Heterogeneous Cell Research Articles

New Anti-Fibrotic Strategies for Keloids: Insights From Single-Cell Multi-Omics.

Keloids are complex pathological skin scars characterised by excessive growth of fibrous tissue and abnormal accumulation of extracellular matrix (ECM). Despite various treatment options available, the treatment of keloids remains a major clinical challenge due to high recurrence rates and inconsistent therapeutic outcomes. By collecting three keloid tissues and three normal skin samples and utilising single-cell RNA sequencing (scRNA-seq), we delved into the cellular heterogeneity and molecular mechanisms of keloids. Our study identified multiple fibroblast subpopulations within keloid tissue. Enrichment and cell-cell communication analyses revealed that POSTN-positive mesenchymal fibroblasts (POSTN+ mesenchymal fibs) are more prevalent in keloids and exhibit higher transforming growth factor β (TGF-β) signalling activity, potentially playing a central role in excessive fibrosis. In contrast, IGFBP2-positive fibroblasts (IGFBP2+ fibs) are more abundant in normal skin, insensitive to TGF-β and Periostin signalling, and possess anti-fibrotic potential, possibly related to limited tissue repair and regenerative capacity. Trajectory analysis inferred the differentiation states and patterns of different fibroblast subpopulations. Additionally, we explored the heterogeneity of endothelial cells, finding an endothelial cell subpopulation (EC10) exhibiting mesenchymal activation characteristics, which may work with specific fibroblasts to promote abnormal angiogenesis and endothelial-to-mesenchymal transition processes. Spatial transcriptomics analysis has shown that the proportion of IGFBP2+ fibroblasts relatively increases in acne keloidalis after hormonal treatment, further demonstrating their value as potential therapeutic targets. Ultimately, we separated these two subpopulations using flow cytometry, highlighting their opposing roles in the secretion of the ECM. These findings provide new insights into the pathogenesis of keloids and lay the theoretical foundation for the development of innovative anti-fibrotic treatment strategies.

Conventional and antibody-enhanced DENV infection of human macrophages induces differential immunotranscriptomic profiles.

Dengue virus (DENV) is a mosquito-borne flavivirus which coexists as four genetically and immunologically distinct serotypes (DENV-1 to -4). In secondary heterologous DENV infection, pre-existing immunity is believed to contribute to severe disease through antibody-dependent enhancement (ADE). Although the elevated pathology observed in ADE conditions has been described, the cell-intrinsic mechanisms governing this process remain unclear. Using single-cell RNA sequencing (scRNAseq), we investigated the transcriptomic profiles of human monocyte-derived macrophages infected by DENV-2 in ADE compared to conventional infection conditions. Unsupervised analysis of scRNAseq data enabled the identification of two distinct cell populations in a heterogeneous cell culture, likely representing infected and bystander/uninfected cells. Differential gene expression and ingenuity pathway analyses revealed a number of significantly upregulated and downregulated genes and gene networks between cells infected by ADE compared to conventional infection. Specifically, these pathways indicated mechanisms such as suppressed interferon signaling and inflammatory chemokine transcription in cells infected via ADE. Further analysis revealed that transcriptomic changes were independent of viral RNA within infected cells, suggesting that the observed changes are reflective of cell-intrinsic responses and not simply a function of per-cell viral burden. The interpreted "bystander" cell population also demonstrated distinct profiles in ADE conditions, indicating an immunologically activated phenotype enriched for the expression of gene networks involved with protein translation, cytokine production, and antigen presentation. Together, these findings support the concept that DENV infection via ADE induces a qualitatively different transcriptomic response in infected cells, contributing to our understanding of ADE as a mechanistic driver of disease and pathogenesis.IMPORTANCEDengue virus (DENV) is a mosquito-borne human pathogen with a significant and growing global health burden. Although correlates of severe dengue disease are poorly understood, pre-existing immunity to DENV has been associated with severe disease risk and known to contribute to an alternative route of viral entry termed antibody-dependent enhancement (ADE). Using single-cell RNA sequencing, we identified distinct transcriptomic processes involved in antibody-mediated DENV entry compared to conventional receptor-mediated entry. These data provide meaningful insight into the discrete processes contributing to DENV pathogenesis in ADE conditions.

