Correction: The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer.

[This corrects the article DOI: 10.18632/oncotarget.25497.].

Development of an orthotopic syngeneic murine model of colorectal cancer for use in translational research.

Improving outcomes in colorectal cancer requires more accurate in vivo modelling of the disease in humans, allowing more reliable pre-clinical assessment of potential therapies. Novel imaging techniques are necessary to improve the longitudinal assessment of disease burden in these models, reducing the number of animals required for translational studies. This report describes the development of an immune-competent syngeneic orthotopic murine model of colorectal cancer, utilising caecal implantation of CT26 cells stably transfected with the luciferase gene into immune-competent BALB/c mice, allowing serial bioluminescent imaging of cancer progression. Luminescence in the stably transfected CT26 cell line, after pre-conditioning in the flank of a BALB/c mouse, accurately reflected cell viability and resulted in primary caecal tumours in five of eight (63%) mice in the initial pilot study following caecal injection. Luminescent signal continued to increase throughout the study period with one mouse (20%) developing a liver metastasis. Histopathological assessment confirmed tumours to be consistent with a poorly differentiated adenocarcinoma. We have now performed this technique in 68 immune-competent BALB/c mice. There have been no complications from the procedure or peri-operative deaths, with primary tumours developing in 44 (65%) mice and liver metastases in nine (20%) of these. This technique provides an accurate model of colorectal cancer with tumours developing in the correct microenvironment and metastasising to the liver with a similar frequency to that seen in patients presenting with colorectal cancer, with serial bioluminescent reducing the murine numbers required in studies by removing the need for cull for assessment of disease burden.

The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer.

Nrf2 is a transcription factor that regulates cellular stress response and irinotecan-metabolising pathways. Its aberrant activity has been reported in a number of cancers, although relatively few studies have explored a role for Nrf2 in colorectal cancer (CRC). This study assessed the expression of Nrf2 in patient CRC tissues and explored the effect of Nrf2 modulation alone, or in combination with irinotecan, in human (HCT116) and murine (CT26) cell lines in vitro and in an orthotopic syngeneic mouse model utilising bioluminescent imaging. Using a tissue microarray, Nrf2 was found to be overexpressed (p<0.01) in primary CRC and metastatic tissue relative to normal colon, with a positive correlation between Nrf2 expression in matched primary and metastatic samples. In vitro experiments in CRC cell lines revealed that Nrf2 siRNA and brusatol, which is known to inhibit Nrf2, decreased viability and sensitised cells to irinotecan toxicity. Furthermore, brusatol effectively abrogated CRC tumour growth in subcutaneously and orthotopically-allografted mice, resulting in an average 8-fold reduction in luminescence at the study end-point (p=0.02). Our results highlight Nrf2 as a promising drug target in the treatment of CRC.

From mice to men: Murine models of colorectal cancer for use in translational research.

Colorectal cancer (CRC) is the third most common carcinoma worldwide and despite advances in treatment, survival for patients with metastatic disease remains poor. With nearly 50% of patients developing metastases, in vivo investigation is essential to improve outcomes for these patients and numerous murine models of CRC have been developed to allow the study of chemoprevention and chemotherapy, in addition to improving our understanding of the pathogenesis of CRC. Selecting the most appropriate murine model for a specific application will maximize the conversion of potential therapies from the laboratory to clinical practice and requires an understanding of the various models available. This review will provide an overview of the murine models currently used in CRC research, discussing the limitations and merits of each and their most relevant application. It is aimed at the developing researcher, acting as a guide to prompt further reading in planning a specific study.

Clinical Relevance of Increased Endothelial and Mesothelial Expression of Proangiogenic Proteases and VEGFA in the Omentum of Patients with Metastatic Ovarian High-Grade Serous Carcinoma.

