Metabolic signatures imaged in cancer-induced cachexia

Abstract

Cancer-induced cachexia is a complex and poorly understood life-threatening syndrome that is characterized by progressive weight loss due to metabolic alterations, depletion of lipid stores, and severe loss of skeletal muscle protein. Gaining the ability to noninvasively image the presence or onset of cachexia is important to better treat this condition, to improve the design and optimization of therapeutic strategies, and to detect the responses to such treatments. In this study, we employed noninvasive magnetic resonance spectroscopic imaging (MRSI) and [(18)F]fluoro-2-deoxy-D-glucose ((18)FDG) positron emission tomography (PET) to identify metabolic signatures typical of cachectic tumors, using this information to analyze the types and extents of metabolic changes induced by the onset of cachexia in normal tissues. Cachexia was confirmed by weight loss as well as analyses of muscle tissue and serum. In vivo, cachexia-inducing murine adenocarcinoma (MAC)16 tumors were characterized by higher total choline (tCho) and higher (18)FDG uptake than histologically similar noncachectic MAC13 tumors. A profound depletion of the lipid signal was observed in normal tissue of MAC16 tumor-bearing mice but not within the tumor tissue itself. High-resolution (1)H magnetic resonance spectroscopy (MRS) confirmed the high tCho level observed in cachectic tumors that occurred because of an increase of free choline and phosphocholine. Higher succinate and lower creatine levels were also detected in cachectic tumors. Taken together, these findings enhance our understanding of the effect of cancer on host organs and tissues as well as promote the development of noninvasive biomarkers for the presence of cachexia and identification of new therapeutic targets.

Figure 1

(a) Normalized weights of MAC13 ( ) and MAC16 (■) tumor-bearing mice over time. (n = 10 per group, *P < 0.05). (b) mRNA expression levels of MuRF1 and Atrogin-1 analyzed by RT-PCR in MAC13 ( ) and MAC16 (■) muscle tissue. Values represent Mean ± SEM (n = 4, ** P < 0.01, *** P < 0.005). (c) Representative immunoblot showing MuRF1 and Atrogin-1 proteins in muscle tissue obtained from mice bearing from MAC13 or MAC16 tumors. GAPDH was used as loading control (n = 3). (d) Lipid analysis of serum from mice bearing MAC13 or MAC16 tumors. Values represent Mean ± SEM (MAC13, n = 8, MAC16, n = 7; *P < 0.05, **P < 0.01).

Figure 2

(a) Representative PET images of ¹⁸FDG uptake in MAC13 and MAC16 tumors (SUV: standardized uptake value). Quantification of the uptake in (b) tumors, (c) brain, (d) lungs and (e) muscle. Values represent Mean ± SEM (MAC13, n = 8, MAC16, n = 5; * P < 0.005).

Figure 3

Representative (a) T₁-weighted images, (b) tCho maps and (c) lactate+lipids maps of a MAC13 (upper panel) and a MAC16 (lower panel) tumor. T₁-weighted images were acquired from the corresponding 4 mm slice used for MRSI using a spin-echo sequence with an echo time of 10 ms, a repetition time of 500 ms, and an in-plane spatial resolution of 62.5 μm. Total choline maps and lactate+lipids maps were generated from the MRSI data and normalized to the water signal to display concentrations in mM units. (d) In vivo tCho concentrations in MAC13 and MAC16 tumors. (e) In vivo lactate+lipids concentration within MAC13 and MAC16 tumors. (f) In vivo concentration of subcutaneous lipids surrounding MAC13 and MAC16 tumors. Values represent Mean ± SEM (n = 10 per group, * P < 0.05).

Figure 4

(a) Cross-sectional T₁-weighted images, (b) cross-sectional lactate+lipids maps, and (c) merged images from (a) and (b) of MAC13 (upper panel) and MAC16 (lower panel) tumor-bearing mice. T₁-weighted images were acquired from the corresponding 4 mm slice used for MRSI using a spin-echo sequence with an echo time of 10 ms, a repetition time of 500 ms, and an in-plane spatial resolution of 125 μm. Lipid maps were generated from MRSI data and normalized to the water signal. Volumes were comparable for the MAC13 (540 mm³) and MAC16 (545 mm³) tumors. (d) In vivo lactate+lipids concentration in MAC13 ( ) and MAC16 (■) tumor-bearing mice (n = 6 per group). Values were obtained from a 4 mm axial slice using a volume coil. Values represent Mean ± SEM. * P < 0.05.

Figure 5

Representative ¹H MR high-resolution spectra of water-soluble (a) MAC13 and (b) MAC16 tumor extracts. Cho (free choline), PC (phosphocholine), GPC (glycerophosphocholine). (c) Concentration of metabolites (Cho, PC, GPC, total choline = Cho+PC+GPC, succinate and creatine) in MAC13 ( ) and MAC16 (■) tumors (n = 6 per group, * P < 0.05, ** P < 0.001). Values represent Mean ± SEM.

Figure 6

mRNA levels of Chk analyzed by RT-PCR in MAC13 ( ) and MAC16 (■) (a) cell and (b) tumor extracts. Values represent Mean ± SEM (n = 5 per group for the cell extract, n = 8 per group for the tumor extracts, * P < 0.05, *** P < 0.005).

Figure 7

Representative ¹H MR high-resolution spectra of lipid-soluble muscle extracts obtained from (a) MAC13 and (b) MAC16 tumor bearing mice. The chemical shift reference peak is tetramethylsilane that is also used as an internal concentration standard. (c) Quantification of lipids in arbitrary units from lipid-soluble extracts of muscle tissue from MAC13 ( ) and MAC16 (■) tumor bearing mice (n = 3 per group). Values represent Mean ± SEM. * P < 0.05.