Dual Energy X-ray Absorptiometry (DEXA) as a Longitudinal ... · 3/9/2020 · 2 24 Abstract (300...

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1 Dual Energy X-ray Absorptiometry (DEXA) as a 2 Longitudinal Outcome Measure of Cancer-3 Related Muscle Wasting in Mice4

5 Calvin L. Cole1,2,3,4, Ph.D., Deja Robinson1,2, Jian Ye3, Ph.D., Bradley Mills3, Ph.D., Scott A. Gerber3,6,7

6 Ph.D., Christopher A. Beck1,2,8 Ph.D., Edward M. Schwarz1,2,5, Ph.D., & David Linehan3,5 MD.

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8 1Department of Orthopaedics, 2Center for Musculoskeletal Research, 3Department of Surgery, 4Cancer

9 Control, 5Wilmot Cancer Institute, 6Department of Microbiology, 7Department of Radiation Oncology,

10 8Department of Biostatistics and Computational Biology, University of Rochester Medical Center,

11 Rochester, New York, 14642,

12 The authors declare no potential conflicts of interest.

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14 Corresponding Author:

15 Calvin L. Cole, Ph.D.

16 University of Rochester Medical Center

17 601 Elmwood Avenue, Box 665

18 Rochester, NY 14642

19 Email: [email protected]

20 Phone: 585-276-5976

21 Fax: 585-276-2177

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23

.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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https://doi.org/10.1101/2020.03.09.983403

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24 Abstract (300 out of 300 Words)

25 Introduction

26 Pancreatic ductal adenocarcinoma (PDAC) is notorious for its associated skeletal muscle wasting

27 (SMW) and mortality. Currently, the relationships between PDAC, SMW, and survival are poorly

28 understood. Thus, there is a great need for a faithful small animal model with a quantitative

29 longitudinal outcome measure that recapitulates clinical PDAC, to define SMW onset and assess

30 progression. Therefore, we aimed to validate dual energy X-ray absorptiometry (DEXA) as a

31 longitudinal outcome of lean body mass, and demonstrate its utility to quantify SMW in the KCKO

32 murine model of PDAC.

33 Methods

34 In vivo body composition of: 1) untreated mice at 5, 8, 12, 18, and 22 weeks of age (n=4), and 2) a

35 cohort of mice with (n=20) and without PDAC (n=10), was determined via DEXA, and lean mass of the

36 lower hind limbs was predicted via a region of interest analysis by two independent observers. Total

37 body weight was determined. Tibialis anterior (TA) muscles were weighed and processed for

38 histomorphometry immediately post-mortem. Statistical differences between groups were assessed

39 using t-tests and ANOVA. Linear regression models and correlation analysis were used to measure the

40 association between TA and DEXA mass, and reproducibility of DEXA was quantified via the intraclass

41 correlation coefficient (ICC).

42 Results

43 Lean mass in growing untreated mice determined by DEXA correlated with TA mass (r2 = 0.94; p

44 <0.0001) and body weight (r2 = 0.89; p <0.0001). DEXA measurements were highly reproducible

45 between observers (ICC = 0.95; 95% CI: 0.89-0.98). DEXA and TA mass also correlated in the PDAC



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46 cohort (r2 = 0.76; p <0.0001). Significant SMW in tumor-bearing mice was detected within 38 days of

47 implantation by DEXA, TA mass, and histomorphometry.

48 Conclusions

49 DEXA is a longitudinal outcome measure of lower limb lean mass in mice. The KCKO syngeneic

50 model is a bona fide model of PDAC-associated SMW that can be quantified with longitudinal DEXA.

