Retinal determination network reactivation drives chemoresistance and blocks myeloid differentiation in acute myeloid leukemia.

Breast cancer is composed of diverse cell populations, and this intratumoral heterogeneity profoundly affects clinical behavior. Here, we leveraged single cell RNA sequencing (scRNA-seq) of 68 breast cancer specimens to dissect tumor heterogeneity at high resolution. Unsupervised clustering identified all major cell types of the tumor microenvironment (TME)—including malignant epithelial cells, fibroblasts, T cells, macrophages, endothelial cells, and others—with striking variability in their proportions across molecular subtypes. For example, a BRCA1-mutant triple-negative breast cancer (TNBC) sample showed dense immune infiltration, whereas an estrogen receptor (ER)-positive tumor was mostly epithelial, consistent with known subtype differences in immunogenicity. We applied inference of copy number variations (inferCNV) to distinguish malignant epithelial cells, identifying ~90,000 tumor cells with significant copy-number aberrations enriched for cancer hallmark pathways. Re-clustering of these malignant cells revealed five discrete subpopulations. Notably, a KRT17-positive subcluster displayed the highest stemness score and a distinctive ETS-family transcription factor (ERG) regulon, suggesting a stem-like phenotype. Using The Cancer Genome Atlas (TCGA) cohort, we found that genes upregulated in this KRT17+ subpopulation, particularly NFKBIA, PDLIM4, and TCP1 stratified patient survival. An 8-gene risk signature derived from the KRT17 program segregated patients into high- and low-risk groups with markedly different outcomes. High-risk tumors were characterized by an immunosuppressive TME enriched in M2-like macrophages, whereas low-risk tumors more often harbored lymphocyte-predominant infiltrates. Focusing on TCP1, a chaperonin subunit upregulated in high-risk tumors, we demonstrate that TCP1 knockdown in breast cancer cell lines substantially impairs cell migration (~50% reduction in wound closure) and invasion (P < 0.01). These findings reveal functionally distinct malignant cell states within breast cancer and identify TCP1 as a promising therapeutic target to disrupt aggressive, stem-like tumor cell programs, ultimately guiding more personalized treatment strategies.

The calyx of Held is a giant axo-somatic synapse classically confined to the auditory brainstem. We recently identified morphologically similar calyx-like terminals in the extended amygdala (EA) that arise from the ventrolateral parabrachial complex and co-express PACAP, CGRP, VAChT, VGluT1, and VGluT2, targeting PKCδ+/GluD1+ EA neurons. Here, we asked whether this parabrachial-EA pathway participates in compensation during acute hypotension. In rats given hydralazine (10 mg/kg, i.p.), we quantified Fos protein during an early phase (60 min) and a late phase (120 min). Early after hypotension, Fos surged in a discrete subpopulation of the parabrachial Kölliker-Fuse (KF) region and in the EA, whereas magnocellular neurons of the supraoptic and paraventricular nuclei (SON/PVN) remained largely silent. By 120 min, magnocellular SON/PVN neurons were robustly Fos-positive. Confocal immunohistochemistry showed that most Fos+ PKCδ+/GluD1+ EA neurons were encircled by PACAP+ perisomatic terminals (80.8%), of which the majority co-expressed VGluT1 (88.1%). RNAscope in situ hybridization further identified a selective KF population co-expressing Adcyap1 (PACAP) and Slc17a7 (VGluT1) that became fos-positive during the early phase. Together, these data suggest that a KF PACAP+/VGluT1+ projection forms calyceal terminals around PKCδ+/GluD1+ EA neurons, providing a high-fidelity route for rapid autonomic rebound to falling blood pressure, while slower endocrine support is subsequently recruited via neurohormone-magnocellular activation. This work links multimodal parabrachial output to temporally layered autonomic-neuroendocrine control.

