Biological Similarities Research Articles

Evaluating cutinase from Fusarium oxysporum as a biocatalyst for the degradation of nine synthetic polymer

Plastic poses a significant environmental impact due to its chemical resilience, leading to prolonged and degradation times and resulting in widespread adverse effects on global flora and fauna. Cutinases are essential enzymes in the biodegradation process of synthetic polymers like polyethylene terephthalate (PET), which recognized organisms can break down. Here, we used molecular dynamics and binding free energy calculations to explore the interaction of nine synthetic polymers, including PET, with Cutinase from Fusarium oxysporum (FoCut). According to our findings, the polymers poly(ethylene terephthalate) (PET), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), poly(butylene succinate) (PBS), poly(butylene adipate-co-terephthalate) (PBAT) and poly(ε-caprolactone) (PCL) can bind to the Cutinase enzyme from F. oxysporum, indicating potential biodegradation activity for these polymers. PET exhibited the highest binding affinity (− 34.26 kcal/mol). Besides PET, the polymers PHBH, PBS, PBAT, and PCL also demonstrated significant affinities for the FoCut enzyme, with binding values of − 18.44, − 29.71, − 22.78, and − 22.26 kcal/mol, respectively. Additionally, analysis of the phylogenetic tree of cutinases produced by different organisms demonstrated that even though the organisms belong to different kingdoms, the cutinase from F. oxysporum (FoCut) showed biological similarity in its activity in degrading polymers with the cutinase enzyme from the bacterium Kineococcus radiotolerans and the fungus Moniliophthora roreri. Furthermore, the phylogenetic analysis demonstrated that the PETase enzyme has a very high similarity with the bacterial cutinase enzyme than with the fungal cutinase, therefore demonstrating that the PETase enzyme from Ideonella sakaiensis can easily be a modified bacterial cutinase enzyme that created a unique feature in biodegrading only the pet polymer through an evolutionary process due to its environment and its biochemical need for carbon. Our data demonstrate that bacterial cutinase enzymes have the same common ancestor as the PETase enzyme. Therefore, cutinases and PETase are interconnected through their biological similarity in biodegrading polymers. We demonstrated that important conserved regions, such as the Ser-Asp-His catalytic triad, exist in the enzyme’s catalytic site and that all Cut enzymes from different organisms have the same region to couple with the polymer structures.

Oronasal mucosal melanoma is defined by two transcriptional subtypes in humans and dogs with implications for diagnosis and therapy.

Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and limited existing therapeutic interventions, in part due to a lack of actionable targets and translational animal models for preclinical trials. Comprehensive data on this tumour type are scarce, and existing data often overlooks the importance of the anatomical site of origin. We evaluated human and canine oronasal mucosal melanoma (OMM) to determine whether the common canine disease could inform the rare human equivalent. Using a human and canine primary OMM cohort of treatment-naive archival tissue, alongside clinicopathological data, we obtained transcriptomic, immunohistochemical, and microbiome data from both species. We defined the transcriptomic landscape in both species and linked our findings to immunohistochemical, microbiome, and clinical data. Human and dog OMM stratified into two distinctive transcriptional groups, which we defined using a species-independent 41-gene signature. These two subgroups are termed CTLA4-high and MET-high and indicate actionable targets for OMM patients. To guide clinical decision-making, we developed immunohistochemical diagnostic tools that distinguish between transcriptomic subgroups. We found that OMM had conserved transcriptomic subtypes and biological similarity between human and canine OMM, with significant implications for patient classification, treatment, and clinical trial design. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Characterization for the Similarity Assessment Between the Proposed Biosimilar SB17 and Ustekinumab Reference Product Using State-of-the-Art Analytical Methods.

