Angiogenin regulates PKD activation and COX-2 expression induced by TNF-α and bradykinin in the colonic myofibroblast.

INTRODUCTION: The myofibroblast is a gastrointestinal stromal cell that is a target of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine strongly implicated in colitis-associated cancer. Crosstalk between TNF-α and other pro-inflammatory mediators amplify inflammatory signaling but the mechanism is unknown. Angiogenin (ANG) is a 14-kDa angiogenesis protein that is regulated in patients with inflammatory bowel disease. However, the role of ANG on inflammatory mediator crosstalk in the myofibroblast is unknown. METHODS: The human colonic myofibroblast cell line 18Co, as well as primary mouse and human colonic myofibroblasts, were exposed to TNF-α (10 ng/ml) and bradykinin (BK, 100 nM). ANG was quantified by ELISA. The expression of cyclo-oxygenase-2 (COX-2) and phosphorylation of PKD was assessed by Western Blot. RESULTS: Primary mouse and human colonic myofibroblasts exposed to TNF-α/BK led to enhanced PKD phosphorylation and synergistic COX-2 expression. 18Co cells secrete high levels of ANG (24h, 265 ± 5 pg/ml). The monoclonal antibody 26-2F, which neutralizes ANG, inhibited TNF-α/BK-mediated PKD phosphorylation and synergistic COX-2 expression in primary human myofibroblasts. Likewise, in primary mouse myofibroblasts that do not express ANG (ANG-KO), TNF-α/BK failed to enhance PKD phosphorylation and COX-2 expression. CONCLUSIONS: TNF-α/BK enhance PKD phosphorylation and COX-2 expression in primary mouse and human colonic myofibroblasts. Angiogenin is produced by the myofibroblast, and inhibition of ANG signaling, either by its absence (ANG-KO) or by pharmacologic inhibition, blocks enhanced PKD phosphorylation and synergistic COX-2 expression induced by TNF-α/BK. ANG mediates crosstalk signaling between TNF-α/BK in the regulation of stroma-derived COX-2 and may be a novel therapeutic target for the management of colitis-associated cancer.

Myofibroblasts Enhance Tumor Growth in a Novel Mouse Model of Colorectal Cancer.

BACKGROUND: Communication between colorectal cancer and stromal cells alters the tumor microenvironment to regulate locoregional disease and cancer progression. However, colon cancer-stromal cell interactions are difficult to study in vivo. Limitations of existing animal models include the use of immunocompromised mice, the inability to genetically modify a cell population in a single organ system, or a lack of anatomic context. Our goal was to develop a novel mouse model of colorectal cancer that is capable of studying tumor-stromal cell interactions in the native colon of immune-competent mice. METHODS: Primary mouse myofibroblasts were isolated from the colon of C57BL/6 mice and were grown in cell culture. Genetically defined (ApcΔ/Δ; Kras G12D/+; Trp53Δ/Δ) primary mouse colon cancer cells were suspended in serum-free media (20 µL) at varying concentrations (5 × 103 to 4 × 104 cells) either alone or in combination with syngeneic myofibroblasts (2 × 105 cells). After isoflurane anesthesia, a colonoscopy was performed on immune-competent 8- to 10-week-old C57BL/6 mice with endoscopic microinjection of the cell suspension into the submucosal space of the colon wall utilizing a small animal colonoscope. Surveillance endoscopy was used to assess for tumor growth, along with histologic analysis. Tumor size is presented on a grading system based on tumor diameter relative to colon circumference. RESULTS: A total of 33 mice were injected with a survival rate of 88% (29/33). Endoscopic microinjection of colorectal cancer cells resulted in dose-dependent tumor growth in the distal mouse colon that could be assessed endoscopically without animal sacrifice. Growth curves varied depending on the concentration of injected colorectal cancer cells, with no growth at the lowest concentration of injected cells (5 × 103 cells), progressive growth over 4 wk using 1-2 × 104 cells, while the highest colorectal cancer cell concentration (4 × 104 cells) led to larger tumors at week 1 followed by a steady decline in tumor growth over the 4-wk time period. Combined microinjection of 2 × 104 colorectal cancer cells with 2 × 105 myofibroblasts resulted in much larger tumors that persisted over the 4-wk time period and which were composed primarily of colorectal cancer cells. Immunofluorescence microscopy after coinjection of colorectal cancer cells with green fluorescent protein positive myofibroblasts confirmed that the injected myofibroblasts are present and remain viable over the 4-wk time period. CONCLUSIONS: Endoscopic submucosal microinjection of primary mouse colorectal cancer cells is feasible and leads to reliable and reproducible short-term growth of colon tumors in immune-competent mice. Coinjection of primary mouse colorectal cancer cells with syngeneic myofibroblasts leads to enhanced tumor growth. Coimplantation of colorectal cancer cells with syngeneic myofibroblasts provides a novel platform to study tumor-stromal interactions in the native colon of immune-competent mice.

