Neuromuscular junctions are stable in patients with cancer cachexia

Abstract

Cancer cachexia is a major cause of patient morbidity and mortality, with no efficacious treatment or management strategy. Despite cachexia sharing pathophysiological features with a number of neuromuscular wasting conditions, including age-related sarcopenia, the mechanisms underlying cachexia remain poorly understood. Studies of related conditions suggest that pathological targeting of the neuromuscular junction (NMJ) may play a key role in cachexia, but this has yet to be investigated in human patients. Here, high-resolution morphological analyses were undertaken on NMJs of rectus abdominis obtained from patients undergoing upper GI cancer surgery compared with controls (N = 30; n = 1,165 NMJs). Cancer patients included those with cachexia and weight-stable disease. Despite the low skeletal muscle index and significant muscle fiber atrophy (P < 0.0001) in patients with cachexia, NMJ morphology was fully conserved. No significant differences were observed in any of the pre- and postsynaptic variables measured. We conclude that NMJs remain structurally intact in rectus abdominis in both cancer and cachexia, suggesting that denervation of skeletal muscle is not a major driver of pathogenesis. The absence of NMJ pathology is in stark contrast to what is found in related conditions, such as age-related sarcopenia, and supports the hypothesis that intrinsic changes within skeletal muscle, independent of any changes in motor neurons, represent the primary locus of neuromuscular pathology in cancer cachexia.

Keywords: Neuromuscular disease; Neuroscience; Oncology.

Figure 1. Atrophy of skeletal muscle fibers in cancer cachexia.

Upper panel shows a box and whisker plot of muscle fiber diameters in control (n = 10 patients), weight-stable (n = 10), and cachectic (n = 10) patients. Bottom panels show representative micrographs of single, teased muscle fibers from control (left), weight-stable (middle), and cachectic (right) patients. Scale bars: 50 μm. Cachectic patients had significantly reduced muscle fiber diameters compared with weight-stable and control cases (control, n = 388; weight stable, n = 362; cachexia, n= 400 muscle fibers). Boxes contain the mean (+) and median (line) muscle-fiber diameters for the group and enclose the central 25th-75th percentile of the data, and whiskers extend from the 10th-90th percentile. Outlying data points are shown beyond the whiskers. ****P < 0.0001, 1-way ANOVA paired with Tukey’s post-hoc test. Individual P values are shown in Supplemental Table 2.

Figure 2. Conservation of NMJ morphology in cancer cachexia.

Confocal micrographs of representative small, medium, and large NMJs from RA in the 3 patient groups. Despite heterogeneity in size and shape of individual NMJs, overall morphology was conserved across all groups, with no evidence of NMJ pathology in either the cachexia or weight-stable groups. Axon and nerve terminals are shown in green (2H3/SV2) and AChRs of the motor endplate in red (α-BTX). Scale bars: 10 μm.

Figure 3. Structural integrity of the NMJ in cancer cachexia.

Morphometric analysis using NMJ-morph revealed that NMJ morphology is conserved in both cachexia and weight-stable disease. Data presented as a pair of charts (scatterplot, above; box and whisker plot, below) for key NMJ variables, including measurements of axon diameter (A) and pre- and postsynaptic architecture (B–F). Scatterplots depict the approximately 40 individual NMJs (data points) for the 10 patients (1 to 10) in each group; the mean NMJ value is given by the red line; the observed heterogeneity is a normal feature of human NMJ morphology. Box and whisker plots constructed using the mean patient data in each group (10 patients; control NMJs, n = 387; weight stable NMJs, n = 386; cachexia NMJs, n = 392). Boxes contain the mean (+) and median (line) values for each NMJ variable and enclose the central 25th-75th percentile of the data; whiskers represent the maximum and minimum values. One-way ANOVA paired with Tukey’s post hoc test.