Introduction: The tumour microenvironment is hypoglycaemic, hypoxic and acidotic. This activates a stress signalling pathway: the unfolded protein response (UPR). The UPR is cytoprotective if the stressor is mild, but may initiate apoptosis if severe. Activation of the UPR in breast carcinoma is induced by microenvironmental stress such as glucose and oxygen deprivation, but may also be linked to oestrogen stimulation. It may be clinically significant as it may alter chemosensitivity to doxorubicin. Methods: 395 human breast adenocarcinomas were immunohistochemically stained for UPR activation markers (glucose-regulated protein (GRP-78 and XBP-1). A model of UPR activation in vitro by glucose deprivation of T47D breast cancer cells was developed to determine how the UPR affects cellular sensitivity to doxorubicin and 5-fluorouracil. Cytotoxicity was assessed using a colorimetric cytotoxicity assay (MTT). The effect of oestrogen stimulation and tamoxifen exposure on UPR activation by T47D cells was determined by western blotting measurement of the key UPR protein, GRP-78. Results: Expression of GRP78 and XBP-1 was demonstrated in 76% and 90% of the breast cancers, respectively, and correlated with oestrogen receptor positivity ( P =0.045 and 0.017, respectively). In vitro UPR activation induced resistance to both doxorubicin and 5-flurouracil, ( P <0.05). Oestrogen stimulation induced GRP78 and XBP1 over-expression on western blotting. Tamoxifen did not block this response and may induce UPR activation in its own right. Conclusions: The UPR is activated in the majority of breast cancers and confers resistance to chemotherapy. In vitro oestrogen stimulates UPR induction. UPR activation may contribute to breast cancer chemoresistance and interact with oestrogen response elements.

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.

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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.