From biogenesis to aptasensors: advancements in analysis for tumor-derived extracellular vesicles research.

Extracellular vesicles (EVs) are enclosed by a nanoscale phospholipid bilayer membrane and typically range in size from 30 to 200 nm. They contain a high concentration of specific proteins, nucleic acids, and lipids, reflecting but not identical to the composition of the parent cell. The inherent characteristics and variety of EVs give them extensive and unique advantages in the field of cancer identification and treatment. Recently, EVs have been recognized as potential tumor markers for the detection of cancer. Aptamers, which are molecules of single-stranded DNA or RNA, demonstrate remarkable specificity and affinity for their targets by adopting distinct tertiary structures. Aptamers offer various advantages over their protein counterparts, such as reduced immunogenicity, the ability for convenient large-scale synthesis, and straightforward chemical modification. In this review, we summarized EVs biogenesis, sample collection, isolation, storage and characterization, and finally provided a comprehensive survey of analysis techniques for EVs detection that are based on aptamers.

Cell-SELEX and application research of a DNA aptamer against esophageal squamous cell carcinoma (ESCC) cell line TE-1.

Esophageal cancer is a common cancer with high morbidity and mortality that severely threatens the safety and quality of human life. The strong metastatic nature of esophageal cancer enables it to metastasize more quickly and covertly, making it difficult for current diagnostic and treatment methods to achieve efficient early screening, as well as timely and effective treatment. As a promising solution, nucleic acid aptamers, a kind of special single-stranded DNA or RNA oligonucleotide selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, can specifically bind with different molecular targets. In this paper, random DNA single-stranded oligonucleotides were used as the initial library. Using TE-1 cells and HEEC cells as targets, specific binding sequences were selected by 15 rounds of the cell-SELEX method, and the aptamer sequence that binds to TE-1 cells with the most specificity was obtained and named Te4. The Te4 aptamer was further validated for binding specificity, binding affinity, type of target, in vitro cytotoxicity when conjugated with DOX(Te4-DOX), and in vivo distribution. Results of in vitro validation showed that Te4 has outstanding binding specificity with a Kd value of 51.16 ± 5.52 nM, and the target type of Te4 was preliminarily identified as a membrane protein. Furthermore, the cytotoxicity experiment showed that Te4-DOX has specific cytotoxicity towards cultured TE-1 cells. Finally, the results of the in vivo distribution experiment showed that the Te4 aptamer is able to specifically target tumor regions in nude mice, showing great potential to be applied in future diagnosis and targeted therapy of esophageal cancer.

Thymus as Incontrovertible Target of Future Immune Modulatory Therapeutics.

Thymus plays a crucial role in cellular immunity by acting as a warehouse for proliferating and differentiating lymphocytes. Thymic stromal cells educate T-cells to differentiate self from non-self antigens while nurse cells and thymoproteasome play a major role in the maturation and differentiation of T-cells. The thymic conditions dictate T-cells to cope with the risk of cancer development. A study was designed to demonstrate potential mechanisms behind the failure to eliminate tumors and impaired immune surveillance as well as the impact of delay in thymus regression on cancer and autoimmune disorders. Scientific literature from Pubmed; Scopus; WOS; JSTOR; National Library of Medicine Bethesda, Maryland; The New York Academy of Medicine; Library of Speech Rehabilitation, NY; St. Thomas' Hospital Library; The Wills Library of Guys Hospital; Repository of Kings College London; and Oxford Academic repository was explored for pathological, physiological, immunological and toxicological studies of thymus. Studies have shown that systemic chemotherapy may lead to micro inflammatory environment within thymus where conventionally and dynamically metastasized dormant cells seek refuge. The malfunctioning of the thymus and defective T and Treg cells, bypassing negative selection, contributes to autoimmune disorders, while AIRE and Fezf2 play significant roles in thymic epithelial cell solidity. Different vitamins, TCM, and live cell therapy are effective therapeutics. Vitamin A, C, D, and E, selenium and zinc, cinobufagin and dietary polysaccharides, and glandular extracts and live cell injections have strong potential to restore immune system function and thymus health. Moreover, the relationship between different ages/stages of thymus and their corresponding T-cell mediated anti-tumor immune response needs further exploration.

Gravity-driven flow control in a fully integrated microfluidic cartridge for molecular point-of-care testing.

