Biology subjects

Stephanou, A.

Publications and source records attributed to Stephanou, A..

11 recordsLinked to original sources

bioRxiv2026

MHC-I downregulation is associated with immune-evasion in breast cancers with heterogeneous activation of epithelial-mesenchymal plasticity

Cancer cell-intrinsic epithelial-mesenchymal plasticity (EMP) drives carcinoma progression through increased intratumoral heterogeneity and an immunosuppressive tumor microenvironment (TME). Specifically, EMP activation in murine mammary carcinomas results in exclusion of CD8+ T-cells and recruitment of immunosuppressive M2-like macrophages, resulting in resistance to anti-CTLA-4 immune checkpoint blockade (ICB) therapy. Notably, mixed E-qM tumors in which only 10% of cancer cells have activated EMP are resistant anti-CTLA-4 immune checkpoint blockade (ICB) therapy. This suggests that minority fractions of qM cancer cells could, in theory, exert immunosuppressive effects on surrounding E cancer and immune cells to shield the entire heterogeneous tumor against immune attack. However, this has not yet been demonstrated. Here, we use spatial transcriptomic profiling to demonstrate that mixed E-qM tumors assemble immunosuppressive cellular niches. Furthermore, E cancer cells within mixed E-qM tumors experience long-term MHC-I downregulation leading to resistance to elimination by CD8+ T-cells. This is associated with the exclusion of T-cells from the tumor core and a dysfunctional phenotype in the remaining CD8+ T-cells. qM cancer cells recruit immunosuppressive M2-like macrophages to mixed tumors, which further downregulates E cancer cell-intrinsic MHC-I levels. Finally, we demonstrate that human triple-negative breast cancers with increased EMP activation exclude T-cells from the tumor core. Together, these findings reveal that qM cancer cells modulate the immune infiltrate of mixed tumors, promoting MHC-I downregulation and evasion of immunological control by CD8+ T-cells.

cancer biology↗

bioRxiv2026

Spatial analysis of canine glioma reveals heterogeneous intratumoral niches characterized by distinct patterns of cell plasticity and immunosuppression

Glioma is a fatal tumor of the central nervous system (CNS) with limited therapeutic options. While immune checkpoint inhibitors (ICI) are gaining traction as a novel alternative treatment for human gliomas (HG), clinical trials have yielded mixed results. A proposed major barrier to ICI therapy in cancer is epithelial-mesenchymal plasticity (EMP)-driven intratumoral immunosuppression. However, studying this mechanism is difficult in ex vivo and murine models, which fail to adequately replicate cancer cell-immune cell interactions. Formalin-fixed, paraffin-embedded (FFPE) patient-derived canine glioma (CG) samples represent an attractive alternative approach. Although mechanistic perturbation is unachievable in archival FFPE tissues, CG is notoriously 'cold' immunologically and is actively being investigated as a comparative model of HG. Moreover, the immune microenvironment in CG advantageously develops under exposure to environmental conditions directly shared with humans. Yet, it remains unknown whether CGs harbor heterogeneous niches combining varied plastic phenotypes and immunosuppressive populations. Defining the immune landscape of CG in relation to EMP is therefore critical to determining its potential as a comparative model for immunotherapy response in HG. By combining standard histology, immunohistochemistry, and tissue microarray-based spatial transcriptomics, we analyzed 30 low-grade (LGr) and high-grade (HGr) FFPE CG cases (n=10 cases/subtype): astrocytomas (4xLGr, 6xHGr), oligodendrogliomas (1xLGr, 9xHGr), and undefined gliomas (3LxGr, 7xHGr). All CG subtypes harbored heterogeneous immunosuppressive niches associated with varied EMP phenotypes. Astrocytomas exhibited a more-mesenchymal identity correlated with recruitment of tumor-associated macrophages (TAMs). Oligodendrogliomas displayed a strong stem-like signature and uniquely upregulated a FOXP3/LAG3/SNAI1 triad, positioned directly at the interface of immunosuppression and EMP. Across all CG subtypes, a more-mesenchymal phenotype co-occurred with TAMs, and intratumoral T-cell dysfunction and exhaustion. We conclude that CG replicates the EMP-immunosuppression heterogeneity seen in humans. Importantly, our results emphasize the existence of subtype-specific mechanisms of EMP and immune evasion, underscoring the major relevance of subtyping for translational applications.

