Quantitative Metabolomics and Lipoprotein Analysis of PDAC Patients Suggests Serum Marker Categories for Pancreatic Function, Pancreatectomy, Cancer Metabolism, and Systemic Disturbances.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to diagnose in the early stages and lacks reliable biomarkers. The scope of this project was to establish quantitative nuclear magnetic resonance (NMR) spectroscopy to comprehensively study blood serum alterations in PDAC patients. Serum samples from 34 PDAC patients obtained before and after pancreatectomy as well as 83 age- and sex-matched control samples from healthy donors were analyzed with in vitro diagnostics research (IVDr) proton NMR spectroscopy at 600 MHz. Uni- and multivariate statistics were applied to identify significant biofluid alterations. We identified 29 significantly changed metabolites and 98 lipoproteins when comparing serum from healthy controls with those of PDAC patients. The most prominent features were assigned to (i) markers of pancreatic function (e.g., glucose and blood triglycerides), (ii) markers related to surgery (e.g., ketone bodies and blood cholesterols), (iii) PDAC-associated markers (e.g., amino acids and creatine), and (iv) markers for systemic disturbances in PDAC (e.g., gut metabolites DMG, TMAO, DMSO2, and liver lipoproteins). Quantitative serum NMR spectroscopy is suited as a diagnostic tool to investigate PDAC. Remarkably, 2-hydroxybutyrate (2-HB) as a previously suggested marker for insulin resistance was found in extraordinarily high levels only after pancreatectomy, suggesting this metabolite is the strongest marker for pancreatic loss of function.

Differential effects of heat-inactivated, secretome-deficient MSC and metabolically active MSC in sepsis and allogenic heart transplantation.

Mesenchymal stem cells (MSCs) are used in various clinical and preclinical models for immunomodulation. However, it remains unclear how the immunomodulatory effect of MSC is communicated. MSC-induced immunomodulation is known to be mediated through both MSC-secreted cytokines and direct cell-cell interactions. Recently, it has been demonstrated that metabolically inactive, heat-inactivated MSCs (HI-MSCs) have similar anti-inflammatory capacities in LPS-induced sepsis compared with viable MSC. To further investigate the immunomodulatory effects of MSC, we introduced MSC and HI-MSC in two animal models with different immunological causes. In the first model, allogeneic hearts were transplanted from C57BL/6 mice to BALB/c recipients. MSC in combination with mycophenolate mofetil (MMF) significantly improved graft survival compared with MMF alone, whereas the application of HI-MSC had no effect on graft survival. We revealed that control MSC dose-dependently inhibited CD3+ and CD8+ T-cell proliferation in vitro, whereas HI-MSC had no effect. In the second model, sepsis was induced in mice via cecal ligation and puncture. HI-MSC treatment significantly improved the overall survival, whereas control MSCs had no effect. in vitro studies demonstrated that HI-MSCs are more effectively phagocytosed by monocytes than control MSCs and induced cell death in particular of activated CD16+ monocytes, which may explain the immune protective effect of HI-MSC in the sepsis model. The results of our study demonstrate that MSC-mediated immunomodulation in sepsis is dependent on a passive recognition of MSC by monocytes, whereas fully functional MSCs are required for inhibition of T-cell-mediated allograft rejection.

The emergence of regenerative medicine in organ transplantation: 1st European Cell Therapy and Organ Regeneration Section meeting.

Regenerative medicine is emerging as a novel field in organ transplantation. In September 2019, the European Cell Therapy and Organ Regeneration Section (ECTORS) of the European Society for Organ Transplantation (ESOT) held its first meeting to discuss the state-of-the-art of regenerative medicine in organ transplantation. The present article highlights the key areas of interest and major advances in this multidisciplinary field in organ regeneration and discusses its implications for the future of organ transplantation.

Gd-EOB-DTPA-enhanced T1 relaxometry for assessment of liver function determined by real-time 13C-methacetin breath test.