Molecular point-of-care testing (POCT) system is crucial for the timely prevention and control of infectious diseases. We recently proposed a gravity-driven microfluidic cartridge for molecular POCT detection, without the need for external sources or actuators, demonstrating the advantages in terms of the reduced cartridge size and low development costs. How to achieve precise control of liquid flow behavior is challenging for the gravity-driven cartridge. In this work, we explored the underlying mechanism of flow control in the cartridge and offered optimized solutions for our cartridge design to achieve precise control of dynamic flow rates and enhance pumping efficiency significantly. Through the computational fluid dynamics simulations, we demonstrated that adopting an asymptotic contraction chamber geometry design and a closed-loop air flow channel design with the cartridge inlet can facilitate stable laminar flow of the liquid in our microfluidic cartridge, enabling precise control of flow velocity. We further optimized the microchannel diameter and the contact angle of the liquid with the microchannel wall. The effectiveness of the optimized cartridge for POCT detection was well validated by the accurate detection of the human papillomavirus type 16 virus in the 120 clinical swab samples.

A Chamber-Based Digital PCR Based on a Microfluidic Chip for the Absolute Quantification and Analysis of KRAS Mutation.

The Kirsten rat sarcoma virus gene (KRAS) is the most common tumor in human cancer, and KRAS plays an important role in the growth of tumor cells. Normal KRAS inhibits tumor cell growth. When mutated, it will continuously stimulate cell growth, resulting in tumor development. There are currently few drugs that target the KRAS gene. Here, we developed a microfluidic chip. The chip design uses parallel fluid channels combined with cylindrical chamber arrays to generate 20,000 cylindrical microchambers. The microfluidic chip designed by us can be used for the microsegmentation of KRAS gene samples. The thermal cycling required for the PCR stage is performed on a flat-panel instrument and detected using a four-color fluorescence system. "Glass-PDMS-glass" sandwich structure effectively reduces reagent volatilization; in addition, a valve is installed at the sample inlet and outlet on the upper layer of the chip to facilitate automatic control. The liquid separation performance of the chip was verified by an automated platform. Finally, using the constructed KRAS gene mutation detection system, it is verified that the chip has good application potential for digital polymerase chain reaction (dPCR). The experimental results show that the chip has a stable performance and can achieve a dynamic detection range of four orders of magnitude and a gene mutation detection of 0.2%. In addition, the four-color fluorescence detection system developed based on the chip can distinguish three different KRAS gene mutation types simultaneously on a single chip.

Aptamers in cancer therapy: problems and new breakthroughs.

Aptamers, a class of oligonucleotides that can bind with molecular targets with high affinity and specificity, have been widely applied in research fields including biosensing, imaging, diagnosing, and therapy of diseases. However, compared with the rapid development in the research fields, the clinical application of aptamers is progressing at a much slower speed, especially in the therapy of cancer. Obstructions including nuclease degradation, renal clearance, a complex selection process, and potential side effects have inhibited the clinical transformation of aptamer-conjugated drugs. To overcome these problems, taking certain measures to improve the biocompatibility and stability of aptamer-conjugated drugs in vivo is necessary. In this review, the obstructions mentioned above are thoroughly discussed and the methods to overcome these problems are introduced in detail. Furthermore, landmark research works and the most recent studies on aptamer-conjugated drugs for cancer therapy are also listed as examples, and the future directions of research for aptamer clinical transformation are discussed.

Identification of PHB2 as a Potential Biomarker of Luminal A Breast Cancer Cells Using a Cell-Specific Aptamer.

