O-glycosides and tricin-oligolignols in all grass species examined, but only show patchy occurrences in unrelated lineages in dicots. More strikingly, tricin is a lignin monomer in grasses and several other angiosperm species, representing one of the “non-monolignol” lignin monomers identified in nature. The unique biological functions of tricin especially as a lignin monomer have driven the identification and characterization of tricin biosynthetic enzymes in the past decade. This review summarizes the current understanding of tricin biosynthetic pathway in grasses and tricin-accumulating dicots. The characterized and potential enzymes involved in tricin biosynthesis are highlighted along with discussion on the debatable and uncharacterized steps. Finally, current developments of bioengineering on manipulating tricin biosynthesis toward the generation of functional food as well as modifications of lignin for improving biorefinery applications are summarized.Tricin  is a specialized metabolite which not only confers stress tolerance and involves in defense responses in plants but also represents a promising nutraceutical. Tricin-type metabolites are widely present as soluble tricin  Flavonoids are a large group of plant-specialized metabolites that are ubiquitous in vascular plants and are also found in non-vascular plant lineages except hornworts . StructuF3H) expression , and lycophytes   and the dicot alfalfa , a prototype in the type III polyketide synthase superfamily, catalyzes sequential condensation of three malonyl-CoAs with chalcone . Chalcond tricin . AlthougO-conjugates and flavone C-glycosides are biosynthesized in separate pathways. Early radiotracer experiments on Lamnaceae plants revealed that 14C-labelled flavanone aglycones could be simultaneously converted into flavone O-glycosides and C-glycosides  and flavone synthase II (FNSII), were expected to convert flavanones into flavones by direct introduction of the C2–C3 double bond  as a model system, FNSII was identified to be the primary enzyme generating the flavone nucleus for tricin biosynthesis in grasses  O-glycosides which are normally not present in wild-type plants , chrysanthemums (chrysanthemum morifolium), and carnations (Dianthus caryophyllus), all of which naturally lack delphinidin-derived anthocyanins  are rarely detected in grasses  C-glycosides, while their production of soluble and lignin-integrated tricin remains unaffected  required for syringyl (S)-lignin biosynthesis in the spikemoss Selaginella moellendorffii. CYP788A1 is involved in both 3- and 5-hydroxylations of phenylpropanoids, but it could only catalyze 5-hydroxylation after 3-O-methylation  or 5-hydroxyconiferaldehyde O-methyltransferases (CAldOMT) as they also show in vitro O-methylation activities toward 5-hydroxyconiferaldehyde, 5-hydroxyferulic acid, and caffeic acid, which are intermediates in the monolignol pathway; hence, they are also involved in S-lignin biosynthesis  and luteolin (non-methoxylated) when compared with wild-type controls -related enzymes could catalyze 3',5'-O-methylation using various flavone substrates -dependent glycosyltransferases  in several putative UGTs were also found to be directly associated with the variations of flavone O-glycoside accumulation in different natural cultivars and/or recombinant inbred lines of rice -guaiacylglycerol-β-tricin ether [(−)-salcolin A], and erythro-(−)-guaiacylglycerol-β-tricin ether [(−)-salcolin B] isolated from Sinocalamus affinis (Poaceae) were determined as 7''S,8''S and 7''R,8''S, respectively --salcolin A and (−)--salcolin B (O–4) type neolignans in Arabidopsis -salcolin A and (+)-salcolin B were isolated -salcolin A and (+)--salcolin B  in typical non-grass vascular plants . The compatibility of tricin with radical coupling was demonstrated by biomimetic oxidations of tricin with monolignols using peroxidase/hydrogen peroxide and silver (I) oxide as oxidants (via the 4'–O–β-coupling mode (O–β)-linked phenylpropane units in the lignin polymers , Medicago legumes and Trigonella foenum-graecum (family: Fabaceae); Artemisia vulgaris (family: Asteraceae), Leucas cephalotes (family: Lamiaceae), and Lonicera japonica (family: Caprifoliaceae)] ] . Meanwhiiaceae)] . Intriguiaceae)] . Hence, Three possible types of dicot enzymes, FNSIs, FNSIIs, and F2Hs, have been described for flavone nucleus formation , but thePetroselinum crispum), FNSIs were long presumed to be confined to Apiaceae  MsFBH-4 and MsFBH-10, are utilized. In in vitro enzyme assays, these CYP proteins catalyze 3'-hydroxylation of different flavonoid classes  and 5'-hydroxylation of their 3'-methoxylated derivatives which include chrysoeriol. Furthermore, apigenin is converted to 3'- and 5'-substituted flavones  when these CYP75B proteins are transiently expressed in Nicotiana benthamiana leaves. Consistent with these findings, M. truncatula MtFBH-4 knockout mutants are completely depleted in tricin O-glycosides, hence establishing an indispensable role of MtFBH-4 in tricin biosynthesis. Basically, the same reaction steps that occur in grasses , which catalyzes the final step in melatonin biosynthesis  or tricetin in vitro  but not isorhamnetin (a 3'-methoxylated flavonol; The enzymes responsible for the 3'- and 5'-avonoids . Howeverynthesis . In addiynthesis , which min vitro . Over-exCereals contribute to more than half of the world population’s daily caloric intake, but the commonly consumed polished grains, which are mainly consisting of endosperms, are poor in phytochemicals and minerals . Their cAlthough tricin and its derivatives have been characterized with many different health-promoting properties , they arMedicago legumes (Functional food crops that are fortified with tricin could be generated by engineering the entire biosynthetic pathway in edible tissues. Previously, transgenic rice seeds that accumulate tricin were generated by expression of genes from multiple species encoding rice PAL, rice CHS, parsley FNSI, soybean FNSII, blue viola F3'5'H, and rice COMT/CAldOMT . Recent  legumes  as well Miscanthus, Erianthus, switchgrass, and bamboo, which show notably high biomass productivity, are attractive lignocellulose feedstocks for various biorefinery applications (Grasses show great potential as a source of lignocellulosic biomass. A large amount of lignocellulose is produced annually as agricultural residues from worldwide cultivation of grass grain crops, including maize, wheat, rice, barley, and sorghum, as well as grass sugar crops, such as sugarcane and sweet sorghum. In addition, grass energy crops, such as ications . Becauseications . HoweverFNSII (A3'H/C5'H (CHS showed increased lignin level and reduced cell wall digestibility in leaves albeit no alteration in either lignin content or cell wall digestibility in stems (CHS-deficient maize leaf cell walls was attributed at least partially to the consequence of the increased carbon flux toward the branching monolignol biosynthesis pathway upon the blockage of the entry of the flavonoid pathway where CHS plays the major role (As tricin could serve as a nucleation site for lignification, reducing the content of tricin used for lignification may result in reduction of lignin content and biomass recalcitrance, which may in turn improve the production of fermentable sugars from biomass in the polysaccharide-oriented biorefinery processes . Indeed,FNSII  or A3'H/3'H/C5'H , 2019a din stems . The altjor role . These sO–β linkages between the tricin- and monolignol-derived lignin polymer units are comparable to the major β–O–4 linkages connecting the internal monolignol-derived lignin polymer units, suggesting that introduction of more tricin units in lignin polymers is unlikely to increase the energy for lignin depolymerization (On the other hand, increasing the levels of tricin serving as initiation sites for lignin polymerization would theoretically reduce the molecular weight of the lignin polymers, which may potentially improve the efficiency of lignin deconstruction in the polysaccharide-oriented biorefinery processes . A recenrization . NonetheMeanwhile, grass crops bioengineered toward high tricin-lignin content could bring benefits in the lignin-oriented biorefinery approaches by amplifying the supply of tricin or tricin-derived aromatic chemicals. It has been estimated that large quantity of tricin could be released from grass lignins . HoweverPL, YT, and CL wrote the manuscript with help from all the other authors. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Prothrombin induced by vitamin K absence II (PIVKA-II) has recently been validated internationally as a diagnostic biomarker for hepatocellular carcinoma (HCC), as part of the GALAD model. However, its role as a treatment response biomarker has been less well explored. We, therefore, undertook a prospective study at a tertiary centre in the UK to evaluate the role of PIVKA-II as a treatment response biomarker in patients with early, intermediate and advanced stage HCC. In a cohort of 141 patients, we found that PIVKA-II levels tracked concordantly with treatment response in the majority of patients, across a range of different treatment modalities. We also found that rises in PIVKA-II levels almost always predated radiological progression. Among AFP non-secretors, PIVKA-II was found to be informative in 60% of cases. In a small cohort of patients undergoing liver transplantation, pre-transplant PIVKA-II levels predicted for microvascular invasion and poorer differentiation. Our results demonstrate the potential utility of PIVKA-II as a treatment response biomarker and in predicting microvascular invasion, in a Western population. PIVKA-II demonstrated improved performance over AFP but, as a single biomarker, its performance was still limited. Further larger prospective studies are recommended to evaluate PIVKA-II as a treatment response biomarker, within the GALAD model. Hepatocellular carcinoma (HCC) is the seventh most common cancer and the third most common cause of cancer-related death worldwide . AlthougCurrent Clinical Practice guidelines recommend HCC surveillance in high risk individuals, to increase the detection of early-stage HCC, increase curative treatments and thus improve survival , 5. HoweProthrombin induced by vitamin K absence II (PIVKA-II), also known as des-gamma-carboxyprothrombin (DCP), is another serum biomarker of HCC that was first reported in 1984 . PIVKA-IThe BALAD , AFP and DCP/PIVKA-II) and subsequent GALAD  scoring systems were developed in an attempt to overcome the insufficient performance of ultrasound imaging for HCC detection, by combining three well characterised and commercially available serum biomarkers  –12. The The landscape of treatment for HCC has changed significantly over the last few years with the approval of several tyrosine kinase inhibitors and immunotherapy, as well as selective internal radiotherapy . ConsequBesides diagnosis, the emerging role of PIVKA-II as a biomarker of treatment response and in predicting microvascular invasion has also been reported –21. At tA total of 141 patients with hepatocellular carcinoma were enrolled between March 2016 and March 2018. Of these, 90 patients had early stage HCC , 16 patients had intermediate stage HCC and 35 patients had advanced stage HCC. Baseline characteristics of these cohorts are summarised in Baseline (pre-treatment) serum PIVKA-II and AFP levels were measured in all patients. Baseline PIVKA-II and AFP levels increased significantly with tumour stage .p = 0.005; p = 0.015; p = 0.865). AFP levels were not significantly associated with the size of the largest HCC lesion .Within the early HCC cohort, the relationships between tumour-related factors and both PIVKA-II and AFP were assessed. PIVKA-II levels showed a significant positive correlation with size of the largest HCC lesion , whilst no significant association with AFP levels were observed (p = 0.635). Higher pre-transplant