Screens of cancer stem cells (CSCs)‐specific agents present significant challenges to conventional cell assays due to the difficulty in preparing CSCs ready for drug testing. To overcome this limitation, developed is a microfluidic single‐cell assay for screening breast cancer stem cell–specific agents. This assay takes advantage of the single‐cell clone‐forming capability of CSCs, which can be specifically inhibited by CSC‐targeting agents. The single‐cell assay is performed on a microfluidic chip with an array of 3840 cell‐capturing units; the single‐cell arrays are easily formed by flowing a cell suspension into the microchip. Achieved is a single cell‐capture rate of ≈60% thus allowing more than 2000 single cells to be analyzed in a single test. Over long‐term suspension culture, only a minority of cells survive and form tumorspheres. The clone‐formation rate of MCF‐7, MDA‐MB‐231, and T47D cells is 1.67%, 5.78%, and 5.24%, respectively. The clone‐forming inhibition assay is conducted by exposing the single‐cell arrays to a set of anticancer agents. The CSC‐targeting agents show complete inhibition of single‐cell clone formation while the nontargeting ones show incomplete inhibition effects. The resulting microfluidic single‐cell assay with the potential to screen CSC‐specific agents with high efficiency provides new tools for individualized tumor therapy.

Cancer stem cells (CSCs) are rare and lack definite biomarkers, necessitating new methods for a robust expansion. Here, we developed a microfluidic single-cell culture (SCC) approach for expanding and recovering colorectal CSCs from both cell lines and tumor tissues. By incorporating alginate hydrogels with droplet microfluidics, a high-density microgel array can be formed on a microfluidic chip that allows for single-cell encapsulation and nonadhesive culture. The SCC approach takes advantage of the self-renewal property of stem cells, as only the CSCs can survive in the SCC and form tumorspheres. Consecutive imaging confirmed the formation of single-cell-derived tumorspheres, mainly from a population of small-sized cells. Through on-chip decapsulation of the alginate microgel, ∼6000 live cells can be recovered in a single run, which is sufficient for most biological assays. The recovered cells were verified to have the genetic and phenotypic characteristics of CSCs. Furthermore, multiple CSC-specific targets were identified by comparing the transcriptomics of the CSCs with the primary cancer cells. To summarize, the microgel SCC array offers a label-free approach to obtain sufficient quantities of CSCs and thus is potentially useful for understanding cancer biology and developing personalized CSC-targeting therapies.

Tumor-derived extracellular vesicle (tEV) biomarkers can reflect cancer cell phenotypes and have great potential for cancer diagnosis and treatment. However, tEVs display high heterogeneity, and rapid and sensitive identification of EV biomarkers remains challenging due to their low expression. Spectral overlap also significantly limits the multiplex analysis of EV biomarkers by fluorescent probes. Herein, we developed a method for highly sensitive tEV phenotyping that uses size-coded microbeads that carry hairpin probes that can bind to aptamers targeting distinct tEV biomarkers. We also designed a microfluidic chip containing spacer arrays that segregate these microbeads in distinct chip regions according to their size to generate location-specific signals indicating the level of different EV biomarkers. The EV biomarker signal on these microbeads was amplified by in situ rolling cyclic amplification (RCA). This strategy permits the simultaneous detection of multiple tEV phenotypes by fluorescence spectroscopy without the limitations of spectral overlap. This study demonstrates that this tEV phenotyping method can rapidly and simultaneously detect six different tEV phenotypes with high sensitivity. Due to the programmability of the sensing platform, this method can be rapidly adapted to detect different tEV phenotype substitutions of the detected biomarkers. Notably, clinical cohort studies show that this strategy may provide new ideas for the precise diagnosis and personalized treatment of cancer patients.

DNA is a biological polymer that encodes and stores genetic information in all living organism. Particularly, the precise nucleobase pairing inside DNA is exploited for the self-assembling of nanostructures with defined size, shape and functionality. These DNA nanostructures are known as framework nucleic acids (FNAs) for their skeleton-like features. Recently, FNAs have been explored in various fields ranging from physics, chemistry to biology. In this review, we mainly focus on the recent progress of FNAs in a pharmaceutical perspective. We summarize the advantages and applications of FNAs for drug discovery, drug delivery and drug analysis. We further discuss the drawbacks of FNAs and provide an outlook on the pharmaceutical research direction of FNAs in the future.