cancer biology↗

bioRxiv2026

Mechanics-Driven Emergence of Mesenchymal Migration Features

Cell migration plays a central role in numerous physiological and pathological processes and emerges from the coordinated interplay between intracellular force generation, adhesion dynamics, and mechanical interactions with the environment. A minimal, mechanistically grounded understanding of these processes is required to disentangle the respective contributions of cell-intrinsic and environmental cues. Here, a two-dimensional in silico cell motility model is introduced to describe mesenchymal migration driven by intracellular traction forces generated within actin-rich protrusions anchored to a substrate. The model explicitly accounts for adhesion nucleation, maturation, force buildup and rupture, and relies on a small set of physically interpretable parameters. A systematic mechanical analysis identifies parameter regimes that permit effective cell translocation and delineates conditions leading to stalled or mobile cells. Within motile regimes, the model reproduces a broad spectrum of cell morphologies and migratory behaviours. In particular, cell trajectories exhibit the statistical features of a persistent random walk, with a crossover from ballistic to diffusive motion that arises solely from adhesion dynamics and force balance, without imposing polarization or directional bias. Cell morphology is shown to strongly regulate migration speed, persistence, and pausing behaviour. Altogether, this model provides a minimal reference framework for cell migration on non-deformable substrates and establishes a baseline for future studies of mechanically driven guidance. By construction, it is well suited for extension to deformable fibrous substrates, where cell-induced matrix remodeling and stiffness feedback are expected to bias migration and regulate cell encounters relevant to tissue morphogenesis and anastomosis.

biophysics↗

bioRxiv2026

Neuroepithelial reprogramming and ERBB vulnerability in canine acanthomatous ameloblastoma

Canine acanthomatous ameloblastoma (CAA) is a locally invasive benign oral neoplasm that is difficult to distinguish from canine oral squamous cell carcinoma (COSCC) due to overlapping clinical, radiologic, and histologic features. Although both tumors exhibit MAPK pathway activation, their mutational landscapes are distinct. Furthermore, previous studies using bulk RNA sequencing (RNA-seq) have demonstrated pronounced differences in programs related to, among others, hypoxia, PI3K-AKT signaling, and cell proliferation. However, these bulk studies lacked the resolution to elucidate the cellular heterogeneity of CAA relative to COSCC. We therefore performed single-nucleus RNA-seq to define the cellular gene expression landscape of CAA, COSCC, and healthy gingiva. Across [~]205,000 nuclei, we identified major epithelial, immune, endothelial, and mesenchymal populations, as well as two epithelial subtypes uniquely enriched in CAA. The CAA-specific keratinocytes exhibited a neuronal-like expression program defined by synaptic regulators, KRAS-associated signaling pathways, and markedly elevated expression of PEG3, ERBB4, GABRB1, MAGI2, and CASK. These findings were validated by bulk RNA-seq, qPCR, and immunohistochemistry, which demonstrated strong nuclear localization of PEG3 exclusively in CAA epithelium. A kinase inhibitor screen independently identified ERBB4 as a candidate therapeutic vulnerability, and pharmacologic inhibition with neratinib was effective. Together, these findings reveal a previously unrecognized neuroepithelial cell state that defines CAA, distinguishes it from COSCC, and reveals unique diagnostic and therapeutic signaling dependencies. Given the molecular and histopathologic parallels between CAA and human ameloblastoma, these data further position CAA as a naturally occurring comparative model for studying ameloblastoma biology and therapeutic vulnerabilities. Significance StatementCanine acanthomatous ameloblastoma (CAA) is a common locally aggressive oral neoplasm often misdiagnosed as canine oral squamous cell carcinoma (COSCC) due to overlapping clinical, radiologic, and histologic features. Using single-nucleus RNA sequencing of CAA, COSCC, and healthy gingiva, we resolve the microenvironment of these tumors and detect two epithelial subpopulations unique to CAA. These CAA-specific keratinocytes exhibit a neuroepithelial-like signature characterized by synaptic regulators, KRAS-associated signaling, and elevated PEG3 and ERBB4 expression. Functional kinase inhibitor screening of patient derived microtumor slices independently converged on ERBB4 as an effective therapeutic target. Together, our study provides a molecular framework for distinguishing CAA from COSCC and establishes a comparative oncology model with translational relevance to human ameloblastoma and other ERBB-driven epithelial neoplasms.