Bacterial proliferation pattern formation

Bacteria can form a great variety of spatially heterogeneous cell density patterns, ranging from simple concentric rings to dynamical spiral waves appearing in growing colonies. These pattern formation phenomena are important as they reflect how cellular processes such as metabolism operate in heterogeneous chemical environments. In the laboratory, they can be studied in simplified setups, where spatial gradients of oxygen and nutrients are externally imposed, and cells are immobilized in a gel matrix. An intriguing example, observed in such setups over 80 years ago, is the sequential formation of narrow bands of high cell density, taking place even for a clonal population. However, key aspects of the dynamics of band formation remained obscure. Using time-lapse imaging of replicate transparent columns in simplified growth media, we first quantify the precision of the positioning and timing of band formation. We also show that the appearance and position of different bands can be modulated independently. This “modularity” is suggested by the observation that different bands differ in their gene expression, and it is reproduced by a theoretical model based on the existence of internal metabolic states and the induction of a pH gradient. Finally, we can also modify the observed pattern formation by introducing genetic modifications that impair selected metabolic pathways. In our opinion, the possibility of precise measurements and controls, together with the simplicity and richness of the “proliferation pattern formation” phenomenon, can make it a model system to study the response of cellular processes to heterogeneous environments. Published by the American Physical Society 2025

Cell–cell heterogeneity in phosphoenolpyruvate carboxylase biases early cell fate priming in Dictyostelium discoideum

Glucose metabolism is a key factor characterizing the cellular state during multicellular development. In metazoans, the metabolic state of undifferentiated cells correlates with growth/differentiation transition and cell fate determination. Notably, the cell fate of the Amoebozoa species Dictyostelium discoideum is biased by the presence of glucose and is also correlated with early differences in intracellular ATP. However, the relationship between early cell–cell heterogeneity, cell differentiation, and the metabolic state is unclear. To address the link between glucose metabolism and cell differentiation in D. discoideum, we studied the role of phosphoenolpyruvate carboxylase (PEPC), a key enzyme in the PEP-oxaloacetate-pyruvate node, a core junction that dictates the metabolic flux of glycolysis, the TCA cycle, and gluconeogenesis. We demonstrate that there is cell–cell heterogeneity in PEPC promoter activity in vegetative cells, which depends on nutrient conditions, and that cells with high PEPC promoter activity differentiate into spores. The PEPC null mutant exhibited an aberrantly high prestalk/prespore ratio, and the spore mass of the fruiting body was glassy and consisted of immature spores. Furthermore, the PEPC null mutant had high ATP levels and low mitochondrial membrane potential. Our results suggest the importance of cell–cell heterogeneity in the levels of metabolic enzymes during early cell fate priming.

Volumetric trans-scale imaging of massive quantity of heterogeneous cell populations in centimeter-wide tissue and embryo.

We established a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide three-dimensional (3D) tissues and embryos. Using a custom-made giant lens system with a magnification of ×2 and a numerical aperture (NA) of 0.25, and a CMOS camera with more than 100 megapixels, we built a trans-scale scope AMATERAS-2, and realized fluorescence imaging with a transverse spatial resolution of approximately 1.1 µm across an FOV of approximately 1.5×1.0 cm2. The 3D resolving capability was realized through a combination of optical and computational sectioning techniques tailored for our low-power imaging system. We applied the imaging technique to 1.2 cm-wide section of mouse brain, and successfully observed various regions of the brain with sub-cellular resolution in a single FOV. We also performed time-lapse imaging of a 1-cm-wide vascular network during quail embryo development for over 24 hr, visualizing the movement of over 4.0×105 vascular endothelial cells and quantitatively analyzing their dynamics. Our results demonstrate the potential of this technique in accelerating production of comprehensive reference maps of all cells in organisms and tissues, which contributes to understanding developmental processes, brain functions, and pathogenesis of disease, as well as high-throughput quality check of tissues used for transplantation medicine.