Precise diagnosis of breast cancer molecular subtypes remains a great challenge in clinics. The present molecular biomarkers are not specific enough to classify breast cancer subtypes precisely, which requests for more accurate and specific molecular biomarkers to be discovered. Aptamers evolved by the cell-systematic evolution of ligands by exponential enrichment (SELEX) method show great potential in the discovery and identification of cell membrane targets via aptamer-based cell membrane protein pull-down, which has been regarded as a novel and powerful weapon for the discovery and identification of new molecular biomarkers. Herein, a cell membrane protein PHB2 was identified as a potential molecular biomarker specifically expressed in the cell membranes of MCF-7 breast cancer cells using a DNA aptamer MF3Ec. Further experiments demonstrated that the PHB2 protein is differentially expressed in the cell membranes of MCF-7, SK-BR-3, and MDA-MB-231 breast cancer cells and MCF-10A cells, and the binding molecular domains of aptamer MF3Ec and anti-PHB2 antibodies to the PHB2 protein are different due to there being no obvious competitions between aptamer MF3Ec and anti-PHB2 antibodies in the binding to the cell membranes of target MCF-7 cells. Due to those four cells belonging to luminal A, HER2-positive, and triple-negative breast cancer cell subtypes and human normal mammary epithelial cells, respectively, the PHB2 protein in the cell membrane may be a potential biomarker for precise diagnosis of the luminal A breast cancer cell subtype, which is endowed with the ability to differentiate the luminal A breast cancer cell subtype from HER2-positive and triple-negative breast cancer cell subtypes and human normal mammary epithelial cells, providing a new molecular biomarker and therapeutic target for the accurate and precise classification and diagnostics and personalized therapy of breast cancer.

Lung Cancer Nodules Detection via an Adaptive Boosting Algorithm Based on Self-Normalized Multiview Convolutional Neural Network.

Lung cancer is the deadliest cancer killing almost 1.8 million people in 2020. The new cases are expanding alarmingly. Early lung cancer manifests itself in the form of nodules in the lungs. One of the most widely used techniques for both lung cancer early and noninvasive diagnosis is computed tomography (CT). However, the intensive workload of radiologists to read a large number of scans for nodules detection gives rise to issues like false detection and missed detection. To overcome these issues, we proposed an innovative strategy titled adaptive boosting self-normalized multiview convolution neural network (AdaBoost-SNMV-CNN) for lung cancer nodules detection across CT scans. In AdaBoost-SNMV-CNN, MV-CNN function as a baseline learner while the scaled exponential linear unit (SELU) activation function normalizes the layers by considering their neighbors' information and a special drop-out technique (α-dropout). The proposed method was trained and tested using the widely Lung Image Database Consortium and Image Database Resource Initiative (LIDC-IDRI) and Early Lung Cancer Action Program (ELCAP) datasets. AdaBoost-SNMV-CNN achieved an accuracy of 92%, sensitivity of 93%, and specificity of 92% for lung nodules detection on the LIDC-IDRI dataset. Meanwhile, on the ELCAP dataset, the accuracy for detecting lung nodules was 99%, sensitivity 100%, and specificity 98%. AdaBoost-SNMV-CNN outperformed the majority of the model in accuracy, sensitivity, and specificity. The multiviews confer the model's good generalization and learning ability for diverse features of lung nodules, the model architecture is simple, and has a minimal computational time of around 102 minutes. We believe that AdaBoost-SNMV-CNN has good accuracy for the detection of lung nodules and anticipate its potential application in the noninvasive clinical diagnosis of lung cancer. This model can be of good assistance to the radiologist and will be of interest to researchers involved in the designing and development of advanced systems for the detection of lung nodules to accomplish the goal of noninvasive diagnosis of lung cancer.

A sample-to-answer, quantitative real-time PCR system with low-cost, gravity-driven microfluidic cartridge for rapid detection of SARS-CoV-2, influenza A/B, and human papillomavirus 16/18.

The pandemic of coronavirus disease 2019 (COVID-19), due to the novel coronavirus (SARS-CoV-2), has created an unprecedented threat to the global health system, especially in resource-limited areas. This challenge shines a spotlight on the urgent need for a point-of-care (POC) quantitative real-time PCR (qPCR) test for sensitive and rapid diagnosis of viral infections. In a POC system, a closed, single-use, microfluidic cartridge is commonly utilized for integration of nucleic acid preparation, PCR amplification and florescence detection. But, most current cartridge systems often involve complicated nucleic acid extraction via active pumping that relies on cumbersome external hardware, causing increases in system complexity and cost. In this work, we demonstrate a gravity-driven cartridge design for an integrated viral RNA/DNA diagnostic test that does not require auxiliary hardware for fluid pumping due to adopted extraction-free amplification. This microfluidic cartridge only contains two reaction chambers for nucleic acid lysis and amplification respectively, enabling a fast qPCR test in less than 30 min. This gravity-driven pumping strategy can help simplify and minimize the microfluidic cartridge, thus enabling high-throughput (up to 12 test cartridges per test) molecular detection via a small cartridge readout system. Thus, this work addresses the scalability limitation of POC molecular testing and can be run in any settings. We verified the analytical sensitivity and specificity of the cartridge testing for respiratory pathogens and sexually transmitted diseases using SARS-CoV-2, influenza A/B RNA samples, and human papillomavirus 16/18 DNA samples. Our cartridge system exhibited a comparable detection performance to the current gold standard qPCR instrument ABI 7500. Moreover, our system showed very high diagnostic accuracy for viral RNA/DNA detection that was well validated by ROC curve analysis. The sample-to-answer molecular testing system reported in this work has the advantages of simplicity, rapidity, and low cost, making it highly promising for prevention and control of infectious diseases in poor-resource areas.