SB17 is being developed as a biosimilar to ustekinumab reference product (RP), a human monoclonal antibody (IgG1 kappa immunoglobulin) that binds to the common p40 subunit of cytokines interleukin (IL)-23 and IL-12. Binding to this subunit prevents interaction with their receptor, resulting in modulation in the immune system responses that play a key role in inflammatory disease. The objective of this study was to demonstrate structural, physicochemical, and biological similarity between ustekinumab RP and SB17 using various state-of-the-art analytical methods. Comprehensive analytical characterization was conducted by the quality range and side-by-side comparison approach for SB17 versus the European Union (EU) and US ustekinumab RP using various analytical methods. Comparisons included purity, product-related impurity, charge heterogeneity, primary structure, posttranslational modification, higher-order structure, quantity, Fab-related biological activities (potency and binding activity), and Fc-related biological activities. On the basis of the analytical similarity assessment, the structural, physicochemical, and biological characterization results demonstrated that SB17 is comparable to the ustekinumab RP. In the structural aspects, it was confirmed that there is no clinically meaningful difference between posttranslational modification profiles and higher-order structures of SB17 compared with the ustekinumab RP. Product-related impurities in the form of aggregates were also confirmed to be similar. SB17 has lower acidic and basic variants compared with ustekinumab RP, owing to the difference in the producing cell line. Ustekinumab RP is expressed in an Sp2/0 cell line, whereas SB17 is expressed in CHO cell line. However, the difference between SB17 and ustekinumab RP in the charge variants is not considered to be clinically meaningful, since equivalent biological activity was observed. In the case of mechanism of action (MoA)-related biological activities, SB17 is highly similar to the EU and US ustekinumab RP with respect to overall critical and noncritical quality attributes analyzed. Moreover, similarity or lack of activity in Fc-related biological activities was also confirmed. On the basis of these results, SB17 is expected to have comparable safety and efficacy as compared with ustekinumab RP. In summary, the overall analytical characterization and similarity assessment results show that SB17 is comparable to the EU and US ustekinumab RP in terms of structural, physicochemical, biophysical, and biological attributes.

Isolation of a Novel Caprine Eimeria christenseni Strain (GC) in Canary Islands and Analysis of Parasitological, Clinical, and Pathological Findings on Experimentally Infected Goat Kids.

Eimeria christenseni is considered among the most pathogenic Eimeria species in goats. The aim of this study was to isolate an E. christenseni strain and to assess its infectivity, pathogenicity, and ability to develop a protective immune response. After previous collection of E. christenseni-positive faeces, purification of oocysts, and amplification in donor animals, an experimental infection was carried out. A total of 19 kids were divided into three groups: primary-infected and challenged, challenge control, and uninfected control. Infections were performed orally with 2 × 105 sporulated oocysts per animal. Oocyst shedding, clinical signs, and production parameters, in addition to haematological and histopathological features, were monitored. The results showed that the Gran Canaria (GC) E. christenseni strain had similar morphological and biological characteristics to those previously described, but no significant clinical signs were observed despite the high oocyst counts here recorded. The novel strain isolated would therefore be of low pathogenicity but still able to develop significant immunoprotective responses upon challenge infections. Its biological similarities to highly pathogenic species such as Eimeria ninakohlyakimovae and Eimeria arloingi might enable comparative studies aimed at developing alternative strategies for drug treatments, including Eimeria species (strain)-specific vaccination strategies for the efficient control of goat coccidiosis.

The clinical utility of circulating human papillomavirus across squamous cell carcinomas.

The similarities in biology, treatment regimens and outcome between the different human papillomavirus (HPV) associated squamous cell carcinomas (SCCs) allow for extrapolation of results generated from one SC tumor type to another. In HPV associated cancers, HPV is integrated into the tumor genome and can consequently be detected in the circulating fragments of the tumor DNA. Thus, measurement of HPV in the plasma is a surrogate for circulating tumor DNA (ctDNA) and holds promise as a clinically relevant biomarker in HPV associated cancers. With the present overview we aim to present the status of circulating HPV studies in SCCs, the clinical potential and the gaps of knowledge, with the overall aim to facilitate the next steps into clinically relevant prospective trials. We reviewed the literature and presented the data for each tumor type as well as analyses of the clinical utility across the SCC. A total of 41 studies were identified in cervical, head and neck and anal SCC and we discuss the common signals from the results across the different tumor sites. Our results not only confirm the strong clinical potential but also emphasize an urgent need to coordinate studies to allow for relevant sample sizes and statistical validations.

Underrepresentation of Small Lymphocytic Lymphoma in Clinical Trials for Chronic Lymphocytic Leukemia.