PIVKA-II levels were also significantly associated with poorer HCC differentiation (p = 0.026), whilst no significant association was observed with AFP (p = 0.198). There were no significant associations observed between pre-transplant levels of either PIVKA-II or AFP and the number of HCC lesions in the explant.Higher pre-transplant PIVKA-II levels were found to be significantly associated with the presence of microvascular invasion on explant histology (p = 0.063). Conversely, serum AFP levels were non-informative, as all five patients were AFP non-secretors.Five patients also had serum PIVKA-II and AFP levels measured post-transplantation, with a median of 372 days between pre- and post-transplant measurements. Reduction in PIVKA-II levels following transplantation was observed in all five patients, from a median of 124.6 mAU/mL to 38.2 mAU/mL .All patients achieved a complete response to their radiofrequency ablation on their post-ablation imaging by mRECIST criteria. In 10/13 patients (77%), the serial PIVKA-II levels showed a concordant response, with a reduction in the PIVKA-II levels post ablation in either the first or subsequent samples . Serum AFour patients developed a recurrence during follow-up at 3, 7, 10 and 17 months post-ablation, respectively. In all four patients the serum PIVKA-II levels showed a concordant response with a rise in PIVKA-II levels  . In all n = 6), no patients developed recurrence during the follow up period. However, for those with PIVKA-II of 125 mAU/mL or more (n = 7), a total of four developed recurrence, giving a Kaplan-Meier estimated recurrence rate of 100% after 17 months of follow up (p = 0.097), likely as a result of low statistical power due to the small sample size. A weaker association between baseline AFP levels and recurrence was detected  vs. AFP non-secretors (n = 6).Patients who developed recurrence tended to have higher baseline PIVKA-II levels, compared to those who did not recur. To assess this further, patients were divided into two groups based on their baseline PIVKA-II levels, using the median as a cut-off value. Of those with baseline PIVKA-II levels of less than 125 mAU/mL . Altogether 58 TACE procedures were performed in this cohort; 18 patients underwent a single TACE procedure, 14 patients underwent two procedures and 4 patients underwent three TACE procedures. The baseline samples were taken a median of 30 days before the first TACE procedure and serial PIVKA-II and AFP levels were measured approximately every three months thereafter, to coincide with routine clinic visits. The median number of samples collected per patient was 3 (range 2–6).In six patients, the time frame between the baseline and the first follow up measurement was greater than four months, thus these patients were excluded from further analysis. An additional patient rapidly progressed clinically and one patient underwent subsequent liver transplantation before any post-TACE imaging, thus these two patients were also excluded from further analysis. Of the remaining 28 patients, following their first TACE procedure, 7 patients (25%) achieved a complete response by mRECIST criteria, 18 patients (64%) achieved a partial response by mRECIST criteria and 3 patients (11%) demonstrated no response by mRECIST criteria.Of the 7 patients who achieved a radiological complete response, the serial PIVKA-II levels showed a concordant response in 6 patients, with decreasing levels on the first or subsequent samples post-TACE . The remFour patients were AFP non-secretors, but in the remaining three patients, the serial AFP levels also demonstrated a concordant response, with a continuous decline post-TACE .Of the 18 patients who achieved a partial response, 11 patients (61%) showed a concordant PIVKA-II response, with continuously decreasing PIVKA-II levels following initial TACE. Three of these partial responders subsequently demonstrated evidence of progressive disease and in all three patients the serum PIVKA-II level demonstrated a corresponding increase, which again predated radiological progression. The AFP response was less concordant among these 11 patients: four patients were AFP non-secretors and only four (50%) of the remaining patients demonstrated a concordant AFP response, with decreasing levels post TACE.Three of the 18 patients (17%) who achieved a partial response demonstrated no change in serial PIVKA-II levels. All three of these patients were also AFP non-secretors.In the remaining four patients who achieved a partial response (22%), a discordant response was observed with rising serum PIVKA-II levels despite radiological improvement. Interestingly, one of these four discordant patients underwent a second TACE and achieved a near complete response by mRECIST criteria and their serum PIVKA-II level decreased correspondingly after their second TACE. The remaining three patients who demonstrated a rising PIVKA-II level also underwent a second TACE procedure but progressed or demonstrated extensive residual active disease; their serum PIVKA-II levels continued to rise post-second TACE, indicating that rising PIVKA-II levels post TACE may predict poor outcomes. Serum AFP levels were again less informative with three of the four PIVKA-II discordant patients being AFP non-secretors and the remaining patient demonstrating discordant falling levels, despite subsequent radiological progression.A total of 35 patients who commenced Sorafenib for advanced HCC were enrolled. The baseline sample was taken a median of 1 day before the commencement of Sorafenib and serial PIVKA-II and AFP levels were measured approximately every month thereafter, to coincide with routine clinic visits. The median number of samples collected per patient was 3 (range 2–10).Five patients achieved stable disease with no subsequent change. Of these, one patient was subsequently lost to follow up and one patient developed liver decompensation and discontinued treatment. The remaining three patients maintained disease stability until the end of follow up and their serial PIVKA-II levels showed a concordant response with stable readings throughout . Serum AAn additional eight patients achieved stable disease as their best response but progressed at a later date. In all eight patients, PIVKA-II levels increased at the time of progression – five patients demonstrated a continuous rise in serum PIVKA-II levels, whereas in three patients the levels were initially stable, concordant with radiological response, but increased just prior to radiological progression . Serum AThe remaining 22 patients achieved progressive disease as their best response. Serial PIVKA-II levels showed a concordant response in all but one patient, with continuously rising levels during follow up . ConversA concordant response was recorded if concordant changes were observed with both PIVKA-II and AFP levels (or if one was a non-secretor). Combining both biomarkers did not improve the predictive accuracy of PIVKA-II alone .Across the three treatment groups, a total of 30/76 patients (39%) were AFP non-secretors . Of thesTo our knowledge, this is the first prospective study evaluating PIVKA-II levels following HCC treatment in a UK population. Our results show that changes in serum PIVKA-II levels track concordantly with treatment response in the majority of patients, across a wide range of treatment modalities. Our results in a Western cohort are consistent with the previous findings reported from Asian cohorts and are also consistent with a recent prospective study from a European cohort , 26, 27.Whilst AFP has been commonly used as a serum biomarker of tumour response for HCC for many years, one of its major limitations is that approximately 30–50% of patients with HCC are AFP non-secretors , 29. We Our results also show the improved performance of PIVKA-II compared to AFP as a biomarker of tumour response. This finding has been observed by other authors and is not surprising as PIVKA-II is intimately linked to cancer development and growth. PIVKA-II binds to the hepatocyte growth factor receptor, c-MET, leading to tumour growth, invasion and tumour metastases via the JAK-1/STAT3 and ERK 1/2 MAPK signalling pathways, , 31 wherNearly all the prospective studies to date have assessed changes in PIVKA-II levels only immediately post treatment. A major strength of our study is the longer duration of follow up (often until disease progression) and, consequently, we were able to observe that rises in PIVKA-II levels almost always predated radiological progression. This has previously been reported for PIVKA-II in the diagnostic setting, where levels of PIVKA-II have been found to be elevated over one year before HCC was detected radiologically, but has not previously been reported in patients on treatment . If thisNevertheless, whilst demonstrating an improvement over AFP, the performance of PIVKA-II as a single serum biomarker is still limited. This was also observed in the diagnostic setting and the best performance (with the highest sensitivity and specificity) has been observed only with the combination of three biomarkers  as part of the GALAD score . One of PIVKA-II has also been shown to be strongly associated with tumour biology, with higher levels significantly related to the presence of vascular invasion, larger tumour size and poorer differentiation , 35. OurIn vitro, PIVKA-II has been shown to induce over-expression of epithelial growth factor receptor and vascular endothelial growth factor, directly stimulating the proliferation and migration of vascular endothelial cells [Microvascular invasion is known to be a major predictor of tumour recurrence after curative liver resection or orthotopic liver transplantation , 21. In al cells –38. We oal cells –21, 39. Patients with early, intermediate or advanced stage HCC (according to the Barcelona-clinic Liver Cancer Classification (BCLC)) who were due to commence standard treatment  were prospectively recruited between March 2016 and March 2018 at the Queen Elizabeth Hospital Birmingham, United Kingdom. All patients were 18 years of age or older with a confirmed diagnosis of HCC, either histologically or radiologically, as per the internationally accepted AASLD criteria. Patients were excluded if they were on the anticoagulant warfarin, had co-existing malignancy, or were pregnant. The study was approved by the National Research Ethics Committee (REC reference 06/Q2707/182) and all patients provided written informed consent before inclusion and any study-related procedures.Data was collected on baseline demographics, aetiology of underlying liver disease, Child-Pugh score, laboratory test results and tumour characteristics. In transplanted patients, histological findings from the liver explant  were also collected.Peripheral blood samples for measurement of serum PIVKA-II and AFP levels were taken before treatment (baseline) and at the time points detailed below following treatment, until progression or the end of follow up (December 2018). The time points were chosen to coincide with routine clinical review:Liver transplantation: at a subsequent follow up appointment following transplantation (before any recurrence).Radiofrequency ablation: at approximately three monthly intervals following ablation.Transarterial chemoembolisation: four to six weeks post TACE and then at approximately three monthly intervals.Sorafenib: at approximately monthly intervals following commencement of Sorafenib.10 ml of whole blood were collected from patients using a serum separator tube and allowed to clot for one hour at room temperature. Samples were then centrifuged for 20 minutes at 3000 g and the serum extracted. Aliquots of serum were immediately frozen and stored at −80°C until analysis.Serum levels of PIVKA-II and AFP were measured using the Tosoh Automated Enzyme Immunoassay Analyzer AIA-900 kindly provided by Tosoh Bioscience, following the manufacturer’s instructions. The Tosoh AIA-900 analyser uses a two-step immunoenzymetric assay. PIVKA-II non-secretors were defined as patients with serum PIVKA-II levels persistently below 40 mAU/mL. AFP non-secretors were defined as patients with serum AFP levels persistently below 20 ng/mL.U tests for dichotomous variables. Nominal variables with more than two categories were analysed using Kruskal-Wallis tests, whilst ordinal variables were analysed using Jonckheere-Terpstra tests. For the subgroup of patients undergoing ablation therapy, recurrence rates were estimated using Kaplan-Meier curves, in order to account for the follow-up time, with comparisons between groups performed using log-rank tests. In patients with serial AFP and PIVKA-II measurements in the post-treatment period, trajectories were visualised with spider plots.Comparisons of AFP and PIVKA-II across cohort characteristics were performed using Spearman’s (rho) correlation coefficients for continuous variables, and Mann-Whitney p < 0.05 deemed to be indicative of statistical significance throughout.All analyses were performed using IBM SPSS 22 , with


