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The abnormal activation of the epidermal growth factor receptor (EGFR) is strongly associated with cancer invasion and metastasis. Tools and methods are required to study and visualize EGFR activation under (patho)physiological conditions. Here, we report the development of a two-step photoaffinity probe (HX101) by incorporation of a diazirine as a photoreactive group and an alkyne as a ligation handle to quantitively study EGFR kinase activity in native cellular contexts and human tissue slices. HX101 is a multifunctional probe based on the pharmacophore of the EGFR tyrosine kinase inhibitor (EGFR-TKI) and can covalently target the EGFR upon photoactivation. The incorporated alkyne serves as a versatile ligation handle and enables HX101 to introduce distinct reporter groups (e.g., fluorophore and biotin) via click chemistry. With variable reporter tags, HX101 enables visualization and target engagement studies of the active EGFR in a panel of cancer cells using flow cytometry, confocal microscopy, and mass spectrometry. Furthermore, as a proof of concept study, we applied HX101 in stochastic optical reconstruction microscopy super-resolution imaging to study EGFR activation in live cells. Importantly, HX101 was also applied to visualize EGFR mutant activity in tumor tissues from lung cancer patients for prediction of EGFR-TKI sensitivity. Altogether, our results demonstrate the wide application of a selective photoaffinity probe in multi-modal assessment/visualization of EGFR activity in both live cells and tissue slices. We anticipate that these diverse applications can facilitate the translation of a strategically functionalized probe into medical use.

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.

Screening for potential drug combinations presents significant challenges to the current microfluidic cell culture systems, due to the requirement of flexibility in liquid handling. To overcome this limitation, we present here an open-access microfluidic tissue array system specifically designed for drug combination screening. The microfluidic chip features a key structure in which a nanoporous membrane is sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach takes advantage of the characteristics of the nanoporous membrane: on one side, this membrane permits the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution; on the other side, it allows diffusion-based media exchange and thus mimics the endothelial layer. In synergy with a liquid-transferring platform, the open-access microfluidic system enables complex multistep operations involving long-term cell culture, medium exchange, multistep drug treatment, and cell-viability testing. By using the microfluidic protocol, a 10 × 10 tissue array was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays indicated that the resultant tumor tissue was faithful to that in vivo. Drug-testing assays showed that the incorporation of the nanoporous membrane further decreased killing efficacy of the anticancer agents, indicating its function as an endothelial layer. Robustness of the microfluidic system was demonstrated by implementing a three-factor, three-level orthogonal screening of anticancer drug combinations, with which 67% of the testing (9 vs. 27) was saved. Experimental results demonstrated that the microfluidic tissue system presented herein is flexible and easy-to-use, thus providing an ideal tool for performing complex multistep cell assays with high efficiencies.

Major degradation pathways such as deamidation, isomerization, oxidation, and glycation may be accelerated after administration of antibody therapeutics to the patient. Tracking in vivo product degradation is important because certain post-translational modifications can inactivate the protein and reduce product efficacy. However, in vivo characterization of protein therapeutics is not routinely performed because of technical challenges and limited sample availability. In vitro models offer several potential advantages, including larger sample supplies, simpler and faster methods for sample preparation and analysis, and the potential to distinguish differences in product degradation from differences in product clearance. In this study, we compared the rates of in vivo product degradation using mAb1 recovered from clinical serum samples with the rates of in vitro product degradation using mAb1 recovered from spiked phosphate buffered saline (PBS) and spiked human serum samples to determine if results from the in vitro model systems could be used to predict the in vivo results. The antibody samples were characterized by peptide mapping or intact mass analysis to quantify multiple quality attributes simultaneously, including deamidation, isomerization, oxidation, N-terminal pyroglutamate formation, and glycation. It was clearly demonstrated that both the spiked PBS and spiked serum models were effective in predicting in vivo results for deamidation, isomerization, N-terminal pyroglutamate formation and glycation, whereas only the spiked serum model was effective in predicting in vivo results for oxidation.

A facile one-step microwave-assisted approach for the preparation of strong fluorescent carbon nitride quantum dots (g-CNQDs) by using guanidine hydrochloride and EDTA as the precursors was developed. Strong chemiluminescence (CL) emission was observed when NaClO was injected into the prepared g-CNQDs, and a novel CL system for direct detection of free chlorine was established. Free residual chlorine in water was sensitively detected with a detection limit of 0.01 μM and had a very wide detection range of 0.02 to 10 μM. On the basis of CL spectral, UV-visible absorption spectral, and electron spin resonance (ESR) spectral studies, as well as investigations on the effects of various free radical scavengers, a possible CL mechanism was proposed. It was suggested that the radiative recombination of oxidant-injected holes and electrons in the g-CNQDs accounted for the CL emission. Meanwhile, (1)O2 on the surface of g-CNQDs, generated from some reactive oxygen species in the g-CNQDs-NaClO system, could transfer energy to g-CNQDs and thus further enhance the CL emission. The CL system is highly sensitive and differentiable, opening a new field for the development of novel CL-emitting species, but also expanding the conventional optical utilizations of g-CNQDs.