| Cancer is one of the major threats to human health.Incurable metastasis is the main reason of cancer-related death,and circulating tumor cells should be responsible to metastasis.Circulating tumor cells(CTCs)escape from the solid tumors,enter in circulatory system,and disseminate to distant organs to form metastasis.CTCs are the biomarker of malignant tumor,and CTC detection is a powerful tool for tumor early diagnosis,monitoring cancer progression and therapy efficiency,surgery prognosis and so on.Isolation of viable CTCs for ex vivo expansion is significant to downstream analysis and drug test,which will greatly promote personalized cancer therapy.However,the occurrence of CTCs is extremely low with that handful CTCs suspending in more than 10~9 blood cells/m L blood,which is a great challenge to CTC detection.It is urgently needed to develop isolation methods of CTCs with high efficiency and high sensitivity.Current isolation techniques includes in vitro and in vivo capture methods.Among which,in vitro methods was isolating CTCs from 1-7.5 m L blood samples,mainly based on physical properties and affinity recognication.These separate devices based on physical properties are able to quickly isolate CTCs.However,it is critically lack of specificity and difficult to obtain the protein expression information.In addition,mechanical damage is inevitable due to the high-flow rate or extrusion during isolating process.Affinity recognition critically relies on expression level of cell-surface specific proteins,which would lose some CTCs due to the heterogeneity and EMT of CTCs.Therefore,new platforms are urgently needed to overcome this problem and isolate CTCs with high sensitivity.The in vitro isolation methods are limited by the 1-7.5 m L blood sample and may lead to false-positive,which doesn’t benefit for tumor early diagnosis.Recently,along with improvement of detection sensitivity,in vivo detetion techniques have been developed including functionalized indwelling needle,intravital flow cytometry,in vivo magnetic enrichment etc.In vivo capture techniques could harvest CTCs from the whole blood,which break through the limitations of small blood volume of in vitro isolation strategies.Therefore,in vivo approaches have greatly improved detection sensitivity.Nonetheless,the slender medical needle provides limited surface area for immobilization of antibody and hinders the efficient contact between CTCs and capture substrate,demonstrating inferior capture performance.And,the biosecurity of nanomaterials is always an inevitable issue when nanomaterials are injected into blood vessel.Current CTC releasing strategies mainly include enzyme degradation,photosensitivity,electrochemical stimulation,thermosensitive materials,chemical-reagent,biological competitive release etc.Among which,the releasing cells via enzyme degradation,thermosensitive materials and biological competitive release are high viability with mediocre efficiency.And releasing CTCs based on photosensitivity,electrochemical stimulation,chemical-reagent need to critically control releasing conditions to aviod damage to cell viability.However,ex vivo expansion of CTCs is still a great challenge duo to the low occurrence,complicated isolation process,poor viability and critical culture conditions.These reasons lead to low efficiency of ex vivo expansion.According to these problems and challenges,in this work,we have prepared the three dimensional PDMS scaffold chip for capture and release of individual and clusters of CTCs;we have designed the CTC-Net for in vivo capture of CTCs;we have prepared the three dimensional cellulose scaffold chip for capture and in situ coculture of CTCs.The main work is listed as following:(1)We developed a microchip embedded with three-dimensional(3D)PDMS scaffold for high-efficient capture of individual and clusters of CTCs.We took use of Ni foam as template to prepare the 3D PDMS scaffold.Then 3D PDMS scaffold were integrated into microfluidic chip to obtained the 3D scaffold PDMS chip,which was functionalized with anti-Ep CAM antibody for capture of CTCs.Spatially distributed 3D scaffold improve the quantity of antibody and compels cells migration from linest chaotic or vortex migration in the channel,thus significantly improve CTCs capture efficiency.The capture efficiency were more than 90%at the flow rate of 100μL/min.The 3D scaffold chip were applied to isolate CTCs from cancer patients’blood samples.1–118 CTCs/m L were identified from 14cancer patients’blood and 5 out of these cancer patients showed 1–14 CTC clusters/m L.(2)We developed a three-dimensional scaffold chip with thermosensitive gelatin for high-efficiency capture and release of individual and clusters of CTCs.Based on our previous work,gelatin was employed to coated on PDMS scaffold and functionalized with anti-Ep AM antibody.The 3D scaffold chip combines specific recognition and physically obstructed effect of 3D scaffold structure for improvement of CTC clusters capture efficiency.Gelatin coated on the scaffold dissolved at 37°C quickly,and the captured cells were released gentlely from the scaffold.At last,the individual and clusters of CTC were collected for global DNA and RNA methylation analysis.(3)We presented an injectable and retractable three dimensional CTC-Net probe for intravascular fishing of CTCs in vivo.The CTC-Net was composed of 3D elastic and transparent scaffold with interconnected and spatially distributed networks,and the capture efficiency was synthetically improved by increased quantity of immobilized antibody and enhanced substrate-cell contact frequency.The elastic CTC-Net could be readily compressed and injected into blood vessel,fully unfolded to form a 3D“fishing-net”structure for capturing CTCs from bloodstream,and retracted for imaging and subsequent analysis of captured CTCs.The significant advantages of the CTC-Net probe were well established by detection of CTCs in a very low abundance from wild type rats,and successful capture of CTCs and CTC clusters before metastasis occurring from tumor bearing rats.(4)We prepared a three dimensional cellulose scaffold chip for capture and in situ culture of CTCs.The free-standing cellulose scaffold was assembled layer by layer,and integrated into microfluidic chip after being biofunctionalized for capture of CTCs.And the capture efficiency was more than 90%.After CTCs were captured in chip,fibroblasts and collagen were introduced into chip for coculture.The cellulose had great biocompatibility,collagen simulated microenvironment,and fibroblasts secreted multiple growth factors,which collectively promoted the quick expansion of CTCs.The cellulose scaffold chip was applied to isolate tumor cells spiked in whole blood,and then the captured cells were cocultured in situ with fibroblasts in collagen.The proliferative capacity of cocultured tumor cells was 3.7 fold higher than that of tumor cell alone.This platform was able to improve ex vivo expansion efficiency of CTCs,which could be applied for downstream analysis of CTCs and drug test for personalized therapy. |