Due to their multifaceted oncological applications and immense translational potential, the bio-nanocarriers and nano-biodevices are being conceived as a futuristic panacea for cancer.Aspects impeding promising prognosis of lung cancer, various nano-biotools, and their plausible benefits over the conventional nanocarriers for lung cancer management have been briefed upon in this review.Research findings from relevant investigations, perspectives, and stipulations for the overall management of lung cancer have also been deliberated. Lung cancer is a complex thoracic malignancy developing consequential to aberrations in a myriad of molecular and biomolecular signaling pathways. It is one of the most lethal forms of cancers accounting to almost 1.8 million new annual incidences, bearing overall mortality to incidence ratio of 0.87. The dismal prognostic scenario at advanced stages of the disease and metastatic/resistant tumor cell populations stresses the requisite of advanced translational interdisciplinary interventions such as bionanotechnology. This review article deliberates insights and apprehensions on the recent prologue of nanobioengineering and bionanotechnology as an approach for the clinical management of lung cancer. The role of nanobioengineered (bio-nano) tools like bio-nanocarriers and nanobiodevices in secondary prophylaxis, diagnosis, therapeutics, and theranostics for lung cancer management has been discussed. Bioengineered, bioinspired, and biomimetic bio-nanotools of considerate translational value have been reviewed. Perspectives on existent oncostrategies, their critical comparison with bio-nanocarriers, and issues hampering their clinical bench side to bed transformation have also been summarized. The global burden of cancer is rising insidiously. With disquietingly high mortality rates (1 in 5 cancer deaths worldwide), lung cancer has been entitled as one of the most lethal forms of cancer . Lung caNanotechnology has provided a platform to design nanocarriers and nanodevices that manifest their activity through various magnetic, thermal, electrical, and optical properties by the virtue of their tunable composition, shapes, structure, and sizes (1–100 nm at least in one dimension) . NanotecOn the other hand, concurrent efforts in the fields of molecular biology, omics, genetic engineering, and cancer biology have revolutionized biotechnology and bioengineering to a significant extent. Sophisticated biotechnology tools have extensively contributed to oncology over recent years toward gathering information about complex cancer biological milieu and biomolecular signaling thereof. However, despite tremendous advances in both of these individual scientific disciplines, the gap between bio-sciences and nanotechnology has undermined their actual potential. The realization of the need to unify these individual disciplines to gain miraculous biomedical outcomes has surfaced up with nanobioengineering. Nanobioengineering is amongst the most challenging domain of bionanotechnology that is advancing rapidly. Nanobioengineering is an applied science that integrates the fundamentals of basic sciences like physics, chemistry, and biology to engineer material properties in nano-dimensions for manifold bio-medical and medical applications. It has assisted contrive novel nanobiotools and strategies for comprehending, managing, and revolutionizing clinical aspects of lung cancer management.Despite the advances in oncological sciences, clinical management of lung cancer is still a perplexing task. The present article scrutinizes abstruseness in the current scenario of lung cancer management and discusses nanobioengineering strategies to overcome the same. A comprehensive overview of the bio-nanotools (bio-nanocarriers and nano-biodevices) for multifaceted applications in lung cancer along with a comparative assessment of the principal advantages and shortcomings has been briefed upon. In the clinical context, disease management comprises four (quadripartite) different aspects: prophylaxis/prevention, diagnosis, treatment/therapeutics, and therapeutic drug monitoring (facilitated through theranostics). A critical appraisal of the research works relating to the application of bio-nanotools for each of these aspects of lung cancer management has been elaborated.. Nano-biocarriers are bioengineered, bioinspired, biogenic, bio-mimicking, and bio-hybrid nanocarriers, cells, or vesicles that employ biological moiety/biomimetic moiety/bioinspired/bioderived moiety as a bioactive or as active targeting vector/ligand. Unlike the other nanocarriers, the nano-biocarriers have distinct advantages like being multi-targeted, highly bio-interactive, biocompatible, intrinsically camouflaged, and scaleable. Additional attributes like tropism and host integration may also aid in personalized therapy, or in enhancing the efficiency and safety of bio-nanocarriers.Nanobioengineering has assisted the development of nano-based cancer vaccines and chemopreventive nanomedicine that can be employed for secondary prophylaxis in lung cancer. Novel bio-nanotools like nano-bio-based contrast and imaging agents, nanoprobes, nanobiosensors, biomarker detection devices, and high-throughput nanodevices have been researched to reinforce early-stage diagnostic techniques for detection, imaging, and molecular imaging at high detection speeds with high specificity and sensitivity. Additionally, the advances in the ‘nano-bioera’ in oncotherapy have resulted in a paradigm shift from conventionally non-targeted therapies to tumor-targeted therapies. Nano-bioengineering has extended its application to radiotherapy, immunotherapy, phototherapy, gene therapy, and combination therapy as well as novel therapies to facilitate multimodal oncotargeting of lung cancer. Nanobioengineering had a late advent in the therapeutic segment of oncotargeting due to delayed evidence of translational and clinical incompetency of a multitude of nanocarrier-based therapeutics reported in the literature. The search for competent targeting strategies has led to the development of novel third-generation nanocarriers termed ‘nano-biocarriers’The multi-functional bio-nanotools have extended their application in theranostics as well. Some researchers have also proposed multi-functional nanoparticles to merge all the facets like diagnosis, therapy as well as prevention with the use of a single nano-modality. Therapeutic drug monitoring, real-time monitoring, and image-guided therapy are some major applications that may be facilitated with the help of theranostics. The contemporary erudition and prospects pertinent to the subject have been summarized as an endnote.Despite the advances in lung cancer diagnostics and therapeutics, lung cancer is still an incurable disease. As stated earlier, metastatic, malignant, resistant, or advanced stage lung cancers are difficult to treat within the precincts of currently available knowledge of lung cancer biology. Chemoprevention and cancer vaccine development have been subject to oncological research for decades, but have not been substantiated through clinical appraisal. While there has been considerable advancement in imaging and screening techniques, the current detection techniques fail to diagnose the early stage of the disease due to a lack of biomarker detection at low concentrations. The other limitation of the prevalent diagnostic option is the lack of identification of the molecular/genetic variant of the tumor sub-type due to high inter-patient diversity. Techniques to preclude the aforementioned disadvantages while facilitating the identification of novel biomarkers and molecular targets are being sought. While considering the therapeutic aspects, combination chemotherapy, nanocarrier-based therapy, biological therapy, and immunotherapy have clinically materialized as adjuvant and neoadjuvant therapy to surgery and radiotherapy. With the advent of novel targeted biological agents and immunotherapeutic agents for the treatment of non-small cell lung cancer (NSCLC), there has been a considerable improvement in therapeutic outcomes & patient survival rates . HoweverAs demonstrated in Table Various bio-nanotechnology-derived tools like nano-biocarriers and nano-bio-devices have recently gained tremendous scientific interest in addressing issues of lung cancer management. The most widely explored bio-nanocarriers for oncological applications can be summarized as: 1) microbiotic nanosystems and bio-nanocarriers, (2) cells and cell membrane-derived nanosystems, (3) ligand-conjugated nanosystems, and (4) nano-bio-devices  are the major factors that have upraised the interest of researches worldwide for tumor targeting . With thA promising scenario can be predicted from the number of ongoing clinical trials for the bacterial treatment of cancer . BionanoBioengineered bacteria are bacteria that are genetically modified for specialized oncological applications. Various tumor-targeted strategies like the release of anticancer agents , genetic transfer , gene triggering strategies , combinations with other treatments , radiation and imaging strategies like bioluminescence, fluorescence, magnetic resonance (MRI), positron emission (PET), etc., have been reported .PAC, developed by EnGeneIc, is a prototype of such targeted minicells, undergoing clinical trials for the treatment of various solid tumors. The minicells were reported to be loaded with chemotherapeutic agents like doxorubicin (DOX), cisplatin, and paclitaxel (PTX) and gene-based therapeutics like siRNA, plasmid DNA encoding the short hairpin RNA (shRNA) .Fi.Fi254].FResearch by Chen et al. has demonstrated the use of bacterial  outer membrane vesicle (BOMVs) for secondary prophylaxis to metastasis and chemo-immunotherapy of lung cancer. This novel nano-biohybrid consists of tegafur-loaded polymeric micelle core, coated over with BOMVs. The BOMVs were designed to induce an immune response by sensitizing the cytotoxic T lymphocytes–mediated immune response to target the tumor cells directly. The antitumor efficacy and antimetastatic activity of BOMVs were apparent from the results of the preclinical in vivo data in a murine model of melanoma. A remarkable increase in the survival rates, tumor inhibition, and antimetastatic potential were observed with the BOMVs, suggesting their use as a versatile nano-biocarrier .An entirely novel bioinspired approach was adapted by Chen et al. for the prevention of pulmonary metastasis of melanoma. The researchers developed a TRAIL-decorated nanovector composed of high-density lipoprotein (HDL) for targeting mesenchymal stem cells (MSCs) that overexpress scavenger receptor B type I (SR-BI). The pDNA that encodes the genetic expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was used as a vector for targeting the