Interleukin-6 and cachexia in ApcMin/+ mice

Interleukin-6 and Cachexia in ApcMin/+ Mice

Kristen A. Baltgalvis1,3, Franklin G. Berger2,3, Maria Marjorette O. Pena2,3, J. Mark Davis1,

Stephanie J. Muga4, and James A. Carson1,3

Integrative Muscle Biology Laboratory, Exercise Science Department1,

Department of Biological Sciences2, and the Center for Colon Cancer Research3,

University of South Carolina, Columbia, South Carolina, 29208

Department of Cellular and Molecular Pharmacology4

Medical University of South Carolina, Charleston, South Carolina

Running title: ApcMin/+ Mice and Cachexia

Corresponding author:

James A. Carson, Ph.D.

University of South CarolinaDepartment of Exercise SciencePublic Health Research Center921 Assembly St., Room 405Columbia, SC 29208

Office Phone: 803-777-0809Lab Phone: 803-777-0142Fax: [email protected]

of 35Articles in PresS. Am J Physiol Regul Integr Comp Physiol (December 5, 2007). doi:10.1152/ajpregu.00716.2007

Copyright © 2007 by the American Physiological Society.

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ABSTRACT

The ApcMin/+ mouse has a mutation in the Apc tumor suppressor gene and develops

intestinal polyps, beginning at 4 weeks of age. This mouse develops cachexia by 6 months,

characterized by significant loss of muscle and fat tissue. The purpose of the present study was

to determine the role of circulating IL-6 and the polyp burden for the development of cachexia in

ApcMin/+ mice. At 26 weeks of age, mice exhibiting severe cachectic symptoms had a 61%

decrease in gastrocnemius muscle weight, complete loss of epididymal fat, a 10-fold increase in

circulating IL-6 levels, and an 89% increase in intestinal polyps compared to mildly cachectic

animals. ApcMin/+ / IL-6-/- mice did not lose gastrocnemius muscle mass or epididymal fat pad

mass, while overall polyp number decreased by 32% compared to ApcMin/+ mice. Plasmid-based

IL-6 over-expression in ApcMin/+ / IL-6-/- mice led to a decrease in gastrocnemius muscle mass,

epididymal fat pad mass, and increased intestinal polyp burden. IL-6 over-expression did not

induce cachexia in non-tumor bearing mice. These data demonstrate that IL-6 is necessary for

the onset of adipose and skeletal muscle wasting in the ApcMin/+ mouse and that circulating IL-6

can regulate ApcMin/+ mouse tumor burden.

KEYWORDS: Cachexia, colorectal cancer, inflammation, polyps, STAT-3

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INTRODUCTION

Cachexia is characterized as an overall state of ill-health, accompanied by a loss of lean

body mass and fat mass, weakness, fatigue, anemia, metabolic abnormalities, inflammation, and

impaired immune function, with or without anorexia (2-4, 24). This condition is associated with

other life-threatening diseases, such as cancer and AIDS, and negatively affects life quality and

survival (2, 26, 36). Almost 50% of all cancer patients experience progressive wasting of

adipose tissue and/or skeletal muscle mass (37), predominantly during the terminal stages of

disease. Cancer patients can lose up to 30% of their original body weight and cachexia accounts

for approximately 20-33% of cancer deaths. In particular, gastrointestinal and lung cancer

patients are extremely susceptible to cachexia (16, 31). The ApcMin/+ mouse is an established

model of colorectal cancer and cachexia (28, 30). This mouse develops intestinal polyps as early

as 4 weeks of age and becomes cachectic between 3 and 6 months of age. Using ApcMin/+ mice

to study cachexia has many advantages in that these mice have a polyp burden that mimics

human colorectal cancer, chronic low levels of circulating IL-6, and lack of anorexia. However,

the mechanisms inducing wasting in these mice are poorly understood.

One potential candidate for inducing cachexia is interleukin-6 (IL-6), a pleiotropic

cytokine that modulates a variety of physiological responses and activates genes associated with

cellular proliferation, differentiation, and apoptosis (20). Elevated circulating IL-6 levels have

been associated with cachexia (21). Intraperitoneal IL-6 treatment (40), intracerebroventricular

IL-6 injections in the lateral ventricle, IL-6 transgenic mice (38), and colon-26 tumor bearing

mice with elevated plasma IL-6 levels (45) all demonstrate approximately a 25% decrease in

gastrocnemius muscle weight. Blocking IL-6 through neutralization antibodies results in

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attenuation of IL-6 induced-muscle wasting in rodents (14, 35, 39, 44). In contrast, some studies

administering exogenous IL-6 to rodents fail to find an association between high circulating IL-6

levels and cachexia (12, 15, 33, 42, 43). The disparities between studies on IL-6 and cachexia

point to indirect effects of IL-6 on skeletal muscle wasting during cancer. One such indirect

mechanism may be IL-6-induced tumor growth. IL-6 is also a potent tumor growth factor (8, 13,

34) and the contribution of IL-6-induced tumor growth to cachexia has not been elucidated.

IL-6 elicits its effect by binding to a plasma membrane receptor complex which contains

the common signal transducing receptor chain, gp130 (glycoprotein 130) (9, 20, 23). Ligation of

this receptor activates STAT-3 (Signal Transducers and Activators of Transcription) (9, 11, 20)

which is generally associated with cell growth, differentiation, development, and survival (6).

Skeletal muscle and myotubes are sensitive to IL-6 and treatment with IL-6 results in

phosphorylation of STAT-3 (1, 17, 19, 32, 41). Skeletal muscle STAT-3 is activated during

atrophy (18, 22), but it is unknown whether muscle STAT-3 activation leads to cachexia.

The purpose of the present study was to determine the relationship between circulating

IL-6 and polyp burden for the development of cachexia in ApcMin/+ mice. We hypothesized that

increased circulating IL-6 would both increase the polyp burden and overall wasting in ApcMin/+

mice. In addition to wild-type and ApcMin/+ mice, ApcMin/+ / IL-6-/- mice were developed to ablate

IL-6. A plasmid-based in vivo electroporation system delivered into skeletal muscle was used to

over-express IL-6 in ApcMin/+ and ApcMin/+ / IL-6-/- mice, as well as wild-type mice.

