Escherichia coli possess virulence genes, adaptability for human colonization and are often associated to resistance genes. We evaluated the prevalence, O-antigen and susceptibility profiles of ST131 among β-lactam resistant E. coli isolates collected in US hospitals.ST131 E. coli isolates collected during 2017 and 2018 were susceptibility tested using reference broth microdilution method. Among these, 1,154 displayed MIC values >1 mg/L against 2 of the following: ceftazidime, ceftriaxone, cefepime or aztreonam or resistance to imipenem or meropenem (CLSI breakpoints) and were submitted to whole genome sequencing (WGS) and data analysis (MLST and O antigen). The genes wzx, wzy, wzm, and wzt were used to identify the O-antigen. Reference guided assembly for gene cluster O-AGC was used to differentiate O25a and O25b.A total of 6,768 E. coli isolates, 627 (54.3%) belonged to ST131 or were single loci variants . A total of 586 (93.5% of ST131 and 50.8% of sequenced isolates) belonged to the O25b serotype. The remaining 41 isolates belonged to serotypes O16 (40 isolates) or O153var1 (1). ST131 isolates and O25b isolates were considerably more resistant to fluoroquinolones  when compared to the overall WGS isolate collection (75.6%-77.0%). ST131-O25b isolates were more resistant to 12 /16 antimicrobial agents analyzed, including all β-lactam agents (0.9%-12.7% more resistant), fluoroquinolones (34.5-41.0%), and aminoglycosides (1.2-37.3%). ST131 (49.0%) and ST131-O25b (51.5%) isolates had higher multi-drug resistant (MDR) rates compared to all E. coli isolates (7.3%), WGS isolates (41.4%), and isolates that did not carry these traits (32.4% for non-ST131 and 12.2% for non-O25b).Among the WGS E. coli isolates were common among β-lactam resistant E. coli from US hospitals. These isolates were significantly more resistant than their counterparts, despite the elevated resistance rates of the overall WGS collection. ST131-O25b E. coli isolates have the potential to present a challenge for antimicrobial treatment. Specific therapies that are effective against these isolates should be investigated.ST131 and ST131-O25b Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  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Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Andrew P. Davis, BS, Bravos Biosciences (Research Grant or Support) Valerie Kantro, BS, Bravos Biosciences (Research Grant or Support) Christopher M. Rubino, Pharm.D., 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Research Grant or Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC Cidara Therapeutics Inc. 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Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti‐HIV‐1 antibodies 2F5 and PG9 as well as the protease inhibitor α1‐antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin‐like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals.The tobacco variant NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.  A1AT, α1‐antitrypsinAc, acetylCDR, complementarity‐determining regionCHO, aldehydeCMK, chloromethylketoneFMK, fluoromethylketoneFP, fluorophosphonatemAb, monoclonal antibodyNb, Nicotiana benthamianaSBT, subtilisin‐like serine proteaseSuc, methoxysuccinylZ, benzyloxycarbonylNicotiana benthamiana [The market for recombinant therapeutic proteins is increasing rapidly . Plants thamiana  due to ithamiana . Furtherthamiana , 7. Thesthamiana  and the thamiana .N. benthamiana as an emerging plant‐based expression platform, a major drawback is still unresolved: recombinant proteins expressed in this plant frequently suffer from proteolytic degradation [1‐antitrypsin (A1AT) have been shown to undergo inadvertent proteolysis in N. benthamiana [Nicotiana species occurs primarily in the apoplast, the pericellular space in plant tissues [Despite all these qualities of radation . Severalthamiana , 13, 14.thamiana . Althougthamiana , 16, con tissues . Unfortu tissues .a priori. So far, the responsible proteases have not been identified in any plant‐based expression system, thus prohibiting targeted approaches to tackle unwanted proteolysis in N. benthamiana and other plant species.Several strategies were proposed to counteract unwanted protein degradation in plants: targeted disruption of protease genes, coexpression of protease inhibitors and downregulation of endogenous protease activities by means of RNA interference or other antisense‐based mechanisms , 20. ForN. benthamiana is known to harbour proteases from all catalytic classes [N. benthamiana apoplastic fluid in vitro [N. benthamiana leaves [N. benthamiana. First, we isolate N. benthamiana SBTs present in the leaf apoplast using activity‐based probes and identify them by mass spectrometry. We then characterize the two most abundant enzymes (NbSBT1 and NbSBT2) and demonstrate their capacity to cleave mAbs and A1AT in vitro. In combination, NbSBT1 and NbSBT2 generate all cleavage products observed upon production of these potential biotherapeutics in plants. Hence, our results implicate NbSBT1 and NbSBT2 in recombinant protein degradation in planta.Although the secretome of  classes , we havein vitro . A recena leaves . SBTs coa leaves . In the Nicotiana benthamiana ∆XT/FT plants deficient in N‐glycan α1,3‐fucosylation and β1,2‐xylosylation [m fluorophosphonate (FP)‐biotin . The sample (2.5 mL) was then chromatographed on a PD‐10 column  equilibrated in 100 mm sodium acetate (pH 5.5). The recovered eluate (3.5 mL) was incubated with 40 µL avidin‐agarose beads  for 16 h at 4 °C under constant agitation. The beads were washed four times with 4 mL 100 mm sodium acetate (pH 5.5) and once with 4 mL 10 mm Tris/HCl (pH 6.8) prior to elution of the bound proteins with 80 µL SDS/PAGE sample buffer . The samples were then subjected to 12.5% SDS/PAGE under reducing conditions prior to staining of the gel with Coomassie Brilliant Blue (CBB) R‐250. The 63–70 kDa bands were excised, S‐carboxamidomethylated and then digested with sequencing‐grade trypsin . The peptides thus generated were fractionated on a Thermo BioBasic C18 separation column  operated using a Dionex UltiMate 3000 system . A gradient from 96.5% solvent A and 3.5% solvent B  to 40% B in 45 min was applied, followed by a 15‐min gradient from 40% B to 95% B, at a flow rate of 6 µL·min−1. Eluted peptides were analysed online on a maXis 4G ETD Q‐TOF mass spectrometer  equipped with an electrospray ionization source and operated in the positive ion mode (m/z range: 150‐2200).sylation  were grosylation  and thenN. benthamiana tissues using the SV Total RNA Isolation Kit (Promega). First‐strand cDNA was synthesized from 1 µg RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and oligo(dT)18 as primer. PCR fragments encompassing the complete ORF of the identified apoplastic SBTs were obtained from total leaf or flower cDNA using suitable primer combinations  and then transferred by Gibson assembly [Agrobacterium tumefaciens strain UIA143 [For ectopic expression of the two most abundant apoplastic SBTs (NbSBT1 and NbSBT2) in assembly  into pEAassembly  after din UIA143 .N. benthamiana plants were inoculated with agrobacterial suspensions diluted to an OD600 of 0.15 with infiltration buffer . Three days after infiltration, the leaves were submerged in extraction buffer  prior to vacuum exposure in a desiccator. Exogenous buffer was removed prior to centrifugation of the leaves for 15 min at 2000 g and 4 °C. The recovered exudate was then supplemented with 20 mm imidazole and loaded on a 1 mL column of Chelating Sepharose  charged with Ni2+ ions. After washing with the same buffer, bound proteins were eluted with 250 mm imidazole in extraction buffer. Protein‐containing eluate fractions were pooled, dialysed twice against 2 L of extraction