Muscle regeneration is disrupted by cancer cachexia without loss of muscle stem cell potential

Abstract

Cancer cachexia is a severe, debilitating condition characterized by progressive body wasting associated with remarkable loss of skeletal muscle weight. It has been reported that cancer cachexia disturbs the regenerative ability of skeletal muscle, but the cellular mechanisms are still unknown. Here, we investigated the skeletal muscle regenerative process in mouse colon-26 (C26) tumor cell-bearing mice as a C26 cancer cachexia model. Although the proliferation and differentiation abilities of muscle stem cells derived from the C26 tumor cell-bearing mice were sustained in vitro, the proliferation and differentiation were severely impaired in the cachexic mice. The numbers of both macrophages and mesenchymal progenitors, which are critical players in muscle regeneration, were reduced in the cancer cachexic mice, indicating that the skeletal muscle regeneration process was disrupted by cancer cachexia. Furthermore, the number of infiltrated neutrophils was also reduced in cancer cachexia mice 24 hours after muscle injury, and the expression of critical chemokines for muscle regeneration was reduced in cancer cachexia model mice compared to control mice. Collectively, although the ability to regeneration of MuSCs was retained, cancer cachexia disturbed skeletal muscle regenerative ability by inhibiting the orchestrated muscle regeneration processes.

Fig 1. Reduced muscle weight in C26-bearing mice.

(A) Body weight (BW), Tibialis anterior (TA), gastrocnemius (GC), and quadriceps (Qu) muscle weights (mg) of #KC (black bar)- or colon26 (C26, white bar)-bearing mice 16 or 19 days after transplantation. (B) Relative tumor weights of #KC (black bar)- and C26 (white bar)- bearing mice 19 days after tumor transplantation. (C) The GC or Qu muscle weights (mg) per body weight (g) of #KC (black bar)- or colon26 (C26, white bar)-bearing mice 16 or 19 days after transplantation. (D) Fat weight (mg) of #KC (black bar)- or C26 (white bar)-bearing mice 19 days after tumor transplantation. *P<0.05, **P<0.01, NS: non-significant.

Fig 2. Regeneration defect in C26-bearing mice.

(A) Time course for analysis of #KC- or C26-bearing mice. (B) H.E. staining of intact or regenerating TA muscle 4 or 7 days after cardiotoxin injection in #KC-, C26-bearing, or non-transplanted (Non) mice. (C) Immunostaining of eMyHC (green) and laminin α2 (LNα2; red) in regenerating TA muscle 4 days after cardiotoxin injection. Nuclei were counterstained with DAPI. Scale bar: 100 μm. Bar graph shows the percentage of eMyHC-positive areas in #KC- or C26-bearing mice. **P<0.01.

Fig 3. Reduced number of myogenic cells, mesenchymal progenitors, and macrophages in C26-bearing mice during muscle regeneration.

(A) Time course of immunohistochemical analyses of #KC- or C26-tumor bearing mice. (B) The sections of TA muscle were stained with anti-M-cadherin (green), laminin α2 (LNα2, red), Pdgfrα (red or white), or F4/80 (green or white) antibodies. Nuclei were stained with DAPI. Scale bar: 100 μm. The graphs indicate the number of each population per field. **P<0.01 (C) Time course of FACS analysis of #KC- or C26-bearing mice. (D) Representative FACS profiles of SM/C-2.6(+)Sca-1(-)CD31(−)CD45(−) cells (myogenic cells), Pdgfrα(+)Sca-1(+)CD31(−)CD45(−) cells (mesenchymal progenitors), and F4/80(+)CD45(+) cells (macrophages) during skeletal muscle regeneration in tumor-bearing mice. TA, GC, Qu muscle were used for these analyses. The graphs indicate the relative number of each population by multiplying the number of mononuclear cell obtained per 1 g muscle and the fraction percentage. *P<0.05, **P<0.01.

Fig 4. Isolated myogenic cells from C26-bearing mice show normal proliferation and differentiation.

(A) Time course of EdU uptake (B) or fusion index (C) of MuSCs derived from TA, GC, Qu muscles of #KC- or C26-bearing mice. (B) Percentage of EdU+ cells (white) in cultured myogenic cells from #KC- or C26-bearing mice. Scale bar: 100 μm. The graph shows the relative number of EdU+ cells per total number of nuclei. (C) Index of the frequency of fusion in freshly isolated myogenic cells from #KC- or C26-bearing mice. Myotubes were stained with anti-α-actinin (green). Scale bar: 100 μm. The graph shows the percentages of multinuclear cell per total number of nuclei.

Fig 5. Severe regeneration defect in C26-bearing mice.

(A) Time course of FACS analysis of neutrophils derived from #KC- or C26-bearing mice. (B) FACS profiles of mononuclear cells derived from #KC- (upper) or C26-bearing (lower) TA, GC, Qu muscles 1 day after CTX injection. The cells were gated for CD11b+ fractions. (C) Giemsa staining of CD11b+Ly6G+Ly6C- fractions. Scale bar: 20 μm. The graph shows the relative cell numbers of CD11b+Ly6G+Ly6C- fractions per 1 g muscle. **P<0.01.

Fig 6. Expression of chemokines inducing neutrophil infiltration.

(A) Time course of mRNA expression of chemokines in #KC- or C26-bearing muscle 1 day after CTX injection. (B) Relative expressions of Ccl2-5 mRNA in injured TA, GC, Qu muscle from #KC (black)- or C26 (white)-bearing mice 24 hours after CTX injection. *P<0.05 (C) Relative expressions of Cxcl1-3, 5 mRNA in injured muscle 24 hours after CTX injection from #KC (black)- or C26 (white)-bearing mice.