The document outlines the musculoskeletal complications associated with cancer and its treatments, highlighting the significant impact of cancer cachexia on muscle mass and function. It emphasizes that cancer-related weight loss is linked to higher mortality rates and worsened chemotherapy outcomes, advocating for strategies to preserve muscle mass to improve survival. Additionally, it discusses the effects of chemotherapy on muscle atrophy and suggests potential therapeutic interventions like bisphosphonates to mitigate these complications.

1. Click to edit Master title style
Click to edit Master subtitle style
2021-11-18 1
Andrea Bonetto, PhD
Musculoskeletal Complications of Cancer
and its Treatments
Associate Professor
Surgery
Indiana University

2. Click to edit Master title style
Click to edit Master subtitle style
2021-11-18 2
Musculoskeletal Complications of Cancer
and its Treatments
An expert presents new research on
cancer and cachexia, and their
complications to anti-cancer treatment.

3. Musculoskeletal complications of
cancer and its treatments
Inside Scientific Webinars
November 18, 2021
Andrea Bonetto, PhD

4. •Frequent in malignancy
•Reduced response to treatments
•Weight loss correlated with mortality
•Accounts for ~1/3 of cancer deaths
•Will cause the deaths of ~150,000
Americans every year
Cancer cachexia
There is currently no cure for cachexia

5.  Preservation of muscle mass prolongs survival in tumor hosts
(Benny Klimek et al., 2010; Zhou et al., 2010)
 Cancer patients with cachexia show severe chemotherapy toxicity
 Patients with higher muscle mass are more resistant to chemotherapy
(Prado et al., 2011, 2013; Antoun et al., 2010)
 Pro-anabolic strategies counteract chemotherapy effects on muscle
(Le Bricon et al., 1995; Damrauer et al., 2008; Garcia et al., 2008; Fanzani et al., 2011; Chen et al., 2015)
The role of skeletal muscle mass in cancer

6. • 1.8 million cases of cancer in the US, 600,000 will die
• Over 19 million cancer survivors by 2024
• Weakness and fatigue affect 70-100% of patients receiving cancer
treatments
• Persistent muscle weakness following chemotherapy
 Poorer QOL, worse outcomes, relevant costs
(American Cancer Society, 2021; Siegel et al., 2021)
Chemotherapy toxicities in cancer: impact on skeletal muscle

7. CANCER
CHEMOTHERAPY
The role of chemotherapy

8. CANCER
CHEMOTHERAPY
?
MUSCLE ATROPHY &
MUSCLE WEAKNESS
How do cancer and chemotherapy affect muscle mass and function?

9. Muscle weights
G
astrocnem
ius
Tibialis
Q
uadriceps
H
eart
0.0
0.2
0.4
0.6
0.8
Weight
/
100mg
BW
***
***
**
**
Organ weights
Liver
Spleen
Fat
0
2
4
6
8
Weight
/
100mg
BW
Control
C26
**
**
***
0 1 3 5 7 9 10 11 12 13 14
0.8
0.9
1.0
1.1
1.2
Days
Variation
vs.
day
0
* **
***
Body weight
Control
C26
C
o
n
t
r
o
l
C
2
6
150
200
250
300
350
Force
(g)
Whole body - Muscle Force
***
0 50 100 150 200
5
10
15
20
25
30
35
Frequency (Hz)
Force
(g)
EDL - Force
*** *** *** *** ***
***
0 50 100 150 200
100
200
300
400
500
Frequency (Hz)
Specific
Force
(kN/m
2
)
EDL - Specific Force
Control
C26
*** *** *** *** ***
***
Control C26
100µm
CANCER
Cancer growth causes body weight loss, along with loss of muscle mass and function
Bonetto et al., JOVE 2016

10. Cancer affects muscle function, both in males and in females
Bonetto Lab, unpublished
1
0
2
5
4
0
6
0
8
0
1
0
0
1
2
5
1
5
0
0
5
1 0
1 5
M a le
F re q u e n c y (H z )
m
N

m
C o ntrol
L L C
* *
* * *
* * * *
1
0
2
5
4
0
6
0
8
0
1
0
0
1
2
5
1
5
0
0 .0
0 .2
0 .4
0 .6
M a le
F re q u e n c y (H z )
m
N

m
/
g
C o ntrol
L L C
*
* *
* *
1
0
2
5
4
0
6
0
8
0
1
0
0
1
2
5
1
5
0
0
5
1 0
1 5
F e m a le
F re q u e n c y (H z )
m
N

m
C o ntrol
L L C
* *
* *
* *
1
0
2
5
4
0
6
0
8
0
1
0
0
1
2
5
1
5
0
0 .0
0 .2
0 .4
0 .6
F e m a le
F re q u e n c y (H z )
m
N