buffer and then concentrated by ultrafiltration using Microsep Advance centrifugal devices .Leaves of 5‐week‐old −1; Polymun Scientific, Klosterneuburg, Austria) or human plasma A1AT  were incubated with concentrated apoplastic fluid (10–250 µg·mL−1), NbSBT1 (1–30 µg·mL−1) or NbSBT2 (3–70 µg·mL−1) in 100 mm sodium acetate (pH 5.5) at 37 °C in the absence or presence of selected protease inhibitors . After incubation for 1–16 h, samples were mixed with an equivalent volume of SDS/PAGE sample buffer and heated at 95 °C for 5 min prior to analysis by western blotting with antibodies to human IgG (Sigma‐Aldrich) or A1AT  as described previously [m glycine/HCl (pH 3.0). Protein‐containing eluate fractions were immediately neutralized by addition of 0.1 m Tris/HCl (pH 8.0), subjected to buffer‐exchange into phosphate‐buffered saline and then concentrated by ultrafiltration using Amicon YM30 centrifugal filter units .mAbs  and then fractionated on a Thermo ProSwift RP‐4H column (250 × 0.20 mm) using a Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific). Native and digested A1AT samples were analysed without prior deglycosylation and reduction. After application of the sample (5 µL), elution was performed at 65 °C and a flow rate of 8 µL·min−1 with a gradient of 20–95% solvent B (80% acetonitrile in 0.01% trifluoroacetic acid) in solvent A (0.05% trifluoroacetic acid) over 40 min as follows: 20–65% B (15 min), 65–95% B (5 min). Eluted polypeptides were analysed online on a maXis 4G ETD Q‐TOF mass spectrometer (Bruker) equipped with an electrospray ionization source and operated in the positive ion mode (m/z range: 400–3800). The analysis files were deconvoluted (Maximum Entropy Method) using DataAnalysis 4.0 (Bruker) and manually annotated.N‐terminal sequence analysis of bands blotted on polyvinylidene difluoride membranes  was performed by Edman degradation on an Applied Biosystems Procise 492 protein sequencer  as described previously . AlternaN. benthamiana leaf apoplast, apoplastic fluid collected from untreated N. benthamiana leaves was incubated with FP‐biotin, a biotinylated activity‐based probe which potently reacts with serine hydrolases [m   correspond to the predicted N termini of the respective mature peptidase domains. As determined by Edman degradation, the major 63 kDa band observed in the NbSBT1 sample was found to be an N‐terminally truncated form of mature NbSBT1 starting with G207  Fig.  was perfEV) Fig. , a pheno07 Table .Nicotiana tabacum phytaspase which selectively cleaves after aspartic acid residues [148) is located within the fragment removed during proteolytic processing, these results indicate that NbSBT1 is cleaved into a two‐chain form consisting of an N‐terminal light chain (T110‐A206) and a C‐terminal heavy chain (G207‐N770) without losing its enzymatic activity  H3 loop [To assess the potential of NbSBT1 and NbSBT2 for protein degradation thamiana . Each mA H3 loop . Interes107↓G108VP were detected by mass spectrometry. In agreement with this result, Edman degradation of the 40 kDa 2F5 fragment revealed a single N terminus starting with G108. 2F5 is cleaved at the same position when the antibody is produced in planta or upon incubation of the antibody with leaf apoplastic fluid in vitro [111↓D112FY, YNYH121↓Y122MD, YHYM123↓D124VW, TTVT133↓V134SS and VTVS135↓S136AS. Cleavage at Y111↓D112 is documented for PG9 extracted from plants [112↓F113YD, DFYD115↓G116YY and HYMD124↓V125WG, demonstrating a notable preference of NbSBT2 for aspartic acid residues at the substrate position preceding the scissile bond , both located in the reactive centre loop of the protease inhibitor. Truncation of A1AT in planta occurs largely by cleavage at the same positions [A1AT is another example of a recombinant protein prone to proteolytic inactivation when expressed in thamiana . When A1ositions . NbSBT2 in vitro cleavage of 2F5 by apoplastic fluid can be completely prevented by the addition of PMSF or FP‐biotin, processing of PG9 is most effectively inhibited by a combination of aminoethylbenzenesulphonyl fluoride (AEBSF) and acetyl (Ac)‐YVAD‐CMK [N. benthamiana, whereas both NbSBT1 and NbSBT2 are involved in the degradation of PG9. In line with previously published inhibition profiles [While YVAD‐CMK . We haveYVAD‐CMK , 30. AEBprofiles , NbSBT1 N. benthamiana SBTs which are capable of cleaving two proteolysis‐prone mAbs  as well as another protease‐susceptible human serum glycoprotein (A1AT) at the same positions as it happens in planta or upon incubation with unfractionated apoplastic fluid. Together, the activities of NbSBT1 and NbSBT2 are sufficient to account for all inadvertent proteolytic events which occur during production of these potential biotherapeutics in N. benthamiana [in planta. However, we cannot rule out that other apoplastic SBTs contribute to mAb and A1AT cleavage in N. benthamiana. The documented catalytic redundancy of plant SBTs [Although many studies have reported instances of unintended proteolysis in plant‐based expression systems , 19, effthamiana , 12. Theant SBTs , 32 suggN. benthamiana [N. benthamiana leaves. Moreover, activity‐based probe profiling (ABPP) confirmed that these two proteases are catalytically active in the apoplast under both conditions [in vitro [N. benthamiana SBTs are upregulated upon infection with agrobacteria and thus enhance the proteolytic potential of apoplastic fluid after agroinfiltration. It should be noted that agroinfiltration has been shown to upregulate the expression of papain‐like cysteine proteinases and legumains [N. benthamiana and related Nicotiana species [Proteomic studies have provided comprehensive evidence that infiltration with agrobacteria modifies the proteolytic landscape of thamiana . Hence, nditions . These fin vitro . Howeveregumains , which h species , 20, 34.Arabidopsis thaliana and N. tabacum. The A. thaliana SBT most similar to NbSBT1 . This CO2‐induced proteolytic activity mediates the repression of stomata formation upon exposure of A. thaliana leaves to elevated atmospheric CO2 concentrations. Mechanistically, AtSBT5.2 has been shown to process epidermal patterning factor 2 (EPF2), the precursor of an extracellular peptide repressing stomatal development [Although NbSBT1 is capable of processing the tomato immune protease Rcr3 , the phyNbSBT5.2 ) is AtSBNbSBT5.2 . Originaelopment . Howeverelopment . This clN. tabacum phytaspase, a constitutively secreted apoplastic SBT promoting programmed cell death in response to viral infection or exposure to abiotic stresses [Solanum lycopersicum Phyt2 [N. benthamiana as long as lines with silenced or disrupted NbSBT1 and NbSBT2 genes are not available.NbSBT2 is a close relative of stresses . Like Nbstresses . Similarum Phyt2 , which dum Phyt2 . Generatum Phyt2 . These rum Phyt2 . This isLM conceived and supervised the study; AP, DM, AG, JB, JT, AC and LM designed experiments; AP, DM, AG, JB, JT and LM performed experiments; AP, DM and LM analysed data; AC and HS provided key reagents; AP and LM wrote the original draft of the manuscript; AP, DM, HS and LM made manuscript revisions.Table S1. Oligonucleotide primers used in this study.Table S2. Sequences of apoplastic NbSBTs.Table S3. NbSBT1 peptides identified by mass spectrometry.Table S4. NbSBT2 peptides identified by mass spectrometry.Table S5. Cleavage sites of NbSBT1 and NbSBT2 in 2F5, PG9 and human α1‐antitrypsin (A1AT).Fig. S1. Location of the identified peptides in the NbSBT1 sequence.Fig. S2. Location of the identified peptides in the NbSBT2 sequence.Fig. S3. Absence of subtilisin‐like serine proteases in empty‐vector controls.Fig. S4. Proteolytic processing of NbSBT1 and NbSBT2.Fig. S5. Processing of 2F5 and PG9 by NbSBT1 and NbSBT2.Click here for additional data file.


