Cellular Behavior Research Articles

Unraveling the Biological Functions of UCEC: Insights from a Prognostic Signature Model

BackgroundUterine corpus endometrial carcinoma (UCEC) is a prevalent gynecological tumor with a bleak prognosis. Anomalous glycosylation plays a pivotal role in tumorigenesis. Currently, there is a lack of prognostic signatures based on glycosylation-related genes for UCEC. Thus, our research aims to construct a predictive model and validate the correlation between relevant genes and biological functions. MethodsUsing the TCGA database, we developed prognostic models and explored their relationships with survival outcomes. We further selected key genes to verify their expression in tissues and assess their impact on cellular behavior. ResultsThe clinical prognosis of the high-risk group was significantly worse than that of the low-risk group. The nomogram model accurately predicted UCEC patient prognosis. Additionally, we identified OLFML1 as a unique signature gene that can inhibit UCEC progression and reduce radiation resistance in vitro. ConclusionsOur model, which is based on glycosylation-related genes in UCEC, effectively identifies high-risk patients and provides valuable prognostic information. In addition, OLFML1 acts as a tumor suppressor in UCEC and enhances radiosensitivity, suggesting a new potential target for improving therapeutic efficacy.

Elucidating the Mechanism of Large-Diameter Titanium Dioxide Nanotubes in Protecting Osteoblasts Under Oxidative Stress Environment: The Role of Fibronectin and Albumin Adsorption.

Large-diameter titanium dioxide nanotubes (TNTs) have shown promise in preserving osteoblast function under oxidative stress (OS) in vitro. However, their ability to enhance osteogenesis in vivo under OS conditions and the underlying mechanisms remain unclear. This study aimed to evaluate the osteogenic potential of 110 nm TNTs (TNT110) compared to 30 nm TNTs (TNT30) in an aging rat model exhibiting OS, and to investigate the mechanisms involved. Surface properties of TNTs were characterized, and in vitro and in vivo experiments were conducted to assess their osteoinductive effects under OS. Transcriptomic, proteomic analyses, and Western blotting were performed to investigate the protective mechanisms of TNT110 on osteoblasts. Protein adsorption studies focused on the roles of fibronectin (FN) and albumin (BSA) in modulating osteoblast behavior on TNT110. In both in vitro and in vivo experiments, TNT110 significantly improved new bone formation and supported osteoblast survival under OS conditions. Subsequent ribonucleic acid sequencing results indicated that TNT110 tended to attenuate inflammatory responses and reactive oxygen species (ROS) expression while promoting endoplasmic reticulum (ER) stress and extracellular matrix receptor interactions, all of which are crucial for osteoblast survival and functionality. Further confirmation indicated that the cellular behavior changes of osteoblasts in the TNT110 group could only occur in the presence of serum. Moreover, proteomic analysis under OS conditions revealed the pivotal roles of FN and BSA in augmenting TNT110's resistance to OS. Surface pretreatment of TNT110 with FN/BSA alone could beneficially influence the early adhesion, spreading, ER activity, and ROS expression of osteoblasts, a trend not observed with TNT30. TNT110 effectively protects osteoblast function in the OS microenvironment by modulating protein adsorption, with FN and BSA synergistically enhancing osteogenesis. These findings suggest TNT110's potential for use in implants for elderly patients.

Cage-like micro-scaffolds fabricated by DLW method for cell investigation

This paper focuses on the use of direct laser writing method in fabricating three-dimensional biocompatible scaffolds that emulate the extracellular matrix. The interaction between HEK 293 cells and these cage-like scaffolds, particularly the effect of pore size on cell invasion, is explored in detail. Our study underscores the influence of scaffold architecture on cellular behavior and highlights the potential of direct laser writing technology in creating complex 3D scaffolds. The insights gleaned from this research could be invaluable in future applications such as tissue engineering, regenerative medicine, and drug delivery.

Locally released dexamethasone and its effects on osteogenic activity at implant-tissue interface.