pulmonary MSCs. For the construction of this bioinspired nanovector, pDNA was electrostatically conjugated to cationic copolymer, composed of polyethyleneimine and lauric acid. The developed nanoparticles had uniform particle size and high in vitro stability in plasma. The nanovectors had high transfection ability and cell uptake by the MSCs targeting B16F10 (melanoma) cells through a highly targeted therapeutic approach. Such an approach not only prevents side effects but also prevents metastasis of the melanoma cells . AnotherThe research works discussed in this section are only a few of the researches that exemplify the emerging prophylactic role of bionanotechnology in pulmonary oncology.. The application of nano-bio-based detection and imaging techniques may facilitate high-throughput screening of biological samples, novel biomarker identification, biomarker detection with high specificity at low detection levels, and molecular mapping [Diagnostics in cancer involve two different aspects: (1) bio-imaging and (2) identification of tumor-associated molecular markers. Conventional diagnosis of lung cancer is done employing diagnostic methods such as sputum cytology, imaging tests like CT scan, thoracic X-ray, PET and biopsy techniques such as bronchoscopy, thoracentesis, endoscopic ultrasound and open biopsy. The prevalent cancer diagnostic methods have limitations like poor image resolution, lack of specificity, low detection sensitivity, low signaling strength, and limited tumor-associated molecular marker identification techniques. In some cases, imaging methods have also been associated with toxicity issues . Several mapping , 272–274While metal-based nanoparticles have significantly contributed to the enhancement of conventional CT scanning techniques, most of them do not facilitate targeted imaging. To facilitate the targeted imaging of lung cancer, a novel nano-bio-hybrid platform was developed by Xu et al. The authors of this research have discussed the development of NJ0001-coated nanomagnetic beads targeted to the SP70 antigen that is overexpressed in the lung adenocarcinoma cells. NJ001 is a monoclonal antibody, directed to the SP70 antigen. Strong signaling intensities were observed in an orthotopic mice model upon a micro-CT scan. The NJ001 can also be labeled with a NIR-florescent dye CF750 to serve as a probe for molecular imaging using an anti-SP70 fluorescent imaging technique. With SP70-targeted bio-nanocarriers, it was possible to detect pulmonary lesions two weeks earlier than the detection in control. Such preclinical findings indicate the NJ0001-labelled probe to be an excellent tool for facilitating early detection of lung cancer through molecular imaging .64Cu and targeted to the tumor cells using the GLT21T aptamer. GLT21T is an aptamer targeted to tyrosine kinase receptor AXL that is overexpressed in lung cancer. GLT21T-conjugated lipid nanocapsules were formulated using self-assembling of the Chlorin e 6 (Ce6)-conjugated lysophosphatidylcholine (LPC). The 64Cu moiety was contained at the center of the Ce6 tetrapyrrole ring, by its self-chelating ability followed by loading of iodixanole into its cavity. The iodixanol loading can be modified to meet the imaging requirements. The bio-nanocarriers thus formed were assessed in the A549-orthotopic mice model and compared to the conventional PET and CT imaging results. It was reported that the microscopic tumors (~ 500 μm) that were undetectable using the conventional PET and CT imaging techniques were detectable at high resolution, contrast, and sensitivity by employing such a dual-mode imaging nano-bio-hybrid tool [Due to the short biological half-life of the conventionally used PET tracers and their constrained diagnostic ability in various types of lung cancer, novel nano-approaches are being explored . Cai et rid tool .+2, 124I, and N-Bromo succinimide can be chelated with MNPs directly to facilitate photoacoustic imaging (PI) also. The nanoprobe displayed assertive results when evaluated for imaging ability through PET, MRI, and PI in the xenograft model of NCI-H69 tumor overexpressing the SSTR2 receptors . With favorable preclinical results, the OC-MNPs nanoprobe may be considered further for clinical trials [Magnetic resonance imaging (MRI) is not a favored diagnostic method for lung tumors due to biological factors like low intrapleural proton density, high mediastinal and thoracic movements, and heterogenic pleural magnetic distribution . A novell trials .Fig. 4 nanoparticles, was developed by Yang et al. to facilitate targeted phototherapy in NIR-II biowindows and MRI imaging of various types of tumors. The developed nano-biohybrid was evaluated for their in vitro and in vivo photothermal activity and MRI imaging ability, wherein they showed excellent tumor targeting, good imaging ability, and photothermal conversion efficiency of 36.7%. A tumor volume reduction of ~ 90%, with insignificant organ toxicity, was observed with the use of this nano-biohybrid as compared to the control group. The observed results present an excellent platform that can be explored further clinically for combining photodynamic therapy and MRI imaging .With the advancing era of microfluidic-based nanomaterials, several nano-biodevices have emerged. Examples of such devices have been reported for the detection and diagnosis of serum biomarkers of lung cancer . Gao et Some other examples of bio-nanotools that have been employed for diagnostics of lung cancer are summarized in Table Nanobioengineering may not only assist targeted drug development as mentioned earlier but also facilitate targeted drug delivery. Idealistic drug delivery is characterized by features like prolonged systemic circulation, controlled release pattern, high biostability, targeting ability across various biological barriers, immuno-stealthing property in addition to protecting the payload. Such idealistic criteria have led to the development of targeted nano-drug carriers that assemble these virtues by the means of their modifiable dimensional properties, fabrication, PEGylation, and surface modification.Nanotechnology has come up with several nanocarriers that have brought a paradigm shift in the field of oncotherapy. Targeted drug delivery in oncology is of utmost importance to overcome challenges associated with tumor targeting. Tumor-targeting may be achieved by the means of ‘passive targeting’ that operate through the EPR effect or by the means of ‘active targeting’ that operates via functionalization using targeting moiety/ligand. Given the complexity of tumor cytoarchitecture and barriers that prevent efficient antitumor activity, several shortcomings of the passive targeting strategies have been identified. These include a lack of selective cellular targeting and transitional hurdles across the tumor stroma. Such findings have led to the conceptualization of active targeting strategy as a futuristic mode of targeted drug delivery. The active targeting strategy can be employed for targeted delivery to extracellular tumor matrix, CTCs, lysosomes as well as mitochondria. Unlike passive drug targeting, active targeting operates via specific ligand–receptor interactions or biochemical mechanisms.However, the real transformation of the active drug targeting approach is difficult due to challenges associated with oncotargeting. The factors such as proximity of the targeting moiety to the target, systemic clearance, unsolicited immune cell response, and immune cell uptake, nanoparticle characteristics, ligand density, non-specific biological/perivascular interactions, juxtaposing ligands at the target site, steric hindrance, unfavorable ligand orientation, etc., play a decisive role in active drug targeting of the nanoparticles .Four generations of nanocarriers have evolved to date. The basic nanovescicular and nanoparticulate systems that aided solubility, conferred sustained/controlled release ability and bioprotection to the loaded cargo are considered as the first-generation nanocarriers. The second-generation nanocarriers are PEGylated nanocarriers that employ PEG as a ‘stealth’ molecule to prevent their immune recognition and RES uptake, thereby prolonging their systemic circulation and conferring passive targeting ability. Third-generation nanocarriers are complex nanocarriers that add to the virtues of the second-generation nanocarriers by offering ‘active targeting ability’, i.e., spatiotemporal delivery aided by the means of stimuli-sensitivity or through cell-targeted moieties. However, the in vivo performance of these active-targeted nanocarriers has been debated due to many factors . The emeNano-biocarriers that combine stimuli-sensitive and tumor micro-environment-sensitive intelligent drug-delivery systems may be employed to facilitate spatiotemporal drug release along with the most accurate biochemical means of active targeting .Approaches like multistage targeting that are difficult for conventional drug delivery systems have been employed using the nanocarriers  and may Overcoming multidrug resistance with the possible use of P-gp inhibitors and nano-biocarriers , 288–291Nano-biocarriers may facilitate ratiometric and synergistic drug delivery  or dosagNano-biocarriers also address recently discovered ‘on target but off the site’ like advanced drug delivery issues, faced by several target-specific drug categories  isolated from inactivated non-replicating Sendai virus. The HJ-E were observed to evoke direct antitumor immune response upon activation of natural killer cells, dendritic cells, and regulatory T-cell suppression through the RIG-I/ MAVS pathway upon their intratumoral administration. Phase I dose escalation tolerability, safety and primary efficiency studies were performed on the patients with chemoresistant malignant pleural mesothelioma upon the intratumoral and s.c administration of HJ-E. The results were affirmative in terms of prevention of relapse at the injected tumor site as well as its safety. Though no serious adverse effects were observed, transient fever and mild site-specific symptoms were observed .−1 Cisplatin in an orthotopic mice model of pleural mesothelioma, the bio-nanocarriers demonstrated significantly higher CD44 expression and subsequently synergistic cellular uptake of Cisplatin in the mesothelioma cells [Sakurai et al. devised a novel bio-nanocarrier composed of cationic liposome and hyaluronic acid lipid conjugate for pleural mesothelioma therapy. The novel bionanocarrier was synthesized through reductive amination process that allowed HA-terminal attachment to the cationic lipids of liposomes containing Cisplatin, while sparing carboxylate group. Upon intrapleural administration of these bio-nanocarriers at a dose of 1.5 mg kgma cells .Singh et al. report the development of a novel injectable/sprayable hydrogel composite with peptide-based microRNA nanoparticles to target pleural mesothelioma cells. The hydrogel serves as therapeutic depot for delivery of miRNA to the tumor cells. For the formation of stable nanoparticles, miRNA was firstly complexed with amphiphilic cationic peptide and these nanoparticles were incorporated into peptide hydrogel vai self-assembly of the amphiphilic cationic peptide to form a composite hydrogel. The formulation was optimized for properties like biopersistence, biostability, loading ability, specific targeting, bio adherenance and integrity. The miRNA nanoparticles were observed to be non-toxic and upon tracking of the Cy3-labelled miRNA, they demonstrated significantly higher clathrin receptor-mediated uptake. In vivo studies in orthotopic mice model demonstrated significantly enhanced anticancer activity, tumor regression, and higher overall survival .Small cell lung cancer (SCLC) is second-most common type of lung cancer associated with very high mortality rates and poor prognosis (only 10% of the diagnosed patients survive for greater than five years).Huang et