cancer biology↗

bioRxiv2025

Spatiotemporal Atlas of Heart Development Reveals Blood-Flow-Dependent Cellular, Structural, Metabolic, and Spatial Remodeling

Embryonic heart development depends on coordinated interactions between cellular programs, molecular signaling, and biomechanical forces, yet how mechanical cues shape cellular and molecular pathways remains incompletely understood. We perturbed cardiac blood flow by partial left or right atrial ligation (LAL/RAL) in chick embryos, generating chamber-specific hemodynamic gain- or loss-of-function states. Using single-cell and unbiased, high-resolution spatial transcriptomics, we generated a spatiotemporal atlas of flow-dependent tissue development, enabling systematic investigations of bidirectional interactions between blood-flow mechanics and tissue development. Spatially resolved analyses recapitulated key features of normal morphogenesis including regional maturation and the cellular neighborhood. We further revealed flow-specific remodeling across molecular, cellular, and architectural levels. Altered flow induced LOX-expressing cardiomyocyte and endocardial states, disrupted ventricular layer organization, and delayed maturation, alongside transient metabolic and ion-transport adaptations. Together, these findings define how redistributed blood flow reshapes developing cardiac tissues and provide a framework for studying flow-dependent remodeling in morphogenesis and malformation.

developmental biology↗

bioRxiv2025

Single-cell transcriptomics reveals targeted modulation of inflammatory repertoire by SOCE blockers

Store-operated calcium entry (SOCE) plays a critical role in regulating intracellular calcium signaling and is essential for immune cell functions. SOCE blockade with pyrazole derivative BTP2 has been explored as an anti-inflammatory strategy in preclinical models and Zegocractin (CM4620) is being investigated in Phase 2 clinical trials as an immunoregulatory agent. However, the mode of action and differential effects of SOCE blockade on diverse immune cell types remain largely unknown, limiting the precision of current therapeutic applications. Here, we used multiplexed single-cell RNA sequencing to investigate the effects of two prototypic SOCE blockers, BTP2 and CM4620, on polyclonally-stimulated, normal human peripheral blood mononuclear cells (PBMCs). The data revealed that SOCE blockade suppresses the expression of cytotoxicity-associated genes in CD8{square} effector T cells and natural killer (NK) cells, restoring them to levels comparable to those in unstimulated cells. Strikingly, SOCE blockade preserved activation-induced expression of anti-inflammatory genes in CD4{square} regulatory T cells, maintaining their tolerance phenotype even after SOCE blockade. These findings suggest that SOCE blockers modulate immune responses with greater selectivity than conventional immunosuppressants by reducing cytotoxicity while preserving tolerance-associated pathways, highlighting their potential for managing immune-mediated conditions, including organ transplantation. KEY POINTSO_LISOCE blockers BTP2 and CM4620 reduce cytotoxic gene expression in CD8{square} T cells and NK cells. C_LIO_LISOCE blockade preserves anti-inflammatory programs in CD4{square} Tregs, supporting selective immune modulation. C_LI

immunology↗

bioRxiv2024

Direct and indirect effects of spliceosome disruption compromise gene regulation by Nonsense-Mediated mRNA Decay

