Progenitor-like cells contributing to cellular heterogeneity in the nucleus pulposus are lost in intervertebral disc degeneration.

The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging. Lineage tracing using Tagln-CreERt2 mice identified peripherally located proliferative NP (PeriNP) cells in developing and postnatal NP that provide a continuous supply of cells to the entire NP. PeriNP cells were diminished in aged mice and absent in puncture-induced degenerated discs. Single-cell transcriptomes of postnatal Tagln-CreERt2 IVD cells indicate enrichment for TGF-ß signaling in Tagln descendant NP sub-populations. Notochord-specific removal of TGF-ß/BMP mediator Smad4 results in loss of Tagln+ cells and abnormal NP morphologies. We propose Tagln+ PeriNP cells are potential progenitors crucial for NP homeostasis.

The Molecular Landscape of Primary CNS Lymphomas (PCNSLs) in Children and Young Adults.

Pediatric brain tumors are often noted to be different from their adult counterparts in terms of molecular features. Primary CNS lymphomas (PCNSLs) are mostly found in elderly adults and are uncommon in children and teenagers. There has only been scanty information about the molecular features of PCNSLs at a young age. We examined PCNSLs in 34 young patients aged between 7 and 39 years for gene rearrangements of BCl2, BCL6, CCND1, IRF4, IGH, IGL, IGK, and MYC, homozygous deletions (HD) of CDKN2A, and HLA by FISH. Sequencing was performed using WES, panel target sequencing, or Sanger sequencing due to the small amount of available tissues. The median OS was 97.5 months and longer than that for older patients with PCNSLs. Overall, only 14 instances of gene rearrangement were found (5%), and patients with any gene rearrangement were significantly older (p = 0.029). CDKN2A HD was associated with a shorter OS (p < 0.001). Only 10/31 (32%) showed MYD88 mutations, which were not prognostically significant, and only three of them were L265P mutations. CARD11 mutations were found in 8/24 (33%) cases only. Immunophenotypically, the cases were predominantly GCB, in contrast to older adults (61%). In summary, we showed that molecular findings identified in the PCNSLs of the older patients were only sparingly present in pediatric and young adult patients.

Suppression of apoptosis impairs phalangeal joint formation in the pathogenesis of brachydactyly type A1.

Apoptosis occurs during development when a separation of tissues is needed. Synovial joint formation is initiated at the presumptive site (interzone) within a cartilage anlagen, with changes in cellular differentiation leading to cavitation and tissue separation. Apoptosis has been detected in phalangeal joints during development, but its role and regulation have not been defined. Here, we use a mouse model of brachydactyly type A1 (BDA1) with an IhhE95K mutation, to show that a missing middle phalangeal bone is due to the failure of the developing joint to cavitate, associated with reduced apoptosis, and a joint is not formed. We showed an intricate relationship between IHH and interacting partners, CDON and GAS1, in the interzone that regulates apoptosis. We propose a model in which CDON/GAS1 may act as dependence receptors in this context. Normally, the IHH level is low at the center of the interzone, enabling the "ligand-free" CDON/GAS1 to activate cell death for cavitation. In BDA1, a high concentration of IHH suppresses apoptosis. Our findings provided new insights into the role of IHH and CDON in joint formation, with relevance to hedgehog signaling in developmental biology and diseases.

In vitro and in vivo models define a molecular signature reference for human embryonic notochordal cells.

Understanding the emergence of human notochordal cells (NC) is essential for the development of regenerative approaches. We present a comprehensive investigation into the specification and generation of bona fide NC using a straightforward pluripotent stem cell (PSC)-based system benchmarked with human fetal notochord. By integrating in vitro and in vivo transcriptomic data at single-cell resolution, we establish an extended molecular signature and overcome the limitations associated with studying human notochordal lineage at early developmental stages. We show that TGF-ß inhibition enhances the yield and homogeneity of notochordal lineage commitment in vitro. Furthermore, this study characterizes regulators of cell-fate decision and matrisome enriched in the notochordal niche. Importantly, we identify specific cell-surface markers opening avenues for differentiation refinement, NC purification, and functional studies. Altogether, this study provides a human notochord transcriptomic reference that will serve as a resource for notochord identification in human systems, diseased-tissues modeling, and facilitating future biomedical research.

Rare Pediatric Cerebellar High-Grade Gliomas Mimic Medulloblastomas Histologically and Transcriptomically and Show p53 Mutations.