Epithelial ovarian cancer (EOC) metastasis to the omentum requires implantation and angiogenesis. We propose that prometastatic changes in the omental endothelium (for angiogenesis) and mesothelium (for implantation) are critical. We investigated the expression of angiogenic proteases [cathepsin D (CD), cathepsin L (CL), and matrix metalloproteinase 2 (MMP2) and MMP9] and vascular endothelial growth factor A (VEGFA) in the mesothelium and endothelium of omentum from patients with EOC with omental metastases and control patients with benign ovarian tumors. Endothelial expression of CL, VEGFA, and MMP9 and mesothelial expression of VEGFA, MMP9, and CD were significantly increased in patients with metastasized EOC. High expression of MMP9 and VEGFA in endothelium and mesothelium and CD in mesothelium was positively associated with poor disease-specific survival (DSS). High MMP9 expression in either endothelium or mesothelium and presence of ascites prospectively showed the greatest risk of shorter DSS [hazard ratio (HR)= 6.16, 95% confidence interval (CI) = 1.76-21.6, P = .0045; HR = 11.42, 95% CI = 2.59-50.35, P = .0013; and HR = 6.35, 95% CI = 2.01-20.1, P = .002, respectively]. High endothelial MMP9 expression and ascites were independent predictors of reduced DSS and overall survival, together resulting in worst patient prognosis. Our data show that omental metastasis of EOC is associated with increased proangiogenic protein expression in the omental endothelium and mesothelium.

Epithelial ovarian cancer-induced angiogenic phenotype of human omental microvascular endothelial cells may occur independently of VEGF signaling.

Epithelial ovarian cancer (EOC) metastasizes transcoelomically to the peritoneum and omentum, and despite surgery and chemotherapy, recurrent disease is likely. Metastasis requires the induction of proangiogenic changes in the omental microenvironment and EOC-induced omental angiogenesis is currently a key therapeutic target. In particular, antiangiogenic therapies targeting the vascular endothelial growth factor A (VEGFA) pathway are commonly used, although, with limited effects. Here, using human omental microvascular endothelial cells (HOMECs) and ovarian cancer cell lines as an in vitro model, we show that factors secreted from EOC cells increased proliferation, migration, and tube-like structure formation in HOMECs. However, EOC-induced angiogenic tube-like formation and migration were unaffected by inhibition of tyrosine kinase activity of VEGF receptors 1 and 2 (Semaxanib; SU5416) or neutralization of VEGFA (neutralizing anti-VEGFA antibody), although VEGFA165-induced HOMEC migration and tube-like structure formation were abolished. Proteomic investigation of the EOC secretome identified several alternative angiogenesis-related proteins. We screened these for their ability to induce an angiogenic phenotype in HOMECs, i.e., proliferation, migration, and tube-like structure formation. Hepatocyte growth factor (HGF) and insulin-like growth factor binding protein 7 (IGFBP-7) increased all three parameters, and cathepsin L (CL) increased migration and tubule formation. Further investigation confirmed expression of the HGF receptor c-Met in HOMECs. HGF- and EOC-induced proliferation and angiogenic tube structure formation were blocked by the c-Met inhibitor PF04217903. Our results highlight key alternative angiogenic mediators for metastatic EOC, namely, HGF, CL, and IGFBP-7, suggesting that effective antiangiogenic therapeutic strategies for this disease require inhibition of multiple angiogenic pathways.

An improved and reliable method for isolation of microvascular endothelial cells from human omentum.

OBJECTIVES: Despite an increasing research demand for human microvascular endothelial cells, isolation of primary endothelial cells from human tissue remains difficult. The omentum, a highly vascular visceral adipose tissue, could provide an excellent source of these cells. METHODS: A reliable method to isolate HOMECs has been developed. It consists of initial enzymatic digestion (to deplete cell contaminants), followed by further digestion, selective filtration, and immunoselection using Dynabeads coated with CD31 antibody. Cultures were characterized for expression of endothelial cell markers and their ability to undergo VEGF-dependent in vitro tube structure formation. RESULTS: Omental-derived cultures of microvascular endothelial cells were achieved with <5% contamination of other cell types. The endothelial origin of cells was confirmed by the constitutive expression of a range of vascular endothelial markers (CD31, CD105, vWF) and internalization of DiI-AcLDL. Furthermore, cultures were negative for lymphatic endothelial markers, underwent in vitro angiogenesis, and exhibited typical endothelial morphology. CONCLUSIONS: This isolation method produces homogeneous HOMEC cultures that can be maintained in vitro for at least six passages without loss of cellular features characterizing endothelial cells.

