Molecular, cellular and physiological characterization of the cancer cachexia-inducing C26 colon carcinoma in mouse

Abstract

Background: The majority of cancer patients experience dramatic weight loss, due to cachexia and consisting of skeletal muscle and fat tissue wasting. Cachexia is a negative prognostic factor, interferes with therapy and worsens the patients' quality of life by affecting muscle function. Mice bearing ectopically-implanted C26 colon carcinoma are widely used as an experimental model of cancer cachexia. As part of the search for novel clinical and basic research applications for this experimental model, we characterized novel cellular and molecular features of C26-bearing mice.

Methods: A fragment of C26 tumor was subcutaneously grafted in isogenic BALB/c mice. The mass growth and proliferation rate of the tumor were analyzed. Histological and cytofluorometric analyses were used to assess cell death, ploidy and differentiation of the tumor cells. The main features of skeletal muscle atrophy, which were highlighted by immunohistochemical and electron microscopy analyses, correlated with biochemical alterations. Muscle force and resistance to fatigue were measured and analyzed as major functional deficits of the cachectic musculature.

Results: We found that the C26 tumor, ectopically implanted in mice, is an undifferentiated carcinoma, which should be referred to as such and not as adenocarcinoma, a common misconception. The C26 tumor displays aneuploidy and histological features typical of transformed cells, incorporates BrdU and induces severe weight loss in the host, which is largely caused by muscle wasting. The latter appears to be due to proteasome-mediated protein degradation, which disrupts the sarcomeric structure and muscle fiber-extracellular matrix interactions. A pivotal functional deficit of cachectic muscle consists in increased fatigability, while the reported loss of tetanic force is not statistically significant following normalization for decreased muscle fiber size.

Conclusions: We conclude, on the basis of the definition of cachexia, that ectopically-implanted C26 carcinoma represents a well standardized experimental model for research on cancer cachexia. We wish to point out that scientists using the C26 model to study cancer and those using the same model to study cachexia may be unaware of each other's works because they use different keywords; we present strategies to eliminate this gap and discuss the benefits of such an exchange of knowledge.

Figure 1

Morphological and histological features of C26 tumor. a) Exposed C26 tumor, three weeks following subcutaneous transplant of a 0.5 mm³ tumor fragment, has grown, becoming a spheroid measuring 0.5-1 cm in diameter. The tumor mass is well-defined and vascularized. Bar 5 mm. b) Photomicrographs of H&E-stained tumor cryosections. The tumor was collected three weeks following tumor transplant for histological analysis. Top, peripheral region showing tumor encapsulation and, bottom, inner region showing anaplastic appearance and lack of a necrotic core. Inset, cell morphology and vascularization at higher magnification. Bar = 100 μm c) Mitoses (arrow) were evaluated on C26 tumor sections, based on cell morphology identified as typical of prophase to telophase, and expressed as percentage of the cells counted in 10 randomly chosen fields for each tumor. An average mitotic index of 5 ± 2% (mean ± SEM) was calculated from data derived by four independent experiments (n = 8). d) TUNEL assay on C26 tumor sections of the same samples used for the mitotic index (the arrow points to a TUNEL+ cell) yielded an apoptotic index of 9 ± 3% (mean ± SEM).

Figure 2

C26 tumor proliferation. a) Tumor mass kinetics, measured upon tumor explant at the indicated times. After a lag time of about two weeks, tumor growth is linear and results in a mass of considerable size (>2 g, i.e. about 10% of the body weight). The mean ± SEM of at least three independent experiments performed in triplicate is shown (for each data point, the number of replicates, "n", is: 9 < n < 22). b) The DNA profile by flow cytometry of PI-labeled cells, at 3 weeks following transplantation, shows: a significant sub-population of cells in the S phase (M3), which indicates that tumor cells are actively proliferating; a haploid, sub-G1 peak (M1) that does not resemble apoptotic cells/debris; lack of polyploidism; M2 and M4 indicate the G1 and G2 sub-populations, respectively. c) BrdU incorporation (red) was detected in the tumor cells at 3 weeks following transplantation, the day after IP injections of two doses (50 mg/Kg of body weight) of BrdU 4 h apart (+ BrdU) though not in the sham-injected mice (-). Nuclei are counterstained with Hoechst (blue). Bar = 100 μm. Inset, confocal phase contrast image merged with the red fluorescence reveals the presence of BrdU+ and negative nuclei (black and white arrow, respectively). Bar = 10 μm d) Flow cytometric analysis of BrdU incorporation in cells enzymatically extracted from C26 tumors at 3 weeks following transplantation. BrdU+ cells were plotted against PI labeling (bottom) and compared to C26 cells from sham-injected mice. Cells above background are in R2 and R3.

