This review paper provides a comprehensive analysis of the advancements, challenges, and future directions of 4D printing in biomedical engineering. We discuss the development of smart materials, including stimuli-responsive polymers, shape-memory materials, and bio-inks, as well as the various fabrication techniques employed, such as direct-write assembly, stereolithography, and multi-material jetting. Despite the promising advances, several challenges persist, including material limitations related to biocompatibility, mechanical properties, and degradation rates; fabrication complexities arising from the integration of multiple materials, resolution and accuracy, and scalability; and regulatory and ethical considerations surrounding safety and efficacy. As we explore the future directions for 4D printing, we emphasise the need for material innovations, fabrication advancements, and emerging applications such as personalised medicine, nanomedicine, and bioelectronic devices. Interdisciplinary research and collaboration between material science, biology, engineering, regulatory agencies, and industry are essential for overcoming challenges and realising the full potential of 4D printing in the biomedical engineering landscape. The advent of 3D printing technology has transformed various fields, including biomedical engineering, by allowing the fabrication of complex structures with high precision and accuracy . Over th3D printing, also known as additive manufacturing, has become an indispensable tool in the field of biomedical engineering over the past few decades. This technology allows for the creation of three-dimensional objects by depositing materials layer-by-layer based on a digital model ,9. The vOne significant application of 3D printing in biomedical engineering is the fabrication of patient-specific implants and prosthetics . Custom-Tissue engineering and regenerative medicine have also benefited from 3D printing technology. By utilising biocompatible materials, such as hydrogels and bio-inks ,13, reseFurthermore, 3D printing has enabled the development of innovative drug delivery systems by allowing for precise control over the shape, size, and composition of drug carriers, such as microcapsules, microneedles, and tablets. These customisable systems can improve drug release kinetics, enhance bioavailability, and enable personalised dosing .Despite the significant advancements achieved with 3D printing in biomedical engineering, there is a growing need for dynamic, stimuli-responsive structures that can better replicate the complex behaviour of living systems. The emergence of 4D printing technology has the potential to address this need and further expand the capabilities of additive manufacturing in the biomedical field.4D printing is an advanced form of additive manufacturing that combines the principles of 3D printing with the use of smart materials that are capable of changing their shape, properties, or functionality over time in response to external stimuli . The terThe significance of 4D printing in biomedical engineering lies in its potential to transform various aspects of healthcare, including tissue engineering, drug delivery systems, medical devices, and diagnostics. The dynamic nature of 4D-printed structures allows for the development of more sophisticated and adaptive devices that can mimic the complex behaviour of living systems, ultimately improving therapeutic outcomes and enabling patient-specific treatments .For instance, in tissue engineering and regenerative medicine, 4D printing can be used to create constructs that transform into the desired shape or structure upon implantation, facilitating better integration with the surrounding tissue and promoting cell growth and differentiation . SimilarMoreover, 4D printing has the potential to enhance the performance of medical devices and prosthetics by enabling self-adjusting, self-assembling, or self-healing capabilities, improving patient comfort, and ensuring a more precise fit. In diagnostics, 4D-printed sensors and devices could adapt to various biological conditions, providing more accurate and reliable detection of biomarkers, pathogens, or other analytes ,19.The primary objective of this review paper is to provide a comprehensive and up-to-date overview of the advancements, challenges, and future directions in the field of 4D printing for biomedical engineering applications. As 4D printing technology continues to evolve, it is crucial for researchers, engineers, and clinicians to understand its implications, opportunities, and limitations in the biomedical field. To achieve this objective, the review paper is structured to provide a detailed analysis of the advancements in 4D printing for biomedical applications, including the development of smart materials, such as stimuli-responsive polymers, shape-memory materials, and bio-inks, and the various fabrication techniques employed, such as direct-write assembly, stereolithography, and multi-material jetting.A thorough exploration of the various biomedical applications of 4D printing, including tissue engineering and regenerative medicine, drug delivery systems, medical devices and prosthetics, and diagnostic tools will be conducted, as well as an examination of the challenges associated with 4D printing in biomedical engineering, such as material limitations related to biocompatibility, mechanical properties, and degradation rates; fabrication complexities arising from the integration of multiple materials, resolution and accuracy, and scalability; and regulatory and ethical considerations surrounding safety, efficacy, and intellectual property. A discussion of the future directions and potential impact of 4D printing on the biomedical engineering field will also be presented, emphasising the need for material innovations, fabrication advancements, and emerging applications such as personalised medicine, nanomedicine, and bioelectronic devices.By providing a comprehensive overview of the current state and prospects of 4D printing in biomedical engineering, this review paper aims to serve as a valuable resource for researchers, engineers, and clinicians interested in harnessing the potential of this transformative technology to improve healthcare and patient outcomes.Over the last few years, advancements in 4D printing have significantly impacted the field of biomedical applications. This innovative technology has the potential to reshape the biomedical sector, offering promising solutions for various challenges. In the following sections, we will discuss various materials and manufacturing processes employed for 4D printing. We will then discuss an array of biomedical applications for 4D printing.Materials and manufacturing techniques for 4D printing are essential for achieving the desired shape-shifting and functionality in printed objects. In 4D printing, smart materials are utilised due to their ability to change properties under specific environmental conditions or stimuli. Advanced manufacturing techniques for 4D printing enable the precise fabrication of complex, multi-material structures with tailored responses to specific stimuli. These techniques have evolved to meet the unique requirements of 4D printing, including the ability to integrate smart materials and achieve high-resolution features. A brief overview of these materials and techniques can be found in the subsequent sections.The development of smart materials that can respond to external stimuli is a critical aspect of 4D printing in biomedical engineering. These materials enable the creation of dynamic structures capable of transforming their shape or properties in response to changes in the environment. Incorporating smart materials into 4D printing processes allows researchers to create dynamic structures with transformative potential across the healthcare field.Shape-memory polymers (SMPs) are one example of smart materials used in 4D printing. SMPs can transition between a deformed state and their original shape upon exposure to a specific stimulus, such as heat or light . This prHydrogels are another example of smart materials in 4D printing. These highly absorbent polymer networks can swell or shrink in response to changes in their surrounding environment, such as temperature or pH ,25. HydrThermo-responsive polymers are a class of smart materials used in 4D printing that exhibit significant changes in their physical properties, such as shape, stiffness, or volume, upon exposure to varying temperatures ,28,29. FElectroactive polymers and magnetically responsive materials, which can alter their shape or properties in response to electrical stimulation or magnetic fields, respectively, are examples of smart materials used in 4D printing with significant potential in the biomedical field. These materials have been employed in the development of advanced medical devices, such as soft robotic prosthetics and targeted drug delivery systems. By harnessing their unique properties, researchers are exploring novel applications in areas such as tissue engineering, bio-inspired devices, and personalised healthcare, paving the way for innovative solutions to complex biomedical challenges ,30,31. FIn addition to these smart materials, bio-inks play a crucial role in 4D printing applications, particularly in the realm of biomedical engineering. Bio-inks are biocompatible materials that often contain living cells, allowing for the creation of structures that can transform and develop into functional tissue over time. By combining bio-inks with the dynamic capabilities of 4D printing, researchers can fabricate complex, multi-layered structures that mimic the natural architecture of human tissues ,33,34. FFor polymer-based additive manufacturing, the rheological properties of polymers play a significant role in the success and feasibility of 4D printing for biomedical applications . The floIn 4D printing for biomedical applications, a wide range of materials are being explored and utilised to achieve desired functionalities and performance. These materials can be categorised into different types, including herbal, bio-based, synthetic, and hybrid materials. Herbal materials, derived from natural sources such as plants or traditional medicines, offer the potential for biocompatibility, biodegradability, and the incorporation of bioactive compounds. Bio-based materials, typically derived from renewable resources, encompass polymers, hydrogels, and composites that exhibit biocompatibility and can mimic the extracellular matrix of tissues. Synthetic materials, such as thermoplastics and shape-memory polymers, offer high mechanical strength, durability, and precise control over shape-memory properties. These materials can be tailored to meet specific requirements, including biocompatibility, degradation rates, and mechanical properties. Hybrid materials combine different types of materials, such as combining natural polymers with synthetic additives or incorporating bioactive agents into synthetic matrices. These hybrids can enhance properties such as mechanical strength, bioactivity, and controlled release capabilities. The choice of material in 4D printing for biomedical applications depends on factors such as the desired functionality, biocompatibility, degradation profile, mechanical requirements, and the targeted tissue or organ. Researchers continue to explore and develop new materials, as well as optimise existing ones, to expand the possibilities and applications of 4D printing in the field of biomedical engineering.Various fabrication techniques have been adapted for 4D printing, with each method offering specific advantages and limitations depending on the materials used and the desired application. Below are the typical manufacturing