Single cell transcriptional profiling reveals distinct subsets of human megakaryocytes in myelofibrosis

Depression and anxiety are prominent contributors to the global disease burden, with significant health system implications particularly in underexamined contexts such as Saudi Arabia. While existing studies often target discrete subpopulations, few have synthesized national data to evaluate mental health prevalence alongside service utilization. This study addresses that gap by analyzing 4 national datasets—the 2024 National Health Survey, the 2024 Woman and Child Health Survey, the 2017 Disability Survey, and the Ministry of Health’s mental health reports—using a framework grounded in WHO standards (ie, the Mental Health Action Plan). We operationalized 8 analytical indicators covering prevalence, symptom frequency, treatment usage, diagnostic distribution, comorbidity patterns, and health system responsiveness. Descriptive statistics and latent class analyses revealed consistently high prevalence of depression and anxiety among adults, children, individuals with disabilities, and healthcare users. Women, adolescents, and chronically ill individuals reported higher symptom severity and lower access to tailored interventions. Mental health service data emphasized diagnostic overrepresentation of psychotic disorders and under-documentation of emotional distress, indicating a systemic diagnostic skew. The findings expose critical gaps between population mental health needs and current diagnostic-service frameworks. By informing SDG 3 (promoting mental health and well-being) and SDG 4 (inclusive education and well-being for children), this study supports enhanced policy design for early identification, equity-focused care, and integration of functional assessment within Saudi Arabia’s mental health system.

Hypothalamic pro-opiomelanocortin (POMC) neurons are classically viewed as mediators of satiety, acting in response to metabolic and hormonal cues and in opposition to Agouti-related protein (AgRP) neurons to maintain energy balance. This model, centered on the appetite-suppressant effects of the POMC-derived neuropeptide α-melanocyte-stimulating hormone (α-MSH) through its activation of melanocortin-4 receptors (MC4R), has shaped our understanding of feeding and body weight regulation for decades. However, recent discoveries have challenged and expanded this traditional view, revealing that POMC neurons are not a uniform population dedicated solely to satiety control. Single-cell transcriptomic analyses have revealed striking molecular heterogeneity, reflected in distinct anatomical distributions, receptor expression profiles, electrophysiological properties, and projection patterns - all supporting the idea of functional specialization within this neuronal population. In this review, we propose a conceptual framework that integrates POMC neuronal heterogeneity with the regulation of appetite, metabolic physiology, and behavior beyond feeding. We highlight emerging evidence showing that discrete POMC neuronal subpopulations respond to specific combinations of interoceptive and environmental cues to orchestrate diverse adaptive responses. This perspective underscores the developmental plasticity and functional versatility of POMC neurons, offering new insights into the mechanisms of obesity and potentially paving the way for novel targeted therapeutic strategies.

Visceral pain and its affective component associated with cystitis remain poorly understood. Here, we delineate the role of central amygdala (CeA) neuronal ensembles in encoding and modulating cystitis-induced bladder pain and its affective components. Utilizing a multidisciplinary approach combining behavioral assays, optogenetic manipulations, whole-cell electrophysiology, and activity-dependent genetic labeling, we identified functionally discrete CeA subpopulations that are selectively recruited during bladder inflammation. Bidirectional optogenetic modulation of these ensembles produced opposing effects on nocifensive and anxiety-like behaviors, indicating their causal involvement. Single-nucleus RNA sequencing of FosTRAP-labeled neurons revealed distinct transcriptional signatures associated with inflammatory activation. Integrating electrophysiological and transcriptomic data, we demonstrate that cystitis drives cell type-specific adaptations in CeA circuits. These findings provide mechanistic insight and uncover a molecularly and functionally defined CeA ensemble that orchestrates the sensory and affective dimensions of visceral pain.

