Giacomazzi, G. | bioRxiv Science

bioRxiv2022

Consistent measurement of LAG-3 expression across multiple staining platforms with the 17B4 antibody clone

ContextAn immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytical precision and interlaboratory reproducibility using a Leica staining platform, but has not been investigated on other IHC staining platforms. ObjectiveTo evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 (ASL-48) and VENTANA BenchMark ULTRA (VBU) compared with Leica BOND-RX (BOND-RX). DesignEighty formalin-fixed paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numerical score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a [≥] 1% cutoff. ResultsLAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Pearson correlation was [≥] 0.88 for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numerical score determined by a pathologist. Correlation increased with a numerical score determined with a digital scoring algorithm (Pearson correlation [≥] 0.93 for all comparisons). Overall percentage agreement was [≥] 77.5% for interplatform and BOND-RX intraplatform comparisons when a [≥] 1% cutoff was used to determine LAG-3 positivity. ConclusionsData from this study demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

pathology↗

bioRxiv2020

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

Neuromuscular junctions (NMJs) ensure proper communication between motor neurons and muscle through the release of neurotransmitters. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy, paralysis and respiratory failure. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to study the effect of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell-derived motor neurons and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of motor neuron neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in a reduced neurite outgrowth and in a decreased NMJ number. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth and the NMJ morphology of FUS-ALS co-cultures, further prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

neuroscience↗