processes used for 4D printing.Fused Deposition Modelling (FDM) is a widely used 3D printing technique that involves the extrusion of a thermoplastic filament through a heated nozzle, which deposits the material layer by layer to create the desired object [d object . Figure d object . For exad object .Stereolithography (SLA) is a 3D printing technique that uses a light source, usually a laser, to selectively cure a photosensitive resin layer by layer [by layer . A sketcby layer ,42.Digital Light Processing (DLP) is similar to SLA, but instead of using a laser, it employs a digital light projector to cure the resin. Selective Laser Sintering (SLS) is a powder-based 3D printing technique that uses a laser to selectively sinter or fuse powdered material layer by layer, as illustrated in Multi-Material Jetting involves the simultaneous deposition of multiple materials during the printing process, allowing for the creation of 4D-printed structures with varying mechanical properties or responsiveness to stimuli [ stimuli . A schem stimuli .Direct Ink Writing (DIW) is an extrusion-based printing technique that involves the deposition of a viscous ink, often containing particles or fibres, to create the desired structure. A schematic view of the DIW process is shown in The successful implementation of additive manufacturing techniques relies on carefully selecting the operational conditions and process parameters, taking into account a multitude of factors such as material selection, geometry of the printed object, and the intended final application. Each additive manufacturing technique requires tailored settings to achieve optimal results. Variables such as print speed, layer thickness, temperature, and energy input must be optimised to ensure the proper adhesion, dimensional accuracy, and mechanical properties of the printed object. Furthermore, factors such as support structures, post-processing requirements, and even environmental conditions may influence the choice of operational conditions. Considering the unique combination of material properties, geometry, and desired application is crucial to determine the most suitable parameters, resulting in high-quality, functional, and customised additive manufacturing outcomes.The emergence of 4D printing technology has brought about remarkable progress in the creation of smart implants and prosthetics. By adding the element of time as the fourth dimension, 4D-printed devices can self-transform or adapt in response to specific stimuli, such as temperature, light, moisture, or magnetic fields. This unique attribute has led to a significant shift in the field of biomedical engineering, particularly in the design and fabrication of customisable and adaptable implants and prosthetics.One example of customisation and adaptability in smart implants and prosthetics is the development of personalised dental implants . By usin4D-printed smart implants and prosthetics have the potential to reduce the need for invasive surgeries and adjustments. Traditional implants and prosthetics often necessitate multiple surgical interventions for installation, replacement, or adjustments, which can be physically and emotionally taxing for patients. In contrast, 4D-printed devices can adapt and adjust themselves according to the body’s changing needs ,55,56. FSmart implants can be designed to change their shape or functionality over time, allowing them to accommodate the patient’s needs better. For example, a 4D-printed spinal implant can gradually change its shape to support the spine as it heals, potentially reducing the risk of complications and promoting faster recovery ,60.4D printing technology has demonstrated remarkable potential in the development of advanced drug delivery systems. These systems offer significant advantages over traditional methods, including controlled release of medication, patient-specific dosing, and targeted delivery, ultimately leading to improved treatment outcomes and patient experiences .Controlled release of medication is a vital aspect of modern drug delivery systems. By utilising 4D-printed materials that respond to specific stimuli, such as temperature or pH changes, researchers can create devices capable of releasing drugs at a precise rate over a predetermined period . For exaAnother example is the development of 4D-printed, personalised drug-eluting implants for cancer treatment. These implants can be designed to release chemotherapeutic agents at a controlled rate in response to specific triggers, such as changes in pH or the presence of particular biomarkers. This targeted drug delivery approach ensures that patients receive the appropriate dosage of medication precisely when and where it is needed, minimising the risk of side effects and increasing the overall effectiveness of the treatment .Patient-specific dosing and targeted delivery are crucial aspects of advanced drug delivery systems. Utilising the potential of 4D printing technology, researchers can develop devices customised to individual patients’ requirements, ensuring optimal dosing and minimising adverse effects. For example, a bioinspired 4D-printed hydrogel capsule designed for smart controlled drug release can respond to environmental stimuli, such as changes in pH or temperature, and release the medication accordingly . AdditioBy creating 4D-printed drug carriers that react to particular cellular or environmental triggers, researchers can ensure that medication is directly administered to the intended site of action. An example is the utilisation of 4D printing to design smart gastroretentive, esophageal, and intravesical drug delivery systems . These 44D printing technology has shown immense promise in the fields of tissue engineering and regenerative medicine ,68. By iBio-inks, which are biocompatible materials containing living cells, are a critical component of 4D printing in tissue engineering . By combFor example, cartilage tissue can be 4D-printed using a bio-ink containing human mesenchymal stromal cells and a swelling-dependent hydrogel . Upon swIn addition to cartilage, 4D printing has also been used to create functional blood vessels. By utilising a bio-ink containing endothelial cells (which form the lining of blood vessels), researchers have successfully 4D-printed sophisticated structures with potential applications in vascularised tissues. An example is the 4D biofabrication of T-shaped vascular bifurcation, which is a critical component of blood vessel networks . Figure The potential for organ transplantation alternatives is a significant area of interest within the realm of tissue engineering and regenerative medicine. Due to the severe shortage of donor organs and the risks associated with transplantation, such as rejection and infection, there is a pressing need for alternative solutions . 4D prinFor instance, A 3D-printed medical model company collaborated with surgeons at Belfast City Hospital in Northern Ireland to successfully create a functional 3D-printed replica of the kidney . By repl4D printing technology has shown immense potential in the development of responsive surgical tools that can adapt to changes in their environment, providing surgeons with greater precision and control during complex procedures. By leveraging the unique properties of 4D-printed materials, these innovative tools can adjust their shape and stiffness in response to specific stimuli, leading to improved surgical outcomes and reduced recovery times for patients .Shape and stiffness adaptability is a crucial aspect of responsive surgical tools, as it allows them to conform to different surgical scenarios and anatomical structures, minimising the risk of damage to surrounding tissues . For exaImproved surgical outcomes and reduced recovery times are important benefits of utilising responsive surgical tools. By providing surgeons with greater control and adaptability during procedures, these 4D-printed devices can help minimise the risk of complications, such as bleeding, infection, or damage to adjacent structures. For example, a 4D-printed microneedle array inspired by the backward-facing barbs found in some natural structures, such as porcupine quills, could be developed to enhance tissue adhesion during medical procedures. These microneedles, when inserted into the tissue, would have barbs that open and lock into the tissue, providing a more secure and stable connection. This innovative microneedle array could potentially improve the performance of transdermal drug delivery systems or be used in wound closure applications, minimising the risk of dislodgement and ensuring more effective treatment outcomes . Further4D printing technology has shown great potential in the development of advanced diagnostic tools that can revolutionise the field of medical diagnostics. By leveraging the unique properties of 4D-printed materials, which can change their shape or properties in response to specific stimuli, researchers can create diagnostic devices that are more sensitive, accurate, and patient-specific, ultimately leading to improved patient care and outcomes.One example of a 4D-printed diagnostic tool is a wearable smart sensor created using thermoplastic polyurethane (TPU) printed on fabric. This sensor can monitor vital signs, such as heart rate, blood pressure, and body temperature, by utilising the stimuli-responsive properties of the TPU material. For instance, a 4D-printed temperature sensor can change its shape or electrical properties in response to fluctuations in body temperature, providing an accurate and continuous measurement that can help detect fever or inflammation. This innovative wearable sensor can enable real-time monitoring and early detection of potential health issues, paving the way for personalised healthcare solutions .Another example of a 4D-printed diagnostic tool is a solid-cured tissue-engineered implant made from photo-polymerisable resins. These resins can be used to create 3D-printed implants with embedded microfluidic channels, enabling the rapid detection of specific biomarkers associated with various diseases, such as cancer or infectious diseases. By incorporating 4D-printed materials that respond to the presence of target molecules, these implants can provide highly sensitive and specific detection, enabling early diagnosis and timely intervention. For example, a 4D-printed implant with integrated microfluidic channels could change its shape or colour upon binding to a specific cancer biomarker, allowing for rapid and easy visualisation of the diagnostic result .Additionally, 4D printing has the potential to revolutionise the field of medical imaging by creating anthropomorphic phantoms, which are physical models used to calibrate and validate imaging equipment for radiotherapy. By using 4D-printed materials that mimic the properties of human tissues and organs, researchers can create phantoms that accurately represent the patient’s unique anatomy, leading to more precise and personalised diagnostic imaging. For example, a 4D-printed deformable lung and liver phantom can be used to assess the accuracy of CT and MR imaging in radiotherapy planning. This enables clinicians to optimise treatment plans by accounting for organ motion during respiration, ultimately resulting in improved patient outcomes and reduced radiation exposure .The advent of 4D printing technology has brought about a paradigm shift in the field of biomedical rehabilitation, introducing a new dimension of possibilities for creating adaptive, responsive, and smart devices. This new approach has the potential to redefine the design, manufacturing, and application of biomedical rehabilitation devices, making them more personalised, efficient, and comfortable for patients.One example of 4D printing