Twitter is a free, open access social media platform that is widely used in medicine by physicians, scientists, and patients. It provides an opportunity for advocacy, education, and collaboration. However, it is likely not utilized to its full advantage by many disciplines in medicine, and pitfalls exist in its use. In particular, there has not been a review of Twitter use and its applications in the field of neurology. This review seeks to provide an understanding of the current use of Twitter in the field of neurology to assist neurologists in engaging with this potentially powerful application to support their work. Though Twitter has been widely used in medicine by physicians, scientists, and patients, its application in the field of neurology remains relatively uncharted.Twitter is a free-to-use social networking and microblogging platform, enabling registered users to post , read, and repost  short messages known as tweets. Tweets are limited to 280 characters—previously 140—and users can share photos, short videos, or website URLs [Here we explore the powerful potential of Twitter in promoting research, education, and health care activities in neurology to relevant stakeholders. We also examine the risks and limitations of Twitter use in a professional capacity.References for this review were identified by searching PubMed, Google Scholar, Embase, MEDLINE, and the Twitter site between March 2006  and July 2020 as well as searching references from relevant articles. The search terms “Twitter,” “neurology,” “journal clubs,” “tweetorials,” “tweet chats,” “misuse,” “unprofessional,” “social media,” “health care,” and “medicine” were used. The search was restricted to English-language articles. The final reference list was generated on the basis of relevance to the topics covered in this review.Twitterverse over the past few years . As with other social media platforms, neurologists, neurology residents, neuroscience researchers, advocacy groups, journals, professional societies, and funding agencies have all asserted their presence in the Twitter hashtags  help connect people with topics of interest. By following specific profiles and hashtags and reading the associated threads, one can quickly establish an overview of the most up-to-date research activities, clinical advances, and future perspectives. This effect is particularly apparent in rare diseases, where following key handles  and hashtags ensures that researchers, physicians, and patients can establish connections; for example, #ALSResearch , @Phelan_McDermid and #PMSF (Phelan McDermid Syndrome Foundation), @thetinman_org (help and awareness for stiff-person syndrome), @yaya4HL , @fragilexsyndrom, and numerous others. Such social media cross talk is often helpful in building clinical registries and biorepositories and may facilitate higher-quality clinical and translational research.Twitter can help promote the visibility of clinical trials, enhancing both recruitment and inclusivity via links to enrollment websites . Some suThe Emergency Laparotomy and Frailty (ELF) study achieved a target enrollment of 500 participants by utilizing a Twitter handle (@ELFStudy) and eye-catching logos on its Twitter profile .The AVERT DOSE trial  posts regular updates on their work evaluating rehabilitation after stroke via their Twitter profile , ensurinNovartis is recruiting participants with advanced malignancies for US clinical trials, and its Twitter profile (@NovartisOncCT) shares the following link for potential participants to join: “http://bit.ly/2LhfJIk! #ClinicalTrialsSM” . Wasilewski et al, in the context of a caregiver study, conducted a secondary analysis of their Twitter recruitment and found that out of the 71 caregivers, 27 were recruited via Twitter. General recruitment tweets were most frequently shared by users .In a study by Sedrak et al, while the majority of the tweets (86.3%) had embedded links to news articles, one tweet led to a patient recruitment site  . HarnessTwitter enables users from around the world to interact with individuals at the forefront of academic research or clinical practice. This interaction may be via tweets, comments, or both—an engagement that might not otherwise have been possible. Conference organizers can also enhance the influence of conference-related discussions beyond those attending in person through social media, including Twitter . FollowiWithin specialties, there are focused spaces for discussion on specific topics or diseases—a global connection for users with a small area of shared interest. For instance, @Microbleeds discusses microbleed research and @ScienceofPD highlights Parkinson disease research . Many alTwitter provides a platform where opinions, queries, and comments on recently published research can be posted, and reacted to, in real time facilitating easy access for clinical neurologists to recent advances. Examples of highly followed journals and societies can be seen in Twitter facilitates patient involvement in care and has the capacity to remove barriers to understanding complex medical conditions through sharing information. By using Twitter, a patient can connect to support groups pertaining to his or her illness, such as the Motor Neuron Disease (MND) Association (@mndassoc)  and ParkTwitter allows for focused reading on a particular topic of interest by providing links to research articles. For instance, someone interested in clinical genetics can follow #GeneTherapy, browse through tweets, and obtain links to pertinent websites, such as the following tweet:Standardizing Cell and #GeneTherapy Remains Bioindustry Goal -- Please Read: http://ow.ly/3fJu50AQgLP; @AVROBIO about characterizing stem cells for gene therapy.Individuals who have or research a specific disease can keep up to date at the level of the individual gene; for example, the inherited neuropathy gene PMP22 has its own hashtag: #PMP22.Conference updates are shared through individual Twitter handles  and via broader platforms using hashtags . For future conferences, links can be utilized for abstract submission , resulting in broader coverage and reach. It is possible to share the latest research presented during and after a conference, increasing accessibility to those unable to travel and attend in person. Consider a neurologist at a conference who replies to a tweet from @GreenJournal; others may respond in real time with critiques and may share related papers, real-life experiences, and links to related conference presentations or other research. This intertwining of experience, expertise, and communication has the potential to advance the reach of health care delivery systems. Virtual conferences have found renewed popularity in the context of the COVID-19 pandemic and benefit from advertising via Twitter; for instance, @WorldStrokOrg recently tweeted the following:#ESOWSO2020 will run as a fully virtual event from 7-9 November. Virtual sessions will allow live interaction and will be available on-demand in the weeks following the Conference.Another example from the Movement Disorder Society (MDS) Twitter handle @movedisorder is as follows:New in #MDSMovingAlong: Looking Forward to the First MDS Virtual Congress. Read more from the MDS Virtual Congress Task Force on plans for this year's big event. There is still time to register at no charge before it begins on September 11. #MDSCongress.Journal clubs on Twitter are an emerging alternative to in-person journal clubs, which are limited by time or geographical location. Twitter provides accessibility and flexibility to involve people globally, without the added pressure of hierarchy, ensuring a broader audience. For example, #NCSTJC is the Twitter hashtag for the Neurocritical Care Society’s journal club. Similar to an in-person journal club, at a Twitter journal club, a paper is selected, sometimes using crowdsourcing to ascertain participant interest. At a specific time, participants log on to Twitter, and there may be a facilitator similar to an in-person setting. Preselected questions and polls may be posted via handles or hashtags to the participants. Though there is potential for textual discussion, it is limited by the 280-character limit, unless a thread is used .Traditionally, journals provide a restricted and moderated platform for research review and comments on recently published data. Twitter provides a more open platform for instant, multidimensional feedback on research quality and validity. On an individual level, reading through such comments may help broaden knowledge and enhance analytical skills. On a larger canvas, it may identify research flaws and knowledge gaps, ultimately leading to better designed and focused research. One can even seek comments from a particular organization or individual using their Twitter handle. Furthermore, there is a distinct advantage of obtaining varied views across the globe for one’s research, rather than the tunnel vision that can exist within smaller communities.Academics and the