Metabolic reprogramming is a hallmark of cancer. Plasma metabolomics offers a minimally invasive approach for identifying metabolic alterations that may provide insights into tumor progression. We aimed to characterize plasma metabolomic profiles in patients with oral squamous cell carcinoma (OSCC) and evaluate their clinical relevance. Plasma samples from 43 OSCC patients and 129 cancer-free controls, matched at a 1:3 ratio based on age, sex, and body mass index, were analyzed using gas chromatography-mass spectrometry (GC-MS). A random forest algorithm was applied to identify key metabolic features distinguishing OSCC from controls. The clinical significance of the top metabolites was assessed and validated in another OSCC cohort (n = 27). A total of 113 compounds were putatively annotated and analyzed based on relative abundances. A ten-feature panel demonstrated good classification performance (area under the curve = 0.87; Matthews correlation coefficient = 0.703). The ten features are maltose, glucose, xylulose, δ-gluconolactone, fructose, indoleacetic acid, α-hydroxybutyric acid, glutamic acid, cysteine, and the monoacylglyceride MG(18:1(9Z)/0:0/0:0), suggesting dysregulated carbohydrate metabolism and oxidative stress as the major plasma metabolomic alterations in OSCC. Notably, α-hydroxybutyric acid levels were elevated in patients with regional lymph node metastasis compared with those without. Our findings underscore the intricate interplay between altered glucose metabolism, redox imbalance, and OSCC. α-hydroxybutyric acid, a marker of oxidative stress and an indicator of insulin resistance, may be associated with metastatic progression.

Cancer stem cells (CSCs) represent a minor but highly adaptable subpopulation within tumors that drives long-term growth, metastasis, and therapy resistance. Their ability to survive and regenerate under metabolic and therapeutic stress relies on a unique integration of energy flexibility, redox balance, and proteostatic programs. While bulk tumor cells typically favor aerobic glycolysis and high protein turnover, CSCs often exhibit elevated mitochondrial activity, fatty acid oxidation, and selective suppression of proteasome function. These metabolic features support quiescence, stress tolerance, and self-renewal. Beyond energy production, metabolic intermediates such as acetyl-CoA, succinate, and lactate serve as epigenetic cofactors, linking nutrient availability to chromatin remodeling and transcriptional plasticity. Reactive oxygen species and antioxidant responses further tune this balance, shaping the transition between glycolytic and oxidative CSC states. These intrinsic programs are continuously influenced by the tumor microenvironment, where hypoxia, cytokine-driven signaling, and metabolic coupling with stromal and immune cells modulate CSC metabolism and reinforce stemness. Despite rapid progress, major conceptual and methodological gaps still limit our understanding of CSC metabolism and this review highlights these unresolved issues and further outline key contextual factors—including tumor-intrinsic, microenvironmental, systemic, and metastatic cues—that shape CSC metabolism and help explain the divergent observations reported across studies. Understanding this network will be essential for designing combinatorial therapies that target CSC metabolism while accounting for their heterogeneity and plasticity.

CDK11 is a cyclin-dependent kinase with a role in transcription and RNA splicing and represents a potential target for cancer treatment. We show that blocking CDK11 activity with the OTS964 inhibitor causes p53 stabilisation through MDM2 downregulation. Under these conditions, p53 activates the expression of its downstream effector CDKN1A (p21WAF1), produced in two isoforms, the canonical p21C and the recently described p21L. We compared the ability of both isoforms to block proliferation and showed that p21L partially lost its inhibitory potential, likely due to the missing cyclin-binding Cy2 and PCNA-interacting motifs and its cytoplasmic localisation. We identified the epitopes of four p21WAF1 antibodies using phage display to determine isoform specificity. Moreover, we show that the trigger for p21L induction is inhibition of the spliceosomal protein SF3B1. CDK11 activates SF3B1 by phosphorylation, and inhibition of either SF3B1 or CDK11 induces p21L. We discovered an isoform similar to human p21L in murine cells, suggesting evolutionary conservation of CDKN1A alternative splicing. Our results uncover an unknown link between RNA splicing and proliferation control involving a novel isoform of a key cell cycle inhibitor.

