositivity in the pre-vaccine launch period; (3) understand the sequences of results for both diagnostic RT-PCR and serologic testing in the pre-vaccine period; (4) identify factors associated with seropositivity in the post-vaccine launch period; and finally (5) use the Pfizer-BioNTech, Moderna, and J&J’s Janssen vaccine data to compute vaccine-induced seropositivity in terms of the rate of and time to seropositivity. As an exploratory aim, we compare seropositivity across age groups 0–18 years in pre- and post-vaccine launch period. Since vaccination was not available for these age groups by the end date of our analytic period (3 May 2021), this comparison gives us a sense of changes in infection-induced immunity in children and adolescents over this time period.Our study sample includes 10,416 individuals who presented to Michigan Medicine for spike protein immunoglobulin G (IgG) SARS-CoV-2 antibody testing between 27 April 2020 and 3 May 2021 . The tests’ Logical Observation Identifiers Names and Codes (LOINC) identifier was “94505-5”. Two time periods were defined: before (pre-vaccine launch) and on/after 14 December 2020 (post-vaccine launch), the date when COVID-19 vaccines roll-out began at Michigan Medicine. In the pre-vaccine launch period (27 April–14 December 2020), 8026 individuals presented for 8220 SARS-CoV-2 antibody tests, while in the post-vaccine launch period (14 December 2020–3 May 2021), 2587 individuals presented for 2712 SARS-CoV-2 antibody tests . For theAdditionally, we utilized a cohort of unmatched controls for comparison. Unmatched controls were defined as individuals without any record of diagnostic (RT-PCR) testing or serologic testing for SARS-CoV-2 at Michigan Medicine. The control cohort was randomly selected at a case -control ratio of 1:3. Individual demographic and clinical characteristics, testing rates, test results, and health outcomes were collected from the electronic medical record (EMR) on 3 May 2021. Sequences of individual-specific test results were derived from laboratory records. This cross-sectional study was approved by the committee for research ethics and compliance at Michigan Medicine and followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines . Study pWe first described the patterns of serological testing for COVID-19 in our study population by summarizing the frequency of daily tests performed between 27 April 2020 and 3 May 2021. Pre- and post-vaccine launch periods were defined relative to the COVID-19 vaccine roll-out date, 14 December 2020. In addition, we compared the seropositivity rates across these two periods. In the COVID-19 post-vaccine launch period, we excluded individuals who had positive serology test results in the pre-vaccine launch period to only capture novel COVID-19 infections that occurred; furthermore, because the vaccination data of unmatched controls were not available, we limited the analysis of the COVID-19 post-vaccine launch period to the individuals who had serology test results before the first vaccination dose or had no documented vaccination. Specifically, we categorized tested people into three groups: children (<12 years), adolescent (≥12 and <18 years), and adults (≥18 years) because vaccination was not available for children and adolescent groups by the end date of our analytic period (3 May 2021).2), sex , race/ethnicity , smoking status , and indicators for enrolling in a COVID-19 research study led by investigators at the University of Michigan. The last variable was included, as this sub-population may have different motivation for having a test than the remaining participants. Additionally, we adjusted for seven pre-existing comorbidities extracted from the EMR: respiratory diseases, circulatory diseases, any cancers, type 2 diabetes, kidney diseases, liver diseases, and autoimmune diseases as yes/no indicators as described previously [In our association analysis, individual characteristics included age (years), body mass index  on all demographic and clinical characteristics listed above. In order to make the selection of unmatched controls more representative and less biased, when fitting the logistic regression, the set of unmatched controls were randomly selected 20 times from the patients who never underwent any COVID-19-related tests, and the case control ratio was fixed as 1:3 each time. We finally pooled 20 estimates from each logistic regression model into a single set of estimates using R package mice . To identify factors associated with serology test positivity in the pre-vaccine launch period, we first compared individual characteristics between positive versus negative serology-tested individuals using a test negative design (TND). Bivariate tests were conducted to evaluate whether the distributions of characteristics differed. In order to avoid selection bias for getting tested, we further compared positive serology-tested individuals versus unmatched controls (case control-(CC)-POS) in a fully adjusted logistic regression model . We regrTo explore relationship between having RT-PCR tests (diagnostic) and serologic tests, we studied the sequences of testing results for each test among individuals with at least one serology test. In this analysis, only test results before vaccination  were included to avoid effects of vaccination. Between 14 December 2020 and 3 May 2021, a large fraction of the serologic testing was likely performed to confirm antibody response to the vaccine. Therefore, we first presented descriptive statistics for all individuals with at least a serologic test and unmatched controls to identify factors associated with having a serology test in the post-vaccine launch period. The adjusted association analyses with unmatched controls were further carried out to identify factors associated with tested individuals in the post-vaccine launch period as described for the analysis of the dataset from the pre-vaccine launch period. To identify factors associated with serology test positivity in the post-vaccine launch period, we first compared individual characteristics between positive versus negative serology-tested individuals using a test negative design (TND). The adjusted association analyses using CC-POS were then carried out to identify factors associated with serology test positivity in the post-vaccine launch period, consistent with the analysis from the pre-vaccine launch period. We used the available vaccination data on Pfizer-BioNTech, Moderna, and J&J/Janssen vaccines, where the first two vaccines required a priming dose, followed by a booster dose for most protection, while the third is a single-dose vaccine. We reviewed the adherence to the recommended vaccination schedule of the CDC. Furthermore, we determined serology positivity rates of individuals vaccinated with Pfizer-BioNTech, Moderna, or J&J/Janssen who never tested positive before vaccination to estimate the vaccine-mediated immunity at different time points . Finally, to have a rough idea of the length of time to develop a positive antibody response after vaccination, we calculated the distributions of time to first positive serologic test after an individual’s first and second doses, respectively.Between 27 April 2020 and 3 May 2021, 10,416 individuals presented to Michigan Medicine for at least a SARS-CoV-2 antibody test . As showSerology testing peaked in the summer of 2020, with 84.6 daily tests on average in August. After August, the number of daily tests decreased greatly . Furtherp-value < 0.05 in bivariate tests of association) and each of them except for BMI and pre-existing type 2 diabetes and kidney diseases remained significantly associated when