Resected Human Specimens Research Articles

EPCO-28. MULTIPLATFORM MOLECULAR AND FUNCTIONAL GENOMIC ANALYSES OF NF1 MUTANT GLIOBLASTOMA IDENTIFY DISTINCT CO-MUTATION PATTERNS AND DRUGGABLE DEPENDENCIES UNDERLYING SELUMETINIB RESPONSE

Abstract The tumor suppressor NF1 is recurrently mutated in sporadic IDH-wildtype glioblastoma and activates Ras/Raf/MEK signaling, motivating the use of targeted MEK inhibition. Here, we combine multiplatform molecular analysis of NF1 mutant glioblastoma human resection specimens with functional genomic CRISPRi screens to the MEK inhibitor selumetinib. METHODS: We identified 123 consecutive patients treated at the University of California San Francisco between 2016-2022 with NF1 mutant, IDH-wildtype glioblastoma. DNA methylation data was processed using the minfi package, and hierarchical clustering, principal component analysis (PCA), and survival analysis was performed in R. Human NF1 mutant GBM43 and mouse NRAS mutant SB28 GBM cells expressing CRISPRi machinery were used for genome wide selumetinib-resistance screens. Immunoblotting was performed to validate CRISPRi screen hits and selumetinib responses in cell lines. RESULTS: DNA sequencing of human glioblastomas identified 12 recurrent alterations in greater than 10% of cases including cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic regulators (ATRX, SETD2), and Ras signaling (EGFR, PDGFRA, PTPN11). Methylation data (n=55 tumors) identified three groups marked by differences in MGMT methylation (p=0.04, chi-square test) and CDKN2A/B loss (p=0.01, chi-square test). CRISPRi screens in GBM43 and SB28 cells identified driver phenotypes in cell cycle regulators (TP53, CDKN2A, CDK4), PI3K signaling (PTEN, MTOR), and Ras signaling (RASA2, PTPN11) consistent with co-occurring mutations in the human NF1 mutant glioblastoma cohort. In contrast, mediators of selumetinib response converged on regulators of Ras/Raf/MEK signaling including the Ras effector SHOC2. Biochemical analysis validated SHOC2 repression is sufficient for selumetinib sensitization. CONCLUSIONS: Using a combination of human tumor genomic analysis, functional genetic screens, and biochemical validation, we identify three epigenetic groups of NF1 mutant glioblastoma with distinct co-mutation patterns and validate compensatory Ras/Raf/MEK activation as a conserved mechanism of selumetinib resistance, suggesting combination molecular therapy as a rational therapeutic combination.

2D carbon network arranged into high-order 3D nanotube arrays on a flexible microelectrode: integration into electrochemical microbiosensor devices for cancer detection

In this work, we develop a new type of mesoporous 2D N, B, and P codoped carbon network (NBP-CNW) arranged into high-order 3D nanotube arrays (NTAs), which are wrapped onto a flexible carbon fiber microelectrode, and this microelectrode is employed as a high-performance carbon-based nanocatalyst for electrochemical biosensing. The NBP-CNW-NTAs synthesized by a facile, controllable, ecofriendly and sustainable template strategy using ionic liquids as precursors possess a high structural stability, large surface area, abundant active sites, and effective charge transport pathways, which dramatically improve their electrocatalytic activity and durability in the redox reaction of cancer biomarker H2O2. Benefiting from these unique structural merits, superb electrochemical activity and good biocompatibility, the NBP-CNW-NTAs-modified microelectrode demonstrates excellent sensing performance toward H2O2 and is embedded in a homemade microfluidic electrochemical biosensor chip for the real-time tracking of H2O2 secreted from different live cancer cells with or without radiotherapy treatment, which provides a new strategy for distinguishing the types of cancer cells and evaluating the radiotherapeutic efficacy of cancer cells. Furthermore, the functional microelectrode is integrated into an implantable probe for the in situ detection of surgically resected human specimens to distinguish cancer tissues from normal tissues. These will be of vital significance for cancer diagnoses and therapy in clinical practice.

Redox state changes of mitochondrial cytochromes in brain and breast cancers by Raman spectroscopy and imaging

This paper presents a non-invasive approach to study redox status of cytochromes in vitro human brain cells of normal astrocytes (NHA), astrocytoma (CRL-1718), glioblastoma (U87-MG) and medulloblastoma (Daoy), and human breast cells of normal cells (MCF10A), slightly malignant cells (MCF7) and highly aggressive cells (MDA-MB-231), in vivo animal models, and ex vivo brain and breast tissues surgically resected human specimens by means of Raman microspectroscopy at 355 nm, 532 nm, 785 nm and endospectroscopic Raman probe at 785 nm. Here we show that the amount of reduced cytochrome becomes abnormally high in human brain tumors and breast cancers. In contrast, the amount of reduced cytochrome c is lower in cancer cells when compared to the normal one at in vitro conditions when the effect of microenvironment is eliminated. Mitochondrial dysfunction and alterations in the chemical composition of the nucleus, mitochondria, lipid droplets, cytoplasm in single cells have been detected by Raman imaging. Incubation in vitro with retinoic acid increases the amount of reduced cytochrome c.

