Antigen retrieval prior to on-tissue digestion of formalin-fixed paraffin-embedded tumour tissue sections yields oxidation of proline residues.

MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Endoscopic management of migrated biliary stent causing sigmoid perforation.

Endoscopically deployed biliary stents are a well established method for dealing with biliary diseases. Perforation of the gut secondary to migrated biliary stent is reported in less than 1% cases. The authors present the first case of a colonic perforation from migrated biliary stent which was managed endoscopically. An 82-year-old female had a biliary stent for a postcholecystectomy bile leak and presented 6 months later with left iliac fossa pain. Barium enema showed a stent perforating the sigmoid colon. In view of the patient's frailty and absence of peritonitis, an endoscopic retrieval of stent was attempted. Flexible sigmoidoscopy showed a stent partially embedded within the sigmoid diverticulum which was successfully removed and the defect was closed endoscopically using three titanium clips. She had an uncomplicated recovery following the procedure and was discharged home on the second day following the procedure.

Novel molecular tumour classification using MALDI-mass spectrometry imaging of tissue micro-array.

The development of tissue micro-array (TMA) technologies provides insights into high-throughput analysis of proteomics patterns from a large number of archived tumour samples. In the work reported here, matrix-assisted laser desorption/ionisation-ion mobility separation-mass spectrometry (MALDI-IMS-MS) profiling and imaging methodology has been used to visualise the distribution of several peptides and identify them directly from TMA sections after on-tissue tryptic digestion. A novel approach that combines MALDI-IMS-MSI and principal component analysis-discriminant analysis (PCA-DA) is described, which has the aim of generating tumour classification models based on protein profile patterns. The molecular classification models obtained by PCA-DA have been validated by applying the same statistical analysis to other tissue cores and patient samples. The ability to correlate proteomic information obtained from samples with known and/or unknown clinical outcome by statistical analysis is of great importance, since it may lead to a better understanding of tumour progression and aggressiveness and hence improve diagnosis, prognosis as well as therapeutic treatments. The selectivity, robustness and current limitations of the methodology are discussed.

Unfolded protein response activation contributes to chemoresistance in hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) has an annual worldwide incidence of 626 000 cases and causes 550 000 deaths per year. Although the mainstay of treatment is surgical resection, for inoperable or metastatic disease, chemotherapy may be offered. The primary agent used is doxorubicin, but response rates are poor (<20%). The unfolded protein response (UPR) is a cytoprotective cellular stress response that enables cells to survive periods of hypoxia and nutrient deprivation. The UPR may confer resistance to anticancer agents and contribute to treatment failure. This study has investigated whether the UPR is activated in HCC and whether this may contribute to doxorubicin resistance. METHODS: Eighty-six human HCCs were immunohistochemically stained for glucose regulated protein 78, the key marker of UPR activation. An in-vitro model of UPR activation in HepG2 HCC cells was developed by glucose deprived culture. UPR activation was confirmed with western blotting and PCR to show overexpression of glucose regulated protein 78. The relative efficacy of doxorubicin chemotherapy on UPR-activated HepG2 cells was compared with normal HepG2 cells by use of an thiazolyl blue tetrazolium bromide colorimetric assay. RESULTS: Expression of glucose regulated protein 78 was shown in 100% of the HCC samples with 66% showing strong staining. In-vitro UPR activation was achieved with glucose deprivation. UPR activation induced significant resistance to doxorubicin: 34% survival under standard culture conditions versus 58% and 63% for UPR-activated cells in 0.5 and 1 mmol glucose respectively (P=0.00928). CONCLUSION: The UPR is activated in HCCs and confers resistance to chemotherapy in vitro. UPR activation may contribute to HCC chemoresistance.

MALDI-ion mobility separation-mass spectrometry imaging of glucose-regulated protein 78 kDa (Grp78) in human formalin-fixed, paraffin-embedded pancreatic adenocarcinoma tissue sections.

MALDI-mass spectrometry imaging (MALDI-MSI) is a technique that allows proteomic information, that is, the spatial distribution and identification of proteins, to be obtained directly from tissue sections. The use of in situ enzymatic digestion as a sample pretreatment prior to MALDI-MSI analysis has been found to be useful for retrieving protein identification directly from formalin-fixed, paraffin-embedded (ffpe) tissue sections. Here, an improved method for the study of the distribution and the identification of peptides obtained after in situ digestion of fppe pancreatic tumor tissue sections by using MALDI-mass spectrometry imaging coupled with ion mobility separation (IMS) is described. MALDI-IMS-MS images of peptide obtained from pancreatic tumor tissue sections allowed the localization of tumor regions within the tissue section, while minimizing the peak interferences which were observed with conventional MALDI-TOF MSI. The use of ion mobility separation coupled with MALDI-MSI improved the selectivity and specificity of the method and, hence, enabled both the localization and in situ identification of glucose regulated protein 78 kDa (Grp78), a tumor biomarker, within pancreatic tumor tissue sections. These findings were validated using immunohistochemical staining.

Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sections.

The identification of proteins involved in tumour progression or which permit enhanced or novel therapeutic targeting is essential for cancer research. Direct MALDI analysis of tissue sections is rapidly demonstrating its potential for protein imaging and profiling in the investigation of a range of disease states including cancer. MALDI-mass spectrometry imaging (MALDI-MSI) has been used here for direct visualisation and in situ characterisation of proteins in breast tumour tissue section samples. Frozen MCF7 breast tumour xenograft and human formalin-fixed paraffin-embedded breast cancer tissue sections were used. An improved protocol for on-tissue trypsin digestion is described incorporating the use of a detergent, which increases the yield of tryptic peptides for both fresh frozen and formalin-fixed paraffin-embedded tumour tissue sections. A novel approach combining MALDI-MSI and ion mobility separation MALDI-tandem mass spectrometry imaging for improving the detection of low-abundance proteins that are difficult to detect by direct MALDI-MSI analysis is described. In situ protein identification was carried out directly from the tissue section by MALDI-MSI. Numerous protein signals were detected and some proteins including histone H3, H4 and Grp75 that were abundant in the tumour region were identified.

The unfolded protein response and cancer: a brighter future unfolding?

Mammalian cells are bathed in an interstitial fluid that has a tightly regulated composition in healthy states. Interstitial fluid provides cells with all the necessary metabolic substrates (oxygen, glucose, amino acids, etc.), and waste molecules are removed by diffusion gradients that are controlled by local vascular perfusion. The health and normal function of all cells within a body is dependent on the maintenance of this microenvironment. However, many disease states cause fluctuations in this, and in some instances, these might be of sufficient severity to stress and/or be toxic to the cell. Cells have developed a number of responses to enable their survival in a hostile environment. This article discusses one such pathway--the unfolded protein response and its relationship to cancer. The molecular signalling cascade, the mechanism of its activation in cancer and the consequences of its activation for a tumour are discussed, as are clinical studies and potential translational approaches for utilising this pathway for tumour targeting.