m
/
g
C o ntrol
L L C
*
* *
*

11. Bonetto Lab, unpublished
Colorectal cancer contributes to reduced activity in mice
S
h
a
m
m
H
C
T
1
1
6
0
5 0
1 0 0
1 5 0
A m b u la to ry
S
e
c
o
n
d
s
S
h
a
m
m
H
C
T
1
1
6
0
2 0
4 0
6 0
S te re o ty p ic
S
e
c
o
n
d
s
S
h
a
m
m
H
C
T
1
1
6
0
5
1 0
1 5
2 0
2 5
R e s tin g
S
e
c
o
n
d
s
S
h
a
m
m
H
C
T
1
1
6
0
5
1 0
1 5
2 0
2 5
R e a rin g E v e n ts
#
*
S
h
a
m
m
H
C
T
1
1
6
0
5
1 0
1 5
2 0
2 5
R e a rin g T im e
S
e
c
o
n
d
s
*
S
h
a
m
m
H
C
T
1
1
6
0
5 0 0
1 0 0 0
1 5 0 0
D is ta n c e
In
c
h
e
s
*
S
h
a
m
m
H
C
T
1
1
6
0
5
1 0
1 5
A v e ra g e S p e e d
S
p
e
e
d
(
in
/s
)
*

12. 0 7 14 21 28 35
0.85
0.90
0.95
1.00
1.05
1.10
Days
Variation
vs.
day
0
*
*
**
***
***
***
Body weight
Vehicle
Folfox
Folfiri
Muscle weights
G
SN
Tibialis
Q
uadriceps
H
eart
0.00
0.05
0.10
0.15
0.20
0.4
0.6
0.8
1.0
Weight
/
100mg
IBW
**
**
***
***
Organ weights
Liver
Spleen
Fat
Kidney
0
2
4
6
Weight
/
100mg
IBW
Vehicle
Folfox
Folfiri
***
*
***
*
**
*
0 50 100 150
0
10
20
30
40
50
Frequency (Hz)
Force
(g)
EDL - Force
*** *** *** *** ***
V
e
h
i
c
l
e
F
o
l
f
o
x
F
o
l
f
i
r
i
0
50
100
150
200
250
Force
(g)
Whole body - Muscle Force
**
0 50 100 150
0
100
200
300
400
500
Frequency (Hz)
Specific
Force
(kN/m
2
)
EDL - Specific Force
Folfox
Vehicle
Folfiri
*
*** *** *** *** ***
***
100µm
CHEMOTHERAPY
Chemotherapy drives loss of muscle mass and function
Barreto et al., Oncotarget 2016
Barreto, Mandili et al., Front Physiol 2016

13. Bonetto Lab, unpublished
1
0
2
5
4
0
6
0
8
0
1
0
0
1
2
5
1
5
0
0
5
1 0
1 5
2 0
F o rc e F re q u e n c y
F re q u e n c y (H z )
m
N

m
C o ntrol
C isplatin
* *
* * *
* * * *
1
0
2
5
4
0
6
0
8
0
1
0
0
1
2
5
1
5
0
0 .0
0 .2
0 .4
0 .6
0 .8
R e la tiv e F o rc e F re q u e n c y
F re q u e n c y (H z )
m
N

m
/
g
C o ntrol
C isplatin
* * * *
1
2
3
4
5
6
7
8
9
1
0
0
5 0
1 0 0
1 5 0
F a tig u e
C o n tra c tio n #
%
C o ntrol
C isplatin
* * * * *
* * * *
Cisplatin affects muscle function in vivo

14. Bonetto Lab, unpublished
C
o
n
t
r
o
l
C
i
s
p
l
a
t
i
n
0
5 0
1 0 0
1 5 0
A m b u la to ry
S
e
c
o
n
d
s
* * *
C
o
n
t
r
o
l
C
i
s
p
l
a
t
i
n
0
2 0
4 0
6 0
8 0
1 0 0
S te re o ty p ic
S
e
c
o
n
d
s
C
o
n
t
r
o
l
C
i
s
p
l
a
t
i
n
0
5 0
1 0 0
1 5 0
R e s tin g
S
e
c
o
n
d
s
* *
C
o
n
t
r
o
l
C
i
s
p
l
a
t
i
n
0
5
1 0
1 5
R e a rin g E v e n ts
#
*
C
o
n
t
r
o
l
C
i
s
p
l
a
t
i
n
0
5
1 0
1 5
2 0
R e a rin g T im e
S
e
c
o
n
d
s
*
C
o
n
t
r
o
l
C
i
s
p
l
a
t
i
n
0
5 0
1 0 0
1 5 0
2 0 0
D is ta n c e
In
c
h
e
s
* * * *
C
o
n
t
r
o
l
C
i
s
p
l
a
t
i
n
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
A v e ra g e S p e e d
S
p
e
e
d
(
in
/s
)
* *
Cisplatin promotes decreased activity in mice