al. developed a novel cytomembrane-mimicking bio-nanocarrier for targeted delivery of anticancer enzyme asparginase (ASP) to SCLC cells. The bio-nanocarriers are self-assembled structures composed of polyethylene glycol 2000 (HA-g-PEG) and α-cyclodextrin conjugated to endogeneous CD44 targeting component. The supramolecular assembly and vesicular encapsulation served as a bioreactor that facilitated loading of ASP in its hollow interior, protected the enzyme and provided targeted delivery. The bio-nanocarrier had explicitly higher stability, enzyme-substrate kinetics, catalytic activity and antitumor efficacy as compared to free ASP. The nanobiocarrier exhibited excellent in vivo safety and biocompatibility and can be employed for the treatment of several other malignancies .in vivo efficacy in orthotopic nude mice model for ensuring safety and efficacy [Whitener et al. developed a unique DNA derivatized gold nanoparticles that can bind to ~1100 daunomycin molecules. The drug was held by the mutating nucleic acid strands of the bio-nanocarrier. The length and sequence of the double-stranded DNA of bio-nanocarrier can be modulated to enhance payload. Moreover, diverse array of aptamers can be used for facilitating binding of the nanocarrier to diverse targets. The highly specific, programmable bio-nanocarrier presents a highly specific, avidity driven nanosystem with high drug loading ability and biocompatibility administrable by intratracheal or intravenous route. The drug release rates and quantity can be modulated by mutating the base pairs of the drug-binding region and changing the sequence. The SCLC-specific aptamer-based bio-nanocarrier needs to be evaluated for efficacy .Bio-nanocarriers may also facilitate alteration in the route of administration  at therapeutic concentrations. The minicells were reported to incorporate vinblastine, cisplatin, and irinotecan that represent amphiphilic, hydrophobic, and hydrophilic prototypic cargo. The in vitro and in vivo evaluations in the A549 xenograft model provide compelling proof of the safety and efficacy of this drug delivery platform. Besides having an excellent antitumor activity (greater than 11 other antitumor treatments), these minicells are highly suitable for facilitating combination chemotherapy .A novel nano-biohybrid drug delivery system, ‘nanofibrin’ was recently reported by Vedakumari et al. for the targeted delivery of erlotinib. The erlotinib-encapsulated nanofibrin was developed through the wet co-precipitation method and was subsequently conjugated to bevacizumab (anti-VEGF antibody) by employing the EDC/NHS method of conjugation. The bio-nanocarriers had a spherical shape with a surface positive charge and a hydrodynamic diameter of ~ 79 nm. The in vitro evaluations in the A549 lung cancer cell line proved the strategy to exhibit enhanced anticancer activity. Effective cell inhibition accompanied by an increase in the sub-G0/G1 phase with a subtle decrease in the G0/G1 phase was observed from the in vitro cytotoxicity assessment .rates in LNP-treated animal groups was higher (90%) as compared to the groups treated with PNPs (80%) vs the cisplatin-treated groups (30%) [In another instance, Chen et al. developed hyaluronic acid (HA) and nitroimidazole (NZ) co-decorated polymeric (PNPs) and lipo-polymeric (LPNs) bio-nanocarriers for targeted delivery of cisplatin. While HA provided targeting to the CD 44 and CD 33 receptors that are overexpressed in lung cancer, NZ facilitated drug delivery to deeper hypoxic regions through bio-reductive prodrug designing and provided an amphiphilic drug delivery platform. The hydrodynamic particle size of LPNs and PNPs was observed to be 185.6 ± 4.7 nm and 136.7 ± 3.5 nm, respectively. In vitro cell uptake of the LPNs in the drug-resistant A549/DDP cell line was significantly greater than the PNPs, indicating the fusogenic property of the lipids to cell membranes. The LPNs and PNPs were observed to have remarkably higher antitumor activity as compared to the pure drug in drug-resistant human lung cancer A549/DDP cells. The in vivo survival ps (30%) .Novel biomimetic bio-nanocarriers composed of platelet cell membrane-derived were developed by Chi et al. for the targeted delivery of Docetaxel to the lung cancer cells. For the construction of these novel bio-nanocarriers, poly (lactide-coglycolide) (PLGA) nanoparticles, encapsulating DTX, were enveloped using the platelet membrane. The developed nano-biohybrid exhibited high drug loading capacity, sustained drug release, excellent stability, and tumor-targeting ability. The results of in vivo studies on A549 xenograft showed increased tumor regression accompanied by reduced adverse effects and toxicity . Some otx), was employed for homotypic cancer cell targeting. Cancer starvation was initiated through glucose oxidase (GO) catalysis that causes glucose breakdown, while the anticancer activity was promoted by the photolysis of hydrogen peroxide to hydroxyl radical. The PAAO-UCNPs- GOx were observed to emit photons of 470 nm on being irradiated by 980 nm laser, which facilitated deep tissue photothermal therapy in the hypoxic tumor milieu. Furthermore, the fluorescent emission of 800 nm generated by the nanoreactor facilitated image-guided therapeutic monitoring. The in vitro and in vivo anticancer efficiency of this formulation was validated employing a 4T1 cell-induced metastatic lung cancer model  micelles-encapsulating NaYF4: Yb, Tm upconversion nanoparticles (UCNPs)-surface-modified using cancer cell membranes, i.e.,  encapsulating the Chlorin e6 (Ce6)-conjugated polydopamine nanoparticles (PD-Ce6) for photodynamic (PD) and photothermal therapy (PT) of lung metastatic melanoma. The MSC-mediated phototherapy was facilitated by the ‘Trojan-horse’ like transit between the cancer cells and the MSCs, characterized by an interplay of endocytosis-exocytosis and re-endocytosis in addition to high tumor penetration and high tumor homing capability. The bio-nanocarriers exhibit no overt toxicity to the MSCs while being highly cytotoxic to the cancer cells. The highly selective cytotoxicity can be achieved after being triggered by NIR for inducing the PD/PT mechanisms. About 60% release of the phototherapeutic agents was observed after 72 h of MSC-PD-Ce6 bio-nanocarriers administration. In vivo evaluations in B16-F10 melanoma, cell-induced metastatic mice model displayed excellent anticancer activity, as apparent from the reduced number of metastatic colonies and survival rates in the animals treated with MSC-PD-Ce6 bio-nanocarriers as compared to the simple PDS-C6-based therapy .2) nanoparticles. MnO2 is an excellent oxygen-producing substrate, employed in the design of antitumoral nanomedicine to respond to hypoxic and acidic pH-stimuli. The MnO2-Ce6 were formed by physical adsorption of the Ce6 moiety to the MnO2 nanoparticles to increase the systemic circulation. The MSCs bound to the MnO2-Ce6 increased the cell uptake significantly while reducing the non-target toxicity. MnO2 released from the MnO2-Ce6 nano-biohybrid catalyzed the reactions that consume the abnormal cancer cell metabolic products like protons (H+) and hydrogen peroxide (H2O2). Consequent formation of O2 resourced the formation of 1O2 upon being irradiated by 633 nm laser light, thereby incapacitating the hypoxic constraint of photodynamic therapy (PDT) for effective antitumor therapy. MnO2 decomposition to Mn2+ representing high T1 relaxivity in the MRI region, followed by its hepatic metabolism and renal excretion. The Mn2+ may greatly facilitate the direct MRI imaging of MSCs and tumor microenvironment (TME), providing a theranostic advantage. The in vivo evaluation in a xenograft mice model of LLC revealed higher survival rates, apoptosis, and tumor cell death in the animals receiving phototherapy with MSCs-MnO2-Ce6 as compared to the phototherapy with MnO2-Ce6 groups, indicating high anticancer activity. The MSCs-MnO2-Ce6 besides being highly biocompatible and biodegradable had no significant systemic toxicity. Some other relevant examples from this category of anticancer activity are discussed in Table Cao et al. developed a novel nano-biohybrid composed of the mesenchymal stem cells (MSCs) encapsulating the Ce6-conjugated manganese dioxide  conjugated to T-cell activators (anti-CD28 and anti-CD3) or checkpoint inhibitors (anti-PD-L1) were developed and evaluated for their efficacy in lung cancer. The IO-FuDex3-nano-biohybrid had an immuno-restorative effect in the immunocompromised patients, mediated by tumor-infiltrating lymphocytes, and tumor targeting was achieved using the external magnetic drive. The nano-biohybrid-based immunotherapy (at dose < 1%) was observed to extend the median survival of animals from 32 to 63 days, with low systemic toxicity in a 4T1-metastatic model of lung carcinoma .−1 GrB. This was accompanied by significantly increase survival rates over the non-targeted and untreated disease control groups, suggesting this approach to be an effective immunotherapeutic modality.Granzyme B is a natural serine protease, secreted by the natural killer cells and cytotoxic T cells. Despite being a highly effective anticancer agent, such peptides lack biostability and have low cellular permeability, which desists their therapeutic effectiveness. Yang et al. developed reduction-responsive polymerosomes loaded with Granzyme B for immunotherapy of lung cancer. The polymerosomes were surface-functionalized with cell-penetrating peptides to facilitate targeted intracellular delivery of the enzyme. The polymerosomes were reported to have a particle size of 82–90 nm, reduction-sensitive drug release property, high cellular internalization, and cytoplasm-specific protein release in the A549 cells. The FITC-labeled cytochrome C-mediated model protein analysis of the developed polymerosomes depicted protein loading of ~ 17.2%w/w. The cell line studies of the polymerosomes on A549 cells demonstrated high anticancer activity (IC50 of 20.7 nM) and high cell-penetrating ability. In vivo evaluation in orthotopic A549-Luc pulmonary tumor-bearing nude murine model demonstrated tumor inhibition in the treatment groups receiving granzyme B-polymerosomes at a dose equivalent to 2.88 nmol kgImmuno-liposomes are a well-known class of nano-biohybrid with multiple oncological applications. Repic et al. developed immunoliposomes, with antibody fragment (Fab) targeted to lung cancer-specific antigen. While covalent coupling is used for the conventional conjugation method of antibody fragments, these novel immunoliposomes were formulated employing a novel formulation strategy that obviated the use of cross-linking agents. For precise conversion of the MAbs to recombinant antibody fragments, P. pastoris. P. pastoris were employed as hosts. The C-terminal of the recombinant Fab antibody fragment was fused to a peptide derived from pulmonary surfactant protein D and incorporated into the liposomal bilayer to fabricate the immunoliposome. The immunoliposomes containing the CD59 and CD48 FAbs were thus observed to have dual specificity. The Fab-labelled immunoliposomes were demonstrated to target the antigen-positive cells with great affinity and specificity . The mul−1) for four weeks, a significant reduction in tumor growth was observed amongst the SCID mice expressing two different P53 with no systemic toxicity. Significant inhibition of the MDM2 and restoration of the P53-downstream signaling pathway were observed in the animal groups receiving the cationic liposomal formulation, suggesting potential anticancer activity. Moreover, the metastatic potential of the H-460 cell was reported to be greatly disrupted by stable bioexpression of the miRNA cells [Numerous bio-nanocarriers have been researched extensively for facilitating gene delivery. It has been well established that the replenishment of the downregulated miR-660 gene has the potential to inhibit the proliferation of lung