The Effect Of Polarized Macrophages On Cell Migration And Invasion And Its Application In Postoperative Tumor Clearance

Objective:In order to solve the crucial scientific problem that the tumor is prone to recurrence after tumor resection in situ,we construct Three-dimensional composite hydrogels to culture M1 type macrophages to extract the decellularized matrix to inhibit tumor recurrence.Methods:1.Three-dimensional composite hydrogel preparation and performance characterization experiment:degradation experiment,swelling experiment,microscopic morphology,biocompatibility experiment.CCK8 test detected cytotoxicity;flow cytometry test detected cell polarity.2.Preparation of decellularized matrix.3.In vitro study of decellularized matrix inhibiting tumors:CCK8 experiment was used to detect tumor suppression effect;the expressions of activated Caspase3 and inducible nitric oxide synthase(i NOS)were detected by Western Blot;the expression of polarity markers of M1 and M2 macrophages were detected by Real-time quantitative PCR(RT-q PCR);enzyme-linked immunosorbent assay(ELISA)detected TNFαsecretion;nitric oxide(NO)kit detected NO secretion.4.In vivo study of decellularized matrix to inhibit tumor recurrence:tumor volume detection and small animal imaging experiments confirmed the effect of decellularized matrix hydrogel in inhibiting recurring tumors;Ki-67immunohistochemical staining detected cell proliferation;H&E staining detected cytotoxicity and tumor lung metastasis;flow cytometry detected immune cell types and macrophage cell types.Results:1.The three-dimensional composite hydrogel was successfully constructed.The performance characterization experiment of the three-dimensional composite hydrogel showed that the concentration of 10 mg/m L was the most suitable for culturing M1 type macrophages.The decellularized matrix was successfully extracted.2.In vitro decellularized matrix inhibited tumor cell proliferation.The decellularized matrix maintained the polarity of M1 macrophages,weakened the polarity of M2 macrophages,and at the same time enhanced the secretion of TNFαand NO by M2macrophages.When M1 and M2 macrophages were co-cultured with tumor cells,the decellularized matrix maintained the polarity of M1 macrophages and reversed the polarity of M2 macrophages.3.The decellularized matrix in vivo significantly inhibited tumor recurrence.The anti-tumor effect of decellularized matrix was achieved by activating mouse autoimmunity and reversing tumor-associated macrophages.The decellularized matrix inhibited tumor proliferation.In addition,the decellularized matrix has no toxic side effects on mice,and inhibits tumor lung metastasis on mice.Conclusion:In summary,we have successfully extracted the decellularized matrix of M1 type macrophages.Through in vivo and in vitro experiments,it has been confirmed that the decellularized matrix can maintain the polarity of M1 type macrophages,weaken or even reverse M2 type macrophages,and increase the number of CD8~+cells,thereby activating the autoimmune response and inhibiting tumor recurrence.The cell migration and invasion behaviors play pivotal roles in tissue regeneration.For the skin repair process,a directed inflammatory response that regulates fibroblasts is critical for efficient wound healing.In this study,the authors present the design and fabrication of a microfluidic-based three-dimensional(3D)microphysiological system and how it impacts in controlling fibroblast migration and invasion under the induction of differently polarized macrophages.The microfluidic device had two chambers on opposite sides of a 1 mm micochannel,providing directed induction and sufficient width for long-term observation.The test cells could be seeded with or without matrix gel,cultured in a 2D or 3D microenvironment according to experiment settings.The microchannel allowed for any sorts of matrix filling and was on-demanding for continuous surveillance.Herein,our microfluidic device reserved the advantages of traditional methods using transwell chamber or scratch wound healing assay.In addition,it even came with more superiority such as retrievability,dynamic observation,and 3D environment simulation.The migration and invasion pattern of NIH3T3 modulated by RAW264.7macrophages in different polarization status was demonstrated as an example.The results of the migration assay corresponded with that of the proliferation and gene expression experiments,verifying that our device was fully capable of restoring in vivo microenvironment and presenting cellular motility behaviors.