Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue.

Colon and rectal tumors, often referred to as colorectal cancer, show different gene expression patterns in studies that analyze whole tissue biopsies containing a mix of tumor and non-tumor cells. To better characterize colon and rectal tumors, we investigated the gene expression profile of organoids generated from endoscopic biopsies of rectal tumors and adjacent normal colon and rectum mucosa from therapy-naive rectal cancer patients. We also studied the effect of vitamin D on these organoid types. Gene profiling was performed by RNA-sequencing. Organoids from a normal colon and rectum had a shared gene expression profile that profoundly differed from that of rectal tumor organoids. We identified a group of genes of the biosynthetic machinery as rectal tumor organoid-specific, including those encoding the RNA polymerase II subunits POLR2H and POLR2J. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3/calcitriol upregulated stemness-related genes (LGR5, LRIG1, SMOC2, and MSI1) in normal rectum organoids, while it downregulated differentiation marker genes (TFF2 and MUC2). Normal colon and rectum organoids share similar gene expression patterns and respond similarly to calcitriol. Rectal tumor organoids display distinct and heterogeneous gene expression profiles, with differences with respect to those of colon tumor organoids, and respond differently to calcitriol than normal rectum organoids.

Vitamin D differentially regulates colon stem cells in patient-derived normal and tumor organoids.

Intestine is a major target of vitamin D and several studies indicate an association between vitamin D deficiency and inflammatory bowel diseases (IBD), but also increased colorectal cancer (CRC) risk and mortality. However, the putative effects of 1α,25-dihydroxyvitamin D3 (calcitriol), the active vitamin D metabolite, on human colonic stem cells are unknown. Here we show by immunohistochemistry and RNAscope in situ hybridization that vitamin D receptor (VDR) is unexpectedly expressed in LGR5+ colon stem cells in human tissue and in normal and tumor organoid cultures generated from patient biopsies. Interestingly, normal and tumor organoids respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. In normal organoids, calcitriol upregulates stemness-related genes, such as LGR5, SMOC2, LRIG1, MSI1, PTK7, and MEX3A, and inhibits cell proliferation. In contrast, in tumor organoids calcitriol has little effect on stemness-related genes while it induces a differentiated phenotype, and variably reduces cell proliferation. Concordantly, electron microscopy showed that calcitriol does not affect the blastic undifferentiated cell phenotype in normal organoids but it induces a series of differentiated features in tumor organoids. Our results constitute the first demonstration of a regulatory role of vitamin D on human colon stem cells, indicating a homeostatic effect on colon epithelium with relevant implications in IBD and CRC.

Plocabulin Displays Strong Cytotoxic Activity in a Personalized Colon Cancer Patient-Derived 3D Organoid Assay.

Plocabulin is a novel microtubule-disrupting antitumor agent of marine origin that is currently undergoing phase II clinical trials. Plocabulin has potent antiproliferative and antiangiogenic actions in carcinoma cell lines and has antitumor activity in xenografted mice. Here, we used three-dimensional (3D) tumor organoids derived from three colorectal cancer (CRC) patients to study the effect of plocabulin in a personalized assay system that ensures dose dependence and high reproducibility. The cytotoxicity of plocabulin was an order of magnitude higher than that of the active irinotecan derivative SN38 (7-ethyl-10-hydroxy-camptothecin) in tumor organoids at different passages. Moreover, plocabulin maintained its strong cytotoxic activity in wash-out experiments, in which a short pulse treatment of tumor organoids was as efficient as continuous treatment. Our data show that plocabulin has a very potent cytotoxic action in CRC patient-derived tumor organoids, supporting ongoing clinical trials with plocabulin and the use of organoid assays to provide personalized validation of antitumor drugs.

Vitamin D and Wnt3A have additive and partially overlapping modulatory effects on gene expression and phenotype in human colon fibroblasts.

The Wnt/ß-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/ß-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)2D3 and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)2D3 and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)2D3 and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)2D3 reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)2D3 and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC.

The human PKP2/plakophilin-2 gene is induced by Wnt/ß-catenin in normal and colon cancer-associated fibroblasts.