The osseointegration of titanium implants within the host tissue holds crucial importance. The introduction of functional coatings at tissue-implant interface enhances the bioactivity of titanium implants, improves their therapeutic outcomes, and enhances the effectiveness of treatments. In this study, we focused on enhancing the bioactivity of titanium-based implant materials by coating the titanium surfaces with chitosan microspheres, which are loaded with osseointegration-promoting agent dexamethasone (DEX). Initially, chitosan microspheres were successfully produced, followed by DEX loading through diffusion, resulting in a drug loading efficiency of around 50.2 (wt %). The subsequent drug release profile displayed a 24-hour duration, releasing approximately 32.6 (wt %) of the loaded DEX. In cell proliferation assays using human osteosarcoma (SAOS-2) cells, Ti surfaces coated with DEX-loaded chitosan microspheres initially exhibited lower cell numbers compared with DEX-free ones. This observation was attributed to transient osteogenic differentiation effects of DEX, since a notable increase in cell proliferation was observed on the 7th day. Von Kossa staining revealed mineralization beginning on the 14th day, particularly evident in DEX-loaded samples. Moreover, alkaline phosphatase (ALP) activity displayed a pattern of initial increase and subsequent decrease, with DEX release from chitosan microspheres showing a clear influence on the osteogenic differentiation, especially on the 7th day. These findings align with literature, highlighting DEX's potential to enhance osteogenic differentiation and cellular behavior on chitosan microsphere-coated titanium surfaces. This study emphasizes the promising implications for functionalizing surfaces of implant materials with DEX-loaded chitosan microspheres to improve their biocompatibility and bioactivity.

Microstructure and compressive behaviour of PLA/PHA-wood lattice structures processed using additive manufacturing

This study investigates the microstructure and compressive behaviour of PLA/PHA-wood lattice structures manufactured using fused filament fabrication (FFF). The primary objective is to evaluate the effects of defects, such as porosity and surface roughness, on the mechanical properties of these lattice structures. X-ray micro-tomography (XRT) and finite element analysis are employed to compare CAD models with real lattice structures, offering insights into defect-induced performance deviations. The novel approach integrates experimental and numerical methods to better understand damage accumulation in porous structures. The methodology includes uniaxial compression testing and image processing for microstructural characterization. Results reveal significant differences in relative density and stress concentration due to manufacturing defects. In particular, 3D printed lattice structures display typical cellular material behaviour with a primary bending deformation mechanism. X-ray tomography reveals that process-induced porosity generate stress heterogeneity, which is not captured from CAD-based model resulting in an overestimation of the stiffness. The study concludes that accounting for these defects can be quantified by shifting the target relative density to compensate lack of performance in 3D-printed lattice materials.

Transcription factor ETS1‑mediated ECT2 expression promotes the malignant behavior of prostate cancer cells.

Prostate cancer remains the most prevalent malignancy diagnosed in men worldwide. Epithelial cell transforming sequence 2 (ECT2) is an oncogene involved in the progression of human tumors. The present study aimed to explore the involvement of ECT2 in prostate cancer and its participation in the malignant progression of prostate cancer. ECT2 expression in prostate cancer cell lines was examined via reverse transcription-quantitative PCR and western blotting. The effects of knockdown of ECT2 expression in PC-3 cells on cellular biological behaviors, including proliferation, migration and invasion, were examined using Cell Counting Kit-8, colony formation, wound healing and Transwell assays. The glycolysis level was determined based on the lactate release, glucose uptake, oxygen consumption rate and extracellular acidification rate. The binding relationship between ECT2 and ETS1 was verified using luciferase reporter and chromatin immunoprecipitation assays. The results indicated that ECT2 was highly expressed in prostate cancer cell lines. Knockdown of ECT2 expression could inhibit cell proliferation, migration, invasion and glycolysis. In addition, the transcription factor ETS1 could directly bind to the ECT2 promoter and positively regulate ECT2 expression. These data were combined with the results of rescue experiments and demonstrated that the inhibitory effects of the knockdown of ECT2 expression on the malignant behavior and glycolysis of prostate cancer cells were partially reversed by ETS1 overexpression. In conclusion, ETS1 induced transcriptional upregulation of ECT2 and enhanced the malignant biological behaviors of prostate cancer cells, thereby promoting the progression of prostate cancer. This evidence provides a theoretical basis for the treatment of prostate cancer.

Mesodermal fibronectin controls cell shape, polarity, and mechanotransduction in the second heart field during cardiac outflow tract development.

