Mesenchymal stromal cells (MSCs) and colorectal cancer - a troublesome twosome for the anti-tumour immune response?

The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis.

Optimization of immunofluorescence protocols for detection of biomarkers in colorectal and breast cancer tissues

Cancer remains an undetermined question for modern medicine. Every year millions of people ranging from children to adult die since the modern treatment is unable to meet the challenge. Research must continue in the area of new biomarkers for tumors. Molecular biology has evolved during last years; however, this knowledge has not been applied into the medicine. Biological findings should be used to improve diagnostics and treatment modalities. In this thesis, human formalin-fixed paraffin embedded colorectal and breast cancer samples were used to optimize the double immunofluorescence staining protocol. Also, immunohistochemistry was performed in order to visualize expression patterns of each biomarker. Concerning double immunofluorescence, feasibility of primary antibodies raised in different and same host species was also tested. Finally, established methods for simultaneous multicolor immunofluorescence imaging of formalin-fixed paraffin embedded specimens were applied for the detection of pairs of potential biomarkers of colorectal cancer (EGFR, pmTOR, pAKT, Vimentin, Cytokeratin Pan, Ezrin, E-cadherin) and breast cancer (Securin, PTTG1IP, Cleaved caspase 3, ki67).

Validity of shape memory NiTi colon ring BioDynamix ColonRing™ (or NiTi CAR 27™) to prevent anastomotic colorectal strictures. Preliminary results

Purpose. Anastomotic strictures occur in 3-30% of colorectal anastomosis and one of the main causes may be a reaction to the presence of the metal staples used for suturing. The aim of this study was to evaluate the efficacy of a compression anastomosis ring using the memoryshaped device in initial, i.e. nickel-titanium alloy (NiTi) for the prevention of colorectal anastomotic strictures. Patients and methods. A compression anastomosis ring device (NiTi CAR 27™) was used to perform compression anastomosis in 20 patients underwent left hemicolectomy and anterior resection of the rectum for carcinoma. An endoscopic check of the anastomosis was carried out at one month and at six months after surgery. Results. In 2 patients (10%) a dehiscence of the anastomosis occurred on the fifth and the eighth postoperative day. No anastomotic strictures were observed in any of the other 18 patients at six months follow-up after surgery. Conclusion. Our preliminary results suggest that the use of a compression anastomosis ring might well be a valid method of preventing anastomotic strictures in colorectal surgery. Further studies involving a larger number of patients are needed in order to confirm these preliminary results.

Anastomotic strictures in colorectal surgery: treatment with endoscopic balloon dilation

The incidence of anastomotic stricture following colorectal surgery has increased in recent years. This complication is observed in 2-5% of all operated patients and is probably due to the greater number of low anastomoses performed with surgical staplers. We observed 31 patients with postoperative stricture, arising from one to nine months post-surgery. All patients had been treated for colorectal cancer and underwent endoscopy either during routine follow-up or for symptoms of stenosis. In 16 patients (group A) the stricture diameter was less than 4 mm and the patients had symptoms attributable to partial bowel obstruction. In the remaining 15 patients (group B), who had difficult bowel movements, the stricture diameter ranged from 4 to 8 mm. All patients were treated with endoscopic dilation using achalasia balloons. The results were considered good when the post-dilation anastomosis diameter achieved was at least 13 mm, fair when it was 9-12 mm and poor when it was less than 9 mm. The short term results (3 weeks) were good in 27 patients (87.2%), fair in 3 patients (9.6%), and poor in 1 patient (3.2%). After several unsuccessful dilations, the latter was treated by surgery. Follow-up at 3-4 months of the remaining 30 patients revealed good results in 20 (66.6%), fair in 6 (20%), and poor in 4 (13.3%). In 1 of these 4 patients, cancer recurrence was observed and a new surgical resection was performed. In 2 patients a self–expandable metal stent was inserted for 4-6 weeks, with satisfactory results. In 1 patient a biodegradable polydioxanone stent was inserted with good results after 6 months. Follow-up at 3-4 months showed good results in 25 patients. After 38 months, cancer recurrence in the area of the anastomosis was observed in 1 patient, who was treated surgically. Endoscopic dilatation should be considered the first therapeutic approach in case of anastomotic strictures, as it is immediately effective, repeatable, and does not preclude surgery if this should become necessary. .