Although chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are the same biologic disease entity and warrant identical treatment approaches, patients with SLL have frequently been excluded from clinical trials in CLL. This study assessed the representation of patients with SLL among Phase II or III clinical trials cited in the 2024 National Comprehensive Cancer Network (NCCN) treatment guidelines. Patients with SLL were explicitly eligible for only 21 (38%) of the 56 clinical trials for CLL, comprising 222 (6%) of the 3440 enrolled patients. Notably, 380 patients with SLL were enrolled in 16 separate non-CLL clinical trials alongside patients with indolent B-cell lymphomas such as follicular lymphoma. In CLL trials, patients with SLL were included in a greater proportion of studies evaluating BTK inhibitors (67%) or BTK/BCL2 inhibitor combinations (67%) compared to BCL2 inhibitors (0%) or chemoimmunotherapy (0%). Although recent and upcoming trials show a promising trend toward the inclusion of patients with SLL, further advocacy is needed to raise awareness of the biological similarities between CLL and SLL and to promote the representation of patients with SLL in CLL/SLL clinical research.

Perceived Biological Bases of Sexual Orientation and Sexual Prejudice: The Moderating Role of Gender and Religious Beliefs

AbstractPrior correlational studies have shown that belief in the biological theory of sexual orientation (BTSO) is associated with more positive attitudes toward homosexuality. However, individuals often interpret scientific evidence in ways that align with their pre-existing beliefs and motivations. This research experimentally investigated whether gender and religiosity moderate heterosexual individuals’ responses to scientific evidence either supporting or refuting BTSO. In two studies, heterosexual men (Study 1, N = 118) and both men and women (Study 2, N = 280) reported their religiosity and were exposed to evidence suggesting either biological differences or similarities between heterosexual and gay individuals. Results showed that, in the biological differences condition, heterosexual women and less religious men were more likely to perceive homosexuality as a natural biological variation and expressed more positive attitudes toward it. In contrast, more religious men interpreted the same evidence as indicating a biological anomaly in gay individuals and displayed more negative attitudes toward homosexuality.

Development and early life stress sensitivity of the rat cortical microstructural similarity network.

Network models derived from neuroimaging have revolutionized our understanding of human brain development but need translation into animal models to interrogate their underlying mechanisms. Rats provide a valuable model due to their complex behaviors and biological similarities to humans; however, in vivo models of individual-level brain networks remain underdeveloped. In this study, we present a novel computational pipeline to construct such networks from in vivo rat structural neuroimaging data. Our findings highlight the dynamic development and experiential sensitivity of fronto-hippocampal systems in rats, offering a reference for cross-species comparisons and mechanistic insights into brain architecture. To support broader research efforts, we include an open release of code and data for rat MIND similarity network analysis.

Disentangled similarity graph attention heterogeneous biological memory network for predicting disease-associated miRNAs

BackgroundThe association between MicroRNAs (miRNAs) and diseases is crucial in treating and exploring many diseases or cancers. Although wet-lab methods for predicting miRNA-disease associations (MDAs) are effective, they are often expensive and time-consuming. Significant advancements have been made using Graph Neural Network-based methods (GNN-MDAs) to address these challenges. However, these methods still face limitations, such as not considering nodes’ deep-level similarity associations and hierarchical learning patterns. Additionally, current models do not retain the memory of previously learned heterogeneous historical information about miRNAs or diseases, only focusing on parameter learning without the capability to remember heterogeneous associations.ResultsThis study introduces the K-means disentangled high-level biological similarity to utilize potential hierarchical relationships fully and proposes a Graph Attention Heterogeneous Biological Memory Network architecture (DiGAMN) with memory capabilities. Extensive experiments were conducted across four datasets, comparing the DiGAMN model and its disentangling method against ten state-of-the-art non-disentangled methods and six traditional GNNs. DiGAMN excelled, achieving AUC scores of 96.35%, 96.10%, 96.01%, and 95.89% on the Data1 to Data4 datasets, respectively, surpassing all other models. These results confirm the superior performance of DiGAMN and its disentangling method. Additionally, various ablation studies were conducted to validate the contributions of different modules within the framework, and’s encoding statuses and memory units of DiGAMN were visualized to explore the utility and functionality of its modules. Case studies confirmed the effectiveness of DiGAMN’s predictions, identifying several new disease-associated miRNAs.ConclusionsDiGAMN introduces the use of a disentangled biological similarity approach for the first time and successfully constructs a Disentangled Graph Attention Heterogeneous Biological Memory Network model. This network can learn disentangled representations of similarity information and effectively store the potential biological entanglement information of miRNAs and diseases. By integrating disentangled similarity information with a heterogeneous attention memory network, DiGAMN enhances the model’s ability to capture and utilize complex underlying biological data, significantly outperforming many existing models. The concepts used in this method also provide new perspectives for predicting miRNAs associated with diseases.