Failure in the elongation of the cardiac outflow tract (OFT) results in congenital heart disease due to the misalignment of the great arteries with the left and right ventricles. The OFT lengthens via the accretion of progenitors from the second heart field (SHF). SHF cells are exquisitely regionalized and organized into an epithelial-like layer, forming the dorsal pericardial wall (DPW). Tissue tension, cell polarity, and proliferation within the DPW are important for the addition of SHF-derived cells to the heart and OFT elongation. However, the genes controlling these processes are not completely characterized. Using conditional mutagenesis in the mouse, we show that fibronectin (FN1) synthesized by the mesoderm coordinates multiple cellular behaviors in the anterior DPW. FN1 is enriched in the anterior DPW and plays a role in OFT elongation by maintaining a balance between pro- and anti-adhesive cell-extracellular matrix (ECM) interactions and controlling DPW cell shape, polarity, cohesion, proliferation, and mechanotransduction.

Effects of phospholipase D1 inhibitory peptide on the growth and metastasis of gastric cancer cells

Phospholipase D1 (PLD1) contributes to cancer development and progression through its effects on cell proliferation, survival, invasion, metastasis, angiogenesis, drug resistance, and modulation of the tumor microenvironment. Its central role in these processes makes it a promising target for novel cancer treatments aimed at inhibiting its activity and disrupting the signaling pathways it regulates. In this study, we aimed to investigate the effect of PLD1 inhibition on gastric cancer cell growth using a novel peptide inhibitor, TAT-TVTSP. PLD1, which plays a role in cancer progression, catalyzes the conversion of phosphatidylcholine into choline and phosphatidic acid through hydrolysis. To effectively target PLD1 in cells, we engineered TAT-TVTSP by fusing a PLD1-inhibitory peptide (TVTSP) with a cell-penetrating peptide (TAT). We observed that TAT-TVTSP effectively inhibited PLD1 activity in AGS gastric cancer cells. Moreover, TAT-TVTSP significantly inhibited the mammalian target of the rapamycin signaling pathway, including the phosphorylation of key downstream targets such as S6K1, AKT, S473, glycogen synthase kinase-3b, and forkhead box O1. TAT-TVTSP did not induce cell death, but it triggered cell cycle arrest by activating p21 and p27 via AKT phosphorylation. Functional assays revealed that TAT-TVTSP significantly impaired the colony-forming ability of AGS cells, thus inhibiting cell proliferation. Transwell and wound-healing assays revealed that this peptide disrupted the cellular behaviors critical to cancer progression, such as migration and invasion. In vivo, TAT-TVTSP significantly reduced tumor growth in the xenograft model of gastric cancer without any toxicity. Overall, our results suggest that TAT-TVTSP is a novel therapeutic agent for PLD1-mediated cancers.

Downregulation of circSTX6 suppresses tumor progression while facilitating radiosensitivity in cervical squamous cell carcinoma

Backgroundcervical squamous cell carcinoma (CSCC) is the second gynecological tumors that seriously threaten women’s life quality. Circular RNA (circRNA) is related with cervical cancer carcinogenesis and radiosensitivity. AimTo investigate the performance of hsa_circ_0007905 (circSTX6) on regulating cellular activities and radiosensitivity in CSCC. MethodsThe relative expression of circSTX6 in different tissue samples was detected by RT-qPCR. The cellular activity influence of circSTX6 in cervical cancer cells was measured by CCK-8 and Transwell assays. The survival fractions of cancer cells were detected after the radiation treatment to explore the relationship between circSTX6 and radiosensitivity of cervical cancer. The downstream miRNAs were predicted and analyzed. Rescue experiments confirmed their targeting relationship. Bioinformatic analysis was performed to identify the potential targets of miR-203a-3p. ResultscircSTX6 was increased and miR-203a-3p was decreased in cervical cancer tissues and radio-resistant tissues. CircSTX6 expression was related to the patient’s survival rates. CircSTX6 absence decreased cervical cancer cell proliferation and invasion while enhancing the sensitivity of cervical cancer cells to radiotherapy by regulating miR-203a-3p. RAB27B may be a target of miR-203a-3p. ConclusioncircSTX6 may be a clinical prognostic biomarker in CSCC. The absence of circSTX6 inhibits cellular behaviors and increases the sensitivity of cervical cancer cells to radiation by modulating miR-203a-3p/RAB27B axis.

Time-varying stimuli that prolong IKK activation promote nuclear remodeling and mechanistic switching of NF-κB dynamics.