Pediatric high-grade gliomas (HGG) of the cerebellum are rare, and only a few cases have been documented in detail in the literature. A major differential diagnosis for poorly differentiated tumors in the cerebellum in children is medulloblastoma. In this study, we described the histological and molecular features of a series of five pediatric high-grade gliomas of the cerebellum. They actually showed histological and immunohistochemical features that overlapped with those of medulloblastomas and achieved high scores in NanoString-based medulloblastoma diagnostic assay. Methylation profiling demonstrated these tumors were heterogeneous epigenetically, clustering to GBM_MID, DMG_K27, and GBM_RTKIII methylation classes. MYCN amplification was present in one case, and PDGFRA amplification in another two cases. Interestingly, target sequencing showed that all tumors carried TP53 mutations. Our results highlight that pediatric high-grade gliomas of the cerebellum can mimic medulloblastomas at histological and transcriptomic levels. Our report adds to the rare number of cases in the literature of cerebellar HGGs in children. We recommend the use of both methylation array and TP53 screening in the differential diagnoses of poorly differentiated embryonal-like tumors of the cerebellum.

Diagnostic Accuracy and Field for Improvement of Frameless Stereotactic Brain Biopsy: A Focus on Nondiagnostic Cases.

BACKGROUND: The diagnostic accuracy of frameless stereotactic brain biopsy has been reported, but there is limited literature focusing on the reasons for nondiagnostic cases. In this study, we evaluate the diagnostic accuracy of frameless stereotactic brain biopsy, compare it with the current international standard, and review the field for improvement. METHODS: This is a retrospective analysis of consecutive, prospectively collected frameless stereotactic brain biopsies from 2007 to 2020. We evaluated the diagnostic accuracy of the frameless stereotactic brain biopsies using defined criteria. The biopsy result was classified as conclusive, inconclusive, or negative, based on the pathologic, radiologic, and clinical diagnosis concordance. For inconclusive or negative results, we further evaluated the preoperative planning and postoperative imaging to review the errors. A literature review for the diagnostic accuracy of frameless stereotactic biopsy was performed for the validity of our results. RESULTS: There were 106 patients with 109 biopsies performed from 2007 to 2020. The conclusive diagnosis was reached in 103 (94.5%) procedures. An inconclusive diagnosis was noted in four (3.7%) procedures and the biopsy was negative in two (1.9%) procedures. Symptomatic hemorrhage occurred in one patient (0.9%). There was no mortality in our series. Registration error (RE) and inaccurate targeting occurred in three trigonal lesions (2.8%), sampling of the nonrepresentative part of the lesion occurred in two cases (1.8%), and one biopsy (0.9%) for lymphoma was negative due to steroid treatment. The literature review suggested that our diagnostic accuracy was comparable with the published literature. CONCLUSION: The frameless stereotactic biopsy is a safe procedure with high diagnostic accuracy only if meticulous preoperative planning and careful intraoperative registration is performed. The common pitfalls precluding a conclusive diagnosis are RE and biopsies at nonrepresentative sites.

Interactive Multi-Stage Robotic Positioner for Intra-Operative MRI-Guided Stereotactic Neurosurgery.

Magnetic resonance imaging (MRI) demonstrates clear advantages over other imaging modalities in neurosurgery with its ability to delineate critical neurovascular structures and cancerous tissue in high-resolution 3D anatomical roadmaps. However, its application has been limited to interventions performed based on static pre/post-operative imaging, where errors accrue from stereotactic frame setup, image registration, and brain shift. To leverage the powerful intra-operative functions of MRI, e.g., instrument tracking, monitoring of physiological changes and tissue temperature in MRI-guided bilateral stereotactic neurosurgery, a multi-stage robotic positioner is proposed. The system positions cannula/needle instruments using a lightweight (203 g) and compact (Ø97 × 81 mm) skull-mounted structure that fits within most standard imaging head coils. With optimized design in soft robotics, the system operates in two stages: i) manual coarse adjustment performed interactively by the surgeon (workspace of ±30°), ii) automatic fine adjustment with precise (<0.2° orientation error), responsive (1.4 Hz bandwidth), and high-resolution (0.058°) soft robotic positioning. Orientation locking provides sufficient transmission stiffness (4.07 N/mm) for instrument advancement. The system's clinical workflow and accuracy is validated with lab-based (<0.8 mm) and MRI-based testing on skull phantoms (<1.7 mm) and a cadaver subject (<2.2 mm). Custom-made wireless omni-directional tracking markers facilitated robot registration under MRI.