public can be biased toward publications in high-impact journals, which may result in excellent research work that is published in less “prestigious” journals, thereby gaining less visibility. Social media outlets, such as Twitter, offer individuals and organizations such as universities a platform to publicize their work. However, expression of one’s expertise and research work through social networking sites, instead of through conventional platforms like journals, can be considered as less thorough or pertinent .Twitter has opened up new dimensions to education and learning. For example, Tracy Milligan (@Tracey1milligan), through #NeurologyMorningReport , shares tweetorials have seen explosive growth , @LGBT_PsychNeuro, and others. Such groups have the potential to reduce isolation and enhance connectivity and visibility, and act as one of the spaces for people to discuss how to best manage the inherent challenges of the working and academic worlds.Work-life balance and healthy working environments are prioritized by health care institutions. Recent studies have shown burnout to be very common among all neurology practice settings and subspecialties, with one study reporting that 60% of respondents had at least one symptom of burnout , and anoMany community-centric health care activities are shared through the Twitterverse. Support groups for a variety of common and, importantly, rare neurological disorders enable relationships beyond the traditional physical space .viral [Public awareness campaigns that use Twitter can reach a broader audience within a shorter time frame than more traditional advertising platforms. For instance, the ALS Ice Bucket Challenge (@IceBucketALS)  was concviral  on sociaviral .A study conducted in 2011 uncovered the stigma associated with epilepsy on Twitter, as 41% of tweets related to “seizure” were seen to be derogatory in nature . SimilarTwitter has been adopted by patient-focused groups  [Shahi et al undertook an exploratory study on COVID-19 misinformation on Twitter. Analysis of 1500 tweets relating to 1274 false and 276 partially false claims revealed that verified Twitter handles, including organizations and celebrities, were also involved in either creating or spreading the misinformation via new tweets or retweets, respectively. They also noted that false claims propagated faster than partially false claims (χ N=1500) . Controv N=1500) . In the  N=1500) .Twitter is experimenting with adding brightly colored labels directly beneath lies or misinformation posted by famous users , F, in aTwitter posts with biased information may be promoted by parties with commercial interests. During the COVID-19 infodemic, the US Food and Drug Administration had to issue warnings to individuals to refrain from promoting or selling colloidal silver on social media profiles . TwitterIn June 2020, an Indian-based Ayurveda pharmacy claimed that it had discovered a potential cure for COVID-19, with an “80 per cent cure rate” . A tweetSimilarly, predatory conferences aggressively solicit conference abstract submissions  and combJust got an invitation for “Neurology 2019”, a world-wide meeting that attracted no less than 60 participants last year in Paris! This gem is organized by @Euroscicon. #Avoid #PredatoryConferences #FakeConferences.identifier hashtags.Strategies to avoid such commercial misuse may include identifying and calling out suspicious activity or insisting on It is possible to be highly influenced by a small group of opinions. Users should take time to consider whether what they see is unbiased, as algorithms suggest potential profiles of interest and posts based on the interests users have already demonstrated. Twitter is an enviable educational resource, but users must always ensure that their practice is appropriately evidence based.research participant data sets, which raises some concerns. In one study, 60% of Twitter users surveyed were unaware that publicly available tweets can be used for research, and 65% felt researchers should not be able to use public tweets without user permission [Twitter provides easy access to potential rmission . There armission .Prevalence of unprofessional social media content among young vascular surgeons was retracted by the Journal of Vascular Surgery [Previous studies on verified medical Twitter users have reported a worrying trend of “unprofessional” tweets, including patient privacy violations, profanity, sexually explicit content, discriminatory statements, inaccurate information, and self-promotion . While m Surgery . The res Surgery ,72. Twit Surgery .Sharing of prepublished content, speaker slides, and pictures taken during lectures beg the question of whether this may qualify as a breach of intellectual property. For users, it may be best to ask for permission from the primary source prior to posting information on Twitter.Trolling refers to deliberate acts of making controversial comments in order to provoke internet users to respond emotionally. Trolls have used tweets to target patients with photosensitive epilepsy with flashing images intended to provoke seizures in the recent past ,75. BefoVarious groups may use the same hashtag and confuse hashtag followers. People may also use a hashtag incorrectly or to spread false information. In addition, people may bog down a hashtag with many posts that are not necessarily relevant or helpful. All of these make it harder to find the desired posts.There are numerous other ethical concerns associated with physicians’ use of Twitter. One critical question is, “Who will regulate its use and how could regulations be enforced?” While Twitter has initiated, and will initiate, changes based on real-life experiences, the onus of patient confidentiality and professionalism lies with individual users. The challenges can be much higher in countries with lower English literacy, since the vast majority of information in the aforementioned resources is presented in English. The issue of language translation should be considered, as the message could be lost or transformed by translation. With this in mind, we propose some tips for using Twitter in How should I use Twitter?:Consider operating separate personal and professional accounts.Make an honest, representative Twitter profile. Identities are periodically verified by Twitter.Before sharing or retweeting, check the authenticity of the information and source. If you should reference another user’s work or tweet, use the retweet function and add a comment or use the relevant hashtag or handle to credit them. Alternatively, provide a reference or a URL to the source material.Healthy critique is positive. Even if offence is not intended, comments that could be interpreted as bullying or discriminatory are ill advised. Consider whether foul language is truly necessary.Educate yourself with the social media guidelines of your institution and strictly adhere to them. Remember that employers often review social media accounts—never tweet anything that could affect your professional standing or employability. In case of complaints and discrepancies, employers, regulatory bodies, or legal agencies can become involved.Use meaningful hashtags. Follow a large number of other accounts and follow your followers back to maximize the number of people who see your tweets.Proudly share your work. If people learn from you, they may like, follow, or retweet you.When following, posting, and retweeting, endeavor to show good judgment and consider why you are saying what you are—ensure it reflects well on you and your colleagues.Carefully choose the people and groups you follow or share information with. Review the privacy settings to regulate who can view your posts.If you are a victim of targeted harassment or abuse, unfollow and end any communication with that user. If the behavior continues, it is recommended to block the user and report them to Twitter.How to maintain social media professionalism:Avoid advertising.Avoid sharing any patient-identifiable information; even if anonymized, ensure you have the proper consent.Demonstrate good judgement—if you should not say it in your workplace, it should remain untweeted. Avoid posting and sharing misinformation, racist or discriminatory statements, or unpleasant or explicit content. Think carefully before tweeting: if necessary, compose your tweet and review it after a short period before posting.The Twitterverse is a vast and exponentially growing expanse of information. Neurologists have taken to Twitter to educate, promote research, share information rapidly, and reach a broader potential global audience. Twitter has added a new dimension to learning and education in neurology in a practical and interactive manner. Multichannel interactions have furthered improvements in patient care and other health care–related activities. This has been particularly beneficial in promoting, furthering, and communicating research. In addition, there is enhanced support available to patients, particularly in the fields of rare and orphan diseases. However, the pros of the Twitterverse must be balanced with the potential risks, which are common to all social media platforms. Greater reach means that misinformation and promotion of vested interests can be too easily and widely disseminated. Unregulated use can result in inappropriate and unprofessional conduct whether intentional or not, highlighting the urgent need for designing and implementing institutional, national, or specialty-specific ethical and legal guidelines on appropriate social media use. Nonetheless, with judicious use, the benefits of Twitter for a neurologist or neuroscientist outweigh the risks; a graphical rendering is provided in Figure S1 in