adjusting for all other variables. For example, female sex, higher population density, and indictors of pre-existing conditions  were associated with an increased probability of having a serologic test, while age, ever-smoking status, and neighborhood SES variables  were associated with a decreased probability of having a serologic test . However, some of the associations were in the reverse direction than expected, questioning the potential bias due to tested individuals affecting both positive and negative groups. For example, circulatory diseases were more common in individuals with all negative tests (66%) than in individuals with at least one positive test result (62%). To account for selection bias and determine patient characteristics that are associated with the risk of a past COVID-19 infection, we fit the fully adjusted logistic regression model in the CC-POS design. We found seven characteristics that are associated in the CC-POS analysis . Specifically, age ) and ever-smoker ) had protective effects on serology test positivity, while non-Hispanic Black ), population density ), respiratory disease ), circulatory disease ), and liver disease ) increased the odds for testing positive . In the post-vaccine launch period, there were 2587 individuals with at least one serology test. Among them, 1456 (56%) received at least one vaccine dose, and 953 (37%) were fully vaccinated b, suggesTo only capture novel COVID-19 infections that occurred in the COVID-19 post-vaccine launch period, we excluded 54 individuals who had positive serology test results in the pre-vaccine launch period; furthermore, we limited the analysis of the COVID-19 post-vaccine launch period to a total of 2024 individuals who had obtained serology test results before the first vaccination dose  or had no documented vaccination . p-value < 0.05 in bivariate tests of association). When adjusting for other variables, age, sex, smoking status, neighborhood SES variables (unemployment and education rates), and pre-existing conditions  remained significantly associated . When determining patient characteristics that are associated with seropositivity in the post-vaccine launch period using a fully adjusted logistic regression model, we observed significant differences in age, smoking status, neighborhood education, respiratory disease, circulatory disease, kidney disease, liver disease, and autoimmune disease . The combination of diagnostic RT-PCR results and antibody test results can lend insight into asymptomatic individuals who never tested positive for active infection but happen to be seropositive. Seroconversion is of interest for many such reasons.In both pre-vaccine and post-vaccine launch period, we found that age, ever-smoking status, and neighborhood SES  were consistently associated with a decreased probability of having a serologic test, while female sex, higher population density, and indictors of pre-existing conditions  were consistently associated with an increased probability of having a serologic test. Seropositivity in the pre-vaccine launch period was 9.2% and increased to 36.2% (after removing tests after vaccination) in the post-vaccine launch period, an increasing trend that was observable in adults (9.1 to 37.1%) but also noted to some extent in adolescents (13.0 to 29.4%) and children (8.6 to 26.2%). The latter two age groups were not eligible for vaccination at the time of this analysis. Earlier studies claims that children made up a small percentage of individuals with COVID-19 . AccordiIn addition, we compared the observed seropositivity with the cumulative incidence of COVID-19 at proximal timeframe. In the pre-vaccine launch period, the number of accumulated reported COVID-19 cases before 14 December 2020 was 0.547 million in Michigan (estimated population of 9.99 million ) and 12,We further compared differences in factors associated with seropositivity in the CC-POS analysis and found five consistent factors in both pre- and post-vaccine launch periods: age and ever-smoking had a decreased odd for testing positive, while pre-existing respiratory, circulatory, and liver diseases increased the odds for testing positive. Particularly, ever-smoking status was associated with both getting serologic tests and receiving negative serologic results when tested. This finding might support an earlier study  that claSimilar results are noted with respect to individual sex and comorbidity burden in an early study of IgM-IgG antibody testing for COVID-19 . DiffereOur study also found that 528 individuals with an initial positive diagnostic test later tested positive for IgG antibodies before being vaccinated, with a median of 45 days (max: 368 days) between the first positive diagnostic test and last positive serologic test. This immunity results were consistent with previous findings ,25. HoweAnother aim of this study was to examine patterns of serologic testing results in a large cohort of individuals receiving the Pfizer-BioNTech, Moderna, or J&J’s Janssen vaccines. Results from early studies on the vaccines showed that serum IgG concentrations and SARS-CoV-2 neutralizing titers increased notably fourteen days after an individual’s second dose ,31. Our Additionally, we note that there are prevailing limitations in using electronic health records for research due to differing purposes for data collection and use. In particular, we cannot confirm whether the patients with no vaccination record were vaccinated elsewhere. Despite these limitations, this report presents a holistic description of serologic testing patterns and results at an academic medical center near a severely affected city  during the pandemic over one year after its onset. Factors associated with seropositivity in the pre- and post-vaccine launch cohorts included age, smoking status, respiratory disease, circulatory disease, and liver disease. The majority of fully vaccinated individuals in this study had developed antibody protection against COVID-19 after two weeks of their final dose.


Photodynamic therapy (PDT) for deep-seated lesion is seriously hindered by the limited depth of visible light penetration. Most recently, researchers have designed a genetically-encoded NanoLuc-miniSOG with internal light source for self-excitation, which is highly beneficial for deep PDT. The optical penetration depth in tissue is strongly dependent on light wavelength4. Even for light irradiation at 700–800 nm, the penetration depth is only around 5–6 mm.Photodynamic therapy (PDT) is a minimally invasive therapeutic modality, which can selectively destroy diseased tissues by reactive oxygen species (ROS) generated through chemical reaction of photosensitizer (PS) that absorbs irradiation light with an appropriate wavelength in the presence of molecular oxygen. However, the clinical PDT applications are still restricted to superficial and endoscopically accessible lesions since PDT for deep-seated neoplasms is seriously hindered by the limited depth of ultraviolet (UV)-visible (VIS) light penetration9. Secondly, the external excitation sources, such as X-ray, γ-ray and acoustic wave were utilized to excite PS based on direct excitation or resonance energy transfer (RET)11. Thirdly, the UV-VIS-NIR light with local light delivery systems was performed for interstitial PDT13. In addition, the internal self-excitation sources, including chemiluminescence, bioluminescence and Cerenkov radiation were investigated to overcome the limited depth of conventional PDT17. Differing from the first three excitation sources mentioned above, the internal self-excitation source not only avoids the need for an external excitation source, but