15. Barreto et al., Oncotarget 2016
Barreto, Mandili et al., Front Physiol 2016
Cancer and chemotherapy activate different signaling pathways

16. Downregulated proteins
Barreto, Mandili et al., Front Physiol 2016
Cancer and chemotherapy cause muscle mitochondrial dysfunctions
Upregulated proteins

17. Vehicle Folfox Folfiri
100μm
V
e
h
i
c
l
e
F
o
l
f
o
x
F
o
l
f
i
r
i
0.0
5.0×107
1.0×108
1.5×108
2.0×108
2.5×108
Intensity
(pixels)
SDH quantification
*
*
O
x
i
d
a
t
i
v
e
G
l
y
c
o
l
i
t
i
c
0
20
40
60
80
100
%
Fiber type
*
***
**
*
**
*
Vehicle
Folfox
Folfiri
O
x
i
d
a
t
i
v
e
G
l
y
c
o
l
i
t
i
c
0
50
100
150
%
Fiber CSA
* *
Chemotherapy causes an oxidative-to-glycolytic shift in fiber composition
Barreto et al., Oncotarget 2016

18. V
e
h
i
c
l
e
F
o
l
f
o
x
F
o
l
f
i
r
i
0.0
0.5
1.0
1.5
mtDNA/Nuclear
DNA
(fold
change)
Chemotherapy-induced cachexia
* *
C
o
n
t
r
o
l
C
2
6
0.0
0.5
1.0
1.5
mtDNA/Nuclear
DNA
(fold
change)
C26 cachexia
**
500nm
Vehicle Folfox Folfiri
Chemotherapy drives muscle mitochondrial abnormalities
Barreto et al., Oncotarget 2016

19. CANCER
CHEMOTHERAPY
ROS
Mitochondrial
abnormalities
Differential activation of
pro-atrophic/pro-catabolic pathways
MUSCLE ATROPHY &
MUSCLE WEAKNESS
Cancer and chemotherapy contribute to development and sustainment of cachexia
Barreto et al., Oncotarget 2016
Barreto, Mandili et al., Front Physiol 2016
Pin et al., FASEB J 2019
Pin et al., JCSM 2019

20. Is it all about the muscle then…?

21. Cancer cachexia is a multi-organ disorder

22. Bone and muscle: a long-lasting relationship
Bonetto and Bonewald, Basic and Applied Bone Biology 2019

23. Unbalances in bone- and muscle-derived biochemical factors
may play a role in the pathogenesis of cachexia
Myokines
IL-6
IGF-1
Myostatin
FGF-2
BAIBA
Irisin
…
Osteokines
Activin A
TGF-β
IGF-1
BMP-2
RANKL
…
Bone
Muscle
Bone and muscle: a long-lasting relationship

24. Vehicle Cisplatin
Essex, Pin et al., Front Endocrinol 2019
V
C
0
10
20
30
BV/TV
%
***
V
C
0.00
0.02
0.04
0.06
0.08
0.10
Tb.Th
mm
V
C
0.0
0.1
0.2
0.3
Tb.Sp
mm
***
V
C
0
1
2
3
4
Tb.N
1/mm
***
Barreto et al., Sci Rep 2017
Vehicle Folfiri
V
e
h
i
c
l
e
F
o
l
f
i
r
i
0
5
10
15
20
BV/TV
%
***
V
e
h
i
c
l
e
F
o
l
f
i
r
i
0
1
2
3
Tb.N
1/mm
***
V
e
h
i
c
l
e
F
o
l
f
i
r
i
0.00
0.02
0.04
0.06
0.08
Tb.Th
mm
***
V
e
h
i
c
l
e
F
o
l
f
i
r
i
0.0
0.2
0.4
0.6
Tb.Sp
mm
***
Chemotherapy causes loss of bone

25. Essex, Pin et al., Front Endocrinol 2019
Generation of marrow-
flushed bone CM (48h)
Myotubes exposed to bone
CM (48h)
Tibiae excised
>48h after last
treatment
Bone-derived soluble factors may participate in muscle wasting

26. Can bone-targeting therapies preserve skeletal muscle in cachexia?

27. Bisphosphonates prevent muscle weakness in a murine model of
breast-cancer induced bone metastases
Waning et al., 2015 Nat Med
Pamidronate improves muscle mass and function in burn children
Borsheim et al., 2015 JBMR; Pin et al., 2019 Front Endocrinol
Bisphosphonates are bone-targeting anti-resorptive agents
Bisphosphonates promote osteoclast apoptosis and inhibit osteoclastic bone resorption
Hughes et al. 1995 J Bone Miner Res; Mundy and Yoneda 1998 N Engl J Med