Cognitive Map Construction Based on Grid Representation

Abstract This paper investigates a grid-representation-based approach to spatial cognition for intelligent agents, aiming to develop an effective neural network model that simulates the functions of the olfactory cortex and hippocampus for spatial cognition and navigation. Despite progress made by existing models in simulating biological nervous system functions, issues such as model simplification, lack of biological similarity, and practical application challenges remain. To address these issues, this paper proposes a neural network model that integrates grid representation, reinforcement learning, and encoding/decoding techniques. The model forms a grid representation by simulating the integration of grid cells in the medial entorhinal cortex (MEC) with perceptual information from the lateral entorhinal cortex (LEC), which encodes and retains spatial location information. By leveraging attractor networks, convolutional neural networks (CNNs), and multilayer perceptrons (MLPs), the model achieves the storage of spatial location and environmental information, as well as the construction of cognitive maps. The experimental results show that after using this model, the map generation accuracy increased by 15%, the navigation accuracy of the agent in complex environments by 20%, and the target localization error was reduced to less than 10%, demonstrating a significant overall performance improvement in the grid-based cognitive map construction.

The Rhesus Macaque as an Animal Model for Human Nutrition: An Ecological-Evolutionary Perspective.

Nutrition is a complex and contested area in biomedicine, which requires diverse evidence sources. Nonhuman primate models are considered an important biomedical research tool because of their biological similarities to humans, but they are typically used with little explicit consideration of their ecology and evolution. Using the rhesus macaque (RM), we consider the potential of nutritional ecology for enriching the use of primates as models for human nutrition. We introduce some relevant aspects of RM evolutionary and social ecology and discuss two examples where they have been used in biomedical research: obesity and aging. We next consider how insights from nutritional ecology can help inform and direct the use of RM as a biomedical model. We conclude by illustrating how conceptual tools might inform the use of RM as a model for human nutrition and extracting insights from RM that might be relevant to broader theoretical considerations around animal model systems.

PD-1 blockade plus cisplatin-based chemotherapy in patients with small cell/neuroendocrine bladder and prostate cancers

Small cell neuroendocrine cancers share biologic similarities across tissue types, including transient response to platinum-based chemotherapy with rapid progression of disease. We report a phase 1b study of pembrolizumab in combination with platinum-based chemotherapy in 15 patients with stage III-IV small cell bladder (cohort 1) or small cell/neuroendocrine prostate cancers (cohort 2). Overall response rate (ORR) is 43% with two-year overall survival (OS) rate of 86% (95% confidence interval [CI]: 0.63, 1.00) for cohort 1 and 57% (95% CI: 0.30, 1.00) for cohort 2. Treatment is tolerated well with grade 3 or higher adverse events occurring in 40% of patients with no deaths or treatment cessation secondary to toxicity. Single-cell and Tcell receptor sequencing of serial peripheral blood samples reveals clonal expansion of diverse Tcell repertoire correlating with progression-free survival. Our results demonstrate promising efficacy and safety of this treatment combination and support future investigation of this biomarker. This study was registered at ClinicalTrials.gov (NCT03582475).

GBNSS: A Method Based on Graph Neural Networks (GNNs) for Global Biological Network Similarity Search

Biological network similarity search plays a crucial role in the analysis of biological networks for human disease research and drug discovery. A biological network similarity search aims to efficiently identify novel networks biologically homologous to the query networks. Great progress has been achieved in biological network similarity searches. However, it remains a challenge to mine the biological network information fully to improve the accuracy of query results without increasing time overheads. In this study, we propose a biological network similarity search method based on graph neural networks named GBNSS, which combines topological and biological information (GO annotations) of biological networks into graph neural networks to find topologically and biologically similar biological networks in the database. Additionally, GBNSS is a topology-free biological network similarity search method with an arbitrary network structure. The experimental results on four benchmark datasets show that GBNSS outperforms the existing methods in terms of computational efficiency and search accuracy. Case studies further demonstrate that GBNSS is capable of searching similar networks in real-world biological networks.