Squamous cell carcinoma of the oral tongue (SCCOT) represents an aggressive malignancy characterized by high metastatic potential and significant heterogeneity in its tumor microenvironment. The tumor‐stroma ratio (TSR) has emerged as a prognostic biomarker, with higher stromal content frequently correlating with worse survival outcomes. Traditional approaches using the standard 50% TSR cutoff may not be optimal for SCCOT, and visual TSR estimation introduces variability during TSR region annotation. This study aimed to develop and validate a dedicated TSR estimation model for SCCOT by incorporating representative TSR regions from the invasive tumor front of whole slide images and to determine the optimal TSR threshold for prognostic stratification. Using hematoxylin and eosin‐stained images from The Cancer Genome Atlas as a discovery cohort and whole slide images from Norrland's University Hospital Umea, Sweden (NUS) as a validation cohort, we developed a computational model to estimate TSR. The model demonstrated a high correlation with pathologist‐based TSR estimation in both discovery ( R = 0.848, p < 0.01) and validation ( R = 0.783, p < 0.01) cohorts. The optimal 55% cutoff identified by the model improved prognostic accuracy over the traditional 50% threshold, with patients having high stroma within the tumor invasive front showing worse overall (log‐rank p = 0.006) and disease‐specific (log‐rank p = 0.016) survival. Our computational TSR model for SCCOT demonstrates that automated TSR estimation enhances prognostic accuracy at an optimal cutoff of 55%, contributing to more precise risk stratification and potentially enabling personalized treatment strategies in SCCOT management.

BACKGROUND:The incidence of squamous cell carcinoma of the oral tongue (SCCOT) among young adults is increasing in several regions of the world. Age-dependent differences in the biology of SCCOT have been suspected. METHODS:We used the Olink Explore 3072 high-throughput platform to comprehensively quantify plasma proteins in 24 young (≤ 40 years of age) and 50 old (> 50 years of age) individuals. Eight young and 20 old individuals were diagnosed with SCCOT, four young and nine old individuals with SCC at other oral subsites (SCCOO), and the remaining 12 young and 21 old individuals were healthy controls. Dimension reduction analysis, differential expression analysis, and functional enrichment analysis were performed to characterize young patient-specific biological signatures. RESULTS:Plasma levels of 2923 proteins were obtained. Principal component analysis indicated age-related expression patterns. Comparing young patients to young controls/old patients/old controls, differential abundance analysis showed that increases in protein levels of Peroxiredoxin 2 (PRDX2) and C-C motif chemokine ligand 26 (CCL26) and a decrease in Kallikrein related peptidase 4 (KLK4) were young patient-specific. Reactome pathway enrichment analysis identified "Cellular response to chemical stress," "Detoxification of reactive oxygen species" and "Cellular responses to stimuli" as the top altered pathways in young patients with SCCOT. CONCLUSIONS:Abnormal cellular stress and aberrant immune regulation could thus be linked to cancer development in young patients. The unique plasma proteomic signature observed in young patients with SCCOT suggests that they constitute a specific group with distinct underlying pathophysiological processes.

TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

Locoregional recurrences and distant metastases are major problems for patients with squamous cell carcinoma of the head and neck (SCCHN). Because SCCHN is a heterogeneous group of tumours with varying characteristics, the present study concentrated on the subgroup of squamous cell carcinoma of the oral tongue (SCCOT) to investigate the use of machine learning approaches to predict the risk of recurrence from routine clinical data available at diagnosis. The approach also identified the most important parameters that identify and classify recurrence risk. A total of 66 patients with SCCOT were included. Clinical data available at diagnosis were analysed using statistical analysis and machine learning approaches. Tumour recurrence was associated with T stage (P=0.001), radiological neck metastasis (P=0.010) and diabetes (P=0.003). A machine learning model based on the random forest algorithm and with attendant explainability was used. Whilst patients with diabetes were overrepresented in the SCCOT cohort, diabetics had lower recurrence rates (P=0.015 after adjusting for age and other clinical features) and an improved 2-year survival (P=0.025) compared with non-diabetics. Clinical, radiological and histological data available at diagnosis were used to establish a prognostic model for patients with SCCOT. Using machine learning to predict recurrence produced a classification model with 71.2% accuracy. Notably, one of the findings of the feature importance rankings of the model was that diabetics exhibited less recurrence and improved survival compared with non-diabetics, even after accounting for the independent prognostic variables of tumour size and patient age at diagnosis. These data imply that the therapeutic manipulation of glucose levels used to treat diabetes may be useful for patients with SCCOT regardless of their diabetic status. Further studies are warranted to investigate the impact of diabetes in other SCCHN subtypes.