OBJECTIVES: To determine whether liver function as determined by intravenous administration of 13C-methacetin and continuous real-time breath analysis can be estimated quantitatively from gadoxetic acid (Gd-EOB-DTPA)-enhanced magnetic resonance (MR) relaxometry. METHODS: Sixty-six patients underwent a 13C-methacetin breath test (13C-MBT) for evaluation of liver function and Gd-EOB-DTPA-enhanced T1-relaxometry at 3 T. A transverse 3D VIBE sequence with an inline T1 calculation based on variable flip angles was acquired prior to (T1 pre) and 20 min post-Gd-EOB-DTPA (T1 post) administration. The reduction rate of T1 relaxation time (rrT1) and T1 relaxation velocity index (∆R1) between pre- and post-contrast images was evaluated. 13C-MBT values were correlated with T1post, ∆R1 and rrT1, providing an MRI-based estimated 13C-MBT value. The interobserver reliability was assessed by determining the intraclass correlation coefficient (ICC). RESULTS: Stratified by three different categories of 13C-MBT readouts, there was a constant increase of T1 post with increasing progression of diminished liver function (p ≤ 0.030) and a constant significant decrease of ∆R1 (p ≤ 0.025) and rrT1 (p < 0.018) with progression of liver damage as assessed by 13C-methacetin breath analysis. ICC for all T1 relaxation values and indices was excellent (> 0.88). A simple regression model showed a log-linear correlation of 13C-MBT values with T1post (r = 0.57; p < 0.001), ∆R1 (r = 0.59; p < 0.001) and rrT1 (r = 0.70; p < 0.001). CONCLUSION: Liver function as determined using real-time 13C-methacetin breath analysis can be estimated quantitatively from Gd-EOB-DTPA-enhanced MR relaxometry. KEY POINTS: • Gd-EOB-DTPA-enhanced T1 relaxometry quantifies liver function • Gd-EOB-DTPA-enhanced MR relaxometry may provide parameters for assessing liver function before surgery • Gd-EOB-DTPA-enhanced MR relaxometry may be useful for monitoring liver disease progression • Gd-EOB-DTPA-enhanced MR relaxometry has the potential to become a novel liver function index.

Outcome of primary percutaneous stent-revascularization in patients with atherosclerotic acute mesenteric ischemia.

Background Patients with acute mesenteric ischemia (AMI) often exhibit severe co-morbidities and significant surgical risks, leading to high perioperative morbidity. Purpose To investigate the feasibility of primary percutaneous stent-revascularization (PPSR) in atherosclerotic AMI and its impact on patients' outcome. Material and Methods Retrospective analysis of 19 consecutive patients (7 women, 12 men; median age, 69 years) with AMI caused by atherosclerotic, non-embolic stenoses/occlusions of the splanchnic arteries and PPSR. Alternative minimally invasive techniques were excluded. Clinical characteristics including the Charlson Comorbidity Index adjusted by age (CCIa) and symptom duration, technical and clinical success of PPSR, clinical course, 30-day mortality, and follow-up were evaluated and compared to literature data for surgical approaches. Technical success was defined as residual stenosis of <30% in diameter. Clinical success was defined as resolution of symptoms of AMI and/or normalization of serum lactate after sole PPSR. Results The majority of patients presented with severe co-morbidities (CCIa >4 in 17 of 19 patients, 89%). Median symptom duration was 50 h. Technical and clinical success rates of PPSR were 95% (21 of 22 arteries) and 53% (10 of 19 patients). Seven patients underwent subsequent laparotomy with bowel resection in four cases. Thirty-day mortality was 42% (8 of 19 patients). Conclusion In our study population of patients with atherosclerotic AMI, severe co-morbidities, prolonged acute symptoms, and significant perioperative risks PPSR of splanchnic stenoses were technically feasible and the clinical outcome was acceptable.

Conversion of Th17 into IL-17A(neg) regulatory T cells: a novel mechanism in prolonged allograft survival promoted by mesenchymal stem cell-supported minimized immunosuppressive therapy.