28. Essex, Pin et al., Front Endocrinol 2019
V
Vehicle
C
Cisplatin
(2.5 mg/kg)
ZA
Zoledronic Acid
(5mg/kg)
C+ZA
Cisplatin +
Zoledronic Acid
C
V ZA C+ZA
V
C
Z
A
C
+
Z
A
0
10
20
30
40
BV/TV
%
*
$$ &&
** $$$
V
C
Z
A
C
+
Z
A
0.00
0.02
0.04
0.06
0.08
Tb.Th
mm
* $$$
&
V
C
Z
A
C
+
Z
A
0.0
0.1
0.2
0.3
Tb.Sp
mm
$$$
**
$$
V
C
Z
A
C
+
Z
A
0
1
2
3
4
5
Tb.N
1/mm
** $$$
**
$$$ &
V
C
Z
A
C
+
Z
A
0
10
20
30
40
Tb.Pf
1/mm
* $$$
$ &
Bisphosphonates preserve trabecular bone in combination with cisplatin

29. Essex, Pin et al., Front Endocrinol 2019
Zoledronate improves muscle mass and strength in cisplatin-treated animals
V
C
Z
A
C
+
Z
A
0.0
0.2
0.4
0.6
0.8
Gastrocnemius
Weight
/
100mg
IBW
***
** &
$$$
V
C
Z
A
C
+
Z
A
0.00
0.05
0.10
0.15
0.20
0.25
Tibialis Anterior
Weight
/
100mg
IBW
***
$
$$
V
C
Z
A
C
+
Z
A
0.0
0.2
0.4
0.6
0.8
1.0
Quadriceps
Weight
/
100mg
IBW
**
$
V
C
Z
A
C
+
Z
A
0.0
0.2
0.4
0.6
Heart
Weight
/
100mg
IBW
***
* $
$$
V
C
Z
A
C
+
Z
A
0
100
200
300
400
500
Whole body grip strength
Force
(g)
Day 1
Day 14
*
$$
$
V
C
Z
A
C
+
Z
A
0
500
1000
1500
2000
Cross-Sectional Area
Fiber
size
(µm
2
)
***
* $ &&
$$$

30. Bonetto et al., Front Physiol 2016
Waning et al., Nat Med 2015
Non-bone metastatic cancers promote loss of muscle with differential effects on bone

31. BMI (kg/m2) 30.5 ± 3.7 33.7 ± 5.2 27.6 ± 4.9b
SMI (cm2/m2) 45.6 ± 6.0 49.8 ± 8.2 39.6 ± 5.1aa bbb
Visceral Fat (cm2) 156.5 ± 59.5 132.7 ± 64.1 90.3 ± 59.5aa
Pin et al., under review
Fearon et al., Lancet Oncol, 2011
Diagnostic Criteria:
1. Weight loss >5% (prior 6 months)
2. Weight loss >2% + BMI <20kg/m2
3. Weight loss >2% + Sarcopenia
Control
OvCa
(no cachexia)
OvCa
(cachexia)
Skeletal muscle
Intramuscular fat
Visceral adipose
Subcutaneous adipose
Ovarian cancer presents with evidence of muscle wasting

32. ES-2 OVARIAN CANCER
Pin et al., JCSM 2018
5
Control
ES-2
C
ontrol
ES-2
-4
-2
0
2
4
Grams
(g)
BW change
***
BW
Muscle
C
o
n
t
r
o
l
E
S
-
2
0.00
0.05
0.10
0.15
0.20
Weight
/
100mg
IBW
Tibialis anterior
***
C
o
n
t
r
o
l
E
S
-
2
0.0
0.2
0.4
0.6
Weight
/
100mg
IBW
Gastrocnemius
***
C
o
n
t
r
o
l
E
S
-
2
0.0
0.2
0.4
0.6
0.8
1.0
Weight
/
100mg
IBW
Quadriceps
***
C
o
n
t
r
o
l
E
S
-
2
0.0
0.2
0.4
0.6
Weight
/
100mg
IBW
Heart
***
Bone
Ovarian cancer is accompanied by loss of muscle and bone
ES-2 tumors recapitulate the
clinical presentation of HGS-OC

