ABSTRACT
We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.
ABSTRACT
Cancer cells rewire metabolism to favour the generation of specialized metabolites that support tumour growth and reshape the tumour microenvironment1,2. Lysine functions as a biosynthetic molecule, energy source and antioxidant3-5, but little is known about its pathological role in cancer. Here we show that glioblastoma stem cells (GSCs) reprogram lysine catabolism through the upregulation of lysine transporter SLC7A2 and crotonyl-coenzyme A (crotonyl-CoA)-producing enzyme glutaryl-CoA dehydrogenase (GCDH) with downregulation of the crotonyl-CoA hydratase enoyl-CoA hydratase short chain 1 (ECHS1), leading to accumulation of intracellular crotonyl-CoA and histone H4 lysine crotonylation. A reduction in histone lysine crotonylation by either genetic manipulation or lysine restriction impaired tumour growth. In the nucleus, GCDH interacts with the crotonyltransferase CBP to promote histone lysine crotonylation. Loss of histone lysine crotonylation promotes immunogenic cytosolic double-stranded RNA (dsRNA) and dsDNA generation through enhanced H3K27ac, which stimulates the RNA sensor MDA5 and DNA sensor cyclic GMP-AMP synthase (cGAS) to boost type I interferon signalling, leading to compromised GSC tumorigenic potential and elevated CD8+ T cell infiltration. A lysine-restricted diet synergized with MYC inhibition or anti-PD-1 therapy to slow tumour growth. Collectively, GSCs co-opt lysine uptake and degradation to shunt the production of crotonyl-CoA, remodelling the chromatin landscape to evade interferon-induced intrinsic effects on GSC maintenance and extrinsic effects on immune response.
Subject(s)
Histones , Lysine , Neoplasms , Protein Processing, Post-Translational , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , Glutaryl-CoA Dehydrogenase/metabolism , Histones/chemistry , Histones/metabolism , Lysine/deficiency , Lysine/metabolism , Neoplasms/drug therapy , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/pathology , RNA, Double-Stranded/immunology , Humans , Animals , Mice , Interferon Type I/immunologyABSTRACT
YAP/TAZ have been identified as master regulators in malignant phenotypes of glioblastoma (GBM); however, YAP/TAZ transcriptional disruptor in GBM treatment remains ineffective. Whether post-transcriptional dysregulation of YAP/TAZ improves GBM outcome is currently unknown. Here, we report that insulin-like growth factor 2 (IGF2) mRNA-binding protein 1 (IGF2BP1 or IMP1) is upregulated in mesenchymal GBM compared with proneural GBM and correlates with worse patient outcome. Overexpression of IMP1 in proneural glioma stem-like cells (GSCs) promotes while IMP1 knockdown in mesenchymal GSCs attenuates tumorigenesis and mesenchymal signatures. IMP1 binds to and stabilizes m6A-YAP mRNA, leading to activation of YAP/TAZ signaling, depending on its m6A recognition and binding domain. On the other hand, TAZ functions as enhancer for IMP1 expression. Collectively, our data reveal a feedforward loop between IMP1 and YAP/TAZ maintaining GBM/GSC tumorigenesis and malignant progression and a promising molecular target in GBM.
Subject(s)
Glioblastoma , Glioma , Humans , Carcinogenesis/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic , Glioblastoma/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , YAP-Signaling ProteinsABSTRACT
BACKGROUND: The RTK/PI3K/AKT pathway plays key roles in the development and progression of many cancers, including GBM. As a regulatory molecule and a potential drug target, the oncogenic role of AKT has been substantially studied. Three isoforms of AKT have been identified, including AKT1, AKT2 and AKT3, but their individual functions in GBM remain controversial. Moreover, it is not known if there are more AKT alternative splicing variants. METHODS: High-throughput RNA sequencing and quantitative reverse transcription-PCR were used to identify the differentially expressed circRNAs in GBM samples and in paired normal tissues. High throughput RNA sequencing was used to identify circ-AKT3 regulated signaling pathways. Mass spectrometry, western blotting and immunofluorescence staining analyses were used to validate AKT3-174aa expression. The tumor suppressive role of AKT3-174aa was validated in vitro and in vivo. The competing interaction between AKT3-174aa and p-PDK1 was investigated by mass spectrometry and immunoprecipitation analyses. RESULTS: Circ-AKT3 is a previously uncharacterized AKT transcript variant. Circ-AKT3 is expressed at low levels in GBM tissues compared with the expression in paired adjacent normal brain tissues. Circ-AKT3 encodes a 174 amino acid (aa) novel protein, which we named AKT3-174aa, by utilizing overlapping start-stop codons. AKT3-174aa overexpression decreased the cell proliferation, radiation resistance and in vivo tumorigenicity of GBM cells, while the knockdown of circ-AKT3 enhanced the malignant phenotypes of astrocytoma cells. AKT3-174aa competitively interacts with phosphorylated PDK1, reduces AKT-thr308 phosphorylation, and plays a negative regulatory role in modulating the PI3K/AKT signal intensity. CONCLUSIONS: Our data indicate that the impaired circRNA expression of the AKT3 gene contributes to GBM tumorigenesis, and our data corroborate the hypothesis that restoring AKT3-174aa while inhibiting activated AKT may provide more benefits for certain GBM patients.