Abstract The precisely organized six-layered architecture of the mammalian neocortex containing diverse types of neurons arises from tightly orchestrated developmental programs. Notably, layer V comprises two principal subtypes of excitatory pyramidal neurons distinguished by their axonal projection targets and stratifies into anatomically distinct sublayers: layer Va and layer Vb. Nevertheless, the mechanisms orchestrating neuronal diversification and their defining molecular markers have yet to be fully elucidated. In this study, we identified two novel molecular markers, Pcp4 (Purkinje cell protein 4, also known as Pep19) and FoxO1 (Forkhead box protein O1), which help define layer Va, characterized by FoxO1+ and Satb2+ neurons; and layer Vb, characterized by Pcp4+ and Ctip2+ neurons, and delineate two functionally discrete subpopulations of layer V projection neurons during late differentiation stages. Meanwhile, our findings were validated in single-cell RNA sequencing (scRNA-seq) data. We then employed two Dicer conditional knockout mouse models and found that microRNA (miRNA) deficiency leads to cortical sublayer disorganization and loss of Pcp4 and FoxO1 expression, demonstrating the essential role of miRNAs in neuronal subtype specification. This study advances our understanding of the molecular mechanisms governing the diversification of layer V neuronal subtypes and their developmental trajectories.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a 5-year survival rate of just 13%. Poor survival is due in part to late detection, as well as resistance to current standard-of-care therapeutics. PDAC tumorigenesis is a result of exocrine cell metaplasia: when stressed, acinar cells transdifferentiate to form metaplastic ducts in a process called acinar-to-ductal metaplasia (ADM). ADM is normally protective, but if metaplastic ductal cells develop a mutation in the proto-oncogene Kras, they can become trapped in the transdifferentiated state and form neoplastic lesions (PanINs), which can eventually develop into PDAC. Metaplastic ducts contain a myriad of cell types, including metaplastic neuroendocrine cells, which have been shown to promote neoplastic growth and PDAC tumorigenesis. In a subset of metaplastic neuroendocrine cells, we have identified upregulation of carbohydrate receptor Clec4f. In humans, Clec4f is upregulated in PDAC tumors compared to normal pancreas and correlates with poor prognosis when elevated in early stage disease. While Clec4f’s function in mice has been primarily associated with glycan recognition for antigen clearance by mouse liver macrophages, its function in PDAC tumor cells is unknown. Since we have identified Clec4f expression in a discrete subpopulation of pancreatic epithelial cells associated with early stage disease, we hypothesize that Clec4f plays a novel role in regulating PDAC tumorigenesis in response to binding glycans in the tumor microenvironment. Ectopic overexpression of Clec4f (Clec4f-oe) in murine PDAC cells revealed prominent reduction of classically mitogenic pathways including MAPK (via phosphorylated ERK) and PI3K (via phosphorylated AKT) as well as elevated levels of phosphorylated c-Myc (S62), which has been shown to play a role in regulating ductal-to-neuroendocrine lineage plasticity and therapeutic resistance. Clec4f-oe also preliminarily protected PDAC tumor cells from cell death via Gemcitabine or a D-galactose-based senescence model. In accordance with other studies in macrophages, Clec4f expression in PDAC tumor cells appears to be niche dependent. We found that, in Clec4f-oe tumor cells, incubation with galactose stabilizes Clec4f expression and Clec4f-associated signaling alterations. These data suggest that carbohydrate binding by Clec4f alters growth and plasticity phenotypes in PDAC tumor cells. Future experiments will investigate the intracellular binding partners for Clec4f to interrogate its signaling function in metaplastic neuroendocrine cells in early PDAC. Citation Format: Bailey A. Bye, R. McKinnon Walsh, Austin E. Eades, Kathleen E. DelGiorno, Michael N. VanSaun. Clec4f is expressed in a subset of PDAC metaplastic neuroendocrine cells and alters tumor cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 241.

