Bioresponsive hydrogel microfibers for neuroblastoma drug evaluation

Bioresponsive hydrogel microfibers for neuroblastoma drug evaluation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 January 2025 V1 Latest version Share on Bioresponsive hydrogel microfibers for neuroblastoma drug evaluation Authors : Taiyu Song 0000-0003-3924-8452 , Qiyang Shen 0000-0001-8963-1244 , Yi Cheng , Yue Zhi , Guangling Liu , Haozhen Ren 0000-0002-6198-2904 , and Jinglin Wang 0000-0002-4349-750X [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173586979.94257316/v1 273 views 156 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Chemotherapy remains the primary systemic treatment for patients with neuroblastoma, but the lack of appropriate in vitro tumor microenvironment models has resulted in suboptimal efficacy of chemotherapeutic agents. In this study, we propose innovative bioresponsive hydrogel microfibers that replicate the mechanical properties of extracellular matrix surrounding neuroblastoma cells for assessing tumor drug responses. These microfibers are composed of an alginate/poly (N-Isopropyl acrylamide) shell and a carboxymethyl cellulose core, fabricated by microfluidic technology. Due to the precise manipulation afforded by microfluidics, it is possible to continuously generate fibers that encapsulate cells with uniform size and precise structure. Additionally, the rapid temperature response characteristics enabled the microfibers to mimic the mechanical properties of the extracellular matrix, thereby regulating the cellular pressure environment and rapidly forming highly active three-dimensional tumor spheroids. Ultimately, our findings demonstrate that neuroblastoma spheres within the microfibers display varying sensitivities to different chemotherapy drugs under distinct external pressure conditions. In conclusion, this biomimetic extracellular matrix microfiber offers a dependable foundation for replicating the neuroblastoma microenvironment and facilitating the assessment of clinical drug efficacy. Introduction Neuroblastoma (NB) is the predominant extracranial malignancy in infants and young children [1] . Despite the administration of intensive chemotherapy, a significant proportion of NB patients still develop chemotherapy resistance [2] . Currently, platforms for evaluating chemotherapy drugs have been presented to guide patient-specific treatment, including cell culture systems and animal models [3] . These platforms aim to mimic the complex in vivo tumor microenvironment (TME) [4] , especially the mechanical effects of the extracellular matrix (ECM) on tumor cells [5] . Traditional 2D or 3D cell culture systems have shown some promising results in drug evaluation [6] , but their similarity to in vivo tissues is limited, hindering their ability to accurately describe physiological responses within a living organism [7] . Additionally, mouse models are often used in drug research for NB, such as xenograft models and genetically engineered mouse models, but they suffer from the drawback of long animal modeling cycles [8] . Hence, it is imperative to establish a streamlined and rapid tumor drug assessment platform capable of replicating the mechanical characteristics of the ECM and its interplay with tumor cells. Here, inspired by the mechanical properties of the NB TME, we propose an innovative bioresponsive hydrogel microfiber designed for assessing tumor drug responses, as shown in Figure 1 . Owing to the precise manipulability of microfluidic technology, submillimeter-scale fluids can be controlled within micrometer or nanometer-scale spaces [9] . Microfluidics technology can continuously produce uniform hydrogel microfibers that mimic the ECM in composition and structure for tumor cell culture [10] . Prominently, by introducing bioactive agents, hydrogel microfibers can achieve functional trigger responses [11] . For example, poly (N-isopropylacrylamide) (PNIPAM) polymer chains with both hydrophilic and hydrophobic groups exhibit temperature-sensitive properties [12] . When the temperature exceeds the critical value of 32°C, the introduction of PNIPAM hydrogel will undergo volume contraction [13] , perfectly mimicking pressure transmission in the TME. Therefore, we believe that by combining microfluidics and smart responsive elements, the prepared hydrogel microfibers can achieve a biomimetic culture of tumor cells and mechanical effects on a single platform, providing a new approach for tumor drug evaluation. In this study, we fabricated a core-shell structured microfiber for NB drug