33. Pin et al., under review
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
BV/TV
*
$$$
#
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0.00
0.05
0.10
0.15
Tb.Th
µm
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0.0
0.1
0.2
0.3
0.4
0.5
Tb.Sp
µm
*
$$$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
1
2
3
4
Tb.N
1/µm
**
$$$
##
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
10
20
30
Tb.Pf
1/µm
*
##
$$$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
100
200
300
Conn.Dn
1/µm
3
$$
1
2
C
2
6
E
S
-
2
A
s
c
i
t
e
s
E
S
-
2
RANKL
* $$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
2
4
6
8
Tnfsf11/Tnfrsf11b
Fold
change
ratio
***
###
$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
100
200
300
RANKL
pg/ml
**
$
##
E
S
-
2
E
S
-
2
+
Z
A
0
1
2
3
4
5
Ascites
Volume
(ml)
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0.0
0.2
0.4
0.6
0.8
1.0
Quad
Weight
/
100mg
IBW
***
##
$$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0.0
0.2
0.4
0.6
GSN
Weight
/
100mg
IBW
***
###
$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0.00
0.05
0.10
0.15
0.20
TA
Weight
/
100mg
IBW
***
###
$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
150
200
250
300
350
Muscle strength
Force
(g)
***
###
$
Muscle
C ZA
ES-2 ES-2 + ZA
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
10
20
30
40
50
BV/TV
%
*
$$$
#
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0.00
0.05
0.10
0.15
Tb.Th
µm
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0.0
0.1
0.2
0.3
0.4
0.5
Tb.Sp
µm
*
$$$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
1
2
3
4
Tb.N
1/µm
**
$$$
##
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
10
20
30
Tb.Pf
1/µm
*
##
$$$
30
100
150
200
RANKL
rg/ml
* $$
C
Z
A
E
S
-
2
E
S
-
2
+
Z
A
0
2
4
6
8
Tnfsf11/Tnfrsf11
Fold
change
ratio
***
###
$
2
3
4
5
Ascites
ume
(ml)
0.4
0.6
0.8
1.0
Quad
/
100mg
IBW
***
##
$$
0.4
0.6
GSN
/
100mg
IBW
***
###
$
0.10
0.15
0.20
TA
/
100mg
IBW
***
###
Bone
Preservation of bone is also beneficial for muscle mass

34. Osteocytes
CTX-I
CTX-I CTX-I
CTX-I
Pin et al., under review
C
2
C
1
2
C
2
6
E
S
-
2
0
10
20
30
RANKL (CM)
pg/ml
* $$
C
o
n
t
r
o
l
E
S
-
2
0
200
400
600
RANKL (plasma)
pg/ml
**
C
2
6
I
D
8
O
V
C
A
R
3
0
5
10
15
50
100
150
Fold
change
RANKL mRNA expression
***
**
Bone homeostasis: the role of RANKL
ZA
E
S-2
ES-
2
+
Z
A
Tb.N
**
$$$
##
C ZA
ES-2
ES-2
+
ZA
0
10
20
30
Tb.Pf
1/µm
*
##
$$$
C ZA
ES-2
ES-2
+
ZA
0
100
200
300
Conn.Dn
1/µm
3
$$
C
Z
A
E
S
-2
E
S
-2
+
ZA
0
2
4
6
8
Tnfsf11/Tnfrsf11b
Fold
change
ratio
***
###
$
C Z
A
E
S
-
2
E
S
-2
+
ZA
0
100
200
300
RANKL
pg/ml
**
$
##

35. Normal ovary Clear cell carcinoma Serous
adenocarcinoma
Serous papillary
adenocarcinoma
Transitional cell
carcinoma
RANKL
Methyl
green
Pin et al., under review
Low RANKL expression
(n=281)
High RANKL expression
(n=92)
C
o
n
t
r
o
l
O
v
C
a
0.0
0.5
1.0
1.5
2.0
2.5
CTX-1
ng/ml
**
C
o
n
t
r
o
l
O
v
C
a
0.0
0.2
0.4
0.6
0.8
RANKL
mol/l
*
0.0 0.5 1.0 1.5 2.0 2.5
0.0
0.2
0.4
0.6
0.8
Correlation
CTX-1
sRANKL
R=0.40**
Ovarian cancer associates with RANKL-dependent bone turnover

36. Activation of RANK signaling in myofibers drives muscle atrophy
and weakness in denervated mice
Dufresne et al., Am J Physiol Cell Physiol 2016
Blockade of RANKL by OPG-Fc improves the muscle phenotype in dystrophic animals
Dufresne et al., Acta Neuropathol Commun 2018
RANKL mediates muscle atrophy and dysfunction in a mouse model of COPD
Xiong et al., Am J Respir Cell Mol Biol 2021
RANKL inhibition improves muscle strength and insulin sensitivity (in mice) and restores bone
mass (in osteoporotic women)
Bonnet et al., JCI 2019
Role of RANKL in skeletal muscle

37. Pin et al., under review
P
B
S
R
A
N
K
L
0
5
1 0
1 5
2 0
H S M M m y o tu b e s iz e
F
ib
e
r
d
ia
m
e
te
r
(
m
m
)
***
P
B
S
R
A
N
K
L
0
5
1 0
1 5
2 0
C 2 C 1 2 m y o tu b e s iz e
F
ib
e
r
d
ia
m
e
te
r
(
m
m
)
**
MyHC
C2C12
PBS
RANKL
HSMM
PBS
RANKL
MyHC
PBS RANKL
207
75
RANKL promotes muscle atrophy