Context Our understanding of population- and ecosystem-level processes commonly considers conspecific individuals to be ecologically equivalent. However, individuals of the same species may use resources differently, supporting the prevalence of individual specialisation or ‘apparent specialisation’. Individuals within a geographically defined population may also exhibit complex subpopulation movements, whereby individuals show philopatry to specific regions that further drives individual variation. Aims White sharks (Carcharodon carcharias) are top predators in temperate to tropical ecosystems. In Australia, two discrete subpopulations of white sharks (an east and a southwest subpopulation) have been proposed based on genetics and limited movement across Bass Strait. We aimed to characterise the extent of ontogenetic divergence in resource–habitat behaviour of white sharks from both regions. Methods We used high-resolution retrospective stable isotope profiles (δ15N and δ13C) of 74 white shark vertebral centra to examine ontogenetic trophic–habitat signatures for individuals sampled from both regions. Key results Our results demonstrate isotopic separation between juvenile–subadult sharks sampled east (−13.7 ± 0.72 δ13C; 14.2 ± 0.8 δ15N, n = 47) and southwest (−14.4 ± 0.6 δ13C; 12.5 ± 1.2 δ15N, n = 27) of Bass Strait, but with strong oscillatory trends across both regions, likely related to seasonal movements. Relative individual niche width revealed apparent specialised behaviour of juvenile–subadult sharks within both regions. Conclusions Retrospective ontogenetic isotopic profiles of vertebrae from Australian white sharks provide evidence to support an ecological two-population model for juvenile and subadult life stages. Implications Given many marine top predators are undergoing systematic population declines, understanding individual variation in diet and movement in the context of population structure and true or apparent specialisation is central to elucidating their ecological roles.

The distinct characteristic of two peritoneal macrophage subsets in a mouse model of hepatocellular carcinoma presents a novel therapeutic strategy.

Resolving Human Hematopoietic Stem Cell Functional Heterogeneity with ATP2B1 Surface Expression

Fluorescently labeled aerolysin (FLAER) is widely used for the identification of paroxysmal nocturnal hemoglobinuria (PNH) clones in peripheral blood (PB) samples. However, there are only a few reports on the differential fluorescent intensity of FLAER in normal bone marrow (BM) cell subpopulations. The purpose of this study was to evaluate FLAER expression during normal and pathological hematopoiesis, to map the critical existence of non-PNH FLAER-dim cells. A total of 54 BM aspirates were prospectively analyzed with FLAER-based flow cytometric (FC) protocols, during their routine work-up. These were obtained from patients with the following diagnoses: PNH (3), infections/reactive (5), myelodysplastic syndromes (MDS) (7), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (4), chronic myelogenous leukemia (CML) (3), acute myelogenous leukemia (AML) at diagnosis (20), AML in measurable residual disease (MRD) assessment (7), and B-cell acute lymphoblastic leukemia (B-ALL) in MRD assessment (5). The applied protocols consisted of FLAER, HLA-DR, CD14, CD33, CD34, CD66b, CD38, CD117, CD64, CD45, and FLAER, CD66c, CD14, CD33, CD34, CD66b, CD123, CD16, CD64, and CD45, respectively. FLAER expression was assessed in CD34++/CD38- and CD34+/CD38+ stem cells, CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, and CD64+/CD14-/HLA-DR+ monocyte precursors but also in mature myeloid cells. All patients revealed an intermediate FLAER intensity in CD34++/CD38- stem cells, with a discrete FLAER-negative subpopulation observed only in maturing CD34+/CD38+ stem cells of patients with PNH. The lowest FLAER intensity was noticed in CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, not only in patients with PNH but also in PNH-negative BM aspirates. An ascending FLAER intensity was further observed during monocytic and granulocytic maturation, with a discrete FLAER-negative population in CD64+/CD14-/HLA-DR+ monocyte precursors and maturing neutrophils and monocytes of patients with PNH only. The maturation pattern of FLAER expression was further confirmed in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid (ATRA), where FLAER was concurrently upregulated with CD66b in a consecutive series of PB samples tested over a 20-day-period after diagnosis. The application of FLAER in PNH-positive and PNH-negative reactive or malignant BM aspirates identified normally expected non-PNH FLAER-dim CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors in all samples. A specific FLAER-associated maturation pattern was observed, which is proposed for further study within MRD and diagnostic protocols.