cancer cells through the restoration of the MDM2-P53 axis. However, the administration of the miRNA has been a challenging task owing to the poor in vivo bioavailability issues. Moro et al. formulated bio-nanocarriers composed of cationic lipid 1, 2-biology-1-3- tri-methylammonium propane (DOTAP) to facilitate targeted delivery of miR-660 gene to lung cancer cells while increasing its bioavailability. In vivo experiments, tumorigenesis was induced in the SCID mice using P53 wild-type patient-derived xenografts (PdX). Upon biweekly administration at a dose of  also effectively caused gene silencing, and GALA (a peptide with 31 amino acids) facilitated the endosomal escape. The developed nano-biocarrier was found to be 40-fold more efficient in inducing gene silencing and higher anti-metastatic potential than the formerly developed membrane-based nanodevice. Effective gene silencing was observed at 0.01 mg siRNA/kg dose, with no reports of other non-targeted toxicity .The research work by Merckx et al. exemplifies how the endogenous surfactant protein b may augment the inhalational delivery of siRNA-encapsulated proteolipid-layered nanogels. Led by the promising results obtained with bio-nanocarriers composed of the Curosurf®, the researchers identified major constitutional components contributing to an improvement in the particle biostability and facilitating in vitro and in vivo siRNA delivery. Wherein, surfactant protein B (SPB) reconstituted in proteolipid-coated nanocomposite/nanogels was identified to be one of the most potent delivery aid Fig. . An effeRadiation therapy is one of the most important oncology modalities. In patients with radio-resistant oncological cell populations, various nanoparticle-mediated radiosensitization strategies have materialized to be a useful therapeutic aid .. Thus, formulated bio-nanocarriers (C@siPLK1-NH) were reported to effectively target the EFGR-positive A549 cells, causing apoptosis by G2/M cell cycle arrest. The C@siPLK1-NH were observed to have excellent translational potential in an orthotopic murine model of lung cancer, which manifested as reduced tumor proliferation and prolonged survival rates . For optimizing the synergy of radiotherapy and targeted delivery, the research group devised cetuximab-conjugated nanoparticles, encapsulating siRNA-directed against polo-like kinase 1 (PLK1)tes Fig. . SimilarIn another instance, Travis et al. developed a nano-biohybrid of gold nanoparticles, surface-modified with cilengitide (a linear/cyclic RGDfV pentapeptide sequence targeted to αvβ3 integrin receptor) to cause radiation sensitization. The radiosensitization capacity of developed nanoparticles and the 137Cs irradiation was evaluated by clonogenic assay on αvβ3-overexpressing tumor cells like H460, HUVEC, and MCF7. The developed nano-biohybrid was observed to induce the dose enhancement factors (DFs) significantly, which was insignificant in the treatment groups receiving radiation therapy alone and the groups treated with radiation and unconjugated gold nanoparticles. Moreover, it was concluded from the observations of the DFs that cyclized RGDfV pentapeptides had higher radiosensitization capability as compared to its linear counterpart and the radiosensitization efficacy bio-nanocarriers was almost equivalent to the radiation therapy with cilengitide .Combination oncotherapy has recently emerged as one of the most feasible therapeutic approaches to tackle complex diseases like cancer. A combination of different therapeutic modalities has led to a paradigm shift in oncology .A unique therapeutic approach, combining the targeting ability of natural cancer cell membrane with the photothermal property of plasmonic gold nanorods and cytotoxic effect of β‑Lapachone (β-LN), was adopted by Marangoni et al. for the treatment of lung cancer. β-LN is a pro-drug like an anticancer agent that is activated by NADP(H): quinine oxidoreductase enzyme, that is intrinsically produced by the cancer cells of solid tumors like lung cancer. Plasmonic nanorods have been established to exhibit excellent photothermal activity upon IR irradiation. This research was undertaken to assemble the advantages of both these therapeutic modalities by fabrication of a multifunctional nano-biohybrid, composed of lung cancer cell (A549)-derived membrane co-loaded with PEGylated plasmonic gold nanorods and β-LN. The system exhibited NIR-triggered release of both the payloads from the cancer cell membrane vesicle. The developed nano-biohybrid demonstrated high in vitro cytotoxicity. In vivo histopathological examination of tumors in cancer-induced animals treated with the nanohybrid revealed excellent antineoplastic potential as compared to the groups receiving only radiation therapy .Bio-nanocarriers combining chemotherapy and gene therapy were developed by Li et al. to combine the anti-angiogenic activity of vascular endothelial growth factor (VEGF)-siRNA with anticancer activity of etoposide (ET) for the treatment of metastatic and malignant forms of lung cancer. The nano-biohybrid constructs are designed using the cationic liposomes co-loaded with (VEGF)-siRNA and ET, enveloped with PEGylated histidine-grafted chitosan-lipoic acid polymer. The developed nano-biohybrid exhibited an excellent tumor penetration and high cellular endocytosis in addition to the pH-triggered release of the siVEGF and ET in an orthotopic mice model of NSCLC. The anticancer and antimetastatic potential of the nano-biohybrid is clear from the in vivo data that supports its high tumor growth suppression and antimetastatic potential .Song et al. have developed bio-nanocarriers to facilitate combination therapy (photodynamic therapy and immunotherapy) for the effective treatment of metastatic lung cancer. Photosensitizer protoporphyrin IX (PIX) was conjugated to a chimeric peptide C16-K(PpIX)-PEG8-KDEVD-1MT (MT) (immune checkpoint inhibitor) via caspase-responsive peptide Asp-Glu-Val-Asp for developing nanoparticles by self-assembling upon dispersion in PBS. The nano-biohybrid thus developed had a high EPR effect and potent antitumor activity. The antitumor activity was mediated by ROS production at 630 nm on NIR irradiation and caspase-3-mediated intratumoral release of MT, thereby evoking an intense CD8+ T cell-induced immune response in lung cancer cells. The strategy was reported to inhibit growth as well as metastasis of lung cancer cells and can be foreseen as a potential clinical-oncotherapy candidate .Some recent and noteworthy examples of bio-nanocarriers, used for targeted delivery of novel categories of anticancer agents to lung cancer, have been discussed in this section. Liu et al. developed graphene oxide (GNO) nanocomposites functionalized with polysaccharide for targeted delivery of a novel anticancer agent SNX-2112 (Hsp90 inhibitor) to lung tumors. The unique physicochemical characteristics of the GNO make them an attractive material for constructing nanocarriers. Surface modification of the GNO-based nanocomposites with polysaccharides like chitosan (CH) and hyaluronic acid (HA) greatly improves its biocompatibility, while targeting the payload to the CD44 positive tumor cells (GNO@CH-HA). SNX-2112 was thereafter loaded in the GNO@CH-HA. SNX-2112 was released at a higher amount and release rate in acidic tumor conditions, conferring an additional TME-targeted property. While being highly cytotoxic to A549 cells, the SNX-2112-loaded GNO@CH-HA nanocomposites showed no cytotoxicity on erythrocytes or normal human bronchial epithelial cells. In vivo toxicity evaluation in Sprague–Dawley rats showed a short-term inflammatory response, but no long-term injury was witnessed. The novel nanocomposite was concluded to be a safe and effective means of targeted onco-therapy .In another instance, Yokoyama et al. developed a nano-biohybrid targeted to EGFR for synergistic anticancer activity through induction of apoptosis and autophagy. To develop a highly targeted formulation, plasmonic magnetic nanoparticles (PMNs) were surface-functionalized with anti-EGFR antibody (Clone 225) that target the NSCLC cells with great selectivity Fig. . Thus, dDespite promising preclinical results, the clinical transformation of these formulations remains questionable due to the challenges associated with their active targeting ability. Due to large hydrodynamic sizes, multi-functionality, non-specific immune interactions, ‘on target but off the site’ response, low bio-stability and difficult nanoparticle conjugation, the efficacy of antibodies and proteins for functionalization is dubious. While the antibody fragments and peptides reduce the size to facilitate their conjugation to the surface of the nanoparticles, their efficacy is challenged by nonspecific biological interactions and peptide geometry. Aptamers have low circulation stability and are prone to degradation by nucleases that are non-specifically attached over the surface of nanoparticles. Small molecules, despite being effective, have a high cost for personalization and targeting. Receptor-negative cell population, genetic mutations, and high inter- and intra-patient diversity play an important role in determining their effectiveness. Moreover, most of the above-stated approaches may be unfeasible to be devised with precision at a large scale . EmployiThe term ‘nanobiotheranostics’ refers to a unified diagnostic and therapeutic application of a single agent. Theranostics not only facilitates concurrent diagnostic and therapeutic aspects but also facilitates image-guided drug delivery and therapeutic drug monitoring. While nanotechnology has significantly contributed to advance this field, bionanotechnology-derived tools are being explored to overcome the limitations of the former. The majority of the nano-biotheranostics that have been employed till now include the inorganic nanoparticles, nanocages, nanoshells, nanoclusters, and metallo-organic frameworks composed of gold, silver, iron nanoparticles, silica, liposomes, dendrimers, micelles, and other bio-polymers conjugated to the bio-molecular ligands. However, with advancing bionanotechnology, several bioinspired bio-nanocarriers such as nanobots and cell-derived nano-biocarriers have emerged . SeveralThe research work by Shevtsov et al. is a unique theranostic approach that combines the immunotherapeutic ability of Granzyme B (Gn B) with the diagnostic ability of superparamagnetic iron oxide nanoparticles (SPIONs) to target many types of cancers including lung cancer. The surface-modified SPIONs target a membrane-bound protein, Hsp 70, that is selectively overexpressed over the tumor cells but is absent over the normal cells. Gn B is a serine protease that is produced by the immune cells like the NK and T cells, that selectively targets the HsP70 receptors. The GnB-functionalized SPIONs (GnB-SPIONs) were reported to undergo selective endocytosis into the tumor cells and provide significantly higher MRI contrast and apoptosis-inducing ability. The therapeutic efficiency of GnB-SPIONs was observed to increase still further by employing a regimen combining magnetic targeting and stereotactic radiotherapy .2S quantum dots entrapping 5-fluorouracil (5-FU). The theranostic quantum dots were observed to overcome the issues of drug resistance and pro-survival mechanisms in A549 cells while facilitating targeted NIR imaging. The novel nanohybrid was observed to induce significant apoptosis in EGFR-positive lung cancer cells by suppression of autophagic mechanisms [Yet another theranostic approach was employed by Duman et al. to facilitate tumor targeting to EGFR-positive cancer cells like lung cancer cells. For facilitating tumor targeting, the research group developed cetuximab-conjugated Agchanisms .2S quantum dots (QD), entrapped in the erythrocyte membrane for combining the benefits of non-invasive sonodynamic therapy with imaging ability. The novel approach employs Ag2S quantum dots for sonosensitization