Volumetric trans-scale imaging of massive quantity of heterogeneous cell populations in centimeter-wide tissue and embryo

We established a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide three-dimensional (3D) tissues and embryos. Using a custom-made giant lens system with a magnification of ×2 and a numerical aperture (NA) of 0.25, and a CMOS camera with more than 100 megapixels, we built a trans-scale scope AMATERAS-2, and realized fluorescence imaging with a transverse spatial resolution of approximately 1.1 µm across an FOV of approximately 1.5×1.0 cm2. The 3D resolving capability was realized through a combination of optical and computational sectioning techniques tailored for our low-power imaging system. We applied the imaging technique to 1.2 cm-wide section of mouse brain, and successfully observed various regions of the brain with sub-cellular resolution in a single FOV. We also performed time-lapse imaging of a 1-cm-wide vascular network during quail embryo development for over 24 hr, visualizing the movement of over 4.0×105 vascular endothelial cells and quantitatively analyzing their dynamics. Our results demonstrate the potential of this technique in accelerating production of comprehensive reference maps of all cells in organisms and tissues, which contributes to understanding developmental processes, brain functions, and pathogenesis of disease, as well as high-throughput quality check of tissues used for transplantation medicine.

Unraveling the tumor microenvironment of esophageal squamous cell carcinoma through single-cell sequencing: A comprehensive review.

Esophageal squamous cell carcinoma (ESCC) is a highly heterogeneous and aggressive malignancy. The progression, invasiveness, and metastatic potential of ESCC are shaped by a multitude of cells within the tumor microenvironment (TME), including tumor cells, immune cells, endothelial cells, as well as fibroblasts and other cell types. Recent advancements in single-cell sequencing technologies have significantly enhanced our comprehension of the diverse landscape of ESCC. Single-cell multi-omics technology, particularly single-cell transcriptome sequencing, have shed light on the expression profiles of individual cells and the molecular characteristics of distinct tumor cell populations. This review summarizes the latest literature on single-cell research in the field of ESCC, aiming to elucidate the heterogeneity of tumor cells, immune cells, and stromal cells at the single-cell level. Furthermore, it explores the impact of cellular interactions within the TME on the progression of ESCC. By compiling a comprehensive overview of single-cell omics research on ESCC, this article aims to enhance our understanding of ESCC diagnosis and treatment by elucidating the intricate interplay within the TME. It explores the cellular composition, spatial arrangement, and functional attributes of the ESCC TME, offering potential therapeutic targets and biomarkers for personalized treatment strategies.

A comprehensive update on the potential of curcumin to enhance chemosensitivity in colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers and a major cause of cancer-related mortality worldwide. The efficacy of chemotherapy agents in CRC treatment is often limited due to toxic side effects, heterogeneity of cancer cells, and the possibility of chemoresistance which promotes cancer cell survival through several mechanisms. Combining chemotherapy agents with natural compounds like curcumin, a polyphenol compound from the Curcuma longa plant, has been reported to overcome chemoresistance and increase the sensitivity of cancer cells to chemotherapeutics. Curcumin, alone or in combination with chemotherapy agents, has been demonstrated to prevent chemoresistance by modulating various signaling pathways, reducing the expression of drug resistance-related genes. The purpose of this article is to provide a comprehensive update on studies that have investigated the ability of curcumin to enhance the efficacy of chemotherapy agents used in CRC. It is hoped that it can serve as a template for future research on the efficacy of curcumin, or other natural compounds, combined with chemotherapy agents to maximize the effectiveness of therapy and reduce the side effects that occur in CRC or other cancers.

Cardiac Heterogeneity Prediction by Cardio-Neural Network Simulation.

