Serum insulin-like growth factor binding protein 2 levels as biomarker for pancreatic ductal adenocarcinoma-associated malnutrition and muscle wasting

Abstract

Background: Malnutrition and muscle wasting are common features frequently observed in pancreatic ductal adenocarcinoma (PDAC) patients with cancer cachexia. They are associated with reduced survival and quality of life. Nutrition therapy is an important part of multimodal cancer care in PDAC. However, due to the complexity of nutrition assessment, only 30-60% of patients with nutritional risks receive nutritional treatment at present. It is important to identify biomarkers that may be used to improve management of PDAC-associated malnutrition. Serum insulin-like growth factor binding protein 2 (IGFBP2) has emerged as a potential serum biomarker in a variety of tumours. However, its association with malnutrition and muscle wasting in PDAC is unclear.

Methods: We evaluated the tumour IGFBP2 expression and serum IGFBP2 level in 98 PDAC patients using immunohistochemistry and enzyme-linked immunosorbent assay and analysed the correlation between them. Furthermore, we explored the relationship between IGFBP2 of both tumour and serum and nutritional status (Patient-Generated Subjective Global Assessment and skeletal muscle index). Pan02 IGFBP2 stable transfection cell lines, Pan02 PLV-IGFBP2 cells, and PLKO-IGFBP2 cells were injected subcutaneously into the flank of C57BL/6 mouse. Serum IGFBP2 levels, food intake, and body weight of these mice were measured. The degree of muscle atrophy is characterized by haematoxylin and eosin, Oil Red O, and Masson's trichrome staining. The mRNA and protein expression of several essential muscle-related signal proteins such as atrogin-1 and muscle RING finger 1 was measured.

Results: Among 98 patients, we found that tumour IGFBP2 expression is related to plasma IGFBP2 levels (r_s = 0.562, P < 0.001), and they significantly increased among patients with Patient-Generated Subjective Global Assessment ≥9 and correlated with overall survival. Moreover, serum IGFBP2 level is negatively correlated with skeletal muscle index (r_s = -0.600, P < 0.001) and Hounsfield units (r_s = -0.532, P < 0.001). In mice injected with Pan02 PLV-IGFBP2 cell, circulating IGFBP2 was elevated while body weight and food intake were decreased when compared with Pan02 PLV-Control group. These mice also exhibited significantly aggravated muscle fibre atrophy, lipid deposition, and increased collagen tissue, and the expression of mRNA and protein of atrogin-1 and muscle RING finger 1 in the gastrocnemius muscle is increased. Conversely, these symptoms were alleviated in the PLKO-IGFBP2 group.

Conclusions: In the current study, there is a significant correlation between serum IGFBP2 levels, malnutrition, and muscle atrophy in PDAC. Our results suggested that serum IGFBP2 level might be a promising biomarker and intervention targets for PDAC-associated severe malnutrition and muscle wasting.

Keywords: Biomarker; Cachexia; IGFBP2; Malnutrition; Muscle wasting; PDAC.

Figure 1

Expression of insulin‐like growth factor binding protein 2 (IGFBP2) is elevated in the tissues of patients with pancreatic ductal adenocarcinoma (PDAC). (A) Representative and quantitation of IGFBP2 immunohistochemical staining in normal pancreatic tissue and PDAC tissues (magnification, ×200). (B) Divided serum levels of IGFBP2 into six groups, the immunohistochemistry (IHC) was divided into nine grades according to the score, and their correlation coefficient was 0.562 (P < 0.001). ELISA, enzyme‐linked immunosorbent assay.

Figure 2

Serum insulin‐like growth factor binding protein 2 (IGFBP2) level is increased in pancreatic ductal adenocarcinoma (PDAC) patients with severe malnutrition and correlates with shorter survival. Serum level of IGFBP2 was measured by enzyme‐linked immunosorbent assay. (A) Serum IGFBP2 levels in healthy control and in patients with PDAC. (B) Higher serum IGFBP2 level concentrations in PDAC patients with Patient‐Generated Subjective Global Assessment (PG‐SGA) ≥9 compared with patients with PG‐SGA < 9. ***P < 0.001. (C) Receiver operating characteristic curve of serum IGFBP2 level for PG‐SGA. (D) Association between serum IGFBP2 levels and overall survival. Significance (P = 0.003) was analysed with the Kaplan–Meier survival curves.

Figure 3

Increasing of serum insulin‐like growth factor binding protein 2 (IGFBP2) levels in tumour‐bearing mouse and emerging the symptom of malnutrition. (A) Serum IGFBP2 level was measured by enzyme‐linked immunosorbent assay after animals were sacrificed by cervical dislocation. (B, C) Body weight and food intake were measured daily per week after subcutaneous tumour transplantation. (D, E) Histopathological detection of gastrocnemius muscle (haematoxylin and eosin ×20) and determined by quantitation of the cross‐sectional area (CSA). ***P < 0.001.

Figure 4

Serum insulin‐like growth factor binding protein 2 (IGFBP2) levels are associated with skeletal muscle index (SMI) and induced muscle wasting. (A) Axial computed tomography images of the third lumbar vertebra region with skeletal muscle highlighted in red [−29 to 150 Hounsfield units (HU)]. (B, C) Scatter plot highlights the relationship between serum IGFBP2 levels and SMI (n = 98; r _s = −0.600; P < 0.001) and HU (n = 98; r _s = −0.532; P < 0.001) among pancreatic ductal adenocarcinoma patients. Results are plotted as mean ± standard deviation.

Figure 5

Overexpression of insulin‐like growth factor binding protein 2 (IGFBP2) cell line induced malnutrition and muscle wasting in mice. (A) Western blotting assessment of IGFBP2 protein expressions after stable transfection with Pan02 PLV‐IGFBP2 and Pan02 PLKO‐IGFBP2. (B) Terminal peripheral blood samples were collected, and IGFBP2 levels in serum were measured using specific enzyme‐linked immunosorbent assay. (C–F) A week after subcutaneous tumour transplantation, body weight and food intake were measured every day. (G–I) The effects of IGFBP2 overexpression and knock‐down on the gastrocnemius muscles as determined by haematoxylin and eosin staining (×20), Oil Red O staining (orange lipid stains), and Masson's trichrome staining (blue collagen stains), which were confirmed by quantitation. Results are plotted as mean ± standard deviation. CSA, cross‐sectional area.

Figure 6

Insulin‐like growth factor binding protein 2 (IGFBP2) induced muscle wasting via regulating muscle RING finger 1 (MuRF1) and atrogin‐1 in C57 mouse. (A) Reverse transcription PCR assessment of MuRF1 and atrogin‐1 mRNA expression in gastrocnemius muscles of PLV‐IGFBP2 mouse and PLKO‐IGFBP2 mice. (B) Western blotting assessment of MuRF1 and atrogin‐1 protein expression in gastrocnemius muscles of PLV‐IGFBP2 mouse and PLKO‐IGFBP2 mice.