in rehabilitation is the creation of dynamic splints and braces for the management of musculoskeletal conditions. These smart devices can be programmed to change their shape or stiffness in response to muscle activation or joint position, offering customised support and promoting healing while minimising the risk of joint stiffness or muscle atrophy. For instance, researchers showed that a 4D-printed wearable system inspired by biological motion mechanisms can automatically adapt to provide dynamic support during specific tasks such as gripping or typing . In anot4D printing can also be used for developing devices that emulate the characteristics of biological tissues, allowing for seamless integration with the human body and providing a natural, comfortable interface for rehabilitation purposes. One notable example is a 4D-printed prosthetic breast for patients who have undergone mastectomy surgery. By mimicking the natural properties of human tissues, this innovative prosthetic would adapt to the wearer’s body shape and movement, providing comfort and support during the recovery process. Moreover, the 4D-printed prosthetic could be designed to respond to changes in the wearer’s body temperature or muscle contractions, ensuring a dynamic fit and personalised experience. This approach not only enhances the wearer’s mobility and confidence, but also reduces the risk of complications associated with traditional prosthetics .Despite the significant advancements in 4D printing for biomedical applications, several challenges remain to be addressed before the widespread adoption and implementation of this technology in clinical settings.The performance of 4D-printed biomedical devices and structures is heavily dependent on the materials used. Smart materials, such as shape-memory polymers, hydrogels, and stimuli-responsive materials, need to exhibit the desired behaviour and response to external stimuli to fulfill their intended function. However, finding materials with the appropriate properties can be challenging. The mechanical properties, such as strength, flexibility, and durability, of these materials may not always meet the requirements for specific biomedical applications. Additionally, it is often difficult to combine multiple smart materials in a single 4D-printed structure, limiting the range of functionalities that can be achieved.Furthermore, the 4D printing process can also impose constraints on the materials used. For instance, some fabrication techniques require materials to withstand high temperatures, mechanical stress, or exposure to light or chemicals during the printing process. These factors can limit the range of materials available for 4D printing in biomedical applications.Biocompatibility is a critical consideration in biomedical engineering, as the materials used must be non-toxic and non-immunogenic to ensure the safety of patients ,89. ThisMoreover, the processing techniques used in 4D printing can also affect the biocompatibility of the final product. For example, some fabrication methods may leave residual chemicals or particles that could cause adverse reactions in patients. Ensuring that the 4D printing process does not compromise the biocompatibility of the printed structure is crucial for successful implementation in biomedical applications.The degradation rate of 4D-printed biomedical devices is a significant concern, particularly in applications such as drug delivery systems, tissue engineering, and implantable devices. The materials used in these applications should degrade at an appropriate rate, allowing the device or structure to perform its intended function without causing harm to the patient. For example, in drug delivery systems, the degradation rate should be controlled to ensure the proper release of the therapeutic agent over a specific period . In tissHowever, achieving the desired degradation rate can be challenging due to the complex interactions between the material, the fabrication process, and the biological environment. Factors such as material composition, structure, and processing conditions can significantly affect the degradation behaviour of 4D-printed devices. Furthermore, the degradation rate may also be influenced by factors specific to the patient, such as the local biological environment, immune response, and individual healing rate. Researchers need to consider these variables when designing 4D-printed biomedical devices to ensure optimal performance and patient safety.To overcome materials limitations in 4D printing for biomedical applications, several potential solutions and strategies can be employed. One approach is the development of novel biomaterials specifically designed for 4D printing, taking into account the unique requirements of the printing process and the desired functionality of the final biomedical device. Researchers can explore the synthesis and characterisation of biocompatible materials with enhanced mechanical properties, shape-memory behaviour, and responsiveness to external stimuli. Another strategy is the utilisation of composite materials, combining different components such as polymers, metals, ceramics, or bioactive agents to achieve desired properties and functionalities. Composite materials can enhance the mechanical strength, biocompatibility, and bioactivity of printed structures. Additionally, optimising the printing process parameters and techniques, such as print speed, temperature, and layer thickness, can improve the accuracy, resolution, and reproducibility of the printed biomedical devices. Thorough investigation and understanding of the interactions between the materials, printing process, and the intended application are crucial to overcome materials limitations and ensure the successful implementation of 4D printing in biomedical engineering. Continuous research and collaboration between materials scientists, engineers, and biomedical professionals are essential in driving advancements and addressing the challenges associated with materials limitations in 4D printing for biomedical applications.One of the key advantages of 4D printing is the ability to create structures with varying mechanical properties and responsiveness to stimuli by incorporating different materials into the printed object. This capability is particularly important in biomedical applications, where devices often need to exhibit a range of properties, such as flexibility, rigidity, or responsiveness to specific stimuli, to function effectively. However, integrating multiple materials in a single 4D-printed structure can be challenging due to the limitations of current printing technologies.Achieving precise control over the distribution and interaction of different materials in a 4D-printed structure is essential to ensure the desired performance and behaviour of the final product. This requires advanced printing techniques that can deposit multiple materials simultaneously while maintaining precise control over their placement and interaction. Additionally, the materials used must be compatible with one another to prevent potential issues, such as delamination or poor adhesion between layers. Addressing these challenges will require the development of new printing techniques and material formulations specifically designed for multi-material 4D printing in biomedical applications.High resolution and accuracy are crucial in 4D printing for biomedical engineering, as the printed structures often need to have intricate features or precise dimensions to function effectively. For example, tissue engineering scaffolds require a high degree of porosity and precise pore geometry to promote cell attachment and growth , while iMany 4D printing techniques struggle to achieve the resolution and accuracy needed for some biomedical applications, particularly when printing with smart materials that can change their shape or properties in response to external stimuli. Factors such as material viscosity, print speed, and printer hardware can affect the resolution and accuracy of the printed structure. Researchers need to develop new printing technologies and optimise printing parameters to achieve the required resolution and accuracy for specific biomedical applications.Scalability is another significant challenge in 4D printing for biomedical engineering, as the printed structures must often be produced in large quantities or at different scales to meet the needs of various patients. Moreover, the manufacturing process should be cost-effective and time-efficient to ensure the widespread adoption of 4D-printed biomedical devices. However, many current 4D printing techniques are time-consuming and have limited scalability, making them unsuitable for large-scale production.Addressing the issue of scalability in 4D printing will require the development of new printing techniques that can produce structures at a faster rate and with minimal material waste. Additionally, researchers need to explore methods for automating the 4D printing process and integrating it with other manufacturing technologies, such as injection moulding or additive manufacturing, to increase production capacity and reduce costs.The introduction of any new medical technology, including 4D printing, requires rigorous testing and approval from regulatory agencies to ensure the safety and efficacy of the devices and structures being produced. In many countries, agencies such as the US Food and Drug Administration (FDA) or the European Medicines Agency (EMA) oversee the regulation of medical products, including those manufactured using 4D printing techniques.Navigating the complex regulatory landscape can be challenging, as the approval process for 4D-printed biomedical devices often involves multiple stages, including preclinical testing, clinical trials, and post-market surveillance. Moreover, given the novelty of 4D printing technology, there may be gaps in existing regulations or the need for new guidelines specifically tailored to address the unique aspects of 4D-printed devices, such as their adaptability and shape-changing properties.Researchers and manufacturers need to work closely with regulatory agencies to develop a clear understanding of the requirements and approval processes for 4D-printed biomedical devices. This includes ensuring that the materials used are biocompatible, the fabrication processes meet quality standards, and the devices demonstrate safety and efficacy in preclinical and clinical testing.The implementation of 4D printing in biomedical engineering raises several ethical questions that must be carefully considered to ensure the responsible development and use of this technology. Some key ethical considerations include patient privacy, informed consent, and equitable access to 4D-printed devices.Patient privacy is a crucial concern, as the development of customised 4D-printed devices often requires access to personal medical data, such as patient-specific anatomy or physiological parameters. Researchers and manufacturers need to develop strategies for protecting patient privacy and ensuring the secure handling of sensitive data throughout the design and manufacturing process.Informed consent is another important ethical consideration, particularly in the context of clinical trials involving 4D-printed devices. Patients participating in such trials should be fully informed of the potential risks and benefits associated with the use of 4D-printed devices, as well as any alternatives to their use.Equitable access to 4D-printed biomedical devices is also a concern, as the high costs associated with the development and manufacturing of these devices may limit their availability to patients in need. Researchers and policymakers need to explore strategies for reducing the costs of 4D