screening through microfluidic technology. The microfiber consisted of carboxymethyl cellulose (CMC) loaded with NB cells as the core, and a hybrid hydrogel shell composed of alginate (ALG) and PNIPAM. The precise fluid manipulation of microfluidics imparted the microfiber with unique uniformity, stability, and controllable dimensions, allowing for the even distribution and growth of tumor cells within it. Due to the excellent biocompatibility of the hydrogel components, the encapsulated tumor cells proliferate rapidly. Besides, the temperature-responsive behavior of PNIPAM facilitated the contraction of the microfiber, providing mechanical performance transmission to the cells and enabling the formation of 3D tumor spheroids. Furthermore, under different external pressure environments, the NB spheroids within the microfiber displayed notable variations in sensitivity to different chemotherapy drugs, offering more accurate drug guidance. In conclusion, the bioresponsive core-shell microfiber serves as a dependable tool for replicating the NB tumor microenvironment and facilitating efficient clinical drug assessment. Figure 1. The schematic of utilizing microfluidic technology to fabricate core-shell microfibers, mimicking the mechanical environment of NB in vivo, for tumor chemotherapy drug evaluation. Results and Discussion In one typical assay, we dedicated to investigating and optimizing the thermo-responsive properties of composite hydrogels, specifically ALG/PNIPAM. A columnar film of ALG/PNIPAM hydrogel was created following curing with Ca 2+ ions to visually assess the contractility effect. As depicted in Figure S1, when the hydrogel was placed on a heating WorkBench set at 37°C, it experienced significant shrinkage, along with the displacement of water. The pronounced change in the size of the composite hydrogels indicated a robust temperature-responsive nature. The study also examined how different volume ratios of ALG to PNIPAM (1:1, 1:2, 1:3) affected the film’s responsiveness. All hydrogel films containing PNIPAM exhibited thermo-triggered deformability. Notably, the hydrogel with a volume ratio of 1:3 demonstrated superior contraction capabilities compared to the 1:1 and 1:2 ratios. Furthermore, the shrinkage efficiency of these ALG/PNIPAM hydrogels was found to decrease with higher concentrations of ALG. This suggests that the hydrophilic, ionically crosslinked ALG remains stable as the temperature increases, thereby diminishing the hydrophobicity of the hydrogel mixture at higher temperatures. As illustrated in Figure S2, the mechanical strength of the columnar hydrogel film was significantly affected by the presence or absence of ionic crosslinks. The study’s findings underscore the importance of the ALG/PNIPAM ratio in dictating the thermo-responsive behavior and mechanical properties of the hydrogel films, offering valuable insights for the development of advanced hydrogel materials. Utilizing a microfluidic device with a concentric design, we fabricated composite microfibers in a single step through ionic polymerization, as depicted in Figure S3a. The process involved simultaneously pumping fluid microchannels with CMC (core flow,1.0 wt%), ALG/PNIPAM (middle flow, 1.0 wt%/3.0 wt%), and CaCl 2 (shell flow, 1.0 wt%). The interaction of these solutions with calcium chloride in the outer phase of the coaxial nozzle and collection pool led to the formation of stiff shells. The hydrogel’s rapid cross-linked capability enabled the encapsulation of CMC within the microfibers, yielding uniformly structured core-shell microfibers, as shown in Figure S3a-S3c. These hydrogel microfibers exhibit a distinct core-shell architecture, with no solubility between the inner and intermediate layers, as illustrated in Figures 2a-2b. The precision of the microfluidic system ensured the microfibers’ excellent monodispersity. Notably, the consistent shell thickness and core diameter across the microfibers highlight the reliability of microfluidic spinning technology for fabricating such materials. High-resolution scanning electron microscopy (SEM) images further corroborate this observation, as seen in Figure 2c. Key parameters concerning the structure and volume of hydrogel microfibers were also controlled. The outer phase flow velocity could be adjusted to regulate the microfiber size. As shown in Figure 2d, an increased flow rate in the outer phase resulted in smaller microfibers. Additionally, maintaining a constant inner phase fluid rate, the overall diameter of the microfibers decreased when the middle