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. 2020 Nov 11;10(1):19501.
doi: 10.1038/s41598-020-75913-w.

Chemoradiation impairs myofiber hypertrophic growth in a pediatric tumor model

Nicole D Paris  1   2   3 , Jacob G Kallenbach  1   4 , John F Bachman  1   5 , Roméo S Blanc  1   2   3 , Carl J Johnston  6 , Eric Hernady  7 , Jacqueline P Williams  2   7   8 , Joe V Chakkalakal  9   10   11   12   13
Affiliations

Chemoradiation impairs myofiber hypertrophic growth in a pediatric tumor model

Nicole D Paris et al. Sci Rep. .

Abstract

Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Characterization of implanted rhabdomyosarcoma tumors from cell line M3-9-M. (a) Representative brightfield images of crystal violet-stained rhabdomyosarcoma (RMS) cells grown for 3 days in culture. Scale 800 µm (left), 600 µm (right). (b) Representative photos of C57BL/6 hindlimbs (top) and mouse (bottom) 1 week following implantation of RMS cells at varying concentrations. (c) RMS tumor morphology by H&E staining of longitudinal sections (left) and cross-sections (right). Scale 100 µm. (d) Representative images of immunostaining of tumor sections to detect muscle-progenitor proteins Myogenin or MyoD (green), DAPI (blue), Laminin (white), and Myosin (red). Scale 100 µm.
Figure 2
Figure 2
Establishment of rhabdomyosarcoma cell line RMS-luc-RFP as a cancer model for hindlimb radiotherapy. (a) Schematic depicting the luciferase-RFP construct stably expressed by RMS-luc-RFP cells. (b) Representative photos of C57BL/6 mice (left) and tumor-bearing gastrocnemius muscles (right) 1 week following implantation of RMS-luc-RFP cells at varying concentrations, with tumor sizes. (c) Representative brightfield/luminescent image and (d) quantification of luminescence by IVIS of RMS-luc-RFP cells implanted at varying concentrations (100–300 K) in PBS with or without matrigel present (1:1 PBS/Matrigel), N = 3 mice.
Figure 3
Figure 3
Targeted hindlimb irradiation during juvenile stages effectively eliminates implanted rhabdomyosarcoma tumors. (a) Schematic describing the method of RMS tumor cell implant into right gastrocnemius, subsequent radiation treatment, and tissue harvest. (b) Representative CT images of two methods of hindlimb targeted radiotherapy, knee-to-ankle (left) and localized to relative tumor location (right). Boxes represent radiation field; red circles label the isocenter. (c) IVIS luminescent detection merged with brightfield image (left) and quantified as average radiance (p/s/cm2/sr) recorded biweekly for 5 weeks post tumor cell implantation (right), n = 6 mice. (d) Quantification of percentages of tumor elimination classified as complete (no tumor), partial (tumor < 12 mm), or none (tumor persisted > 12 mm) based on gross measurement at time of harvest, n = 6 mice/group. (e) Ratio of weight at time of harvest to weight at four weeks old was quantified. Tibialis anterior (TA) mass shown as (f) normalized to mouse body weight or (g) absolute mass, n = 3 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001, to CTL or Comp CL. One-way ANOVA, Tukey’s test. Comp complete, Part partial, CL contralateral leg, RL irradiated leg.
Figure 4
Figure 4
Juvenile hindlimb irradiation in conjunction with chemotherapy treatment effectively eliminates RMS tumors and is a pre-clinical mouse model of pediatric RMS. (a) Schematic describing the timeline of RMS tumor cell implant into 3.5–4-week-old mice, Vincristine (VIN) or vehicle treatment, subsequent radiation treatment, and tissue harvest at 8 weeks old. (b) IVIS bioluminescent detection quantified as average radiance (p/s/cm2/sr) recorded for 5 weeks post tumor cell implantation. (c) Mouse body weight at time of VIN or vehicle injection until time of harvest was quantified. N = 6 mice. *P < 0.05, ***P < 0.001, VIN Rad to Veh Rad, Two-way ANOVA, Tukey’s multiple comparisons test. CL contralateral leg, RL irradiated leg.
Figure 5
Figure 5
Juvenile irradiated mice experience muscle atrophy four weeks following treatment which is exacerbated by chemotherapy. (a) Representative images of contralateral and irradiated, vehicle or Vincristine treated DAPI-stained fixed EDL single fibers, Scale = 200 μm. (b) EDL myofiber average cross-sectional area (CSA). (c) EDL myofiber volume (CSA × Length). (d) Representative images of contralateral and irradiated, vehicle or Vincristine treated DAPI-stained fixed Soleus (SOL) single fibers, Scale = 200 μm. (e) SOL myofiber average cross-sectional area (CSA). (f) SOL myofiber volume (CSA × Length). **P < 0.01, ***P < 0.001, to CL. &P < 0.05, $P < 0.01, #P < 0.001, %P < 0.0001 to Vehicle CL or RL. Two-way ANOVA, Tukey’s multiple comparisons test. CL contralateral leg, RL irradiated leg. 50 myofibers/muscle, N = 3 mice/group.
Figure 6
Figure 6
Chemotherapy treatment causes a synergistic decline of EDL myonuclear domain and results in decreased SOL myonuclear number with no effect on SC number. (a) EDL myonuclei number per mm of myofiber length. (b) EDL myonuclear domain ((CSA × Length)/Total MN). (c) SOL myonuclei number per mm of myofiber length. (d) SOL myonuclear domain ((CSA × Length)/Total MN). Representative Pax7 (red), DAPI (blue) immunofluorescent images and quantification of cross-sections of (e,g) EDL and (f,h) SOL. Scale = 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001, to CL. &P < 0.05, #P < 0.001, to Vehicle CL or RL (a–d) or to CL of same condition (g,h). Two-way ANOVA, Tukey’s multiple comparisons test. CL contralateral leg, RL irradiated leg. N = 3 mice/group.
Figure 7
Figure 7
Chemoradiation treatment results in worsened performance in a Rotarod test. (a) Schematic describing the timeline of RMS tumor cell implant into 3.5–4-week-old mice, Vincristine (VIN) or vehicle treatment, subsequent radiation treatment, and rotarod training and endurance fatigue test at 8 weeks old. (b) Rotarod endurance fatigue test results quantification of average number of cumulative falls over one-hour testing period for each condition. (c) Frequency distribution of rotarod endurance fatigue test performance for each condition. (d) Quantification of percent of mice in each condition to pass (< 7 falls) or fail (> 7 falls) the rotarod endurance fatigue test. N = 7 mice.
Figure 8
Figure 8
Working model of muscle-specific late effects of juvenile cancer therapies. Schematic demonstrating the different phenotypes associated with either EDL or SOL muscles when chemotherapy, targeted radiation, or both are administered to mice during the juvenile growth period. CSA cross-sectional area, MN myonuclear number, MD myonuclear domain.
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