MATERIALS AND METHODS

Animals. C57BL/6, ApcMin/+, and ApcMin/+ / IL-6-/- mice were originally purchased from Jackson

Laboratories (Bar Harbor, ME) and breeding was maintained at the University of South

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Carolina’s animal resource facility as previously described (28). ApcMin/+ mice (n=12) were

individually housed in cages with voluntary activity wheels and sacrificed at 26-weeks of age

(See activity protocol below). ApcMin/+ / IL-6-/- mice were genotyped for the mutant Apc allele,

IL-6, and neomycin resistance. The primer sequences for IL-6 were: Forward: 5' – TTC CAT

CCA GTT GCC TTC TTG G – 3' and Reverse: 5' – TTC TCA TTT CCA CGA TTT CCC AG –

3'. The reverse primer sequence for neomycin resistance was: Reverse: 5’ – CCG GAG AAC

CTG CGT GCA ATC C – 3’. ApcMin/+ / IL-6-/- mice (n=8), ApcMin/+ mice (n=7), and C57BL/6

mice (n=5) were sacrificed at 26-weeks of age to examine a cachectic time point. Another group

of ApcMin/+ / IL-6-/- mice (n=6), ApcMin/+ mice (n=6), and C57BL/6 mice (n=5) were sacrificed at

13-weeks of age to examine a pre-cachectic time point. To increase circulating IL-6 levels, 16-

wk-old C57BL/6 (Control; n=10 and + IL-6; n=10), ApcMin/+ (Control; n=8 and + IL-6; n=10)

and ApcMin/+ / IL-6-/- mice (Control; n=8 and + IL-6; n=6) were used for IL-6 over-expression

experiments (See procedure below). The room was maintained on a 12:12 light:dark cycle with

the light period starting at 0700. Mice were provided standard rodent chow (Harlan Teklad

Rodent Diet, #8604, Madison, WI) and water ad libitum. Body weights and food intake were

measured weekly. All animal experimentation was approved by the University of South

Carolina’s Institutional Animal Care and Use Committee.

Voluntary wheel running activity. ApcMin/+ mice (n=12) were individually housed with 9.5-in

diameter stainless steel activity wheels at 4-wks of age (Mini Mitter, Bend, OR). Running

activity was monitored from 4 to 26 weeks of age. Bicycle computers (Specialized, Morgan Hill,

CA) using magnetic sensors measured running distance, and the data were recorded daily. Food

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intake and body weight were recorded weekly. Activity wheels were fixed 18 hours prior to

sacrifice.

Scoring cachexia. A strength of ApcMin/+ mice as a cachectic model is variability in the

development of cachectic symptoms between mice. To take advantage of this variability,

ApcMin/+ mice were categorized on their amount of wasting at the study’s end. A cachexia

severity score (Mild/None=1, Moderate=2, or Severe=3) based on body weight, gastrocnemius

muscle weight, and epididymal fat pad weight was assigned to each animal that had access to a

voluntary activity wheel. The weight ranges for each variable were based on the averages of

C57BL/6 mice of the same age. Each individual score (body weight + gastrocnemius weight +

epididymal fat pad weight) was averaged for a total cachexia score for each animal. A score of

“1” was within 1 standard deviation of the C57BL/6 mean and these mice had mild/no cachexia.

A score of “3” was more than 2 standard deviations away from the C57BL/6 mean, and these

mice had severe cachexia.

IL-6 over-expression. An IL-6 plasmid was used to increase circulating IL-6 levels in C57BL/6,

ApcMin/+, and ApcMin/+ / IL-6-/- mice. Electroporation of plasmid DNA into the quadriceps muscle

was used for endogenous IL-6 production and subsequent secretion into circulation. Fifty

micrograms of the IL-6 plasmid driven by the CMV promoter, or empty control vector was

injected intramuscularly. Mice were anesthetized with a 2% mixture of isoflurane and oxygen (1

L/min). The leg was shaved and a small incision was made over the quadriceps muscle. Fat was

dissected away from the muscle and the plasmids were injected in a 50 µl volume of PBS. A

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series of eight 50 ms, 100 V pulses was used to promote uptake of the plasmid into myofibers,

and then the incision was closed with a wound clip. Mice were electroporated every 2 weeks to

maintain circulating IL-6 levels, using alternate quadriceps muscles. C57BL/6 and ApcMin/+ / IL-

6-/- mice were sacrificed after 10 weeks of IL-6 over-expression. ApcMin/+ mice were sacrificed

after 4 weeks of IL-6 over-expression since they spontaneously undergo cachexia by 6 months of

age. It should be noted that the quadriceps muscle was not used for any analyses, but rather the

gastrocnemius muscle to examine the effect of circulating IL-6 on muscle mass.

Plasma IL-6. Plasma IL-6 levels were measured with a mouse specific ELISA (Biosource,

Carlsbad, CA) by taking blood samples under brief isoflurane anesthesia from the retroorbital

eye sinus 7 days following each electroporation and at sacrifice to ensure validity of the

procedure. The IL-6 level for each animal was averaged for all time points assessed over the

experimental period. In previous studies using this kit, we have detected 3-30 pg IL-6/ml

ApcMin/+ mouse plasma (28, 29).

Tissue collection. Mice were injected with a ketamine/xylazine/acepromazine cocktail (1.4

ml/kg BW), and gastrocnemius muscles, epididymal fat pads, tibias, and intestines were excised.

Tibia length was measured as an indicator of animal body size. The gastrocnemius muscle was

rinsed in PBS, snap frozen in liquid nitrogen, weighed, and stored at -80 °C until further

analysis. The small intestines were removed as described previously (29). Briefly, the intestines

were dissected, cleared of mesentery adipose tissue, and fixed in 10% buffered formalin for 24

hours. Plasma was collected via the inferior vena cava with heparinized needles, stored on ice,

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centrifuged at 1,000 g for 10 min at 4 °C, and the plasma was stored at -80 °C until further

analysis.

Polyp counts. Polyp counts were performed as described previously (29). Briefly, formalin-

fixed intestinal sections from all animals were rinsed in deionized water, stained in 0.1%

methylene blue, and counted by a single investigator who was blinded to the treatments. Polyps

were counted under a dissecting microscope, using tweezers to pick through the intestinal villi

and identify polyps. Polyps were categorized as >2 mm, 1-2 mm, and <1 mm in the small and

large intestine.

Total RNA Isolation and cDNA Synthesis. Total RNA was isolated from the gastrocnemius

muscles using TRIzol reagent (Invitrogen, Carlsbad, CA) as previously described (27).

Extracted RNA was treated with 10 units of DNase I to degrade any residual contaminating

genomic DNA. Complementary DNA (cDNA) was reverse transcribed (RT) from 3 µg of total

RNA using 1 µl of random hexamers and 50 units of Superscript III reverse transcriptase

(Invitrogen, Carlsbad, CA) in a final volume of 20 µl at 25°C for 10 min, followed by 42°C for

60 min, and 70°C for 15 min.