The bidirectional interactions between brain and heart through autonomic nervous system is the prime focus of neuro-cardiology community. The computer models designed to analyze brain and heart signals are either complex in terms of molecular and cellular interactions or not capable of representing the complex ion channel dynamics. Therefore, scientists are unable to extract the overall behavior of organs by electrical response of heterogeneous cells of brain and heart. In this study, a unified model of excitable cells is proposed that can be modulated by adrenergic features. By implementing the proposed model, a network of one thousand sparsely coupled cardio-neural network is simulated. The major findings of study include i. cardiac heterogeneity in electrical behavior of cardiac myocytes is the prime factor of heart rate variability ii. Brain-heart interplay through electrical pulses holds the necessary information of brain and heart signals that can be analyzed through spiking neural networks iii. Heart rate variability can be predicted and monitored by spiking neural networks from electrophysiological recordings of brain and heart iv. Heart rate variability related to tachycardia and bradycardia depends upon the polarization protocols of cardiac myocytes during plateau phase of action potential. This study provides the modeling and simulation phase of brain-heart interface to predict the morbidity at early stages. The recent advancements in nano-electronics will make is possible to develop brain-heart interface as nano-chip to deploy in subject to stimulate the brain-heart interplay through electrophysiological signals.

Superloaded Multiplexed scRNA-seq Data Preserves Primary Immune Cell Heterogeneity but Necessitates Stringent Doublet Removal

ABSTRACT Background Single-cell RNA sequencing (scRNA-seq) has improved our ability to characterize rare cell populations. In practice, cells from different tissues or donors are simultaneously loaded onto the instrument (multiplexed) at the recommended (standard loading) or higher (superloading) numbers to save time and money. Although cost-effective, superloading can stymie computational analyses owing to high multiplet rates and sample complexity. Methods We compared the effects of superloading on multiplexed single-cell gene expression and T cell receptor (TCR) data generated from human thymus and blood samples from different donors. Results Minimal transcriptomic differences were observed between the data generated by either standard or superloading. Irrespective of the loading cell number, we found that over 50% of the T cells expressing multiple TCR chains were doublets. Conclusion Multiple samples can be run simultaneously without compromising data quality and subsequent analyses. However, an additional doublet removal step based on TCR configuration may improve the accuracy of T cell analysis.

Abstract TP346: Investigating Transcriptomic Profiles of Peripheral Blood Mononuclear Cells in Moyamoya Disease Using Single-Cell RNAseq

Objectives: Moyamoya Disease (MMD) is a complex cerebrovascular disease marked by progressive narrowing in the cerebral arteries. Previous studies have suggested that dysregulated genes in the peripheral blood of MMD patients are involved in extracellular matrix organization and immune responses. However, the mechanism by which the immune response contributes to MMD progression is unclear. In this study we investigated the immune landscape in the peripheral blood mononuclear cells (PBMCs) of MMD patients using single-cell RNA sequencing (scRNA-seq). Methods: Blood samples were collected from 3 bilateral MMD patients and 2 healthy controls. PBMCs were isolated and processed for 10x single-cell RNA sequencing. Immune cell heterogeneity and differentially expressed genes (DEGs) in each cell type were identified. Top biological pathways were determined using Ingenuity Pathway Analysis (IPA). Flow cytometry with an 11-fluorochrome panel was use to validate key immune populations and fluorescence-activated cell sorting (FACS) was used to isolate specific immune populations for transcriptome analysis. Results: Preliminary analysis revealed notable increase of cell percentages in certain cell types in MMD, such as CD8+T (9.21% vs 7.11%), B cells (6.94% vs 3.45%) and CD4+NKT (5.78% vs 3.22%), and decrease in CD8+NKT (9.59% vs 15.74%), compared to controls. Higher transcriptome dynamics were observed in certain cell type in MMD, with 2402 DEGs in classical monocytes, 1379 DEGs in CD4+T, 1312 DEGs in CD8+NKT and 997 DEGs in B cells. Interestingly, many cell types, except for classical and non-classical monocytes, displayed a higher number of downregulated DEGs in MMD, particularly in T cells, B cells, and NKT cells. Pathway analysis indicated that CD4+T, CD8+NKT, and B cells show predicted inhibition in several pathways, including T cell receptor and mTOR signaling, interleukins and CXCR4 signaling, as well as cell surface interactions at the vascular wall. Conclusion: These preliminary findings point to a distinct immunological profile in MMD, suggesting significant alterations in both immune cell activity and interaction within the vascular environment. Ongoing studies utilize FACS to validate specific immune populations and their transcriptomes in a larger cohort. These findings reveal unique transcriptomic signatures of peripheral blood immune cells in MMD and highlight key immune cell types that may contribute to its pathogenesis.