38. Pin et al., under review
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
R A N K L
p
g
/m
l
***
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
2 0 0
4 0 0
6 0 0
8 0 0
O P G
p
g
/m
l
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0
1 0 0
1 5 0
2 0 0
R A N K L /O P G ra tio
F
o
ld
c
h
a
n
g
e
***
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .1 0
0 .1 5
0 .2 0
0 .2 5
0 .3 0
T A
W
e
i
g
h
t
/
1
0
0
m
g
I
B
W
* *
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .4 5
0 .5 0
0 .5 5
0 .6 0
0 .6 5
0 .7 0
0 .7 5
G S N
W
e
i
g
h
t
/
1
0
0
m
g
I
B
W
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
C S A
µm
2
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
6
8
1 0
1 2
1 4
F o rc e
m
N
×m
*
l
i
n
e
w
e
e
k
w
e
e
k
w
e
e
k
e
e
k
0 .0 4 0
0 .0 4 5
0 .0 5 0
0 .0 5 5
0 .0 6 0
B M D
g
/
c
m
2
* * *
e
l
i
n
e
w
e
e
k
w
e
e
k
w
e
e
k
e
e
k
0 .2 5
0 .3 0
0 .3 5
0 .4 0
0 .4 5
0 .5 0
B M C
g
A A V -N U L L
A A V -R A N K L
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
R A N K L
p
g
/m
l
***
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
2 0 0
4 0 0
6 0 0
8 0 0
O P G
p
g
/m
l
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0
1 0 0
1 5 0
2 0 0
R A N K L /O P G ra tio
F
o
ld
c
h
a
n
g
e
***
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .1 0
0 .1 5
0 .2 0
0 .2 5
0 .3 0
T A
W
e
i
g
h
t
/
1
0
0
m
g
I
B
W
**
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .4 5
0 .5 0
0 .5 5
0 .6 0
0 .6 5
0 .7 0
0 .7 5
G S N
W
e
i
g
h
t
/
1
0
0
m
g
I
B
W
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
C S A
µm
2
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
6
8
1 0
1 2
1 4
F o rc e
m
N
×m
*
e
l
i
n
e
w
e
e
k
w
e
e
k
w
e
e
k
e
e
k
0 .0 4 0
0 .0 4 5
0 .0 5 0
0 .0 5 5
0 .0 6 0
B M D
g
/
c
m
2
** *
e
l
i
n
e
w
e
e
k
w
e
e
k
w
e
e
k
w
e
e
k
0 .2 5
0 .3 0
0 .3 5
0 .4 0
0 .4 5
0 .5 0
B M C
g
A A V -N U L L
A A V -R A N K L
*
AAV-NULL AAV-RANKL
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0 0
1 0 0 0
1 5 0 0
p
g
/
m
l
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
2 0 0
4 0 0
6 0 0
p
g
/
m
l
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0
1 0 0
1 5 0
F
o
ld
c
h
a
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .1 0
0 .1 5
0 .2 0
0 .2 5
W
e
ig
h
t
/
1
0
0
* *
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .4 5
0 .5 0
0 .5 5
0 .6 0
0 .6 5
W
e
ig
h
t
/
1
0
0
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
C S A
µm
2
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
6
8
1 0
1 2
1 4
F o rc e
m
N
×m
*
B
a
s
e
l
i
n
e
1
w
e
e
k
2
w
e
e
k
3
w
e
e
k
4
w
e
e
k
0 .0 4 0
0 .0 4 5
0 .0 5 0
0 .0 5 5
0 .0 6 0
B M D
g
/
c
m
2
* * *
B
a
s
e
l
i
n
e
1
w
e
e
k
2
w
e
e
k
3
w
e
e
k
4
w
e
e
k
0 .2 5
0 .3 0
0 .3 5
0 .4 0
0 .4 5
0 .5 0
B M C
g
A A V -N U L L
A A V -R A N K L
*
A
A
V
-
N
U
L
L
A
V
-
R
A
N
K
L
0
5
1 0
1 5
2 0
2 5
B V /T V
%
* *
A
A
V
-
N
U
L
L
A
V
-
R
A
N
K
L
0 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
T b .T h
µm
** *
A
A
V
-
N
U
L
L
A
V
-
R
A
N
K
L
0 .0
0 .1
0 .2
0 .3
0 .4
0 .5
T b .S p
µm
**
A
A
V
-
N
U
L
L
A
V
-
R
A
N
K
L
0
1
2
3
4
T b .N
1
/µm
* **
A
A
V
-
N
U
L
L
A
V
-
R
A
N
K
L
0
5
1 0
1 5
2 0
2 5
T b .P f
1
/µm
A
A
V
-
N
U
L
L
A
V
-
R
A
N
K
L
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
C o n n .D n
1
/µm
AAV-NULL AAV-RANKL
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
R A N K L
p
g
/m
l
***
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
2 0 0
4 0 0
6 0 0
8 0 0
O P G
p
g
/m
l
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0
5 0
1 0 0
1 5 0
2 0 0
R A N K L /O P G ra tio
F
o
ld
c
h
a
n
g
e
***
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .1 0
0 .1 5
0 .2 0
0 .2 5
0 .3 0
T A
W
e
ig
h
t
/
1
0
0
m
g
I
B
W
**
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
0 .4 5
0 .5 0
0 .5 5
0 .6 0
0 .6 5
0 .7 0
0 .7 5
G S N
W
e
ig
h
t
/
1
0
0
m
g
I
B
W
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
C S A
µm
2
*
A
A
V
-
N
U
L
L
A
A
V
-
R
A
N
K
L
6
8
1 0
1 2
1 4
F o rc e
m
N
×m
*
B M D B M C
High RANKL is sufficient to cause muscle and bone loss