Real-time PCR. Real-time PCR was performed using reagents from Applied Biosystems (Foster

City, CA). Gene expression for IL-6 (FAM dye) was carried out in 25 µl reactions consisting of

2x Taqman Universal PCR master mix (AmpliTaq Gold DNA Polymerase, Buffer, dNTP mix,

AmpErase UNG, MgCl2), 1.0 µl cDNA, RNase-free water, and 1 µl of primer. Gene expression

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for cyclophilin (SYBR dye) was carried out in 25 µl reactions consisting of 2x SYBR green PCR

buffer (AmpliTaq Gold DNA Polymerase, Buffer, dNTP mix, AmpErase UNG, MgCl2), 0.1 µl

cDNA, RNase-free water, and 60 nM of each primer. The sequence for the primers were as

follows: Cyclophilin Forward: 5’ – TGT GCC AGG GTG GTG ACT T – 3’; Cyclophilin

Reverse, 5’ – TCA AAT TTC TCT CCG TAG ATG GAC TT – 3’. Samples were analyzed on

an ABI 7300 Sequence Detection System. Reactions were incubated for 2 min at 50°C and 10

min at 95°C, followed by 50 cycles consisting of a 15-s denaturing step at 95°C and 1-min

annealing/extending step at 60°C. Data were analyzed by ABI software using the cycle

threshold (CT), which is the cycle number at which the SYBR or FAM fluorescence emission is

midway between detection and saturation of the reaction. The 2-∆∆ CT method (25) was used to

determine changes in gene expression between treatment groups with the cyclophilin CT as the

correction factor.

Western Blotting. Western blotting was performed as previously described (28). Briefly, frozen

gastrocnemius muscle was homogenized in Mueller buffer and protein concentration determined

by the Bradford method (7). Crude muscle homogenate (30-60 µg) was fractionated on 8%-12%

SDS-polyacrylamide gels. Gels were transferred to PVDF membranes overnight. Membranes

were Ponceau stained to verify equal loading of each gel. Membranes were blocked overnight in

5% milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T). Primary antibodies for total and

phosphorylated STAT-3 (TYR 705) were purchased from Cell Signaling (Danvers, MA).

Antibodies were diluted 1:1000 to 1:2000 in 5% BSA in TBS-T followed by overnight

incubation with membranes at 4 ºC. Anti-rabbit IgG horseradish-peroxidase conjugated

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secondary antibodies (GE Healthcare Life Sciences, Piscataway, NJ) were incubated with the

membranes at 1:2000 to 1:5000 dilutions for 2 hours in 5% milk in TBS-T. Enhanced

chemiluminescence (ECL) (GE Healthcare Life Sciences, Piscataway, NJ) was used to visualize

the antibody-antigen interactions. Film was digitally scanned and blots were quantified by

densitometry using scientific imaging software (Scion Image, Frederick, MD). The Ponceau

stained membranes were also digitally scanned and the 45 kDa actin bands were quantified by

densitometry and used as a protein loading correction factor for each lane.

Statistical analysis. Repeated measures ANOVA was used to examine changes in body weight,

food intake, and wheel running activity over time in mice categorized by stage of cachexia. One-

way ANOVAs or independent t-tests were used to determine significance for all other variables.

Post-hoc analyses were performed with Student-Newman-Keuls methods. When the assumption

of normality failed, a Kruskal-Wallis one-way ANOVA on ranks was performed. Post-hoc

analyses were performed with Dunn’s method. Significance was set at p<0.05.

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RESULTS

Voluntary activity and body weight. Body weight was traced over the entire study in ApcMin/+

mice that had access to voluntary activity wheels. There were no differences in body weight

between mildly and severely cachectic ApcMin/+ mice at the beginning of the study (Figure 1A;

p=0.904). The severely cachectic mice started to decrease in body weight by 18-weeks of age

(p=0.015) and differed in body weight by the completion of the study at 26-weeks of age

(p<0.001). Body weights in severely cachectic ApcMin/+ mice were reduced 20% compared to

mildly cachectic mice at the study’s end (26.2 ± 0.3 g vs. 20.9 ± 0.6 g; p<0.001). Food intake

was also measured weekly; there were no differences between the mildly cachectic and severely

cachectic mice at any time point (Figure 1B; p=0.115). All of these mice had access to voluntary

activity wheels which was used as an indicator of each animal’s health. Running distance per

day was not different between the 2 groups at the beginning of the study (Figure 1C; p=0.975).

However, by 15-18 weeks of age, the severely cachectic mice started to decrease running wheel

distance (p<0.001) and were running significantly less than the mildly cachectic group through

the completion of the study.

Muscle and fat pad mass. Skeletal muscle weights were measured to determine the amount of

skeletal muscle wasting in ApcMin/+ mice stratified by their degree of cachexia. Severely

cachectic ApcMin/+ mice had a 61% decrease in gastrocnemius muscle weight and complete

elimination of the epididymal fat pad compared to mildly cachectic mice (Figure 2A). Other

hindlimb muscles were examined. In severely cachectic mice, soleus muscle mass was reduced

30% (p=0.003), plantaris muscle mass reduced 57% (p<0.001), and tibialis anterior muscle mass

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reduced 53% (p=0.002), when compared to mildly cachectic mice. However, tibia length, an

indicator of body frame size, did not differ by degree of cachexia (16.4 ± 0.0 vs. 16.5 ± 0.1 mm;

p=0.297).

Polyp number. To determine whether the intestinal tumor burden varied with cachexia severity,

polyp counts were also performed in these mice. Total polyp number was 89% higher in

severely cachectic mice, compared to mildly cachectic mice (Figure 2B; p<0.001). However,

when stratified by polyp size, polyps 1-2 mm in diameter and >2 mm in diameter were greater in

severely cachectic mice compared to mildly cachectic mice.

Plasma IL-6 and muscle STAT-3 activation. Plasma IL-6 levels were increased ~10-fold in mice

with severe cachexia (Figure 2C; p=0.020). Overall, there was a strong negative correlation

(Figure 2D) between tumor number and plasma IL-6 (r=-0.893, p<0.001). To determine if

downstream targets of IL-6 signaling were activated in cachectic muscle, western blotting was

performed to assess STAT-3 activation in mildly and severely cachectic ApcMin/+ mouse

gastrocnemius muscle (Figure 2E). The ratio of phosphorylated STAT-3 to total STAT-3

increased 4.5-fold in severely cachectic mice compared to mildly cachectic mice (p=0.004).