printing technologies and ensuring that the benefits of this innovative approach to healthcare are accessible to all, regardless of their socioeconomic status.In addition to the general limitations of 4D printing in biomedical engineering mentioned above, it is important to recognise that each specific application within this field may entail its own unique set of challenges. The requirements and objectives of different projects vary greatly, necessitating tailored approaches and problem-solving strategies. Each combination of materials, techniques, and applications presents its own set of challenges that necessitate thorough investigation. Factors such as the desired functionality, compatibility with biological systems, structural complexity, and scalability can all introduce specific hurdles that need to be thoroughly investigated. Therefore, it is essential to approach each application individually, conducting comprehensive research and analysis to identify and address the specific challenges associated with that particular project. By doing so, we can effectively navigate the limitations of 4D printing and unlock its full potential in the diverse landscape of biomedical engineering.As 4D printing technology continues to mature, there is immense potential for further advancements in biomedical engineering. This section highlights future directions for 4D printing in the field, offering novel solutions to pressing clinical challenges.4D printing involves the creation of smart materials that can change their properties or shape over time in response to external stimuli. As research and development in this field progress, several emerging trends and areas of research are expected to shape the future of 4D printing in biomedical engineering.One promising area of research in 4D printing involves the integration of sensors and electronics into the printed structures . By embeCombining living cells with 4D-printed structures can lead to the development of biohybrid systems that exhibit the desired characteristics of both biological tissues and engineered materials. Researchers are exploring the possibility of creating 4D-printed constructs with living cells , which cThe development of self-healing materials for 4D printing is another exciting area of research that could have significant implications for biomedical engineering. By incorporating self-healing properties into 4D-printed devices, researchers can create structures that can repair themselves in response to damage, extending their lifespan and improving their reliability . This co4D printing has the potential to play a significant role in the growing field of personalised medicine. By enabling the fabrication of patient-specific devices and structures, 4D printing can help tailor treatments and interventions to the unique needs of individual patients. This includes the development of customised implants and prosthetics that fit the patient’s anatomy perfectly , as wellAs concerns over the environmental impact of materials and waste disposal grow, there is increasing interest in developing environmentally friendly and biodegradable materials for 4D printing in biomedical engineering. Researchers are exploring the use of naturally derived materials, such as cellulose or chitosan, as well as biodegradable synthetic polymers for 4D printing applications . The useResearchers are investigating the potential of multi-stimuli responsive materials in 4D printing for biomedical applications. These materials can respond to multiple external stimuli, such as temperature, light, pH, or magnetic fields, and exhibit different behaviours depending on the specific stimulus. This adaptability can lead to the development of 4D-printed devices and structures with enhanced functionality and versatility. For instance, a drug delivery system made from multi-stimuli responsive materials could adjust the release rate and targeting of therapeutic agents based on the patient’s body temperature, pH, or other biological factors, resulting in more effective and personalised treatments .The field of biomimicry, which involves the imitation of natural systems and structures, has inspired the development of biomimetic materials for 4D printing in biomedical engineering. These materials mimic the properties and functions of biological tissues, providing potential advantages in terms of biocompatibility, functionality, and adaptability. Researchers are exploring the use of biomimetic materials in various applications, including tissue engineering, regenerative medicine, and the fabrication of implants and prosthetics that closely resemble the patient’s native tissues. By incorporating biomimetic materials into 4D-printed structures, researchers can create devices that integrate seamlessly with the body and promote more effective healing and recovery ,97.The focus in this area will be on the design, synthesis, and application of materials and devices at the nanoscale. Advancements in 4D printing techniques will enable the creation of sophisticated, responsive nanostructures and drug-delivery systems that target specific cells, tissues, or physiological conditions. These nanoscale constructs can improve therapeutic efficacy, reduce side effects, and enable personalised medicine . As reseAdvancements in fabrication techniques are crucial for driving innovation in 4D printing and other emerging technologies. As the field evolves, researchers continue to explore new methods and refine existing ones to create more sophisticated, efficient, and precise fabrication processes. Some notable advancements in fabrication techniques include:Multi-material and multi-process printing is a pivotal advancement in fabrication techniques, enabling the creation of complex, multifunctional structures by combining different materials and processes simultaneously. This innovation is particularly significant for 4D printing, where materials with diverse responsiveness to stimuli are often merged to achieve desired dynamic behaviours. Multi-material printing allows the design of objects with distinct regions exhibiting varying mechanical, thermal, or electrical properties, broadening the scope for advanced materials that respond to specific environmental stimuli. Multi-process printing enhances this concept by integrating multiple fabrication techniques within a single build, generating structures with diverse fabrication-induced characteristics. This progress in multi-material and multi-process printing has impacted 4D printing applications in biomedical engineering, soft robotics, and smart materials. Overcoming remaining challenges, such as advanced printing systems and material compatibility, will further expand the potential of 4D printing technology ,100.High-resolution printing has made significant strides in fabrication techniques, allowing for the creation of structures with intricate geometries and features at the micro- or even nanoscale. This precision is crucial for applications such as tissue engineering and drug delivery systems, where fine control over structure and material properties is essential. Developments in high-resolution printing techniques, such as two-photon polymerisation , have exHybrid fabrication techniques, which combine multiple fabrication methods, have emerged as a key advancement in fabrication technology. These techniques unlock novel structures and functionalities by merging methods such as 3D printing with electrospinning or bioprinting with microfluidics. This synergy results in 4D-printed constructs with enhanced mechanical, biological, and stimuli-responsive properties. These hybrid techniques can create constructs with diverse characteristics, paving the way for new applications in biomedical engineering. For example, combining 3D printing with electrospinning can produce scaffolds with tunable mechanical properties and tailored pore structures, which are ideal for tissue engineering ,103. ConSelf-assembly and self-folding techniques are emerging as promising fabrication methods for creating complex 3D and 4D structures. By leveraging the inherent properties of materials and precise control of environmental conditions, these techniques enable the formation of dynamic constructs without the need for external manipulation or assembly. Self-assembly relies on the spontaneous organisation of materials into ordered structures, driven by molecular interactions and surface chemistry. Self-folding, on the other hand, involves inducing shape changes in materials through programmed stress patterns or stimuli-responsive elements ,104,105.Embedded sensors and actuators in 4D-printed constructs offer real-time monitoring and control of their behaviours, enabling the creation of smart structures that respond to various stimuli ,106. AdvBiofabrication techniques, such as bioprinting  and cellMachine learning and AI-driven design have become increasingly important in the development and optimisation of fabrication processes, including 4D printing. These technologies can predict the behaviour of 4D-printed materials and structures, guiding the selection of materials, geometries, and fabrication parameters to achieve desired functionalities. Incorporating AI and machine learning into the design process allows for the exploration of vast design spaces, identification of optimal solutions, and prediction of material properties and performance. This leads to more efficient and effective fabrication processes and improved 4D-printed constructs. As research in AI and machine learning progresses, their integration into fabrication techniques will enhance the capabilities of 4D printing technology, enabling the creation of increasingly complex, functional, and responsive materials and structures across various applications and industries ,109,110.Scalability and automation are essential for translating 4D printing from research labs to real-world applications. Advancements in high-throughput manufacturing methods, automated quality control systems, and standardised processes and protocols will accelerate the large-scale production of 4D-printed constructs. By improving scalability and automation, 4D printing technology can become more accessible and cost-effective, paving the way for widespread adoption in biomedical engineering. As research continues, further innovations in scalable and automated fabrication techniques will play a crucial role in unlocking the full potential of 4D printing and driving its integration into various applications.This review paper has provided a comprehensive overview of the advancements, challenges, and future directions for 4D printing in biomedical engineering. 4D printing technology has emerged as a promising approach to address various unmet clinical needs and revolutionise healthcare practices by integrating the advantages of 3D printing with the added dimension of time-dependent shape transformations.The paper has discussed the significant advancements in 4D printing for biomedical applications, including the development of stimuli-responsive materials, innovations in fabrication techniques, and successful implementation in various fields such as tissue engineering, drug delivery, and medical devices. Moreover, the review has highlighted several exciting future directions for 4D printing in biomedical engineering, such as the development of advanced smart materials, integration of bioprinting and 4D printing techniques, personalised medicine, nanomedicine, and bioelectronic devices. Addressing these challenges and exploring these future directions will be essential for realising the full potential of 4D printing technology in various biomedical applications.As 4D printing technology continues to advance, it is poised to make significant contributions to biomedical engineering, ultimately improving patient outcomes and transforming healthcare practices. By fostering interdisciplinary collaboration, promoting public engagement and education, and encouraging industry partnerships, 4D printing has the potential to transform the way we approach the design and development of medical devices, treatments, and therapies. It is clear that 4D printing technology holds immense promise for the future of biomedical engineering, and continued research and innovation in this field will pave the way for innovative advancements in healthcare.