ABSTRACT
In the published article [1], an error was noticed in Fig. 6B. The western blot results were reversed between the overexpression group and the knockdown group of circ-AKT3. The corrected and updated Fig. 6 is provided below. This error does not affect the findings or conclusions of the article.
ABSTRACT
Cancers commonly reprogram translation and metabolism, but little is known about how these two features coordinate in cancer stem cells. Here we show that glioblastoma stem cells (GSCs) display elevated protein translation. To dissect underlying mechanisms, we performed a CRISPR screen and identified YRDC as the top essential transfer RNA (tRNA) modification enzyme in GSCs. YRDC catalyzes the formation of N6-threonylcarbamoyladenosine (t6A) on ANN-decoding tRNA species (A denotes adenosine, and N denotes any nucleotide). Targeting YRDC reduced t6A formation, suppressed global translation and inhibited tumor growth both in vitro and in vivo. Threonine is an essential substrate of YRDC. Threonine accumulated in GSCs, which facilitated t6A formation through YRDC and shifted the proteome to support mitosis-related genes with ANN codon bias. Dietary threonine restriction (TR) reduced tumor t6A formation, slowed xenograft growth and augmented anti-tumor efficacy of chemotherapy and anti-mitotic therapy, providing a molecular basis for a dietary intervention in cancer treatment.
Subject(s)
Glioblastoma , Threonine , Glioblastoma/genetics , Glioblastoma/pathology , Glioblastoma/metabolism , Humans , Animals , Mice , Threonine/metabolism , Threonine/genetics , Protein Biosynthesis , Neoplastic Stem Cells/metabolism , Cell Line, Tumor , Codon/genetics , RNA, Transfer/genetics , Xenograft Model Antitumor Assays , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/metabolismABSTRACT
Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain-containing gene, specifically Kelch domain containing 8A (KLHDC8A) with a previously unknown function as an epigenetically driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound chaperonin-containing TCP1 (CCT) to promote the assembly of primary cilia to activate hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and hedgehog signaling. Combinatorial targeting of Aurora B/C kinase and hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as what we believe to be a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.
Subject(s)
Brain Neoplasms , Glioblastoma , Glioma , Humans , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Glioblastoma/pathology , Glioma/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Neoplastic Stem Cells/pathology , Signal TransductionABSTRACT
Activated by its single ligand, hepatocyte growth factor (HGF), the receptor tyrosine kinase MET is pivotal in promoting glioblastoma (GBM) stem cell self-renewal, invasiveness and tumorigenicity. Nevertheless, HGF/MET-targeted therapy has shown limited clinical benefits in GBM patients, suggesting hidden mechanisms of MET signalling in GBM. Here, we show that circular MET RNA (circMET) encodes a 404-amino-acid MET variant (MET404) facilitated by the N6-methyladenosine (m6A) reader YTHDF2. Genetic ablation of circMET inhibits MET404 expression in mice and attenuates MET signalling. Conversely, MET404 knock-in (KI) plus P53 knock-out (KO) in mouse astrocytes initiates GBM tumorigenesis and shortens the overall survival. MET404 directly interacts with the MET ß subunit and forms a constitutively activated MET receptor whose activity does not require HGF stimulation. High MET404 expression predicts poor prognosis in GBM patients, indicating its clinical relevance. Targeting MET404 through a neutralizing antibody or genetic ablation reduces GBM tumorigenicity in vitro and in vivo, and combinatorial benefits are obtained with the addition of a traditional MET inhibitor. Overall, we identify a MET variant that promotes GBM tumorigenicity, offering a potential therapeutic strategy for GBM patients, especially those with MET hyperactivation.
