Cellular Responses to Extracellular Vesicles as Potential Markers of Colorectal Cancer Progression.

The development of novel screening tests aims to support early asymptomatic diagnosis and subtyping patients according to similar traits in the heterogeneous cancer cohort. Extracellular vesicles (EVs) are promising candidates for the detection of disease markers from bodily fluids, but limitations in the standardisation of isolation methods and the intrinsic EV heterogeneity obtained from liquid biopsies are currently obstacles to clinical adoption. Here, cellular responses to cancer EVs were initially explored as potential complementary biomarkers for stage separation using colorectal cancer (CRC) SW480 and SW620 cell line models. A pilot study on a small cohort of CRC patients and controls was then developed by performing a multivariate analysis of cellular responses to plasma-derived EVs. Several cell activities and markers involved in tumour microenvironment pathways were influenced by the treatment of cell line EVs in a stage-dependent manner. The multivariate analysis combining plasma EV markers and cellular responses to plasma EVs was able to separate patients according to disease stage. This preliminary study offers the potential of considering cellular responses to EVs in combination with EV biomarkers in the development of screening methods.

Metachronous Colorectal Adenomas Occur Close to the Index Lesion.

GOALS: The aim of this study is to assess the spatial relationship between index and metachronous colorectal adenoma location. BACKGROUND: After the complete excision of a human sporadic colorectal adenoma, patients are at elevated risk of developing a further metachronous adenoma. Data regarding the occurrence site of a metachronous colorectal adenoma relative to the index adenoma are scarce. STUDY: Prospectively maintained databases were interrogated to identify all colonoscopies and adenoma excisions performed over a 10-year period at a single university teaching hospital. Data for the colonic segments at which adenoma removal were reported at index and all subsequent colonoscopies were extracted and 2 allied data sets merged. RESULTS: A total of 15,121 colonoscopies and 4759 polyp events were recorded. Four hundred fifty-two patients [296 male, 156 female, median (range) age 75 (32 to 100) y] developed at least 1 metachronous adenoma at follow-up colonoscopy. When single index events only are considered (ie, synchronous adenoma cases excluded), over 61% of metachronous adenomas were recorded in the same or an adjacent colonic segment. When the full span of the colon is considered, metachronous adenomas were more likely to occur in a section of the colon proximal to that of the index adenoma (41%±5%) than the same (39%±5%) or distal segment (20%±5%; P =0.006; 1-way χ 2 test). CONCLUSIONS: A metachronous human sporadic colorectal adenoma is more likely to be found in the same colonic segment to that of the index adenoma or 1 immediately adjacent. These data suggest a shared origin of metachronous adenoma with preceding lesions, supporting the existence of precancerous fields.

Imaging of Light-Enhanced Extracellular Vesicle-Mediated Delivery of Oxaliplatin to Colorectal Cancer Cells via Laser Ablation, Inductively Coupled Plasma Mass Spectrometry.

Extracellular vesicles (EVs) are lipid bilayer structures released by all cells that mediate cell-to-cell communication via the transfer of bioactive cargo. Because of the natural origin of EVs, their efficient uptake by recipient cells, capacity to stabilize and transport biomolecules and their potential for cell/tissue targeting and preferential uptake by cancer cells, they have enormous potential for bioengineering into improved and targeted drug delivery systems. In this work, we investigated the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as a tool to measure the loading of platinum-based chemotherapeutic agents. The EV loading of oxaliplatin via co-incubation was demonstrated, and LA-ICP-MS imaging showed greater efficiency of delivery to colorectal cancer cells compared to free oxaliplatin, leading to enhanced cytotoxic effect. Further, the impact of EV co-loading with a porphyrin (C5SHU, known as 'C5') photosensitizer on oxaliplatin delivery was assessed. Fluorescence analysis using nano-flow cytometry showed dose-dependent EV loading as well as a trend towards the loading of larger particles. Exposure of OXA-C5-EV-treated colorectal cancer cells to light indicated that delivery was enhanced by both light exposure and porphyrins, with a synergistic effect on cell viability observed between oxaliplatin, EVs and light exposure after the delivery of the co-loaded EVs. In summary, this work demonstrates the utility of LA-ICP-MS and mass spectrometry imaging in assessing the loading efficiency and cellular delivery of platinum-based therapeutics, which would also be suitable for agents containing other elements, confirms that EVs are more efficient at delivery compared to free drugs, and describes the use of light exposure in optimizing delivery and therapeutic effects of EV-mediated drug delivery both in combination and independently of porphyrin-based photosensitizers.