Laparoscopic colorectal resections performed over a seven-years period in a single Italian Centre

Introduction. Laparoscopic approach for treatment of colorectal lesion is gaining acceptance gradually. Evidence from numerous randomised controlled trials has shown the short-term benefits of laparoscopic colon resection over open surgery, and its long-term outcomes also does not differ considerably from those of open surgery. This study aims at a retrospective analysis of operative and short term outcomes of patients. Patients and methods. All laparoscopic colon and rectal resections performed between September 2004 and September 2011 were included. The clinical parameters, operative parameters and short-term outcome details of laparoscopic colorectal surgery patients were collected from the retrospectively reviewed database. Results. A total of 347 patients, median age 71 years (range 32 to 96), underwent laparoscopic resection of the colon and rectum. The median Body Mass Index (BMI) was 26.5. The majority of the procedures were performed for malignant disease (97,1%) and the most common procedure was right colectomy (41%). The median duration of surgery was 202,3 minutes, with conversion to open surgery in 40 patients (11.5%). Complications occurred in 23 patients (6.6%). The median length of hospital stay was 8.9 days. In patients with malignant disease, the median number of lymph nodes removed was 14.9. Conclusion. Our results show that laparoscopic approach for colon-rectal lesions is safe, feasible and produces favourable results. The most important aspect of surgery for malignant disease is the ability to remove radically the disease. However all data are still related to the experience of the operator.

Benefits of laparoscopic colorectal surgery in the geriatric patient

Background. Our aim is the retrospective valuation of results in over 75 year-old patients, with colorectal cancer, treated with laparoscopic and laparotomic surgery, considering how laparoscopic surgery has improved these patients’ outcome. Patients and methods. We took all over 75 year-old patients, affected by colorectal cancer, treated with colectomy. Patients has been divided into two groups: laparotomy group and laparoscopy group. Data concerning patients, i.e., age, sex, BMI, ASA, comorbidities, were collected with data concerning the operation (surgical time, conversion percentage). Postoperative outcomes – i.e., gas evacuation, bowel movements, solid and liquid feeding, need to ICU, complications, re-surgery, hospitalization and type of discharge, mortality – were evaluated. Results. A total of 145 patients are included: laparotomy 80 and laparoscopy 51. Two groups are homogeneous for age, sex, BMI, ASA, comorbidities. Surgical times are the same. Need to Intesive Care Unit (ICU) is lower in laparoscopy. Gas evacuation and bowel movements are earlier in laparoscopy. Liquid and solid diet is earlier in laparoscopy. Hospitalization was earlier after laparoscopy. Discharge at home is more frequent in laparoscopy. Major and minor complications are lower in laparoscopy. Post-operative mortality is lower in laparoscopy. Conclusions. Laparoscopy improves over 75 year-old patients’ outcomes, after elective surgery for colorectal cancer. Surgery trauma, anaesthesia, nutritional and hemodynamic alterations, are factors that break the old patients’ fragile physiologic balance. Less traumatic surgery improves old patients’ outcomes.

Colorectal retained foreign bodies per anum introduced. Three years retrospective study at Emergency Surgery Unit

Colorectal foreign bodies per anum introduced are not exceptional. They can be classified as high-lying or low-lying, depending on their location relative to the recto-sigmoid junction. High-lying rectal foreign bodies sometimes require surgery; low-lying ones are often palpable by digital examination and can removed at bedside. No reliable data exist regarding the frequency of inserted rectal foreign bodies and the literature is largely anecdotal. We review our experience on patients almost all males and heterosexual with retained colorectal foreign bodies and their outcome in Surgical Emergency Unit of a Southern Italy University hospital.

One-stage laparoscopic procedure for a patient with bilateral colorectal tumours and renal carcinoma

We describe a case of a patient with synchronous bilateral colorectal tumours and renal carcinoma who underwent one-stage laparoscopic surgery procedure with right transperitoneal nefrectomy, right hemicolectomy and sigmoidectomy. One-stage laparoscopic procedure can be used safely and successfully for a patient with multiple primary tumours.