We focus on the characterization of the transforming growth factor-β (TGFβ) signaling pathway in B acute lymphoblastic leukemia (ALL) and in resistance to the multi-kinase inhibitor dasatinib in order to provide a better understanding of the molecular and functional mechanisms underlying leukemic transformation and the development of drug resistance. We provide evidence that TGFβ signaling is an important negative regulator of cell growth in B-cell precursor- ALL as well as in generated dasatinib-resistant ALL cells.+/E2A-PBX1+ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFβ pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Hence, dasatinib blocks, at least partially, TGFβ-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Activation of the TGFβ signaling pathway by TGF-β1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFβ signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The TGFβ signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFβ signaling pathway in our previously generated human dasatinib-resistant pre-BCR Acute lymphoblastic leukemia (ALL) represents the most common cancer in children and adolescents, accounting for 25% of all childhood malignancies, and derives in the majority of cases from B-cell precursors (BCP) . Althoug+/E2A-PBX1+ALL. Pre-BCR+ ALL occurs in 10–15% of BCP-ALL cases and is characterized by the expression of functional pre-BCR mediating survival during early B-cell development. Half of pre-BCR+ ALL cases are associated with the chromosomal translocation t  coding for the oncogenic transcription factor E2A-PBX1 (TCF3-PBX1) ..+/E2A-PBell line c,d, amonh clones e,f. We aV clones . Both thPhosphorylated SMAD proteins are central effectors of TGFβ signaling as they act both as signal transducers downstream of a hetero-tetrameric receptor complex, consisting of type I and II receptors, and as transcriptional regulators at the nuclear level ,24. To f+ 697 cells were moderately sensitive to dasatinib (IC50 100 nM–1 μM). In contrast, the E2A-HLF+ HAL-01, the MLL-AF4+ SEM and the TEL-AML1+ REH cell lines were resistant to dasatinib (IC50 > 1 μM) [In order to validate our findings and to identify possible underlying molecular features, we used several BCP-ALL cell lines with different cytogenetic backgrounds for studying the effects of dasatinib and TGF-β1 on TGFβ signaling activity in BCP-ALL. According to our previous studies, the E2A-PBX1 > 1 μM) . Using i+/E2A-PBX1+ cell lines 697, in both dasatinib-sensitive and -resistant RCH-ACV, and moreover in the pre-BCR−/E2A-PBX1− REH cell line, which all displayed an intermediate increase in TGF-β1-induced phosphorylation levels, which could be partially blocked by dasatinib  as well as HAL-01 (E2A-HLF+) showed the highest sensitivity to TGF-β1 growth inhibitory effects compared to SEM (MLL-AF4+) and REH (TEL-AML1+). Our results indicate that cell lines arising from BCP-ALL retain at least partial responsiveness to TGF-β1 growth-inhibitory effects  inhibitors p15Ink4b (CDKN2B) and p21Cip1 (CDKN1A) [-MYC expression was downregulated by TGF-β1 in dasatinib-resistant and -sensitive RCH-ACV clone 1 cells. Consistent with this, we observed TGF-β1-induced transcriptional activation of p15Ink4b. Unexpectedly, p21Cip1 expression was downregulated following TGF-β1 treatment in dasatinib-sensitive but not in -resistant RCH-ACV cells  . Target CV cells . These d+/E2A-PBX1+, using gene ontology (GO) analysis [+/E2A-PBX1+ ALL cells. Different members of the TGFβ and BMP subfamilies were found to be upregulated; however, knowing their role in cancer and cell proliferation regulation, we pursued further functional studies investigating the role of the TGFβ/SMAD signaling pathway in the preclinical model of dasatinib-resistance in pre-BCR+/E2A-PBX1+ ALL, as well as in several BCP-ALL cell lines with different cytogenetics. We cannot exclude the involvement of BMP signaling in contributing to the resistant phenotype of RCH-ACV cells to dasatinib, which requires additional investigation in future research.In previous studies, we identified several enriched and depleted KEGG signaling pathways in a model of secondary dasatinib resistance in a distinct subtype of BCP-ALL, i.e., pre-BCRanalysis . In the + HAL-01 cells showed the highest sensitivity to TGF-β1 in terms of induction of SMAD2/3 phosphorylation as well as cytostatic effects. In contrast, the MLL-AF4+ SEM cell line presented the lowest increase in SMAD2/3 phosphorylation levels following stimulation with TGF-β1. Moreover, they were the least sensitive to TGF-β1 anti-proliferative effects. Conversely, the pre-BCR+/E2A-PBX1+ cell lines RCH-ACV and 697 as well as the TEL-AML1+ REH cells displayed significant responsiveness to growth-inhibitory effects mediated by TGF-β1. Buske et al. reported high inter-individual heterogeneity in anti-proliferative effects within primary blasts from patients with common B-cell ALL treated in vitro with TGF-β1; however differences in cytogenetic background or possible underlying molecular features were not addressed in the study [Our functional studies involving SMAD2/3 phosphorylation analysis and cell proliferation assays suggest that the examined BCP-ALL cell lines retain at least partial responsiveness to TGF-β1 cytostatic effects. However, they exhibit heterogeneous basal as well as TGF-β1-induced SMAD2/3 phosphorylation levels, which might have an impact on the TGF-β1-mediated anti-proliferative response. Among the examined cell lines, E2A-HLFhe study . +/E2A-PBX1+ ALL cells, retaining its growth-inhibitory function. This is supported by a proliferation decrease observed after the treatment with TGF-β1 in several B-ALL cell lines with different karyotypes, including the TEL-AML1+ REH ALL cell line. In contrast, Ford et al. described previously that TGF-β1 did not further reduce cell proliferation of the murine pro-B cell line, Ba/F3, transduced with TEL-AML1 [+/E2A-PBX1+ RCH-ACV ALL cell lines show growth difference in response to TGF-β1. BCP-ALLs respond heterogeneously to TGF-β1, as shown by cell proliferation assays and pSMAD2/3 phosphorylation studies. The mechanisms that determine differences in growth inhibition in dasatinib-sensitive or -resistant cell lines and in BCP-ALL with different karyotypes in response to TGF-β1 were not systematically investigated in the study. Further studies are needed to elucidate the role of the TGFβ signaling pathway in dasatinib resistance.As previously described , dasatinTEL-AML1 . Neither+ CML and ALL [We showed that signaling activation via phosphorylation of SMAD2/3 proteins in response to TGF-β1 is blocked in a heterogeneous manner by dasatinib in different BCP-ALL cell lines, as well as in the dasatinib-resistant RCH-ACV cells, and that inhibition of SMAD2/3 phosphorylation does not correlate with sensitivity to dasatinib . Dasatin and ALL ,29. Inde and ALL , which w and ALL ; however and ALL ,32. Our -MYC and upregulation of the CDK inhibitor p15Ink4b in both dasatinib-sensitive and -resistant RCH-ACV cells. Inhibition of cell proliferation mediated by TGF-β1 has been mainly related to cell cycle arrest at the G0/G1 stage, whereas mechanisms regulating programmed cell death in response to TGF-β1 remain largely unknown [+/E2A-PBX1+ ALL cells are dependent on hyperactivated PI3K/AKT signaling [p15Ink4b and p21Cip1 [+/E2A-PBX1+ ALL cells might have additive effects to antiproliferation mediated by TGF-β1. Future studies highlighting the complex web of interactions between TGFβ and other signaling pathways are needed to gain further insight into these questions.Cell cycle analysis by flow cytometry revealed growth inhibition by TGF-β1 via induction of G0/G1 cell cycle arrest and increase in apoptotic subG0 cell fraction. However, target gene expression studies showed downmodulation of c unknown . Differe unknown ,22,33,34 unknown ,22,25. Fignaling ,9,35,36, p21Cip1 ,38. TherIn addition, cellular senescence and autophagy as alternative mechanisms of inhibiting cell proliferation have been suggested to be induced by TGFβ signaling, but these have not been investigated in this study .+ RCH-ACV cells. Although dasatinib shows promising early preclinical effects for the treatment of pre-BCR+/E2A-PBX1+ ALL, its ability to target a broad spectrum of different kinases might contribute to the onset of secondary drug resistance. For almost two decades, the TGFβ signaling pathway has not been in the spotlight of hematological cancer research, and approaches to target this pathway in various diseases remain challenging. Therefore, further characterization of the complex regulatory actions by which TGFβ signaling mediates its functions during normal lymphopoiesis and in its malignant counterparts should be the focus of future research in order to improve our understanding of this signaling pathway, as well as to develop novel treatment approaches in BCP-ALL. In this study, we show that TGFβ signaling plays an important role in the negative regulation of cell proliferation in BCP-ALL cell lines as well as in dasatinib-resistant E2A-PBX1


The pig IPEC-J2 and chicken SL-29 cell lines are of interest because of their untransformed nature and wide use in functional studies. Molecular characterization of these cell lines is important to gain insight into possible molecular aberrations. The aim of this paper is to provide a molecular and epigenetic characterization of the IPEC-J2 and SL-29 cell lines, a cell-line reference for the FAANG community, and future biomedical research. Whole genome sequencing, gene expression, DNA methylation, chromatin accessibility, and ChIP-seq of four histone marks  and an insulator (CTCF) are used to achieve these aims. Heteroploidy (aneuploidy) of various chromosomes was observed from whole genome sequencing analysis in both cell lines. Furthermore, higher gene expression for genes located on chromosomes with aneuploidy in comparison to diploid chromosomes was observed. Regulatory complexity of gene expression, DNA methylation, and chromatin accessibility was investigated through an integrative approach.  •Untransformed, nontumorigenic IPECJ2 and SL-29 cell lines show some aneuploidy•Aneuploid chromosomes result in increased gene expression•Positive correlation between hypomethylation, H3K4me1, H3K27ac, and gene expression•Identified enhancer and promoter regions play an important role as a reference Cell biology Cell lines directly derived from tissues or organs of an animal are referred to as primary cell lines. Such cells can then either continue growing indefinitely or die off after a certain number of cell divisions.The IPEC-J2 cell line in pigs and the chicken SL-29 cell line are of interest for the animal genomics community because of the untransformed nature and wide use in functional studies in these cells. We have analyzed both the pig and the chicken cell lines with a range of whole genome-based assays. We first report the results from the pig IPEC-J2 cell line followed by the chicken SL-29 cell line. For each of these cell lines, we first focus on chromosome level structural variation followed by an in-depth analysis of the expression, chromatin accessibility, and methylation of the cell line genomes.The whole genome sequence data were investigated in different ways to determine the structure of the genome. Chromosomal abnormalities such as aneuploidy and heteroploidy can occur in cell lines that grow indefinitely. We therefore first investigated the chromosomal structures and possible changes in aneuploidy. Aneuploidy events can be detected using whole genome sequencing (WGS) data by examining both the read-depth and the ratio of reads that support the alternative allele for heterozygous SNPs; this should be around 0.5 for diploid chromosomes D, whereaSome chromosomes, e.g. chromosome 17 D, show cWe further investigated additional structural variants within the genome of this cell line with Manta A for theFor small variants, deletions are the most abundant type of variant for all chromosomes, and relatively more insertions and deletions are observed on chromosome 17. For large variants, the number of deletions and duplications vary between the chromosomes, and the chromosomes showing evidence of triploidy  have a rRNA-seq data provide insight into gene expression levels across the genome. These data can provide insight into elements that regulate gene expression like promotors, enhancers, and epigenetic marks, as well as chromosomal abnormalities affecting them. Of the 31,907 genes tested, 10,412 were expressed (transcript per million [TPM] >1).Interestingly, the expression levels on chromosomes 2, 5, and 17 , with histone mark H3K4me3 sharing the lowest number of peaks most likely resulting from the lower number of reads for this mark in the first experiment. We assessed the relationship of the shared peaks and investigated the similarities between the two experiments, by calculating Pearson correlations . A high MESD and TLNRD1 TSS  related to gene expression. The peaks called for H3K4me3, H3K27ac, and H3K4me1 were provided to Homer for identification of enriched TF involved in gene expression in IPEC-J2. The significant transcription factors identified were Sp100 and IRF for the peak regions of H3K4me3, E2F and ZNF449 for H3K27ac, and FOS and TEAD for H3K4me1. We identified 36,638 enhancer regions, and motif analysis for these enhancers is shown in Gene expression is negatively correlated with DNA methylation; therefore, investigating the methylome provides further insight into specific characteristics of the cell line’s genome characteristics. The methylome was investigated using both reduced representation bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (WGBS) .Table 3AMost DNA methylation is observed at CpG sites . We therefore investigated whether all sites identified by RRBS are also covered by WGBS. To do so, we disregarded any sites with a coverage <10 for both types of data as not being sufficiently informative. In total, 23,952 sites covered by RRBS are not covered by WGBS. Further details on possible functional relevance of these RRBS-specific sites is done for the chicken cell line  and CTCF are shown in The histone modifications associated with promoter and enhancer regions  show patterns as expected with a positive correlation with gene expression. Histone mark H3K27me3, which is associated with gene suppression, shows a negative correlation with gene expression B. As expThe methylation levels at the TSS and within the gene-body for RRBS and WGBS A follow As with the pig cell line, WGS for the chicken cell line provided a comprehensive insight into genetic variation, chromosomal abnormalities, and structural variation at the global genome level. Multiple chromosomal abnormalities such as aneuploidy and heteroploidy are observed for the chicken SL-29 cell line .Figure 7A higher read-depth B, suggesRelative abundance of copy-number variants is higher for some of the small micro-chromosomes , whereas the macro-chromosomes have relatively fewer copy-number variants. Intron variants  and intergenic variants  are the most abundant consequences from the copy-number variants in this cell line. The effects, as determined by VEP, of both copy-number variants and large structural variants identified through CNVnator are shown in The expression profile for this cell line was investigated to obtain further insight into the genes expressed and interaction of regulatory elements, aneuploidy, and CNVs affecting gene expression. We tested the expression of 24,356 genes, of which 13,546 were expressed (TPM > 1).Higher levels of gene expression are observed on chromosomes 20, 25, 27, and 33 . This shAs described for pigs, histone modifications are of importance to investigate chromatin accessibility and provide further insight into regulatory elements. The peak calling results for the chicken cell line are shown in The peaks called show much overlap between the replicates for H3K4me1, H3K4me3, and H3K27ac. The two ChIP-seq experiments were compared using Pearson correlations . High coFKBP5 (MICAL1 and TULP1).Various chromatin states were identified, through identification of presence or absence of histone marks using ChromHMM, which provides insight into interactions between different histone marks. Histone marks H3K4me3 and H3K27ac are enriched around the TSS of expressed genes, with H3K4me3 enriched at approximately 20% of the TSS and H3K27ac enriched at >5% of the TSS . The chrFKBP5 B; enrichAnnotation of the peak regions to genomic features provides insight into functional elements related to the histone modifications. As for the histone/CTCF marks in the pig cell line, most marks are found in intron and intergenic regions of the genome. Furthermore, a large percentage (16%–21%) of the H3K4me3, CTCF, and H3K27ac histone modifications are found within promoter regions , and 11%Significant consensus sequences for known motifs identified the TFs IRF1, ZNF711, YY2, AP-2alpha, Atf3, and Pax8 for the three histone marks  . In the Chromatin accessibility was profiled in the chicken SL-29 cell line using ATAC-seq data, from which 86,983 peak regions were identified. To infer the functional significance of accessible regions that were identified, consensus peaks were characterized by genomic localization. Annotation of ATAC-seq showed most accessible (open) chromatin is found in the intron and intergenic regions of the genome, , with 12Overall, consensus peaks identified for PAX6 recognition sites as most significant, with about 1.74% of accessible regions harboring this consensus motif . Roles oWe also determined the average methylation levels for cytosine for the chicken cell line, calculated from both RRBS and WGBS data .Table 7AAs expected, average methylation (>10 reads) is observed at CpG sites (37%–59%), and slightly higher average non-CpG methylation (1%–2%) is observed. Average chromosome level methylation levels estimated from WGBS fluctuate around 0.2–0.6 . The aveAs stated earlier for the pig, it is of importance to investigate sites covered by RRBS data while not covered by WGBS (and vice versa), as WGBS is considered as the gold standard for insight into whole genome DNA methylation; 926,495 sites were identified by RRBS and not by WGBS in the chicken cell-line . We further investigated if these sites specifically covered by RRBS overlayed regions within predicted promoters and enhancer regions (from the ChIP-seq data). Examples of such regions (chromosome 1 and 2) covered by only RRBS data including predicted active enhancers/promoters are shown in The transcription factor motif analysis of identified enhancers that overlap with the identified specific RRBS regions is shown in An integrative approach was used to gain insight into the dynamics of methylation and histone modifications for regulation of gene expression . As expeThe correlations between the 4 histone marks , ATAC-seq, and distinct classes of gene expression levels are visualized in The positive correlation observed for H3K4me3 and H3K27ac with the gene expression B is highCell lines provide an ethical approach for research in animal production, and thus, molecular characterizations are necessary for functionally relevant cell lines. The pig IPEC-J2 and chicken SL-29 cell lines have never been characterized using an integrative genomics approach. These two cell lines were chosen specifically for their usability and application to research in animal production as well as biomedical research. Using different omics data , the genome structure, transcriptome, methylome, and chromatin accessibility were investigated and characterized for these cell lines; this provides a reference of the genome architecture of these cell lines for future functional studies using these cell lines as well as for farm animal research community.,Results for both pig and chicken cell lines show that aneuploidy is common in both cell lines, as we observed various chromosomes that were either (partly) monoploid, triploid, and even tetraploid. More aneuploidy as well as more structural variants were observed for the chicken SL-29 cell line in comparison with the pig IPEC-J2 cell line. For SV this could be due to the additional filtering of common structural variants from the pig cell line. Previous studies have shown that more chromosomal abnormalities, as well as structural variants occur when cell lines are maintained in culture for a longer time (more passages),,cis-regulatory elements, promoters, and enhancers.,,The increase in ploidy leads to an increase in expression of the genes on these chromosomes, likely affecting functional relevant aspects of these cell lines. A comparison of the results from the pig cell line with similar tissue type and organoid samples showed pronounced higher rates of expression of the genes on the triploid chromosome 17 of the pig cell line. From this comparison it is evident that these cell lines show higher rates of gene expression on all chromosomes, followed by organoids, and with tissue showing the lowest rates of gene expression; this is in agreement with studies showing that organoids resemble gene expression levels and physiology of tissue more closely than cell lines.,,The results from the WGS and RNA-seq show the potential of using these assays to detect chromosomal abnormalities, in addition to investigation of variation in the genomeChromatin accessibility and histone modifications were investigated to gain further insight into the genome architecture of both cell lines using ChIP-seq data for CTCF and four histone modifications: H3K4me1, H3K4me3, H3K27ac, H3K27me3. The importance of using a standardized protocol for comparative ChIP-seq studies was explored using two technical replicates of the ChIP-seq experiments for three histone marks, performed in two different laboratories and using different inputs as a background ; this provides insight into the reproducibility of results between different laboratories and the use of DNA or IgG as an input. We conclude there is a high consensus between overlapping peaks of the experiments, and secondly, the read coverage between experiments showed medium to high consensus between experiments. However, signal values show little correlation between experiments, suggesting that the confidence related to the high number of overlapping peaks is limited and should therefore be used with care if the signal values are used for comparative analysis. A reason for this low correlation of signal values could be the differences in background signal in the two experiments. ChIP-seq guidelines and practices from (mod)ENCODE have found that an IgG control mimics a ChIP experiment more closely than a DNA input control. Cases of strong sonication bias are rarely observed, but this can potentially affect peak calling.,,,,,Peak regions identify possible binding sites of proteins associated with DNA (protein-chromatin interactions) and provide insight into regulatory regions and elements. The number of histone marks identified are different between