39. GAPDH (37 kDa)
RANKL (32 kDa)
1- Marker
2- rmRANKL
3- C26
4- C26pR
1 2 3 4
C
2
6
C
2
6
p
R
0
50
100
150
200
RANKL
rg/ml
**
C
2
6
C
2
6
p
R
0
5
10
15
20
25
FBW
Grams
(g)
*
C
2
6
C
2
6
p
R
0
5
10
15
20
25
FBW - Tumor
Grams
(g)
*
C
2
6
C
2
6
p
R
0
5
10
15
Carcass
Grams
(g)
*
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
0.8
Quadriceps
Weight
/
100mg
IBW
*
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
Heart
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0
2
4
6
Liver
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
0.8
1.0
Spleen
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.5
1.0
1.5
WAT
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0
500
1000
1500
RANKL
rg/ml
**
C
2
6
C
2
6
p
R
0
200
400
600
800
OPG
rg/ml
C
2
6
C
2
6
p
R
0.00
0.05
0.10
0.15
0.20
0.25
TA
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
GSN
Weight
/
100mg
IBW
**
C
2
6
C
2
6
p
R
0
50
100
150
200
RANKL
rg/ml
**
C
2
6
C
2
6
p
R
0
5
10
15
20
25
FBW
Grams
(g)
*
C
2
6
C
2
6
p
R
0
5
10
15
20
25
FBW - Tumor
Grams
(g) *
C
2
6
C
2
6
p
R
0
5
10
15
Carcass
Grams
(g)
*
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
0.8
Quadriceps
Weight
/
100mg
IBW
*
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
Heart
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0
2
4
6
Liver
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
0.8
1.0
Spleen
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.5
1.0
1.5
WAT
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0
500
1000
1500
RANKL
rg/ml
**
C
2
6
C
2
6
p
R
0
200
400
600
800
OPG
rg/ml
C
2
6
C
2
6
p
R
0
2
4
6
8
RANKL/OPG
Fold
change
**
C
2
6
C
2
6
p
R
0.00
0.05
0.10
0.15
0.20
0.25
TA
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
GSN
Weight
/
100mg
IBW
**
I
B
W
d
a
y
7
d
a
y
9
d
a
y
1
0
d
a
y
1
1
80
85
90
95
100
105
Body weight change
%IBW
C26
C26pR
*
*
L
e
a
n
m
.
F
a
t
m
.
0
5
10
15
20
25
EchoMRI
Grams
(g)
C26
C26pR
*
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
0.8
Tumor
Grams
(g)
C
2
6
C
2
6
p
R
0.00
0.02
0.04
0.06
BMD
g/cm
2
***
C
2
6
C
2
6
p
R
0.0
0.1
0.2
0.3
0.4
BMC
Grams
(g)
*
C
2
6
C
2
6
p
R
0
10
20
30
BV/TV
Grams
(g)
*
C
2
6
C
2
6
p
R
0.00
0.02
0.04
0.06
0.08
0.10
Tb.Th
µm
C
2
6
C
2
6
p
R
0.00
0.05
0.10
0.15
0.20
0.25
Tb.Sp
µm
*
Tb.N Tb.Pf Conn.Dn
C
2
6
C
2
6
p
R
0
50
100
150
200
RANKL
rg/ml
**
C
2
6
C
2
6
p
R
0
5
10
15
20
25
FBW
*
C
2
6
C
2
6
p
R
0
5
10
15
20
25
FBW - Tumor
Grams
(g)
*
C
2
6
C
2
6
p
R
0
5
10
15
Carcass
Grams
(g)
*
C
2
6
C
26
p
R
0.0
0.2
0.4
0.6
0.8
Quadriceps
Weight
/
100mg
IBW
*
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
Heart
C
2
6
C
2
6
p
R
0
2
4
6
Liver
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
0.8
1.0
Spleen
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.5
1.0
1.5
WAT
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0
500
1000
1500
RANKL
rg/ml
**
C
2
6
C
2
6
p
R
0
200
400
600
800
OPG
rg/ml
C
2
6
C
2
6
p
R
0
2
4
6
8
RANKL/OPG
Fold
change
**
C
2
6
C
2
6
p
R
0.00
0.05
0.10
0.15
0.20
0.25
TA
Weight
/
100mg
IBW
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
GSN
Weight
/
100mg
IBW
**
I
B
W
d
a
y
7
d
a
y
9
d
a
y
1
0
d
a
y
1
1
80
85
90
95
100
105
Body weight change
%IBW
C26
C26pR
*
*
L
e
a
n
m
.
F
a
t
m
.
0
5
10
15
20
25
EchoMRI
Grams
(g)
C26
C26pR
*
C
2
6
C
2
6
p
R
0.0
0.2
0.4
0.6
0.8
Tumor
Grams
(g)
C
2
6
C
2
6
p
R
0.00
0.02
0.04
0.06
BMD
g/cm
2
***
C
2
6
C
2
6
p
R
0.0
0.1
0.2
0.3
0.4
BMC
Grams
(g)
*
C
2
6
C
2
6
p
R
0
10
20
30
BV/TV
Grams
(g)
*
C
2
6
C
2
6
p
R
0.00
0.02
0.04
0.06
0.08
0.10
Tb.Th
µm
C
2
6
C
2
6
p
R
0.00
0.05
0.10
0.15
0.20
0.25
Tb.Sp
µm
*
C
26
C
26pR
0
500
1000
1500
RANKL
rg/ml
**
C
26
C
26pR
0
200
400
600
800
OPG
rg/ml
C
26
C
26pR
0
2
4
6
8
RANKL/OPG
Fold
change
**
C
26
C
26pR
0.00
0.05
0.10
0.15
0.20
0.25
TA
Weight
/
100mg
IBW
C
26
C
26pR
0.0
0.2
0.4
0.6
GSN
Weight
/
100mg
IBW
**
80
85
90
95
100
105
%IBW
Lean
m
.
Fat m
.
0
5
10
15
20
25
EchoMRI
Grams
(g)
C26
C26pR
*
0.0
0.2
0.4
0.6
0.8
Grams
(g)
C
26
C
26pR
0.00
0.02
0.04
0.06
BMD
g/cm
2
***
C
26
C
26pR
0.0
0.1
0.2
0.3
0.4
BMC
Grams
(g)
*
C
26
C
26pR
0
10
20
30
BV/TV
Grams
(g)
*
C
26
C
26pR
0.00
0.02
0.04
0.06
0.08
0.10
Tb.Th
µm
C
26
C
26pR
0
1
2
3
4
Tb.N
1/µm
*
C
26
C
26pR
0
50
100
150
200
250
Tb.Pf
1/µm
C26 C26pR
RANKL worsens cancer-induced muscle and bone loss
Pin et al., under review

