Cachexia-associated adipose tissue morphological rearrangement in gastrointestinal cancer patients

Abstract

Background and aims: Cachexia is a syndrome characterized by marked involuntary loss of body weight. Recently, adipose tissue (AT) wasting has been shown to occur before the appearance of other classical cachexia markers. We investigated the composition and rearrangement of the extracellular matrix, adipocyte morphology and inflammation in the subcutaneous AT (scAT) pad of gastrointestinal cancer patients.

Methods: Surgical biopsies for scAT were obtained from gastrointestinal cancer patients, who were signed up into the following groups: cancer cachexia (CC, n = 11), weight-stable cancer (WSC, n = 9) and weight-stable control (non-cancer) (control, n = 7). The stable weight groups were considered as those with no important weight change during the last year and body mass index <25 kg/m(2). Subcutaneous AT fibrosis was quantified and characterized by quantitative PCR, histological analysis and immunohistochemistry.

Results: The degree of fibrosis and the distribution and collagen types (I and III) were different in WSC and CC patients. CC patients showed more pronounced fibrosis in comparison with WSC. Infiltrating macrophages surrounding adipocytes and CD3 Ly were found in the fibrotic areas of scAT. Subcutaneous AT fibrotic areas demonstrated increased monocyte chemotactic protein 1 (MCP-1) and Cluster of Differentiation (CD)68 gene expression in cancer patients.

Conclusions: Our data indicate architectural modification consisting of fibrosis and inflammatory cell infiltration in scAT as induced by cachexia in gastrointestinal cancer patients. The latter was characterized by the presence of macrophages and lymphocytes, more evident in the fibrotic areas. In addition, increased MCP-1 and CD68 gene expression in scAT from cancer patients may indicate an important role of these markers in the early phases of cancer.

Keywords: Adipose tissue; Extracellular matrix; Fibrosis, Inflammation, Cachexia.

Figure 1

Morphological characteristics of subcutaneous adipose tissue depot in cachexia and control patients. Haematoxylin and eosin stained sections of subcutaneous tissue from (A) weight‐stable subjects and control, (B) weight‐stable cancer (WSC) patients and (C) cancer cachexia (CC) patients. Morphometric analysis of sectional area (D) and (E) perimeter of adipocytes from different experimental groups. Photomicrographs (A–C) illustrate the most representative images considering data related to morphological analysis (D, E). Values are mean ± SEM. * P < 0.05 vs. control subjects.

Figure 2

Picro sirius red staining sections of subcutaneous adipose tissue in cachexia and control patients. Collagen fibres are presented in different colours. Type I collagen fibres are orange to red, whereas the thinner type III collagen fibres appear yellow to green from (A, B) weight‐stable subjects and control, (C, D) weight‐stable cancer (WSC) patients and (E, F) cancer cachexia (CC) patients. Total collagen quantification in cachexia and control patients of (G) type I collagen and (H) types I–III ratio. Values are mean ± SEM. * P < 0.05 vs. control subjects.

Figure 3

Identification of different immune cell types present in subcutaneous adipose tissue obtained from cancer cachexia patients. Serial sections of weight‐stable subjects and control, weight‐stable cancer (WSC) patients and cancer cachexia (CC) patients were stained with markers of macrophages (CD68), T‐lymphocytes (CD3) and for neutrophils (CD15). Nuclei were stained with haematoxylin (blue staining). HE, haematoxylin and eosin staining.

Figure 4

Immunohistochemistry for immune cells from weight‐stable subjects and control, weight‐stable cancer (WSC) patients and cancer cachexia (CC) patients. Subcutaneous adipose tissue (scAT) macrophages localize to crown‐like structures (CLS) around individual adipocytes, which increase in frequency with cancer cachexia. Light microscopy of scAT of CC patients showing CD68 immunoreactive macrophages (brown colour) aggregated to numerous (C, CC patients) CLS among unilocular adipocytes. Note that almost all CD68 immunoreactive macrophages are organized to form CLS. CD3 immunoreactive T‐lymphocyte shows positive cells in fibrotic areas, stained with DAB (brown colour) (arrow). Nuclei were stained with haematoxylin (blue labelling). Bar (µm): 50 µm for A and B; 25 µm for C and D.

Figure 5

Expression levels of genes involved in the inflammation of subcutaneous adipose tissue depots in subjects from different experimental groups. Real‐time PCR analysis of RNA isolated from weight‐stable subjects and control, weight‐stable cancer (WSC) patients and cancer cachexia (CC) patients. mRNA levels of target genes were normalized to 18 S. Values are mean ± SEM for five to nine samples per group. * P < 0.05 vs. control subjects.