also makes the limitless treatment depths achievable for PDT19. Therefore, the internal light source for deep PDT has recently received great attention.As shown in Table Light: Science & Applications20, entitled “Genetically-encoded BRET-activated photodynamic therapy for the treatment of deep-seated tumors”, by Proshkina’s group from Russian Academy of Sciences, reports a proof of concept for the genetically-encoded bioluminescence RET (BRET)-activated PDT19. To achieve this, they synthesized genetic NanoLuc-miniSOG BRET pair with the combination of NanoLuc luciferase flashlight and phototoxic flavoprotein miniSOG, and the ROS can be efficiently generated under luciferase substrate injection. The BRET ratio for NanoLuc-miniSOG pair was quantified to be around 0.74 ± 0.05, which indicates high efficiency was obtained for energy transfer in the BRET pair. The BRET-mediated cytotoxicity for the incubated BT-474/NanoLuc-miniSOG cells with membrane localization was obviously observed in the presence of furimazine and Riboflavin mononucleotide (RF), which is correlated with furimazine concentration and reaches 71% at 75 μM furimazine. To further demonstrate BRET-activated PDT in vivo, engineered BT-474 cells stably expressing NanoLuc-miniSOG BRET pair were used to generate tumors in BALB/c nu/nu mice, and the greatest tumor growth inhibition (TGI) of 72% for the BRET- PDT mice was achieved without external light irradiation. More importantly, HER2-specific lentiviruses (LVs) were chosen as the carriers to directly deliver NanoLuc-miniSOG gene into mice bearing tumor. As a result, the mice bearing BT-474 xenograft tumors treated with HER2-specific LV-NanoLuc-miniSOG have the average TGI value of 67%. These obtained results demonstrate that the genetically encoded NanoLuc-miniSOG pair offers a new paradigm for effectively initiating PDT for deep lesions.A recent research paper in The development of the genetically encoded PSs with internal light source provides a less complicated and cost-efficient strategy for efficient BRET-activated PDT, which has great potential for depth-independent PDT and would significantly expand the PDT scope of clinical indications. Moreover, the genetically encoded PSs can be modified with genetic therapy modalities and targeted with specific motifs to particular cellular organelle and cell type, providing the possibility for combination of PDT and immunotherapy.21, while the cytotoxicity of furimazine and its oxidized product furimamide should be minimized. As a consequence, further improvement should focus on the efficiency of the self-illuminating materials and BRET for higher ROS production. Second, given the physiological complexity of deep-seated neoplasms, more detailed investigations on the biological mechanisms of activation of NanoLuc-miniSOG pair are highly needed. Third, more dosimetric parameters are required to be optimized and standardized for achieving a satisfactory outcome since the treatment efficiency of NanoLuc-miniSOG pair for deep PDT has been complicated by the additional use of furimazine and RF.Despite the genetically encoded PSs with internal light source are rapidly developed in the past decade, there are still several technological challenges need to be thoroughly addressed before clinical translation. First, the ROS quantum yield for miniSOG as PS during photosensitization is extremely low in biological microenvironments


In December 2020, the vaccine was granted Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) as well as an interim recommendation for use among persons aged ≥16 years by the Advisory Committee on Immunization Practices (ACIP) (The Pfizer-BioNTech COVID-19 vaccine (BNT162b2) is a lipid nanoparticle-formulated, nucleoside mRNA vaccine encoding the prefusion spike glycoprotein of SARS-CoV-2, the virus that causes COVID-19. Vaccination with the Pfizer-BioNTech COVID-19 vaccine consists of 2 intramuscular doses  occurring in the 7 days after vaccination  were more likely to be reported among vaccine recipients (10.7%) than placebo recipients (2.3%) (relative risk = 4.7) . Among vhttps://www.cdc.gov/vaccines/acip/recs/grade/covid-19-pfizer-biontech-vaccine.html.From the GRADE evidence assessment, the level of certainty for the benefits of Pfizer-BioNTech COVID-19 vaccination among persons aged ≥16 years was type 1 (high certainty) for the prevention of symptomatic COVID-19, type 2 (moderate certainty) for prevention of hospitalization and death attributed to COVID-19, and type 4 (very low certainty) for prevention of asymptomatic SARS-CoV-2 infection . Regardihttps://www.cdc.gov/vaccines/acip/recs/grade/covid-19-pfizer-biontech-etr.html.Data reviewed within the EtR framework support the use of the Pfizer-BioNTech COVID-19 vaccine. The COVID-19 Vaccines Work Group concluded that COVID-19 remains an important public health problem and that the desirable effects of disease prevention via vaccination with Pfizer-BioNTech COVID-19 vaccine in persons aged ≥16 years are large and outweigh the potential harms. The Work Group determined that the vaccine is acceptable to vaccine providers and that implementation of vaccination is feasible. Moreover, full FDA approval might increase acceptability of the vaccine among unvaccinated persons. The Work Group also acknowledged that vaccine-eligible persons aged ≥16 years probably considered the desirable effects of vaccination to be favorable compared with the undesirable effects; however, there is likely important variability in vaccine acceptance within this age group, especially among those who are currently unvaccinated. The Work Group had varying opinions regarding the impact that a standard ACIP recommendation for Pfizer-BioNTech COVID-19 vaccine would have on health equity; most felt the impact might vary by subpopulation. The evidence used to guide the EtR is available at In summary, after 8 months of use under an FDA EUA and ACIP interim recommendation, the Pfizer-BioNTech COVID-19 vaccine, Comirnaty, now has full FDA approval and is recommended by ACIP for use in persons aged ≥16 years in the United States. Comirnaty has the same formulation and can be used interchangeably with the Pfizer-BioNTech COVID-19 vaccine used under EUA without presenting any safety or effectiveness concerns. ACIP considered new information beyond what was available at the time of the interim recommendation: an additional 4 months of follow-up of phase II/III clinical trial participants, 26 observational vaccine effectiveness studies involving hundreds of thousands of vaccinated persons, and two postauthorization safety monitoring systems that encompassed millions of vaccinated persons in the United States. The additional information increased certainty that the benefits of Pfizer-BioNTech COVID-19 vaccine outweigh vaccine-associated risks. The Pfizer-BioNTech COVID-19 vaccine continues to have FDA authorization for emergency use and ACIP interim recommendation for use in adolescents aged 12–15 years  that uses text messaging and online surveys to provide near real-time health check-ins after receipt of a COVID-19 vaccine. Adult vaccine recipients can register in v-safe, and parents or guardians can register their adolescent children in v-safe and complete the health surveys on their behalf. CDC’s v-safe call center follows up on reports to v-safe that include possible medically significant health events to collect additional information for completion of a VAERS report. Information on v-safe is available at https://www.cdc.gov/vsafe.Providers are required to report adverse events  that occur after receipt of any COVID-19 vaccine to VAERS  outweigh vaccine-associated risks.


Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that block immune inhibitory pathways. Administration of ICIs augments T cell-mediated immune responses against tumor, resulting in improved overall survival in cancer patients. It has emerged that the intestinal microbiome can modulate responses to ICIs via the host immune system and that the use of antibiotics can lead to reduced efficacy of ICIs. Recently, reports that fecal microbiota transplantation can lead to ICI therapy responses in patients previously refractory to therapy suggest that targeting the microbiome may be a viable strategy to reprogram the tumor microenvironment and augment ICI therapy. Intestinal microbial metabolites may also be linked to response rates to ICIs. In addition to response rates, certain toxicities that can arise during ICI therapy have also been found to be associated with the intestinal microbiome, including in particular colitis. A key mechanistic question is how certain microbes can enhance anti-tumor responses or, alternatively, predispose to ICI-associated colitis. Evidence has emerged that the intestinal microbiome can modulate outcomes to ICI therapies via two major mechanisms, including those that are antigen-specific and those that are antigen-independent. Antigen-specific mechanisms occur when epitopes are shared between microbial and tumor antigens that could enhance, or, alternatively, reduce anti-tumor immune responses via cross-reactive adaptive immune cells. Antigen-independent mechanisms include modulation of responses to ICIs by engaging innate and/or adaptive immune cells. To establish microbiome-based biomarkers of outcomes and specifically modulate the intestinal microbiome to enhance efficacy of ICIs in cancer immunotherapy, further prospective interventional studies will be required. Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that antagonize immune inhibitory pathways known as checkpoints. The most widely used ICIs are anti-cytotoxic T lymphocyte antigen-4 (CTLA-4), anti-programmed death protein-1 (PD-1), and anti-programmed death-ligand 1 (PD-L1) antibodies . These dThe human body harbors trillions of resident microbes that comprise the microbiome, which plays a vital role in many aspects of human health and disease. The intestinal microbiome plays a particularly important role in shaping systemic immune responses , 15. In Preclinical models have shown that the intestinal microbiome composition regulates efficacy of cancer therapies such as chemotherapy –18. InteBacteroides fragilis, Bacteroides thetaiotaomicron, Burkholderia cepacia, or the combination of B. fragilis and B. cepacia led to restoration of anti-CTLA-4 antibody-mediated anti-tumor responses [Bifidobacterium was associated with superior anti-tumor effects of anti-PD-L1 antibody [Bifidobacterium improved anti-tumor effects of ICIs by augmenting dendritic cell function and activating cytotoxic CD8+ T cells. Heat inactivation of Bifidobacterium before oral administration abrogated the effects on tumor and T cell responses, suggesting that specific live commensal bacteria may modulate responses to ICIs against tumor cells. Thus, these studies have successfully demonstrated that mouse-derived commensal bacteria can support anti-tumor effects in ICI therapy.Pre-clinical mouse models have been a critically important tool for studying the potential effects of microbes on ICIs. By comparing the anti-tumor efficacy of anti-CTLA-4 antibody in mice maintained under conditions in specific-pathogen-free (SPF) that are free of particular pathogens, and germ-free (GF) that harbor no living microorganisms, a pioneering study found that anti-CTLA-4 antibody treatment controlled tumor progression in SPF but not in GF animals . The antesponses . Anotherantibody . In addiFaecalibacterium prausnitzii and Gemmiger formicilis were associated with a positive response to anti-CTLA-4 antibody whereas Bacteroides were associated with poor response [Akkermansia muciniphila and Enterococcus hirae in patients with advanced non-small cell lung cancers or urothelial carcinomas [Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium were more abundant in melanoma patients responding to anti-PD-1 antibody whereas Ruminococcus obeum and Roseburia intestinalis were more abundant in non-responding patients [+ T cells in the intestine [+ T cell-dependent manner in a mouse model. Recently, the potential role of B cells within tertiary lymphoid structures in the response to ICIs was also reported in patients with melanoma and renal cell carcinoma [Subsequent studies focused on the human-derived commensal microbiome to identify specific bacteria that can improve anti-tumor efficacy. Metagenomic studies of patient stool samples have revealed significant associations between the intestinal microbiome and clinical responses in different solid cancers. In a study of metastatic melanoma patients, Chaput et al. showed that response . Gopalakresponse . Routy ercinomas . Matson patients . GF micepatients –28. Usinntestine . These barcinoma . HoweverAltogether, these studies indicate that the intestinal microbiome in both mice and humans can be important modulators of tumor responses to ICIs via modulation of the host immune system. Lack of consistency, however, in the key bacteria subgroups identified to be associated with tumor response in these studies, has been a major limitation and explanations for this inconsistency have yet to be firmly identified. While sequencing center heterogeneity in technical and computational procedures are known to impact substantially on microbiome readouts , 32, thiThere are many environmental and life style factors including dietary habits, smoking, and alcohol consumption that can influence the intestinal microbiome and immune system . SmokingTargeting the intestinal microbiome as a therapeutic strategy to augment ICI is still in its infancy, but progress has been recently reported. Two early single-arm clinical studies of FMT were found to potentially result in improved responses ICI therapy , 39. A pAs described above, preclinical experiments in mice have indicated that use of antibiotics can lead to reduced efficacy of ICIs , 24, 27.Intestinal microbial metabolites are thought to link the intestinal microbiome to systemic immunity. Researchers have begun to explore the relationship between host, diet, the intestinal microbiome, and microbial-derived metabolites in treatment with ICIs. A major metabolic activity of the intestinal microbiome is the conversion of ingested dietary fiber and mucosal glycans into short-chain fatty acids (SCFAs), which include acetate, propionate, and butyrate . SCFAs cTryptophan is an essential amino acid for humans and tryptophan metabolites are known to be bioactive compounds that play important roles in cancer and immune regulation. Most of dietary-derived tryptophan is taken up in the small intestine, but a fraction reaches the colon and is catabolized by the intestinal microbiome . Tryptop+ T cells and enhanced anti-tumor responses. A metabolic analysis found increased levels of several molecules, including mevalonate, dimethylglycine and inosine, in both cecal contents and the serum of GF mice inoculated with the consortium, compared to GF mice [Bifidobacterium pseudolongum-monocolonized GF mice who exhibited enhanced anti-tumor responses by ICIs [2AR signaling specifically in T cells.It also has emerged that the purine nucleoside inosine produced by the intestinal microbiome is associated with ICI therapy responses. In a preclinical model, Tanoue et al. demonstrated that GF mice inoculated with an 11-member bacterial consortium had increased IFNγ positive CD8 GF mice . What ro by ICIs . They fuIn contrast, some metabolites have been reported to have detrimental effects on ICI efficacy. Fecal levels of organic compounds such as 2-pentanone (ketone) and tridecane were associated with early progression of tumor in 11 