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52 Introduction

53 Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas, and

54 is the fourth leading cause of cancer-related deaths, with its incidence expected to increase over the

55 coming decade (1, 2). Despite advances in the treatment of PDAC, the 5-year survival rate remains

56 below 10% (3). Additionally, treatment intolerance and/or discontinuation of treatment continue to

57 present challenges for PDAC patients and caregivers. Most notable among the detractors of quality of

58 life for PDAC patients is sarcopenia, also known as skeletal muscle wasting (SMW), which is a growing

59 burden among cancer survivors (4, 5). As such, SMW is prognostic of treatment failure, radiotherapy

60 toxicity, and a shorter time to tumor progression related to survival (6-9). SMW can be defined as any

61 loss of muscle tissue, function, and/or strength due to aging, chronic diseases, low protein-energy intake

62 and physical inactivity (10). Importantly, a large percentage of patients with PDAC experience cancer-

63 related SMW (10), and these patients have reduced physical function, increased postoperative morbidity,

64 reduced response to chemotherapy, and shorter life expectancy (11). Furthermore, SMW has been

65 identified as a prognostic factor in pancreatic cancer (12) and is an independent predictor of infectious

66 disease and postoperative mortality in resected patients (13),(14). Thus, reductions in the incidence of

67 SMW in patients with pancreatic cancer may reduce disease and treatment-related complications, which

68 adversely affect the dose and length of treatment.

69 Currently, the time of onset of SMW following a cancer diagnosis is poorly understood.

70 However, clinical studies suggest that once cancer-related SMW is initiated, it is irreversible (15).

71 Therefore, a priority in the treatment of PDAC-related muscle wasting must be determining when it is

72 initiated and preventing its establishment. Clinically, this is relevant because identifying the onset of

73 cancer-related SMW in an animal model may lead to the discovery of paracrine factors that are emitted

74 in the early stages of disease that perpetuate SMW. The anatomical distance between tumor cells and

75 sites of SMW posit that inflammatory cytokines may transmit systemic signals that potentiate muscle



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76 wasting through the alteration of myofibrillar intracellular pathways regulated by both hormones and

77 cytokines that slow protein synthesis and accelerate catabolism (16). Unfortunately, further elucidations

78 of PDAC-related SMW and identification of treatable targets have been challenging due to the absence

79 of small animal models with longitudinal outcomes.

80 Early recognition of SMW may also aid clinicians in devising an appropriate dosing algorithm to

81 reduce treatment toxicity, while improving treatment tolerance and related outcomes of the cancer

82 diagnosis. In the context of non-metastatic disease, the only possible cure for PDAC is surgical

83 resection (17). However, less than 20% of PDAC patients meet the criteria for resection due to the

84 locally advanced or metastatic nature of their diagnosis (9). Recent research suggests that neoadjuvant

85 treatment (NAT) may help to improve the resectability rates among patients with PDAC, but may have

86 an adverse effect on body composition that worsen post-surgical outcomes or reduce resection

87 opportunities (9). Among many other reasons, the adverse effect of NAT may be the result of an

88 incorrect dosing regimen that is based on body weight or body mass index (BMI). Indeed, studies have

89 shown the measurement of lean mass to be a superior indicator of treatment toxicity and dosing response

90 (18) in patients who experience cancer-related SMW, when compared to body weight and BMI.

91 Therefore, it may be beneficial to monitor a patient’s body composition before, during, and after

92 neoadjuvant treatment to determine the early need for additional intervention.

93 Dual-energy X-ray absorptiometry (DEXA) has emerged as a viable, non-invasive method of

94 serial in vivo body composition analysis in small animals, due to its feasibility, accuracy, and

95 reproducibility(19). Additionally, DEXA is widely used for body composition measurements in humans

96 for both clinical and research purposes. Thus, DEXA outcomes in an animal model of disease may

97 recapitulate clinical outcomes. Furthermore, use of a non-invasive form of lean mass (LM)

98 measurement is vital during PDAC-related SMW to better understand the onset of this phenomenon. In

99 a homeostatic myocellular environment, pathways that regulate protein synthesis and breakdown



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100 function to prevent unnecessary protein cycling. However, in an environment of SMW, a dysregulation

101 of anabolic and catabolic systems exists that results in a net loss of protein. A determination of the

102 timing of this dysfunction may play a key role in understanding the mechanism(s) that lead to PDAC-

103 related SMW. Therefore, we aimed to validate dual energy X-ray absorptiometry (DEXA) as a

104 longitudinal outcome of lean body mass, and demonstrate its utility to quantify SMW in the KCKO

105 murine model of PDAC (20).