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/ß-catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD-18Co cells. A set of genes encoding inhibitors of the Wnt/ß-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin-2 is a novel direct transcriptional target of Wnt/ß-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by ß-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/ß-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/ß-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells.

Vitamin D receptor expression and associated gene signature in tumour stromal fibroblasts predict clinical outcome in colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is a major health concern. Vitamin D deficiency is associated with high CRC incidence and mortality, suggesting a protective effect of vitamin D against this disease. Given the strong influence of tumour stroma on cancer progression, we investigated the potential effects of the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on CRC stroma. DESIGN: Expression of vitamin D receptor (VDR) and two 1,25(OH)2D3 target genes was analysed in 658 patients with CRC with prolonged clinical follow-up. 1,25(OH)2D3 effects on primary cultures of patient-derived colon normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) were studied using collagen gel contraction and migration assays and global gene expression analyses. Publicly available data sets (n=877) were used to correlate the 1,25(OH)2D3-associated gene signature in CAFs with CRC outcome. RESULTS: High VDR expression in tumour stromal fibroblasts was associated with better overall survival (OS) and progression-free survival in CRC, independently of its expression in carcinoma cells. 1,25(OH)2D3 inhibited the protumoural activation of NFs and CAFs and imposed in CAFs a 1,25(OH)2D3-associated gene signature that correlated with longer OS and disease-free survival in CRC. Furthermore, expression of two genes from the signature, CD82 and S100A4, correlated with stromal VDR expression and clinical outcome in our cohort of patients with CRC. CONCLUSIONS: 1,25(OH)2D3 has protective effects against CRC through the regulation of stromal fibroblasts. Accordingly, expression of VDR and 1,25(OH)2D3-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Therefore, treatment of patients with CRC with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.

Negative-Pressure Therapy to Reduce the Risk of Wound Infection Following Diverting Loop Ileostomy Reversal: An Initial Study.

OBJECTIVE: To evaluate if the application of a negative-pressure therapy system (Prevena Incision Management System, Kinetics Concepts Inc, [KCI] an Acelity Company, San Antonio, Texas) on ileostomy-closure surgical wounds would reduce surgical site infections (SSIs) in comparison with conventional closure and dressing. DESIGN: Prospective interventional pilot study. SETTING: La Paz University Hospital, tertiary care academic hospital in Madrid, Spain. PATIENTS: The Prevena device was applied on the wounds of 17 consecutive patients undergoing ileostomy reversal. Control subjects were 43 patients undergoing the same procedure, in which conventional dressings were used for the wound. INTERVENTION: The device was applied on the wound immediately after surgery (under sterile conditions) and maintained for 5 to 7 days. Patients were evaluated daily, and on the seventh postoperative day, the device was removed and wounds carefully inspected. Another evaluation was performed a month after the surgical intervention in the outpatient clinic. MAIN OUTCOME MEASURE: The primary end point of the study was the detection of SSI (defined according to the Centers for Disease Control and Prevention definitions). Other intervention-related complications were also registered. MAIN RESULTS: There were no significant differences in demographic variables between groups. In the control group, 9 patients (21%) presented SSI, with statistical significance (P < .038) when compared with the intervention group (0%). There were no complications associated with the application of the Prevena device. Other complications (for example, ileus or obstruction) occurred in 30% of patients. CONCLUSIONS: The negative-pressure Prevena System was safe and easy to use and may prevent SSIs in dirty wounds, such as those from ileostomy closure.

Galactomannan Downregulates the Inflammation Responses in Human Macrophages via NFκB2/p100.

We show that galactomannan, a polysaccharide consisting of a mannose backbone with galactose side groups present on the cell wall of several fungi, induces a reprogramming of the inflammatory response in human macrophages through dectin-1 receptor. The nuclear factor kappa-light-chain-enhancer of activated B cells 2 (NFκB2)/p100 was overexpressed after galactomannan challenge. Knocking down NFκB2/p100 using small interfering RNA (siRNA) indicated that NFκB2/p100 expression is a crucial factor in the progression of the galactomannan-induced refractoriness. The data presented in this study could be used as a modulator of inflammatory response in clinical situations where refractory state is required.