phase fluid rate was increased from 45 to 65 μL/min, as depicted in Figure 2e. Similarly, keeping the CaCl 2 fluid rate constant and increasing the inner CMC fluid rate from 2 to 10 μL/min led to an expansion of the core layer diameter and a reduction in microfiber thickness, as documented in Figure 2f. By manipulating the flow velocities of the inner and middle phases in varying proportions while maintaining a constant outer phase flow rate, hydrogel microfibers of different thicknesses can be produced, as demonstrated in Figure S4. Incorporating distinct fluorescent nanospheres into the inner and middle phase solutions allowed for more direct visualization of the changes in microfiber thickness due to varying middle/inner phase velocity ratios, as shown in Figure 2g. Figure 2. Preparation of ALG/PNIPAM/CMC microfibers.A typical core-shell microfiber was recorded from both Top-view (a) and Cross-sectional view (b). An freeze-dried microfiber was observed through SEM images (c-i): its surface (c-ii), as well as the CMC core (c-iii). The microfiber size is shown to be dependent on the external flow rate (d) and the middle flow rate (e). The influence of the inner flow rate on microfiber size is depicted in (f). Fluorescent images of microfibers was presented in (g), where ALG/PNIPAM and CMC solutions was added with fluorescent nanoscale particles L4655 (green) or L3280 (red). The scale bars represent 300μm in (a), 200μm in (b), (c-i), and (g), 100μm in (c-ii), and 10μm in (c-iii). To evaluate the contractile capabilities of the composite microfibers in response to temperature variations, a hot stage maintained at a consistent temperature was employed. The temperature-responsive behavior of the microfibers was monitored using an optical microscope, as illustrated in Figure 3a. The contraction of PNIPAMupon temperature elevation results from the aggregation of its hydrophilic groups, which decreases solubility and imparts hydrophobic properties. In contrast, a drop in temperature triggers a structural transformation in PNIPAM, dispersing its hydrophilic groups and enhancing water solubility, as depicted in Figure 3b. Notably, the microfibers exhibited a remarkable ability to return to their original morphology and color following the cessation of heating, as shown in Figure 3c. Analysis over the time span of 0-500 seconds revealed that the overall length of the composite microfibers contracted, with both transverse and longitudinal diameters reducing by approximately 20% after 500 seconds of heating (Figures 3d-3e). Furthermore, the microfibers demonstrated the capability to revert to their original dimensions after undergoing ten heating-cooling cycles, indicating their thermal durability and reproducibility, as evidenced in Figure 3f. This consistent behavior reinforces the potential application of these temperature-responsive composite microfibers in various fields, including soft robotics and smart materials. Figure 3. Thermal Responsiveness of ALG/PNIPAM/CMC Hydrogel Microfibers. The schematic diagrams in (a-i) and (a-ii) illustrate the original state and network structure of the hydrogel microfibers, respectively. The transverse (a-iii) and longitudinal diameters (a-iv) are shown before heating. The schematic diagrams in (b-i) and (b-ii) depict the contraction state and network structure of the microfibers during heating. The transverse (b-iii) and longitudinal diameters (b-iv) are displayed when the microfibers are heated. The schematic diagrams in (c-i) and (c-ii) represent the recovery state and network structure of the microfibers after returning to room temperature (RT). The transverse (c-iii) and longitudinal diameters (c-iv) are shown once the microfibers have recovered. The graphs in (d) and (e) demonstrate the transverse and longitudinal shrinkage ratios of the composite microfibers as a function of heating time. Figure (f) shows the volume change of the composite microfibers under ten cycles of heating (45°C) on/off. The scale bars are 400μm for (c-iii) and 500μm for (c-iv). The composite microfiber scaffolds were evaluated for their capacity to support biological activity by encapsulating the SN-SY5Y NB cell line and monitoring cellular behavior over a period of nine days, as elaborated in Figure 4a. Tumor cells were observed to initiate aggregation within a 24-hour timeframe. Over subsequent days, the cells exhibited robust viability and rapid proliferation, culminating in the organization of distinct spheroid structures, as shown in Figure 4b. Fluorescence quantitative analysis confirmed the