106 Materials and Methods

107 Experimental Model

108 Aged Mice

109 The University of Rochester Medical Center University Committee on Animal Resources

110 (UCAR) has approved of all animal work conducted herein. Female C57BL/6 mice were purchased

111 from the Jackson Laboratory (stock number 000664) at 4, 7, 11, 17, and 21 weeks of age. Mice were

112 aged in a pathogen-free facility for one week, under an IACUC approved protocol. Anesthesia and

113 Euthanasia were performed in accordance with our approved protocol. Briefly, on the day of sacrifice,

114 mice were anesthetized using 10µl of ketamine per gram of bodyweight. After sedation, mice

115 underwent a full body DEXA scan. Mice were sacrificed immediately after the DEXA scan and their

116 lower hind limb muscles were harvested for analysis.

117 Murine orthotopic model of pancreatic cancer

118 We utilized the murine syngeneic-orthotopic model of PDAC as previously described (20). As there is

119 no known sexual dimorphism in this model, only female C57BL/6J mice were used. They were

120 obtained at 6-8 weeks of age from the Jackson Laboratory (stock number 000664) and maintained in a

121 pathogen-free facility under an IACUC approved protocol. As the development of this model has been

122 described elsewhere (20), we will only briefly describe the model. After a 1-week acclimation period,



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123 mice were randomized to one of two groups: PDAC (n=5) or no tumor control (NTC) (n=5). Mice in

124 the PDAC group were anesthetized and injected in the tail of the pancreas with 2×105 KCKO-luc cells

125 suspended in a 1:1 PBS to Matrigel (Corning) mixture. NTC mice received no surgery and were

126 sacrificed at a ratio of 1:1 with mice of the PDAC group. Mice were maintained in standard isolation

127 cages with a 12hr light: dark cycle, and given ad libitum access to water and standard chow.

128 Longitudinal DEXA scanning was performed on days 14, 35, 42, 49, and 56. The total length of this

129 experiment was 56 days. Tumor-bearing mice were sacrificed when they developed end-stage disease

130 defined by exhibiting three or more characteristics defined by the Institutional Animal Care and Use

131 Committee (IACUC) as “failure to thrive”. Characteristics of “failure to thrive” included, but were not

132 limited to self-isolation, hunched over appearance, lack of or reduced cage activity, lack of or no

133 resistance to scruffing, mangled hair appearance after scruffing, failure to eat or drink, and/or visual

134 signs of breathing difficulty. To determine if these characteristics were being exhibited, animals were

135 checked twice daily, 30 days after tumor inoculation. This is the time point in which untreated animals

136 reach an advanced stage of disease (20). Mice in the non-tumor-bearing group were sacrificed

137 concordantly to allow for intergroup comparisons. Following euthanasia, skeletal muscles (quadriceps,

138 Extensor Digitorum Longus (EDL), Soleus (SOL), and Tibialis Anterior (TA)) and PDAC tumors were

139 harvested for histology, and cardiac puncture was performed to obtain serum for Luminex assay.

140 Dual-energy X-Ray Absorptiometry (DEXA)

141 Body composition was assessed in all mice using a DEXA scanner (PIXImus2; Lunar, Madison, WI).

142 Each mouse was anesthetized for the duration of the procedure (5 min) with an i.p. injection of 100

143 mg/kg ketamine. Each mouse was placed on the scanner bed in the prone position, with the limbs and

144 tail stretched away from the body. The PIXImus employs a cone beam X-ray source generating energies

145 at 35 and 80 keV with a current of 0.5 mA for both energy levels. The detector is flat (100 × 80 mm) and

146 comprised of individual pixels of 0.18 × 0.18 mm. Based on the attenuation of two energy levels, the



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147 system provides quantitative data on the fat tissue content, the lean tissue content, and the total tissue

148 mass within the region of interest (ROI). One scan per mouse was performed and analyzed with