Whilst the underlying mechanisms of IDO1-mediated dormancy are elusive, we summarize the IDO1 pathways which potentially contribute to dormancy in this review. Critically, distinct IDO1 activities are involved in dormancy initiation and maintenance; factors outside the well-studied IDO1/kynurenine/aryl hydrocarbon receptor axis, including the mammalian target of rapamycin and general control nonderepressible 2, appear to be implicated in dormancy. We also discuss various strategies for cancer treatment  Cancer cells often remain unaltered and hidden until appropriate conditions for further progression are met. This status can be explained by tumour dormancy which is defined as a stage during which dormant cancer cells reversibly enter a G0 phase of the cell cycle and exhibit neither proliferative nor apoptotic characteristics; at the population level, it is characterised as a quiescent state of equilibrium in which a balance is maintained between tumour cell division and death , 2. DormPrevious literature has extensively investigated factors associated with the dormancy mechanisms, and indoleamine 2,3-dioxygenase 1 (IDO1) has been identified as one of the players . IDO1 avia targeting these interactions.Despite that IDO1 blockers have shown encouraging effects towards tumour suppression in preclinical settings and phase I/II trials, they are far from satisfactory in phase III clinical development via JAK/STAT, NF-κB, or other signalling pathways  are signalling proteins expressed, primarily by cytotoxic T lymphocytes, NK cells, and plasmacytoid dendritic cells (pDCs), in response to viral infection . They caHowever, stem-like TRCs are capable of hijacking such mechanisms for protection against the immune system , 12. ThrThe IFN-stimulated IDO1/Kyn/AhR cascade is favoured over the JAK/STAT pathway in TRCs rather than in differentiated tumour cells. This difference between tumour subpopulations might be explained by their distinct expression profiles that TRCs exhibit higher levels of IDO1 and AhR than differentiated tumour cells do , 12. EveAdditionally, SOX2, which acts upstream of IDO1 and downstream of IFN-γ, is identified as another potential key involved in TRC dormancy . As a weWhilst the positive correlation of activities between IDO1 and IFN-γ is strong in colorectal cancers, for example, it is relatively weak in ovarian or kidney cancers such that they may show great IDO1 expression but less activity of IFN-γ. Thus, researchers should consider the variation of IDO1 and IFN-γ activities in different cancer cell lines, because the inconsistent relations between these two factors have implied (1) the presence of other IDO1 inducers and (2) multiple stages of dormancy captured , 10. ImpCollectively, IFN signalling appears to be crucial for initiating tumour dormancy. Yet, distinct mechanisms have been revealed, at least, between the initiation and maintenance of tumour dormancy, or between different tumour cells. One should pay special attention when attempting to alter the dormancy state through IDO pathway regulation. Merely targeting the IFN pathways seems to be insufficient for effectively controlling the IDO-dependent tumour dormancy.via inhibition of integrin β3  are now considered as potential contributors to cancer development and IDO1-dependent tumour dormancy , 30. COXgnalling . Therefognalling –38. Unlignalling –42.Apparently, the PGE2 signalling serves a crucial role in balancing apoptosis and growth in tumours, at least partly, through IDO1 mediation. Future research should examine how the interplay between IDO1 and PGE2 may impact on the initiation and maintenance of tumour dormancy. We also anticipate a systematic investigation of different cancer cell lines which should guarantee a more advanced understanding of the underpinnings of tumour dormancy.Aiming at the cancer-associated events triggered by IDO1 pathways, most researchers focused on the enzymatic effects of IDO1. Nonetheless, previous literature has demonstrated the noncatalytic activity of IDO1, particularly in plasmacytoid DCs, for induction of immunosuppression –45. SpecIn line with these studies, TGF-β promotes immunoregulatory events, including the differentiation of the regulatory T cells (Tregs) , 47. At As discussed in the previous section, the AhR signalling is activated by the IDO1-mediated conversion of kynurenine from tryptophan , 12. TheOther than the IDO1/Kyn/AhR signalling, IDO1-mediated immunosuppression can also be achieved through the mammalian target of rapamycin (mTOR) signalling , 54. TheThe general control nonderepressible 2 (GCN2) is identified as another sensor of tryptophan sufficiency, and always works together with the mTOR pathways to regulate T cell activity , 62. WhiOwing to the critical role in tumour-associated immunosuppression, IDO1 appears to be a potential target for cancer treatment , 65–70. Van den Eynde et al. have summarised possible reasons and corresponding solutions specifically for the unsatisfactory clinical trial of Epacadostat . For exavia the Kyn/AhR signalling may not always be IDO1 specific.Targeting IDO1-associated dormancy yet lacks practicality, and few IDO inhibitors are tested against tumour dormancy. We expect future research to work on the impacts driven by IDO1 regulation, particularly on the dormancy mechanisms in addition to the active tumours. This should be crucial for the success of IDO1 treatment development, as tumour dormancy has been strongly associated with metastasis and later resistance to cancer treatments , 75. It via aggressive treatments, the pro-dormancy approaches may suppress tumour cell growth and enhance cancer patients’ quality of life in a gentler manner  nanoparticle loaded with PD-L1 trap synergises with chemotherapy . Such a Previous literature has suggested IDO1 should be connected to immunosuppression and tumour dormancy. However, there is still much room for understanding how exactly these IDO1 pathways result in dormancy. One of the keys falls on the IDO1 expression patterns, and constitutive rather than simply inducible IDO1 expressions may be critical for entering the dormancy state. Furthermore, initiation and maintenance of dormancy appear to involve distinct mechanisms such that they require different molecular factors to participate. Having a more thorough understanding of these mechanisms should provide novel insights into dormancy regulation and cancer treatment. Regarding the perspective on IDO1-centered dormancy regulation, while current research has devoted to the development of IDO1 inhibitors for active tumour elimination, how these inhibitors may be applied in dormancy models needs further attention. On the other hand, pro-dormancy therapy through IDO1 activation may serve as an alternative strategy for cancer treatment, yet it requires additional investigation in the future. Finally, given the potential of nanotechnology, we foresee an ever-rising use of nanomaterials for providing accurate delivery of treatments targeting IDO1-associated dormancy and enhancing cancer therapy.CY and C-TN conceived and designed this manuscript. DL and ZL have read, revised and approved the manuscript. All authors contributed to the article and approved the submitted version.The work was supported by the Natural Science Foundation of Jiangsu Province, China (Grant No. BK20190960), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (Grant No. 19KJB180014), China Postdoctoral Science Foundation (2019M663456 and 2019TQ0044), Xinglin Scholar Research Promotion Project of Chengdu University of TCM (BSH2019008), Sichuan Province Science and technology innovation seedling project (2020091), Open Research Fund of Chengdu University of Traditional Chinese Medicine Key Laboratory of Systematic Research of Distinctive Chinese Medicine Resources in Southwest China (2020BSH004) and Canada Mitacs Fellowship (IT18262).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the published version of this article, a grant number was omitted from the section ‘Funding Information’. The section should have read:This work was funded by an MRC grant (MR/S001026/1) to M. H., while S. C. was funded by the China Scholarship Council. G. R. F. C., C. B. and P. R. were funded by FAPESP .The authors apologise for any inconvenience caused.


