Subcellular Fractionation for Pancreatic Cancer Biomarkers

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(Last Updated On: April 23, 2017)
Immunohistochemical staining of patient tumor tissue with respective antibodies

Immunohistochemical staining of patient tumor tissue with respective antibodies. Image: McKinney et al. 2011

Pancreatic cancer is one of the highly aggressive malignant diseases with survival rate 6 months or less. This is because, pancreatic cancer is asymptomatic and don’t show symptoms unless the disease reaches to the incurable stage. The current strategy of diagnosing pancreatic cancer is based on the use of molecular marker CA19-9. However, CA19-9 is not specific to pancreatic cancer because it is not expressed at a high level early in the disease progression.

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Therefore, the current treatment has been limited with only successful treatment option surgical resection of the tumor at its early stage. However, the chance of recurrence in patients is high and it gets easily spread over the different parts of the pancreas. There are also some complications in the surgical treatments, such as pancreatic cancer patients are not suitable for surgical removal of the tumor due to their proximity to the superior mesenteric artery.

Therefore, there is a need to identify a more specific molecular marker for pancreatic cancer that has higher sensitivity than the current molecular marker. The use of mass spectrometric techniques in the identification and quantification of proteins has emerged as a new approach for the discovery of biomarkers. Discovering proteins that can be a potent biomarker for the disease can provide a new insight into the molecular pathogenesis and progression of pancreatic cancer.

However, there is a major challenge in the proteomic studies for the discovery of the biomarkers because there is a large dynamic range of proteins existing in the tissue sample. To solve this problem, subcellular fractionation can be employed that separates the proteins based on their location within the cell. Subcellular fractionation decreases the complexity of the sample and allows the researchers to delve into the lower abundant proteome that makes easy for the identification of the disease-related proteins.

Now, researchers have used subcellular fractionation to discover the biomarkers for pancreatic cancer, during which they compared the proteomic profile from non-tumor and disease tissue. They found a subset of proteins that are being upregulated in cancer. Among these proteins, they choose four proteins; Biglycan (BGN), Pigment Epithelium-derived Factor (PEDF), Thrombospondin-2 and TGF-β-induced protein ig-h3 precursor (βIGH3) for further validation by immunohistochemistry and western blotting.

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For this purpose, researchers took five patients with pancreatic cancer. During the surgical resection, the pancreas was obtained and sent to the pathology for staging. Pancreas was dissected for a part of the tumor as well as a part of the non-tumor adjacent pancreas tissue from each of the patients and snap froze on liquid nitrogen. These tissue samples were labeled as 30N, 44N, 47N, 101N, and 69N for non-tumor pancreatic tissues while, for tumor tissues, samples were labeled as 30T, 44T, 47T, 101T, and 69T.

Subcellular fractionation of about 50 mg tissue from snap frozen non-tumor tissue and tumor from the five patients was carried out followed by the western blotting using fraction-specific antibodies. During subcellular fractionation, four fractions were obtained; fraction-1 (cytosol enriched fraction), fraction-2 (membrane enriched fraction), fraction-3 (nuclear enriched fraction) and fraction-4 (cytoskeleton enriched fraction).

Subcellular fractionation products from two pairs (represented by 44N/T and 69N/T) were chosen for liquid chromatography coupled with mass spectrometry (LC-MS/MS) based on their optimal protein concentration after subcellular fractionation. These pairs of tissues were from male patients with similar age and identical stage of pancreatic cancer. These pairs of tissues were subjected to subcellular fractionation and four fractions were obtained for each of the samples.

30 µg of protein was loaded into the SDS-PAGE and electrophoresed. After electrophoresis, gel lanes were dissected into 20 slices. After destaining and dehydrating, these gel pieces were rehydrated in ammonium bicarbonate buffer containing trypsin. Peptides from the gel pieces were extracted and subjected to the reverse phase LC-MS/MS. Proteins that were identified as significantly changed in cancer were compared to a database of blood plasma proteins and additional comparison was made across the biological samples (such as 44 vs. 69) as well as pathological status (such as the non-tumor vs. tumor) for each of the subcellular fractions.

Results

Mass spectrometric analysis of the subcellular fractionation resulted into 2031 total proteins in patient 44 (among which 351 were specific to non-tumor while 512 were unique to cancer) and 1963 total protein identifications in patient 69 (among which 507 were specific for non-tumor while 476 were specific for tumor). Combining these data to a non-redundant data set, researchers identified a total of 2393 proteins in non-tumor and pancreatic cancer tissue among which 408 proteins were unique to the non-tumor pancreatic tissues while 622 proteins were unique to the pancreatic cancer tissue samples and 1363 proteins were common to both tissue types.

Using PLGEM statistical modeling software, researchers have identified cancer associated up-regulation of proteins; Biglycan (BGN), Pigment Epithelium-derived Factor (PEDF), Thrombospondin-2 and TGF-β-induced protein ig-h3 precursor (βIGH3). All of these proteins are related with cancer progression and play important roles in the tumor microenvironment, cell proliferation, and angiogenic processes.

PEDF is an anti-angiogenic factor that is associated with inhibition of the pancreatic cancer cell proliferation. This seems to be odd with the results. However, researchers have observed that in non-tumor pancreatic tissue, PEDF is expressed in the cytosol while in cancerous cell PEDF is expressed more prominently in the nucleus.

Thus, subcellular fractionation can be employed as a powerful tool to screen and detect the protein biomarker of pancreatic cancer. And the validity of the use of subcellular fractionation can be strengthened by the fact that all of the proteins chosen for validation have been published in connection with pancreatic cancer. As for example, Biglycan which is a component of the extracellular matrix is known to be a target of TGF-β in pancreatic cancer cells. In the same way, expression of throbospondin-2 by pancreatic stellate cells has been found to promote the pancreatic cancer invasion in vitro. Therefore, these proteins can be considered as biomarkers for pancreatic cancers due to their significant role of fibrosis in the pathogenesis of the disease.

βIGH3 is also involved in the TGF-β signaling and has been identified in pancreatic cancer as well as in lung and colorectal carcinomas while PEDF has been identified as an inhibitor of the caspase-independent apoptosis that is triggered by the AID translocation into the nucleus and is interrupted by the PEDF up-regulation of the Bcl-2.

In conclusion, subcellular fractionation in combination with gel electrophoresis and LC-mass spectrometric analysis is a powerful tool that can be used to identify potential biomarkers of disease.

Reference: Journal of Proteomics

Article doi: 10.1016/j.jprot.2010.08.006

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