A high-throughput and low-waste viability assay for microbes.

Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across disciplines, but it is time-intensive and resource-consuming. Here we describe the geometric viability assay (GVA) that replicates CFU measurements over 6 orders of magnitude while reducing over 10-fold the time and consumables required. GVA computes a sample's viable cell count on the basis of the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with Gram-positive and Gram-negative planktonic bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis), biofilms and fungi (Saccharomyces cerevisiae). Laborious CFU experiments such as checkerboard assays, treatment time-courses and drug screens against slow-growing cells are simplified by GVA. The ease and low cost of GVA evinces that it can replace existing viability assays and enable viability measurements at previously impractical scales.

High Throughput Viability Assay for Microbiology.

Counting viable cells is a universal practice in microbiology. The colony forming unit (CFU) assay has remained the gold standard to measure viability across disciplines; however, it is time-intensive and resource-consuming. Herein, we describe the Geometric Viability Assay (GVA) that replicates CFU measurements over 6-orders of magnitude while reducing over 10-fold the time and consumables. GVA computes a sample's viable cell count based on the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with gram-positive and -negative planktonic bacteria, biofilms, and yeast. Laborious CFU experiments such as checkerboard assays, treatment time-courses, and drug screens against slow-growing cells are simplified by GVA. We therefore screened a drug library against exponential and stationary phase E. coli leading to the discovery of the ROS-mediated, bactericidal mechanism of diphenyliodonium. The ease and low cost of GVA evinces it can accelerate existing viability assays and enable measurements at previously impractical scales.

Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.