Construction Of Droplet Microfluidic Based Single-Cell Culture Platform And Application In Expansion Of Colorectal Cancer Stem Cells

Colorectal cancer（CRC）has one of the highest incidence and mortality rates among malignancies of the digestive system.Despite progress in therapy improvements in CRC,most patients develop metastasis and recurrence after treatments.Recently,cancer stem cells（CSCs）are deemed to drive tumor growth and are responsible for resistance to chemo-and radiotherapy.The isolation of CSCs and the readout of their biological informations are conducive to guiding the clinical diagnosis and therapy.However,CSCs are extremely rare,accounting for only 1-5%of the total cancer cell population.The effective method,which is used for isolating and expanding CSCs,is urgently needed for the CSCs research and development of CSCs-targeted therapies.In this work,we developed a microfluidic single-cell culture approach for expanding and recovering colorectal CSCs from both cell lines and tumor tissues.The single-cell culture approach takes advantage of the self-renewal property of stem cells,as only the CSCs can survive the single-cell culture and form tumorspheres.The main contents included:（1）Formation of alginate microgel supported single-cell array by using a droplet microfluidic device.A multilayered microfluidic chip with 13 droplet generators was used for the highly efficient cell encapsulation.The cell suspension and the carrier fluid from respective inlets are forced to enter the cross-shaped droplet generators.Encapsulating cells delivered to the droplet-generation nozzle at random is a process that yields a resulting population of droplets with a Poisson-distributed cell occupancy.The number of cells encapsulated in a droplet can be≤1by adjusting the flow speed and the input cell density.With a flow-rate ratio(Q_oil/Q_aqu)at 3 that is beneficial to maintain cell viability,simultaneous working of the 13 droplet generators formed 128,000 droplet arrays in～5 min.With the optimized input cell density of 1×10⁶·m L^-1,～30%（～35,000）of the droplets occupied cancer cell（s）.Then,the microfluidic chip integrated steps of droplet gelation and single-cell culture.The microfluidic chip also integrated steps of droplet gelation and single-cell culture.After the cell culture,the cells were released from the alginate microgel upon EDTA solution treatment.The cell recovery rate was determined to be61.65±7.59%.The number of cells harvested in a single run is～6000,thus meets the requirements of most bioassays.（2）Alginate microgel single-cell culture for selective expansionIn the continuous single-cell culture,the viability of colorectal cancer cells decreased with the extension of time.By day 14,only 12.48±5.15%and 18.17±2.37%of the droplets contained viable HCT116 and HT29 cells.Time-lapse imaging confirmed that the formation of single cell-derived tumorspheres.The first division mostly occurred on day 2,and the second division mostly occurred on day 4.Besides,a significant number of single cells showed significantly delayed or no division,indicating the difference in individual cells’division potential.Our results evidenced that the small-sized cells have a higher sphere formation rate,suggesting that the alginate microgel single-cell culture selectively expanded those small-sized cells and maintained their properties.This group of small-sized cells could have been stem cell-like cancer cells.（3）The characterization of cells derived from the single-cell cultureCells recovered from the microfluidic chip showed the characteristics as follow:（a）small-sized;（b）with expressions of intrinsic genes maintaining stemness;（c）containing a high proportion of cells positive for stemness markers.Moreover,these SCC cells showed enhanced self-renewal capability and stably maintained stemness phenotypes.All of these are the characteristics of CSCs,we conclude that the microfluidic single-cell culture approach selectively expands stem cell-like colorectal cancer cells.It is also demonstrated the power and utility of the single-cell culture approach in the context of CSCs biology.（4）Expanding cancer stem cells from patient-derived colorectal cancer tissueColorectal cancer tissues harbor high proportions of Ki67（+）,CK20（+）,and CDX2（+）cells but rare CD133（+）and LGR5（+）cells.With the input of primary cells,the formation of single-cell-derived tumorspheres was observed after 14 days of culture.The flow cytometry analysis showed significantly increased proportions of LGR5（+）and CD133（+）cells in SCC cells derived from colorectal cancer tissues.Similar to the result obtained from the cell lines,the biological processes,including cell growth（proliferation and development）,cellular structure organization（protein expression）,and cellular response（metabolism,immune response,anti-apoptosis,drug resistance,and metastasis）in the SCC cells are significantly different from the primary cancer cells.These expression-altered genes may indicate the characteristic biological process of the stem-like cancer cells.The immunofluorescence assay also showed the tumorspheres were highly positive for CD133 and LGR5.To find the potential value of the single-cell sphere formation assay,we conducted the gene set enrichment analysis（GSEA）,which reveals the stemness-specific gene sets upregulated in the SCC cells compared to the primary cancer cells.These altered gene sets involve the Wnt/β-catenin,Hedgehog,Notch and KRAS signaling pathways.As was estimated by the Kaplan-Meier method,alterations in Wnt/β-catenin and Hedgehog signaling pathway correlate to lower median OS.Thus,intervention on these pathways,either as single agents or in combination with other chemotherapeutic agents,may effectively improve patient survival through targeted the CSCs population.In conclusion,we established a microfluidic approach that incorporates droplet microfluidics with alginate hydrogel.By taking advantage of this approach,high-density microgel single-cell culture arrays were established on a microfluidic chip.We confirmed the formation of single cell-derived tumorspheres in alginate microgels by single-cell tracking.The single cell-derived tumorspheres were recovered from the microfluidic chip and characterized as stem cell-like cancer cells.Therefore,this microfluidic approach can achieve CSCs enrichment through selective single-cell expansion.The potential value of this approach in clinical application was also investigated by the identification of CSC-specific therapeutic targets using comparative transcriptomics.The alginate microgel single-cell culture offers a label-free approach to obtain sufficient quantities of CSCs,thus is of great value in understanding cancer biology and developing personalized CSCs-targeting cancer therapies.