Unveiling the role of ACTL6A in uveal melanoma metastasis and immune microenvironment.

To predict and evaluate the possible mechanisms and clinical value of ACTL6A in the prognosis and development of UM. Bioinformatics analyze the relationship between ACTL6A and immunity in UM, which derived from TCGA, Gene Expression Omnibus (GEO) databases. Tumor-infltrated immune cells were demonstrated using QUANTISEQ and MCP-counter. Furthermore, scRNA-seq was used to detect ACTL6A expression, distribution, immune infiltration and revealing the gene expression profile of UM. The expression of ACTL6A was lower in UM compared with pantumor in TCGA databases. Kaplan-Meier analysis revealed that downregulated ACTL6A was associated with poor OS, and ACTL6A was associated with cancer stem cells (CSCs) and immune infiltration. Moreover, ACTL6A might act as a chemotherapy resistance gene and closely relate- to epithelial-mesenchymal transition. Analysis in 8 GSE databases showed that IL13, TPTE, IL17B and CCL22 genes were significantly overexpressed in metastatic UM. Furthermore, the single-cell transcriptomic profling identified a new cell cluster - as a unique type of immune cell, which associating with malignant cell heterogeneity and complexity, and further revealing that the metastasis of UM is mainly associated with CD4 Tconv, B , CD8 Tex, and Plasma cells. Downregulated ACTL6A acts as a risk factor for poor prognosis in UM, which implies as an potential prognostic marker for independent targeted immunotherapy.

CNS resident macrophages exhibit region-specific states and immunogenic responses during Rbpj-deficient brain arteriovenous malformation

Microglia are heterogeneous macrophage cells that serve as the central nervous system’s resident immune cells. During neuro-related diseases, CNS resident macrophages change their molecular, cellular, and functional properties—that collectively define “states”—in response to specific neural perturbations. Neurovascular diseases elicit state changes, by promoting increased vascular permeability among microvessels and thus altering blood–brain barrier integrity. Here, we used a mouse model of brain arteriovenous malformation (bAVM)—mediated by endothelial loss of Recombination signal binding protein for immunoglobulin kappa J region (Rbpj)—to investigate changes to brain resident macrophage states during neurovascular disease pathogenesis. We found increased area of Ionized calcium-binding adapter molecule 1 (Iba1) expression in Rbpj-deficient bAVM tissue, as well as Iba1 + cell hypertrophy, increased cell number, and hyperproliferation within areas of increased Iba1 + density. Hypertrophic cells had increased cell body areas and decreased process length, suggesting a transition in surveillance state. Gene expression data revealed region-specific molecular changes to Iba + cells, suggestive of altered metabolic activity. CNS resident macrophages isolated from cortical and cerebellar regions showed profiles consistent with cytokine-associated immunogenic responses and an immunovigilant pathogen-recognition response, respectively. Thus, our findings demonstrate region-specific changes to CNS resident macrophages during Rbpj-deficient bAVM.

Epithelial cell diversity and immune remodeling in bladder cancer progression: insights from single-cell transcriptomics.

