Naomi J. Guppy

Finding cancer stem cells: are aldehyde dehydrogenases fit for purpose?

Despite many years of intensive effort, there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison, the study of cancer stem cells is still in its infancy; so, unsurprisingly, there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self‐renewal, clonogenicity, multipotentiality, adherence to the niche, and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs), probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules, and contributing to drug resistance. Antibodies are available against the ALDH enzyme family, but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours, but notably breast cancer, cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour‐initiating activity (presumed cancer stem cells) in immunodeficient mice, and indeed the frequency of so‐called ALDHbri cells in many tumours can be an independent prognostic indicator. Copyright

Cellular pathways to β‐cell replacement

In the twenty‐first century, diabetic patients are likely to be one of the major beneficiaries from the advancement of regenerative medicine through cellular therapies. Though the existence of a specific self‐renewing stem cell within the pancreas is still far from clear, a surprising variety of cells within the pancreas can differentiate towards a β‐cell phenotype: ductular cells, periductular mesenchymal cells and β‐cells themselves can all give rise to new β‐cells. Extra‐pancreatic adult somatic stem cells, in particular, those originating from bone marrow may also be capable of differentiating to β‐cells, though equally well the beneficial effects of bone marrow cells may reside in their contribution to the damaged islet vasculature. Forced expression of the β‐cell‐specific transcription factor Pdx1 in hepatocytes also holds promise as a therapeutic strategy to increase insulin levels in diabetic individuals. Embryonic stem (ES) cells are clearly another possible source for generating β‐cells, but ES cells are beyond the scope of this review, which focuses on adult stem and progenitor cells capable of producing β‐cells. Despite considerable endeavour, we still have much to learn in the field of pancreatic regeneration prior to any clinically applicable therapy based upon adult stem cells. Copyright

EGFR inhibitors identified as a potential treatment for chordoma in a focused compound screen

Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well‐characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µm), based on their growth‐inhibitory effect. Their half‐maximal effective concentration (EC50) values were determined in chordoma cells and normal fibroblasts. Twenty‐seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho‐EGFR and its downstream pathways in a dose‐dependent manner. Analysis of substituent patterns suggested that EGFR‐inhibitors with small aniline substituents in the 4‐position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U‐CH1 xenograft and a patient‐derived xenograft, SF8894). One of the resistant cell lines (U‐CH2) was shown to express high levels of phospho‐MET, a known bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p‐)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo.

C-terminal tensin-like gene functions as an oncogene and promotes cell motility in pancreatic cancer.

Objectives C-terminal tensin-like gene (CTEN, also known as TNS4) localizes to focal adhesions and is reported to function as an oncogene in colonic, breast, lung, and gastric cancers. Its role in pancreatic cancer is unknown and was thus investigated in this study. Methods C-terminal tensinlike gene expression was evaluated by immunohistochemistry in a series of pancreatic cancers. Functional activity of the CTEN was tested by manipulating cellular CTEN levels using a dual approach of gene knockdown/forced expression. Results The CTEN is overexpressed in 31 (70.45%) of 44 pancreatic cancers. Functionally, changes in CTEN level did not alter cellular proliferation, but CTEN levels were positively associated with enhanced colony-forming efficiency in both Panc-1 and PSN-1 cell lines. Forced CTEN expression in Panc-1 cells stimulated cell motility, whereas knockdown of CTEN in PSN-1 inhibited cell motility in both transwell migration and wound-healing assays. Evaluation of downstream targets demonstrated that alterations in CTEN levels induced changes in focal adhesion kinase and E-cadherin, whereas integrin-linked kinase (ILK) remained unchanged. Conclusions These are the first data showing an oncogenic role for CTEN in pancreatic cancer through promotion of colony formation and cell motility. The latter may be mediated by signaling through focal adhesion kinase and inhibiting E-cadherin.

Trefoil factor family peptides in normal and diseased human pancreas.

Objectives Trefoil factor family (TFF) peptides promote wound healing in the gut. Recent evidence has suggested that TFF3 may be a pancreatic mitogen, an unusual role for TFF peptides. We sought to clarify human pancreatic TFF and mucin expression and performed in vitro experiments to see how pancreatic cell lines respond to TFF3 in particular. Methods Samples of normal and diseased pancreas (chronic pancreatitis, pancreatic intraepithelial neoplasia, neuroendocrine tumors, and pancreatic ductal adenocarcinoma [PDAC]) were studied by immunohistochemistry and in situ hybridization. Pancreatic cell lines were challenged with TFF2 and TFF3 in wound and migration assays. Results In normal islets, colocalization of insulin or glucagon with TFF3 was common. All TFF messenger RNAs were seen in ductal epithelium. Adenocarcinomas expressed all TFF messenger RNAs. Normal ducts were mucin free; MUC5AC was strongest in pancreatic intraepithelial neoplasia and chronic pancreatitis but was reduced in PDAC. TFF2 induced Panc-1 migration and accelerated wound closure in Capan-2 and COLO-357. Double immunohistochemistry for insulin or TFF3 and Ki67 colabeled only very rare islet cells. TFF3-positive PDAC ducts showed some Ki67 colocalization. Conclusions No correlation between TFF3 or insulin and Ki67 was seen without ductal hyperplasia. TFF2 may assist pancreatic tumor cell movement, but TFF3 may not be a pancreatic mitogen. Abbreviations CP - chronic pancreatitis EGF - epidermal growth factor GKN2 - gastrokine 2 IHC - immunohistochemistry ISH - in situ hybridization NET - neuroendocrine tumor PDAC - pancreatic ductal adenocarcinoma TFF - trefoil factor family

The Impact of Diet-Induced Weight Loss on Biomarkers for Colorectal Cancer: An Exploratory Study (INTERCEPT)

The aim of this study was to explore the potential effects of diet‐induced weight loss on molecular biomarkers of colorectal cancer risk in serum and colorectal tissue.