There was a significant inverse correlation between STAT-3 activation (Pi/total STAT-3) and

gastrocnemius muscle weight (r=-0.831; p=0.002). Gastrocnemius muscle IL-6 mRNA did not

change with severity of cachexia (38.40 ± 0.35 vs. 38.22 ± 0.25 CT; p=0.91), suggesting that

circulating IL-6 rather than muscle-produced IL-6 was contributing to STAT-3 activation.

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IL-6 ablation. The effect of IL-6 ablation on cachexia development and progression was

examined with ApcMin/+ / IL-6-/- mice. At 3 months of age, ApcMin/+ mice had already begun

epididymal fat pad loss (Figure 3A), but there were no differences in gastrocnemius muscle mass

(Figure 3B) between the three strains. At 26-weeks of age, ApcMin/+ / IL-6-/- mice did not

demonstrate wasting, and had gastrocnemius muscle and epididymal fat pad masses similar to

C57BL/6 mice. Maintenance of fat and muscle mass was not due to increased caloric intake

since daily food intake was actually reduced 11% in ApcMin/+ / IL-6-/- mice compared to ApcMin/+

mice compared to (0.147 ± 0.003 vs. 0.165 ± 0.002 g food/g BW; p<0.001).

To determine if skeletal muscle was sensitive to circulating IL-6 levels, western blotting

was performed to determine STAT-3 activation (Figure 3C). The ratio of phosphorylated to total

STAT-3 was lowered 83% in ApcMin/+ / IL-6-/- mice compared to ApcMin/+ mice.

IL-6 over-expression. Mice were electroporated in the quadriceps muscle with either an IL-6

expression plasmid or control plasmid. C57BL/6 mice and ApcMin/+ / IL-6-/- were electroporated

every 2 weeks for a total of 10 weeks, starting at 16-weeks of age. ApcMin/+ mice were only

electroporated for a total of 4 weeks since they undergo cachexia spontaneously by 6-months of

age (Figure 4A). Average plasma IL-6 levels were significantly elevated in mice receiving the

IL-6 plasmid compared to mice receiving the control plasmid in C57BL/6 mice, ApcMin/+ mice,

and ApcMin/+ / IL-6-/- mice (Table 1). The plasma IL-6 levels obtained with over-expression were

similar to humans with cachexia prior to death (21).

There were no differences in body weight at the beginning of the study between the

different treatment groups and within any strain (Table 1). ApcMin/+ mouse body weight was

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decreased 11% by IL-6 over-expression (p=0.010) and ApcMin/+ / IL-6-/- mouse body weight

decreased 21% (p=0.012) compared to mice receiving the control plasmid. ApcMin/+ mice

receiving IL-6 had a 70% reduction in epididymal fat pad mass (p=0.028) and ApcMin/+ / IL-6-/-

mice had a 63% decrease in epididymal fat pad mass (p=0.030) compared to control mice

(Figure 4B). IL-6 over-expression in the circulation also reduced gastrocnemius muscle mass

23% in ApcMin/+ mice (p=0.004) and 32% in ApcMin/+ / IL-6-/- mice (Figure 4C; p=0.007).

Plasmid-based IL-6 over-expression induced cachexia in mice with a polyp burden, but not in

wild-type mice. The body weights of C57BL/6 mice were not affected by IL-6 over-expression.

C57BL/6 epididymal fat pad mass and gastrocnemius muscle weight were not affected by

circulating IL-6 over-expression. Food intake was not affected by IL-6 over-expression within

any of the strains (data not shown).

During IL-6 over-expression, phosphorylated STAT-3 was induced in all strains of mice

(Figures 4D). It appears that skeletal muscle is sensitive to circulating IL-6 levels and STAT-3 is

activated even in the absence of muscle mass loss. Since IL-6 can act in a paracrine manner,

gastrocnemius IL-6 mRNA was measured to determine if IL-6 produced by the muscle was

responsible for STAT-3 activation. Gastrocnemius muscle IL-6 mRNA did not change with IL-6

over-expression in wild-type mice (p=0.943) or in ApcMin/+ mice (p=0.34). Thus, changes in

skeletal muscle STAT-3 activation appear to be due to circulating IL-6 rather than muscle-

produced IL-6.

Polyp burden and IL-6. We examined if ApcMin/+ mouse intestinal polyp burden was sensitive to

circulating IL-6 levels. Total polyp number was 32% lower in the 26-wk-old ApcMin/+ / IL-6-/-

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compared to ApcMin/+ mice (Figure 5A; p=0.014). Polyp size was also affected in the ApcMin/+ /

IL-6-/- mouse with polyps >1 mm in diameter being reduced by 32% compared to ApcMin/+ mice

(Figure 5B; p=0.023).

IL-6 over-expression increased both intestinal and colon polyp number and size. Total

polyp number was 64% greater with IL-6 electroporation in ApcMin/+ / IL-6-/- mice (Figure 5C).

Polyps >1 mm in diameter also increased 61% with IL-6 over-expression (Figure 5D; p=0.030).

Similar results were found with IL-6 over-expression in ApcMin/- mice. ApcMin/+ mouse total

polyp number increased 82% with IL-6 over-expression (55 ± 13 vs.100 ± 5 polyps; p=0.005).

Polyp size also increased with IL-6 over-expression in polyps >1 mm in diameter by 98% (47 ±

14 vs. 92 ± 5; p=0.005). Overall, these results indicate that changes in circulating IL-6 are

associated with a greater tumor burden and a greater loss of adipose and muscle tissue.

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DISCUSSION

A strength of the ApcMin/+ mouse as a cachectic model is the variable wasting seen

between mice. This variable wasting rate may be related to significant differences in tumor

burden between mice. To examine different stages of muscle wasting we classified ApcMin/+

mice by the degree of wasting they exhibited. ApcMin/+ mice with the most severe cachectic

symptoms also had the highest circulating IL-6 levels and the largest intestinal polyp burden.

The importance of IL-6 was also demonstrated by the lack wasting and a lower overall tumor

burden in ApcMin/+ / IL-6-/- mice when compared to age-matched ApcMin/+ mice. Systemic IL-6

over-expression in ApcMin/+ and ApcMin/+ / IL-6-/- mice induced wasting and polyp formation.

However, IL-6 over-expression was not sufficient to induce skeletal muscle wasting in non-

tumor bearing mice. These data suggest that systemic IL-6 is essential for the development of

cachexia in ApcMin/+ mice, and is associated with a greater tumor burden.

