Adipose tissue fibrosis in human cancer cachexia: the role of TGFβ pathway

Abstract

Background: Cancer cachexia is a multifactorial syndrome that dramatically decreases survival. Loss of white adipose tissue (WAT) is one of the key characteristics of cachexia. WAT wasting is paralleled by microarchitectural remodeling in cachectic cancer patients. Fibrosis results from uncontrolled ECM synthesis, a process in which, transforming growth factor-beta (TGFβ) plays a pivotal role. So far, the mechanisms involved in adipose tissue (AT) re-arrangement, and the role of TGFβ in inducing AT remodeling in weight-losing cancer patients are poorly understood. This study examined the modulation of ECM components mediated by TGFβ pathway in fibrotic AT obtained from cachectic gastrointestinal cancer patients.

Methods: After signing the informed consent form, patients were enrolled into the following groups: cancer cachexia (CC, n = 21), weight-stable cancer (WSC, n = 17), and control (n = 21). The total amount of collagen and elastic fibers in the subcutaneous AT was assessed by histological analysis and by immunohistochemistry. TGFβ isoforms expression was analyzed by Multiplex assay and by immunohistochemistry. Alpha-smooth muscle actin (αSMA), fibroblast-specific protein (FSP1), Smad3 and 4 were quantified by qPCR and/or by immunohistochemistry. Interleukin (IL) 2, IL5, IL8, IL13 and IL17 content, cytokines known to be associated with fibrosis, was measured by Multiplex assay.

Results: There was an accumulation of collagen and elastic fibers in the AT of CC, as compared with WSC and controls. Collagens type I, III, VI, and fibronectin expression was enhanced in the tissue of CC, compared with both WSC and control. The pronounced expression of αSMA in the surrounding of adipocytes, and the increased mRNA content for FSP1 (20-fold) indicate the presence of activated myofibroblasts; particularly in CC. TGFβ1 and TGFβ3 levels were up-regulated by cachexia in AT, as well in the isolated adipocytes. Smad3 and Smad4 labeling was found to be more evident in the fibrotic areas of CC adipose tissue.

Conclusions: Cancer cachexia promotes the development of AT fibrosis, in association with altered TGFβ signaling, compromising AT organization and function.

Keywords: Adipose tissue; Cancer cachexia; Extracellular matrix; Fibrosis; TGFβ.

Fig. 1

Adipose tissue remodeling is triggered during cancer cachexia. a Haematoxilin and eosin sections of subcutaneous adipose tissue. b Collagen detection by Picro Sirius Red staining. c Localization of elastic fibers performed with Verhoeff’s Van Gieson staining. Photomicrographs of the representative images from each group: Control (n = 5), Weight-stable cancer (WSC; n = 5), Cancer cachexia (CC; n = 5). Ad- Illustrates preserved adipocytes. Arrows indicate intense labeling. F- Indicates areas with excessive ECM deposition (fibrotic areas)

Fig. 2

Cachexia induces overexpression of collagen type I, III, VI and fibronectin in the adipose tissue. a-d Sections of human subcutaneous adipose tissue were immunostained with (a) COL1A1, (b) COL3A1, (c) COL6A1, and (d) Fibronectin (FN1). Control (n = 5), Weight-stable cancer (WSC; n = 5), Cancer cachexia (CC; n = 5). Note the positive labeling for all groups, with stronger intensity in CC. Immunostaining for COL6A1 in Control, WSC and CC samples was found around the adipocytes, whereas, in CC, positive staining in several fibrotic areas were observed (F). Ad- Illustrates preserved adipocytes. Blue staining (Mayer’s Haematoxylin) represents a non-reactive nucleous

Fig. 3

TGFβ and Myofibroblast presence contributes to adipose tissue fibrosis in cancer cachexia. a Immunohistochemistry for myofibroblasts with αSMA antibody. Note that almost immunoreactivity in WSC is in vessel walls, whereas CC shows positive cells among adipocytes and in (F) fibrotic areas. b-c TGFβ immunohistochemical analysis in subcutaneous AT illustrates its possible role in inducing cachexia-associated fibrosis, whereas (c) Smad4 shows activation of this pathway. Counterstaining (blue) with Mayer’s Haematoxylin was performed. The groups were identified as Control (n = 5), Weight-stable cancer (WSC; n = 5), Cancer cachexia (CC; n = 5). Fibrotic areas are indicated (F). Arrows indicate positive labeling. d-f qPCR analysis of fibroblast marker (d) FSP1 (S100A4) (control, n = 13; WSC n = 6; CC n = 12), (e) Smad3 (control, n = 9; WSC n = 6; CC n = 7), and (f) Smad4 (control, n = 11; WSC n = 7; CC n = 12). Data presented as median and 1st and 3st quartile. *p < 0.05 CC vs control; # WSC vs control

Fig. 4

TGFβ pathway is activated in the fibrotic subcutaneous AT in cancer cachexia. a-c Multiplex assay analysis of activated (a) TGFβ1, (b) TGFβ2, and (c) TGFβ3 in whole subcutaneous AT. Control (n = 14), Weight-stable cancer (WSC) (n = 12), Cancer cachexia (CC) (n = 14). d-f Adipocytes also contribute for the enhanced observed TGFβ levels, as shown by Multiplex analysis of (a) TGFβ1, (b) TGFβ2, and (c) TGFβ3 from isolated adipocytes of subcutaneous AT. Control (n = 5), Weight-stable cancer (WSC) (n = 5), Cancer cachexia (CC) (n = 5). Data presented as median and 1st and 3st quartile. *p < 0.05, CC vs control; ***p < 0.003 CC vs control. g Immunofluorescence staining in subcutaneous AT for Smad3 (red) and DAPI (blue) for nuclei, which showed higher density for Smad3 labeling in cancer cachexia. Control (n = 5), Weight-stable cancer (WSC; n = 5), Cancer cachexia (CC; n = 5)