tissues, stages of development, and number of reads sequenced. ENCODE standards have shown that the number of peaks that can be identified ranges from thousands to tens of thousands of peaks.,,To evaluate the quality of the experiments and the success thereof, we compared the consensus motif for the CTCF sites identified for pig and chicken with the human CTCF consensus sequence. There was good similarity between the consensus motifs for both pig and chicken and human. Furthermore, this motif was also identified in the human K562 cell line as a CTCF mark. CTCF is a highly conserved protein in mammals (between pig and mouse) as well as in vertebrates,,,,Further investigation into motifs identified for the histone marks in both cell lines, together with comparison of similarities thereof to known motifs, provides insight into possible TFs. Firstly, interferon receptor factor (IRF) was identified in both cell lines (pig IRF2 and chicken IRF1). IRF is part of a TF family that is found in humans as super enhancer TFs and is highly conserved within species.,,Open chromatin was investigated using ATAC-seq data for the chicken cell line with a number of regions identified, slightly lower than expected when following ENCODE recommendations, which suggests >100,000 peaks. However, this is similar to the number of enriched regions identified by other studies in animals.,,,,Further insight into gene expression and the molecular characteristics of both cell lines was obtained through characterization of the methylome, which ensures comprehensive characterization of the functional genome. DNA methylation is an epigenetic mark that is found in most species and has been found to be inherited and influenced by environmental factors .,Finally, we attempted to integrate the various epigenetic marks together with gene expression to show how the functional genome regulates expression levels. Correlations between the expression data and methylation indicate that promoter methylation has a reverse relationship with gene expression, with, as expected, methylated sites inhibiting gene expression. Interestingly, the methylation level within the gene-body in the pig cell line increases slightly with higher expression levels, whereas the methylation in the gene-body of the chicken cell line decreases slightly with increased expression levels; this is similar to what has been observed within the gene-body in earlier studies of the methylome in pig and avian species.in vivo research. We propose these cell lines as a reference for future functional and comparative studies in animals, whereby knowledge of ploidy, expression profile, chromatin landscape, and methylome provide the backbone for the comparison.This paper is the first to describe the molecular characteristics (structure) of the pig IPEC-J2 and chicken SL-29 cell lines. The genomic approaches provided an insight into the different levels of the epigenome influencing gene expression in these cell lines, as well as provided a description of the architecture of the epigenome. Chromosomal abnormalities, copy-number variations, and aneuploidy, typical for a cell line, were identified for several chromosomes for both cell lines. These cell lines are referred to as nontumorigenic and nontransformed; however, as these cells go through many passages, aneuploidy events do occur. Future researchers should note the characteristics of these cell lines and proceed with caution for interpretation of results. Epigenetic marks such as histone modifications, chromatin accessibility, and DNA methylation were integrated with expression data for both cell lines; this provided insight into the interactions between the epigenetic marks and gene expression. The characteristics as described in this paper for these cell lines will be similar for cells cultured using the same protocol and cells grown for the same number of passages. Deviations from these guidelines/methodologies are expected to result in different genomic and epigenomic characteristics. Understanding these cell lines and the (epi)genetic makeup thereof can provide a better understanding of the limitations of these cell lines as a model for The ATAC-seq data were unavailable for the pig IPEC-J2 cell line. Further investigation using traditional methods for confirmation chromosomal abnormalities e.g. karyotypes staining would be beneficial.jani.devos@wur.nl).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jani de Vos  from pig IPECJ2 (PRJEB59474) and chicken CRL (PRJEB59475) cell lines have been deposited at ENA and are publicly available as of the date of publication. Accession numbers are listed in the •https://doi.org/10.5281/zenodo.7274310) and is publicly available as of the date of publication. DOIs are listed in the All original code has been deposited at Zenodo  cell line was obtained from the cell repository at DSMZ,MSPI restriction enzyme followed by a 20-250 bp fragment size selection and library preparation using the Ovation RRBS library (NuGEN). Samples were pooled and sequenced with the TruSeq SBS sequencing kit version 4 on the HiSeq 2500 (Illumina). A biological replicate was also sequenced following the same procedure for both pig IPECJ2 cell line. For WGBS genomic DNA was spiked with lambda DNA, fragmented by sonication to 200-400 bp with Covaris S220 , followed by end repair and A-ligation. Cytosine-methylated barcoded adapters were ligated to the sonicated DNA. The DNA bisulfite conversion was performed using the EZ DNA Methylation Gold Kit . DNA fragments were size selected and amplified using the KAPA HiFi HotStart ReadyMix (2X) . Library concentration was quantified using a Qubit 2.0 fluorometer  and qPCR . Libraries were sequenced using the HiSeqX S4 flow cell with PE150 strategy.These cells were then used for whole genome sequencing (WGS), RNA sequencing, reduced representation bisulphite sequencing (RRBS), whole genome bisulphite sequencing (WGBS), ChIP-seq and ATAC-seq. DNA and RNA were isolated from the cell lines using the All Prep DNA/RNA Mini Kit (Qiagen) following manufacturer’s instructions. WGS libraries of ∼ 300-400 bp fragments were prepared using Illumina paired-end kits  and 150 bp paired-end sequenced with Illumina HiSeqX. RNA-seq library preparation and sequencing was done as described in van der Hee et al.ChIP-seq for both cell lines was performed for the insulator Anti-CTCF  and histone marks H3K4me1 , H3K4me3 , H3K27ac  and H3K27me3 . These histone marks were chosen as they provide insight into transcriptional activation and the location of enhancers and promoters. ChIP-seq data sets were generated in two different laboratories  for each cell-line. As input control in the first experiment, an IgG “mock” control was used, whereas in experiment two, an “input” DNA control was used for the ChIP-seq studies. In the second experiment only three histone marks  were assayed. The same ChIP-seq protocol was applied in both laboratories. Chromatin preparation was performed where cells (cultured in petri-dishes) were cross-linked with 1.1% formaldehyde for 10 min, stopped by adding 1/10 vol of 1.25 M Glycine for 2 min and washed with cold PBS. Cells were harvested by scraping, incubated with different buffers and finally resuspended in an incubation buffer with PIC with a final concentration of 15 million cells/ml. Shearing of the cells was performed in 300 μl cell suspension /tube with 10 cycles 30 seconds on and 30 seconds off at 4°C using the Bioruptor Pico sonicator (Diagenode). Lastly the sonicated material was divided into aliquots and stored at -80 °C. The overnight immunoprecipitation step with the different antibodies at 4°C was performed on the chromatin using 4.5 million cells as input per antibody. Immunoprecipitated chromatin was incubated overnight with a 50:50 mix of PureProteome Protein A and G magnetic beads (Millipore). The beads were washed (6 washes with 4 wash buffers), rotated and de-cross-linked. The de-cross-linked DNA was finally purified (MinElute PCR Purification columns (Qiagen)), and DNA quantities were determined with Qubit fluorometric quantification (ThermoFisher Scientific). A qPCR analysis of ChIP DNA was performed with iQ SYBR Green Supermix (Bio-Rad) on a CFX96 Real-Time System C1000 Thermal Cycler (Bio-Rad). Library prep was performed using the Kapa Hyper Prep Kit for Illumina sequencing using the manufacturers protocol, with the following adjustments. DNA was used as an input together with NextFlex adapters (Bioo Scientific), followed by PCR amplification. Post-amplification cleanup was performed using QIAquick MinElute columns (Qiagen) and library size selection (300-bp fragments) was performed using the E-gel iBase (Invirtogen). Thereafter the quality and quantity of the library was examined using a High Sensitivity DNA Chip on a Bioanalyzer 2100 system (Agilent). Finally, the libraries were paired-end sequenced using Illumina high-throughput sequencing protocol on a HiSeq2000 (Illumina). For the second experiment sequencing was performed on a HiSeq4000 (Illumina)., 2016, with the following exceptions we used 25k cells as input and the standard Illumina Nextera primers for library amplification. These libraries were sequenced on a HiSeq4000 (Illumina), paired-end with 150bp.Lastly ATAC-sequencing was completed following the Fast-ATAC-sequencing protocol as described in Corces et al.Gallus gallus GRCg6a) reference genomes, together with ENSEMBL annotations  were utilized for all data analyses of our study. Default settings were used unless otherwise stated, and a brief overview of the data analyses is shown in Pig (Sus Scrofa 11.1) and chicken Structural variant analysis of both cell lines was completed using Manta (v1.4.0).Stranded RNA-seq datasets for both cell lines (pig IPECJ2 and chicken SL-29) were trimmed for adapters, quality and minimum length using TrimGalore v0.6.4 a wrapper for Cutadapt v1.18Additional raw RNA-sequencing data was downloaded from ENA from the PRJNA610529 project: two pig jejunum organoid samples grown for different time periods (3 weeks (ENA:SAMN14300031) and 12 weeks (ENA:SAMN14300021), a 5 week old pig jejunum tissue sample (ENA:SAMN14300018), cell lines IPECJ87, an IPECJ2 cell line grown for 87 passages (ENA:SAMN14300016), and IPECJ91, an IPECJ2 cell line grown for 91 passages (ENA:SAMN14299997). We trimmed, aligned, and completed gene quantification of this data following the same procedure as the above procedure used for the IPECJ2 cell line used in this study. These samples were used to compare the average gene expression level per chromosome in jejunum tissue, organoid and IPECJ2 cell line.Raw reads were trimmed with TrimGalore v0.6.4 a wrapper for Cutadapt v 1.18,,,Peak calling for the respective marks, was completed using MACS2 (v2.7.1),,,Trimming and alignment of the ATAC-seq reads were completed as described above for ChIP-seq reads. PCR duplicates were removed using picard (v2.23.9), and only unique, paired-end reads were kept for further analysis.RRBS and WGBS raw reads were trimmed as described above for ChIP-seq reads, with an additional --rrbs parameter for RRBS data. Genome index was built using BSseeker2 v 2.1.8,Finally, the number of sites identified by RRBS data and not by WGBS data was investigated using the following approach. Firstly, the methylation calls were filtered for only CG sites and for a coverage of more than 10. Thereafter bedtools (v2.30.0)An integrative approach was used to investigate the relationship between WGS, expression data (RNA-seq), methylation status and ChIP-seq marks. Output files from the homer annotate called peaks for the respective marks, gene expression values from RSEM, and methylation calls from CGmaptools were used. For this investigation, correlations, scatterplots, and boxplots were created from these files using an in-house python script.Pearson correlations between the read coverage and signal value of peaks for histone marks H3K4me1, H3K4me3 and H3K27ac of each experiment was done using bedtools (v2.30.0),


