Biology subjects

Spencer, A.

Publications and source records attributed to Spencer, A..

16 recordsLinked to original sources

bioRxiv2026

ProtoPure: Literature Mining and Analysis of Protein Purification Protocols at Proteome Scale

Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA, or RNA. A variety of protein purification strategies exist to address desired scale, throughput, and downstream applications. However, selecting an effective experimental procedure remains a largely empirical process that a quick literature review can help guide. Researchers typically survey on the order of a dozen publications for proteins with similar biochemical properties, assessing conditions on a case-by-case basis, leaving no systematic framework for comparison and decision-making at the scale of entire protein families. As protein engineering and design evolve, identifying effective purification conditions remains critical for obtaining sufficient quantities of properly folded and functional proteins. To address this gap, we developed ProtoPure, an LLM-based workflow and database that automates the extraction, standardization and analysis of protein purification conditions from peer-reviewed literature at scale. Given a user-defined UniProt table-enabling flexible selection of proteins by family, organism, or proteome. ProtoPure retrieves associated publications, classifies them based on the presence of purification protocols, and extracts a standardized set of 12 purification parameters (e.g., buffer composition, elution conditions). By transforming dispersed experimental descriptions into a structured resource, ProtoPure makes it possible to compare purification strategies across thousands of proteins. The resulting dataset revealed quantitative convergence toward a limited set of recombinant expression practices, with BL21(DE3)-based systems dominating protein production and recurrent combinations of expression host, inducer, growth medium, and plasmid occurring substantially more frequently than expected by chance. In contrast, downstream purification parameters exhibited greater diversity, revealing a workflow architecture that combines standardized expression infrastructure with more flexible biochemical components.

biophysics↗

bioRxiv2026

Menin regulates oncogenic cell identity transcriptional networks in multiple myeloma

Multiple myeloma (MM) is a heterogenous cancer that remains mostly incurable. A unifying feature of MM cells is a highly interconnected network of transcription factors and co-factors that coordinate the activity of super-enhancers to enforce myeloma cell identity. Targeting this network offers a promising avenue for therapeutic intervention across genetically diverse myeloma sub-types. Using integrated molecular and functional genomic approaches we identified Menin as a key driver of oncogenic gene expression in MM cells. Menin and its co-factor KMT2A bind at super-enhancers and maintain expression of essential myeloma genes, including IRF4. We demonstrate that Menin inhibitors, which have recently been approved for treatment of acute myeloid leukaemia, are highly active in myeloma cell lines and in vivo models. Combining Menin inhibitors with other super-enhancer targeting therapies such EP300/CREBBP inhibitors or immunomodulatory drugs (IMiDs) overcomes epigenetic plasticity in cells resistant to single agent treatment.

Cancer Biology↗

bioRxiv2025

A periplasmic protein complex mediates cell envelope integrity and intrinsic multidrug resistance in Mycobacterium tuberculosis

The intrinsic drug resistance of Mycobacterium tuberculosis (Mtb) is a major barrier to effective tuberculosis (TB) treatment, largely due to its complex, impermeable cell envelope. We identified a periplasmic protein complex comprising FecB and Rv3035 that is essential for maintaining envelope integrity and mediating intrinsic multidrug resistance in Mtb. FecB interacts with Rv3035, forming a stable heterodimer that associates with the cell envelope biosynthesis protein AftB. We report the structures of Rv3035 alone and in complex with FecB and identify critical residues for complex formation and function. Co-essentiality and genetic interaction analyses support a functional link between FecB, Rv3035 and AftB, an arabinofuranosyltransferase which synthesizes arabinogalactan and lipoarabinomannan. Loss of FecB or Rv3035 disrupted AftB-mediated arabinan synthesis, suggesting that these proteins support AftBs enzymatic activity. Importantly, FecB is required for Mtb virulence in mice, underscoring its physiological relevance. These findings highlight FecB, Rv3035 and AftB as promising therapeutic targets.

microbiology↗

bioRxiv2024

Mapping activity and functional organisation of the motor and visual pathways using ADC-fMRI in the human brain