Increased postoperative complications after protective ileostomy closure delay: An institutional study.

AIM: To study the morbidity and complications associated to ileostomy reversal in colorectal surgery patients, and if these are related to the time of closure. METHODS: A retrospective analysis of 93 patients, who had undergone elective ileostomy closure between 2009 and 2013 was performed. Demographic, clinical and surgical variables were reviewed for analysis. All complications were recorded, and classified according to the Clavien-Dindo Classification. Statistical univariate and multivariate analysis was performed, setting a P value of 0.05 for significance. RESULTS: The patients had a mean age of 60.3 years, 58% male. The main procedure for ileostomy creation was rectal cancer (56%), and 37% had received preoperative chemo-radiotherapy. The average delay from creation to closure of the ileostomy was 10.3 mo. Postoperative complications occurred in 40% of the patients, with 1% mortality. The most frequent were ileus (13%) and wound infection (13%). Pseudomembranous colitis appeared in 4%. Increased postoperative complications were associated with delay in ileostomy closure (P = 0.041). Male patients had more complications (P = 0.042), mainly wound infections (P = 0.007). Pseudomembranous colitis was also associated with the delay in ileostomy closure (P = 0.003). End-to-end intestinal anastomosis without resection was significantly associated with postoperative ileus (P = 0.037). CONCLUSION: Although closure of a protective ileostomy is a fairly common surgical procedure, it has a high rate of complications, and this must be taken into account when the indication is made. The delay in stoma closure can increase the rate of complications in general, and specifically wound infections and colitis.

Cirugía transanal a través de puerto único (TAMIS). Revisión frente otras técnicas de excisión endoscópica de lesiones rectales / Transanal surgery through single port (TAMIS). Revision front of the techniques of endoscopic excision of rectal lesions

Introducción: los programas de screening y la mejora en las técnicas diagnósticas han aumentado el porcentaje de tumores rectales que se pueden tratar en estadios iniciales. La morbimortalidad asociada a la cirugía radical de la neoplasia rectal, así como la tendencia a una cirugía menos agresiva, ha hecho que se desarrollen las técnicas de abordaje transanal, aplicando las últimas tecnologías disponibles. Material y métodos: se realizó una revisión de la literatura, considerando las diferentes técnicas de excisión endoscópica. Resultados: la cirugía endoscópica transanal, en todas sus variantes, ha demostrado ser un abordaje seguro para el tratamiento de adenomas y tumores rectales en estadios iniciales (pT1N0). Conclusión: son necesarios más estudios que permitan demostrar la seguridad de ampliar esta técnica para el tratamiento de lesiones avanzadas, asociada a quimio-radioterapia neoadyuvante.

Background: screening programs and improvement of diagnostic techniques have increased the percentage of rectal tumors that can be treated in an early stage. Radical surgery of the rectum is associated with high morbimortality, and the general tendency towards a less aggressive surgery has led to the development of transanal techniques, adjusting the latest technologies available. Methods: a review of the literature, including the different types of endoscopic surgery available. Results: transanal endoscopic microsurgery, in all its variants, has proven to be a safe and effective method for treating rectal adenomas and early stage cancer (pT1N0). Conclusion: further studies are required to prove the safeness of these techniques on more advanced lesions, in association with neoadjuvant chemoradiation therapy.

Cirugía abierta vs. laparoscópica: calidad de vida y sintomatología ansioso-depresiva en cáncer colorrectal / Laparoscopic vs. open surgery: quality of life and anxious-depressive symptomatology in colorectal cancer