The current study reports that severity of cachexia in ApcMin/+ mice is associated with

both a greater number and larger intestinal polyps. While others have suggested that cachexia-

inducing tumors are not necessarily large tumors (37), our data suggests that a critical tumor

burden is necessary in order to induce cachexia in the ApcMin/+ mouse. The ApcMin/+ mouse

primarily develops adenomas, but there is not metastasis (30). It is possible that the increase in

adenoma number or growth creates an environment that promotes muscle wasting. Additionally,

we had previously shown that plasma IL-6 levels were elevated in cachectic ApcMin/+ mice

compared to age-matched, wild-type mice (28). The current study extended this finding by

showing that in 26-wk-old ApcMin/+ mice, increased plasma IL-6 levels were also associated with

increased polyp number and size. Since IL-6 is a potent tumor growth factor (8, 13, 34), IL-6

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may be leading to an increase in polyp burden, promoting the cachectic environment driven by

the tumor. While the association between high circulating IL-6 levels and muscle wasting has

been demonstrated previously (21, 38, 40, 45), the current study shows that even low, chronic

levels of IL-6 are associated with muscle mass loss. This is important because the levels of

circulating IL-6 seen in ApcMin/+ mice are similar to humans with cachexia (21).

To examine the importance of IL-6 for development of cachexia and intestinal/colon

polyps, we generated ApcMin/+ / IL-6-/- mice. Unlike 26-wk-old ApcMin/+ mice, age-matched

ApcMin/+ / IL-6-/- mice did not exhibit wasting of muscle and fat pad mass. This demonstrates the

dependence of IL-6 on the induction of cachexia. These data coincide with other models of

cachexia showing that blocking the effects of IL-6 have positive effects on skeletal muscle mass

(14, 35, 39, 44). In the current study, ApcMin/+ / IL-6-/- mice also had a lower intestinal tumor

burden than ApcMin/+ mice. While tumor growth is associated with cachexia, many studies that

delay or reverse cachexia through the inhibition of IL-6 or other inflammatory cytokines fail to

detect a change in tumor growth (5, 10, 35). This may be related to the tumor model used to

induce cachexia. An advantage of the ApcMin/+ mouse is the ability to count and classify by

polyp size throughout the intestine and colon. Therefore, it is possible that the ablation of IL-6,

which leads to preservation of muscle and fat mass in ApcMin/+ mice, is mediated through the

inhibition of tumor growth. One limitation of the ApcMin/+ / IL-6-/- mouse model is that IL-6 is

ablated in both skeletal muscle and tumors, making it impossible to determine the source of IL-6

that is important for the induction of cachexia. Nevertheless, this has important implications for

clinicians treating cachectic patients because methods for controlling the increase in plasma IL-6

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seen during cancer may be beneficial for both suppressing tumor growth and maintaining

skeletal muscle and adipose tissue mass.

To determine if IL-6 was sufficient to induce cachexia, circulating IL-6 was increased in

wild-type, ApcMin/+, and ApcMin/+ / IL-6-/- mice. While the circulating levels obtained through

over-expression were physiological, it should be noted that these were at least 10-fold higher

than those seen in cachectic ApcMin/+ mice. Over-expression of circulating IL-6 induced wasting

in both ApcMin/+and ApcMin/+ / IL-6-/- mice, but not in wild-type mice. Others have also shown

that IL-6 induces cachexia in mice with tumors (33, 35, 43), similar to our finding in ApcMin/+

mice. Additionally, ApcMin/+and ApcMin/+ / IL-6-/- mice receiving IL-6 also had a greater tumor

burden than mice receiving the vector control. An additional finding of the current study was

that elevated circulating IL-6 induced wasting and polyp growth in the absence of tissue level

expression. ApcMin/+ / IL-6-/- mice lacked IL-6 expression in intestinal polyps, adipose tissue, and

skeletal muscle, and underwent cachexia in the presence of high circulating IL-6. It has been

suggested that tumor-produced IL-6 is essential for cachexia (44), but the current study does not

support tumor production of IL-6 as necessary for the induction of cachexia. In addition, IL-6

did not induce wasting in wild-type, non-tumor bearing mice, suggesting other factors related to

the overall tumor environment may also be involved in the regulation of cachexia. Further work

is needed to discover the specific interaction between the tumor and IL-6 that mediates wasting.

IL-6 signaling at the cellular level involves the phosphorylation and dimerization of

STAT-3. The current study examined STAT-3 phosphorylation in wasting skeletal muscle.

Skeletal muscle STAT-3 activation has been detected in other forms of atrophy, such as

sarcopenia (18) and following crush injury (22), but STAT-3 activation has not been studied

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19R-00716-2007.R2

during cancer cachexia. Greater muscle STAT-3 activation was associated with more muscle

mass loss in ApcMin/+ mice. Over-expression of circulating IL-6 in ApcMin/+ mice did induce

STAT-3 activation. However, STAT-3 activation also occurred in wild-type mice that did not

have muscle wasting. Although skeletal muscle STAT-3 phosphorylation is sensitive to

circulating IL-6, this signaling pathway does not appear to be sufficient to induce skeletal muscle

mass loss. This adds further support that indirect mechanisms of IL-6, possibly related to other

factors related to the overall tumor burden of the animal, are important for inducing muscle mass

loss during cancer.

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PERSPECTIVES AND SIGNIFICANCE

This study supports a role of systemic IL-6 in the development of cachexia and intestinal

polyp growth in the ApcMin/+ mouse. ApcMin/+ mice exhibiting a greater severity of cachexia and

a larger intestinal tumor burden also had the highest circulating IL-6 levels. Circulating IL-6

over-expression also accelerated ApcMin/+ mouse cachexia and increased intestinal polyp number.

Wasting was not induced by circulating IL-6 over-expression in wild-type mice, which have no

intestinal tumors. Together, these data suggest an interaction between intestinal tumors and high

circulating IL-6 levels, which create an environment favoring catabolism of skeletal muscle and

adipose tissue. This was further demonstrated by the absence cachexia in tumor-bearing mice

that lack IL-6, and adding back systemic IL-6 induced cachexia in these mice. These findings

have important implications for treatment of cancer and the associated cachexia. The ability to

inhibit specific targets of IL-6 regulation may serve beneficial to both tumor progression and the

maintenance of total body mass. Future work will attempt to identify muscle specific and other

systemic related changes induced by IL-6 that promote adipose tissue and muscle catabolism.