In contrast to blood-oxygenation-level-dependent (BOLD) functional MRI (fMRI), which relies on changes in blood flow and oxygenation levels to infer brain activity, diffusion fMRI (DfMRI) investigates brain dynamics by monitoring alterations in the Apparent Diffusion Coefficient (ADC) of water. These ADC changes may arise from fluctuations in neuronal morphology, providing a distinctive perspective on neural activity. The potential of ADC as an fMRI contrast (ADC-fMRI) lies in its capacity to reveal neural activity independently of neurovascular coupling, thus yielding complementary insights into brain function. To demonstrate the specificity and value of ADC-fMRI, both ADC-and BOLD-fMRI data were collected at 3T in human subjects during visual stimulation and motor tasks. The first aim of this study was to identify an acquisition design for ADC that minimises BOLD contributions. By examining the timings in responses, we report that ADC 0/1 timeseries (acquired with b-values of 0 and 1 ms/{micro}m2) exhibit residual vascular contamination while ADC 0.2/1 timeseries (with b-values of 0.2 and 1 ms/{micro}m2) show minimal BOLD influence and higher sensitivity to neuromorphological coupling. Second, a General Linear Model was employed to identify activation clusters for ADC 0.2/1 and BOLD, from which average ADC and BOLD responses were calculated. The negative ADC response exhibited a significantly reduced delay relative to the task onset and offset as compared to BOLD. This early onset further supports the notion that ADC is sensitive to neuromorphological rather than neurovascular coupling. Remarkably, in the group-level analysis, positive BOLD activation clusters were detected in the visual and motor cortices, while the negative ADC clusters mainly highlighted pathways in white matter connected to the motor cortex. In the averaged individual level analysis, negative ADC activation clusters were also present in the visual cortex. This finding confirmed the reliability of negative ADC as an indicator of brain function, even in regions with lower vascularisation such as white matter. Finally, we established that ADC-fMRI timecourses yield the expected functional organisation of the visual system, including both gray and white matter regions of interest. Functional connectivity matrices were used to perform hierarchical clustering of brain regions, where ADC-fMRI successfully reproduced the expected structure of the dorsal and ventral visual pathways. This organisation was not replicated with the b=0.2 ms/{micro}m2 diffusion-weighted time courses, which can be seen as a proxy for BOLD (via T2-weighting). These findings underscore the robustness of ADC time courses in functional MRI studies, offering complementary insights to BOLD-fMRI regarding brain function and connectivity patterns. KeypointsO_LIThe functional time course of the Apparent Diffusion Coefficient (ADC), specifically measured with alternating b-values of 0.2 and 1 ms/{micro}m2 at 3T, appears to be minimally affected by BOLD contamination. C_LIO_LIIn the activity maps, the location of negative ADC clusters suggests neural activity in WM tracts that are connected to the motor cortex, which is not detected with positive BOLD. C_LIO_LIFunctional Connectivity analysis utilising ADC is better able to detect the organisation of the dorsal and ventral visual streams than diffusion- and T2-weighted time courses. C_LI

neuroscience↗

bioRxiv2024

Apparent Diffusion Coefficient fMRI shines a new light on white matter resting-state connectivity, as compared to BOLD

Resting-state functional magnetic resonance imaging (fMRI) is used to derive functional connectivity (FC) between brain regions. Typically, blood oxygen level-dependent (BOLD) contrast is used. However, BOLDs reliance on neurovascular coupling poses challenges in reflecting brain activity accurately, leading to reduced sensitivity in white matter (WM). WM BOLD signals have long been considered physiological noise, although recent evidence shows that both stimulus-evoked and resting-state WM BOLD signals resemble those in gray matter (GM), albeit smaller in amplitude. We introduce apparent diffusion coefficient fMRI (ADC-fMRI) as a promising functional contrast for GM and WM FC, capturing activity-driven neuromorphological fluctuations. Our study compares BOLD-fMRI and ADC-fMRI FC in GM and WM, showing that ADC-fMRI mirrors BOLD-fMRI connectivity in GM, while capturing more robust FC in WM. ADC-fMRI displays higher average clustering and average node strength in WM, and higher inter-subject similarity, compared to BOLD. Taken together, this suggests that ADC-fMRI is a reliable tool for exploring FC that incorporates gray and white matter nodes in a novel way.

neuroscience↗

bioRxiv2023

The molecular and cellular anatomy of a fetal programming defect: the impact of low protein diet on the developing kidney.