The progression of bladder cancer (BC) from non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC) significantly increases disease severity. Although the tumor microenvironment (TME) plays a pivotal role in this process, the heterogeneity of tumor cells and TME components remains underexplored. We characterized the transcriptomes of single cells from 11 BC samples, including 4 NMIBC, 4 MIBC, and 3 adjacent normal tissues. Bulk RNA-seq data were used to validate the clinical features of characteristic cells, and protein levels of these cells were further confirmed through immunohistochemistry (IHC) and multiplex immunofluorescence. Bladder cancer progression was associated with distinct transcriptomic features in the TME. Tumor cells in MIBC displayed enhanced glycolytic activity and downregulation of chemokines and MHC-II molecules, reducing immune cell recruitment and facilitating immune evasion. This highlights glycolysis as a potential therapeutic target for disrupting tumor progression. We identified a T cell exhaustion pathway from naive CD8 + T cells (CD8 + TCF7) to terminally exhausted CD8 + STMN1 cells, with progressively declining immune surveillance. Targeting intermediate exhaustion states may restore T cell function and improve anti-tumor immunity. Macrophages polarized toward a pro-tumorigenic phenotype, while VEGFA + mast cells promoted angiogenesis in early-stage BC, suggesting their role as potential targets for therapeutic intervention in NMIBC. Furthermore, conventional dendritic cells (DCs) transformed into LAMP3 + DCs, contributing to an immunosuppressive microenvironment and enabling immune evasion. This study reveals dynamic changes in the TME during BC progression, including enhanced glycolysis, T cell exhaustion, and immune cell remodeling, which contribute to immune evasion and tumor progression. These findings identify critical pathways and cell populations as potential therapeutic targets, offering new strategies to improve treatment outcomes in BC patients.

Single-cell sequencing reveals cell heterogeneity and aberrantly activated pathways associated with microvascular invasion in hepatocellular carcinoma

IntroductionHepatocellular carcinoma (HCC) is the most common primary liver cancer, with microvascular invasion (MVI) identified as a major predictor of early recurrence. However, the intratumor cellular heterogeneity of MVI, the identification of pertinent biomarkers, and the role of intercellular signalling interactions in MVI progression are unclear. This study aims to explore these aspects using single-cell transcriptomic analysis.MethodsThe present study utilized single-cell transcriptomic data from public databases to conduct an in-depth transcriptome analysis of tumour tissues and adjacent nontumor tissues from five patients with hepatocellular carcinoma, with a particular focus on samples from three patients exhibiting microvascular invasion. Bioinformatics tools were employed to analyze gene expression patterns and signalling pathways.ResultsThe findings indicated that MVI-positive malignant cells activate multiple signalling pathways to facilitate invasion and metastasis. Specific malignant cell subtypes strongly associated with MVI were identified, exhibiting distinctive gene expression patterns related to proliferation, invasion, and metabolic reprogramming of tumour cells. Further analysis revealed that the laminin and VEGF signalling pathways are crucial for remodelling the tumour microenvironment and angiogenesis associated with MVI. The MARCKSL1 gene was predominantly expressed in MVI-positive malignant cells and may contribute to MVI progression by interacting with the PTN signalling network. Additionally, MARCKSL1 is linked to tumour resistance to multiple anticancer drugs.DiscussionThis study sheds light on the molecular characteristics and functional heterogeneity of MVI-associated malignant cell subpopulations. The single-cell transcriptome and bioinformatics analyses provided insights into the mechanisms driving MVI, potentially aiding the development of targeted diagnostic and therapeutic strategies. Future research should further validate the role of MARCKSL1 in MVI progression and explore its potential clinical applications.

Microenvironmental determinants of endothelial cell heterogeneity.