TP73 is a member of the TP53 gene family and produces N- and C-terminal protein isoforms through alternative promoters, alternative translation initiation and alternative splicing. Most notably, p73 protein isoforms may either contain a p53-like transactivation domain (TAp73 isoforms) or lack this domain (ΔTAp73 isoforms) and these variants have opposing or independent functions. To date, there is a lack of well-characterised isoform-specific p73 antibodies. Here, we produced polyclonal and monoclonal antibodies to N-terminal p73 variants and the C-terminal p73α isoform, the most common variant in human tissues. These reagents show that TAp73 is a marker of multiciliated epithelial cells, while ΔTAp73 is a marker of non-proliferative basal/reserve cells in squamous epithelium. We were unable to detect ΔNp73 variant proteins, in keeping with recent data that this is a minor form in human tissues. Most cervical squamous cell carcinomas (79%) express p73α, and the distribution of staining in basal cells correlated with lower tumour grade. TAp73 was found in 17% of these tumours, with a random distribution and no association with clinicopathological features. These data indicate roles for ΔTAp73 in maintaining a non-proliferative state of undifferentiated squamous epithelial cells and for TAp73 in the production of differentiated multiciliated cells.

The field cancerization concept indicates the presence of pre-cancerous changes in clinically normal tissue surrounding the tumor. In squamous cell carcinoma of the oral tongue (SCCOT) which is infrequently linked to human papillomavirus infection, we have previously reported that clinically normal tongue contralateral to tumor (NTCT) is molecularly abnormal. Here, combining our transcriptomic and genomic data, we aimed to investigate the contribution of molecular changes in NTCT to cancer development. Microarray gene expression data of 14 healthy controls, 23 NTCT and 29 SCCOT samples were investigated to characterize transcriptional profiles in NTCT. Whole exome sequencing and RNA-sequencing data of paired NTCT and tumor samples from 15 SCCOT patients were used to study correlation between copy number variation and differential gene expression. Using supervised multivariate partial least squares discriminant analysis, a total of 61 mRNAs that distinguish NTCT from healthy tongue were selected. Functional enrichment analysis of the 22 upregulated genes showed increased “positive regulation of nitrogen compound metabolic process” in NTCT. All 12 genes involved in this process have roles in apoptosis (anti- and/or pro-apoptotic). Compared to healthy controls, Zinc Finger Protein 395 (ZNF395), a pro-apoptotic tumor suppressor located on chromosome 8p, was the only gene showing increased mRNA level in NTCT whereas decreased in SCCOT. Given the frequent loss of chromosome 8p in SCCOT, the impact of ZNF395 copy number variation on gene expression was further examined, revealing a positive correlation between copy number and mRNA level (correlation coefficient = 0.572, p < 0.001). NTCT is susceptible to malignant transformation, where tissue homeostasis is maintained at least partly through regulation of apoptosis. Loss of the pro-apoptotic gene ZNF395 could thus initiate cancer development.