The ultimate goal in transplantation medicine is the promotion of operational tolerance. Although Th cells of the Th17 type have been predominantly associated with rejection of allogeneic solid organ grafts, regulatory T (T(reg)) cells appear to foster operational tolerance. Induced T(reg) and Th17 cells have a higher lineage plasticity than has been recognized thus far. We found that when mesenchymal stem cells (MSCs) were used to induce long-term acceptance of allogeneic heart grafts in mice, the induction of T(reg) cells was preceded by development of a CD11b(hi)Gr1(int) myeloid-derived immunosuppressive cell-mediated Th17 response. Substantial suppression of Foxp3(+) T(reg) cell generation from retinoic acid receptor-related orphan receptor γ(-/-) T cells by MSCs revealed that retinoic acid receptor-related orphan receptor γ is a common factor in the differentiation of T(reg) and Th17 cells. Immunosuppressant mycophenolate mofetil treatment of enriched IL-17A(+) cells from MSC-primed allograft mouse recipients resulted in a reduction of IL-17A production and an increase in the Foxp3(+) T(reg) cell fraction. Furthermore, identification of IL-17A(+) Foxp3(+) double-positive and ex-IL-17-producing IL-17A(neg)Foxp3(+) T cells strongly argues for direct conversion of Th17 cells into T(reg) cells as the underlying mechanism of immune regulation in MSC-mediated allograft survival. The Th17 into T(reg) conversion identified in this study constitutes an important immunological mechanism by which MSC-induced myeloid-derived immunosuppressive cells mediate operational transplant tolerance. The possibility to create T(reg) cell-regulated operational tolerance in the absence of complete immune suppression provides strong clinical implications for cell therapy-assisted minimization protocols.

Mesenchymal stromal cells for immunoregulation after liver transplantation: the scene in 2016.

PURPOSE OF REVIEW: The current review presents an update on the existing preclinical and human experience of mesenchymal stromal cell (MSC) therapies for post-transplant immunomodulation. RECENT FINDINGS: Although results from early clinical studies have demonstrated that the application of autologous and allogeneic MSC to be both safe and feasible in a solid organ transplantation setting, for example in liver, the efficacy of MSC immunotherapy demonstrated in preclinical models has yet to be replicated in human clinical trials. SUMMARY: Eagerly awaited results from the second generation of solid organ transplantation clinical trials, many of which are nearing completion, will perhaps establish the effectiveness of combining MSCs and low-dose pharmacological immunosuppression in promoting graft acceptance. At present, the question of whether infusional cell products based on MSCs will have a significant clinical impact in the field of liver transplantation remains open.

Caveats of mesenchymal stem cell therapy in solid organ transplantation.

In the past decade, therapeutic use of mesenchymal stem cells (MSCs) has increased dramatically. The weight of existing evidence supports that the short-term application of MSCs is safe and feasible; however, concerns remain over the possibility of unwanted long-term effects. One fundamental difference between MSCs and pharmacotherapy is that, once applied, the effects of cell products cannot be easily reversed. Therefore, a carefully considered decision process is indispensable before cell infusion. In addition to unwanted interactions of MSCs with the host immune system, there are concerns that MSCs may promote tumor progression or even give rise to cancer themselves. As animal models and first-in-man clinical studies have provided conflicting results, it is challenging to estimate the long-term risk of individual patients. In addition, most animal models, especially rodents, are ill-suited to adequately address questions over long-term side effects. Based on the available evidence, we address the potential pitfalls for the use of MSCs as a therapeutic agent to control alloimmune effects. The aim of this review was not to discourage investigators from clinical studies, but to raise awareness of the intrinsic risks of MSC therapy.

Double deficiency for RORγt and T-bet drives Th2-mediated allograft rejection in mice.

Although Th1, Th2, and Th17 cells are thought to be major effector cells in adaptive alloimmune responses, their respective contribution to allograft rejection remains unclear. To precisely address this, we used mice genetically modified for the Th1 and Th17 hallmark transcription factors T-bet and RORγt, respectively, which allowed us to study the alloreactive role of each subset in an experimental transplant setting. We found that in a fully mismatched heterotopic mouse heart transplantation model, T cells deficient for T-bet (prone to Th17 differentiation) versus RORγt (prone to Th1 differentiation) rejected allografts at a more accelerated rate, indicating a predominance of Th17- over Th1-driven alloimmunity. Importantly, T cells doubly deficient for both T-bet and RORγt differentiated into alloreactive GATA-3-expressing Th2 cells, which promptly induced allograft rejection characterized by a Th2-type intragraft expression profile and eosinophilic infiltration. Mechanistically, Th2-mediated allograft rejection was contingent on IL-4, as its neutralization significantly prolonged allograft survival by reducing intragraft expression of Th2 effector molecules and eosinophilic allograft infiltration. Moreover, under IL-4 neutralizing conditions, alloreactive double-deficient T cells upregulated Eomesodermin (Eomes) and IFN-γ, but not GATA-3. Thus, in the absence of T-bet and RORγt, Eomes may salvage Th1-mediated alloimmunity that underlies IL-4 neutralization-resistant allograft rejection. We summarize that, whereas Th17 cells predictably promote allograft rejection, IL-4-producing GATA-3(+) Th2 cells, which are generally thought to protect allogeneic transplants, may actually be potent facilitators of organ transplant rejection in the absence of T-bet and RORγt. Moreover, Eomes may rescue Th1-mediated allograft rejection in the absence of IL-4, T-bet, and RORγt.