Introducción: El objetivo del presente estudio es evaluar la calidad de vida, y la sintomatología ansioso-depresiva, en una muestra de pacientes de cáncer colorrectal sometidos a tratamiento quirúrgico, por vía abierta o laparoscópica. Material y Métodos: 60 pacientes fueron evaluados en Calidad de vida, mediante el cuestionario de la EORTC QLQ-C30, y sintomatología ansioso-depresiva mediante la escala de Ansiedad y Depresión Hospitalaria (HADS). Además se realizaron análisis en función de tres variables sociodemográficas: edad, género y nivel socioeducativo. Resultados: No se encontraron diferencias significativas en función de la modalidad quirúrgica en calidad de vida y sintomatología ansioso-depresiva. La mayor parte de los pacientes presenta niveles de calidad de vida muy satisfactorios, si bien un 27% presentan problemas de calidad de vida. Las medias en sintomatología ansioso-depresiva se encuentran en los rangos normales, no obstante el 18% presenta sintomatología depresiva y el 22% ansiosa. Discusión y Conclusiones: Se puede concluir que los dos tipos de cirugía, a medio y largo plazo, tienen efectos similares sobre la calidad de vida y la sintomatología ansioso-depresiva de los pacientes. No obstante, aun estando libres de tratamiento activo y habiendo transcurrido más de un año de media desde la cirugía, se ha encontrado que hay pacientes que presentan problemas en su calidad de vida, y sintomatología ansioso-depresiva. Por ello es necesario que éstos sean evaluados psicológicamente para prestarles la atención necesaria, y así optimizar la calidad asistencial.

Background: The aim of this study was to assess the quality of life, and anxious-depressive symptoms in a sample of colorectal cancer patients undergoing surgical treatment, open or laparoscopic approach. Material and Method: 60 patients were assessed on quality of life, through the questionnaire of the EORTC QLQ-C30, and anxious-depressive symptomatology scale by Hospital Anxiety and Depression (HADS). Further analyzes were conducted according to three demographic variables: age, gender and socio-educational level. Results: No significant differences were found related to surgical type in quality of life and anxious-depressive symptomatology. Most of the patients have very satisfactory quality of life levels, while 27% of them have problems about it. Means in anxious-depressive symptoms are at normal range, despite of 18% whom presented depressive symptoms and 22% anxiety. Conclusion: It can be concluded that the two types of surgery, in a medium and long term, have similar effects on quality of life and anxious-depressive symptoms of patients. However, even free of active treatment and over a year after surgery, we found that there are patients who have problems in their quality of life and anxious-depressive syndrome. Therefore it is necessary to evaluate and to give them the attention needed and optimize quality of care.

[Doppler-guided transanal haemorrhoidal dearterialisation. An alternative treatment for haemorrhoids]. / Desarterialización hemorroidal transanal guiada por Doppler. Una alternativa en el tratamiento de las hemorroides.

INTRODUCTION: The frequency of haemorrhoid disease and the deterioration in the quality of life in the immediate post-operative period has led to the appearance of new techniques in an attempt to obtain improve patient satisfaction. PATIENTS AND METHOD: A prospective study was carried out in which 50 consecutive patients with a diagnosis of Goligher grade III haemorrhoids were intervened. To perform the haemorrhoid dearterialisation, a device called THD R was used (designed by TKC SRL and distributed by Palex Medical). The technique consisted of, a reduction in arterial flow using ligation of the terminal branches above the anorectal ring, starting in the anterior position, it was carried out in a clockwise direction: 1, 3, 5, 7, 9, 11. Follow up was carried out at one week, 1 month, 3 months, 6 months and 1 year. RESULTS: We intervened 50 consecutive patients with a diagnosis of grade III haemorrhoids. The mean age was 45 years (range, 25-78). The surgical indication was, pain-discomfort, 40 (80%); bleeding, 35 (70%), prolapse 6 (12%). The procedure was always performed under local/regional anaesthesia. The mean duration of the procedure was 25 minutes (range, 20-35). Analgesia was required by 90% of the patients during the first 24 hours, decreasing to 15% for those who continued to require it until the third day and only 2 (4%) patients continued for one week. Pain was resolved 48 hours after surgery, in all patients who consulted for this reason, except for one patient (2.5%) who had a recurrence in the pain as well as in his prolapse. This meant that patients could re-start their daily living within 48-72 hours. CONCLUSIONS: Pending for randomised studies, we can say that in our experience, Doppler guided transanal haemorrhoidal dearterialisation is a technique that should be offered to the patient with haemor-rhoidal disease.