During development, endothelial cells (ECs) undergo an extraordinary specialization by which generic capillary microcirculatory networks spanning from arteries to veins transform into patterned organotypic zonated blood vessels. These capillary ECs become specialized to support the cellular and metabolic demands of each specific organ, including supplying tissue-specific angiocrine factors that orchestrate organ development, maintenance of organ-specific functions and regeneration of injured adult organs. Here, we illustrate the mechanisms by which microenvironmental signals emanating from non-vascular niche cells induce generic ECs to acquire specific inter-organ and intra-organ functional attributes. We describe how perivascular, parenchymal and immune cells dictate vascular heterogeneity and capillary zonation, and how this system is maintained through tissue-specificsignalling activatedbyvasculogenic and angiogenic factors and deposition of matrix components. We also discuss how perturbation of organotypic vascular niche cues lead to erasure of EC signatures,contributing to the pathogenesis of disease processes. We alsodescribe approaches that use reconstitution of tissue-specific signatures of ECs to promote regeneration of damaged organs.

Understanding the Dual Role of Macrophages in Tumor Growth and Therapy: A Mechanistic Review.

Macrophages are heterogeneous cells that are the mediators of tissue homeostasis. These immune cells originated from monocytes and are classified into two basic categories, M1 and M2 macrophages. M1 macrophages exhibit anti-tumorous inflammatory reactions due to the behavior of phagocytosis. M2 macrophages or tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME) and have a basic role in tumor progression by interacting with other immune cells in TME. By the expression of various cytokines, chemokines, and growth factors, TAMs lead to strengthening tumor cell proliferation, angiogenesis, and suppression of the immune system which further support invasion and metastasis. This review discusses recent and updated mechanisms regarding tumor progression by M2 macrophages. Moreover, the current therapeutic approaches targeting TAMs, their advantages, and limitations are also summarized, and further treatment approaches are outlined along with an elaboration of the tumor regression role of macrophages. This comprehensive review article possibly helps to understand the mechanisms underlying the tumor progression and regression role of macrophages in a comparative way from a basic level to the advanced one.

PCMMD: A Novel Dataset of Plasma Cells to Support the Diagnosis of Multiple Myeloma

Multiple Myeloma (MM) is a cytogenetically heterogeneous clonal plasma cell proliferative disease whose diagnosis is supported by analyses on histological slides of bone marrow aspirate. In summary, experts use a labor-intensive methodology to compute the ratio between plasma cells and non-plasma cells. Therefore, the key aspect of the methodology is identifying these cells, which relies on the experts’ attention and experience. In this work, we present a valuable dataset comprising more than 5,000 plasma and non-plasma cells, labeled by experts, along with some patient diagnostics. We also share a Deep Neural Network model, as a benchmark, trained to identify and count plasma and non-plasma cells automatically. The contributions of this work are two-fold: (i) the labeled cells can be used to train new practitioners and support continuing medical education; and (ii) the design of new methods to identify such cells, improving the results presented by our benchmark. We emphasize that our work supports the diagnosis of MM in practical scenarios and paves new ways to advance the state-of-the-art.

Abstract B008: Understanding myeloid and lymphoid cell heterogeneity following radiotherapy and its impact on metastasis development in breast cancer