Temporal properties of molecules within signaling networks, such as sub-cellular changes in protein abundance, encode information that mediate cellular responses to stimuli. How dynamic signals relay and process information is a critical gap in understanding cellular behaviors. In this work, we investigate transmission of information about changing extracellular cytokine concentrations from receptor-level supramolecular assemblies of IκB kinases (IKK) downstream to the nuclear factor κB (NF-κB) transcription factor (TF). In a custom robot-controlled microfluidic cell culture, we simultaneously measure input-output (I/O) encoding of IKK-NF-κB in dual fluorescent-reporter cells. When compared with single cytokine pulses, dose-conserving pulse trains prolong IKK assemblies and lead to disproportionately enhanced retention of nuclear NF-κB. Using particle swarm optimization, we demonstrate that a mechanistic model does not recapitulate this emergent property. By contrast, invoking mechanisms for NF-κB-dependent chromatin remodeling to the model recapitulates experiments, showing how temporal dosing that prolongs IKK assemblies facilitates switching to permissive chromatin that sequesters nuclear NF-κB. Remarkably, using simulations to resolve single-cell receptor data accurately predicts same-cell NF-κB time courses for more than 80% of our single cell trajectories. Our data and simulations therefore suggest that cell-to-cell heterogeneity in cytokine responses are predominantly due to mechanisms at the level receptor-associated protein complexes.

REVIEW OF LITERATURE ON NANOSCALE TOPOGRAPHIC MODIFICATION, MAGNETIC NANOPARTICLES, AND THEIR ROLES IN OSSEOINTEGRATION AND BONE TISSUE ENGINEERING

Here, we will review together the roles of nanoscale modification of titanium substrates and use of magnetic nanoparticles in osseointegration and bone tissue engineering. The technological advances in nanotechnology that has been achieved create new measures to increase the implants-biological systems for an optimal osseointegration through improving important cell-specific functions. We systematically review nanoscale surface modification techniques of titanium implants, covering their physicochemical properties, influence on cellular behaviors and clinical relevances. The report also lengthy discusses the clinical applicability of magnetic nanoparticles for drug and gene delivery systems, targeted cancer therapy as well bone tissue engineering regulting their intrinsic superparamagnetism properites, biocompatibility with other therapies etc. We present detailed review of these technologies and their potential applications in clinical settings.

Impact of calcium propionate on cellular behavior in A549 and DMS114 lung cancer cell lines

Abstract Objectives To investigate the cellular and apoptotic effects of food additive calcium propionate by in vitro methods. Methods Cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, intracellular Glutathione (GSH) by luminometric method, mitochondrial membrane potential (MMP) by fluorometry, apoptosis by dual Acridine Orange/Ethidium Bromide (AO/EB) fluorescent staining were studied in lung cells. Results It has been observed that calcium propionate reduced GSH and MMP levels significantly while increased cytotoxicity, apoptosis, and reactive oxygen species (ROS) levels in both A549 and DMS114 cancer cells in a dose-dependent manner. Conclusions Cytotoxic effects of calcium propionate were more pronounced in cancer cells compared to healthy cells, suggesting its potential suitability as a chemo-preventive agent.

From Bioink to Tissue: Exploring Chitosan-Agarose Composite in the Context of Printability and Cellular Behaviour

This study presents an innovative method for producing thermosensitive bioink from chitosan hydrogels saturated with carbon dioxide and agarose. It focuses on a detailed characterisation of their physicochemical properties and potential applications in biomedicine and tissue engineering. The ORO test approved the rapid regeneration of the three-dimensional structure of chitosan–agarose composites in a unidirectional bench press simulation test. The diffusion of dyes through the chitosan–agarose hydrogel membranes strongly depended on the share of both polymers in the composite and the molecular weight of the dyes. Glucose, as a nutrient marker, also diffused through all membranes regardless of composition. Biocompatibility assessment using MTT tests on 46BR.1N fibroblasts and HaCaT keratinocytes confirmed the safety of the bioink. The regenerative potential of the bioink was confirmed by efficient cell migration, especially HaCaT. Long-term viability studies showed that chitosan–agarose scaffolds, unlike the agarose ones, support cell proliferation and survival, especially 14 days after bioink extrusion. Experiments in a skin wound model in mice confirmed the biocompatibility of the tested dressing and the beneficial action of chitosan on healing. Studies on vessel formation in chicken embryos highlight the potential of the chitosan–agarose composition to enhance proangiogenic effects. This composition meets all entry criteria and possesses excellent biological properties.

Water-Soluble Aromatic Nanobelt with Unique Cellular Internalization.