rapid proliferation of cells throughout the cultivation process, validating the suitability of the culture system for NB cells, as illustrated in Figures S5b-S5c. The tumor spheroids formed within the composite microfibers maintained uniform sizes, as shown in Figure S5d. Further observations indicated that NB cells within ALG/CMC microfibers retained good proliferation capabilities, stable cell activity, and uniform cell sizes, as demonstrated in Figure S5a. To assess the impact of shell contraction on pressure transmission within internal tumor spheroids, a computational simulation was conducted using COMSOL Multiphysics simulation software. The simulation outcomes demonstrated that the microfiber shell, via volumetric phase transitions, consistently promoted the transfer of pressure from the external environment to the internal milieu, thereby maintaining a persistent pressure environment for the inner tumor spheroid, as elaborated in Figures 4c-4e. Figure 4. Generation of NB tumor spheroids encapsulated in composite hydrogel microfibers and simulation of tumor compression microenvironment. (a) This illustration depicts the process of encapsulating tumor cells within composite hydrogel microfibers and simulating the state of the tumor compression microenvironment. (b) Light microscopy and live/dead fluorescence images of the SY5Y cell line are presented after encapsulation in PNIPAM microfibers for 1, 3, 7, and 9 days. These images provide insights into the cellular viability and proliferation over time. (c-e) Numerical simulations are used to analyze the shrinkage effect of ALG/PNIPAM/ CMC microfibers. (c) The simulation image shows the formation of Von Mises Stress in the thermo-correspondences of ALG/PNIPAM hydrogels. (d) The simulation illustrates the process by which stress is transferred into the inner microfibers from a cross-sectional perspective. (e) The overall distance strain of the microfibers is simulated, providing a comprehensive view of the mechanical behavior under stress. The scale bars represent 200 µm for light microscopic images and 100 µm for fluorescent live/dead cells images. To investigate the biological behavior of tumor cells in two distinct extracellular matrix environments, hematoxylin and eosin (HE) staining was performed on tumor spheroids derived from two types of composite microfibers. The staining results revealed that tumor spheroids within ALG shell microfibers exhibited loosely arranged cells, a higher presence of extracellular matrix components, and lightly stained nuclei, as depicted in Figure 5a. Conversely, tumor spheroids within ALG/PNIPAM shell microfibers displayed a more compact cell arrangement, reduced extracellular matrix components, and darker stained nuclei. These observations suggest differences in the proliferation rates of neuroblastoma cells and the final size of tumor spheroids under varying extracellular pressure conditions, as illustrated in Figure 5b. The expression of tumor-relevant biological molecules was further analyzed by immunoblotting following the formation of tumor spheroids. The CD133 marker, commonly used to identify cancer stem cells in NB cells, was assessed [14] . Nestin, a neuronal biomarker was also evaluated [15] . Vimentin, a phenotypic protein found in interstitial cells and indicative of epithelial-mesenchymal transition (EMT), was analyzed for its expression levels. Elevated expression of vimentin can lead to structural and functional abnormalities in the adhesion molecules of epithelial-derived tumor cells, promoting tumor migration and resistance to chemotherapy [16] . Compared to traditional 2D cell culture models, this innovative neuroblastoma cell culture model exhibits enhanced stem cell properties and EMT capabilities, as shown in Figures 5c-5e and S6. Additionally, ki-67, a marker of cell proliferation, indicated that tumor cells encapsulated within microfibers were proliferative, as demonstrated in Figure 5e and S6. These findings confirm that SN-SY5Y cells embedded within the composite microfibers exhibit properties similar to those of orthotopic tumors, making them suitable as an appropriate model to drug testing. Real-time immunofluorescence also highlighted changes in cellular protein content when encapsulated in ALG/PNIPAM microfibers, as shown in Figures 5f-5g. Figure 5. Histological characteristics of tumor spheroids encapsulated in hydrogel microfibers. (a) HE staining is shown for both cross-sectional and longitudinal sections of NB tumor spheroids encapsulated in ALG/CMC or ALG/PNIPAM/CMC microfibers (b) A comparative statistical chart illustrates the proliferation of NB cells under two distinct culture conditions. (c) Quantitative analysis of protein expression levels for NB cell-related indicators under the two culture conditions is presented. (d) Western blotting analysis was employed to compare the protein expression levels of Nestin and CD133 between 2D culture and ALG/PNIPAM culture conditions. (e) Western blotting was utilized to evaluate the expression levels of Vimentin and Ki67 under these same culture conditions. (f) Confocal fluorescent imaging of NB tumor spheroids stained with CD133 and Nestin at day 3 and day 9 is displayed. (g) Confocal fluorescent imaging of NB tumor spheroids stained with ki-67 and Vimentin at day 3 and day 9. Scale bars: 100 μm (a- longitudinal sections), 50 μm (a- cross-sectional), 20 μm (f and g, high-magnification) and 10 μm (f-g, Low-magnification). In mammals, the YAP and TAZ transcription factors interact with cytoskeletal proteins, typically sequestered in the cytoplasm, which prevents them from entering the nucleus and activating transcription, thereby regulating organ size and volume [17] . In solid tumors, the ECM interacts with tumor cells, and mechanical forces generated by ECM components and stiffening activate YAP/TAZ [18] . This triggers a mechanotransduction mechanism that regulates the phenotype of solid tumor cells, influencing tumor proliferation, migration, angiogenesis, immune evasion, stemness, and resistance to treatment [19] , as illustrated in Figure 6a. In this experiment, tumor cells grown in hydrogels with thermal responsive contraction show a predominantly nuclear distribution of the YAP transcription factor. In contrast, under simple ALG gel and ECM-free culture conditions, YAP is primarily cytoplasmic, as depicted in Figure 6b and S7. Flow cytometry results indicate that the ECM’s pressure environment can affect the stem cell characteristics and EMT ability of tumor cells. Compared to soft ECM or ECM-free conditions, NB tumor cells exhibit enhanced tumor stemness (CD133 and Nestin) and EMT behavior (Vimentin) in a stiff ECM, as elaborated in Figures 6c-6e. The tumor spheroids were subjected to treatment with Carboplatin, Methotrexate, and Etoposide, adhering to established clinical drug protocols. Subsequent analysis demonstrated a reduction in cell viability correlating with elevated drug concentrations and prolonged exposure durations, as shown in Figures 6g-6i. This suggests that the evaluated pharmacological agents effectively induce apoptosis in NB tumor cells in a manner dependent on both dosage and exposure duration. Furthermore, the influence of extrusion effect on drug sensitivity was assessed, revealing that tumor spheroids enclosed in microfibers with ALG/PNIPAM shells exhibit increased resistance, especially at high concentrations of chemotherapy drugs, as demonstrated in Figures 6f-6h. This resistance may be due to increased external mechanical pressure promoting the nuclear entry of more YAP transcription factors, which participate in the expression of cell stemness and drug resistance-related proteins [20] . These findings suggest that composite hydrogel microfibers with thermo-responsive properties can mimic actual tumor environments and have the potential for use in more precise drug screening processes. Figure 6. YAP1 signaling pathway and tumor cell response to ECM rigidity. (a) A schematic diagram depicts the transition of tumor cells from a soft ECM to a rigid ECM, illustrating the changes in cellular behavior and adaptation . (b) Confocal immunofluorescence images show the localization of YAP in human SN-SY5Y cells cultured on Free-ECM, ALG, and ALG/PNIPAM hydrogels. (c-e) Flow cytometry is a commonly employed technique to identify stem cell-associated protein expression in tumor cells exposed to varying degrees of ECM rigidity. (f-h) The response of tumor spheroids under three different culture conditions to drug treatments is detailed: (f) 72-hour treatment with Carboplatin, (g) 72-hour treatment with Methotrexate, and (h) 72-hour treatment with Etoposide. Scale bar: 50 μm (b). In summary, we have effectively developed composite hydrogel microfibers comprising ALG/PNIPAM shells and CMC cores through the utilization of Microfluidic platform. These scaffolds have demonstrated potential for the formation of tumor spheroids and applications in drug screening. The microfibers produced showcased precise 3D structures and uniformity, allowing for size regulation through the adjustment of core and shell flow rates. Our