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ACKNOWLEDGEMENTS

The authors would like to thank Tia Davis for technical assistance, and Dr. John Baynes

for critical review of the manuscript.

The research described in this report was supported by NIH Grant P20 RR-017698 from

the National Center for Research Resources. Its contents are solely the responsibility of the

authors and do not necessarily represent the official views of the NIH.

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REFERENCES

1. Al-Khalili L, Bouzakri K, Glund S, Lonnqvist F, Koistinen HA, and Krook A.

Signaling specificity of interleukin-6 action on glucose and lipid metabolism in skeletal muscle.

Molecular endocrinology (Baltimore, Md 20: 3364-3375, 2006.

2. al-Majid S, and McCarthy DO. Cancer-induced fatigue and skeletal muscle wasting:

the role of exercise. Biol Res Nurs 2: 186-197, 2001.

3. Ardies CM. Exercise, cachexia, and cancer therapy: a molecular rationale. Nutr Cancer

42: 143-157, 2002.

4. Argiles JM, Busquets S, and Lopez-Soriano FJ. Cytokines in the pathogenesis of

cancer cachexia. Curr Opin Clin Nutr Metab Care 6: 401-406, 2003.

5. Barton BE, and Murphy TF. Cancer cachexia is mediated in part by the induction of

IL-6-like cytokines from the spleen. Cytokine 16: 251-257, 2001.

6. Battle TE, and Frank DA. The role of STATs in apoptosis. Current molecular medicine

2: 381-392, 2002.

7. Bradford MM. A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254,

1976.

8. Brozek W, Bises G, Girsch T, Cross HS, Kaiser HE, and Peterlik M. Differentiation-

dependent expression and mitogenic action of interleukin-6 in human colon carcinoma cells:

relevance for tumour progression. Eur J Cancer 41: 2347-2354, 2005.

9. Croker BA, Krebs DL, Zhang JG, Wormald S, Willson TA, Stanley EG, Robb L,

Greenhalgh CJ, Forster I, Clausen BE, Nicola NA, Metcalf D, Hilton DJ, Roberts AW, and

of 35

23R-00716-2007.R2

Alexander WS. SOCS3 negatively regulates IL-6 signaling in vivo. Nat Immunol 4: 540-545,

2003.

10. Davis TW, Zweifel BS, O'Neal JM, Heuvelman DM, Abegg AL, Hendrich TO, and

Masferrer JL. Inhibition of cyclooxygenase-2 by celecoxib reverses tumor-induced wasting. J

Pharmacol Exp Ther 308: 929-934, 2004.

11. Duval D, Reinhardt B, Kedinger C, and Boeuf H. Role of suppressors of cytokine

signaling (Socs) in leukemia inhibitory factor (LIF) -dependent embryonic stem cell survival.

Faseb J 14: 1577-1584, 2000.

12. Espat NJ, Auffenberg T, Rosenberg JJ, Rogy M, Martin D, Fang CH, Hasselgren

PO, Copeland EM, and Moldawer LL. Ciliary neurotrophic factor is catabolic and shares with

IL-6 the capacity to induce an acute phase response. Am J Physiol 271: R185-190, 1996.

13. Fenton JI, Hursting SD, Perkins SN, and Hord NG. Interleukin-6 production induced

by leptin treatment promotes cell proliferation in an Apc (Min/+) colon epithelial cell line.

Carcinogenesis 27: 1507-1515, 2006.

14. Fujita J, Tsujinaka T, Yano M, Ebisui C, Saito H, Katsume A, Akamatsu K, Ohsugi

Y, Shiozaki H, and Monden M. Anti-interleukin-6 receptor antibody prevents muscle atrophy

in colon-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin-

dependent proteolytic pathways. Int J Cancer 68: 637-643, 1996.

15. Garcia-Martinez C, Lopez-Soriano FJ, and Argiles JM. Interleukin-6 does not

activate protein breakdown in rat skeletal muscle. Cancer Lett 76: 1-4, 1994.

16. Giordano A, Calvani M, Petillo O, Carteni M, Melone MR, and Peluso G. Skeletal

muscle metabolism in physiology and in cancer disease. J Cell Biochem 90: 170-186, 2003.

of 35

24R-00716-2007.R2

17. Glund S, Deshmukh A, Long YC, Moller T, Koistinen HA, Caidahl K, Zierath JR,

and Krook A. Interleukin-6 directly increases glucose metabolism in resting human skeletal

muscle. Diabetes 56: 1630-1637, 2007.

18. Haddad F, and Adams GR. Aging Sensitive Cellular and Molecular Mechanisms

Associated with Skeletal Muscle Hypertrophy. J Appl Physiol 100: 1188-1203, 2006.

19. Haddad F, Zaldivar FP, Cooper DM, and Adams GR. IL-6 Induced Skeletal Muscle

Atrophy. J Appl Physiol 98: 911-917, 2005.

20. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, and Schaper

F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374: 1-

20, 2003.

21. Iwase S, Murakami T, Saito Y, and Nakagawa K. Steep elevation of blood interleukin-

6 (IL-6) associated only with late stages of cachexia in cancer patients. Eur Cytokine Netw 15:

312-316, 2004.

22. Kami K, and Senba E. In vivo activation of STAT3 signaling in satellite cells and

myofibers in regenerating rat skeletal muscles. J Histochem Cytochem 50: 1579-1589, 2002.

23. Kamimura D, Ishihara K, and Hirano T. IL-6 signal transduction and its physiological

roles: the signal orchestration model. Rev Physiol Biochem Pharmacol 149: 1-38, 2003.

24. Khalfoun B, Thibault F, Watier H, Bardos P, and Lebranchu Y. Docosahexaenoic

and eicosapentaenoic acids inhibit in vitro human endothelial cell production of interleukin-6.

Adv Exp Med Biol 400B: 589-597, 1997.

of 35

25R-00716-2007.R2

25. Livak KJ, and Schmittgen TD. Analysis of relative gene expression data using real-

time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif 25: 402-

408, 2001.

26. McCarthy DO. Rethinking nutritional support for persons with cancer cachexia. Biol

Res Nurs 5: 3-17, 2003.

27. McClung JM, Mehl KA, Thompson RW, Lowe LL, and Carson JA. Nandrolone

decanoate modulates cell cycle regulation in functionally overloaded rat soleus muscle. Am J

Physiol Regul Integr Comp Physiol 288: R1543-1552, 2005.

28. Mehl KA, Davis JM, Berger FG, and Carson JA. Myofiber degeneration/regeneration

is induced in the cachectic ApcMin/+ mouse. J Appl Physiol 99: 2379-2387, 2005.