while utilizing the endogenous erythrocytic catalase-mediated O2 as sonodynamic therapeutic. This is the first instance of employing sonodynamic therapy as an oncological modality, which is difficult to be employed otherwise due to hypoxic tumor conditions. To overcome challenges associated with spatial tumor characteristics, the research groups utilized a novel theranostic formulation that had an intrinsic source for O2 production. To increase the sensitivity of sonodynamic therapy through increased intracellular O2 production, phenethyl isothiocyanate (PEITC) was administered orally. The theranostic agent can be image-guided to the tumor tissue by employing fluorescence-NIR II-aided imaging technique and subsequently subjected to ultrasound to evoke reactive oxygen species (ROS) generation and cause subsequent cell death. The in vivo evaluation of the nano-biohybrid demonstrated long systemic circulation, exceptional biocompatibility accompanied by high anticancer efficacy in a xenograft mice model [Cheng et al. developed a nano-biohybrid composed of Agce model .c-Met human single-chain variable fragment (scFv) antibody as a vector. The anti-c-Met scFv fragment was attached over the surface of PEGylated liposomes encapsulating doxorubicin. The nano-biohybrid offered excellent targetability while facilitating in vivo fluorescent imaging and tumoral cell uptake as apparent from the tumor to normal ratio. Thus, devised immunoliposomes demonstrated high antitumor activity in the H460 xenograft mice model, suggesting its potential as a theranostic agent [c-MET (hepatocyte growth factor receptor) aberrations have been well reported in many forms of cancers including lung cancer and metastatic malignancies. Lu et al. have developed a novel nano-biohybrid employing anti-ic agent .An innovative approach for theranostic relegation of progression and metastasis of lung adenocarcinoma has been reported by Chan et al. The authors of this research work have developed novel nanomaterial by employing silica shell-based nanocarrier, which serves as near infrared-persistent luminescence nanoparticles (NIR PNs). The NIR PNs were optimized for their physicochemical properties to attain a prolonged NIR luminescence. For an additional therapeutic effect, afatinib (AT) was conjugated to the PNs, and was subsequently functionalized with MAGE-A3 (Map) to obtain C). MAGE-A3 is a DNA aptamer, targeted against tumor-specific melanoma-associated peptide antigen (MAGE-A3111-125), that is overexpressed in lung cancer and many metastatic forms of cancer. Due to the advantages like highly modifiable structure and specific/targeted tumor targeting, this nano-biohybrid may be envisioned as a future of image-guided theranostics .With several positive outcomes, the application of doctrines of nano-bioscience and bio-nanotechnology has presented an immense potential to redefine the current oncological scenario. An entire armada of different forms of bio-modified nanocarriers is being researched to subjugate various hurdles in the way of oncotargeting. Nanobiomedicines, by the virtue of their three-dimensional, multicomponent array exhibit certain distinct colloidal characteristics that bear an immense potential to transfigure the current state of oncotherapy. These future ‘nano’ tools have been supposed to cast their idealism upon the forthcoming preventive, diagnostic, therapeutic as well as theranostic aspects of several forms of neoplasms including lung cancer. Various novel nanosystems have been extensively explored to solve various issues confronted by conventional therapies like poor tumor bioavailability, nonspecific drug targeting, drug resistance, off-target side effects as well as detection of cancer cell progression and metastasis. However, the prognostic significance of nanotechnology and biotechnology in the oncological context is constrained and demands remarkable improvement.An entirely different perspective on oncology awaits unanimous efforts in the field of nano-biosciences and bio-nanotechnology. Bio-nanotechnology tools may help envision and discover several novel oncological aspects by providing kaleidoscopic vision through molecular diagnosis and advanced bioimaging techniques. Several novel targets may be identified and novel therapeutic strategies be devised for reinforcement of oncotherapy. Revisiting the principles of nano-biosciences may help leverage the oncological sciences to a considerable extent by devising novel strategies that smoothen the trajectory to target the tumors. Nano-biocarriers may be explicitly designed to assist the delivery of various types of cargoes to the tumor tissue while overcoming various physicochemical and biological barriers. Additionally, the nano-biocarriers are a useful means for addressing tumor heterogeneity through personalized and tailored-drug delivery. The concept of combination therapy for targeting as well as theranostic purpose may also be facilitated using several forms of nano-biocarriers. Bionanotechnology-derived theranostic and other multi-functional arsenal has an immense potential to empower the oncology and expand the horizon toward optimistic outcomes.However, the factual translation of nanomedicine may be made possible only by overpowering the challenges associated with them. The major challenges faced by bionanotechnology-based approaches are related to the lack of comprehensive interdisciplinary understanding amongst researchers of different scientific disciplines. Some of the other challenges associated with the use of nanomedicines relate to their biological repercussions, pharmacoeconomic impact, scale-up, immunological and toxicological concerns due to inter-patient bio-variance and modulated pharmacokinetics and biodistribution. Subtle alterations in the pharmacokinetic behavior of nanoparticles may often impede the in vivo biodistribution and alter the tumor bioavailability of the drug, thereby reflecting their impact on its pharmacodynamics. Bio-phenomenon like nano-biointeractions, bio-molecular corona formation, RBC hitchhiking, hepatic sequestration of nanomedicine for prevention of metastasis, etc., that have been less explored also needs to be extensively researched to deduce the complete therapeutic serendipity of the nano-biocarriers . BjörnmaStreamlined efforts of the researchers from mathematical, engineering, chemistry, physics, and biology disciplines may help devise models for predictive analysis of the raw research data and findings gathered through bionanotechnology. Besides this, regulatory guidelines on the validation, functionalization, processing, safety, and toxicity assessments of nano-biocarriers and nano-biodevices also need to be devised. While there is a long way to safeguard the clinical applicability of bionanotechnology-derived tools, pacing up data integration and facilitating applications of validated predictive analytical and biomodeling tools may significantly pace up the process of their regulatory approval. With compelling shreds of evidence from current advances in the bionanotechnology-derived tools, their application in oncology proclaims a promising futuristic scenario.


This study evaluates the performance of the antigen-based anterior nasal screening programme implemented in all Austrian schools to detect SARS-CoV-2 infections. We combined nationwide antigen-based screening data obtained in March 2021 from 5,370 schools (Grade 1–8) with an RT-qPCR-based prospective cohort study comprising a representative sample of 244 schools. Considering a range of assumptions, only a subset of infected individuals are detected with the programme (low to moderate sensitivity) and non-infected individuals mainly tested negative (very high specificity). Early recognition and isolation of infected individuals are crucial in containing the coronavirus disease COVID-19) pandemic. Rapid antigen tests deliver timely results, can be performed without laboratory resources, and are widely used in screening programmes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 9 pandemi. As tranThe present study aimed to evaluate the performance of the antigen-based screening programme for detecting SARS-CoV-2 infections in Austrian schools. The design of the study is outlined in The first component used aggregate data from the nationwide rapid antigen test-based screening programme  providedThe screening programme used two rapid antigen tests licenced for anterior nasal self-testing , (ii) from a previously tested negative sample and (iii) a no-template control. Further details on study design and methodology have been published previously [The second component of this study used data from the Austrian School-SARS-CoV-2 study , a prospeviously . Our analysis focused on Round 3 of the Austrian School-SARS-CoV-2 study, which was conducted between 1 and 18 March 2021 (Weeks 9–11) and included 6,423 pupils and 1,100 teachers (from 244 schools) who had previously tested negative in the rapid antigen test-based screening programme. Key findings on the prevalence of RT-qPCR-detected SARS-CoV-2 infection are shown in the We modelled sensitivity and specificity of the antigen-based screening programme at Austrian schools by (i) combining findings from the two data sources, (ii) assuming a range of positive predictive values for the rapid antigen test (20–100%) and (iii) using point estimates and 95% confidence interval limits in the Austrian School-SARS-CoV-2 study as estimates of the negative predictive value . For insEthical approval was not required for the data from the nationwide rapid antigen test-based screening programme because we used aggregate data only. The Austrian School-SARS-CoV-2 study received ethics approvals by the ethics committees of the Medical University of Graz (no. 32–672 ex 19/20), Medical University of Innsbruck (no. 1319/2020), the Johannes Kepler University of Linz (no. 1222/2020), and the University of Vienna (no. 00591/2020). Participants or their legal representative provided written informed consent.The present study scrutinised the antigen-based screening programme introduced at Austrian schools in early 2021 to detect SARS-CoV-2 cases. While self-collected anterior nasal swabs can be easily performed, were well accepted by the children in our study and help increase testing capacities, knowledge about their performance in real-world settings are required to guide further decision-making.While several studies exist on sensitivity and specificity of rapid antigen tests in adults , a recenOur study has strengths and limitations. By combining nationwide data on the antigen-based screening programme with RT-qPCR testing in a carefully chosen representative sample of schools, we provide a unique insight into the potential and limitations of one of the key measures to mitigate SARS-CoV-2 transmission in schools. However, our findings may not be generalisable to other countries and antigen tests. Also, for reasons of data protection, only aggregate data were available from the screening programme, precluding more detailed analyses. Furthermore, health authorities could not provide data on how frequently a positive rapid antigen test was confirmed with RT-qPCR. To account for this limitation, our modelling involved a range of positive predictive values.Our study indicates that only a subset of infected individuals are detected with the antigen-based screening programme at Austrian schools (low to moderate sensitivity). Non-infected individuals were largely tested negative (very high specificity). Given the low-to-moderate sensitivity of antigen-based anterior nasal self-testing particularly in children, additional measures such as face masks or ventilation are important to prevent secondary cases, especially in periods with high incidence. Furthermore, as a mitigation measure, switching SARS-CoV-2 screening in schools to RT-qPCR based approaches should be preferred, where logistically feasible.