A water-soluble aromatic nanobelt was synthesized, and its cellular uptake behavior in HeLa cells was investigated. The late-stage functionalization of the parent methylene-bridged [6]cycloparaphenylene ([6]MCPP) provided an easily accessible alkyne-functionalized nanobelt in a single reaction step. The alkyne-substituted [6]MCPP was subjected to Cu-catalyzed azide-alkyne cycloaddition by using a dye-attached azide to obtain a water-soluble aromatic nanobelt. Cell-imaging experiments on the synthesized nanobelt in HeLa cells revealed stop-and-go cellular uptake dynamics. Similar experiments with control molecules and theoretical studies indicated that the unique dynamics of the nanobelt was derived from the belt-shaped structure.

Water‐Soluble Aromatic Nanobelt with Unique Cellular Internalization

A water‐soluble aromatic nanobelt was synthesized, and its cellular uptake behavior in HeLa cells was investigated. The late‐stage functionalization of the parent methylene‐bridged [6]cycloparaphenylene ([6]MCPP) provided an easily accessible alkyne‐functionalized nanobelt in a single reaction step. The alkyne‐substituted [6]MCPP was subjected to Cu‐catalyzed azide–alkyne cycloaddition by using a dye‐attached azide to obtain a water‐soluble aromatic nanobelt. Cell‐imaging experiments on the synthesized nanobelt in HeLa cells revealed stop‐and‐go cellular uptake dynamics. Similar experiments with control molecules and theoretical studies indicated that the unique dynamics of the nanobelt was derived from the belt‐shaped structure.

Biomimetic concentric microgrooved titanium surfaces influence bone marrow-derived mesenchymal stem cell osteogenic differentiation via H3K4 trimethylation epigenetic regulation.

Material surface micromorphology can modulate cellular behavior and promote osteogenic differentiation through cytoskeletal rearrangement. Bone reconstruction requires precise regulation of gene expression in cells, a process governed by epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling. We constructed osteon-mimetic concentric microgrooved titanium surfaces with different groove sizes and cultured bone marrow-derived mesenchymal stem cells (BMSCs) on the material surfaces to study how they regulate cell biological behavior and osteogenic differentiation through epigenetics. We found that the cells arranged in concentric circles along the concentric structure in the experimental group, and the concentric microgrooved surface did not inhibit cell proliferation. The results of a series of osteogenic differentiation experiments showed that the concentric microgrooves facilitated calcium deposition and promoted osteogenic differentiation of the BMSCs. Concentric microgrooved titanium surfaces that were 30 μm wide and 10 μm deep promoted osteogenic differentiation of BMSC by increasing WDR5 expression via H3K4 trimethylation upregulation.

Chemofunctionalization of knitted silk to improve interface connection in a nano/micro scaffold based on polycaprolactone-chitosan-multi-walled carbon nanotube/silk.

Nano/micro hybrid scaffolds in long-term healing tissue engineering can simultaneously offer both mechanical and biological properties. In this study, a hybrid scaffold was fabricated through electrospinning of polycaprolactone (PCL)-chitosan (Cs)/ multi-walled carbon nanotubes (MWCNTs) based nanofibers onto a chemically functionalized knitted silk substrate (F-Silk) and the scaffold were evaluated with regard to morphology, chemical and crystalline structure, hydrophilicity, mechanical properties, bioactivity, biodegradability, and cellular behavior. Chemical functionalization of silk using N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) resulted in greater integrity in the formation of nanofibers onto the microfibers. The presence of MWCNTs significantly reduced the contact angle of the scaffolds from 79.72°±2.72 to 68.92°±5.63. Chemical functionalization of silk, the presence of nanofiber coating, and the presence of MWCNTs increased the ultimate tensile strength of the hybrid scaffolds by 18%, 20%, and 30% compared to raw silk fabric, respectively. The presence of MWCNTs and chemical functionalization of knitted silk increased the bioactivity and reduced the degradation rate of hybrid scaffolds. The increase in the amount of carboxyl groups as a result of adding 0.5wt% of MWCNTs significantly improved the adhesion, growth and proliferation of chondrocyte cells on the hybrid scaffolds as observed through cell morphology. According to the obtained results, hybrid scaffold based on PCL-Cs-MWCNTs/F-silk can be a suitable option for further research in cartilage tissue engineering.