29. Mehl KA, Davis JM, Clements JM, Berger FG, Pena MM, and Carson JA.

Decreased intestinal polyp multiplicity is related to exercise mode and gender in ApcMin/+ mice.

J Appl Physiol 98: 2219-2225, 2005.

30. Moser AR, Pitot HC, and Dove WF. A dominant mutation that predisposes to multiple

intestinal neoplasia in the mouse. Science 247: 322-324, 1990.

31. Price SA, and Tisdale MJ. Mechanism of inhibition of a tumor lipid-mobilizing factor

by eicosapentaenoic acid. Cancer Res 58: 4827-4831, 1998.

32. Rotter Sopasakis V, Larsson BM, Johansson A, Holmang A, and Smith U. Short-

term infusion of interleukin-6 does not induce insulin resistance in vivo or impair insulin

signalling in rats. Diabetologia 47: 1879-1887, 2004.

of 35

26R-00716-2007.R2

33. Soda K, Kawakami M, Kashii A, and Miyata M. Manifestations of cancer cachexia

induced by colon 26 adenocarcinoma are not fully ascribable to interleukin-6. Int J Cancer 62:

332-336, 1995.

34. Steiner H, Godoy-Tundidor S, Rogatsch H, Berger AP, Fuchs D, Comuzzi B,

Bartsch G, Hobisch A, and Culig Z. Accelerated in vivo growth of prostate tumors that up-

regulate interleukin-6 is associated with reduced retinoblastoma protein expression and

activation of the mitogen-activated protein kinase pathway. Am J Pathol 162: 655-663, 2003.

35. Strassmann G, Fong M, Kenney JS, and Jacob CO. Evidence for the involvement of

interleukin 6 in experimental cancer cachexia. J Clin Invest 89: 1681-1684, 1992.

36. Tisdale MJ. Cachexia in cancer patients. Nat Rev Cancer 2: 862-871, 2002.

37. Tisdale MJ. The 'cancer cachectic factor'. Support Care Cancer 11: 73-78, 2003.

38. Tsujinaka T, Ebisui C, Fujita J, Kishibuchi M, Morimoto T, Ogawa A, Katsume A,

Ohsugi Y, Kominami E, and Monden M. Muscle undergoes atrophy in association with

increase of lysosomal cathepsin activity in interleukin-6 transgenic mouse. Biochem Biophys Res

Commun 207: 168-174, 1995.

39. Tsujinaka T, Fujita J, Ebisui C, Yano M, Kominami E, Suzuki K, Tanaka K,

Katsume A, Ohsugi Y, Shiozaki H, and Monden M. Interleukin 6 receptor antibody inhibits

muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice. J Clin Invest

97: 244-249, 1996.

40. Wallenius V, Wallenius K, Ahren B, Rudling M, Carlsten H, Dickson SL, Ohlsson

C, and Jansson JO. Interleukin-6-deficient mice develop mature-onset obesity. Nat Med 8: 75-

79, 2002.

of 35

27R-00716-2007.R2

41. Weigert C, Hennige AM, Brodbeck K, Haring HU, and Schleicher ED. Interleukin-6

acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by

phosphorylation of Ser473 of Akt. Am J Physiol Endocrinol Metab 289: E251-257, 2005.

42. Williams A, Wang JJ, Wang L, Sun X, Fischer JE, and Hasselgren PO. Sepsis in

mice stimulates muscle proteolysis in the absence of IL-6. Am J Physiol 275: R1983-1991, 1998.

43. Yasumoto K, Mukaida N, Harada A, Kuno K, Akiyama M, Nakashima E, Fujioka

N, Mai M, Kasahara T, Fujimoto-Ouchi K, and et al. Molecular analysis of the cytokine

network involved in cachexia in colon 26 adenocarcinoma-bearing mice. Cancer Res 55: 921-

927, 1995.

44. Zaki MH, Nemeth JA, and Trikha M. CNTO 328, a monoclonal antibody to IL-6,

inhibits human tumor-induced cachexia in nude mice. Int J Cancer 111: 592-595, 2004.

45. Zhou W, Jiang ZW, Tian J, Jiang J, Li N, and Li JS. Role of NF-kappaB and cytokine

in experimental cancer cachexia. World J Gastroenterol 9: 1567-1570, 2003.

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FIGURE LEGENDS

Figure 1. Body weight, food intake, and activity wheel distance in 26-wk-old male ApcMin/+ mice

stratified by degree of cachexia. A. Body weight. B. Food intake. C. Activity wheel distance.

Values are means ± SE. Data were analyzed by two-way repeated measures ANOVA.

Significance was set at p<0.05. *Signifies different from Mild/None.

Figure 2. Muscle size, polyp burden, and IL-6 in 26-wk-old male ApcMin/+ mice stratified by

degree of cachexia. A. Fat and muscle weights. B. Polyp counts. C . Plasma IL-6. D.

Pearson correlation between polyp number and plasma IL-6 (r=-0.831; p=0.002). E.

Representative Western blot and ponceau stain of gastrocnemius phosphorylated (TYR 705) and

total STAT-3. Fat=Epididymal fat. Gastroc=Gastrocnemius. Sol=Soleus. Plan=Plantaris.

TA=Tibialis Anterior. Values are means ± SE. Polyp counts were analyzed with independent t-

tests within each polyp size category. Significance was set at p<0.05. *Signifies different from

Mild/None.

Figure 3. IL-6 depletion rescues gastrocnemius muscle and epididymal fat loss in ApcMin/+ mice.

A. Epididymal fat pad weight. B. Gastrocnemius muscle weight. C. Representative Western

blot and ponceau stain of gastrocnemius muscle phosphorylated (TYR 705) and total STAT-3

during IL-6 depletion in 26-wk-old mice. Values are means ± SE. Weight data were analyzed

by two-way ANOVA with Student-Newman-Keuls post-hoc analyses. STAT-3 was analyzed by

a one-way ANOVA. Post-hoc analyses were performed with Student-Newman-Keuls methods.

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Significance was set at p<0.05. *Signifies different from C57BL/6. †Signifies different from

ApcMin/+ / IL-6-/-. ‡Signifies different from 13-wk-old.