Purpose: A major issue in radiotherapy is the relative resistance of hypoxic cells to radiation. Historic approaches to this problem include the use of oxygen mimetic compounds to sensitize tumour cells, which were unsuccessful. This review looks at modern approaches aimed at increasing the efficacy of targeting and radiosensitizing hypoxic tumour microenvironments relative to normal tissues and asks the question of whether non-targeted effects in radiobiology may provide a new “target”. Novel techniques involve the integration of recent technological advancements such as nanotechnology, cell manipulation, and medical imaging. Particularly, the major areas of research discussed in this review include tumour hypoxia imaging through PET imaging to guide carbogen breathing, gold nanoparticles, macrophage-mediated drug delivery systems used for hypoxia-activate prodrugs, and autophagy inhibitors. Furthermore, this review outlines several features of these methods, including the mechanisms of action to induce radiosensitization, the increased accuracy in targeting hypoxic tumour microenvironments relative to normal tissue, preclinical/clinical trials, and future considerations. Conclusions: This review suggests that the four novel tumour hypoxia therapeutics demonstrate compelling evidence that these techniques can serve as powerful tools to increase targeting efficacy and radiosensitizing hypoxic tumour microenvironments relative to normal tissue. Each technique uses a different way to manipulate the therapeutic ratio, which we have labelled “oxygenate, target, use, and digest”. In addition, by focusing on emerging non-targeted and out-of-field effects, new umbrella targets are identified, which instead of sensitizing hypoxic cells, seek to reduce the radiosensitivity of normal tissues. One of the most difficult barriers encountered when treating the majority of solid tumours is attributed to the scattered microregions within the tumour characterized by the lack of oxygen. This is known as tumour hypoxia . In ordeOne of the earliest techniques used to control tumour hypoxia was hyperbaric oxygen (HBO). 2 and 95% O2 to create a gaseous compound known as carbogen [The uncertainty surrounding HBO treatment for combating tumour hypoxia has shifted to using a combination of 5% COcarbogen . Recent carbogen ,27. Althcarbogen . Moreovecarbogen . To facicarbogen . Howevercarbogen . Despite18F-fluoromisonidazole (18F-FMISO) has garnered the most success and has been extensively studied [18F-FMISO. 18F-FMISO is a lipophilic compound; thus, it is readily available to passively diffuse into cells, subsequently, reduction of this compound via the nitroreductase enzyme (NTR), results in the production of R-NO2 radicals [2 < 10 mmHg), these radicals are unable to be reoxidized, leading to further reduction of R-NO2 radicals to form R-NHOH molecules that can bind to cellular components, allowing for tumour hypoxia imaging [18F-Fluoroazomycin arabinoside (18F-FAZA), has been gaining more popularity relative to 18F-FMISO [18F-FAZA, in comparison to 18F-FMISO, is less lipophilic due to the presence of an additional sugar moiety [18F-FAZA has a faster diffusion and clearance rate relative to 18F-FMISO, allowing for an improved tumour-to-background (T/B) ratio, thus, this compound has been gaining more interest as a PET radiotracer [18F-FAZA as a PET radiotracer for tumour hypoxia imaging, researchers are beginning to investigate the use of PET imaging to mediate oxygen-based manipulation therapies, such as carbogen breathing. Thus, tumour hypoxia imaging serves as a promising method to identify hypoxic tumour microenvironments relative to normal tissue, ultimately improving the efficacy of oxygen-based manipulation therapies. In relation to the non-targeted effect theme of this review, which is discussed in detail later, it is likely that increased oxygen will increase oxidative stress and lead to initiation of both bystander signalling and genomic instability phenotypes. To what extent this impacts the therapeutic ratio is not known and is an area for further research.As previously stated, in order to facilitate effective tumour hypoxia imaging, an “ideal” PET radiotracer is required and must meet a number of biochemical characteristics . Althoug studied . The mecradicals . Further imaging ,34,35,368F-FMISO . Studiesr moiety ,37,38. Miotracer ,40. Over18F-FAZA has been gaining recent attention as a PET radiotracer, in relation to the popular counterpart, 18F-FMISO, due to increased efficacy in imaging. An experiment conducted by Tran et al. [18F-FAZA PET, as a tool for guiding treatments that target tumour hypoxia, such as carbogen breathing. In this study, Tran et al. [18F-FAZA with a volume of 500 μL. Within both tumour models, a control group was set aside and was not subjected to IR; in addition, rats with 9L-glioma were exposed to 40 Gy [18F-FAZA PET was carried out prior to irradiation. Moreover, treatment groups  were randomly organized into two distinct groups, breathing in air or carbogen [18F-FAZA, and stayed under these conditions until the end of the study [18F-FAZA administration [2) within tumours, EPR oximetry was used. Using this approach, it was confirmed that both rat models displayed pO2 < 10 mmHg confirming that they are hypoxic [2 levels less than 10 mmHg to a T/B ratio greater than 1.7 when using 18F-FAZA, whereby hypoxic tumour microregions remained radioresistant. The significance of 10 mmHg serves as a threshold for distinguishing between radioresistivity  or radiosensitivity . Furthermore, a T/B ratio of 1.7 serves as a benchmark to determine the efficacy of radiotherapy following tumour hypoxia therapeutics such as carbogen breathing [18F-FAZA using PET imaging and EPR oximetry [p < 0.05) for both tumour models; however, rats with 9L-gliomas experienced a greater response to carbogen breathing relative to rats with rhabdomyosarcoma. Furthermore, through EPR oximetry measurements, rats with 9L-gliomas demonstrated a greater increase in intracellular levels of oxygen following carbogen breathing in comparison to rats with rhabdomyosarcoma, which did not surpass the threshold of pO2 for displaying radiosensitivity [18F-FAZA serves as an attractive PET tracer for PET tumour hypoxia imaging in order to dictate oxygen manipulation-based therapeutics such as carbogen breathing. In addition, the results from this study display promising evidence that tumour hypoxia imaging via PET imaging can potentially become a standardized diagnostic approach for identifying hypoxic tumour microenvironments in order to improve anticancer therapies. Moreover, future studies should analyze the efficacy of using 18F-FAZA with PET for tumour hypoxia imaging in order to measure the effectiveness of anticancer therapies outside of oxygen manipulation-based therapies. Overall, although this method has yet to be translated to a clinical setting and other forms of cancer, this study serves as an exciting gateway for further exploring the role of 18F-FAZA as a PET tracer and its connections to mitigating tumour hypoxia, and as a novel approach to better detect hypoxic tumour sites.Due to the ongoing barrier of tumour hypoxia causing resistance to IR, there has been an increase in research regarding tumour hypoxia imaging using PET scans in order to modify treatment methods, such as carbogen breathing . As prevn et al.  exploredn et al.  used twoto 40 Gy . In contcarbogen . Rats suhe study . Subsequstration . In addi hypoxic . In orde hypoxic . A previ hypoxic  has linkreathing . Moreoveoximetry . Based ooximetry  producedsitivity . Moreovesitivity  analyzedsitivity  have detDue to the concerns associated with HBO treatment, researchers began to investigate compounds that mimic oxygen, which also allows for the radiosensitization of hypoxic tumour cells. Aside from the classic compounds used as radiosensitizers, GNPs have demonstrated promising results as novel hypoxic cell radiosensitizers. Following exposure to IR within hypoxic cells subjected to GNPs, these materials undergo three distinct interactions with IR, physical, chemical, and biological, to induce radiosensitization . First, 2 to produce free radicals [Following physical interactions with GNPs, chemical interactions soon take effect. Although the mechanisms underlying these interactions have not been completely elucidated, studies suggest that chemical enhancement occurs through two different pathways . The firradicals . Accordiradicals , due to radicals . Moreoveradicals .2−), hydroxyl radicals (OH), and hydrogen peroxide (H2O2) interact with cellular components to induce cellular damage through two different mechanisms. Her and colleagues [The biological interactions between GNPs and IR occur through three different pathways, oxidative stress, disruption of the cell cycle, and inhibition of DNA repair . One of lleagues  state thlleagues . Althouglleagues . Severallleagues ,69 suggelleagues  indicatelleagues . Moreovelleagues . Mackey lleagues  have elulleagues ,72,73,74lleagues . The inalleagues . Using clleagues ,75,76,77lleagues  state thlleagues . Overalllleagues ,81,82,83Furthermore, research has also been conducted on developing various methods for nano-drug delivery systems (NDDS) in order to effectively deliver radiosensitizers to hypoxic regions of a tumour . One suc®, which is comprised of pegylated silica core-gold shell nanoparticles used within photothermal therapy for head and neck cancers [Despite the high potential of GNPs as novel radiosensitizers, these compounds have not yet been approved for clinical use. However, several clinical trials have demonstrated the efficacy and potential of using these compounds in tandem with radiotherapy to treat tumour hypoxia . Chen et cancers . Kumthek cancers  conducte cancers  have com cancers  have proOverall, all four studies demonstrate the potential for GNPs as novel radiosensitizers; however, there is a general consensus that many questions regarding the translation of GNPs to a clinical setting have not yet been answered. A review by Schuemann et al.  suggestsN-oxide and has shown compelling results both in vitro and in vivo [Aside from radiosensitizing drugs, alternative drugs classified as hypoxia-activated prodrugs (HAPs) were designed to preferentially kill hypoxic tumour cells through the generation of free radicals ,90. The  in vivo . However in vivo . In addi in vivo ,93. Simi in vivo . Particu in vivo . Macroph in vivo . One rea in vivo ,96,97,98 in vivo ,96,97,98 in vivo  state thAs stated earlier, current research has been focused on advancing drug delivery of HAPs to hypoxic tumour regions mediated by macrophages due to the possibility of HAPs entering normoxic cells and causing detrimental effects on the cell. Macrophages are responsible for the production of inflammatory and antimicrobial cytokines, as well as the removal of pathogens, and are the predominant phagocyte within the immune system . DependiIn order to use macrophage-mediated delivery for HAPs, the process requires four distinct phases; “cargo” loading , maintaining cargo integrity, motility of macrophage in vivo to the target site, and cargo expulsion . In ordeFollowing the release of HAPs from a macrophage carrier, HAPs undergo a series of chemical reactions to activate the drug. Moreover, HAPs display inefficient selective behaviour, which is apparent in one of the most extensively studied HAPs, TPZ . In prinC12. Evans and colleagues [C12 displayed a 3.7 reduction in tumour size and weight, relative to mice treated solely with TPZ and TPZ-C12.Although several HAPs, most notably, TPZ, have successfully advanced to clinical trials, several shortcomings have prevented the approval of HAP in clinical settings . Recent lleagues  conductelleagues  concludeOverall, this study provides compelling evidence for the use of macrophage-mediated drug delivery systems for HAPs as a novel technique for treating tumour hypoxia. In addition, the conclusions drawn from this study strongly suggest that one of the major shortcomings of TPZ, the inability to effectively target hypoxic tumour microenvironments relative to normal tissues, can be overcome through macrophage-mediated drug delivery. Although this technique has only recently emerged, this study serves as an exciting gateway for exploring extensions of this technique to other HAPs or chemotherapeutic drugs and other types of cancers. Unsurprisingly, as this technique is still in its early developments, much more extensive research through preclinical trials is required prior to the translation to clinical settings.Another method is the use of drugs that target tumour metabolism. Tumours demonstrate a growth advantage through a shift in metabolism, specifically, from oxidative phosphorylation to glycolytic metabolism, which is driven by HIF-1–pyruvate dehydrogenase kinase 1 (PDK1) . ConsequAs previously mentioned, recent studies on tumour metabolism have evolved from analyzing the shift in oxidative to glycolytic processes within tumours to the role of autophagy in tumour progression and the subsequent development of autophagy inhibitors. In particular, the most notable autophagy inhibitors are CQ and HCQ, which are classified as 4-aminoquinoline agents and were first intended as anti-malarial drugs . HCQ is The mechanism of autophagy and its relationship to radiation and NTE has been well studied ,148,149.As recent research has shifted away from tumour metabolism to autophagy, many preclinical and clinical trials are examining the use of autophagy inhibitors, namely CQ and HCQ, to treat tumour hypoxia. Due to the extensive clinical trials conducted on CQ and HCQ, this review will focus on the early clinical trials and current developments. Early clinical trials conducted by Briceño and colleagues  examinedHowever, one of the major shortcomings of solely using CQ or HCQ is the inability to determine changes in autophagy, such as increases or decreases in activity, termed as “autophagy flux”; hence, current research is aimed at identifying autophagy biomarkers that detect these levels ,162. NotAlthough CQ and HCQ are currently the only autophagy inhibitors being investigated for clinical use, future objectives should be aimed at uncovering potentially more potent and efficient autophagy inhibitor drugs. In addition, the cellular mechanisms and relationship between autophagy biomarkers, such as LC3II and sequestosome 1, have yet to be completely elucidated. Hence, additional research must be conducted to examine currently known autophagy biomarkers, as well as identifying novel biomarkers in relation to responses to CQ, HCQ, and potentially novel autophagy inhibitor drugs. Despite the potential for developing acute kidney injury resulting from CQ-based anticancer therapies, validating the cause-and-effect relationship may not be possible . The uncWhile most work aiming to improve the therapeutic ratio focuses on sensitizing the tumour tissue, an equally valid approach is to increase the resistance of normal tissue. One approach to this is to manipulate bystander signaling. The bystander effect refers to effects in cells that were not themselves irradiated but that were in receipt of signals from irradiated cells . Such siThe aim of manipulating NTE would be either to turn the mechanism ON in hypoxic tumour cells or OFF in normal cells. Identifying ways to do this requires detailed knowledge of how NTE are initiated and perpetuated in cells. Except for one very recent paper from Zhang et al. , who fouA very different set of papers in the hypoxia literature uses the term “bystander effect” It is widely understood that tumour hypoxia has proved to be a major barrier for treating the majority of solid tumours. In response to this longstanding problem, despite extensive research on early tumour hypoxia targeted therapeutics, these treatments have faced several limitations, including the ineffective targeting to hypoxic microregions relative to normal tissue. This complex issue led researchers to integrate novel technological advancements, such as nanotechnology, medical imaging, and cell manipulation to create novel approaches. Furthermore, the shift in perspectives led to the development of modern approaches including, imaging using PET to mediate carbogen breathing, GNPs, macrophage-mediated drug delivery for HAPs, and indirectly targeting tumour metabolism through autophagy inhibitors. All four novel approaches aim to improve anticancer therapeutics, primarily radiotherapy outcomes, as well as increasing the efficacy of targeting hypoxic tumour microenvironments relative to normal tissue. These therapeutics are still within the early stages of development, some reaching preclinical trials such as carbogen breathing guided by PET tumour hypoxia imaging and macrophage-mediated drug delivery, while others have reached clinical trials and await clinical translation, such as GNPs and CQ/HCQ. Furthermore, the knowledge affiliated with these novel therapeutics has seen a surge in attention; however, the biological mechanisms and the road to clinical translation have yet to be completely elucidated. Thus, in order to further potentiate the use of these approaches as novel tumour hypoxia therapeutics, thorough investigations must continue. Each of these novel approaches may be impacted differently by the presence of NTE, and several ways that NTE could be harnessed to improve the therapeutic ratio are discussed; however, this review found little evidence that this approach is being considered, and we suggest it is a novel and important avenue for research in the future.This review was centered around outlining the evolution of the methods for radiosensitizing hypoxic tumour cells and improving the targeting of hypoxic tumour microregions relative to normal tissue. Moreover, this review provides a comprehensive summary of the molecular mechanisms that allow for enhanced targeting and radiosensitization of hypoxic tumour microenvironments relative to normal tissue, preclinical/clinical studies examining the efficacy, and future considerations for clinical translations.


Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment.We performed TIL analysis and T-cell analysis by IHC on the pretreatment and ‘On-treatment’ samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation.n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10–3) but not TILs (p = 0.1) in their ‘On-treatment’ tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 +  and CD4 +  T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p < 0.01).In our sample cohort (The immune system may be ‘primed’ prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.The online version contains supplementary material available at 10.1007/s10549-021-06244-1. HER2-positive breast cancer accounts for approximately 20% of all breast cancers and prior to the clinical development of trastuzumab, had the worst outcome of any breast cancer subtype . HoweverTrastuzumab, a humanized monoclonal antibody, is known to have both cytotoxic and immunological effects on tumour cells , 2. In tUsing these tumour samples, we conducted TIL analysis and assessed the impact of a single dose of TCH/L chemotherapy treatment on the numbers of infiltrating lymphocytes in breast tumours. For the first time, our study identifies that immune contexture is significantly modulated in breast tumours after only 1 cycle of TCH/L chemotherapy, and this may provide clues as to how and why some patients achieve a subsequent pathological complete response (pCR).www.clinicaltrials.gov. pCR was determined in the TCHL clinical trial by the absence of invasive carcinoma. Of the 88 patients enrolled we were able to obtain lymphocyte information for 68 patients. Of those 68 patients, 20 had a core biopsy taken by an interventional radiologist, 20-days post-cycle 1 of either TCH/TCHL therapy (On-treatment samples). Samples were snap frozen and stored at −80 °C until required. Full clinicopathological details of patients involved in this study are including in Table TCHL ICORG10-05) (NCT01485926) is a Phase-II neo-adjuvant study run by Cancer Trials Ireland (formerly All Ireland Co-Operative Oncology Research Group (ICORG)) assessing TCH  and TCHL (TCH and lapatinib) in stage II-III HER-2-positive breast cancer patients [0-05 . Haematoxylin and Eosin (H&E) staining was performed on 3 µM sections of biopsies and assessed for invasive tumour epithelial cellularity by a Histopathologist. Only samples with greater than 10% tumour cellularity were used for further analysis. On-treatment samples were embedded in optical coherence tomography and the samples were cryo-sectioned. A single 3 µM section was taken for H&E staining and analysis, and the adjacent ten 10 µm sections were cut and stored in a chilled cryovial. Following this, a second 3 µM section was then cut for H&E staining. Cut sections were stored at −80 °C.2) were selected in each case. CD45 + cells were counted in each of the four areas. Cytokeratin AE1/3 was used to assess the location of tumour cells relative to the CD45 + cells in each of the areas counted. These IHC stains were completed on FFPE baseline biopsy samples (n = 68/88) and on fresh frozen (FF) biopsies taken 20-days post-cycle 1 (Day-20) of TCH/TCHL (n = 20/88). A lymphocyte was counted as a TIL if it was observed to be in direct contact with an invasive tumour epithelial cell [H&E staining was performed on a Thermo Shandon Varistain Gemini stainer using Harris haematoxylin  and alcoholic eosin Y  before being cover slipped (Thermo Shandon Consul). 4 µm serial tissue sections were cut using a Leica RM2135 microtome. IHC analysis was carried out on a Bond-III immunostainer . Primary antibodies CD45, Common Leukocyte Antigen  and Cytokeratin  were diluted in Bond Primary Antibody Diluent  at 1/500 and 1/400, respectively. Pre-treatment of samples was carried out on the Bond-III using Bond Epitope Retrieval Solution I  for 30 min (CD45) and Bond Enzyme Pre-treatment solution  for 10 min (AE1/3). Detection and visualisation of stained cells was achieved using the Bond Polymer Refine Detection Kit  with Bond DAB Enhancer . Tissues were counterstained with haematoxylin and cover slipped. Slides were scanned at 40X using a Philips 2.0 scanner, viewed with Philips Image Management System 2.2 and analysed as per current guidelines [We had previously shown from MCP counter analysis  a small The non-parametric Wilcoxon signed-rank test was used to determine if there was a significant difference between pathological complete response (pCR) and no-pCR for the three comparison groups . The test was paired when comparing baseline and on-treatment groups. The paired test was also used when comparing pre versus On-Treatment CD3 + , CD4 + and CD8 + counts. T-cell markers and tumour content were correlated using the non-parametric Spearman’s rank correlation. Tumour content versus T-cell markers was plotted and loess regression was used to fit a smooth line to illustrate the relationship between the two variables. P-values of less than 0.05 were considered statistically significant.p = 0.05) in their baseline pre-treatment tumour samples, relative to those patients who did not achieve a pCR post-chemotherapy . While larger studies have shown estrogen receptor status is predictive of pCR, it did not have an impact on rates of pCR in the TCHL study (p = 0.2141) [We determined the number of both SLs and TILs in the baseline pretreatment FFPE tumors of 68/88 patients who were recruited to the TCH/L trial Fig. a, b. Ourapy Fig. c. We als 0.2141) .Fig. 2AnWe have previously shown that tumour epithelial cells are undetectable in the day-20 On-treatment biopsies of some patients who go on to achieve pCR at subsequent surgery . Tumour p = 0.14). Of the 20 available on-treatment biopsy samples, eight (which includes a sample from the no-pCR group) had no residual tumour left in the biopsy after 20-days of starting neo-adjuvant treatment. Upon excluding these cases in which the immune response is possibly already subsiding, we observed that OL (p = 9.09 × 10–3) counts were significantly higher in on-treatment tumour biopsies from patients who subsequently achieved a pCR relative to those who failed to achieve a pCR at subsequent surgery  in their On-treatment tumour biopsy samples than those patients who did not achieve a subsequent pCR, but this effect was not seen for TILs (p = 0.1).In our pCR group we observed that after 1 cycle of therapy 70% (7/10) of biopsies had no residual tumour remaining . When we compared TIL counts in the pCR group we observed a non-significant trend whereby TIL numbers were lower in the biopsies with no residual tumour relative to the remaining biopsies where residual tumour remained (n = 9 vs No-pCR n = 7), irrespective of residual tumour status, determined that 1 cycle of neo-adjuvant TCHL treatment was associated with changes in levels of infiltrating lymphocytes in patient tumours when they were stratified on the basis of subsequent pCR. There was no consistent significant difference in TIL, SL or OL levels between baseline and day-20 tumour biopsies when the tumours in the group that attained a pCR at subsequent surgery were analysed   Fig. b, c.Fig.Given the key role of T-cells in regulating the immune response, we performed IHC analysis on 13/16 paired pre- and on-treatment fresh frozen samples for which we had sufficient material. The T-cell markers CD3 (pan T-cell), CD4 (T helper cells) and CD8 (cytotoxic T-cell) were examined. No distinction was made between stromal and tumour infiltrating T-cell populations. When analysing all patients, we observed that levels of CD3 + , CD4 + or CD8 + T-cells did not significantly increase or change from the baseline to the On-treatment tumour biopsy samples .To further analyse the effect of neo-adjuvant treatment on T-cell numbers we observed the changes in CD3 + , CD4 + and CD8 + T-cells in the matched baseline and day-20 On-treatment tumour biopsy samples of individual patients  and CD4 +  T-cells was associated with decreased residual tumour content post-1 cycle of treatment. We also observed a similar positive trend for CD8 + T-cells, but the results did not reach statistical significance . We, however, did not see a positive trend for OLs .As outlined above, neo-adjuvant TCH/L-based treatment results in a reduction of tumour volume after 1 cycle of treatment, and this tumour reduction correlates with a greater chance of a patient achieving a pCR . HoweverLong-term outcomes for women with HER2–driven early-stage breast cancer have significantly improved since the advent of HER2-targeted therapies including trastuzumab and lapatinib . In our n = 1256) [p = 0.0634)). Importantly, we classified these lymphocytes in accordance with Salagado et al. [TILs are proven to have positive prognostic implications in the outcome of early-stage breast cancer ; with el = 1256) . This ef = 1256) . In our  = 1256)  and Vina = 1256) . In the o et al. , being do et al. , 16. The. [p = 0.066) in the pCR group relative to the non-pCR group [In a limited number of the day-20 on-treatment biopsies available, we determined SL and TIL levels in patients who achieved a pCR  and those who failed to achieve a pCR at subsequent surgery. We determined that SL numbers were significantly increased at day-20 in the pCR group. However, we also found, by comparing matched lymphocyte levels between baseline and on-treatment tumour biopsy samples, that levels of lymphocytes do not increase in the group that achieve a pCR at subsequent surgery. This was in contrast to the non-pCR group, where both TIL and OL counts were significantly increased in the tumour after 1 cycle of neo-adjuvant chemotherapy treatment. Our findings support the hypothesis that in patients who achieve a pCR the immune microenvironment which already surrounds the tumour at baseline likely plays an important role in response to subsequent therapy. Hamy et al.  identifi. . HurvitzCR group . Therefo. [Interestingly, in the only other neo-adjuvant HER2-positive breast cancer study to collect matched on-treatment biopsy samples (PAMELA), Nuciforo et al.  found thMany studies have identified the importance of baseline lymphocyte numbers as a positive prognostic factor in determining subsequent pCR –22. To dOur analysis sheds light on the modulation of the immune response that occurs early during neo-adjuvant chemotherapy. In on-treatment tumour biopsy samples, lymphocyte counts increase after 1 cycle of neo-adjuvant therapy (in particular in tumours that do not end in pCR at subsequent surgery), but a reduction in T-cell counts occurs in some tumours which correlates with a lower tumour burden in day-20 on-treatment tumour biopsies. The latter is associated with a higher likelihood of pCR at subsequent surgery . The resSupplementary file1 (TIF 92 kb)Below is the link to the electronic supplementary material.