Serum IgG2 and tissue IgG2 plasma cell elevation in orbital IgG4-related disease (IgG4-RD): Potential use in IgG4-RD assessment.

Aims To determine the role of serum and tissue IgG2 in orbital biopsies with the histological features of IgG4-related disease (IgG4-RD) in comparison with non-IgG4-related orbital inflammatory disorders (OID), including autoimmune disorders. Methods This is an international (Sheffield, UK, and Singapore) collaborative, retrospective case review of 69 patients (38 from Singapore National Eye Centre and 31 from Royal Hallamshire Hospital, Sheffield) with orbital inflammatory biopsies between 2002 and 2016. Clinical information and histology were reviewed and cases were classified into three groups: Group 1: IgG4-RD orbital inflammation (n=43); Group 2: idiopathic OID (n=12) and Group 3: autoimmune OID (n=14). Serum IgG1, IgG2, IgG3 and IgG4 levels were collated where available and immunohistochemistry (IHC) for tissue IgG2 plasma cells was performed. Results Dual IHC showed IgG2 plasma cells as a distinct population from IgG4 plasma cells. Significant (twofold) serum IgG2 elevation was noted among IgG4-RD (group 1), idiopathic (group 2) and autoimmune OID (group 3). Similarly, significant elevation of tissue IgG2 plasma cells was also seen among IgG4-RD (group 1), idiopathic and autoimmune OID (groups 2 and 3). Conclusions Significant elevations of serum IgG2 and tissue IgG2 plasma cells are present in orbital IgG4-RD in comparison with non-IgG4 orbital inflammation (idiopathic and autoimmune OID), suggesting that IgG2 may play a role in IgG4-RD. A serum IgG2 cut-off >5.3 g/L was found to be 80% sensitive and 91.7% specific for orbital IgG4-RD, with an accuracy of 0.90. Tissue IgG2 and IgG4 subclass reporting may provide additional insight regarding the ‘IgG4-RD’ pathogenesis.

ABC Transporters, Aldehyde Dehydrogenase, and Adult Stem Cells

Despite many years of intensive effort, there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. So-called markers of stem cells have been varied but can be broadly categorized into molecular determinants that govern self-renewal, clonogenicity, multipotentiality, adherence to the niche and longevity. This chapter describes two specific attributes of many stem cells that appear to be the main determinants of stem cell survival, namely either an ability to detoxify many potentially cytotoxic molecules by virtue of high aldehyde dehydrogenase (ALDH) activity or an ability to actively efflux a wide variety of cytotoxic agents by virtue of the presence of one or more ATP-binding cassette transporters (ABC transporters), and indeed many stem cells may be endowed with both properties. Antibodies are available against the ALDH enzyme family and ABC transporters, but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these molecules. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence-activated cell sorting (FACS), the latter technique is also being used to isolate cells with high ABC transporter activity after incubation with a fluorescent dye (usually Hoechst 33342) that is actively effluxed from these cells giving rise to a fluorescent dull population known as the “side population”. Since both ALDH and ABC transporter activities are cytoprotective strategies, it is not surprising that many cancer stem cells have these mechanisms working robustly, but they are also present in normal adult stem cells. This chapter critically reviews ALDH activity and the SP as markers of normal adult stem cells.

Validation of Immune Cell Modules in Multicellular Transcriptomic Data

Numerous gene signatures, or modules have been described to evaluate the immune cell composition in transcriptomes of multicellular tissue samples. However, significant diversity in module gene content for specific cell types is associated with heterogeneity in their performance. In order to rank modules that best reflect their purported association, we have generated the modular discrimination index (MDI) score that assesses expression of each module in the target cell type relative to other cells. We demonstrate that MDI scores predict modules that best reflect independently validated differences in cellular composition, and correlate with the covariance between cell numbers and module expression in human blood and tissue samples. Our analyses demonstrate that MDI scores provide an ordinal summary statistic that reliably ranks the accuracy of gene expression modules for deconvolution of cell type abundance in transcriptional data.

Tumor Necrosis Factor (TNF) Bioactivity at the Site of an Acute Cell-Mediated Immune Response Is Preserved in Rheumatoid Arthritis Patients Responding to Anti-TNF Therapy

The impact of anti-tumor necrosis factor (TNF) therapies on inducible TNF-dependent activity in humans has never been evaluated in vivo. We aimed to test the hypothesis that patients responding to anti-TNF treatments exhibit attenuated TNF-dependent immune responses at the site of an immune challenge. We developed and validated four context-specific TNF-inducible transcriptional signatures to quantify TNF bioactivity in transcriptomic data. In anti-TNF treated rheumatoid arthritis (RA) patients, we measured the expression of these biosignatures in blood, and in skin biopsies from the site of tuberculin skin tests (TSTs) as a human experimental model of multivariate cell-mediated immune responses. In blood, anti-TNF therapies attenuated TNF bioactivity following ex vivo stimulation. However, at the site of the TST, TNF-inducible gene expression and genome-wide transcriptional changes associated with cell-mediated immune responses were comparable to that of RA patients receiving methotrexate only. These data demonstrate that anti-TNF agents in RA patients do not inhibit inducible TNF activity at the site of an acute inflammatory challenge in vivo, as modeled by the TST. We hypothesize instead that their therapeutic effects are limited to regulating TNF activity in chronic inflammation or by alternative non-canonical pathways.