non-small lung cell carcinoma patients treated with anti-PD-1 antibody . In anotTogether, these clinical studies and preclinical experiments indicate that not only the commensal bacteria but also microbial-derived metabolites may be important in impacting on the efficacy of ICIs Table . Thus, cBacteroides fragilis and Burkholderia cepacia, abrogated ICI-associated colitis in SPF mice via promoting the proliferation of inducible T cell co-stimulator (ICOS) positive regulatory T cells (Tregs) in the lamina propria. In another murine study using dextran sulfate sodium for induction of colitis, it was shown that Bifidobacterium could largely rescue mice treated with ICIs from immunopathology while preserving anti-tumor effects [ICIs are associated with increased T cell activation and effective anti-tumor immune responses in a subset of patients. However, this treatment in some patients can also trigger serious immune-related adverse effects . One of  effects . One lim effects . For thaFaecalibacterium prausnitzii and Gemmiger formicilis, have been demonstrated to be associated with a higher incidence of colitis [In humans, ICI-associated colitis is more common during CTLA-4 blockade than during PD-1/PD-L1 blockade . Dubin e colitis . Interes colitis .Clostridium difficile-associated colitis and inflammatory bowel disease [+ T cells in both patients and an increase in infiltrating Tregs in one patient. These data suggested that modulation of the intestinal microbiome with FMT may abrogate ICI-associated colitis. However, because of the limited sample size, further studies with larger cohorts are required to confirm FMT efficacy in ICI-associated colitis. Thus, the intestinal microbiome may be an attractive therapeutic target for the treatment and prevention of ICI-associated colitis. However, despite its possible efficacy, FMT therapy still remains a controversial treatment due to potential drawbacks, including the challengs of securing a healthy microbiome donor and ensuring that FMT products are free of potential pathogens.Evidence for potential benefits of reconstituting the intestinal microbiome of patients by introducing a FMT from a healthy donor has been well-demonstrated in randomized clinical studies of other types of colitis, including recurrent  disease , 61. In  disease . These tCurrently, mechanisms underlying modulation of ICI therapy by the intestinal microbiome remain unclear. A key mechanistic question is how certain microbial species can modulate immunity. Evidence has emerged that the intestinal microbiome can modulate the efficacy of ICIs via two general categories of mechanisms, including those that are antigen-independent and those that are antigen-specific Fig. .Fig. 1TLactobacillus rhamnosus GG, one of the most well-characterized and used probiotics, promoted innate immune responses against tumor via activation of DCs when combined with anti-PD-1 antibody in a preclinical model [Researchers have found that differences in the composition of the intestinal microbiome can induce changes in mucosal and systemic immune responses. The differentiation of naïve CD4 T cells into various effector subsets, including T helper type (Th) 1, Th2, Th17, Tregs, and T follicular helper cells, has been particularly well-demonstrated to be modulated by microbes. Th1 responses can arise via activation of dendritic cells and cytotoxic T cell responses across the intestinal barrier , 29, 40.al model . Thus, vBifidobacterium breve, a commensal intestinal bacterial species, could augment anti-tumor response by amplifying T cells recognizing a Bifidobacteria antigen with a similar epitope to that of a tumor antigen [Enterococcus hirae and demonstrated that Enterococcus hirae harboring this prophage induced a TMP-specific MHC-restricted CD8+ T cell response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies in a mouse tumor model [Another group of mechanisms can be termed antigen-specific, where antigen mimicry between microbial and tumor antigens can impact on anti-tumor immune responses. Neoantigens, known to arise from mutations in tumor cells as part of the process of carcinogenesis, can be targeted by T cells and are likely critical to successful eradication of tumors by ICI therapy , 68. One antigen , indicator model . They alor model . These dIntestinal microbes and microbial-derived metabolites can clearly have impactful effects on host immunity. Recent studies indicate that avoiding both an unfavorable microbiome as well as antibiotic-associated dysbiosis help to optimize ICI results, and a baseline intestinal microbiome assessment could be one approach to help predict responses associated with ICIs. Currently, clinical trials to elucidate the association between microbes, microbial-derived metabolites, and ICI efficacy or toxicity are ongoing Table . AlthougThus, the composition of the intestinal microbiome may impact on not only the strength and durability of anti-tumor effects but also unfavorable intestinal inflammation in patients treated with ICIs. More studies are needed to achieve a better understanding of the interactions between the host and the intestinal microbiome, with a goal of identifying microbiome-based biomarkers of outcomes and designing strategies to target the microbiome to enhance the efficacy and safety of cancer immunotherapy.


Nucleolin (NCL) is a multifunctional nucleolar phosphoprotein harboring critical roles in cells such as cell proliferation, survival, and growth. The dysregulation and overexpression of NCL are related to various pathologic and oncological indications. These characteristics of NCL make it an ideal target for the treatment of various cancers. AS1411 is a synthetic quadruplex-forming nuclease-resistant DNA oligonucleotide aptamer which shows a considerably high affinity for NCL, therefore, being capable of inducing growth inhibition in a variety of tumor cells. The high affinity and specificity of AS1411 towards NCL make it a suitable targeting tool, which can be used for the functionalization of therapeutic payloaddelivery nanosystems to selectively target tumor cells. This review explores the advances in NCL-targeting cancer therapy through AS1411-functionalized delivery nanosystems for the selective delivery of a broad spectrum of therapeutic agents. Human nucleolin (NCL) is a 707-amino acid multifaceted nucleolar phosphoprotein with an approximate molecular weight of 76 kDa. It is highly expressed in exponentially growing eukaryotic cells in a proliferation-dependent manner. Its presence in the dense fibrillar core and granular regions of the nucleolus is regarded to be higher than any other protein , 2. NCL NCL dysregulation has been observed in numerous pathological conditions such as autoimmune disorders, Alzheimer and Parkinson’s disease, and numerous cancer types –8. It haNCL plays substantially imperative tasks in the modulation of cell proliferation, survival, and growth, cytokine production, and nuclear biogenesis , 15. FurAptamers are approximately 20- to 60-nucleotide deoxyribonucleic acid, ribonucleic acid, or xeno nucleic acid constructs capable of binding craved molecules with robust a capability and specificity by folding into tertiary structures . They ar14 to 1016 single-stranded random 40 to 100 long oligonucleotides are incubated with the target of interest. These oligonucleotides exhibit different binding affinities to the target of interest. Therefore, throughout the process, the oligonucleotides that have a low binding affinity to the target are excluded and, in the end, only a number of oligonucleotides remain which have a high level of binding affinity for the target of interest. Recently, various SELEX-based methods have been proposed and utilized for the development of aptamers for novel targets with considerable specificity and sensitivity. These techniques include capture-SELEX, capillary electrophoresis-SELEX (CE-SELEX), cell-SELEX, atomic