Combined endo-laparoscopic surgery (CELS) for benign colon polyps: a single institution cost analysis.

BACKGROUND: Endoscopic removal of benign colon polyps is not always possible, even with advanced endoscopic techniques. Segmental colectomy has been the traditional therapy but is associated with an increased risk of complications and may be unnecessary since fewer than 20% of these polyps harbor malignancy. Combined endo-laparoscopic surgery (CELS) has emerged as an alternative method to address these polyps. While feasibility, safety, and improved short-term patient outcomes have been demonstrated, there has never been an evaluation of cost comparing these two approaches within a single institution. METHODS: In this observational cohort study, we compared short-term outcomes and costs of 11 patients who underwent CELS for right colon polyps with 11 patients who underwent a laparoscopic right colectomy between April 2014 and November 2017. The cost analysis covered the perioperative period from operating room to hospital discharge. RESULTS: A total of 11 patients underwent an attempted CELS procedure for right colon polyps with a success rate of 90% (10/11). The median length of stay (LOS) for CELS patients was 1 day. LOS for patients who underwent a laparoscopic right colectomy at TMC was 3.82 days. The median OR time for CELS was 166.73 (± 57.88) min, compared to 204.73 (± 51.49) min for a laparoscopic right colectomy. The calculated total cost for a CELS patient was $5523.29, compared to $12,626.33 for a laparoscopic right colectomy, for a cost-savings of $7103.04 per patient. CONCLUSIONS: CELS procedures are associated with good short-term outcomes and are performed at a lower cost compared to traditional laparoscopic colectomy, with the most significant cost saver being shorter hospital LOS. This is the first study to directly compare the cost of CELS to traditional laparoscopic colectomy in the surgical management of benign colon polyps within a single institution.

A CRISPR reimagining: New twists and turns of CRISPR beyond the genome-engineering revolution.

Despite its explosive applications in genome engineering, CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) has been developed into a versatile tool beyond its well-known nuclease function. In this prospect article, we summarize a few exciting "off-label" applications of CRISPR including manipulating DNA sequences, visualizing chromosomal loci in living cells, and modulating transcription and chromatin structures. These novel applications will likely elevate CRISPR tools into yet another level of sophistication and diversity, leading to many more exciting cell biological discoveries.

Case Studies of Community-Academic Partnerships Established Using the Give-Get Grid Model.

While partnerships for health delivery and improvement are frequently described by their structure, goals, and plans, less attention is paid to the interactive relationships among partners or for larger stakeholder groups' coalition memberships. The Give-Get Grid group process tool can be used to assess each stakeholders' expected benefits ("gets") and contributions ("gives") needed to establish and maintain long-term, mutually advantageous community-academic partnerships. This article describes three case study experiences using the Give-Get Grid in real-world context to understand and generate ideas to address contemporary health promotion opportunities among a variety of stakeholders. The case studies address three distinct community health promotion opportunities: prevention of school-based adolescent obesity disparities, higher education health professions training programs in rural community-based settings, and methods for engaging community coalitions in state Comprehensive Cancer Control Programs. The case studies demonstrate the Give-Get Grid's utility in both planning and evaluating partnerships and documenting key elements for progress in health promotion initiatives built on long-term community-academic relationships. Steps are explained with practical lessons learned in using the Grid.