Impaired glycine neurotransmission causes adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting millions of adolescents worldwide, but it lacks a defined theory of etiopathogenesis. Because of this, treatment of AIS is limited to bracing and/or invasive surgery after onset. Preonset diagnosis or preventive treatment remains unavailable. Here, we performed a genetic analysis of a large multicenter AIS cohort and identified disease-causing and predisposing variants of SLC6A9 in multigeneration families, trios, and sporadic patients. Variants of SLC6A9, which encodes glycine transporter 1 (GLYT1), reduced glycine-uptake activity in cells, leading to increased extracellular glycine levels and aberrant glycinergic neurotransmission. Slc6a9 mutant zebrafish exhibited discoordination of spinal neural activities and pronounced lateral spinal curvature, a phenotype resembling human patients. The penetrance and severity of curvature were sensitive to the dosage of functional glyt1. Administration of a glycine receptor antagonist or a clinically used glycine neutralizer (sodium benzoate) partially rescued the phenotype. Our results indicate a neuropathic origin for "idiopathic" scoliosis, involving the dysfunction of synaptic neurotransmission and central pattern generators (CPGs), potentially a common cause of AIS. Our work further suggests avenues for early diagnosis and intervention of AIS in preadolescents.

Recommendations for intervertebral disc notochordal cell investigation: From isolation to characterization.

Background: Lineage-tracing experiments have established that the central region of the mature intervertebral disc, the nucleus pulposus (NP), develops from the embryonic structure called "the notochord". However, changes in the cells derived from the notochord which form the NP (i.e., notochordal cells [NCs]), in terms of their phenotype and functional identity from early developmental stages to skeletal maturation are less understood. These key issues require further investigation to better comprehend the role of NCs in homeostasis and degeneration as well as their potential for regeneration. Progress in utilizing NCs is currently hampered due to poor consistency and lack of consensus methodology for in vitro NC extraction, manipulation, and characterization. Methods: Here, an international group has come together to provide key recommendations and methodologies for NC isolation within key species, numeration, in vitro manipulation and culture, and characterization. Results: Recommeded protocols are provided for isolation and culture of NCs. Experimental testing provided recommended methodology for numeration of NCs. The issues of cryopreservation are demonstrated, and a pannel of immunohistochemical markers are provided to inform NC characterization. Conclusions: Together we hope this article provides a road map for in vitro studies of NCs to support advances in research into NC physiology and their potential in regenerative therapies.

Identifying predictors of glioma evolution from longitudinal sequencing.

Clonal evolution drives cancer progression and therapeutic resistance. Recent studies have revealed divergent longitudinal trajectories in gliomas, but early molecular features steering posttreatment cancer evolution remain unclear. Here, we collected sequencing and clinical data of initial-recurrent tumor pairs from 544 adult diffuse gliomas and performed multivariate analysis to identify early molecular predictors of tumor evolution in three diffuse glioma subtypes. We found that CDKN2A deletion at initial diagnosis preceded tumor necrosis and microvascular proliferation that occur at later stages of IDH-mutant glioma. Ki67 expression at diagnosis was positively correlated with acquiring hypermutation at recurrence in the IDH-wild-type glioma. In all glioma subtypes, MYC gain or MYC-target activation at diagnosis was associated with treatment-induced hypermutation at recurrence. To predict glioma evolution, we constructed CELLO2 (Cancer EvoLution for LOngitudinal data version 2), a machine learning model integrating features at diagnosis to forecast hypermutation and progression after treatment. CELLO2 successfully stratified patients into subgroups with distinct prognoses and identified a high-risk patient group featured by MYC gain with worse post-progression survival, from the low-grade IDH-mutant-noncodel subtype. We then performed chronic temozolomide-induction experiments in glioma cell lines and isogenic patient-derived gliomaspheres and demonstrated that MYC drives temozolomide resistance by promoting hypermutation. Mechanistically, we demonstrated that, by binding to open chromatin and transcriptionally active genomic regions, c-MYC increases the vulnerability of key mismatch repair genes to treatment-induced mutagenesis, thus triggering hypermutation. This study reveals early predictors of cancer evolution under therapy and provides a resource for precision oncology targeting cancer dynamics in diffuse gliomas.

Scoliosis in osteogenesis imperfecta: identifying the genetic and non-genetic factors affecting severity and progression from longitudinal data of 290 patients.