BackgroundLow nephron number has a direct impact on the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood. MethodsWe conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development to affect nephron number. ResultsSingle cell analysis at E14.5 and P0 revealed differences in the expression of genes and pathways involved in metabolism, cell cycle, epigenetic regulators and reciprocal inductive signals in most cell types analyzed, yielding imbalances and shifts in cellular energy production and cellular trajectories. In the nephron progenitor cells, LPD impeded cellular commitment and differentiation towards pre-tubular and renal vesicle structures. Confocal microscopy revealed a reduction in the number of pre-tubular aggregates and proliferation in nephron progenitor cells. We also found changes in branching morphogenesis, with a reduction in cell proliferation in the ureteric tips as well as reduced tip and tip parent lengths by optical projection tomography which causes patterning defects. ConclusionsThis unique profiling demonstrates how a fetal programming defect leads to low nephron endowment which is intricately linked to changes in both branching morphogenesis and the commitment of nephron progenitor cells. The commitment of progenitor cells is pivotal for nephron formation and is significantly influenced by nutritional factors, with a low protein diet driving alterations in this program which directly results in a reduced nephron endowment. Significance StatementWhile a mothers diet can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal developmental programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.

developmental biology↗

bioRxiv2023

Potent immunogenicity and protective efficacy of a multi-pathogen vaccination targeting Zaire ebolavirus, Sudan ebolavirus, Marburg and Lassa viruses

Viral haemorrhagic fevers (VHF) pose a significant threat to human health. In recent years, VHF outbreaks caused by Ebola, Marburg and Lassa viruses have caused substantial morbidity and mortality in West and Central Africa. In 2022, an Ebola disease outbreak in Uganda caused by Sudan ebolavirus resulted in 164 cases with 55 deaths. In February 2023, a Marburg disease outbreak was confirmed in Equatorial Guinea resulting in 15 confirmed and 23 suspected cases to date, with a second outbreak occurring concurrently in Tanzania. There are no clearly defined correlates of protection against these VHF, impeding targeted subunit vaccine development. Any vaccine developed should therefore induce strong and preferably long-lasting humoral and cellular immunity against these viruses. Ideally this immunity should also cross-protect against viral variants, which are known to circulate in animal reservoirs and cause human disease. We have utilized two viral vectored vaccine platforms, an adenovirus (ChAdOx1) and Modified Vaccinia Ankara (MVA), to develop a multi-pathogen vaccine regime against three filoviruses (Zaire ebolavirus, Sudan ebolavirus, Marburg) and an arenavirus (Lassa). These platform technologies have consistently demonstrated the capability to induce robust cellular and humoral antigen-specific immunity in humans, most recently in the rollout of the licensed ChAdOx1-nCoV19 /AZD1222. Here, we show that our multi-pathogen vaccines elicit strong cellular and humoral immunity, induce a diverse range of chemokines and cytokines, and most importantly, confers protection after lethal Zaire ebolavirus, Sudan ebolavirus and Marburg virus challenges in a small animal model. Author summaryOutbreaks caused by Ebola and Lassa viruses have made headlines worldwide in recent years. Most recently, in 2023 a Marburg virus outbreak has claimed tens of lives with a high case fatality rate. As yet, no licensed vaccine exists to protect against this and other viral haemorrhagic fevers. An ideal vaccine would induce long-lasting immunity to, and protection from, viruses causing viral haemorrhagic fevers. We developed vaccines which can target multiple strains of Ebolavirus, the closely related Marburg virus and Lassa virus. The geographical ranges of these viruses overlap in West and Central Africa. We used viral vector platform technologies to generate these vaccines; ChAdOx1 has now been administered worldwide as part of COVID-19 vaccine rollouts, and MVA has been used in numerous clinical trials thus far. We found that both long lasting, antigen specific T cell and antibody responses were induced after vaccination. Lastly, we demonstrated these vaccines could protect small animals against challenge with Zaire ebolavirus, Sudan ebolavirus and Marburg virus.

immunology↗

bioRxiv2023

ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface

Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.

microbiology↗

bioRxiv2023

The androgen receptor does not directly regulate the transcription of DNA damage response genes

The clinical success of combined androgen deprivation therapy (ADT) and radiation therapy (RT) in prostate cancer (PCa) created interest in understanding the mechanistic links between androgen receptor (AR) signaling and the DNA damage response (DDR). Convergent data have led to a model where AR both regulates, and is regulated by, the DDR. Integral to this model is that the AR regulates the transcription of DDR genes both at steady state and in response to ionizing radiation (IR). In this study, we sought to determine which immediate transcriptional changes are induced by IR in an AR-dependent manner. Using PRO-seq to quantify changes in nascent RNA transcription in response to IR, the AR antagonist enzalutamide, or the combination of the two, we find that enzalutamide treatment significantly decreased expression of canonical AR target genes but had no effect on DDR gene sets in PCa cells. Surprisingly, we also found that the AR is not a primary regulator of DDR genes either in response to IR or at steady state in asynchronously growing PCa cells. Our data indicate that the clinical benefit of ADT and RT is not due to the direct regulation of DDR gene transcription by AR.