149 PIXImus software (2.10; GE/Lunar). The head was excluded from calculation using a manual ROI. The

150 PIXImus was calibrated with an aluminum/lucite phantom (corresponding to bone mineral density =

151 0.0592 g/cm2 and 12.5% fat) on each day of testing according to the manufacturer's instructions. Lean

152 mass was calculated using the lower hindlimbs as an ROI to exclude the measurement of the tumor

153 burden. Lean mass was calculated as an index of total mass minus fat mass using the following

154 equation: total mass – ((% fat x total mass)/100). For each mouse, the lean mass was calculated for the

155 lower right and left limb independently, and the average of both measurements was used as the final lean

156 mass for the animal.

157 Antibodies

158 The following antibodies were used: laminin (rat, 1:1500, Sigma-Aldrich, L0663) and DAPI (1:3000).

159 Muscle Histology

160 TA, SOL, and EDL muscles were harvested for histology. TA muscles were cut from the most distal and

161 proximal TA tendon attachment, cleaned of extraneous tissue, blotted, and weighed. After which, TA,

162 muscles were stored in a 30% mixture of sucrose in PBS for 24 hours, at which time, muscles were

163 embedded in OCT (Tissue Tek), flash-frozen using dry ice and 2-methylbutane (Sigma-Aldrich), and

164 processed for fresh-frozen histology as previously described (21). 10µm sections were cut and stained

165 with H&E and representative micrographs were obtained for descriptive analyses.

166 Immunohistochemistry for laminin (extracellular matrix) and nuclei determination were also performed

167 as previously described (21, 22), in which TA muscles were cryosectioned at 10 μm, to obtain transverse

168 sections. Muscle sections were fixed in 4% paraformaldehyde (PFA) for 10 min. Sections were

169 permeabilized with PBS-T (0.2% Triton X-100 in PBS) for 10 min, blocked in 10% normal goat serum



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170 (NGS, Jackson ImmunoResearch) for 30 min at room temperature. If a mouse primary antibody was

171 used, sections were blocked in 3% AffiniPure Fab fragment goat anti-mouse IgG (H+L) (Jackson

172 ImmunoResearch) with 2% NGS in PBS at room temperature for 1 h. Primary antibody incubation was

173 performed in 2% NGS/PBS at 4°C overnight. Secondary antibody incubation was carried out in 2%

174 NGS/PBS at room temperature for 1 h. After washing in PBS, sections were counter-stained with DAPI

175 to label myonuclei. All slides were mounted with Fluoromount-G (SouthernBiotech). Fluorescent

176 microscopy was performed using a Zeiss Imager: M1m microscope with AxioVision SE64 software, and

177 representative images were used to quantify muscle cell area.

178 Fixed Single Fiber Staining

179 Single myofiber size and myonuclear analysis was performed, as previously described (21). For single

180 myofiber size and myonuclear analysis, whole limbs (n=10) were fixed in 4% PFA for 48 h prior to

181 EDL and SOL muscle dissection. Fixed muscles were incubated in 40% NaOH for 2 h to induce

182 dissociation, and single myofibers were gently titrated and washed in PBS prior to staining with DAPI.

183 For quantification, the cross-sectional area (CSA) of 100 fibers per mouse was determined manually

184 from digitally-photographed DAPI stained fibers at 100X, and averaged as the CSA for the mouse.

185 Statistical Analysis

186 Myofiber CSA was determined using ImageJ software. The diameter of the fiber was measured at three

187 points along the fiber to get an average CSA. TA cell area was calculated by drawing an ROI around the

188 extracellular space of 200 individual cells and taking the average of the sum of those cells. Results are

189 presented as mean±sd. Statistical significance was determined using t-tests for single comparisons and

190 one-way and two-way ANOVA for multiple comparisons. Pearson correlation coefficient was

191 calculated to measure the association between TA weight vs. DEXA lean mass and body weight vs.