Gastric-cancer is a heterogeneous type of neoplastic disease and it lacks appropriate therapeutic options. There is an urgent need for the development of innovative pharmacological strategies, particularly in consideration of the potential stratified/personalized treatment of this tumor. All-Trans Retinoic-acid (ATRA) is one of the active metabolites of vitamin-A. This natural compound is the first example of clinically approved cyto-differentiating agent, being used in the treatment of acute promyelocytic leukemia. ATRA may have significant therapeutic potential also in the context of solid tumors, including gastric-cancer. The present study provides pre-clinical evidence supporting the use of ATRA in the treatment of gastric-cancer using high-throughput approaches.ATRA in 27 gastric-cancer cell-lines and tissue-slice cultures from 13 gastric-cancer patients. We performed RNA-sequencing studies in 13 cell-lines exposed to ATRA. We used these and the gastric-cancer RNA-sequencing data of the TCGA/CCLE datasets to conduct multiple computational analyses.We evaluated the anti-proliferative action of ATRA-sensitivity.  The model predicts that 45% of the TCGA gastric-cancers are sensitive to ATRA. RNA-sequencing studies performed in retinoid-treated gastric-cancer cell-lines provide insights into the gene-networks underlying ATRA anti-tumor activity. In addition, our data demonstrate that ATRA exerts significant immune-modulatory effects, which seem to be largely controlled by IRF1 up-regulation. Finally, we provide evidence of a feed-back loop between IRF1 and DHRS3, another gene which is up-regulated by ATRA.Profiling of our large panel of gastric-cancer cell-lines for their quantitative response to the anti-proliferative effects of ATRA indicate that approximately half of the cell-lines are characterized by sensitivity to the retinoid. The constitutive transcriptomic profiles of these cell-lines permitted the construction of a model consisting of 42 genes, whose expression correlates with ATRA is endowed with significant therapeutic potential in the stratified/personalized treatment gastric-cancer. Our data represent the fundaments for the design of clinical trials focusing on the use of ATRA in the personalized treatment of this heterogeneous tumor. Our gene-expression model will permit the development of a predictive tool for the selection of ATRA-sensitive gastric-cancer patients. The immune-regulatory responses activated by ATRA suggest that the retinoid and immune-checkpoint inhibitors constitute rational combinations for the management of gastric-cancer.The online version contains supplementary material available at 10.1186/s13046-023-02869-w. Intestinal”, “Diffuse” and “Mixed”  [51 [51IRF1,pathways . Finallyoprotein , which moprotein . In gastapeutics . Thus, AF1 Fig. S.Overall, the data obtained in this study suggest that pharmacological agents targeting the two proteins may be used for the design of innovative therapeutic strategies in retinoid-sensitive gastric-cancers.The present study provides data supporting the therapeutic potential of ATRA in the stratified/personalized treatment of gastric-cancer. In this context, the gene-expression model that we generated will permit the development of a predictive clinical tool for the selection of patients who may benefit from ATRA-based therapeutic strategies. Overall, our data represent the fundaments for the design/organization of clinical trials focusing on the use of ATRA in the treatment of this heterogeneous type of tumor. In particular, the strong immune-regulatory responses activated by the retinoid suggest that ATRA and immune checkpoint inhibitors constitute rational combinations in the management of gastric-cancer.Additional file 1: Supplementary Methods. Supplementary Table S1. Characteristics and source of the gastric-cancer cell-lines. Supplementary Table S2. Structure of the double stranded DNAs used for the construction of the shRNA plasmid constructs. Figure S1. Growth curves of the gastric cancer cell-lines exposed to increasing concentrations of ATRA. Figure S2. Ki67 immune-histochemistry in tissue-slice cultures of representative primary gastric-cancers exposed to ATRA. Figure S3. Effects of ATRA on the body weight of SCID mice transplanted with LMSU and NCI-N87 cells. Figure S4. HALLMARK pathway analysis of the RNA-seq results obtained following treatment of the indicated gastric cell lines with ATRA. Figure S5. KEGG pathway analysis of the RNA-seq results obtained following treatment of the indicated gastric cell lines with ATRA. Figure S6. Number of genes modulated by ATRA in retinoid-sensitive G-INT and G-DIFF gastric cancer cell-lines.Figure S7. Effects of ATRA on IRF1 protein levels and cell-growth in the retinoid-sensitive LMSU cell-line.Figure S8. Effects of ATRA on IRF1 protein expression in retinoid resistant gastric cancer cells and IRF1 over-expression in AGS cells. Original Western blots.Additional file 2: Table S3. Characteristics of the gastric-cancer patients used for the studies involving tissue-slice cultures. The table summarizes the clinical characteristics of the 13 patients considered.Additional file 3: Table S4. RNA-sequencing data. The table contains the processed RNA-seq data obtained with our panel of gastric cancer cell-lines exposed to vehicle and ATRA.Additional file 4: Table S5. Effects of ATRA on the expression of the RNAs derived from endogenous retroviruses. The table contains the levels of endogenous retroviral RNAs determined by RNA-seq data obtained from the indicated cell lines exposed to vehicle and ATRA.


Despite rapid developments in immunotherapy and targeted therapy, dacarbazine (DTIC)-based chemotherapy still has been placed at the first-line for advanced melanoma patients who are after failure of immunotherapy or targeted therapy. However, the limited response rate and survival benefit challenge the DTIC-based chemotherapy for advanced melanoma patients.Two melanoma cell lines, A375 and SK-MEL-28 were cultured with PA and DTIC over a range of concentrations for 72 h and the cell viabilities were detected by CCK8 assay. The Bliss model and ZIP model were used for calculating the synergistic effect of PA and DTIC. DNA double-strand breaks in the two cell lines were examined by the Comet assay, and cell apoptosis was analyzed by flow cytometry. The short hairpin RNA (shRNA)-mediated knockdown, Real-time polymerase chain reaction (RT-PCR) and Western blot were performed for molecular analysis.In the present study, we report that Protocatechuic aldehyde (PA) synergistically enhances the cytotoxicity of DTIC to two melanoma cell lines, A375 and SK-MEL-28. The combination of PA and DTIC augments DNA double-strand breaks and increases cell apoptosis. Further mechanism study reveals that PA destabilizes MGMT protein (O-6-Methylguanine-DNA Methyltransferase) through the ubiquitin-proteasome process and directly repairs DTIC-induced genetic lesions. Knockdown of MGMT compromises the synergistic effect between PA and DTIC.Our study demonstrates that the bioactive compound, Protocatechuic aldehyde, synergistically promotes the cytotoxicity of DTIC to melanoma cells through destabilization of MGMT protein. It could be a potential candidate for melanoma chemotherapy.The online version contains supplementary material available at 10.1186/s12906-023-03933-w. Cutaneous melanoma is the most aggressive and lethal skin cancer. Although there has been huge progression in immune checkpoint inhibitors (ICI) and BRAF or MEK targeted therapy, dacarbazine (DTIC)-based chemotherapy still stands at the first-line for advanced melanoma patients who are after failure of immunotherapy or targeted therapy , 2. The DTIC or its analogue temozolomide (TMZ) is an alkylating agent that refers alkyl groups to O6 position of guanine forming O6-methylguanine (O6-MeG). DNA containing O6-MeG is prone to be mismatched with T at the opposite strand. Regardless of whether the O6-MeG is matched with T or C, the DNA mismatch repair system recognizes them as mismatched nucleotides, and proceeds to excise and degrade the strand opposite to the O6-MeG. The continuous excision process will generate DNA double-strand breaks and trigger cell apoptosis . MGMT  is a bioactive compound isolated from a traditional Chinese herb, iorrhiza . Severaliorrhiza –18. We piorrhiza . In this2.Human cutaneous melanoma cell lines A375 and SK-MEL-28 were obtained from the Cell Bank of the Chinese Academy of Sciences. Cells were maintained in high glucose DMEM medium  supplemented with 10% fetal bovine serum  at 37◦C with 5% COProtocatechuic aldehyde  and Dacarbazine  were dissolved in dimethyl sulfoxide (DMSO) as stock solutions at the concentrations of 200mM and 50mM. Cycloheximide  was dissolved in PBS at the concentration of 15 mg/ml. MG132  was dissolved in DMSO as a stock solution at the concentration of 50mM.A375 and SK-MEL-28 cells were planted in 96-well plates at a density of 2000 cells per well. After the 72 h treatments with PA, DTIC or their combination. Cell viability was detected by the Cell Counting Kit-8 assay (CCK8) .https://synergyfinder.fimm.fi) was used for evaluating synergistic effects and calculating synergy scores following the online instructions.SynergyFinder 3.0 software , MGMT, γ-H2AX(Ser139), caspase-3, cleaved caspase-3.The RNA isolation and Real-Time PCR assay were described previously . 18s expThe sequences of primers used for Real-Time PCR are as follows.MGMT Forward: GCTGAATGCCTATTTCCACCMGMT Reverse: ACTTCTCCGAATTTCACAACC18 S Forward: TGACGGAAGGGCACCACCAG18 S Reverse: GCACCACCACCCACGGAATCNeutral comet assay was performed by the comet assay kit  following the manufacturer’s manual . Slides A375 and SK-MEL-28 cells were stained with Annexin V-FITC and propidium iodide (PI) provided by the apoptosis-detection kit . Cell apoptosis was detected by BD FACSAriaIII  and the data were analyzed by FlowJo V10.https://www.addgene.org/protocols/plko/. The constructs containing shRNAs were confirmed by Sanger sequencing and were transfected into 293T cells by the Lipofectamine 2000  with the packing plasmids psPAX2 and pMD2 to produce lentivirus.Two different MGMT-targeted shRNAs and scramble oligos were cloned into PLKO.1-neo vector, respectively.  following the Addegen online protocol at A375 and SK-MEL-28 cells were co-cultured with the lentivirus for 24 h and then selected with 1 mg/mL G418 for 10 days. The knockdown efficiency was determined by Western blotting. The shRNA oligos used in the study are listed below:shMGMT-1: GCTGCTGAAGGTTGTGAAATTshMGMT-2: GGAGGAGCAATGAGAGGCAATScramble: CCTAAGGTTAAGTCGCCCTCGP < 0.05 was considered statistically significant.All experiments were performed at least three times. Statistical comparisons were conducted by the GraphPad Prism 8.0 software and analyzed by Student t-test or multiple comparisons were performed by one-way ANOVA with Tukey’s post-hoc tests. 50 of DTIC for A375 and SK-MEL-28 were 15.40 ± 1.39 µM and 309.55 ± 5.73 µM. By contrast, the IC50 of DTIC for the 2 cell lines were 0.28 ± 0.07 µМ and 98.20 ± 0.37 µM in the presence of PA   and non-homologous end joining (NHEJ), it is rational to focus on additional DNA repair proteins potentially regulated by PA .Our study demonstrated that the bioactive compound, Protocatechuic aldehyde, synergistically promoted the cytotoxicity of DTIC to melanoma cells through the destabilization of MGMT protein. It could be a potential candidate for melanoma combinational chemotherapy.Below is the link to the electronic supplementary material.Additional file 1.