Detection of PIK3CA hotspot mutations in canine mammary tumors using droplet digital PCR: tissue validation and liquid biopsy feasibility

Domestic dogs (Canis lupus familiaris) serve as valuable translational models for human cancer research due to their biological similarities. Canine mammary tumors (CMTs), frequently diagnosed in female dogs, share various characteristics with human breast cancers. This study investigates the PIK3CA (H1047R) mutation in CMTs using droplet digital PCR (ddPCR) and explores the potential of liquid biopsy for non-invasive detection. We analyzed 80 formalin-fixed, paraffin-embedded (FFPE) CMT tissue samples and compared ddPCR results with next-generation sequencing (NGS) data, achieving high concordance. Plasma and serum samples were also assessed for mutation concordance with tissue results. Our findings indicate a higher frequency of the PIK3CA (H1047R) mutations in benign and grade I malignant CMTs compared to more aggressive malignancies. The ddPCR assay demonstrated high sensitivity and specificity, with plasma testing showing 78.6% sensitivity and 87.5% specificity, and serum testing showing 66.7% sensitivity and 90.0% specificity. These results highlight the viability of liquid biopsy as a minimally invasive method for monitoring PIK3CA mutations in canine patients. The study suggests that liquid biopsy techniques hold significant promise for improving the early detection and monitoring of canine cancers, warranting further research to refine these methods and explore their applications in canine cancer diagnostics and treatment.

RECAPITULATING THE POST-SURGICAL BRAIN MICROENVIRONMENT OF ATYPICAL TERATOID RHABDOID TUMOURS TO IDENTIFY MEMBRANE PROTEINS FOR TARGETED THERAPY

Abstract AIMS Atypical Teratoid Rhabdoid Tumours (AT/RT) are rare aggressive tumours primarily arising in the hind brain of children under three, conferring median survival of <12 months. Surgery remains the only standard treatment; however tumours recur from post-surgical residual disease. Decellularised human brain cerebellum aim to recapitulate AT/RT post-surgical brain microenvironments which retain extracellular matrix (ECM) ligands, and where AT/RT plasma membrane proteins are hypothesised to represent viable molecular therapy targets. METHOD AT/RT, astrocyte and human brain cerebellum (decellularised and non-decellularised) membrane protein fractions were extracted and analysed using liquid chromatography-mass spectrometry. Proteomic analysis tools were integrated to identify differential expressed proteins and identify shared correlations. RESULTS Decellularised cerebellar ECM exhibited an enriched membrane proteome relative to total proteome, evidence of complete removal of intracellular components. Furthermore, decellularised and non-decellularised cerebellar tissue expressed comparable levels of membrane proteins and protein networks, indicative of ECM ligand retention. AT/RT and astrocyte KEGG Pathway analysis revealed specific oncology-based pathway expression in membrane protein compared to total protein, reinforcing membrane proteins as more visible therapeutic targets. Between the top 20 expressed proteins, ATP1A1 was the only membrane protein shared exclusively between all AT/RT cells and astrocytes, suggesting a critical functional role. Membrane proteins showing shared expression when normalised against each AT/RT cell line included ATP1A1, HSPD1, GNA13 and SLC3A2, indicating biological similarities between AT/RT molecular subtypes, which may offer candidate ubiquitous therapy targets. Membrane proteins expressed similarly in AT/RT-MYC molecular subtypes include ATP1A1, SLC7A5 and EEF1A1. STRING membrane pathway analysis also identified shared proteins between AT/RT and astrocyte cell populations. CONCLUSION AT/RT membrane protein pathways provide ideal therapeutic targets directly amenable for drug repurposing. Future work prioritises proteomic analysis utilising 3D AT/RT spheroids and cerebellum brain tissue to maximise accurately recapitulating the AT/RT post-surgical microenvironment. Cerebellum decellularisation still permits retention of essential proteins and ligands.

P19-61 Combining chemical, biological similarity and metabolite data in read-across approach

P19-61 Combining chemical, biological similarity and metabolite data in read-across approach

Carvacrol-loaded premixed calcium phosphate bone cements with exceptional osteogenic and antibacterial properties to heal infected bone defects

The healing of infected bone fractures represents a significant clinical challenge in orthopedic surgery. Calcium phosphate cements (CPCs) are attractive materials, highly applicable in bone healing due to their satisfactory biological properties and chemical similarity to bone minerals. However, manual mixing is often required before localized application of conventional CPCs, increasing the risk of infection. Moreover, their antibacterial properties are often insufficient for treating infected fractures. In this study, antimicrobial two-paste premixed CPCs loaded with carvacrol were developed. The influence of the carvacrol on the setting properties and mechanical strength of the cement was studied. In vitro studies demonstrated the biocompatibility, osteogenic potential, and broad-spectrum antimicrobial efficacy of the premixed bone cements, including effectiveness against gram-positive, gram-negative, and drug-resistant bacteria. Notably, in vivo studies revealed exceptional antimicrobial and osteogenic properties of the carvacrol-loaded cements, confirming the promising potential of the premixed cements for the healing of infected bone defects.