force microscopy-SELEX (AFM-SELEX), artificially expanded genetic information system-SELEX (AEGIS-SELEX), and immunoprecipitation-coupled SELEX (IP-SELEX)  [Chemotherapy and radiotherapy are regarded as the most prevalent cancer treatment approaches but they can lead to serious side effects in cancer patients . In the therapy] –46. Eventherapy] –46. On tIn this review, we discuss different types of AS1411 aptamer-functionalized nanosystems for the delivery of chemotherapeutic agents, therapeutic nucleotides, and therapeutic proteins to different types of malignant cells overexpressing NCL. We also briefly discuss how these nanosystems can be exploited for enhancing photodynamic therapy (PDT) . UltimatCombinational micelle therapeutic agent delivery platforms equipped with the anti-NCL aptamer can be applied for the precise delivery of different chemotherapeutics including doxorubicin (DOX) to various malignant cells. The elaborate system described in this section somehow manages to tackle the poor drug-loading capacity and micelle stability issues through the development of an AS1411-modified hybrid system consisting of Pluronic F127 and beta-cyclodextrin-linked poly(ethylene glycol)-b-polylactide (β-CD-PELA) block copolymers as co-carriers of the anticancer drug . PluroniAnother investigation has described the establishment of a cancer-specific delivery system for the selective cytoplasmic release of paclitaxel (PTX) in ovarian malignant cells based on a combinatorial micellar construct. This construct is made up of (A) a pH-responsive copolymer synthesized by a condensation polymerization reaction in the presence of tocopheryl polyethylene glycol 1000 succinate (TPGS)-diacrylate macromonomer and (B) an AS1411 aptamer-decorated TPGS polymer (AS1411-TPGS). As proposed by Zhang et al. , the incFurthermore, other researchers have proposed a novel pH-reactive micelle-based delivery system for the delivery of DOX for effective breast cancer therapy . These DAnother contribution to the topic has been conducted by Mohammadzadeh et al.  as they AS1411-aptamer-mediated targeted delivery can also be used for the delivery of an antimiR for the inhibition of miRNA-221 which has been known to be involved in the cancer development of papillary thyroid carcinoma in which it is highly expressed , 73. A NRB gene or both allele inactivation, which impacts the cell cycle and apoptosis regulators of immature retinal cells [The onset of cancer causes cancer cells to overexpress a variety of proteins known as inhibitors of apoptosis (IAPs), which enable them to increase their life-span while minimizing the chance of apoptosis occurrence. Survivin (Sur), as an example of IAPs, which is highly overexpressed in the nucleus, cytoplasm, and cellular organelles such as the mitochondria of various cancer types including retinoblastoma (RB), is responsible for mediating various oncogenic signaling pathways, inhibition of apoptosis, rapid cell growth, and eventually as the name indicates the survival of tumor cells –78. RB ial cells . Becauseal cells –83.An aptamer-mediated NCL-targeting approach for the delivery of survivin DNAzyme (Sur_Dz) can act as a way of specific gene-targeting therapy to suppress the progression of RB cancer. A chimeric conjugate composed of the AS1411 aptamer (NCL-APT) and Sur_Dz with poly T linker at the 5’ end of the aptamer followed by complementary bases to Sur_Dz has been developed by Subramanian et al . This coSince both Sur and NCL are overexpressed in a variety of cancers, the chimerization of NCL-APT with Sur_Dz for the development of a targeted therapeutic platform can be functional on a wide spectrum of cancer types. Furthermore, Sur_Dz has only been chosen due to its dose-dependent manner catalytic reactivity towards Sur mRNA which has been proven in pancreatic carcinoma and its overexpression profile in RB tumor cells , 85. MorNon-small cell lung cancer (NSCLC) is one of the cancer types that causes a high mortality rate in lung cancer patients due to its early metastasis events which are composed of several consecutive stages including epithelial-mesenchymal transition (EMT), cancer cell migration, invasion, intravasation into the systemic circulation, eventual adhesion to endothelial cells, and extravasation and colonization of distant organs as well as induction of angiogenesis –88. The NCL aptamer-siRNA (AS1411-siRNA) chimeras specific for SLUG (aptNCL-SLUGsiR) and NRP1 (aptNCL-NRP1siR) can be used for specific tumor invasion and angiogenesis suppression in only metastatic tumor cells without blocking the signaling pathways of cells that are under physiologic conditions . AptNCL-BRAF gene, such as BRAFV599E, and the activation of the RAS/RAS/MAPK pathway which happens to be essential for melanoma cell viability and transformation [BRAF gene expression makes it a suitable target for the treatment of melanomas through siRNA-mediated gene silencing approaches. Since liposomes are non-viral carriers employed for the development of successful drug delivery systems, some of which have already been approved by the US FDA for medical use, they can be employed for the efficient delivery of gene-modifying agents into cells [BRAF gene and inhibit melanoma growth [Additionally, melanoma is a common type of skin cancer that is developed from the pigment-producing cells melanocytes and is difficult to treat due to the high rate of its early-stage metastasis, poor prognosis, and resistance to conventional radiotherapy , 98. In to cells , 101. PEa growth .Additionally, another study has reported the development of redox-reactive gelatin/silica-based nanogels functionalized with AS1411 for targeted siRNA delivery (Apt-GS/siRNA) which were transiently conjugated to smart nanogels via a disulfide linker . Since tIn addition to the mentioned studies, an AS1411 aptamer-functionalized nanoliposome-based delivery system has been developed for the co-delivery of PTX and polo-like kinase 1-targeted siRNA (PLK1-targeted siRNA) to breast cancer cells . PLK1 isSplice-switching oligonucleotides (SSOs) are short synthetic single-stranded oligonucleotides capable of binding to a splice site or splicing enhancer of a pre-mRNA, thus preventing the endogenous splicing machinery from binding to those splice sites which can eventually result in the disruption of the normal splicing repertoire and the generation and subsequent translation of alternative versions of a mature mRNA –115. SSOO-methyl-phosphorothioate [Since the ability of AS1411 for internalization into multiple cancer cells and migration to their nucleus has been evident, the therapeutic applicability of SSOs can be further exercised by their targeted delivery to the nuclei of specific cells of interest , 122. Thothioate , 122. Heothioate .Modulation of nuclear events through the enhancements in splice correction of pre-mRNA can be achieved using aptamer-splice-switching oligonucleotide chimeras . It has Fe) in combination with multimodal imaging efficacy of Fe3O4 NPs which are hereafter referred to as bovLfn-Fe3O4 [3O4 to achieve tumor-specific uptake of the constructs. This mentioned nanoformulation (bovLfn-Fe3O4 nano-constructs) achieved considerably encouraging tumor rejection in triple-positive  colon cancer xenograft mouse models inducing higher survival rates in comparison with non-targeted nano-constructs which demonstrates the importance of nano-constructs functionalization with the anti-NCL and anti-EpCAM aptamers [3O4 nano-constructs occurs as the extracellular death domain receptors of TRAIL and Fas mediate the phosphorylation and subsequent activation of p53 and the Notch pathway inhibition [3O4 nano-constructs combine the anticancer potential of bovLfn with the efficacious multimodal imaging capabilities of Fe3O4 as they can completely inhibit tumor growth without exhibiting signs of adverse events alongside showing immunomodulatory benefits through increasing red blood