Subject(s)
Brain Neoplasms , Glioblastoma , Animals , Mice , Glioblastoma/metabolism , Proto-Oncogene Proteins c-met/metabolism , RNA, Circular/genetics , Brain Neoplasms/metabolism , Cell Transformation, Neoplastic/genetics , Carcinogenesis/genetics , Cell Line, TumorABSTRACT
Chlorfortunones A (1) and B (2), two novel sesquiterpenoid dimers, were isolated from the roots of Chloranthus fortunei. Their structures were elucidated by spectroscopic analysis and X-ray diffraction analysis. Compounds 1 and 2 represent a new type of sesquiterpenoid dimer possessing an unprecedented 3/5/6/6/6/5 hexacyclic system with a unique dispiro[4,2,5,2]pentadecane-6,10,14-trien moiety. A plausible biosynthetic pathway of 1 and 2 was proposed. Compound 1 showed transforming growth factor (TGF)-ß inhibitory activity in MDA-MB-231 cells.
ABSTRACT
Glioblastoma (GBM) is a highly aggressive primary brain tumour and is resistant to nearly all available treatments, including natural killer (NK) cell immunotherapy. However, the factors mediating NK cell evasion in GBM remain largely unclear. Here, we report that EZH2-92aa, a protein encoded by circular EZH2, is overexpressed in GBM and induces the immune evasion of GBM stem cells (GSCs) from NK cells. Positively regulated by DEAD-box helicase 3 (DDX3), EZH2-92aa directly binds the major histocompatibility complex class I polypeptide-related sequence A/B (MICA/B) promoters and represses their transcription; it also indirectly represses UL16-binding protein (ULBP) transcription by stabilizing EZH2. The downregulation of NK group 2D ligands (NKG2DLs, including MICA/B and ULBPs) in GSCs mediates NK cell resistance. Moreover, stable EZH2-92aa knockdown enhances NK cell-mediated GSC eradication in vitro and in vivo and synergizes with anti-PD1 therapy. Our results highlight the immunosuppressive function of EZH2-92aa in inhibiting the NK cell response in GBM and the clinical potential of targeting EZH2-92aa for NK-cell-directed immune therapy.
Subject(s)
Enhancer of Zeste Homolog 2 Protein , Glioblastoma , NK Cell Lectin-Like Receptor Subfamily K , Tumor Escape , Enhancer of Zeste Homolog 2 Protein/genetics , GPI-Linked Proteins/genetics , Glioblastoma/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Ligands , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Receptors, Natural Killer CellABSTRACT
The seed of Myristica fragrans Houtt (nutmeg) is one of the important spices that have been extensively used in the culinary, food, beverage, and also in medicinal products industry. Previous phytochemical studies on nutmeg were mainly focused on lignans and neolignans. However, the other constituents have been poorly studied. In this study, 11 new monoterpene-conjugated phenolic derivatives, named myrifratins A-K (1-11), and five known compounds were isolated from nutmeg. The novel neolignan-diarylnonanoid-monoterpene conjugates (1 and 2) were first isolated in nature. Compounds 3-7 were rarely monoterpene-diarylnonanoid-conjugated derivatives, and 8-11 were the first examples of monoterpene-neolignan conjugates. Compounds 4-6, 12, and 13 showed potent autophagy inhibitory activities in a concentration-dependent manner. Our findings showed an uncommon class of monoterpene-conjugated phenolic derivatives in nature and reported their autophagy inhibition activities for the first time, which may give a new insight into the benefits or safety of nutmeg in foods.
Subject(s)
Lignans , Myristica , Autophagy , Lignans/chemistry , Monoterpenes/analysis , Monoterpenes/pharmacology , Myristica/chemistry , Phenols/analysis , Phenols/pharmacology , Seeds/chemistryABSTRACT
Nickel bioaccumulation capacity of a marine Brevibacterium sp., designated as X6, was evaluated to explore its potential application in the bioremediation of Ni2+ pollutants in marine environments. The minimum Ni2+ inhibitory concentration and maximum Ni2+ bioaccumulation of X6 were 1000 mg/L and 100.95 mg/g, respectively, higher than most reported strains. Among the co-existing metal ions in seawater, K+ caused a slight adverse impact on Ni2+ uptake, followed by Na+ and Ca2+, whereas Mg2+ drastically inhibited Ni2+ bioaccumulation. Other heavy metals such as Co2+, Zn2+ and Cd2+ moderately affected Ni2+ binding, but the adverse effect of Cu2+ was severe. The investigation of the mechanism of Ni2+ bioaccumulation revealed that 66.34% of the accumulated Ni2+ was bound to the cell surface. Carboxylic, hydroxyl, amino and thiol groups participated in Ni2+ binding, while carboxylic group contributed the most, while thiol group may be more involved in Ni2+ binding at low Ni2+ concentrations.
