Activated mast cells in skeletal muscle can be a potential mediator for cancer-associated cachexia

Abstract

Background: Eighty per cent of United States advanced cancer patients faces a worsened prognosis due to cancer-associated cachexia. Inflammation is one driver of muscle atrophy in cachexia, and skeletal muscle-resident immune cells could be a source of inflammation. This study explores the efficacy of cancer activated skeletal muscle-resident mast cells as a biomarker and mediator of cachexia.

Methods: Individual gene markers for immune cells were assessed in a publicly available colon carcinoma cohort of normal (n = 3), moderate cachexia (n = 3), and severe cachexia (n = 4) mice. Lewis lung carcinoma (LL/2) cells induced cachexia in C57BL/6 mice, and a combination of toluidine blue staining, immunofluorescence, quantitative polymerase chain reaction, and western blots measured innate immune cell expression in hind limb muscles. In vitro measurements included C2C12 myotube diameter before and after treatment with media from primary murine mast cells activated with LL/2 conditioned media. To assess translational potential in human samples, innate immune cell signatures were assessed for correlation with skeletal muscle atrophy and apoptosis, dietary excess, and cachexia signatures in normal skeletal muscle tissue. Gene set enrichment analysis was performed with innate immune cell signatures in publicly available cohorts for upper gastrointestinal (GI) cancer and pancreatic ductal adenocarcinoma (PDAC) patients (accession: GSE34111 and GSE130563, respectively).

Results: Individual innate immunity genes (TPSAB1 and CD68) showed significant increases in severe cachexia (weight loss > 15%) mice in a C26 cohort (GSE24112). Induction of cachexia in C57BL/6 mice with LL/2 subcutaneous injection significantly increased the number of activated skeletal muscle-resident degranulating mast cells. Murine mast cells activated with LL/2 conditioned media decreased C2C12 myotube diameter (P ≤ 0.05). Normal human skeletal muscle showed significant positive correlations between innate immune cell signatures and muscle apoptosis and atrophy, dietary excess, and cachexia signatures. The mast cell signature was up-regulated (positive normalized enrichment score and false discovery rate ≤ 0.1) in upper GI cachectic patients (n = 12) compared with control (n = 6), as well as in cachectic PDAC patients (n = 17) compared with control patients (n = 16).

Conclusions: Activated skeletal muscle-resident mast cells are enriched in cachectic muscles, suggesting skeletal-muscle resident mast cells may serve as a biomarker and mediator for cachexia development to improve patient diagnosis and prognosis.

Keywords: Cancer-associated cachexia; Degranulation; Innate immunity; Mast cells; Skeletal muscle.

Figure 1

Increased expression of innate immune cell genes in cachectic mice skeletal muscle. (A) Expression of adaptive immunity‐related genes in quadriceps muscles of C26 colon carcinoma‐bearing mice [GSE24112, normal (n = 4), moderate cachexia (n = 3), and severe cachexia (n = 4)]. (B) Expression of innate immunity‐related genes. Detailed analyses of (C) tryptase alpha/beta 1 (TPSAB1), (D) tryptase beta 2 (TPSB2), and (E) CD68 gene expression from (B). Mean ± SEM. ^* P ≤ 0.05, ^** P ≤ 0.01 (one‐way ANOVA, Tukey's multiple comparisons test).

Figure 2

Murine model of LL/2 induced cachexia. Murine lung cancer LL/2 cells or cell culture medium without cells (sham) were inoculated into C57BL/6 mice subcutaneously (sham n = 10, LL/2 n = 7). Body weight, food intake, tumour growth, and muscle function were assessed after 3 weeks. (A) Tumour free body mass of LL/2 and sham‐bearing mice at baseline and termination of experiment. (B) Longitudinal measurement of daily food intake of LL/2 and sham‐bearing mice. (C) Correlative comparison of excised tumour mass with percentage of body mass lost. (D–G) Comparison of muscle mass among sham‐bearing mice and LL/2 bearing mice losing <10% or ≥10% baseline body mass at termination of experiment. Mean ± SEM, ^** P ≤ 0.01, ^**** P ≤ 0.0001 vs. sham‐injected group (one‐way ANOVA, Bonferroni's multiple comparisons test). (H, I) Hind limb muscle function [(H) maximal tetanic force and (I) time to fatigue] was measured by force transduction at termination of experiment. Mean ± SEM, ^** P ≤ 0.01 vs. sham‐injected group (Student's t‐test).

Figure 3

Cachexia shifts muscle fibre size distribution and increases cachexia‐related genes in in vivo murine model. Analysis of muscle fibre cross‐sectional area and cachexia‐related gene expression in muscles obtained from animals in Figure 2. (A) Representative images of wheat germ agglutinin (WGA) stained TA muscles. ×10. Bar = 100 μm. (B–E) Quantification of muscle fibre cross‐sectional area percent distribution. Mean ± SEM, ^** P ≤ 0.01, ^*** P ≤ 0.001, ^**** P ≤ 0.0001 vs. sham‐injected group (two‐way ANOVA, Tukey's multiple comparisons test). (F–I) tripartite motif containing 63 (TRIM63) and (J–M) F‐box protein 32 (FBXO32) mRNA expression of muscles. Mean ± SEM. ^** P ≤ 0.01, ^*** P ≤ 0.001, ^**** P ≤ 0.0001 vs. sham‐injected group (Student's t‐test).