The expression pattern of the 70-kDa heat shock protein Hspa2 in mouse tissues.

The highest expression level of a 70-kDa heat shock protein family member Hspa2 is detected specifically in meiotic and post-meiotic male germ cells, which is reflected by original name of this protein, i.e., testis-specific Hsp70. However, this chaperon protein could be also detected in certain somatic tissues. Here, the extra-testicular expression pattern of mouse Hspa2 was analyzed. We found expression of Hspa2 in various epithelial cells including lining of bronchioles and oviduct, columnar epithelium of endometrium, epithelial reticular cells of thymus, transitional epithelium of the urinary bladder, or ependymal cells covering walls of the ventricular system of the brain. Surprisingly, Hspa2 was a putative secretory protein in intestine, endometrial glands and subcommissural organ. Hspa2 was detected in central and peripheral nervous system: in neuron's bodies and fiber tracts, in the subventricular zone of the lateral ventricles, in the dentate gyrus of the hippocampus, in enteric ganglia of the gastrointestinal tract. Hspa2 was also expressed in smooth muscles and at low level in immune system (in germinal centers associated with B-lymphocyte production). In addition to somatic tissues listed above, Hspa2 was detected in oocytes arrested at diplotene of the first meiotic division.

Heat shock transcription factor 1 down-regulates spermatocyte-specific 70 kDa heat shock protein expression prior to the induction of apoptosis in mouse testes.

Expression of constitutively active heat shock transcription factor 1 (HSF1) in mouse spermatocytes induces apoptosis and leads to male infertility. We report here that prior to the onset of massive apoptosis caused by expression of active HSF1 in spermatocytes a marked reduction in spermatocyte-specific Hsp70.2 mRNA and protein levels occurs. In addition, HSP70.2 protein relocalizes from a predominant cytoplasmic to a nuclear position in developing spermatocytes that express active HSF1. Later in the developmental stages, cells undergoing HSF1-induced apoptosis essentially lack the HSP70.2 protein. The down-regulation of Hsp70.2 gene expression by HSF1 is paradoxical because HSF1 is the prototypical activator of HSP genes. Furthermore, HSF1-mediated repression neither involved a heat shock element (HSE)-like sequence adjacent to the Hsp70.2 gene nor were Hsp70.2 promoter sequences associated directly with HSF1. Interestingly, other spermatocyte- and spermatid-specific transcripts are also down-regulated in testes of transgenic mice expressing active HSF1, suggesting involvement of a putative HSF1-dependent block of development of spermatogenic cells. Importantly however, transcription of the Hsp70.2 gene is down-regulated in testes of wild-type mice subjected to a hyperthermia that induces transient activation of HSF1, indicating that the spermatocyte-specific activity of HSF1 might misdirect a network of transcription factors required for proper regulation of Hsp70.2.

The GC-box is critical for high level expression of the testis-specific Hsp70.2/Hst70 gene.

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

Inducible 70 kDa heat shock protein does not protect spermatogenic cells from damage induced by cryptorchidism.

Accumulation of inducible heat shock proteins (e.g. Hsp70i) during cellular stress confers thermotolerance, reduces the consequences of damage and facilitates cellular recovery, while abrogation of Hsp70i expression renders sensitivity to apoptosis. Testis translocation into abdominal cavity, which results in temperature elevation, does not induce expression of the Hsp70i proteins. Despite constitutive expression of testis-specific Hsp70 proteins, spermatocytes are very sensitive to damage at elevated temperatures. To test whether Hsp70i protein could protect testes from heat-induced damage, we have engineered transgenic mice that over-express this protein selectively in spermatocytes and spermatids. We demonstrate that the testes of cryptorchid transgenic mice, like those of wild-type mice, exhibit reduced weight and smaller sizes of their seminiferous tubules, disorganization of their germinal epithelium structures, appearance of multinucleated giant cells, and reduced populations of germ cells. The data show that constitutive expression of Hsp70i does not protect the seminiferous epithelium against cryptorchidism-induced damage.