BACKGROUND: Scoliosis is widely prevalent among osteogenesis imperfecta (OI) patients, and is progressive with age. However, factors affecting scoliosis in OI are not well known. METHODS: We retrospectively retrieved longitudinal radiographic and clinical records of consecutive OI patients seeking treatments at our hospital from 2014 to 2022, graded their pre-operative spinal conditions into four outcome groups, estimated their progression rates, and descriptively and inferentially analyzed the genetic and non-genetic factors that may affect the outcomes and progression rates. RESULTS: In all, 290 OI patients met the inclusion criteria, where 221 had genetic records. Of these 221, about 2/3 had mutations in COL1A1 or COL1A2, followed by mutations in WNT1 (9.0%), IFITM5 (9.0%) and other OI risk genes. With an average age of 12.0 years (interquartile range [IQR] 6.9-16.1), 70.7% of the cohort had scoliosis (Cobb angle > 10°), including 106 (36.5%) mild (10°-25°), 40 (13.8%) moderate (25°-50°), and 59 (20.3%) severe (> 50°) scoliosis patients. Patients with either COL1A1 and COL1A2 were strongly biased toward having mild or no scoliosis, whereas patients with mutations in IFITM5, WNT1 and other recessive genes were more evenly distributed among the four outcome grades. Lower-limb discrepancy, bone mineral density (BMD) and age of first drug used were all significantly correlated with severity outcomes. Using multivariate logistic regression, we estimated that each year older adds an odds ratio of 1.13 (95% confidence interval [CI] 1.07-1.2) in progression into advanced stages of scoliosis. We estimated a cohort-wide progression rate of 2.7 degrees per year (95% CI 2.4-3.0). Early-onset patients experienced fast progressions during both infantile and adolescent stages. Twenty-five of the 59 (42.8%) patients with severe scoliosis underwent spinal surgeries, enjoying an average Cobb angle reduction of 33° (IQR 23-40) postoperatively. CONCLUSION: The severity and progression of scoliosis in osteogenesis imperfecta were affected by genetic factors including genotypes and mutation types, and non-genetic factors including age and BMD. As compared with COL1A1, mutations in COL1A2 were less damaging while those on IFITM5 and other recessive genes conferred damaging effects. Progression rates were the fastest in the adolescent adult age-group.

Hypertrophic chondrocytes at the junction of musculoskeletal structures.

Hypertrophic chondrocytes are found at unique locations at the junction of skeletal tissues, cartilage growth plate, articular cartilage, enthesis and intervertebral discs. Their role in the skeleton is best understood in the process of endochondral ossification in development and bone fracture healing. Chondrocyte hypertrophy occurs in degenerative conditions such as osteoarthritis. Thus, the role of hypertrophic chondrocytes in skeletal biology and pathology is context dependent. This review will focus on hypertrophic chondrocytes in endochondral ossification, in which they exist in a transient state, but acting as a central regulator of differentiation, mineralization, vascularization and conversion to bone. The amazing journey of a chondrocyte from being entrapped in the extracellular matrix environment to becoming proliferative then hypertrophic will be discussed. Recent studies on the dynamic changes and plasticity of hypertrophic chondrocytes have provided new insights into how we view these cells, not as terminally differentiated but as cells that can dedifferentiate to more progenitor-like cells in a transition to osteoblasts and adipocytes, as well as a source of skeletal stem and progenitor cells residing in the bone marrow. This will provide a foundation for studies of hypertrophic chondrocytes at other skeletal sites in development, tissue maintenance, pathology and therapy.

The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc.

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1-Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12 h ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

Utilization, surgical populations, centers, coverages, regional balance, and their influential factors of deep brain stimulation for Parkinson's disease: a large-scale multicenter cross-sectional study from 1997 to 2021.

BACKGROUND: Deep brain stimulation (DBS) is an emerging and effective therapy for Parkinson's disease (PD). However, little is known about its utilization, surgical populations, centers, coverages, regional balance, and influential factors. MATERIALS AND METHODS: This large-scale multicenter cross-sectional study was conducted using a national census involving 74 Chinese centers. National DBS populations and centers for PD were investigated in 1997-2021, and regional sociodemographic features, surgical populations, related resources, and insurance policies in 2020 were explored. RESULTS: Since the first DBS surgery in 1997, a total of 38 122 PD patients from 349 centers underwent DBS by 2021, which covered 1.118% (1.108-1.129) of patients and 0.954% (0.933-0.976) of centers. Significant upward trends in the annual surgical population and coverages were observed with rapid climbing rates, while the annual surgical centers and their coverage showed two growth peaks in 2002-2006 and 2010-2018, correlating with clinical approvals and new technologies. A total of 103 070 (51 165-154 975) PD patients [2.088% (1.351-2.825) coverage] and 603 (72-1134) centers [1.356% (1.126-1.586) coverage] are predicted to conduct DBS by 2030. The new remotely programmed DBS technology was recoded as the first application in 2015 and rapidly increased to 2771 (47.39%, 46.11-48.67) patients with 10 507 remote programming sessions annually in 2021. Provinces in the eastern and central regions had better economic status, more surgical patients, higher insurance affordability, and more related resources than those in the western and northeastern regions. Higher gross domestic product per capita ( ß =5.041, 3.324-6.758 and ß =0.008, 0.004-0.012; all P <0.001) and more functional neurosurgery doctors ( ß =3.596, 0.353-6.839; P =0.031 and ß =0.010, 0.002-0.017; P =0.013) positively influenced surgical populations and coverages, while higher insurance levels ( ß =128.888, 64.702-193.075; P <0.001) positively influenced surgical coverages. CONCLUSION: Although surgical populations, centers, and coverages of DBS for PD have rapidly improved and are predicted to show future increases, this is still insufficient to cover potential eligible patients. Regionally imbalanced health coverage should be given attention to promote coordinated development.