Figure 4

Degranulating skeletal muscle‐resident mast cells increase in in vivo murine cancer‐associated cachexia model. Examination of skeletal muscle‐resident mast cells (MCs) in muscles obtained from animals in Figure 2. (A) Representative images of toluidine blue stained TA muscle. ×20 and ×40. Bar = 25 μm. (B–I) Quantification of total number of muscle‐resident (B–E) mast cells and (F–I) activated degranulating mast cells in hind limb skeletal muscles. (J–M) Relative mRNA expression of tryptase alpha/beta 1 (TPSAB1) in the muscles. Mean ± SEM, ^* P ≤ 0.05, ^** P ≤ 0.01 vs. sham‐injected group (Student's t‐test).

Figure 5

No change in skeletal muscle‐resident macrophages and neutrophils in murine cancer‐associated cachexia model. Examination of skeletal muscle‐resident CD68 (macrophage marker) positive cells and neutrophil elastase (NE) (neutrophil marker) positive cells in muscles obtained from animals in Figure 2. (A) Representative immunofluorescence images of CD68 and DAPI stained TA muscle. ×20. Bar = 50 μm. (B–E) Quantification of number of positive CD68 cells per muscle cross section. (F–I) Relative mRNA expression of CD68 in muscle. (J) Representative immunofluorescence images of NE and DAPI stained TA muscle ×20. Bar = 50 μm. (K–N) Quantification of number of positive NE cells per muscle cross section. (O–R) Relative mRNA expression of neutrophil elastase gene (ELANE) in muscle. Mean ± SEM. ^* P ≤ 0.05, ^** P ≤ 0.01, ^**** P ≤ 0.0001 vs. sham‐injected group (Student's t‐test).

Figure 6

LL/2 altered expression of innate immune cell markers in skeletal muscles, but did not affect protein expression of muscle protein specific E3 ubiquitin ligases. Representative western blot of tryptase, Mas‐related gene X2 (MrgX2), CD68, myeloperoxidase (MPO), muscle‐specific RING finger‐1 (MuRF1), muscle atrophy F‐box (MAFbx), and fast myosin skeletal heavy chain (MyHC) on (A) TA, (B) GA, (C) EDL, and (D) SOL muscles obtained from animals in Figure 2. GAPDH was used for loading control.

Figure 7

LL/2 altered expression of innate immune cell markers in skeletal muscles, but did not affect protein expression of muscle protein specific E3 ubiquitin ligases. Quantifications of western blots in Figure 6. (A) tryptase, (B) Mas‐related gene X2 (MrgX2), (C) CD68, (D) myeloperoxidase (MPO), (E) muscle‐specific RING finger‐1 (MuRF1), (F) muscle atrophy F‐box (MAFbx), and (G) fast myosin skeletal heavy chain (MyHC). Mean ± SEM. ^* P ≤ 0.05, ^** P ≤ 0.01, ^*** P ≤ 0.001 vs. sham‐injected group (Student's t‐test).

Figure 8

LL/2 activated mast cells decrease differentiated Myotube diameter. (A) Representative histogram of LAMP2 expression in P815 cells with no treatment, control conditioned media (CM), or LL/2 CM. (B) IL‐6 levels at 24 h in (i) LL/2 CM, (ii) bone marrow‐derived MC (BMMC) CM, and (iii) BMMC CM treated with LL/2 CM (LL/2 / BMMC CM). Mean ± SEM. ^**** P ≤ 0.0001 (one‐way ANOVA, Tukey's multiple comparisons test). (C) Differentiated murine C2C12 myotubes were treated with (i) control CM, (ii) LL/2 CM, (iii) BMMC CM, or (iv) LL/2/BMMC CM for 24 h. The average C2C12 myotube diameter was measured under the microscope. Mean ± SEM. ^* P ≤ 0.05 (one‐way ANOVA, Tukey's multiple comparisons test). (D) Representative western blot of fast myosin skeletal heavy chain (MyHC) expression in differentiated murine C2C12 myotubes treated with (i) control CM, (ii) LL/2 CM, (iii) BMMC CM, or (iv) LL/2/BMMC CM for 24 h. (E) Quantification of western blot in Figure 8D. Mean ± SEM. (one‐way ANOVA, Tukey's multiple comparisons test).

Figure 9

Innate immune cell signatures positively correlate with skeletal muscle atrophy and apoptosis signatures. Spearman's correlation of muscle atrophy gene signature and (A) mast cell, (B) macrophage, and (C) neutrophil gene signatures in human skeletal muscle tissue from the genotype‐tissue expression (GTEx) project. Correlation of muscle apoptosis signatures and (D) mast cell, (E) macrophage, and (F) neutrophil gene signatures.

Figure 10

Innate immune cell signatures positively correlate with dietary excess and cachexia signatures. Spearman's correlation of dietary excess gene signature and (A) mast cell, (B) macrophage, and (C) neutrophil gene signatures in human skeletal muscle tissue from the genotype‐tissue expression (GTEx) project. Correlation of cachexia signatures and (D) mast cell, (E) macrophage, and (F) neutrophil gene signatures.

Figure 11

Enriched mast cell gene signature in muscles of cachectic upper GI cancer and PDAC patients. Gene set enrichment analyses of (A) adaptive immunity, (B) innate immunity, (C) mast cell, (D) macrophage, and (E) neutrophil gene signatures expressed in the quadriceps muscles obtained from upper gastrointestinal (GI) cancer patients from GSE34111 with 7% weight loss (n = 12, PreOp) and normal controls (n = 6, normal). Enrichment plots of (F) adaptive immunity, (G) innate immunity, (H) mast cell, (I) macrophage, and (J) neutrophil gene signatures expressed in the rectus abdominis muscles obtained from pancreatic ductal adenocarcinoma (PDAC) patients from GSE130563 with cachexia (n = 17, cachectic) or non‐cancer controls (n = 16, non‐cancer). FDR, false discovery rate; NES, normalized enrichment score; FWER, family‐wise error rate.