This study aimed to determine the correlation between intraoperative 5-ALA-induced fluorescence and preoperative MET-PET signals of gliomas. We quantitatively measured the fluorescence intensity from tumor samples and calculated the MET-PET uptake by the tumor. Our study showed that strong tumor fluorescence correlated with high MET-PET uptake and cellular proliferation. Our findings might be valuable to rapidly provide information on tumor biology at the time of surgery in circumstances where MET-PET is inaccessible.In malignant brain tumor surgery, precise identification of the tumor is essential. 5-Aminolevulinic acid (5-ALA) labels tumor cells with red fluorescence to facilitate tumor resection. On the other hand, the nuclear medicine imaging technique, positron emission tomography with 11C-methionine (MET-PET) represents the activity of brain tumors with precise boundaries but is not readily available. We hypothesized that quantitative 5-ALA-induced fluorescence intensity might correlate with MET-PET uptake in gliomas. Methods: Adult patients with supratentorial astrocytic gliomas who underwent preoperative MET-PET and surgical tumor resection using 5-ALA were enrolled in this prospective study. The regional tumor uptake of MET-PET was expressed as the ratio of standardized uptake volume max to that of the normal contralateral frontal lobe. A spectrometric fluorescence detection system measured tumor specimens’ ex vivo fluorescence intensity at 635 nm. Ki-67 index and IDH mutation status were assessed by histopathological analysis. Use of an antiepileptic drug (AED) and contrast enhancement pattern on MRI were also investigated. Results: Thirty-two patients, mostly with Glioblastoma IDH wild type (46.9%) and anaplastic astrocytoma IDH mutant (21.9%), were analyzed. When the fluorescence intensity was ranked into four groups, the strongest fluorescence group exhibited the highest mean MET-PET uptake and Ki-67 index values. When rearranged into fluorescence Visible or Non-visible groups, the Visible group had significantly higher MET-PET uptake and Ki-67 index compared to the Non-visible group. Contrast enhancement on MRI and IDH wild type tumors were more frequent among the Visible group. AED use did not correlate with 5-ALA-induced fluorescence intensity. Conclusions: In astrocytic glioma surgery, visible 5-ALA-induced fluorescence correlated with high MET-PET uptake, along with a high Ki-67 index.Background: 5-Aminolevulinic acid (5-ALA) is widely employed to assist fluorescence-guided surgery for malignant brain tumors. Positron emission tomography with  The most common primary malignant brain tumor in adults is glioblastoma (GBM). This cancer, along with other tumor types of the same lineage , has a dismal prognosis despite the current intensive treatment strategies ,2. GBM, To facilitate maximal safe reduction of the tumor, fluorescence-guided surgery (FGS) has become a widely employed technique to identify the presence of tumor cells intraoperatively ,6,7. In 11C-methionine (MET), has been reported to delineate brain tumors with better sensitivity [Preoperatively, tumor localization and its boundary must be vigorously investigated predominantly by imaging studies to assist brain tumor surgery. Magnetic resonance imaging (MRI) with gadolinium contrast enhancement is the most accepted technique for diagnosing this actively proliferating tumor. In general, GBM exhibits solid enhancement on the rim and minor or no enhancement in the necrotic tissue core (ring enhancement) due to the BBB breakdown in and around the tumor. More recently, positron emission tomography (PET) imaging with metabolic tracers, especially amino acid tracers such as sitivity  in the psitivity . Furthersitivity  and reflsitivity , MET-PETsitivity  with morsitivity . Despitesitivity .Given these backgrounds, we hypothesized that quantitative 5-ALA-induced fluorescence intensity might correlate with MET-PET uptake as both parameters reflect the tumor cell malignancy. Thus, the purpose of this study is to elucidate the relationship between preoperative MET-PET uptake and intraoperative 5-ALA-induced fluorescence intensity quantified from ex vivo tumor specimens, adding a new axis to the 5-ALA-induced fluorescence in glioma surgery.The patients were enrolled in this prospective study at the Department of Neurosurgery, Tokyo Medical and Dental University, from May 2016 to August 2019. The inclusion criteria were age >20, a diagnosis of supratentorial malignant glioma (newly diagnosed or recurrent), eligibility for surgical resection of the tumor using 5-ALA, and availability of the MET-PET data taken prior to the surgery. Biopsy cases were excluded. Because oligodendrogliomas are known to have higher MET-PET uptake relative to astrocytomas , only asPositron emission tomography measurements were determined using a PET scanner  and presented as the equilibrated radioactivity 20 min after intravenous injection of MET (250–350 MBq), as described previously . The traThe patients received an oral administration of 5-ALA  at 20 mg/kg body weight three hours before elective craniotomy as part of the routine procedures for fluorescence-guided resection of gliomas. Tumor removal was conducted using standard neurosurgical procedures. Tumor samples were obtained from a MET uptake-high region of the tumor according to the image-navigation system using the preoperative images, including Gd-enhanced MRI and MET-PET . PreoperThe ex vivo fluorescence intensity from the tumor specimens was measured immediately after collecting the sample in the operating room. As previously described , we usedAll tumor specimens were subject to histopathological analysis by neuropathologists who were blinded to the presence of the fluorescence. Hematoxylin and eosin (H&E) staining and immunohistochemistry  were used as part of routine procedures to make final diagnosis according to 2016 WHO Classification of Tumors of the Central Nervous System. t test and one way ANOVA test compared two and multiple groups, respectively. In addition, the Pearson Chi-square test was used to test the frequency of the two variables. All p-values were two-sided, and p-values less than 0.05 were considered statistically significant. All statistical analyses were conducted using JMP version 14.2.0 .Fluorescence intensity was compared by non-parametric analysis. The Unpaired A total of 52 adult patients with supratentorial tumors suspected of malignant glioma underwent surgical resection using 5-ALA, and 32 patients were included in the final analysis after excluding those without adequate MET-PET data or those diagnosed other than astrocytic tumors . Fresh surgical specimens from the tumor indicated by the navigation system were collected and subject to fluorescence measurement in the operating room. Samples were cut into pieces approximately 10 mm in diameter before scanning the fluorescence spectrum. A quantitative spectroscopic fluorescence excitation and detection system was used as previously described , with a Two illustrative cases are shown in We next compared the four tumor groups of varying intraoperatively measured fluorescence intensities for the levels of preoperative MET-PET uptake a. We alsp < 0.001 for MET-PET uptake and p = 0.0091 for Ki-67 index, Unpaired t tests). We next rearranged the tumors into two groups: Visible  or not: Non-visible , because we noted that the fluorescence from ex vivo tumors was obviously visible when the intensity is above 5000 a.u. Our further analysis of the relationship between the MET-PET uptake or Ki-67 index and fluorescence intensity showed that the Visible group had significantly higher MET-PET uptake c and Ki-p = 0.0007). IDH wild type tumors were more common in the subjects with strong fluorescence than non-fluorescent tumors; conversely, IDH-mutant tumors were more frequent in the non-fluorescent tumors than fluorescent tumors (p = 0.0434). In contrast, AED use did not correlate with the intensity of 5-ALA-induced fluorescence (p = 0.6996).Lastly, we investigated the association between other parameters  and tumor 5-ALA fluorescence using mosaic plots and Pearson Chi-square test . Ring orThere are only a few reports regarding combining 5-ALA and MET-PET. 5-ALA-induced fluorescence and MET-PET correlated to cell density or Ki-67 index but did not correlate with each other ,38. MET-11C-methionine and 5-ALA  has not been understood. The expression of L-Type amino acid transporter 1 (LAT1: SLC7A5) in human glioma correlates with MET uptake [+- and Cl−-dependent taurine transporter (TAUT: SLC6A6) [The mechanism behind the correlation between T uptake . On the  SLC6A6) ,46,47. T SLC6A6) . AlthougA limitation of this study is that the location of the tumor specimen might not have been precisely matched with the preoperative MET-PET images due to the brain shift that occurred during surgery. Therefore, when available, we utilized the specimen exhibiting the strongest fluorescence to correlate with the SUVmax of the MET-PET. Secondly, because of the limited dynamic range of the spectrometer relative to the marked brightness of 5-ALA-induced fluorescence in some cases, our fluorescence intensity data could not be used as a continuous value. Consequently, the data points were distributed somewhat unevenly among the fluorescence intensity groups, making statistical analysis challenging. On another note, the MET-PET uptake was calculated as the SUV max of the lesion over the SUV max of the normal region. Accordingly, the calculated values may look smaller compared with reports in which the SUV max of the lesion is divided by the SUV mean of the normal region . NeverthTogether, the present study demonstrated a correlation between the preoperative MET-PET uptake and Ki-67 index versus intraoperative ex vivo fluorescence intensity in astrocytic glioma surgery. The visible 5-ALA-induced fluorescence may suggest the locus of high MET-PET high uptake. Given the broad clinical availability of the 5-ALA technique, our findings might be valuable to provide information on tumor biology rapidly  at the time of surgery in circumstances where MET-PET is inaccessible. Additionally, intraoperative quantitative measurement of excised tumors can inform the surgeon of the presence of viable tumors cells at the resection margins and assist a decision-making process towards maximizing the extent of resection during astrocytic brain tumor surgery.In astrocytic glioma surgery, visible 5-ALA-induced fluorescence correlates with high MET-PET uptake, along with a high Ki-67 index. This quantitative, biological insight might add a new axis to the utilities of fluorescence-guided brain tumor surgery using 5-ALA.