The circadian system coordinates multiple behavioral outputs to ensure proper temporal organization. Timing information underlying circadian regulation of behavior depends on a molecular circadian clock that operates within clock neurons in the brain. In Drosophila and other organisms, clock neurons can be divided into several molecularly and functionally discrete subpopulations that form an interconnected central clock network. It is unknown how circadian signals are coherently generated by the clock network and transmitted across output circuits that connect clock cells to downstream neurons that regulate behavior. Here, we have exhaustively investigated the contribution of clock neuron subsets to the control of two prominent behavioral outputs in Drosophila: locomotor activity and feeding. We have used cell-specific manipulations to eliminate molecular clock function or induce electrical silencing either broadly throughout the clock network or in specific subpopulations. We find that clock cell manipulations produce similar changes in locomotor activity and feeding, suggesting that overlapping central clock circuitry regulates these distinct behavioral outputs. Interestingly, the magnitude and nature of the effects depend on the clock subset targeted. Lateral clock neuron manipulations profoundly degrade the rhythmicity of feeding and activity. In contrast, dorsal clock neuron manipulations only subtly affect rhythmicity but produce pronounced changes in the distribution of activity and feeding across the day. These experiments expand our knowledge of clock regulation of activity rhythms and offer the first extensive characterization of central clock control of feeding rhythms. Despite similar effects of central clock cell disruptions on activity and feeding, we find that manipulations that prevent functional signaling in an identified output circuit preferentially degrade locomotor activity rhythms, leaving feeding rhythms relatively intact. This demonstrates that activity and feeding are indeed dissociable behaviors, and furthermore suggests that differential circadian control of these behaviors diverges in output circuits downstream of the clock network.

Addiction is a complex behavioral disorder characterized by compulsive drug-seeking and drug use despite harmful consequences. The prefrontal cortex (PFC) plays a crucial role in cocaine addiction, involving decision-making, impulse control, memory, and emotional regulation. The PFC interacts with the brain’s reward system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc). The PFC also projects to the lateral habenula (LHb), a brain region critical for encoding negative reward and regulating the reward system. In the current study, we examined the role of PFC-LHb projections in regulating cocaine reward-related behaviors. We found that optogenetic stimulation of the PFC-LHb circuit during cocaine conditioning abolished cocaine preference without causing aversion. In addition, increased c-fos expression in LHb neurons was observed in animals that received optic stimulation during cocaine conditioning, supporting the circuit’s involvement in cocaine preference regulation. Molecular analysis in animals that received optic stimulation revealed that cocaine-induced alterations in the expression of GluA1 subunit of AMPA receptor was normalized to saline levels in a region-specific manner. Moreover, GluA1 serine phosphorylation on S845 and S831 were differentially altered in LHb and VTA but not in the PFC. Together these findings highlight the critical role of the PFC-LHb circuit in controlling cocaine reward-related behaviors and shed light on the underlying mechanisms. Understanding this circuit’s function may provide valuable insights into addiction and contribute to developing targeted treatments for substance use disorders.

To inform the management of wild fish populations, it is equally important to understand both the ecological connectivity of habitat patches, apparent at annual and seasonal scales, and the genetic connectivity, emerging at evolutionary scales across generations. Ecological connectivity indicates the potential for rapid recolonization upon local depletion, while genetic connectivity informs about the conservation needs related to the evolution of subpopulations and ecotypes in metapopulations. We combined acoustic biotelemetry and pooled-genome sequencing to study a northern pike (Esox lucius) population as a model of a freshwater piscivore that inhabits a network of shallow brackish lagoons in the southern Baltic Sea. We found limited ecological connectivity among genetically similar subpopulations of pike, suggesting a metapopulation structure characterized by discrete local subpopulations with infrequent migrations between them. Connectivity of different lagoons increased during spawning, suggesting directed spawning migrations to either freshwater rivers or low salinity patches in connected lake-like bays. Spawning site fidelity to either brackish or freshwater spawning sites was observed, further contributing to the reproductive isolation of certain subpopulations. The genetic population structure aligned with salinity gradients and geographical distance and was significant between pairs of rivers draining into the lagoon network, but it was unrelated to ecological connectivity. The results collectively suggest that local subpopulations may not rapidly replenish upon local depletion and that even weak connectivity among subpopulations was sufficient to maintain genetic homogeneity across lagoons with similar salinity levels. Effective management and conservation of species forming metapopulations, such as the coastal northern pike studied here, necessitate localized approaches that adapt fishing mortality to local abundance and promote access to specific habitats, especiallyrivers, during spawning to conserve the entire genetic biodiversity and foster resilience of the metapopulation.

Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Clear cell renal cell carcinomas (ccRCCs) epitomize the most formidable clinical subtype among renal neoplasms. While the impact of tumor-associated fibroblasts on ccRCC progression is duly acknowledged, a paucity of literature exists elucidating the intricate mechanisms and signaling pathways operative at the individual cellular level. Employing single-cell transcriptomic analysis, we meticulously curated UMAP profiles spanning substantial ccRCC populations, delving into the composition and intrinsic signaling pathways of these cohorts. Additionally, Myofibroblasts were fastidiously categorized into discrete subpopulations, with a thorough elucidation of the temporal trajectory relationships between these subpopulations. We further probed the cellular interaction pathways connecting pivotal subpopulations with tumors. Our endeavor also encompassed the identification of prognostic genes associated with these subpopulations through Bulk RNA-seq, subsequently validated through empirical experimentation. A notable escalation in the nFeature and nCount of Myofibroblasts and EPCs within ccRCCs was observed, notably enriched in oxidation-related pathways. This phenomenon is postulated to be closely associated with the heightened metabolic activities of Myofibroblasts and EPCs. The Myofibroblasts subpopulation, denoted as C3 HMGA1+ Myofibroblasts, emerges as a pivotal subset, displaying low differentiation and positioning itself at the terminal point of the temporal trajectory. Intriguingly, these cells exhibit a high degree of interaction with tumor cells through the MPZ signaling pathway network, suggesting that Myofibroblasts may facilitate tumor progression via this pathway. Prognostic genes associated with C3 were identified, among which TUBB3 is implicated in potential resistance to tumor recurrence. Finally, experimental validation revealed that the knockout of the key gene within the MPZ pathway, MPZL1, can inhibit tumor activity, proliferation, invasion, and migration capabilities. This investigation delves into the intricate mechanisms and interaction pathways between Myofibroblasts and ccRCCs at the single-cell level. We propose that targeting MPZL1 and the oxidative phosphorylation pathway could serve as potential key targets for treating the progression and recurrence of ccRCC. This discovery paves the way for new directions in the treatment and prognosis diagnosis of ccRCC in the future.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

Cholangiocarcinoma (CCA) stands as an aggressive malignancy of the biliary tract. The interplay between the tumor and immune system plays a pivotal role in disease progression and treatment outcomes. Hence, the present study aimed to extensively explore the immunogenomic landscape of CCA, with the objective of unveiling unique molecular and immunological signatures that could guide personalized therapeutic approaches. The study collected data from The Cancer Genome Atlas databases, performed gene set variation analysis for the chemokine ligand 5 (CCL5) high/low expression group, conducted principal component analysis, gene set enrichment analysis enrichment and mutation pattern analysis, generated a heatmap, and performed cox regression analysis. The two discrete subpopulations were found to exhibit contrasting mutational and immunogenomic characteristics, emphasizing the heterogeneity of CCA. These subsets also showed pronounced discrepancies in the infiltration of immune cells, indicating diverse interactions with the tumor immune microenvironment. Furthermore, the dissimilarities in mutational patterns were observed within the two CCA subgroups, with PBRM1 and BAP1 emerging as the most frequently mutated genes. In addition, a prognostic framework was formulated and validated utilizing the expression profiles of COX16 and RSAD2 genes, effectively segregating patients into high-risk and low-risk cohorts. Furthermore, the connections between immune-related parameters and these risk groups were identified, underscoring the potential significance of the immune microenvironment in patient prognosis. In vitro experiments have shown that COX16 promotes the proliferation and metastasis of CCA cells, whereas RSAD2 inhibits it. The present study provides an intricate depiction of the immunogenomic landscape of CCA based on CCL5 expression, thereby paving the way for novel immunotherapy strategies and prognostic assessment.