Stress responses play a vital role in cellular survival against environmental challenges, often exploited by cancer cells to proliferate, counteract genomic instability, and resist therapeutic stress. Heat shock factor protein 1 (HSF1), a central transcription factor in stress response pathways, exhibits markedly elevated activity in cancer. Despite extensive research into the transcriptional role of HSF1, the mechanisms underlying its activation remain elusive. Upon exposure to conditions that induce protein damage, monomeric HSF1 undergoes rapid conformational changes and assembles into trimers, a key step for DNA binding and transactivation of target genes. This study investigates the role of HSF1 as a sensor of proteotoxic stress conditions. Our findings reveal that purified HSF1 maintains a stable monomeric conformation independent of molecular chaperones in vitro. Moreover, while it is known that heat stress triggers HSF1 trimerization, a notable increase in trimerization and DNA binding was observed in the presence of protein-based crowders. Conditions inducing protein misfolding and increased protein crowding in cells directly trigger HSF1 trimerization. In contrast, proteosynthesis inhibition, by reducing denatured proteins in the cell, prevents HSF1 activation. Surprisingly, HSF1 remains activated under proteotoxic stress conditions even when bound to Hsp70 and Hsp90. This finding suggests that the negative feedback regulation between HSF1 and chaperones is not directly driven by their interaction but is realized indirectly through chaperone-mediated restoration of cytoplasmic proteostasis. In summary, our study suggests that HSF1 serves as a molecular crowding sensor, trimerizing to initiate protective responses that enhance chaperone activities to restore homeostasis.

The p53 family of proteins evolved from a common ancestor into three separate genes encoding proteins that act as transcription factors with distinct cellular roles. Isoforms of each member that lack specific regions or domains are suggested to result from alternative transcription start sites, alternative splicing or alternative translation initiation, and have the potential to exponentially increase the functional repertoire of each gene. However, evidence supporting the presence of individual protein variants at functional levels is often limited and is inferred by mRNA detection using highly sensitive amplification techniques. We provide a critical appraisal of the current evidence for the origins, expression, functions and regulation of p53-family isoforms. We conclude that despite the wealth of publications, several putative isoforms remain poorly established. Future research with improved technical approaches and the generation of isoform-specific protein detection reagents is required to establish the physiological relevance of p53-family isoforms in health and disease. In addition, our analyses suggest that p53-family variants evolved partly through convergent rather than divergent evolution from the ancestral gene.

BACKGROUND Interpretable machine learning (ML) for early detection of cancer has the potential to improve risk assessment and early intervention. METHODS Data from 261 proteins related to inflammation and/or tumor processes in 123 blood samples collected from healthy persons, but of whom a sub-group later developed squamous cell carcinoma of the oral tongue (SCCOT), were analyzed. Samples from people who developed SCCOT within less than 5 years were classified as tumor-to-be and all other samples as tumor-free. The optimal ML algorithm for feature selection was identified and feature importance computed by the SHapley Additive exPlanations (SHAP) method. Five popular ML algorithms (AdaBoost, Artificial neural networks [ANNs], Decision Tree [DT], eXtreme Gradient Boosting [XGBoost], and Support Vector Machine [SVM]) were applied to establish prediction models, and decisions of the optimal models were interpreted by SHAP. RESULTS Using the 22 selected features, the SVM prediction model showed the best performance (sensitivity = 0.867, specificity = 0.859, balanced accuracy = 0.863, area under the receiver operating characteristic curve [ROC-AUC] = 0.924). SHAP analysis revealed that the 22 features rendered varying person-specific impacts on model decision and the top three contributors to prediction were Interleukin 10 (IL10), TNF Receptor Associated Factor 2 (TRAF2), and Kallikrein Related Peptidase 12 (KLK12). CONCLUSION Using multidimensional plasma protein analysis and interpretable ML, we outline a systematic approach for early detection of SCCOT before the appearance of clinical signs.

Supplementary Figure 2 from Differential Contextual Responses of Normal Human Breast Epithelium to Ionizing Radiation in a Mouse Xenograft Model

Supplementary Figure 1 from Differential Contextual Responses of Normal Human Breast Epithelium to Ionizing Radiation in a Mouse Xenograft Model