192 DEXA lean mass. Linear regression models were used to evaluate DEXA as a predictor of TA weight



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193 and body weight with and without adjustment for the effects of time (treated as a categorical covariate to

194 avoid assuming linearity over time), with predictability measured by the model’s square root of mean

195 squared error (rMSE). Reproducibility of DEXA was assessed via the intraclass correlation coefficient

196 (ICC) along with its 95% CI. Analyses were performed using GraphPad Prism software (GraphPad

197 Software, San Diego, CA, USA) version 7.2 and 8.0, R version 3.5.1, and SAS version 9.4. P<0.05 was

198 considered significant (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

199 Results

200 In vivo DEXA measures are reliable and reproducible in predicting the body mass of growing

201 mice

202 To assess the utility of longitudinal DEXA to assess in vivo murine body mass, we completed DEXA

203 scans on 5, 8, 12, 18, and 22 week old mice (Figure 1). DEXA revealed a significant increase in lower

204 limb lean mass of growing mice from week 5 to week 18 (Figure 2A). Lower limb mass was measured

205 by two independent observers and the reproducibility of DEXA proved to be excellent. The intra-class

206 correlation coefficient (ICC) analysis completed on these measures shows strong agreement between the

207 two separate observations (Figure 2B). TA and body weight analysis confirmed the DEXA results, as

208 both averages increased with age in this cohort of growing mice (Figure 2C,E). In addition, regression

209 modeling revealed substantial variation due to time, such that adding time as a covariate improved the

210 predictability of lean mass by DEXA. Furthermore, this analysis determined that DEXA has an rMSE of

211 1.89 mg when predicting TA weights. Likewise, a Pearson correlation analysis demonstrated a strong

212 relationship between DEXA and TA and body weight (Figure 2D,F).

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Figure 1: In vivo visualization and quantification of lean mass in a lower limb region of interest (ROI). A representative dual energy x-ray absorptiometry (DEXA) scan image of a mouse is shown to illustrate segmentation of the head (red oval), body (green outline), and the ROI for lean mass analysis (green box) of the (A) right and (B) left lower hind limbs as described in Material and Methods. Figure 2. Validation of DEXA as an in vivo measure of lean mass in lower limbs of growing mice. Dual energy X-ray absorptiometry (DEXA) was performed on female mice at 5, 8, 12, 18 and 22 weeks of age (n=4 per group), postmortem, mice Tibialis Anterior (TA) muscles were dissected and weighed immediately. (A) The cross-sectional lower limb mass for each mouse was determined by DEXA at each time point by two independent observers as described in Figure 1, and the average mass of the two readings is presented with the mean for each group. (B) The intraclass correlation coefficient (ICC) was determined to assess the between reader variability of the DEXA measurements. (C) average TA weights of each group is presented as the mean ±SD. (D) A Pearson correlation analysis was performed to determine the relationship between DEXA lean mass vs. TA weight. (E) Average body weight of each group is presented as the mean ±SD. (F) A Pearson correlation analysis was performed to determine the relationship between DEXA lean mass vs. body weight.



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216 DEXA predicts skeletal muscle wasting in a murine model of PDAC

217 To assess the onset and progression of SMW in PDAC-bearing mice, we performed longitudinal, in vivo

218 DEXA analyses normalized to total body weight (TBW) (weight including the tumor), and compared the

219 findings to those in no tumor controls (NTC). DEXA revealed a decrease in the lower limb mass of

220 tumor-bearing mice beginning at day 38, resulting in a significant decrease vs. NTC mice at day 56

221 (Figure 3B), a decrease that could not be detected by the measurement of TBW (Figure 3A).

222 Postmortem examination of each animal determined that TBW is an unreliable measure of the onset of

223 SMW because the growing tumor mass compensates for the loss of viable tissue. Thus, the TBW of the

224 tumor-bearing mice was significantly greater than their net weight (NW) (weight – tumor weight)

225 (Figure 3C), and this increase was commensurate with primary tumor weight, such that there were no

226 differences in TBW between the PDAC and NTC groups. Smaller TA muscles, as determined by

227 weight, in the tumor-bearing mice when compared to the NTC group is further evidence of PDAC-

228 associated SMW (Figure 3D). A Pearson correlation analysis, performed on a larger cohort of PDAC

229 mice (n=15), confirmed a strong relationship between DEXA lean mass and TA weight (Figure 3E) in

230 this cohort of mice as well.