results demonstrate that cells encapsulated in these microfibers can self-assemble into 3D tumor masses that maintain consistent morphology and exhibit high viability. Moreover, the composite microfibers with PNIPAM shells displayed significant thermo-responsive volume shrinkage, effectively simulating the mechanical pressures found in actual tumor environments. Furthermore, SN-SY5Y cell spheroids encapsulated within the composite hydrogel scaffolds, particularly those incorporating PNIPAM shells, exhibited differential sensitivities to identical chemotherapeutic agents when contrasted with spheroids in traditional hydrogel microfibers. These findings imply that the fabrication of microfibers that replicate mechanical extrusion processes may offer novel perspectives for the development of more precise drug screening platforms. Methods Materials: Na-alginate (ALG), Poly-N-Isopropylacrylamide (PNIPAM), calcium chloride (CaCl 2 ), polyoxymethylene (PFA) and A low-viscosity type of carboxymethyl cellulose (CMC) was purchased from Macklin. Carboplatin, Methotrexate and Etoposide were purchased from MedChemExpress (MCE). Phosphate Buffer Solution were purchased from Pricella. Bovine Serum Albumin (BSA) were bought from MERCK. A variety of capillaries were purchased from Wuhan Gairdner Tech Co., Ltd. Alginase and FluoSpheres were purchased from Sigma-Aldrich. The LIVE/DEAD cell staining kit, goat serum and 4’,6-diamidino-2-phenylindole were purchased from Beyotime Tech Co., Ltd. The 96-Well White Opaque Plates were obtained from Beyotime Tech Co., Ltd, the CellTiter-Glo kits were purchased from Promega Corporation Co., Ltd, and all antibodies were purchased from Proteintech (WuHan, CHN). SN-SY5Y Cell Line was ordered from Pricella. Fabrication of composite microfibers: The composition of the outer flow consisted of 1.0 wt% CaCl 2 , the middle (shell) flow consisted of 1 wt% ALG, PNIPAM (5wt%), and the inner (core) fluid consisted of 1.0 wt% CMC solution. Phase fluids were introduced into a microfluidic device consisting of circular fluid inlets, syringe pumps, and a microfiber fabrication unit. The inner, middle, and outer capillaries had transverse diameters of about 100 μm, 300 μm, and 1000 μm, respectively. The microfluidic capillary and the collecting bath containing 1.0 wt% CaCl 2 were connected to a voltage power supply with positive and negative terminals. Subsequently, the microfibers were then cross-linked with Ca 2+ under the collecting solution after shearing. SN-SY5Y cell spheroids culture: The SN-SY5Y cells were dissociated into single cell suspensions and mixed with CMC (1.0wt%) was encapsulated within composite microfibers and cultured in a complete medium consisting of Minimum Essential Medium, 15% fetal bovine serum, and Gentamicin-Penicillin-Streptomycin Solution (100X). The cells were then incubated in Esco CelMate cell incubator at 37℃ with 5% CO2. SY5Y cells spheroids viability: The cell viability of SN-SY5Y cells within hydrogel scaffolds was assessed using the Calcein AM/PI kit at 1, 3, 5, and 9 days post-seeding. Following remove the supernatant gently, the cell-laden microfibers were incubated within LIVE/DEAD cell staining solution (1:1000 in MEM) in the dark for 45 minutes. The proportion of viable cells was then measured by quantitatively. cell-laden microfibers were dissolved through 50% vol CellTiter-Glo solution, placed into 96-well plates, and shaken for 30 minutes at room temperature. Subsequently, the Luminescence of the wells was measured using a multifunctional microplate reader set to detect luminescence at an integration value of 500. Hematoxylin-eosin staining: Add appropriate amount of 4% polyoxymethylene to the microfibers encaspulating NB tumor spheroids aggregates for fixation. Take out the microfibers containing t tumor spheroids, and perform gradient dehydration in gradient ethanol. After dehydration is completed, place them in xylene for transparency treatment. After the transparency treatment is completed, immediately transfer them to a wax cylinder for embedding, slicing, and staining with the routine HE staining method for observation under a microscope. Western Blot:Cells were extracted and cleaned, followed by the addition of cell lysis buffer containing PMFS. The cell fragments and lysate were subsequently transferred to a 1.5 mL centrifuge tube and subjected to centrifugation at 12,000 rpm for 5 minutes at 4°C. Following centrifugation, the supernatant was carefully extracted. A protein standard curve was prepared, and the protein content of the sample was