Subject(s)
Brevibacterium , Metals, Heavy , Water Pollutants, Chemical , Bays , Bioaccumulation , China , Metals, Heavy/analysis , Nickel/analysis , Water Pollutants, Chemical/analysisABSTRACT
Fifteen undescribed dimeric sesquiterpenoids, linderanoids A-O along with one known lindenane-type sesquiterpenoid dimer, lindenaneolide F, were isolated and identified from the roots of Lindera aggregata. Their structures and absolute configurations were elucidated using spectroscopy and electronic circular dichroism (ECD) analysis. All the isolated compounds were screened for transforming growth factor (TGF)-ß inhibitory activity, and the results showed that linderanoid E significantly inhibited the TGF- ß induced smad2 phosphorylation at a concentration of 25 µM.
Subject(s)
Lindera , Sesquiterpenes , Plant Roots , Sesquiterpenes/pharmacologyABSTRACT
BACKGROUND: Aberrant activation of the Hedgehog pathway drives tumorigenesis of many cancers, including glioblastoma. However, the sensitization mechanism of the G protein-coupled-like receptor smoothened (SMO), a key component of Hedgehog signaling, remains largely unknown. RESULTS: In this study, we describe a novel protein SMO-193a.a. that is essential for Hedgehog signaling activation in glioblastoma. Encoded by circular SMO (circ-SMO), SMO-193a.a. is required for sonic hedgehog (Shh) induced SMO activation, via interacting with SMO, enhancing SMO cholesterol modification, and releasing SMO from the inhibition of patched transmembrane receptors. Deprivation of SMO-193a.a. in brain cancer stem cells attenuates Hedgehog signaling intensity and suppresses self-renewal, proliferation in vitro, and tumorigenicity in vivo. Moreover, circ-SMO/SMO-193a.a. is positively regulated by FUS, a direct transcriptional target of Gli1. Shh/Gli1/FUS/SMO-193a.a. form a positive feedback loop to sustain Hedgehog signaling activation in glioblastoma. Clinically, SMO-193a.a. is more specifically expressed in glioblastoma than SMO and is relevant to Gli1 expression. Higher expression of SMO-193a.a. predicts worse overall survival of glioblastoma patients, indicating its prognostic value. CONCLUSIONS: Our study reveals that SMO-193a.a., a novel protein encoded by circular SMO, is critical for Hedgehog signaling, drives glioblastoma tumorigenesis and is a novel target for glioblastoma treatment.
Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , Hedgehog Proteins/genetics , RNA, Circular/genetics , Signal Transduction/genetics , Smoothened Receptor/genetics , Animals , Brain Neoplasms/pathology , Cell Proliferation , Cell Transformation, Neoplastic , Disease Models, Animal , Female , Glioblastoma/pathology , HEK293 Cells , Hedgehog Proteins/metabolism , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Patched Receptors/metabolism , Smoothened Receptor/metabolism , Stem CellsABSTRACT
Activated EGFR signalling drives tumorigenicity in 50% of glioblastoma (GBM). However, EGFR-targeting therapy has proven ineffective in treating patients with GBM, indicating that there is redundant EGFR activation. Circular RNAs are covalently closed RNA transcripts that are involved in various physiological and pathological processes. Herein, we report an additional activation mechanism of EGFR signalling in GBM by an undescribed secretory E-cadherin protein variant (C-E-Cad) encoded by a circular E-cadherin (circ-E-Cad) RNA through multiple-round open reading frame translation. C-E-Cad is overexpressed in GBM and promotes glioma stem cell tumorigenicity. C-E-Cad activates EGFR independent of EGF through association with the EGFR CR2 domain using a unique 14-amino-acid carboxy terminus, thereby maintaining glioma stem cell tumorigenicity. Notably, inhibition of C-E-Cad markedly enhances the antitumour activity of therapeutic anti-EGFR strategies in GBM. Our results uncover a critical role of C-E-Cad in stimulating EGFR signalling and provide a promising approach for treating EGFR-driven GBM.