Retrograde stapling of a free cervical jejunal interposition graft: a technical innovation and case report.

BACKGROUND: Free jejunal interposition is a useful technique for reconstruction of the cervical esophagus. However, the distal anastomosis between the graft and the remaining thoracic esophagus or a gastric conduit can be technically challenging when located very low in the thoracic aperture. We here describe a modified technique for retrograde stapling of a jejunal graft to a failed gastric conduit using a circular stapler on a delivery system. CASE PRESENTATION: A 56 year-old patient had been referred for esophageal squamous cell carcinoma at 20 cm from the incisors. On day 8 after thoracoabdominal esophagectomy with gastric pull-up, an anastomotic leakage was diagnosed. A proximal-release stent was successfully placed by gastroscopy and the patient was discharged. Two weeks later, an esophagotracheal fistula occurred proximal to the esophageal stent. Cervical esophagostomy was performed with cranial closure of the gastric conduit, which was left in situ within the right hemithorax. Three months later, reconstruction was performed using a free jejunal interposition. The anvil of a circular stapler (Orvil®, Covidien) was placed transabdominally through an endoscopic rendez-vous procedure into the gastric conduit. A free jejunal graft was retrogradely stapled to the proximal end of the conduit. Microvascular anastomoses were performed subsequently. The proximal anastomosis of the conduit was completed manually after reperfusion. CONCLUSIONS: This modified technique allows stapling of a jejunal interposition graft located deep in the thoracic aperture and is therefore a useful method that may help to avoid reconstruction by colonic pull-up and thoracotomy.

Rationale and prospects of mesenchymal stem cell therapy for liver transplantation.

PURPOSE OF REVIEW: Despite their potential to supplement the donor organ pool, expanded donor criteria grafts are associated with an elevated risk of graft failure and increased early mortality. Likewise, attempts to promote operational graft tolerance through conventional immunosuppressive therapy have demonstrated significant safety-related drawbacks. Because of their potent regenerative and immunomodulative potential, adjunct mesenchymal stem cell (MSC) therapy represents an innovative approach to both of these clinical problems. RECENT FINDINGS: Recent studies have begun to delineate the benefit and mechanisms of short-term therapy combining MSCs and low-dose immunosuppressive drugs in promoting graft acceptance and potentially regeneration. SUMMARY: The current review presents our rationale for the first-in-man clinical trial in liver transplantation utilizing a mesenchymal cell product (MultiStem, Athersys, Cleveland, Ohio, USA). The long-term objective of this program is to safely minimize the dose of complementary immunosuppressive drugs while achieving long-term allograft survival and operational tolerance. The use of adjunct cellular therapy as a means of reducing long-term pharmacotherapy would represent a major advancement in the field of liver transplantation.

Establishing a New Platform to Investigate the Efficacy of Oncolytic Virotherapy in a Human Ex Vivo Peritoneal Carcinomatosis Model.