Ileostomy for chronic constipation: a good idea or just asking for more trouble?

AIM: A defunctioning stoma may be an option for a small group of patients with chronic constipation who have exhausted all forms of conservative management and remain symptomatic. We investigated this group in terms of stoma-related complications and whether they regretted the intervention. METHODS: Patients presenting to Sheffield Teaching Hospitals Pelvic Floor Unit over a 7-year period with chronic constipation unresponsive to conservative management and who had undergone a loop ileostomy for management were interviewed using the decision regret scale. Details about subsequent stoma-related surgery were recorded. RESULTS: Thirty-seven of 38 female patients identified (median age 49 years, range 24-86) completed the decision regret scale. Median follow-up was 34 months (range 7-74). About half (49%) had no regret and a further 27% had minimal regret about the decision for a stoma. Fifty-five per cent of patients had further operations related to the stoma, some undergoing up to five operations. CONCLUSION: A small group of patients with intractable constipation may benefit from a loop ileostomy but are likely to need subsequent surgery to the stoma. Despite this most patients who have had a stoma do not regret the decision.

Clozapine-induced stercoral colitis: a surgical perspective.

We describe a case of a 46-year-old man with schizophrenia treated with clozapine who presented as an emergency with abdominal pain on the background of a 1 month history of constipation. The initial presenting symptoms were vague and a diagnosis was difficult to establish. Initial CT of the abdomen and pelvis demonstrated only minor abnormalities. He continued to deteriorate until a further CT scan revealed worsening stercoral colitis. He subsequently underwent an emergency total colectomy and ileostomy formation and had a complicated prolonged postoperative recovery. This case highlights the risks that clozapine can have on slowing bowel transit and the dangerous consequences that can occur if not identified early.

Solitary juvenile polyp as a cause of elevated faecal calprotectin in an adult.

Faecal calprotectin (FCP) levels are commonly measured in both primary and secondary care as an adjunct to the diagnosis of inflammatory bowel disease (IBD). Juvenile polyps are a rare form of colonic polyp found in both adults and children. We present a case of an adult patient who presented with a very high FCP level, which subsequently normalised following removal of a solitary colonic juvenile polyp. There was no evidence of IBD. Elevation of FCP levels due to this type of colonic pathology have not previously been described in the literature.

Neuropilins: expression and roles in the epithelium.

Initially found expressed in neuronal and then later in endothelial cells, it is well established that the transmembrane glycoproteins neuropilin-1 (NRP1) and neuropilin-2 (NRP2) play essential roles in axonal growth and guidance and in physiological and pathological angiogenesis. Neuropilin expression and function in epithelial cells has received little attention when compared with neuronal and endothelial cells. Overexpression of NRPs is shown to enhance growth, correlate with invasion and is associated with poor prognosis in various tumour types, especially those of epithelial origin. The contribution of NRP and its ligands to tumour growth and metastasis has spurred a strong interest in NRPs as novel chemotherapy drug targets. Given NRP's role as a multifunctional co-receptor with an ability to bind with disparate ligand families, this has sparked new areas of research implicating NRPs in diverse biological functions. Here, we review the growing body of research demonstrating NRP expression and role in the normal and neoplastic epithelium.

Nonelevation of serum CA 19-9 level in a true nonparasitic splenic cyst.

The diagnosis and management of true nonparasitic splenic cysts has markedly changed in recent years. The use of serum CA 19-9 has been increasingly advocated for diagnosis, while the advent of minimally invasive surgery has radically altered surgical management. We present the first case of a true nonparasitic splenic cyst in which serum CA 19-9 was not elevated. Treatment was by laparoscopic cyst decapsulation utilising the endoscopic Ligasure.

Interstitial cell cyclooxygenase-2 expression is associated with increased angiogenesis in human sporadic colorectal adenomas.