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236 Immunohistochemistry confirms PDAC-related SMW

Figure 3. Longitudinal quantification of lean mass via DEXA demonstrates commencement of PDAC-related muscle wasting within 38 days of tumor implantation, which cannot be detected by assessment of total body weight. Mice were randomized to two groups prior to orthotopic tumor injections: 1) NTC; (n=5), and 2) PDAC tumor bearing; (n=5). Longitudinal DEXA and total body weight (TBW) measurements were performed as described in Materials and Methods. (A) Longitudinal TBW is presented for the NTC and PDAC mice that were sacrificed on day 56. (B) Longitudinal lower limb mass determined by DEXA is presented (**p<0.01). (C) The total body weight (TBW) (g) and (D) TA weights (mg) of the PDAC bearing mice was determined on the day of sacrifice, and the data are presented as TBW (+ tumor) before primary tumor harvest and net weight (- tumor) after primary tumor harvest for each mouse with the mean +/- SD (***p=0.0005) (****p<0.0001. (E) A Pearson correlation analysis was performed on a separate and larger cohort of PDAC tumor bearing mice (n=15) to determine the relationship between DEXA lean mass and TA weight.



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237 To confirm SMW is this model of PDAC, we performed histomorphometry on fast twitch (EDL) and

238 slow twich (SOL) skeletal muscle fibers from the PDAC-bearing and NTC mice (Figure 4A,B). We

239 found a significant decrease in the CSA of both muscle fiber types in the tumor-bearing mice, and that

240 the 38% and 33% decrease is similar to reported decreases in EDL and SOL muscles respectively, in

241 mice with age and disease-related sarcopenia (23). Histomorphemetry of TA sections (Figure 4E-H)

242 show an atrophied appearance, while quantification of the CSA of these muscle fibers (Figure 4I)

243 substantiate SMW. Collectively, these results formally establish SMW in this PDAC model, which can

244 be longitudinally assessed via DEXA scanning.

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249 Discussion

250 To better understand the mechanisms of PDAC-related SMW and identification of treatable targets,

251 there remains a need for the development of small animal models with longitudinal outcomes. Thus, we

252 utilized in vivo DEXA scans to predict lean muscle mass in a selected region of interest (ROI) of

253 growing and PDAC mice. The lower portion of the hind limbs were selected as the ROI to omit the

254 mass of the tumor from analysis. Using this method of analysis, we found strong, significant

255 correlations between DEXA predicted lean mass and whole body and TA weight, in both the growing

256 and PDAC mice. These findings suggest that DEXA can be used as a longitudinal outcome measure of

257 PDAC-related SMW in a murine model.

Figure 4: Histologic confirmation of skeletal muscle fiber atrophy in terminal mice with PDAC. Following euthanasia, the EDL and SOL muscles were harvested from PDAC baring and NTC mice and stained with DAPI for calculation of CSA (100 fibers per mouse). TA muscles were dissected and weighed. After which, TA muscles were frozen and stained with Laminin and H&E, and CSA was measured. Photomicrographs (10x) show representative DAPI stained (A) EDL and (B) SOL muscle fibers at the time of sacrifice from NT and PDAC baring mice (n=5), with quantification of (C) EDL and (D) SOL CSA (mean ± SD ****p<0.0001). Images (10x) of laminin and H&E stained TA cross-sections of (E,F) NTC and PDAC (G,H) are shown, respectively, along with quantification of TA (I) CSA (µm2) (n=5) and are presented with the mean ± SD (**p<0.01).



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258 Because of its high reproducibility and accuracy for measurement of lean body mass, DEXA has

259 emerged as one of the most promising longitudinal assessment tools for the direct measurement of lean

260 mass and diagnosis of sarcopenia (18). Currently, DEXA is widely used in clinical studies to diagnose

261 sarcopenia, and is recognized as the gold standard (18).