Oncolytic virotherapy constitutes a promising treatment option for many solid cancers, including peritoneal carcinomatosis (PC), which still represents a terminal stage of many types of tumors. To date, the in vitro efficacy of oncolytic viruses is mostly tested in 2D-cultured tumor cell lines due to the lack of realistic 3D in vitro tumor models. We have investigated the feasibility of virotherapy as a treatment option for PC in a human ex vivo peritoneum co-culture model. Human HT-29 cancer cells stably expressing marker genes GFP and firefly luciferase (GFP/luc) were cultured on human peritoneum and infected with two prototypic oncolytic viruses (GLV-0b347 and MeV-DsRed). Both viral constructs were able to infect HT-29 cells in patient-derived peritoneum with high tumor specificity. Over time, both GFP signal and luciferase activity decreased substantially, thereby indicating successful virus-induced oncolysis. Furthermore, immunohistochemistry stainings showed specific virotherapeutic infections of HT-29 cells and effective tumor cell lysis in infected co-cultures. Thus, the PC model established here provides a clinically relevant screening platform to evaluate the therapeutic efficacy of virotherapeutic compounds and also to investigate, in an autologous setting, the immunostimulatory potential of oncolytic viruses for PC in a unique human model system superior to standard 2D in vitro models.

How to Make Sense out of 75,000 Mesenchymal Stromal Cell Publications?

Mesenchymal stromal cells have been the subject of an expanding number of studies over the past decades. Today, over 75,000 publications are available that shine light on the biological properties and therapeutic effects of these versatile cells in numerous pre-clinical models and early-phase clinical trials. The massive number of papers makes it hard for researchers to comprehend the whole field, and furthermore, they give the impression that mesenchymal stromal cells are wonder cells that are curative for any condition. It is becoming increasingly difficult to dissect how and for what conditions mesenchymal stromal cells exhibit true and reproducible therapeutic effects. This article tries to address the question how to make sense of 75,000, and still counting, publications on mesenchymal stromal cells.

Pharmacologic Targeting of MMP2/9 Decreases Peritoneal Metastasis Formation of Colorectal Cancer in a Human Ex Vivo Peritoneum Culture Model.

BACKGROUND: Matrix metalloproteinases (MMPs) play a crucial role in tumour initiation, progression, and metastasis, including peritoneal carcinosis (PC) formation. MMPs serve as biomarkers for tumour progression in colorectal cancer (CRC), and MMP overexpression is associated with advanced-stage metastasis and poor survival. However, the molecular mechanisms of PC from CRC remain largely unclear. METHODS: We investigated the role of MMPs during peritoneal colonisation by CRC cell lines in a human ex vivo peritoneum model and in patient-derived CRC and corresponding PC samples. MMP2 and MMP9 were inhibited using the small-molecule inhibitors batimastat and the specific MMP2/9 inhibitor III. RESULTS: MMP2 and MMP9 were strongly upregulated in patient-derived samples and following peritoneal colonisation by CRC cells in the ex vivo model. MMP inhibition with batimastat reduced colonisation of HT29 and Colo205 cells by 36% and 68%, respectively (p = 0.0073 and p = 0.0002), while MMP2/9 inhibitor III reduced colonisation by 50% and 41%, respectively (p = 0.0003 and p = 0.0051). Fibronectin cleavage was enhanced in patient-derived samples of PC and during peritoneal colonisation in the ex vivo model, and this was inhibited by MMP2/9 inhibition. CONCLUSION: MMPs were upregulated in patient-derived samples and during peritoneal attachment of CRC cell lines in our ex vivo model. MMP2/9 inhibition prevented fibronectin cleavage and peritoneal colonisation by CRC cells. MMP inhibitors might thus offer a potential treatment strategy for patients with PC.

Triple Targeting of HER Receptors Overcomes Heregulin-mediated Resistance to EGFR Blockade in Colorectal Cancer.

Current treatment options for patients with advanced colorectal cancers include anti-EGFR/HER1 therapy with the blocking antibody cetuximab. Although a subset of patients with KRAS WT disease initially respond to the treatment, resistance develops in almost all cases. Relapse has been associated with the production of the ligand heregulin (HRG) and/or compensatory signaling involving the receptor tyrosine kinases HER2 and HER3. Here, we provide evidence that triple-HER receptor blockade based on a newly developed bispecific EGFR×HER3-targeting antibody (scDb-Fc) together with the HER2-blocking antibody trastuzumab effectively inhibited HRG-induced HER receptor phosphorylation, downstream signaling, proliferation, and stem cell expansion of DiFi and LIM1215 colorectal cancer cells. Comparative analyses revealed that the biological activity of scDb-Fc plus trastuzumab was sometimes even superior to that of the combination of the parental antibodies, with PI3K/Akt pathway inhibition correlating with improved therapeutic response and apoptosis induction as seen by single-cell analysis. Importantly, growth suppression by triple-HER targeting was recapitulated in primary KRAS WT patient-derived organoid cultures exposed to HRG. Collectively, our results provide strong support for a pan-HER receptor blocking approach to combat anti-EGFR therapy resistance of KRAS WT colorectal cancer tumors mediated by the upregulation of HRG and/or HER2/HER3 signaling.