Evaluating ECM stiffness and liver cancer radiation response via shear-wave elasticity in 3D culture models

BackgroundThe stiffness of the tumor microenvironment (TME) directly influences cellular behaviors. Radiotherapy (RT) is a common treatment for solid tumors, but the TME can impact its efficacy. In the case of liver cancer, clinical observations have shown that tumors within a cirrhotic, stiffer background respond less to RT, suggesting that the extracellular matrix (ECM) stiffness plays a critical role in the development of radioresistance.MethodsThis study explored the effects of ECM stiffness and the inhibition of lysyl oxidase (LOX) isoenzymes on the radiation response of liver cancer in a millimeter-sized three-dimensional (3D) culture. We constructed a cube-shaped ECM-based millimeter-sized hydrogel containing Huh7 human liver cancer cells. By modulating the collagen concentration, we produced two groups of samples with different ECM stiffnesses to mimic the clinical scenarios of normal and cirrhotic livers. We used a single-transducer system for shear-wave-based elasticity measurement, to derive Young’s modulus of the 3D cell culture to investigate how the ECM stiffness affects radiosensitivity. This is the first demonstration of a workflow for assessing radiation-induced response in a millimeter-sized 3D culture.ResultsIncreased ECM stiffness was associated with a decreased radiation response. Moreover, sonoporation-assisted LOX inhibition with BAPN (β-aminopropionitrile monofumarate) significantly decreased the initial ECM stiffness and increased RT-induced cell death. Inhibition of LOX was particularly effective in reducing ECM stiffness in stiffer matrices. Combining LOX inhibition with RT markedly increased radiation-induced DNA damage in cirrhotic liver cancer cells, enhancing their response to radiation. Furthermore, LOX inhibition can be combined with sonoporation to overcome stiffness-related radioresistance, potentially leading to better treatment outcomes for patients with liver cancer.ConclusionsThe findings underscore the significant influence of ECM stiffness on liver cancer’s response to radiation. Sonoporation-aided LOX inhibition emerges as a promising strategy to mitigate stiffness-related resistance, offering potential improvements in liver cancer treatment outcomes.

A Scalable Method to Fabricate 2D Hydrogel Substrates for Mechanobiology Studies with Independent Tuning of Adhesiveness and Stiffness.

Mechanical signals from the extracellular matrix are crucial in guiding cellular behavior. Two-dimensional hydrogel substrates for cell cultures serve as exceptional tools for mechanobiology studies because they mimic the biomechanical and adhesive characteristics of natural environments. However, the interdisciplinary knowledge required to synthetize and manipulate these biomaterials typically restricts their widespread use in biological laboratories, which may not have the material science expertise or specialized instrumentation. To address this, we propose a scalable method that requires minimal setup to produce 2D hydrogel substrates with independent modulation of the rigidity and adhesiveness within the range typical of natural tissues. In this method, norbornene-terminated 8-arm polyethylene glycol is stoichiometrically functionalized with RGD peptides and crosslinked with a di-cysteine terminated peptide via a thiol-ene click reaction. Since the synthesis process significantly influences the final properties of the hydrogels, we provide a detailed description of the chemical procedure to ensure reproducibility and high throughput results. We demonstrate examples of cell mechanosignaling by monitoring the activation state of the mechanoeffector proteins YAP/TAZ. This method effectively dissects the influence of biophysical and adhesive cues on cell behavior. We believe that our procedure will be easily adopted by other cell biology laboratories, improving its accessibility and practical application.

Deciphering the Impact of Collagenase Treatment Duration on Primary Breast Cancer Cell Proteome: A Comprehensive Study

Primary cell isolation is essential for studying cellular behavior and disease mechanisms, with collagenase-mediated tissue dissociation playing a critical role in the process. However, the impact of collagenase treatment duration on the proteome of primary cells, particularly in breast cancer research, remains underexplored. This study aims to investigate the effects of collagenase II treatment duration on the proteomic profiles of primary breast cancer cells. Breast cancer tissues from patients diagnosed with infiltrating ductal carcinoma were treated with collagenase II for either 1 or 3 hours. Subsequent proteomic analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identified proteins were subjected to bioinformatic analyses to determine the functional implications of the proteomic changes induced by the different treatment durations. Bioinformatic analyses showed that 1-hour treatment predominantly affected proteins involved in cytoskeletal organization and cell adhesion, with significant enrichment in actin cytoskeleton dynamics and structural molecule activity. In contrast, 3-hour treatment led to significant metabolic reprogramming, with enhanced regulation of pathways involved in energy production, including the TCA cycle and glycolysis. This study reveals for the first time that, collagenase II treatment duration significantly alters the proteomic profile of primary breast cancer cells, with shorter durations affecting structural proteins and longer durations inducing metabolic changes. Optimizing treatment time is crucial for targeted proteomic studies.