Plasmon enhanced broadband photoelectrochemical response of ZnO/CdTe/Bi nanoarrays for quantitative analysis of nasopharyngeal carcinoma in a recyclable microfluidic biosensing chip.

Traction and quantitative detection of trace amount of target cells inside of biochip system in real-time has been a challenge for biomedical and clinical researchers. In this manuscript, we report fabrication of a photoelectrochemical platform that has integrated both biometric recognition and signal acquisition through microfabrication technology. In this chip, a ternary ZnO/CdTe/Bi nanorod array is fabricated, which significantly extends the absorption wavelength from the UV to the visible and even near-infrared regions for both photocarrier generation and surface plasmon resonance, ultimately achieving the amplification of initial photocurrent responses. The artificially designed aptamers with amino groups are assembled on the surface of the outermost Bi nanoparticles, which are used as signal probes due to the specific recognition to the nasopharyngeal carcinoma 5-8F cell. We demonstrate that different concentration of 5-8F cells is captured by aptamers, and the signal changes accordingly with the amount of the cells that have been trapped. As a result, the proposed biochip demonstrates rapid response in a wide linear range of 102-107 cells·mL-1 with the detection limit as low as 32 cells·mL-1 and provides a potential useful model for a variety of biological analysis including clinical point-of-care testing.

Rapid Capturing and Chemiluminescent Sensing of Programmed Death Ligand-1 Expressing Extracellular Vesicles.

Cancer specific extracellular vesicles (EVs) are of significant clinical relevance, for instance programmed death ligand-1 (PD-L1) expressing EVs (PD-L1@EVs) have been shown to be ideal biomarker for non-invasive diagnosis of cancer and can predate the response of cancer patients to anti-PD-1/PD-L-1 immunotherapy. The development of sensitive and straightforward methods for detecting PD-L1@EVs can be a vital tool for non-invasive diagnosis of cancer. Most of the contemporary methods for EVs detection have limitations such as involvement of long and EV's loss prone isolation methods prior to detection or they have employed expensive antibodies and instruments to accomplish detection. Therefore, we designed an ultracentrifugation-free and antibody-free sensing assay for PD-L1@EV by integrating Titanium oxide (TiO2) coated magnetic beads (Fe3O4@TiO2) rapid capturing of EVs from undiluted serum with aptamers specificity and chemiluminescence (CL) sensitivity. To accomplish this we used Fe3O4@TiO2 beads to rapidly capture EVs from the undiluted patient serum and added biotin labelled PD-L1 aptamer to specifically recognize PD-L1@EVs. Later, added streptavidin-modified Alkaline phosphates (ALP) taking advantage of biotin-streptavidin strong binding. Addition of CDP-star, a chemiluminescent substrate of ALP, initiates the chemiluminiscense that was recorded using spectrophotometer. The sensing assay showed high sensitivity with limit of detection (LOD) as low as 2.584×105 EVs/mL and a wider linear correlation of CL intensity (a.u.) with the concentration of PD-L1@EVs from 105 to 108 EVs/mL. To examine the clinical utility of sensing assay we used undiluted serum samples from lung cancer patients and healthy individuals and successfully discern between healthy individuals and lung cancer patients. We are optimistic that the sensing assay can ameliorate our ability to be able to diagnose lung cancer non-invasively and can be helpful to predate the patient's response to anti-PD-1/PD-L1 immunotherapy.

Programmable Biosensors Based on RNA-Guided CRISPR/Cas Endonuclease.