Abstract Breast cancer (BC) is the most frequent malignancy in women worldwide and the gold standard for its treatment currently entails resection of the primary tumor, followed by radiotherapy (RT), chemotherapy and/or immunotherapy. Specifically, adjuvant RT directed at the breast has been successfully employed to improve the risk of local recurrences, but the main obstacle to full remission of most BC patients remains the development of metastasis. RT directed at the primary tumor (pRT) acts via a combination of direct cytotoxicity and immunomodulatory effects on the local tumor microenvironment (TME), whereas its impact at distal sites and on the metastatic TME is still poorly understood. Thus, we hypothesized that pRT-driven systemic effects could change the nature and function of immune cell populations at distal sites, affecting survival and growth of disseminated tumor cells and ultimately influencing the development of metastasis. Analysis of scRNAseq data from murine lungs at the pre-metastatic stage showed that pRT towards a tumor-bearing breast induces alterations within the lung immune landscape, particularly suggesting a dual role of neutrophils and alterations in their interactions with the adaptive immune system. Moreover, we demonstrated that in a mouse model of metastasis, experimentally induced via intravenous (IV) injection of 4T1 cells, pRT directed to a tumor-bearing breast induces a significant increase in lung metastasis, accompanied by numerical and functional differences in lymphoid and myeloid cells which collectively suggest the establishment of a local immunosuppressive microenvironment. Interestingly, the metastatic increase observed in irradiated mice resembled the phenotype of mice IV injected with 4T1 cells in absence of a primary tumor, thus when there is no pre-activation of the immune system prior to the IV injection. This result suggested that pRT could alter systemic anti-tumor immunity in our model, resulting in metastatic growth. Therefore, current experiments are focusing on elucidating the specific immune populations involved in this effect by selectively depleting cell types of interest, such as neutrophils and Treg cells. Moreover, we are further investigating the mechanisms influencing DTCs’ outgrowth via scRNAseq of lungs bearing niche-labelling metastasis, which allow to identify changes occurring specifically in the metastatic niche. Finally, we are confirming the effects observed in our experimentally-induced metastatic model also in a clinically relevant mouse model, comprising both the spontaneous metastatic spread of 4T1 cells and the resection of the primary tumor prior to RT, resulting in a therapeutic protocol more similar to current clinical practice. Our data show a pro-metastatic effect of RT, possibly driven by alterations in the activation of the immune system. Results from this project could better inform on RT’s influence on immune cell behavior in the metastatic TME, allowing to identify relevant targets for novel immunotherapy approaches against metastatic disease. Citation Format: Alessandra Perini, Marti Brucoli, Sophie Acton, Luigi Ombrato. Understanding myeloid and lymphoid cell heterogeneity following radiotherapy and its impact on metastasis development in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B008.

Heterogeneous focal adhesion cytoskeleton nanoarchitectures from microengineered interfacial curvature to oversee nuclear remodeling and mechanotransduction of mesenchymal stem cells

BackgroundInterfacial heterogeneity is widely explored to reveal molecular mechanisms of force-mediated pathways due to biased tension. However, the influence of cell density,, curvature, and interfacial heterogeneity on underlying pathways of mechanotransduction is obscure.MethodsPolydimethylsiloxane (PDMS)-based stencils were micropatterned to prepare the micropores for cell culture. The colonies of human mesenchymal stem cells (hMSCs) were formed by controlling cell seeding density to investigate the influences of cell density, curvature and heterogeneity on mechanotransduction. Immunofluorescent staining of integrin, vinculin, and talin-1 was conducted to evaluate adhesion-related expression levels. Then, immunofluorescent staining of actin, actinin, and myosin was performed to detect cytoskeleton distribution, especially at the periphery. Nuclear force-sensing mechanotransduction was explained by yes-associated protein (YAP) and laminA/C analysis.ResultsThe micropatterned colony of hMSCs demonstrated the coincident characters with engineered micropores of microstencils. The cell colony obviously developed the heterogeneous morphogenesis. Heterogeneous focal adhesion guided the development of actin, actinin, and myosin together to regulate cellular contractility and movement by integrin, vinculin, and talin-1. Cytoskeletal staining showed that actin, actinin, and myosin fibers were reorganized at the periphery of microstencils. YAP nuclear translocation and laminA/C nuclear remodeling were enhanced at the periphery by the regulation of heterogeneous focal adhesion (FA) and cytoskeleton arrangement.ConclusionsThe characters of the engineered clustering colony showed similar results with prepared microstencils, and colony curvature was also well adjusted to establish heterogeneous balance at the periphery of cell colony. The mechanism of curvature, spreading, and elongation was also investigated to disclose the compliance of FA and cytoskeleton along with curvature microarrays for increased nuclear force-sensing mechanotransduction. The results may provide helpful information for understanding interfacial heterogeneity and nuclear mechanotransduction of stem cells.