40. IgG2a R Ab ES-2 + IgG2a ES-2 + R Ab
Pin et al., under review
E
S
-
2
E
S
-
2
+
R
A
b
0
5
1 0
M y o tu b e s iz e
F
ib
e
r
d
ia
m
e
te
r
(
m
m
)
**
MyHC
ES-2
ES-2+R Ab
Anti-RANKL treatments preserve muscle and bone mass in cancer

41. Cancer and chemotherapy promote the development of cachexia,
accompanied by loss of muscle and bone mass
The muscle/bone crosstalk is affected by cancer and chemotherapy
Anti-resorptive strategies protect from muscle and bone loss
in cancer or following chemotherapy
Growth of non-metastatic cancers can result in bone loss
RANKL is a new regulator of muscle size in cachexia
Conclusions

42. Acknowledgments
Dept. Anatomy, Cell Biology & Physiology -
ICMH
Lynda Bonewald
Lilian Plotkin
Dept. Otolaryngology – Head & Neck Surgery
Thomas O’Connell
Dept. Surgery
Teresa Zimmers
Ashok Narasimhan
Rafael Barreto
abonetto@iu.edu
@A_Bonetto_PhD

43. Click to edit Master title style
Click to edit Master subtitle style
2021-11-18 43
Andrea Bonetto, PhD
Associate Professor
Surgery
Indiana University
Thank you for participating!
CLICK HERE to learn more and
watch the webinar