Highly infectious illnesses caused by pathogens constitute severe threats to public health and lead to global economic loss. The use of robust and programmable clustered regularly interspaced short palindromic repeat and CRISPR-associated protein (CRISPR-Cas) systems, repurposed from genome-engineering applications has markedly improved traditional nucleic acid detection for precise identification, independently enabling rapid diagnostics of multiplex biomarker with genetic and mutation related to tumors, and microbial pathogens. In this review, we delineate the utility of the current CRISPR-Cas enzyme as biosensors by which these effector toolkits achieve recognition, signaling amplification, and finally, accurate detection. Additionally, we discuss the details of the dominance and hurdles related to expanding this revolutionary technology into an effective and convenient contraption crucial for improving the rational redesign to CRISPR/Cas biosensing. Overall, this review provides an insight into the current status of rapid and POC diagnostic systems by CRISPR/Cas tools.

Selection of a High-Affinity DNA Aptamer for the Recognition of Cadmium Ions.

Cadmium ions as toxic metallic pollutants have been dramatically risen due to industrial production, which causes serious environmental damages and potential health risks. Thus, developing sensitive and specific probes to recognize cadmium ions is vital for human and environmental safety. In this work, the aptamer as a capture probe was selected for the identification of cadmium ions. The modified SELEX method based on immobilizing ssDNA libraries on streptavidin magnetic beads was used to the high-affinity aptamer selection for binding cadmium ions. After 9 rounds of selection, 4 ssDNA sequences with the highest enrichment were obtained, and the Cd-1 aptamer exhibited the highest affinity to cadmium ions. The dissociation constant Kd value of the Cd-1 aptamer was 81.39 µM for cadmium ions. Later, we also investigated the binding specificity of Cd-1 aptamer toward other heavy metal ions. Given the availability of immobilizing ssDNA library on matrix, we believe the strategy also applies to discover other ions in a reproducible manner.

HACE1-mediated NRF2 activation causes enhanced malignant phenotypes and decreased radiosensitivity of glioma cells.

HACE1, an E3 ubiquitin-protein ligase, is frequently inactivated and has been evidenced as a putative tumor suppressor in different types of cancer. However, its role in glioma remains elusive. Here, we observed increased expression of HACE1 in gliomas related to control subjects, and found a strong correlation of high HACE1 expression with poor prognosis in patients with WHO grade III and IV as well as low-grade glioma (LGG) patients receiving radiotherapy. HACE1 knockdown obviously suppressed malignant behaviors of glioma cells, while ectopic expression of HACE1 enhanced cell growth in vitro and in vivo. Further studies revealed that HACE1 enhanced protein stability of nuclear factor erythroid 2-related factor 2 (NRF2) by competitively binding to NRF2 with another E3 ligase KEAP1. Besides, HACE1 also promoted internal ribosome entry site (IRES)-mediated mRNA translation of NRF2. These effects did not depend on its E3 ligase activity. Finally, we demonstrated that HACE1 dramatically reduced cellular ROS levels by activating NRF2, thereby decreasing the response of glioma cells to radiation. Altogether, our data demonstrate that HACE1 causes enhanced malignant phenotypes and decreased radiosensitivity of glioma cells by activating NRF2, and indicate that it may act as the role of prognostic factor and potential therapeutic target in glioma.

Applications of Aptamer-Bound Nanomaterials in Cancer Therapy.

Cancer is still a major disease that threatens human life. Although traditional cancer treatment methods are widely used, they still have many disadvantages. Aptamers, owing to their small size, low toxicity, good specificity, and excellent biocompatibility, have been widely applied in biomedical areas. Therefore, the combination of nanomaterials with aptamers offers a new method for cancer treatment. First, we briefly introduce the situation of cancer treatment and aptamers. Then, we discuss the application of aptamers in breast cancer treatment, lung cancer treatment, and other cancer treatment methods. Finally, perspectives on challenges and future applications of aptamers in cancer therapy are discussed.

Rapid Detection of DNA Methylation with a Novel Real-Time Fluorescence Recombinase-Aided Amplification Assay.

Researchers have conducted in-depth research on DNA methylation mechanism, which is related to various diseases such as deficiency of imprinted gene and occurrence of tumors. This study provides a novel rapid quantitative detection assay and real-time fluorescence recombinase-aided amplification assay (RAA) for DNA methylation. Firstly, specific sequence of methylation genes was chosen and primers and fluorogenic probe for RAA experiment were designed and synthesized. Lastly, these amplification products were proven by sequencing and analysis. Results showed that the amplification efficiency and template concentration of RAA had linear dependent (R² > 95%) when the concentration range was 4.64×108 copies/µL˜4.64×104 copies/µL. The test assay can also detect positive samples when the template concentration is below 4.64×104 copies/µL. Remarkably, the entire experiment process only takes 15-20 minutes, so it is beneficial for rapid bedside simple screening of some special DNA methylation sites, such as detection of resistance genes. In a word, this method has very great potential for diseases with DNA methylation in clinical settings, especially if methylation analysis needs to be done quickly and easily.