Biological ageing and colorectal cancer: fetuin A, sirtuins and telomeres at the interface between inflammation, metabolism and cancer

Colorectal cancer is a common, age-associated disease with significant comorbidity and mortality. Biomarkers of ageing may have prognostic or predictive value in colorectal cancer. Fetuin A, members of the sirtuin family of proteins and telomeres have shown promise as potential biomarkers of ageing. AIM: To evaluate these potential biomarkers in the context of colorectal cancer. METHODS: Two cohorts of patients were used. Telomere length was measured in peripheral blood leukocytes (PBL), and for a subset of patients, in normal colorectal and colorectal tumour tissue. Serum fetuin A was measured for these patients and data on clinico-pathological factors of accepted significance in colorectal cancer was collected prospectively. Telomere length in the matched samples of leukocytes, normal colorectal and colorectal tumour tissue was compared. Associations between telomere length in the different tissues, serum fetuin A and clinico-pathological factors of accepted significance in colorectal cancer were evaluated. A systematic review of the literature was performed to examine the evidence for correlation between telomere length in different tissues in humans. Tissue from colorectal tumours from the second cohort patients was mounted in a tissue microarray (TMA) and stained for sirtuin proteins (SIRT2-SIRT7). This TMA also contained tissue from a subset of matched samples of adjacent normal colorectal mucosa. Staining of normal colorectal and colorectal tumour tissue was evaluated by the weighted Histoscore method and compared. The effect of staining in tumour tissue on cancer-specific survival was examined. Associations between Histoscores and clinico-pathological factors of accepted significance in colorectal cancer were assessed. RESULTS: Systematic review of the literature did not show robust evidence of correlation between telomere length in different tissues in humans. Telomere length in peripheral blood leukocytes did not show correlation with telomere length in normal colorectal mucosa, or in colorectal tumour tissue. PBL telomere length was potentially related to the presence of distant metastases. Fetuin A was inversely associated with markers of systemic inflammation and with T stage. Novel nuclear localisation was described for SIRT4 and SIRT5. Protein expression of the sirtuins was reduced in tumour tissue in comparison to normal colorectal mucosa, apart from SIRT3 cytoplasmic and nuclear and SIRT6 nuclear stainng. Lowest and highest quartile SIRT2 expression was associated with worse survival. Sirtuin protein expression levels and localisation correlate with increased systemic inflammation and pathological markers of poor prognosis in tumour tissue. Intercorrelations between sirtuin expression levels in normal tissue are not seen in tumour tissue, possibly indicating a breakdown of signalling and control.

ANTICANCER MECHANISM OF TOLFENAMIC ACID IN COLORECTAL CANCER

Colorectal cancer (CRC) is the third leading cause of cancer-related death in the United States. Chemopreventive therapies could be effective way to treat CRC. Tolfenamic acid, one of the NSAIDs, shows anti-cancer activities in several types of cancer. Aberrant Wnt/β-catenin regulation pathway is a major mechanism of colon tumorigenesis. Here, we sought to better define the mechanism by which tolfenamic acid suppresses colorectal tumorigenesis focusing on regulation of β-catenin pathway. Treatment of tolfenamic acid led to a down-regulation of β-catenin expression in dose dependent manner in human colon cancer cell lines without changing mRNA. MG132 inhibited tolfenamic acid-induced downregulation of β-catenin and exogenously overexpression β-catenin was stabilized in the presence of tolfenamic acid. Tolfenamic acid induced an ubiquitin-mediated proteasomal degradation of β-catenin. In addition, tolfenamic acid treatment decreased transcriptional activity of β-catenin and expression of Smad2 and Smad3 while overexpression of Smad 2 inhibited tolfenamic acid-stimulated transcriptional activity of β-catenin. Moreover, tolfenamic acid decreased β-catenin target gene such as vascular endothelial growth factor (VEGF) and cyclin D1. In summary, tolfenamic acid is a promising therapeutic drug targeting Smad 2-mediated downregulation of β-catenin in CRC.