cancer biology↗

bioRxiv2021

A Comparison of Persistence of SARS-CoV-2 Variants on Stainless Steel

The survival of newer variants of SARS-CoV-2 on a representative surface has been compared to the established UK circulating isolate to determine whether enhanced environmental stability could play a part in their increased transmissibility. Stainless-steel coupons were inoculated with liquid cultures of the three variants, with coupons recovered over seven days and processed for recoverable viable virus using plaque assay. After drying, there was no significant difference in inactivation rates between variants. Indicating there is no increased environmental persistence from the new variants.

microbiology↗

bioRxiv2021

Persistence of SARS-CoV-2 virus and viral RNA on hydrophobic and hydrophilic surfaces and investigating contamination concentration

The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used RT-PCR analysis to detect SARS-CoV-2 RNA on surfaces but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered, and whether virus viability is concentration dependant. Viable virus persisted for the longest time on surgical mask material and stainless steel with a 99.9% reduction in viability by 124 and 113 hours respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 hours. Viability on cotton was reduced second fastest, with 99.9% reduction in 72 hours. RNA on all the surfaces exhibited a one log reduction in genome copy recovery over 21 days. The findings show that SARS-CoV-2 is most stable on non-porous hydrophobic surfaces. RNA is highly stable when dried on surfaces with only one log reduction in recovery over three weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within two days. Therefore, when RNA is detected on surfaces it does not directly indicate presence of viable virus even at high CT values. ImportanceThis study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface. Unless sampled immediately after contamination it is difficult to align RNA copy numbers to quantity of viable virus on a surface.

microbiology↗

bioRxiv2020

An Age-Specific Atlas for Delineation of White Matter Pathways in Children Aged 6-8 Years

Diffusion MRI allows non-invasive assessment of white matter maturation in typical development and of white matter damage due to brain injury or pathology. Probabilistic white matter atlases provide delineation of white matter tracts, allowing diffusion metrics to be measured in specific white matter pathways. However, given the known age-dependency of developmental change in white matter it may not be optimal to use an adult template when assessing data acquired from children. This study develops an age-specific probabilistic white matter atlas for delineation of 12 major white matter tracts in children aged 6-8 years. By comparing to subject-specific tract tracing in two validation cohorts, we demonstrate that this age-specific atlas gives better overall performance than simply registering to the Johns Hopkins University adult white matter template. Specifically, when normalising diffusion data acquired from children to an adult template, estimates of fractional anisotropy (FA) values for corticospinal tract, uncinate fasciculus, forceps minor, cingulate gyrus part of the cingulum and anterior thalamic radiation were all less accurate than those obtained when using an age-specific atlas, potentially leading to false negatives when performing group comparisons. We then applied the newly developed atlas to compare FA between children treated with therapeutic hypothermia for neonatal encephalopathy and age-matched controls, which revealed significant reductions in the fornix, the left superior longitudinal fasciculus, and both the hippocampal and cingulum parts of the left cingulate gyrus. To our knowledge, this is the first publicly available probabilistic atlas of white matter tracts for this age group.

neuroscience↗

bioRxiv2020

ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in December 20191,2 and is responsible for the COVID-19 pandemic3. Vaccines are an essential countermeasure urgently needed to control the pandemic4. Here, we show that the adenovirus-vectored vaccine ChAdOx1 nCoV-19, encoding the spike protein of SARS-CoV-2, is immunogenic in mice, eliciting a robust humoral and cell-mediated response. This response was not Th2 dominated, as demonstrated by IgG subclass and cytokine expression profiling. A single vaccination with ChAdOx1 nCoV-19 induced a humoral and cellular immune response in rhesus macaques. We observed a significantly reduced viral load in bronchoalveolar lavage fluid and respiratory tract tissue of vaccinated animals challenged with SARS-CoV-2 compared with control animals, and no pneumonia was observed in vaccinated rhesus macaques. Importantly, no evidence of immune-enhanced disease following viral challenge in vaccinated animals was observed. ChAdOx1 nCoV-19 is currently under investigation in a phase I clinical trial. Safety, immunogenicity and efficacy against symptomatic PCR-positive COVID-19 disease will now be assessed in randomised controlled human clinical trials.