A biotin-avidin-system-based virus-mimicking nanovaccine for tumor immunotherapy.

Virus is a nanosized pathogen and mainly composed of viral protein and nucleic acids. Under the pressure of long-term selection, mammals have gradually evolved effective immune mechanisms to defend themselves against viruses. In addition to recognizing viral proteins, immune system can also respond to viral sequence-specific nucleic acids, including CpG ODN, single- and double- strand RNA, and thereby enhancing the ability to remove infected viruses. Inspired by these immune mechanisms, we have attempted to develop a tracing virus-mimicking nanovaccine for tumor immunotherapy. This nanovaccine mainly consists of nucleic acids (CpG ODN), proteins (including tumor-associated antigen, and neutravidin (nAvidin) as skeleton materials for constructing nanovaccine and carriers for loading tumor-associated antigen and CpG ODN), and the dye molecules for assembling nAvidin to form nanoparticles comparable in size to viruses and tracing the vaccine in vitro and in vivo. The as-prepared nanovaccine efficiently induces the maturation of dendritic cell, the enhancement of antigen cross-presentation ability, and amplification of cytokine production in vitro. Furthermore, in vivo analysis clearly shows that it targets lymph nodes, successfully presents antigens to generate tumor-antigen-specific CD8+ T cells and induces a Th1-biased immune response. Most notably, this virus-mimicking nanovaccine significantly inhibits the growth of antigen-expressed tumor and prolongs the survival time of the antigen-expressed tumor bearing mice.

Synergistic activation of mutant TERT promoter by Sp1 and GABPA in BRAFV600E-driven human cancers.

The activating TERT promoter mutations and BRAFV600E mutation are well-established oncogenic alterations in human cancers. Coexistence of BRAFV600E and TERT promoter mutations is frequently found in multiple cancer types, and is strongly associated with poor patient prognosis. Although the BRAFV600E-elicited activation of ERK has been demonstrated to contribute to TERT reactivation by maintaining an active chromatin state, it still remains to be addressed how activated ERK is selectively recruited to mutant TERT promoter. Here, we report that transcription factor GABPA mediates the regulation of BRAFV600E/MAPK signaling on TERT reactivation by selectively recruiting activated ERK to mutant TERT promoter, where activated ERK can phosphorylate Sp1, thereby resulting in HDAC1 dissociation and an active chromatin state. Meanwhile, phosphorylated Sp1 further enhances the binding of GABPA to mutant TERT promoter. Taken together, our data indicate that GABPA and Sp1 synergistically activate mutant TERT promoter, contributing to tumorigenesis and cancer progression, particularly in the BRAFV600E-driven human cancers. Thus, our findings identify a direct mechanism that bridges two frequent oncogenic alterations together in TERT reactivation.

Advances in Aptamer Screening and Drug Delivery.

Chemotherapy is the most commonly used treatment for cancer. However, due to their short circulation time and lack of specific biological distribution, chemotherapeutic drugs cause severe systemic toxicity. Using the agents specifically binding to the targeted molecules might resolve this dilemma. Aptamers can directly connect with the drugs or couple with the nano-carrier to reduce systemic toxicity. In this review, we elucidated the definition, characteristics and screening process of aptamers. The methods of drug delivery by aptamers were illustrated in details. Furthermore, clinical application of aptamers in recent years was briefly summarized and its long-term prospects were put forward.

A mini-review of embedded 3D printing: supporting media and strategies.

Embedded 3D printing is an additive manufacturing method based on a material extrusion strategy. Its distinctive feature is that the printing process is carried out in a supporting medium, and the printed ink filaments can be embedded in the supporting medium. It makes the printing process almost undisturbed by gravity, and no additional support needs to be planned before the object is printed. In recent years, embedded 3D printing has been used in sensors, software robots, tissue engineering, dosage forms, and organ models, showing strong potential. This article summarizes embedded 3D printing's latest progress on supporting media and strategies in a short space.