Figure 4. IL-6 over-expression induces cachexia in ApcMin/+ and ApcMin/+ / IL-6-/- mice, but not

wild-type mice. A. Description of IL-6 over-expression experiments. C57BL/6, ApcMin/+, and

ApcMin/+ / IL-6-/- mice were administered an IL-6 over-expression plasmid or empty plasmid

control through electroporation. Experimentation started at 16-weeks of age. Mice were

electroporated bi-weekly for a total of 10-weeks (C57BL/6 and ApcMin/+ / IL-6-/-) or 4-weeks

(ApcMin/+). B. Epididymal fat pad weight. C. Gastrocnemius muscle weight. D. Representative

Western blot and ponceau stain of gastrocnemius muscle phosphorylated (TYR 705) and total

STAT-3 during IL-6 over-expression. Values are means ± SE. Data were analyzed with

independent t-tests within each strain. Significance was set at p<0.05. *Signifies different from

Control. EP=Electroporation.

Figure 5. ApcMin/+ mouse polyp burden varies with changes in circulating IL-6. A. Total polyp

burden in 26-wk-old ApcMin/+ and ApcMin/+ / IL-6-/- mice. B. Polyp burden stratified by size in

26-wk-old ApcMin/+ and ApcMin/+ / IL-6-/- mice. C. Total polyp burden in ApcMin/+ / IL-6-/- mice

following IL-6 over-expression. D. Polyp burden stratified by size in ApcMin/+ / IL-6-/- mice

following IL-6 over-expression. Values are means ± SE. Data were analyzed with independent t-

tests. Significance was set at p<0.05. *Signifies different from ApcMin/+ mice or Control.

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C57BL/6 ApcMin/+ ApcMin/+ / IL-6-/-

Control + IL-6 Control + IL-6 Control + IL-6

(n=10) (n=10) (n=8) (n=10) (n=8) (n=6)

Plasma IL-6 1.7 ± 0.8 152.5 ± 17.9* 9.7 ± 2.8 181.1 ± 29.6* 0 ± 0 317.4 ± 143.8*

(pg/ml)

Pre BW 27.7 ± 1.0 27.3 ± 0.4 24.0 ± 0.5 24.6 ± 0.4 26.0 ± 0.3 25.0 ± 0.8

(g)

Post BW 27.1 ± 1.0 28.9 ± 0.4 24.0 ± 0.5 21.8 ± 0.5*† 27.1 ± 0.7 21.4 ± 2.1*†

(g)

Table 1. IL-6 over-expression reduces body weight in mice with a polyp burden, but not in wild-type

mice. Plasma IL-6 was measured after each electroporation and averaged for each mouse. Values are

means ± SE. Changes in body weight were analyzed with a repeated measures one-way ANOVA.

Changes in plasma IL-6 were analyzed with independent t-tests. Significance was set at p<0.05.

*Signifies different from Control. †Signifies different from Pre.

of 35

A.

B.

C.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

6 8 10 12 14 16 18 20 22 24 26

Bo

dy w

eight

(g)

Age (Weeks)

Mild/None Severe

***** ****

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

6 8 10 12 14 16 18 20 22 24 26

Dai

ly F

oo

d I

nta

ke

(g F

oo

d/g

BW

)

Age (Weeks)

Mild/None Severe

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

4 to 7 8 to 10 11 to 14 15 to 18 19 to 22 23 to 26

Dis

tance

(km

/d)

Age (Weeks)

Mild/None Severe

*

**

Figure 1. of 35

A.

B.

C.

0

20

40

60

80

100

120

140

160

180

Fat Gastroc Sol Plan TA

Tis

sue

wei

ght

(mg)

Mild/None Severe

*

*

* **

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

Mild/None Severe

Pla

sma

IL-6

(p

g/m

l)

Degree of cachexia

*

D.

0.00

1.00

2.00

3.00

4.00

5.00

6.00

Mild/None Severe

Pi/

tota

l S

TA

T-3

(N

orm

aliz

ed I

OD

)

Degree of cachexia

*

0

20

40

60

80

100

120

140

160

180

Total <1 1-2 >2

Po

lyp

num

ber

Polyp diameter (mm)

Mild/None Severe*

*

*

P-STAT-3 (TYR 705)

STAT-3

PONCEAU STAIN

Figure 2.

E.

0

20

40

60

80

100

120

140

160

180

200

0 10 20 30 40 50

Po

lyp

num

ber

Plasma IL-6 (pg/ml)

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A.

B.

C.

0.0

5.0

10.0

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20.0

25.0

30.0

35.0

40.0

Pi/

tota

l S

TA

T-3

(N

orm

aliz

ed I

OD

)


*

*†

ApcMin/+ ApcMin/+ / IL-6-/-

P-STAT-3 (TYR 705)

STAT-3

C57BL/6

PONCEAU STAIN

Figure 3.

0

50

100

150

200

250

300

350

400

450

500

13 26

Ep

idid

ym

al f

at w

eight

(mg)

Age (weeks)

**†


*†

0

20

40

60

80

100

120

140

13 26

Gas

tro

c w

eight

(mg)

Age (weeks)

*†


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0

20

40

60

80

100

120

140

160

Gas

tro

c w

eight

(mg)

Control

+ IL-6

* *

ApcMin/+ / IL-6-/-ApcMin/+C57BL/6

0

50

100

150

200

250

300

350

400

450

500

Ep

idid

ym

al f

at w

eight

(mg)

Control

+ IL-6

ApcMin/+ / IL-6-/-

*

*

ApcMin/+C57BL/6

A.

D.

B.

C.

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Pi/

tota

l S

TA

T-3

(N

orm

aliz

ed I

OD

)

Control

+ IL-6*

*

*

ApcMin/+ / IL-6-/-ApcMin/+C57BL/6

1st

EP

Age (weeks)

16 2624222018

2nd

EP

3rd

EP

4th

EP

5th

EP

Tissue collection

from ApcMin/+

mice

Tissue collection

from ApcMin/+ /

IL-6-/- mice

Tissue collection

from C57BL/6

mice

P-STAT-3 (TYR 705)

STAT-3

PONCEAU STAIN

ApcMin/+

Figure 4. of 35

A. C.

0

20

40

60

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120

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Po

lyp

num

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ApcMin/+ ApcMin/+/ IL-6-/-

*

0

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120

<1 >1

Po

lyp

num

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Polyp diameter (mm)

*

ApcMin/+

ApcMin/+ / IL-6-/-

0

20

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Control + IL-6

Po

lyp

num

ber

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ApcMin/+ / IL-6-/- ApcMin/+ / IL-6-/-

0

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120

<1 >1

Po

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*

ApcMin/+ / IL-6-/- ApcMin/+ / IL-6-/-

Control + IL-6

D.B.

Figure 5. of 35

Interleukin-6 and cachexia in ApcMin/+ mice

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