detected. Subsequently, SDS-PAGE and transfer were performed, followed by overnight incubation with primary antibody dilution. The primary antibody was then recovered, the strip was washed several times, and secondary antibody incubation was added. Chemiluminescence was used for detection, followed by image development, fixation, and analysis. Immunofluorescence of SN-SY5Y cells spheroids: The cell-laden microfibers were fixation in a 10% formalin solution for 2 hours, permeabilization with 0.1%vol Cell permeabilization fluid (Triton-X100), and incubation with 5% goat serum for 60 minutes. Subsequently, the goat serum was removed, the cell-laden microfibers were incubated with the primary antibodies diluted in normal goat serum overnight. After removal of the primary antibodies, cell-laden microfibers were rinsed with cold balanced salt solution, incubated with secondary antibodies for 1 hour. The spheroids, which were labeled with primary and secondary antibodies, were then stained with 4’,6-diamidino-2-phenylindole solution (1:1000) for 8 minutes. Subsequently, the fluorescence images of the cellular spheroids were analyzed using ImarisViewer 10.1.0. Flow cytometry: NB tumor single cell suspension was prepared, PBS resuspended and washed cells, 4% paraformaldehyde fixed cells, permeabilized buffer resuspended cells, 3% BSA incubated cells for 30 min, relevant primary antibodies were added and incubated in dark for 0.5 hour, singel cells were rinsed with balanced salt solution, followed by resuspension in a solution of fluorescent dye-coupled secondary antibodies diluted in PBS buffer. The cells were then incubated in darkness for 30 minutes before being washed again with PBS buffer. The resuspended cells were then analyzed using flow cytometry. The relative data obtained from the cellular spheroids was analyzed using FlowJo_10.8.1_VL. Drug evaluation: The Carboplatin, Methotrexate, and Etoposide working solutions were freshly prepared and added to the complete medium at a consistent concentration. Tumor spheroids were then treated with these chemotherapeutic agents for 72 h, and The proportion of viable cells was then measured using the CellTiter-Glo kit. A control group of tumor spheroids was treated with 0.1% dimethyl sulfoxide (DMSO). The treated spheroids were subsequently analyzed for absorbance using a multifunctional microplate reader. The final data is analyzed by the origin software. Characteristics: The microfibers were lyophilized, gold-plated, and further examined under SEM. Subsequently, the composite microstructures of microfibers were analyzed using scanning electron microscopy (SEM, JSM-IT200, Japan). Subsequently, Statistical Analyses : The experimental results were reported as means ± standard deviation (M±SD). Relative significance was assessed using the student’s t-test, with the statistical differences presented as follows: * < 0.05, ** < 0.01, *** 0.05. Sample numbers (n) are indicated in the corresponding figure legends. Acknowledgements Supplementary Information accompanies this paper is available. Competing financial interests: The authors declare no competing financial interests. Author contributions： T.Y.S, Y.C and J.L.W conceived the idea, T.Y.S, Y.Z and Q.Y.S designed and carried out the experiments. T.Y.S., J.L.W. analyzed the data and wrote the paper. J.L.W, H.Z.R, G.L.L. contributed to the scientific discussion of the article. Funding information： This work was supported by the National Natural Science Foundation of China (82303960), The Talent introduction project from Nanjing Drum Tower Hospital (HH7070202101). Reference: [1] B. Qiu, K. K. Matthay, Nature reviews. 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Keywords bioresponsive chemotherapy microfiber microfluidic neuroblastoma Authors Affiliations Taiyu Song 0000-0003-3924-8452 Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Qiyang Shen 0000-0001-8963-1244 Children's Hospital of Nanjing Medical University View all articles by this author Yi Cheng Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Yue Zhi Southeast University View all articles by this author Guangling Liu Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Haozhen Ren 0000-0002-6198-2904 Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Jinglin Wang 0000-0002-4349-750X [email protected] Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Metrics & Citations Metrics Article Usage 273 views 156 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Taiyu Song, Qiyang Shen, Yi Cheng, et al. 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