cells, hemoglobin, and zinc levels [Lactoferrin is an innocuous natural multifaceted glycoprotein, primarily identified in bovine and human milk, with various characteristics such as immunomodulatory and anticancer properties which has been regarded safe for consumption –127. Nowfn-Fe3O4 . Chitosafn-Fe3O4  for the aptamers . The tumhibition . The acthibition . Eventuahibition . Furtherhibition . In a nuc levels .Ai et al.  have invAnother study has proposed a similar method with minor differences. In this novel strategy, as a drug carrier to target malignant cells for PDT, several molecules of porphyrin derivative are physically conjugated to AS1411 to form the apt-TMP complex . PorphyrMore specificity to eliminate malignant cells via photodamage could also be achieved through controlling the light irradiation for the activation of the photosensitizer . Additio2-labeled ATP-binding aptamer (termed BHQ2-ATP-apt), and the complementary oligonucleotide c-TA for ATP-apt to make their delivery platform (Apt-HyNP/BHQ2) [Moreover, Shen et al.  have shoNP/BHQ2) .2, Apt-HyNP/BHQ2 is fluorescence and PDT “off” in the beginning [2 in the lysosomes of the cells [2-ATP-apt, therefore, cause them to be released from Apt-HyNP/BHQ2 which leads to turning “on” both fluorescence and PDT with significant recovery of both fluorescence emissions and 1O2 production capacity [1O2 generation which in turn would lead to rapid lysosome rupture and ultimate tumor cell death (Due to the quenching effect of BHQeginning . After ehe cells . The higcapacity . Therefoll death . As a reFurthermore, various studies have combined other approaches such as targeted-delivery of drugs and photosensitizers. In this regard, Xu et al.  have proIn this article, we reviewed the studies using the AS1411 aptamer for functionalizing and redirecting NPs loaded with chemotherapeutic agents or therapeutic nucleotides towards various types of NCL-overexpressing cancer cells. We also discussed how these aptamer-functionalized nanosystems can selectively deliver their payload only to tumor cells, therefore, reducing the off-target delivery-associated toxicities of conventional cancer therapy treatment modalities. Furthermore, several studies have shown that chemical modifications performed on AS1411 can result in augmented functionality, increased tumor cell targeting affinity, enhanced S-phase cell cycle arrest capability, and improved DNA replication and cancer cell growth prohibition ability of the aptamer , 136. Th


Acute myeloid leukemia (AML) is the second most common leukemia among children. Although significant progress in AML therapy has been achieved, treatment failure is still associated with poor prognosis, emphasizing the need for novel, innovative therapeutic approaches. To address this major obstacle, extensive knowledge about leukemogenesis and the complex interplay between leukemic cells and their microenvironment is required. The tremendous role of this bone marrow microenvironment in providing a supportive and protective shelter for leukemic cells, leading to disease development, progression, and relapse, has been emphasized by recent research. It has been revealed that the interplay between leukemic cells and surrounding cellular as well as non-cellular components is critical in the process of leukemogenesis. In this review, we provide a comprehensive overview of recently gained knowledge about the importance of the microenvironment in AML whilst focusing on promising future therapeutic targets. In this context, we describe ongoing clinical trials and future challenges for the development of targeted therapies for AML. Acute myeloid leukemia (AML) is a malignant, hematologic disease that accounts for about one-fifth of all childhood leukemia cases. In recent decades, the prognosis of AML patients has improved thanks to advances in distinct diagnostic and therapeutic tools. Although the vast majority of children initially achieve complete remission, overall long-term survival is still limited by refractory disease and relapse, which occur in about one-third of children with AML and are linked to poor prognosis ,2. To da11 cells each day, making the hematopoietic system one of the most regenerative tissues in humans , C-X-C motif chemokine [CXCL]-12 abundant reticular [CAR] cells, leptin-receptor-positive [LepR+] cells, and endothelial cells) and structural components . Various non-cellular factors ), C-X-C motif chemokine [CXCL]-12, CXCL-4, transforming growth factor beta [TGF-β], vascular endothelial growth factor [VEGF], and vascular endothelial cadherin [VE-Cad], thrombopoietin [TPO], Notch, and its ligand Jagged-1) expressed by the cellular components contribute to hematopoietic regulation in the BMM. Since primitive HSCs have first been isolated from the inner bone surface, close to osteolineage cells, the endosteal region was identified as the HSC niche ,28. As tIn different mouse models, parathyroid-hormone signaling and lack of bone morphogenetic protein (BMP) receptor-1A stimulated osteoblastic cells, which in turn increased HSC numbers through Notch ligand Jagged-1- or N-cadherin-mediated cell adhesion to HSCs, respectively ,33. FurtThe BMM is traversed by an intricate vascular system consisting of arterioles that adjoin the endosteum and branches, creating an intricate venous–sinusoidal network in the central bone cavity . RecentlIn the perisinusoidal niche, CXCL-12-abundant reticular (CAR) and leptin-receptor-positive (LepR+) cells direct the mobilization and maintenance of HSCs expressing high amounts of CXCL-12 and SCF ,45,68. OIn vivo imaging identified different subpopulations of stromal cells characterized by green fluorescent protein (GFP) expression under the control of the nestin promoter (Nes-GFP) . WhereasIn addition to mesenchymal-derived stromal cells, ECs serve as critical sources of regulatory niche factors and importantly contribute to hematopoietic regulation ,58. GiveSinusoidal ECs are known to importantly contribute to the niche-dependent HSC regulation through vascular endothelial (VE)-cadherin and vascular endothelial growth factor (VEGF) receptors 2 and 3 ,71. ThisConditional deletion of VEGF-C in endothelial and stromal cells leads to decreased hematopoietic cell and niche regeneration. Conversely, exogenous administration of VEGF-C leads to improved hematopoietic niche recovery after irradiation, suggesting that VEGF is important for maintaining the HSC niche . AdhesioIn the perisinusoidal region, a major subset of megakaryocytes is reported to positively direct HSC quiescence and maintenance by expression of mediators such as CXCL-4, TPO, and transforming growth factor beta (TGF-β)1 ,77,78. AAs the hematopoietic BMM is vastly innervated and controlled by the sympathetic nervous system , non-myeA relevant subset of adipocytes localized in the perisinusoidal region has been recently identified as sympathetically innervated and regulated . InitialThe above-discussed results, in part, are indicative of the idea of a vascular sub-niche that plays a binary role within the hematopoietic BMM. Whereas periarteriolar localized parts seem to rather protect the quiescent HSC pool ,95, peri2) levels reported to range from 0.1% and 6% in different areas within the bone marrow ) as well as non-cellular mediators , including related cellular interaction and signaling (vascular cell adhesion molecule [VCAM)]-1 and very late antigen , C-X-C motif chemokine [CXCL]-12 abundant reticular [CAR] cells, leptin-receptor-positive [LepR+] cells, and endothelial cells) and structural components , certain non-clonal (epi-)genetic aberrations have been revealed and differ from mutations found in hematopoietic cells ,109,110.In contrast, multiple reports indicate that donor-derived leukemia following allogenic stem cell transplantation for different hematopoietic malignancies (reviewed by ) is a poContrary to the concept of a transformed microenvironment that