Safety and feasibility of third-party multipotent adult progenitor cells for immunomodulation therapy after liver transplantation--a phase I study (MISOT-I).

BACKGROUND: Liver transplantation is the definitive treatment for many end-stage liver diseases. However, the life-long immunosuppression needed to prevent graft rejection causes clinically significant side effects. Cellular immunomodulatory therapies may allow the dose of immunosuppressive drugs to be reduced. In the current protocol, we propose to complement immunosuppressive pharmacotherapy with third-party multipotent adult progenitor cells (MAPCs), a culture-selected population of adult adherent stem cells derived from bone marrow that has been shown to display potent immunomodulatory and regenerative properties. In animal models, MAPCs reduce the need for pharmacological immunosuppression after experimental solid organ transplantation and regenerate damaged organs. METHODS: Patients enrolled in this phase I, single-arm, single-center safety and feasibility study (n = 3-24) will receive 2 doses of third-party MAPCs after liver transplantation, on days 1 and 3, in addition to a calcineurin-inhibitor-free "bottom-up" immunosuppressive regimen with basiliximab, mycophenolic acid, and steroids. The study objective is to evaluate the safety and clinical feasibility of MAPC administration in this patient cohort. The primary endpoint of the study is safety, assessed by standardized dose-limiting toxicity events. One secondary endpoint is the time until first biopsy-proven acute rejection, in order to collect first evidence of efficacy. Dose escalation (150, 300, 450, and 600 million MAPCs) will be done according to a 3 + 3 classical escalation design (4 groups of 3-6 patients each). DISCUSSION: If MAPCs are safe for patients undergoing liver transplantation in this study, a phase II/III trial will be conducted to assess their clinical efficacy.

Hepatobiliary procedures in patients undergoing cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: The long-term prognosis of patients with peritoneal malignancies has greatly improved since the introduction of cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Nevertheless, CRS can be associated with high postoperative morbidity. In this retrospective study, we analyzed the influence of hepatobiliary surgery as part of CRS on postoperative short-term patient outcome. METHODS: Between 2005 and 2008, a total of 63 (25%) of 252 patients with peritoneal surface malignancies undergoing CRS and HIPEC required hepatobiliary surgery. Liver resection was performed in 22, resection of Glisson capsule in 39, and bile duct resection in 2 patients. The mean age of the study population was 49.3 years. Thirty-four patients (54%) were women. RESULTS: Complete macroscopic cytoreduction (CC-0/1) was reached in 59 patients (93.7%). The median hospital stay was 18 days. Twenty-two patients developed minor complications (35%), such as moderate fever, pain, or secondary wound healing. In 21 patients (33%), severe complications occurred, most commonly pancreatitis and abdominal abscess. Three patients (4.8%) developed a biliary leakage. Of these, 2 had to be reoperated. CONCLUSIONS: In our experience, hepatobiliary procedures have to be performed in up to one-third of patients and are associated with a low rate of specific complications, such as biliary leakages.

Intestinal ischemia: current treatment concepts.