Subject(s)
Antigens, CD/metabolism , Brain Neoplasms/enzymology , Cadherins/metabolism , Glioblastoma/enzymology , RNA, Circular/metabolism , STAT3 Transcription Factor/metabolism , Animals , Antigens, CD/genetics , Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cadherins/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Mice, Nude , Neoplasm Invasiveness , RNA, Circular/genetics , STAT3 Transcription Factor/genetics , Signal Transduction , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The metabolic role of micropeptides generated from untranslated regions remains unclear. Here we describe MP31, a micropeptide encoded by the upstream open reading frame (uORF) of phosphatase and tensin homolog (PTEN) acting as a "circuit breaker" that limits lactate-pyruvate conversion in mitochondria by competing with mitochondrial lactate dehydrogenase (mLDH) for nicotinamide adenine dinucleotide (NAD+). Knocking out the MP31 homolog in mice enhanced global lactate metabolism, manifesting as accelerated oxidative phosphorylation (OXPHOS) and increased lactate consumption and production. Conditional knockout (cKO) of MP31 homolog in mouse astrocytes initiated gliomagenesis and shortened the overall survival of the animals, establishing a tumor-suppressing role for MP31. Recombinant MP31 administered intraperitoneally penetrated the blood-brain barrier and inhibited mice GBM xenografts without neurological toxicity, suggesting the clinical implication and application of this micropeptide. Our findings reveal a novel mode of MP31-orchestrated lactate metabolism reprogramming in glioblastoma.
Subject(s)
Lactic Acid/metabolism , Peptides/metabolism , Phosphoric Monoester Hydrolases/metabolism , Tensins/metabolism , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout , Peptides/deficiencyABSTRACT
BACKGROUND: Aberrant epidermal growth factor receptor (EGFR) activation is observed in over 50% of cases of adult glioblastoma (GBM). Nevertheless, EGFR antibodies are ineffective in clinical GBM treatment, suggesting the existence of redundant EGFR activation mechanisms. Whether circular RNA (circRNA) encodes a protein involved in EGFR-driven GBM remains unclear. We reported an unexpected mechanism in which circular EGFR RNA (circ-EGFR) encodes a novel EGFR variant to sustained EGFR activation. METHOD: We used RNA-seq, Northern blot, and Sanger sequencing to confirm the existence of circ-EGFR. Antibodies and a liquid chromatograph tandem mass spectrometer were used to identify circ-EGFR protein products. Lentivirus-transfected stable cell lines were used to assess the biological functions of the novel protein in vitro and in vivo. Clinical implications of circ-EGFR were assessed using 97 pathologically diagnosed GBM patient samples. RESULTS: The infinite open reading frame (iORF) in circ-EGFR translated repeating amino acid sequences via rolling translation and programmed -1 ribosomal frameshifting (-1PRF) induced out-of-frame stop codon (OSC), forming a polymetric novel protein-complex, which we termed rolling-translated EGFR (rtEGFR). rtEGFR directly interacted with EGFR, maintained EGFR membrane localization and attenuated EGFR endocytosis and degradation. Importantly, circ-EGFR levels correlated with the EGFR signature and predicted the poor prognosis of GBM patients. Deprivation of rtEGFR in brain tumor-initiating cells (BTICs) attenuated tumorigenicity and enhanced the anti-GBM effect. CONCLUSION: Our findings identified the endogenous rolling-translated protein and provided strong clinical evidence that targeting rtEGFR could improve the efficiency of EGFR-targeting therapies in GBM.
Subject(s)
Brain Neoplasms , ErbB Receptors/genetics , Glioblastoma , Signal Transduction , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , ErbB Receptors/metabolism , Glioblastoma/genetics , Humans , RNA, CircularABSTRACT
Three-dimensional (3D) printing is a new developing technology for printing individualized materials swiftly and precisely in the field of biological medicine (especially tissue-engineered materials). Prior to printing, it is necessary to scan the structure of the natural biological tissue, then construct the 3D printing digital model through optimizing the scanned data. By searching the literatures, magazines at home and abroad, this article reviewed the current status, main processes and matters needing attention of confocal laser scanning microscope (LSCM) in the application of soft tissue fine structure 3D scanning, empathizing the significance of LSCM in this field.