Antioxidant and detoxify genes polymorphisms in colorectal cancer predisposition

Colorectal cancer (CRC) results from histologic and gene alterations can lead to a massive cellular proliferation. Most of the authors assume multifactorial causes to CRC genesis. Low physical activity, a fat diet poor in fibers and smoking habits seems to have an important role in CRC. However, there are also genetic causes associated with CRC risk. It has been described that oxidative stress levels could influence CRC development. Thus, cellular balance reactive species and defense enzymes involved in oxidative stress are crucial to maintain a good tissue function and avoid neoplasic process. Therefore, genome variations on these defense enzymes, such as MNSOD, SOD3, GSTP1, GSTT1 and GSTM1, could be important biomarkers to colorectal adenocarcinomas. We intend to determine frequencies distribution of most common polymorphisms involved on oxidative stress regulation (MNSOD, SOD3, GSTP1, GSTT1 and GSTM1) in patients with sporadic colorectal adenocarcinoma (SCA) and in healthy controls, evaluation their possible correlation with SCA risk. Samples common polymorphisms of antioxidant and detoxify genes (MNSOD T175C, SOD3 R213G, GSTP1 A105G, GSTP1 C114T, GSTT1del and GSTM1del) analysis was done by PCR-SSP techniques. In this study we found a higher prevalence of MNSOD 175CC (55% vs 2%; p<0.0001; OR: 58.5; CI 13.3 to 256.7), SOD3 213GG (31% vs 2%; p<0.0001; OR: 21.89; CI 4.93 to 97.29), GSTP1 105GG (46% vs 12%; p<0.0001; OR: 6.14; CI 2.85 to 13.26), GSTP1 114TT (38% vs 0%; p<0.0001; OR: Infinity) and GSTT1 null (75% vs 28%; p<0.0001; OR: 7.71; CI 3.83 to 15.56) mutated genotypes among SCA patients, while the normal genotypes were associated with SCA absence. Furthermore, we found GSTP1 114TT mutated genotype (52% vs 27%; p=0.003; OR: 2.88; CI: 1.41 to 5.89) and GSTT1 null genotype (87% vs 65%; p=0.003; OR: 3.66; CI 1.51 to 8.84) associated with colon samples. These findings suggest a positive association between most of common polymorphisms involved on oxidative stress regulation and SCA prevalence. Dysregulation of MNSOD, SOD3, GSTP1, GSTT1 and GSTM1 genes could be associated with an increase of ROS in colon and rectum tissue and p53 pathway deregulation, induced by oxidative stress on colonic and rectal cells. The present study also provides preliminary evidence that MNSOD 175C, SOD3 213G, GSTP1 105G, GSTP1 114T and GSTT1 null polymorphisms, may be involved in SCA risk and could be useful to clarify this multifactorial disorder.

Investigating the role of N-WASP in the development of colorectal cancer

Colorectal cancer (CRC) is the second most common cancer in Europe, with the second highest mortality rate. Although prognosis is improving, survival rates remain poor for those presenting with the most advanced stages of the disease. There is therefore a need for improved early diagnosis and thus a greater understanding of the early stages of the development of colorectal tumours is desirable. Additionally, as most deaths in colorectal cancer are due to advanced metastatic disease, it is of great interest to explore any potential mechanisms by which metastatic disease can be inhibited. N-WASP is a ubiquitously expressed protein with multiple intracellular roles including actin regulation and maintaining stability of epithelial cell-cell junctions. Through its role as an actin regulator, it has been implicated in the processes of invasion and metastasis of multiple cancer types. Its role in the development and progression of colorectal cancer however has not been fully explored. This thesis will present a series of in vitro and in vivo studies that were carried out with the aim of answering the following questions: • Does N-Wasp have a role in normal intestinal homeostasis? • Does N-Wasp knockout affect the development of tumours in a mouse model of intestinal tumourigenesis? • Does N-Wasp knockout affect the invasive properties of intestinal cancer in vitro? • Does N-WASP correlate with prognosis or other indicators in human colorectal cancer TMAs? Findings from the in vivo experiments, using an inducible, gut-specific knockout model, have uncovered potential roles for N-Wasp in regulating differentiation and migration of intestinal epithelial cells. Although it had no effect in short term models of intestinal hyperproliferation, N-Wasp knockout increased tumour burden and decreased survival in an established in vivo model of intestinal tumourigenesis, in which there is heterozygous loss of Apc (Apcfl/+). No effect was seen on tumour development or survival when additional N-WASP knockout was introduced into a more rapid model, with heterozygous loss of Apc and mutation of Kras (Apcfl/+ KrasG12D/+). N-WASP expression in human colorectal cancer was assessed using immunohistochemical staining of two tissue microarrays. Low levels of N-WASP expression were found to be associated with presence of MMR deficiency. There was no statistically significant difference in overall or cancer specific survival based on N-WASP expression. Collectively, the data presented here suggest a previously unreported role for N-WASP in regulation of intestinal epithelial differentiation and indicate that it may act as a tumour suppressor against development of benign precursor lesions of colorectal cancer. Further research is warranted to delineate the mechanisms underlying these processes.