Pre-mRNA splicing, carried out in the nucleus by a large ribonucleoprotein machine known as the spliceosome, is functionally and physically coupled to the mRNA surveillance pathway in the cytoplasm called nonsense mediated mRNA decay (NMD). The NMD pathway monitors for premature translation termination signals, which can result from alternative splicing, by relying on the exon junction complex (EJC) deposited on exon-exon junctions by the spliceosome. Recently, multiple genetic screens in human cell lines have identified numerous spliceosome components as putative NMD factors. Using publicly available RNA-seq datasets from K562 and HepG2 cells depleted of 18 different spliceosome components, we find that natural NMD targeted mRNA isoforms are upregulated when members of the catalytic spliceosome are reduced. While some of this increase could be due to widespread pleiotropic effects of spliceosome dysfunction (e.g., reduced expression of NMD factors due to mis-splicing of their mRNAs), we identify that AQR, SF3B1, SF3B4 and CDC40 may have a more direct role in NMD. We also test the hypothesis that increased production of novel NMD substrates may overwhelm the pathway to find a direct correlation between the amount of novel NMD substrates detected and the degree of NMD inhibition observed. Finally, similar transcriptome alterations and NMD substrate upregulation are also observed in cells treated with spliceosome inhibitors and in cells derived from retinitis pigmentosa patients with mutations in PRPF8 and PRPF31. Overall, our results show that regardless of the cause, spliceosome disruption upregulates a broad set of NMD targets, which could contribute to cellular dysfunction in spliceosomopathies. AUTHOR SUMMARYDuring gene expression, a complex cellular machine known as spliceosome removes extraneous non-coding sequences from precursor RNAs to produce messenger RNA (mRNA) with a contiguous code for protein sequence. To guard against splicing errors that may interrupt protein coding sequence, splicing is linked to a mRNA surveillance pathway known as nonsense mediated mRNA decay (NMD). In this work, we follow up on recent findings from multiple genetic screens that several spliceosome components are necessary for efficient NMD. Our analysis of transcriptomes of lymphoblast K562 cells depleted of 18 spliceosome factors show that NMD based regulation is compromised in cells lacking catalytic spliceosome proteins. Four of these spliceosome proteins may have a direct effect on NMD even though spliceosome disruption in general also causes other changes in gene expression that indirectly affect NMD. Our results suggest that defective NMD based regulation contributes to cellular dysfunction in spliceosomopathies, a collection of human genetic disorders caused by mutations in spliceosome factors.

molecular biology↗

bioRxiv2024

Single-cell multi-omic analysis of fibrolamellar carcinoma reveals rewired cell-to-cell communication patterns and unique vulnerabilities

Fibrolamellar carcinoma (FLC) is a rare malignancy disproportionately affecting adolescents and young adults with no standard of care. FLC is characterized by thick stroma, which has long suggested an important role of the tumor microenvironment. Over the past decade, several studies have revealed aberrant markers and pathways in FLC. However, a significant drawback of these efforts is that they were conducted on bulk tumor samples. Consequently, identities and roles of distinct cell types within the tumor milieu, and the patterns of intercellular communication, have yet to be explored. In this study we unveil cell-type specific gene signatures, transcription factor networks, and super-enhancers in FLC using a multi-omics strategy that leverages both single-nucleus ATAC-seq and single-nucleus RNA-seq. We also infer completely rewired cell-to-cell communication patterns in FLC including signaling mediated by SPP1-CD44, MIF-ACKR3, GDF15-TGFBR2, and FGF7-FGFR. Finally, we validate findings with loss-of-function studies in several models including patient tissue slices, identifying vulnerabilities that merit further investigation as candidate therapeutic targets in FLC.

cancer biology↗

bioRxiv2024

Spatial organization of mediated-macrophage chemoprotective niches in solid tumors: A mathematical analysis

Acquired resistance is one of the major causes of failure of standard therapies in cancer patients. Chemotherapeutic agents are still widely used and the understanding of the mechanisms leading to secondary resistance to these molecules are still puzzling. Recently, the role of the tumor immune microenvironment has been recognized. Among the cells potentially involved, macrophages seem to be the perfect culprits. In a previous work, we have shown that hypoxic macrophages are able to provide strong protection against 5-fluorouracil, a first-line chemotherapeutic agent in digestive cancers. In the present work, we use mathematical modeling to explore the spatiotemporal aspects of the treatment-induced organization of the tumor environment. Based on analytical and numerical analysis, we propose that macrophage-driven protection against chemotherapy under treatment does not rely solely on biochemical degradation, but is enhanced by the emergence of spatially structured chemotherapeutic protective niches. This work paves the way for the development of new therapeutic strategies that rely on targeting the spatial organization of tumors as a way to control treatment resistance.

systems biology↗

bioRxiv2023

A computational model of the spatiotemporal adaptation of tumor cells metabolism in a growing spheroid