Figure 3

C26-induced cachexia. a) Gross appearance of control (left) compared with tumor-bearing mice (right) at 3 weeks following tumor implant. b) Carcass weight loss, defined as the total weight minus the tumor weight at given time points and reported as a percentage of the initial weight of each mouse. Non-significant weight loss is observed for up to two weeks of tumor burden, followed by 30% weight loss in the third week. c) If compared with a healthy mouse (left), the hindlimb of a tumor-bearing mouse (right) is severely atrophied at 3 weeks following transplantation. d) Dorsal and abdominal view of subcutaneous fat pads in control (left panels, arrows) and C26-bearing mice (right panels) at 3 weeks following transplantation. The subcutaneous fat is virtually absent in cachexia. e) The weight of several internal organs is unaffected 3 weeks following tumor transplant, while the spleen is hypertrophic. p > 0.01 by Student's t test if compared with the weight of matching organs from control animals. The mean ± SEM of two experiments performed in triplicate is shown.

Figure 4

Muscle fiber atrophy in C26-bearing mice. a) Esterase staining of TA cross-sections from control (left) and tumor-bearing (right) mice, three weeks following transplant. Fiber atrophy is evident in different fiber types in the absence of lysosome accumulation. The intensely stained neuromuscular junctions are also visible. Bar = 50 μm. b) NADH-transferase staining can be used to differentially analyze larger, glycolytic fibers (arrow) and smaller, oxidative fibers (arrowhead), in TA cross-sections from control (top) and tumor-bearing (bottom) mice. Intermediate fibers are also visible. The fiber cross-sectional area (CSA) was measured, and the distribution is shown for both glycolytic and slow fibers, in control (black bars) and C26-bearing (gray bars) mice. The average size ± SEM of each fiber size class calculated from replicate experiments is shown, along with the medians of each distribution. C26-induced fiber atrophy is detectable in both fiber types. c) Immunostaining for laminin (green) on TA cross-sections from control (left) and tumor-bearing (right) mice, three weeks following transplant, showing alterations in the basement membrane. Bar = 100 μm. d) Immunostaining for laminin (red) performed on enzymatically isolated fibers, obtained from EDL of control (left) and tumor-bearing (right) mice, and longitudinally depicted by confocal microscopy. Nuclei are counterstained with TO-PRO and pseudo-colored in blue. Bar = 30 μm.

Figure 5

Ultrastructural characterization of sarcomeres in ultrathin cross-sections of TA from control (top) and tumor-bearing (bottom) mice observed by transmission EM. A disarrangement of the myofilament hexagonal organization at the level of the A-band is observed upon tumor burden. Bar = 250 nm.

Figure 6

Protein ubiquitination and fatigue induced by C26. RT-PCR (a) and WB (b) analyses show the upregulation of E3 ligase (Atrogin-1)-mediated protein ubiquitination in the TA of tumor-bearing mice (C26). GAPDH is shown as loading control. c) The functional analysis of the EDL from control (open bar) and C26-bearing (solid bar) mice shows unaltered specific force (i.e. tetanic force normalized by muscle mass) and reduced fatigue time (i.e. time required to halve titanic force upon repeated stimuli) in cachectic muscles. p > 0.05 by Student's t test. The mean ± SEM of two experiments performed in triplicate is shown.