Beyond the petri dish: Model organisms in non-clinical exploration

Animal models have been integral to scientific research for decades, offering invaluable insights into complex biological processes, advancing medical knowledge, and aiding the development of new treatments. This review explores the significance of animal models in various research fields, emphasizing their biological similarity to humans as a key factor. Animals, ranging from mice and rats to primates, fruit flies, and worms, share fundamental physiological, anatomical, and genetic characteristics with humans, making them crucial proxies for studying diseases and biological pathways. This review underscores the essential role of animal models in preclinical research, particularly in evaluating drug candidates for safety, efficacy, and potential side effects before human trials. The controlled experimental environment provided by animal models allows researchers to manipulate variables, isolate factors, and establish cause-and-effect relationships, enhancing the understanding of complex genetic disorders.

The EphA2 Receptor Regulates Invasiveness and Drug Sensitivity in Canine and Human Osteosarcoma Cells.

Osteosarcoma is an aggressive bone cancer affecting both humans and dogs, often leading to pulmonary metastasis. Despite surgery and chemotherapy being the primary treatment modalities, survival rates remain low in both species, underscoring the urgent need for more efficacious therapeutic options. Accumulating evidence indicates numerous biological and clinical similarities between human and canine osteosarcoma, making it an ideal choice for comparative oncological research that should benefit both species. The EphA2 receptor has been implicated in controlling invasive responses across different human malignancies, and its expression is associated with poor prognosis. In this study, we utilized a comparative approach to match EphA2 functions in human and canine osteosarcoma models. Our objectives were to assess EphA2 levels and its pro-malignant action in osteosarcoma cells of both species. We found that EphA2 is overexpressed in most of both canine and human osteosarcoma cell lines, while its silencing significantly reduced cell viability, migration, and invasion. Moreover, EphA2 silencing enhanced the sensitivity of osteosarcoma cells to cisplatin, a drug commonly used for treating this cancer. Furthermore, inhibition of EphA2 expression led to a significant reduction in tumor development capability of canine osteosarcoma cells. Our data suggest that these EphA2 effects are likely mediated through various signaling mechanisms, including the SRC, AKT, and ERK-MAPK pathways. Collectively, our findings indicate that EphA2 promotes malignant behaviors in both human and canine osteosarcoma and that targeting EphA2, either alone or in combination with chemotherapy, could offer potential benefits to osteosarcoma patients.

Promoting biological similarity by collagen microfibers in 3D colorectal cancer-stromal tissue: Replicating mechanical properties and cancer stem cell markers

The extracellular matrix (ECM) of cancer tissues is rich in dense collagen, contributing to the stiffening of these tissues. Increased stiffness has been reported to promote cancer cell proliferation, invasion, metastasis, and prevent drug delivery. Replicating the structure and mechanical properties of cancer tissue in vitro is essential for developing cancer treatment drugs that target these properties. In this study, we recreated specific characteristics of cancer tissue, such as collagen density and high elastic modulus, using a colorectal cancer cell line as a model. Using our original material, collagen microfibers (CMFs), and a constructed three-dimensional (3D) cancer-stromal tissue model, we successfully reproduced an ECM highly similar to in vivo conditions. Furthermore, our research demonstrated that cancer stem cell markers expressed in the 3D cancer-stromal tissue model more closely mimic in vivo conditions than traditional two-dimensional cell cultures. We also found that CMFs might affect an impact on how cancer cells express these markers. Our 3D CMF-based model holds promise for enhancing our understanding of colorectal cancer and advancing therapeutic approaches. Statement of significanceReproducing the collagen content and stiffness of cancer tissue is crucial in comprehending the properties of cancer and advancing anticancer drug development. Nonetheless, the use of collagen as a scaffold material has posed challenges due to its poor solubility, hindering the replication of a cancer microenvironment. In this study, we have successfully recreated cancer tissue-specific characteristics such as collagen density, stiffness, and the expression of cancer stem cell markers in three-dimensional (3D) colorectal cancer stromal tissue, utilizing a proprietary material known as collagen microfiber (CMF). CMF proves to be an ideal scaffold material for replicating cancer stromal tissue, and these 3D tissues constructed with CMFs hold promise in contributing to our understanding of cancer and the development of therapeutic drugs.