PURPOSE: Mesenteric ischemia is a condition well-known among physicians treating patients with abdominal symptoms. Even so, mortality rates have not decreased significantly over the last decades. The purpose of this article is to review current treatment concepts of acute and chronic mesenteric ischemia. RESULTS: Early diagnosis is one of the most important features that determine a patient's prognosis. Conventional angiography and multidetector computed tomography are therefore appropriate to quickly diagnose mesenteric ischemia, the latter being commonly more available. Once a patient presents with signs of peritonitis, instant laparotomy is indicated, and infarcted bowel segments need to be resected, followed by a second-look operation if necessary. If bowel necrosis is clinically not suspected, different approaches should be applied according to source and nature of mesenteric ischemia. Besides established surgical treatment concepts, more and more interventional procedures are developed and evaluated. However, superiority of these new techniques could only be shown for selected patient groups so far. In chronic mesenteric ischemia, interventional approaches seem to be an attractive alternative in patients who are in a condition too bad to undergo surgery. Patients with colonic ischemia are treated best in a conservative manner and by resolving the underlying cause, if identified. CONCLUSION: Patients with acute mesenteric ischemia are still at highest risk for a fatal course of disease. New diagnostic and therapeutic developments have not been tested in larger studies yet, neither has any of these methods led to an increased survival in studies published so far. Taken together, mesenteric ischemia requires high awareness, earliest possible diagnosis, and treatment by an experienced interdisciplinary team of gastroenterologists, radiologists, and surgeons.

Three dimensional cultivation increases chemo- and radioresistance of colorectal cancer cell lines.

Although 2D cell cultures are commonly used to predict therapy response, it has become clear that 3D cultures may better mimic the in vivo situation and offer the possibility of tailoring translational clinical approaches. Here, we compared the response of 2D and 3D colorectal cancer (CRC) cell lines to irradiation and chemotherapy. Classic 2D cultures and 3D spheroids of CRC cell lines (CaCo2, Colo205, HCT116, SW480) were thoroughly established, then irradiated with doses of 1, 4, or 10 Gy, using a clinical-grade linear accelerator. The response was assessed by immunohistochemistry, flow cytometry, and TUNEL assays. Upon irradiation, CRC 3D spheroids were morphologically altered. After irradiation with 10 Gy, annexin V/PI staining revealed a 1.8- to 4-fold increase in the apoptosis rate in the 2D cell cultures (95% CI 3.24±0.96), and a 1.5- to 2.4-fold increase in the 3D spheroids (95% CI 1.56±0.41). Irradiation with 1 Gy caused 3- and 4-fold increases in TUNEL positive cells in the CaCo2 and HCT116 (p = 0.01) 2D cultures, respectively, compared with a 2-fold increase in the 3D spheroids. Furthermore, the 2D and 3D cultures responded differently to chemotherapy; the 3D cultures were more resistant to 5-FU and cisplatin, but not to doxorubicin and mitomycin C, than the 2D cultures. Taken together, CRC cells cultured as 3D spheroids displayed markedly higher resistance to irradiation therapy and selected chemotherapeutic drugs than 2D cultures. This in vitro difference must be considered in future approaches for determining the ideal in vitro systems that mimic human disease.

A human ex vivo coculture model to investigate peritoneal metastasis and innovative treatment options.

OBJECTIVES: Peritoneal metastasis (PM) is commonly observed in patients with colorectal cancer (CRC). The outcome of these patients is poor, with an average survival of only six months without therapy, which requires a better understanding of PM biology and new treatment strategies. METHODS: We established and characterized a human ex vivo peritoneal model to investigate the mechanisms of peritoneal seeding and possible treatment options. For this, CRC cell lines and patient-derived tumor organoids were cultured together with human peritoneum to investigate the invasion of malignant cells and the effects of local chemotherapy. RESULTS: Fresh human peritoneum was cultured for up to three weeks in a stainless steel ring system, allowing for survival of all peritoneal structures. Peritoneal cell survival was documented by light microscopy and immunohistochemical staining. Further, immunohistological characterization of the tissue revealed CD3-positive T-lymphocytes and vimentin-positive fibroblasts within the peritoneum. In addition, extracellular matrix components (collagens, matrix metalloproteinases) were localized within the tissue. Coculture with CRC cell lines and patient-derived CRC organoids revealed that cancer cells grew on the peritoneum and migrated into the tissue. Coculture with CRC cells confirmed that hyperthermal treatment at 41 °C for 90 min significantly enhanced the intracellular entry of doxorubicin. Moreover, treatment with mitomycin C under hyperthermic conditions significantly reduced the amount of cancer cells within the peritoneum. CONCLUSIONS: This human ex vivo peritoneal model provides a stringent and clinically relevant platform for the investigation of PM and for further elucidation of possible treatment options.