Oral cancer is a paradigm of Slaughter's concept of field cancerization, where tumors are thought to originate within an area of cells containing genetic alterations that predispose to cancer development. The field size is unclear but may represent a large area of tissue, and the origin of mutations is also unclear. Here, we analyzed whole exome and transcriptome features in contralateral tumor-distal tongue (i.e. distant from the tumor, not tumor-adjacent) and corresponding tumor tissues of 15 patients with squamous cell carcinoma of the oral tongue. The number of point mutations ranged from 41 to 237 in tumors and from one to 78 in tumor-distal samples. Tumor-distal samples showed mainly clock-like (associated with aging) or tobacco smoking mutational signatures. Tumors additionally showed mutations that associate with cytidine deaminase AID/APOBEC enzyme activities or a UV-like signature. Importantly, no point mutations were shared between a tumor and the matched tumor-distal sample in any patient. TP53 was the most frequently mutated gene in tumors (67%), whereas a TP53 mutation was detected in only one tumor-distal sample, and this mutation was not shared with the matched tumor. Arm-level copy number variation (CNV) was found in 12 tumors, with loss of chromosome (Chr) 8p or gain of 8q being the most frequent events. Two tumor-distal samples showed a gain of Chr8, which was associated with increased expression of Chr8-located genes in these samples, although gene ontology did not show a role for these genes in oncogenic processes. In situ hybridization revealed a mixed pattern of Chr8 gain and neutral copy number in both tumor cells and adjacent nontumor epithelium in one patient. We conclude that distant field cancerization exists but does not present as tumor-related mutational events. The data are compatible with etiologic field effects, rather than classical monoclonal field cancerization theory. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The 2023 Annual Review Issue of The Journal of Pathology , Recent Advances in Pathology, contains 12 invited reviews on topics of current interest in pathology. This year, our subjects include immuno‐oncology and computational pathology approaches for diagnostic and research applications in human disease. Reviews on the tissue microenvironment include the effects of apoptotic cell‐derived exosomes, how understanding the tumour microenvironment predicts prognosis, and the growing appreciation of the diverse functions of fibroblast subtypes in health and disease. We also include up‐to‐date reviews of modern aspects of the molecular basis of malignancies, and our final review covers new knowledge of vascular and lymphatic regeneration in cardiac disease. All of the reviews contained in this issue are written by expert groups of authors selected to discuss the recent progress in their particular fields and all articles are freely available online ( https://pathsocjournals.onlinelibrary.wiley.com/journal/10969896 ). © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Breast cancers are a heterogeneous group of tumors classified according to their histological growth patterns and receptor expression characteristics. Intratumor heterogeneity also exists, with subpopulations of cells with different phenotypes found in individual cancers, including cells with stem or progenitor cell properties. At least two types of breast cancer stem cells (CSCs) exist, the epithelial and the basal/mesenchymal subtypes, although how these phenotypes are controlled is unknown. Delta Np63 is a basal cell marker and regulator of stem/progenitor cell activities in the normal mammary gland and is expressed in the basal-like CSC subpopulation in some estrogen receptor-positive (ER+) and/or human epidermal growth factor receptor 2-positive (HER2(+)) breast adenocarcinomas. Whilst p63 is known to directly impart CSC properties in luminal breast cancer cells, how p63 is regulated and induced in these cells is unknown. We initially confirmed the existence of a small subpopulation of Delta Np63(+) cells in lymph node metastases of ER+ human ductal adenocarcinomas, indicating together with previous reports that Delta Np63(+) tumor cells are present in approximately 40% of these metastases. Notably, Delta Np63(+) cells show a preferential location at the edge of tumor areas, suggesting possible regulation of Delta Np63 by the tumor microenvironment. Subsequently, we showed that the high levels of Delta Np63 in basal non-transformed MCF-10A mammary epithelial cells rely on insulin in their culture medium, whilst Delta Np63 levels are increased in MCF-7 ER+ luminaltype breast cancer cells treated with insulin or insulin-like growth factor 1 (IGF-1). Mechanistically, small molecule inhibitors and siRNA gene knockdown demonstrated that induction of Delta Np63 by IGF-1 requires PI3K, ERK1/2, and p38 MAPK activation, and acts through FOXO transcriptional inactivation. We also show that metformin inhibits Delta Np63 induction. These data reveal an IGF-mediated mechanism to control basal-type breast CSCs, with therapeutic implications to modify intratumor breast cancer cell heterogeneity and plasticity.

Supplementary Table 3 from Indirect Macrophage Responses to Ionizing Radiation: Implications for Genotype-Dependent Bystander Signaling

Supplementary Table 1 from Indirect Macrophage Responses to Ionizing Radiation: Implications for Genotype-Dependent Bystander Signaling