262 Using a DEXA instrument for small animals to longitudinally quantify skeletal muscle mass in a cohort

263 of PDAC mice (n=5), we found a significant reduction in skeletal muscle mass compared to their NTC

264 littermates. This decrease in skeletal muscle mass was concomitant with “failure to thrive,” and

265 subsequent mandatory sacrifice of the animals. TBW was not different between groups at any of the

266 time-points. Therefore, identification of the onset of SMW may be vital for the survival of animal

267 models and patients with cancer, if it can be halted at that time. Clinical researchers agree that once

268 muscle loss reaches a point of clinically obvious detriment, it is irreversible (15).

269 Indeed, the SMW and cachexia that are experienced by patients with cancer have been defined on a

270 continuum that begins with pre-cachexia and ends with refractory cachexia, which unfortunately cannot

271 be relieved (15, 24). Therefore, it is important to understand when these disorders begin during disease

272 progression and the mechanisms involved when considering viable treatment options. To our

273 knowledge, this is the first study to assess the validity of the syngeneic KCKO-luc cell orthotopic model

274 of PDAC as a model of PDAC-associated SMW, and the utility of longitudinal DEXA analysis to assess

275 SMW in tumor bearing mice. The longitudinal quantification of lean mass proved to be predictive of

276 “failure to thrive,” which was an indication for euthanasia. Thus, the diagnosis and attenuation of

277 PDAC-associated SMW in this model can ultimately translate to improving the quality of life and

278 survival in patients with cancer.

279 In addition, patients who experience SMW have a lower tolerance for treatment and higher drug toxicity

280 (18, 25, 26), which often results in treatment discontinuation. Treatment-associated toxicity in these

281 patients may be the result of improper drug dosing regimens that are based on antiquated methodology



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282 (18). Recent clinical evidence suggests the use of body composition measurement to improve drug

283 dosing, reduce toxicity, and establish early intervention in patients with cancer (7, 9). Therefore, we

284 conclude that the use of DEXA as a tool to characterize SMW to improve dosing is an area that warrants

285 further study. Recent research reports that SMW is independently prognostic of lower survival in

286 patients with gastrointestinal cancers (6). Martin et al.(27) showed similar results in a large cohort of

287 patients with cancer. This study concluded that survival in sarcopenic patients was 1/3 of their non-

288 sarcopenic counterparts, regardless of body weight. In addition, increases in lean mass during

289 neoadjuvant treatment (NAT) has been shown to be independently associated with progression to

290 resection surgery in patients with PDAC (9), even when these patients experienced loss of adipose

291 tissue. Notably, patients who experienced decreases in lean mass during or after NAT underwent

292 surgical exploration, but not resection.

293 Not surprisingly, we found a significant difference in TBW of animals (with tumor), compared to the

294 animals’ normalized weight without tumor. This finding suggests that TBW is a poor indicator of

295 survival and general health due to the continuous growth of the tumor, which mask the loss of viable

296 lean and fat mass. These findings are significant because they specify that the longitudinal

297 quantification of lean mass is a more appropriate prognostic indicator than the measure of TBW in this

298 model of murine PDAC.

299 Conclusion

300 We have demonstrated the efficacy of DEXA as a longitudinal outcome measure of PDAC-related

301 SMW. PDAC is a disease with an extremely high mortality rate. Survival is further decreased by the

302 onset of SMW. Early detection and treatment of SMW in affected patients may improve quality of life

303 and survival. Therefore, more research is needed to understand the mechanism(s) that lead to a

304 dysregulation in myocellular homeostasis. Research of this nature requires a pre-clinical model that

305 provides longitudinal outcome measures of body composition and disease progression. In this study, we



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306 used DEXA as a longitudinal outcome measure to assess SMW in growing mice and in an established

307 murine PDAC model. Utilizing this technique, we were able to detect the onset of SMW which was

308 commensurate with “failure to thrive” and was confirmed through analysis of TA and body weight and

309 histomorphometry.

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