Dual base editor catalyzes both cytosine and adenine base conversions in human cells.

Although base editors are useful tools for precise genome editing, current base editors can only convert either adenines or cytosines. We developed a dual adenine and cytosine base editor (A&C-BEmax) by fusing both deaminases with a Cas9 nickase to achieve C-to-T and A-to-G conversions at the same target site. Compared to single base editors, A&C-BEmax's activity on adenines is slightly reduced, whereas activity on cytosines is higher and RNA off-target activity is substantially decreased.

Blocking STAT3 by pyrvinium pamoate causes metabolic lethality in KRAS-mutant lung cancer.

Signal transducer and activator of transcription 3 (STAT3) exerts a profound role in regulating mitochondrial function and cellular metabolism. Mitochondrial STAT3 supports RAS-dependent malignant transformation and tumor growth. However, whether pharmacological blockade of STAT3 leads to metabolic lethality in KRAS-mutant lung cancer remains unclear. Pyrvinium pamoate, a clinical antihelminthic drug, preferentially inhibited the growth of KRAS-mutant lung cancer cells in vitro and in vivo. Mechanistic study revealed that pyrvinium dose-dependently suppressed STAT3 phosphorylation at tyrosine 705 and serine 727. Overexpression mitochondrial STAT3 prominently weakened the therapeutic efficacy of pyrvinium. As a result of targeting STAT3, pyrvinium selectively triggered reactive oxygen species release, depolarized mitochondrial membrane potential and suppressed aerobic glycolysis in KRAS-mutant lung cancer cells. Importantly, the cytotoxic effects of pyrvinium could be significantly augmented by glucose deprivation both in vitro and in a patient-derived lung cancer xenograft mouse model in vivo. The combined efficacy significantly correlated with intratumoural STAT3 suppression. Our findings reveal that KRAS-mutant lung cancer cells are vulnerable to STAT3 inhibition exerted by pyrvinium, providing a promising direction for developing therapies targeting STAT3 and metabolic synthetic lethality for the treatment of KRAS-mutant lung cancer.

Separation and dual detection of prostate cancer cells and protein biomarkers using a microchip device.

Current efforts for the detection of prostate cancer using only prostate specific antigen are not ideal and indicate a need to develop new assays - using multiple targets - that can more accurately stratify disease states. We previously introduced a device capable of the concurrent detection of cellular and molecular markers from a single sample fluid. Here, an improved design, which achieves affinity as well as size-based separation of captured targets using antibody-conjugated magnetic beads and a silicon chip containing micro-apertures, is presented. Upon injection of the sample, the integration of magnetic attraction with the micro-aperture chip permits larger cell-bead complexes to be isolated in an upper chamber with the smaller protein-bead complexes and remaining beads passing through the micro-apertures into the lower chamber. This enhances captured cell purity for on chip quantification, allows the separate retrieval of captured cells and proteins for downstream analysis, and enables higher bead concentrations for improved multiplexed ligand targeting. Using LNCaP cells and prostate specific membrane antigen (PSMA) to model prostate cancer, the device was able to detect 34 pM of spiked PSMA and achieve a cell capture efficiency of 93% from culture media. LNCaP cells and PSMA were then spiked into diluted healthy human blood to mimic a cancer patient. The device enabled the detection of spiked PSMA (relative to endogenous PSMA) while recovering 85-90% of LNCaP cells which illustrated the potential of new assays for the diagnosis of prostate cancer.

Concurrent Detection of Cellular and Molecular Cancer Markers Using an Immunomagnetic Flow System.

We report a detection system for simultaneous measurement of cellular and molecular markers of cancer. Magnetic beads conjugated with antibodies against a specific antigen are used to capture both free molecules and whole cells overexpressing the antigen. The target-bound beads then flow through a microfluidic chamber where they are drawn to a glass surface by an external magnetic field. The cells and molecules captured on the surface are quantitatively analyzed using fluorescent microscopy. The system was characterized by detecting free folate receptor (FR) and an FR+ cancer cell line (KB) in culture media. The system detected as low as 10 pM of FR and captured 87% of the spiked KB cells at a volumetric throughput of 3 mL/min. We further demonstrated the detection of 100 KB cells and 200 pM FR spiked into healthy human blood to simulate detection of rare cells and protein biomarkers present in a cancer patient's blood sample. The FR concentration was measured to be 244 pM (including the intrinsic FR present in the blood), and the total number of KB cells in the sample was estimated to be 98. The potential of this approach in clinical diagnostics was also demonstrated by detecting both FR+ cells and free FR in an ascites sample obtained from an ovarian cancer patient. Because of the system's capability to detect multiple targets at the same time, its high throughput, and its overall simplicity, we expect it to be highly useful in a wide range of research settings.

Circulating tumor cell detection using a parallel flow micro-aperture chip system.

We report on-chip isolation and detection of circulating tumor cells (CTCs) from blood samples using a system that integrates a microchip with immunomagnetics, high-throughput fluidics and size-based filtration. CTCs in a sample are targeted via their surface antigens using magnetic beads functionalized with antibodies. The mixture is then run through a fluidic chamber that contains a micro-fabricated chip with arrays of 8 µm diameter apertures. The fluid runs parallel to the microchip while a magnetic field is generated underneath to draw the beads and cells bound to them toward the chip surface for detection of CTCs that are larger than the apertures and clear out free beads and other smaller particles bound to them. The parallel flow configuration allows high volumetric flow rates, which reduces nonspecific binding to the chip surface and enables multiple circulations of the sample fluid through the system in a short period of time. In this study we first present models of the magnetic and fluidic forces in the system using a finite element method. We then verify the simulation results experimentally to determine an optimal flow rate. Next, we characterize the system by detecting cancer cell lines spiked into healthy human blood and show that on average 89% of the spiked MCF-7 breast cancer cells were detected. We finally demonstrate detection of CTCs in 49 out of 50 blood samples obtained from non-small cell lung cancer (NSCLC) patients and pancreatic cancer (PANC) patients. The number of CTCs detected ranges from 2 to 122 per 8 mL s of blood. We also demonstrate a statistically significant difference between the CTC counts of NSCLC patients who have received therapy and those who have not.

On-demand weighing of single dry biological particles over a 5-order-of-magnitude dynamic range.

We report a simple and highly versatile system to select and weigh individual dry biological particles. The system is composed of a microtweezer to pick and place individual particles and a cantilever-based resonator to weigh them. The system can weigh entities that vary from a red blood cell (~10(-11) g) to the eye-brain complex of an insect (~10(-6) g), covering a 5-order-of-magnitude mass range. Due to its versatility and ease of use, this weighing method is highly compatible with established laboratory practices. The system can provide complementary mass information for a wide variety of individual particles imaged using scanning electron microscopy and determine comparative weights of individual biological entities that are attached to microparticles as well as weigh fractions of individual biological entities that have been subjected to focused ion beam milling.