drives leukemia according to microenvironment-mediated malignancy, an opposite but not exclusive notion assumes malignant hematopoietic cells mediate malignancy-associated changes in the microenvironment , as MDS cells have been shown to induce malignant/MDS-like transformation in healthy MSCs . SimilarIn in vivo AML models, LSCs lead to transcriptional changes in healthy MSCs, affecting the expression of various molecular mediators involved in intercellular crosstalk . InteresThese results suggest that leukemic cells differentially remodel the previously healthy microenvironment to their favor, indicative of a secondary induced malignant microenvironment that may further support disease progression. In this context, microenvironmental-mediated malignancy and malignancy-mediated microenvironment do not seem to mutually exclude each other, but rather to complementarily cooperate in a bidirectional manner, favoring hematopoietic insufficiency and leukemogenesis.Numerous studies point to a self-reinforcing, bidirectional interplay between leukemic cells and their microenvironment that favors further disease progression  and resThe retention of LSCs in the leukemic bone marrow niche depends on cellular interactions, which are mediated by CXCR-4/CXCL-12 signaling and other pathways. In addition, AML blasts secrete high amounts of SCF, a retention factor, that importantly attracts HSCs into less suitable areas within the leukemic BMM, from where they can only be mobilized with difficulty, which serves to impede healthy hematopoiesis .Malignant, leukemic cells express certain adhesion molecules to an increased extent, such as CD44, that interact with ECs expressing E-selectin, resulting in promoted chemoresistance and upregulated viability and proliferative capacity . The celLeukemic cells increasingly produce several proinflammatory cytokines, such as Il-1 that induce surrounding ECs and stromal cells to secrete growth factors such as CXCL-12 or colony-stimulating factors, which are proposed to result in unlimited cell growth and proliferation of leukemic blasts . In pediDespite the healthy microenvironment, TGF-β restricts hematopoietic proliferation, and in the leukemic microenvironment, TGF-β has also been implicated in leukemogenesis . In thisGiven that MKs and leukemic blasts abundantly express TGF-β, resulting in markedly increased TGF-β levels in the DS-AMKL BMM, and TGF-β has been shown to induce early-stage marrow fibrosis in DS-AMKL , TGF-β aIn AML, insulin-like growth factor (IGF)-1 and its same-named receptor IFG-R1 significantly contribute to leukemogenesis, according to evidence that IGF-1/IGF-1R signaling exerts pro-leukemic effects on AML cells in vitro via activation of downstream PI3K/Akt and the extracellular signal-regulated kinase (Erk) pathways ,145. In In an MLL-AF9 AML model, it was demonstrated that leukemic infiltration leads to sympathetic neuropathy, which is presumably due to damaged β-adrenergic signaling and includes disruption of quiescent perisinusoidal nestin-positive cells, resulting in an impaired physiological niche function with reduced HSC-maintaining NG2+ periarteriolar cells but increased leukemia-supportive pericytes . This prIn patient-derived AML cells, VEGF and its receptor VEGFR2 have been reported to be overexpressed  and assoHypoxia-mediated signaling through the transcription factor HIF-1α is required for the maintenance of leukemia-initiating cells in vitro  and in v2 in physiological bone marrow samples (46.05 mmHg [6.1%] and 54.9 mmHg [7.2%]) ) ,165. In  [7.2%]) ), the me [7.2%]) ,168,169. [7.2%]) .Along with the above-mentioned ontogenic changes in hematopoietic development, there are noteworthy age-related differences in the fetal, neonatal, and adult hematopoietic and leukemic BMMs. The healthy fetal liver provides a unique environment that ensures enormous HSC-expansion without malignant transformation. The results of various studies focusing on the fetal liver niche suggest an important role of fetal liver stromal cells in supporting the massive expansion of early HSCs through the expression and secretion of pivotal hematopoietic mediators, including angiopoietin-like 2 and 3, TPO, and IGF-2 . ThIn the postnatal period, it has been reported that HSCs possess increased CXCL-12 expression and high cell cycling activity, which are linked to an engraftment defect that can be reversed by antagonizing the CXCL-12/CXCR-4 interplay . IntriguBased on the high impact of this remodeled leukemia-supportive microenvironment in the pathogenesis of AML, relevant molecular structures in the BMM need to be considered as therapeutic targets for efficiently eradicating leukemic blasts and preventing relapsing/non-responding unfavorable courses of disease. To date, several cellular interactions that protect AML blasts within the highjacked leukemic microenvironment have been approved as potential therapeutic targets for disrupting this leukemia-protecting interplay.Regarding the aforementioned physiological effects of the CXCL-12–CXCR-4 axis on CD34-positive HSC mobilization , adminisThe novel CXCR-4 antagonist BL8040, which has inverse agonism activity, more efficiently inhibits CXCR-4 with increased binding affinity and thus represents a next-generation CXCR-4–directed agent . A combiThe E-Selectin antagonist Uproleselan (GMI-1271) reduces niche-mediated chemoresistance, survival, and regeneration of leukemic cells by sensitizing them to chemotherapy while similarly reinforcing healthy hematopoiesis . Based oIn the leukemic microenvironment, the cellular crosstalk between VLA-4 and VCAM-1 has been proposed to mediate increased blast proliferation and to reduce therapy-induced apoptosis, contributing to a poor prognosis . ConsistAS101 is a non-toxic VLA-4 antagonist that reconstitutes sensitivity to chemotherapy by disrupting the underlying PI3K/Akt/Bcl-2 pathway, and concomitant administration with cytarabine demonstrated promising anti-leukemic activity in pre-clinical trials . The effThe peptide FNIII14, which disturbs VLA-4–mediated activation by disrupting the interaction between fibronectin and the beta1-subunit, exhibited synergistic anti-leukemic effects when given with cytarabine, contributing to significantly improved survival in investigated mouse models without increasing adverse myelosuppressive effects . InhibitRegarding the previously described leukemia-supporting impact of IGF–IGF-1R signaling in leukemogenesis in AML , inhibitFurthermore, administration of the IGF-1R inhibitor picropodophyllin (PPP) was able to abrogate the proliferative capacity of treated LSCs, which was attributed to downregulation of IGF-2 and Nanog expression . TreatmeGiven that, in contrast to HSCs, LSCs express a markedly decreased level of the tumor-suppressing IGF-BP7, this secreted factor appears may represent a promising therapeutic agent in AML . In a prDespite these first promising clinical results, studies assessing the utility of novel therapeutic approaches targeting the microenvironmental interaction in pediatric AML are rare, and available clinical data are limited by low incidence, emphasizing the necessity of strong international collaboration.In this review, the major mechanisms of cell–cell interaction in the BMM that contribute to healthy hematopoiesis and leukemogenesis have been outlined. Microenvironmental crosstalk involves various cellular and non-cellular components that mutually influence each other in a multidirectional manner, resulting in a complex leukemia-supportive niche. Considering the high impact of this BMM in leukemogenesis, targeting such microenvironmental crosstalk may provide a promising future strategy to cure AML. The first auspicious BMM-directed therapies are already being tested, but clinical studies in pediatric AML are still limited. Further research is necessary to deepen our knowledge about the BMM and identify novel approaches to eradicate leukemic blasts.