The Warburg effect, commonly depicted as an inherent metabolic trait of cancer in literature, is under intensive investigation to comprehend its origins. However, while the prolonged presence of excessive lactic acid production in tumors has been noted, it merely constitutes a fraction of the potential metabolic states cancer cells can adopt. This study aimed to elucidate the emergence of spatiotemporal diversity in tumor energy metabolism by expanding an existing model based on experimental facts. The resulting hybrid model integrates discrete formulations for individual cells and their processes, along with continuous elements for metabolism and the diffusion of crucial environmental substrates like oxygen, glucose, lactate, and the often underestimated acidity. This model enables simulation of a tumor spheroid, a standard experimental model, composed of numerous cells which can have distinct traits. By subjecting the spheroid to alterations of the environment such as cyclic hypoxia, acid shocks, or glucose deprivation, novel insights into metabolic regulation were obtained. The findings underscore the significance of the pyruvate-lactate interaction in governing tumor metabolic routes. Integrating aciditys impact into the model, revealed its pivotal role in energy pathway regulation. Consequently, the conventional portrayal of a respiration/fermentation dichotomy proves inaccurate, as cells continuously and spatially adjust the ratio of these energy production modes, in contrast to abrupt, irreversible switches. Moreover, a cooperative cellular behavior akin to the reverse Warburg effect has emerged. This implies that the Warburg effect is not universally inherent to tumor metabolism, but a contextual, transient metabolic expression. Ultimately, the dynamic cellular-environment metabolic landscape influences cells survival under external conditions, with epigenetic regulations shaping their mobility potential within this landscape. While genetic mutations within tumor cells are undoubtedly present, this study shows they are not invariably essential for extreme metabolic modes or pathological characteristics to arise. Consequently, this research paves the way for innovative perspectives on metabolism, guiding tailored therapeutic strategies that consider not just patient-specific tissue attributes but also treat tumors as intricate ecosystems beyond their genetic diversity. Author SummaryFor years, scientists have been intrigued by the peculiar energy consumption patterns of cancer cells, such as the Warburg effect characterized by excessive lactic acid production. This study aimed to decipher the underlying reasons for the varying energy behaviors observed in different parts of tumors. Using a computational model, we simulated the collaborative dynamics of cells within tumors. The results revealed compelling insights. Two molecules, pyruvate and lactate, were identified as influential players in shaping energy utilization. Remarkably, the surrounding acidity was also found to exert a significant impact. Interestingly, tumor cells display a certain flexibility in their energy production strategies, adjusting according to prevailing conditions to maintain their survival and adaptability. Interestingly, cellular cooperation challenges the Warburg effect as an omnipresent phenomenon and reveals a transient nature. Our study underscores the significance of environmental influences, shedding light on the interplay between genetic modifications and the tumor environment in shaping cellular behavior. These findings hold promise for transforming cancer comprehension and devising treatments that tailor to both patients and the distinctive characteristics of their tumors.

cancer biology↗

bioRxiv2019

Why the tumor cell metabolism is not that abnormal

The cell energy metabolism is a multifactorial and evolving process that we address with a theoretical approach in order to decipher the functioning of the core system of the glycolysis-OXPHOS relationship. The model is based on some key experimental observations and well established facts. It emphasizes the role of lactate as a substrate, as well as the central role of pyruvate in the regulation of the metabolism. The simulations show how imposed environmental constraints and imposed energy requirements push the cell to adapt its metabolism to sustain its needs. The results highlight the cooperativeness of the two metabolic modes and allows to revisit the notions of metabolic switch and metabolic reprogramming. Our results thus tend to show that the Warburg effect is not an inherent characteristic of the tumor cell, but a spontaneous and transitory adaptation mechanism to a disturbed environment. This means that the tumor cell metabolism is not fundamentally different from that of a normal cell. This has implications on the way therapies are being considered. The quest to normalize the tumor acidity could be a good strategy. Author SummaryCancer cells metabolism focuses the interest of the cancer research community. Although this process is intensely studied experimentally, there exists very few theoretical models that tackle this issue. One main reason is the extraordinary complexity of the metabolism that involves many inter-related regulation networks which makes it illusory to recreate computationally this complexity. In this study we propose a simplified model of the metabolism which focuses on the interrelation of the three main energetic metabolites that are oxygen, glucose and lactate with the aim to better understand the dynamic of the core system of the glycolysis-OXPHOS relationship. However simple, the model highlights the main rules that allow the cell to dynamically adapt its metabolism to its changing environment. It moreover allows to address this impact at the tissue scale. Simulations performed in a spheroid exhibit non-trivial spatial heterogeneity of the energy metabolism. It further reveals that the metabolic features that are commonly assigned to cancer cells are not necessarily due to cell intrinsic abnormality. They can emerge spontaneously because of the disregulated over-acidic environment.

cell biology↗