Cyclooxygenase (COX)-2 plays an important role in intestinal tumorigenesis and angiogenesis in animal models. In superficial areas of human sporadic colorectal adenomas, COX-2 is expressed predominantly by interstitial macrophages, in close proximity to microvessels. The aim of this study was to investigate the association between microvessel density (MVD) and COX-2 expression in human sporadic colorectal adenomas. Immunohistochemistry and immunofluorescence for CD31 and COX-2 were performed on a well-characterized series of human sporadic colorectal adenomas (n = 37). The mean MVD and COX-2 expression level (scored 0-3) in superficial and deep interstitial cells of adenomas were assessed by two independent observers. Superficial MVD was increased in COX-2-positive adenomas, compared with COX-2-negative adenomas (p = 0.037). There was a significant correlation between superficial MVD and increasing superficial interstitial cell COX-2 expression score (p = 0.048). COX-2-expressing interstitial cells aggregated in areas of high MVD. No relationship was evident between MVD and COX-2 expression in either deep interstitial cells or epithelial cells. Multivariate analysis demonstrated that only adenoma size (p = 0.005) was a significant independent predictor of MVD. COX-2 protein expression by superficial interstitial cells in human sporadic colorectal adenomas is associated with increased angiogenesis. Promotion of angiogenesis may play a role in the pro-tumourigenic activity of COX-2 during growth of human colorectal adenomas.

Paracrine cyclooxygenase-2-mediated signalling by macrophages promotes tumorigenic progression of intestinal epithelial cells.

In human colorectal adenomas or polyps, cyclooxygenase-2 is expressed predominantly by stromal (or interstitial) macrophages. Therefore, we tested the hypothesis that macrophage cyclooxygenase-2 has paracrine pro-tumorigenic activity using in vitro models of macrophage-epithelial cell interactions. We report that macrophages can promote tumorigenic progression of intestinal epithelial cells (evidenced by decreased cell-cell contact inhibition, increased proliferation and apoptosis, gain of anchorage-independent growth capability, decreased membranous E-cadherin expression, up-regulation of cyclooxygenase-2 expression, down-regulation of transforming growth factor-beta type II receptor expression and resistance to the anti-proliferative activity of transforming growth factor-beta(1)) in a paracrine, cyclooxygenase-2-dependent manner. Pharmacologically relevant concentrations (1-2 microM) of a selective cyclooxygenase-2 inhibitor had no detectable, direct effect on intestinal epithelial cells but inhibited the macrophage-epithelial cell signal mediating tumorigenic progression. Cyclooxygenase-2-mediated stromal-epithelial cell signalling during the early stages of intestinal tumorigenesis provides a novel target for chemoprevention of colorectal cancer (and other gastro-intestinal epithelial malignancies, which arise on a background of chronic inflammation, such as gastric cancer) and may explain the discrepancy between the concentrations of cyclooxygenase inhibitors required to produce anti-neoplastic effects in vitro and in vivo.

Analysis of cyclooxygenase expression in human colorectal adenomas.

INTRODUCTION: Evidence from rodent intestinal tumorigenesis models suggests that both cyclooxygenase-1 and cyclooxygenase-2 may play important roles in the development and progression of human sporadic colorectal adenomas. However, previous studies of cyclooxygenase isoform expression in human colorectal adenomas have produced conflicting data. Cyclooxygenase-1 expression has been poorly studied, and cyclooxygenase-2 positivity of adenomas has been variable depending on the detection technique used. It also remains unclear whether villous adenomas express cyclooxygenase-2. METHODS: Cyclooxygenase isoform expression in human sporadic colorectal adenomas was analyzed by reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry. RESULTS: Variable cyclooxygenase-1 expression was detected in all adenomas (n = 9) by both reverse transcription-polymerase chain reaction and Western blot analysis. Cyclooxygenase-2 expression was detected in eight (89 percent) of nine adenomas by reverse transcription-polymerase chain reaction and immunohistochemistry. Cyclooxygenase-2 protein was not detected by Western blot analysis in any adenoma. Cyclooxygenase-2 was expressed by all histopathologic types of adenoma and localized predominantly to superficial interstitial cells, in which it was associated with increased adenoma size. CONCLUSION: Cyclooxygenase-1 is expressed at variable levels by all adenomas. Cyclooxygenase-2 is expressed by the majority of adenomas, including those of the villous type, at levels below the sensitivity of Western blot analysis.