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.

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.

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.

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.

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.

Identification of selective inhibitors of RET and comparison with current clinical candidates through development and validation of a robust screening cascade.

RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series.  Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.

Ileo-right hemi-colonic cervical pull-up on a non-supercharged ileocolic arterial pedicle: A technical and case report.

Esophageal reconstruction can be challenging when stomach and colon are not anatomically intact and their use as esophageal substitutes is therefore limited. Innovative individual approaches are then necessary to restore the intestinal passage. We describe a technique in which a short stump of the right hemicolon and 25 cm of ileum on a long, non-supercharged, fully mobilized ileocolic arterial pedicle were used for esophageal reconstruction to the neck. In this case, a 65 year-old male patient had accidentally indigested hydrochloric acid which caused necrosis of his upper digestive tract. An emergency esophagectomy, gastrectomy, duodenectomy, pancreatectomy and splenectomy had been performed in an outside hospital. A cervical esophagostomy and a biliodigestive anastomosis had been created and a jejunal catheter for enteral feeding had been placed. After the patient had recovered, a reconstruction of his food passage via the left and transverse colon failed for technical reasons due to an intraoperative necrotic demarcation of the colon. Our team then faced the situation that only a short stump of the right hemi-colon was left in situ when the patient was referred to our center. After intensified nutritional therapy, we reconstructed this patient's food passage with the right hemicolon-approach described herein. After treatment of a postoperative pneumonia, the patient was discharged from hospital on the 26(th) postoperative day in a good clinical condition on an oral-only diet. In conclusion, individual approaches for long-segment reconstruction of the esophagus can be technically feasible in experienced hands. They do not always require arterial supercharging or free intestinal transplantation.

Anilinoquinazoline inhibitors of the RET kinase domain-Elaboration of the 7-position.

We have previously reported a series of anilinoquinazoline derivatives as potent and selective biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished cellular potency. Herein we describe further optimisation of the series through modification of their physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved challenging.

The discovery of 2-substituted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity.

Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR.

Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation.

Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development.

The immunomodulatory properties of mesenchymal stem cells and their use for immunotherapy.

There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohn's disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

ERK1/2, but not ERK5, is necessary and sufficient for phosphorylation and activation of c-Fos.

Growth factor-stimulated expression and activation of c-Fos is regulated by the ERK1/2 pathway. However, recent reports have also suggested a prominent role for the closely related ERK5 pathway in regulating the expression, transcriptional activation and nuclear localization of c-Fos. Here we have compared the role of ERK1/2 and ERK5 in regulating c-Fos using a combination of conditional protein kinases, selective biochemical inhibitors and ERK5 null fibroblasts. We demonstrate that activation of the ERK1/2 pathway, but not ERK5, is sufficient for c-Fos phosphorylation and transcriptional activation. Furthermore, growth factor-dependent expression of c-Fos is blocked by low doses of PD184352 that selectively inhibit the ERK1/2 pathway but proceeds normally in ERK5-/- 3T9 cells; in addition, nuclear localization of c-Fos is normal in ERK5-/- cells. ERK5-/- cells are, however, defective for c-Jun expression but this is reversed by re-expression of ERK5. In addition to ERK5, neither the JNK nor p38 pathways can substitute for ERK1/2 in the regulation of c-Fos transcriptional activity. These results demonstrate that c-Fos transcriptional activity is not regulated by the ERK5 pathway; rather, of all the MAPKs and SAPKs, c-Fos activation appears to be predominantly linked to the ERK1/2 pathway.

[Organ doses from CT examinations in children]. / Evaluation des doses d'irradiation aux organes en scanographie pédiatrique.

PURPOSE: To estimate radiation doses from routine pediatric CT scans (body) and to propose dose reduction protocols. MATERIAL AND METHODS: [corrected] Study performed with a phantom equivalent to the body of 5 year old child with evaluation of doses delivered to breast, gonads, bone marrow (sternum, T12) and thyroid for CT examinations of the chest, abdomen, pelvis and spine. Extrapolation is made to estimate the doses for 1 year old and 10 year old children. Finally, dose reduction protocols are evaluated. RESULTS: CT of the chest delivers significant doses to breast tissue and bone marrow, CT of the abdomen and pelvis delivers significant doses to the ovaries and CT pf the spine delivers significant doses to thyroid and bone marrow. Optimization can be achieved without degradation of the image quality, by reducing Kv and mAs within reasonable limits. This study may be used in order to evaluate the doses delivered by multi-detector CT units.

[Primary pulmonary hypertension arterial and Basedow disease]. / Hypertension artérielle pulmonaire primitive et maladie de Basedow.

We report a case of a fifty year old woman with Graves' disease with positive AntiTPO antibodies and positive AntiTSH receptor antibodies, who was hospitalized with a right cardiac failure. A pulmonary hypertension was discovered on echocardiography. After adequate antithyroid therapy, the right cardiac failure regressed rapidly and pulmonary pressure normalised. An autoimmune process has often been proposed to explain the association between pulmonary hypertension and hyperthyroidism. We report the arguments supporting this autoimmune etiopathogenesis. We also discuss an other hypothesis based on a direct effect of thyroid hormones on the pulmonary circulation and the effects of high cardiac output associated with hyperthyroidism.

[Infundibulitis, an unusual case of central diabetes insipidus]. / L'infundibulite, une cause rare (?) de diabète insipide central.

We report the case of a 53 year old patient who was admitted with polyuria, polydipsia associated with fatigue, depression and sexual dysfunction. Central diabetes insipidus with hypogonadotrophic hypogonadism was diagnosed by a water restriction test and different static and dynamic hormonal dosages. Nodular thickening of the pituitary stalk was noted on the MRI and the biopsy permitted a histological diagnosis of infundibulitis.

Stubby prostheses: an alternative to conventional prosthetic devices.

Stubby prostheses offer potential advantages over conventional prosthetic devices in terms of safety, stability, and energy efficiency. Although cosmesis is compromised in the process, these short nonarticulated pylon prostheses may be a viable option to consider in bilateral A-K or knee disarticulation amputee patients under the following circumstances: (1) as a training tool to determine whether progression to full-length articulated devices is feasible; (2) as permanent prostheses for the patient whose primary need for ambulation is within his own home; (3) for the elderly bilateral amputee in whom ambulation is feasible but safety and energy efficiency are of particular importance; and (4) as a definitive device in the patient who expresses a preference for them. Two patients who have become successful users of stubby prostheses are presented to illustrate these points.