microbiology↗

bioRxiv2020

Biomechanical Regulation of Breast Cancer Metastasis and Progression

Physical activity has been consistently linked to decreased incidence of breast cancer and a substantial increase in the length of survival of patients with breast cancer. However, the understanding of how applied physical forces directly regulate breast cancer remains limited. We investigated the role of mechanical forces in altering the chemoresistance, proliferation and metastasis of breast cancer cells. We found that applied mechanical tension can dramatically alter gene expression in breast cancer cells, leading to decreased proliferation, increased resistance to chemotherapeutic treatment and enhanced adhesion to inflamed endothelial cells and collagen I under fluidic shear stress. A mechanistic analysis of the pathways involved in these effects supported a complex signaling network that included Abl1, Lck, Jak2 and PI3K to regulate pro-survival signaling and enhancement of adhesion under flow. Studies using mouse xenograft models demonstrated reduced proliferation of breast cancer cells with orthotopic implantation and increased metastasis to the skull when the cancer cells were treated with mechanical load. Using high throughput mechanobiological screens we identified pathways that could be targeted to reduce the effects of load on metastasis and found that the effects of mechanical load on bone colonization could be reduced through treatment with a PI3K{gamma} inhibitor.

cancer biology↗

bioRxiv2019

The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology

Tauopathies are neurodegenerative diseases that affect millions of people worldwide including those with Alzheimers disease. While many efforts have focused on understanding the role of tau protein in neurodegeneration, there has been little done to systematically analyze and study the structures within taus encoding RNA and their connection to disease pathology. Knowledge of RNA structure can provide insights into disease mechanisms and how to affect protein production for therapeutic benefit. Using computational methods based on thermodynamic stability and evolutionary conservation, we identified structures throughout the tau pre-mRNA, especially at exon-intron junctions and within the 5' and 3' untranslated regions (UTRs). In particular, structures were identified at twenty exon-intron junctions. The 5' UTR contains one structured region, which lies within a known internal ribosome entry site. The 3' UTR contains eight structured regions, including one that contains a polyadenylation signal. A series of functional experiments were carried out to assess the effects of mutations associated with mis-regulation of alternative splicing of exon 10 and to identify regions of the 3' UTR that contain cis-regulatory elements. These studies defined novel structural regions within the mRNA that affect stability and pre-mRNA splicing and may lead to new therapeutic targets for treating tau-associated diseases.

bioinformatics↗

bioRxiv2017

Network analysis of mass spectrometry imaging data from colorectal cancer identifies key metabolites common to metastatic development.

A deeper understanding of inter-tumor and intra-tumor heterogeneity is a critical factor for the advancement of next generation strategies against cancer. The heterogeneous morphology exhibited by solid tumors is mirrored by their metabolic heterogeneity. Defining the basic biological mechanisms that underlie tumor cell variability will be fundamental to the development of personalized cancer treatments. Variability in the molecular signatures found in local regions of cancer tissues can be captured through an untargeted analysis of their metabolic constituents. Here we demonstrate that DESI mass spectrometry imaging (MSI) combined with network analysis can provide detailed insight into the metabolic heterogeneity of colorectal cancer (CRC). We show that network modules capture signatures which differentiate tumor metabolism in the core and in the surrounding region. Moreover, module preservation analysis of network modules between patients with and without metastatic recurrence explains the inter-subject metabolic differences associated with diverse clinical outcomes such as metastatic recurrence.\n\nSignificanceNetwork analysis of DESI-MSI data from CRC human tissue reveals clinically relevant co-expression ion patterns associated with metastatic susceptibility. This delineates a more complex picture of tumor heterogeneity than conventional hard segmentation algorithms. Using tissue sections from central regions and at a distance from the tumor center, ion co-expression patterns reveal common features among patients who developed metastases (up of > 5 years) not preserved in patients who did not develop metastases. This offers insight into the nature of the complex molecular interactions associated with cancer recurrence. Presently, predicting CRC relapse is challenging, and histopathologically like-for-like cancers frequently manifest widely varying metastatic tendencies. Thus, the methodology introduced here more robustly defines the risk of metastases based on tumor biochemical heterogeneity.\n\nAuthor contributionsP.I., Z.T., R.C.G.: designed the study, developed the workflow, analyzed the data, interpreted the results, wrote the paper; N.S. collected the MS, performed the H...E staining, wrote the paper; L.D.: interpreted the results, wrote the paper; A.M.: collected the MS; A.S.: histological assessment; L.P.: collected the tissue specimens and clinical metadata; A.D.: collected the MS; H.K.: performed the H...E staining; R.M.: collected the tissue specimens and clinical metadata. R.G.: histological assessment; J.K.N: designed the study, edited the paper.

cancer biology↗