Hypochondroplasia gain-of-function mutation in FGFR3 causes defective bone mineralization in mice.

Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis.

A Semi-Autonomous Stereotactic Brain Biopsy Robotic System With Enhanced Surgical Safety and Surgeon-Robot Collaboration.

OBJECTIVE: Despite benefits brought by recent neurosurgical robots, surgical safety and surgeon-robot collaboration remain significant challenges. In this article, we analyze and address these problems in the context of brain biopsy, by proposing a semi-autonomous system. METHODS: A robotic module is designed for the automation of all the brain biopsy procedures, and a biopsy cannula with tissue blocker is developed to avoid tissue excess and haemorrhage. In addition, two methods are proposed for surgical safety and surgeon-robot collaboration enhancement. First, a priority-based control framework is proposed for neuronavigation with simultaneous optical tracking line-of-sight maintenance and surgeon avoidance. Second, after neuronavigation, an adaptive reconfiguration method is developed to optimize the arm angle of KUKA robot based on the surgeon's pose, for workspace interference minimization, high robot dexterity, and joint-limit avoidance. RESULT: Effectiveness of the proposed solution demonstrated by simulations and experiments. CONCLUSION: The system can perform automatic navigation with simultaneous optical tracking maintenance and surgeon avoidance, autonomous brain biopsy, and adaptive reconfiguration for workspace interference minimization. SIGNIFICANCE: This work improves existing neurosurgical systems, in terms of autonomy level from mechanical guidance to task autonomy, surgical safety, and surgeon-robot collaboration.

An optofluidic platform for interrogating chemosensory behavior and brainwide neural representation in larval zebrafish.

Studying chemosensory processing desires precise chemical cue presentation, behavioral response monitoring, and large-scale neuronal activity recording. Here we present Fish-on-Chips, a set of optofluidic tools for highly-controlled chemical delivery while simultaneously imaging behavioral outputs and whole-brain neuronal activities at cellular resolution in larval zebrafish. These include a fluidics-based swimming arena and an integrated microfluidics-light sheet fluorescence microscopy (µfluidics-LSFM) system, both of which utilize laminar fluid flows to achieve spatiotemporally precise chemical cue presentation. To demonstrate the strengths of the platform, we used the navigation arena to reveal binasal input-dependent behavioral strategies that larval zebrafish adopt to evade cadaverine, a death-associated odor. The µfluidics-LSFM system enables sequential presentation of odor stimuli to individual or both nasal cavities separated by only ~100 µm. This allowed us to uncover brainwide neural representations of cadaverine sensing and binasal input summation in the vertebrate model. Fish-on-Chips is readily generalizable and will empower the investigation of neural coding in the chemical senses.

scONE-seq: A single-cell multi-omics method enables simultaneous dissection of phenotype and genotype heterogeneity from frozen tumors.

Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity. Most previous single-cell whole-genome RNA sequencing (scWGS-RNA-seq) methods demonstrate utility with intact cells from fresh samples. Among them, many are not applicable to frozen samples that cannot produce intact single-cell suspensions. We have developed scONE-seq, a versatile scWGS-RNA-seq method that amplifies single-cell DNA and RNA without separating them from each other and hence is compatible with frozen biobanked samples. We benchmarked scONE-seq against existing methods using fresh and frozen samples to demonstrate its performance in various aspects. We identified a unique transcriptionally normal-like tumor clone by analyzing a 2-year frozen astrocytoma sample, demonstrating that performing single-cell multi-omics interrogation on biobanked tissue by scONE-seq could enable previously unidentified discoveries in tumor biology.