The Role of PME-1 in Cancer: Therapeutic Implication

Protein phosphatase 2A (PP2A) plays a major role in maintaining cellular signaling homeostasis in human cells by reversibly affecting the phosphorylation of a variety of proteins. Protein phosphatase methylesterase-1 (PME-1) negatively regulates PP2A activity by reversible demethylation and active site binding. Thus far, it is known that overexpression of PME-1 in human gliomas contributes to ERK pathway signaling, cell proliferation, and malignant progression. Whether PME-1-mediated PP2A inhibition promotes therapy resistance in gliomas is unknown. Specific PP2A targets regulated by PME-1 in cancers also remain elusive. Additionally, whether oncogenic function of PME-1 can be generalized to various human cancers needs to be investigated. This study demonstrated that PME-1 expression promotes kinase inhibitor resistance in glioblastoma (GBM). PME-1 silencing sensitized GBM cells to a group of clinically used indolocarbazole multikinase inhibitors (MKIs). To facilitate the quantitative evaluation of MKIs by cancer-cell specific colony formation assay, Image-J software-plugin ‘ColonyArea’ was developed. PME-1-silencing was found to reactivate specific PP2A complexes and affect PP2A-target histone deacetylase HDAC4 activity. The HDAC4 inhibition induced synthetic lethality with MKIs similar to PME-1 depletion. However, synthetic lethality by both approaches required co-expression of a pro-apoptotic protein BAD. In gliomas, PME-1 and HDAC4 expression was associated with malignant progression. Using tumor PME-1, HDAC4 and BAD expression based stratification signatures this study defined patient subgroups that are likely to respond to MKI alone or in combination with HDAC4 inhibitor therapies. In contrast to the oncogenic role of PME-1 in certain cancer types, this study established that colorectal cancer (CRC) patients with high tumor PME-1 expression display favorable prognosis. Interestingly, PME-1 regulated survival signaling did not operate in CRC cells. Summarily, this study potentiates the candidacy of PME-1 as a therapy target in gliomas, but argues against generalization of these findings to other cancers, especially CRC.

NOD2 mutations and colorectal cancer - Where do we stand?

Due to the overwhelming burden of colorectal cancer (CRC), great effort has been placed on identifying genetic mutations that contribute to disease development and progression. One of the most studied polymorphisms that could potentially increase susceptibility to CRC involves the nucleotide-binding and oligomerization-domain containing 2 (NOD2) gene. There is growing evidence that the biological activity of NOD2 is far greater than previously thought and a link with intestinal microbiota and mucosal immunity is increasingly sought after. In fact, microbial composition may be an important contributor not only to inflammatory bowel diseases (IBD) but also to CRC. Recent studies have showed that deficient NOD2 function confers a communicable risk of colitis and CRC. Despite the evidence from experimental models, population-based studies that tried to link certain NOD2 polymorphisms and an increase in CRC risk have been described as conflicting. Significant geographic discrepancies in the frequency of such polymorphisms and different interpretations of the results may have limited the conclusions of those studies. Since being first associated to IBD and CRC, our understanding of the role of this gene has come a long way, and it is tempting to postulate that it may contribute to identify individuals with susceptible genetic background that may benefit from early CRC screening programs or in predicting response to current therapeutic tools. The aim of this review is to clarify the status quo of NOD2 mutations as genetic risk factors to chronic inflammation and ultimately to CRC. The use of NOD2 as a predictor of certain phenotypic characteristics of the disease will be analyzed as well.