Abstract B95: PD-L1-mediated nanotherapeutic targeting of glioma-infiltrating myeloid cells synergizes with radiotherapy for glioblastoma

Abstract Massive infiltration of tumor-associated myeloid cells (TAMCs), reaching 30-50% of the tumor mass, is a distinctive characteristic of glioblastoma (GBM). These cells are a critical player in generating highly immunosuppressive microenvironment in GBM. Thus, therapeutic targeting of TAMCs might dampen the immunosuppression and improve the outcome of antiglioma therapies. In this work, we developed a novel nano-immunotherapy platform to actively and specifically target glioma-infiltrating TAMCs in vivo. Based on our finding that glioma-infiltrating TAMCs exhibit the highest expression of PD-L1 among all the immune cell infiltrates, we designed a lipid nanoparticle system (LNP) with surface-engineered anti-PD-L1 therapeutic antibody. Decoration of anti-PD-L1 antibody rendered our LNP highly capable of specifically targeting TAMCs and, interestingly, routing PD-L1 to lysosomal degradation. Co-encapsulation of dinaciclib, a cyclin-dependent kinase inhibitor, into LNP caused a dramatic elimination of TAMCs and inhibition of immunosuppression in glioma. Notably, the targeting efficiency of TAMC-targeted LNP was further increased when combined with radiation therapy (RT), a standard care of GBM. The superior therapeutic efficacy in mice treated by RT+LNP combination over the RT or LNP monotherapy was demonstrated in two syngeneic murine glioma models, GL261 and CT2A. We also demonstrated that our LNP successfully targeted TAMCs harvested from human resection specimens, which further validated the translational potential and clinical relevance of our nano-immunotherapy approach. In conclusion, our work established a new nanotechnology-based immunotherapeutic approach with great potential to improve the clinical outcome in patients with GBM. Citation Format: Peng Zhang, Jason Miska, Catalina Lee-Chang, Aida Rashidi, Wojciech K. Panek, Shejuan An, Markella Zannikou, Aurora Lopez-Rosas, Yu Han, Ting Xiao, Irina V. Balyasnikova, Maciej S. Lesniak. PD-L1-mediated nanotherapeutic targeting of glioma-infiltrating myeloid cells synergizes with radiotherapy for glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B95.

Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging.

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.

Insights into the Pathogenesis of Pancreatic Cystic Neoplasms.

With the current epidemic of diagnosed pancreatic cystic neoplasms on the rise, a substantial amount of work has been done to unravel their biology, thus leading to implications on clinical decision making. Recent genetic profiling of resected human specimens has identified alterations in signaling pathways involving KRAS and GNAS signaling as early events in the pathogenesis of intraductal pancreatic mucinous neoplasms. Progressively, mutations in genes such as TP53, SMAD4, RNF43, and others are thought to characterize invasive and advanced lesions. The role of inflammation in fueling the growth and transformation of these cysts has also begun to be studied with greater interest. A number of promising clinical studies have attempted to integrate these genetic insights into classifying these cysts and treating patients. We have reviewed existing literature on similar lines besides commenting on some useful animal models that recapitulate molecular and phenotypic progression of these cysts.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions.

Organ fibrosis or "scarring" is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic. To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first "organ" culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's <em>Ex Vivo</em> Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or “scarring” is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic. To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first “organ” culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Abstract 5272: Multimodality interrogation of systemic RNA interference-defective-1 transmembrane family member 1 (SIDT1) identifies axon guidance protein interactions.

Abstract Introduction SIDT1 is an 11-transmembrane domain channel protein that conveys small double stranded RNA (dsRNA) molecules between cells. SIDT1 is highly expressed in human pancreatic stellate cells and disruption of SIDT1 function attenuates synthesis of components of the desmoplastic response that is typical of pancreatic cancer. We sought to identify the intra- and extracellular protein interaction partners of SIDT1 using an unbiased multimodality screening approach. Methods SIDT1 was used as ‘bait’ in a membrane-directed split ubiquitin yeast 2-hybrid (Y2H) screen. Candidate interacting partners identified in multiple biological replicates using a stringent selection approach were validated using immunoprecipiation and the functional effects of disrupting candidate interacting partner protein expression were determined using lentiviral short hairpin RNA-based RNA interference. Gene ontology (GO) term analysis was performed. A high-throughput flow cytometry-based assay was used to quantify the effects of depleting SIDT1 interacting partners on fluorescent dsRNA transfer mediated by SIDT1. Potential SIDT1 ectodomain interactors were probed using cell surface phage display. SIDT1-tGFP transfected HEK293 cells were probed, with tGFP transfectants serving as a counter-selection control population. Phage display hits were deconvoluted using BLASTP. Fluorescently labeled peptides were synthesized and interactions assessed by epifluorescence microscopy using human pancreatic ductal adenocarcinoma cells. Enzymatic quantification of biotinylated synthetic peptide colocalisation was performed. Immunohistochemical and clincopathological correlation using resected pancreatic adenocarcinoma specimens was undertaken. Results The Y2H screen identified a shortlist of consistent SIDT1 interacting partners in which axon guidance proteins were significantly overrepresented, based on GO-term analysis. Disruption of Reticulon 4 expression resulted in impairment of SIDT1 intercellular small dsRNA transfer function. Phage display identified a SIDT1-binding 12-mer with sequence homology to human Growth arrest-specific 6 (GAS6). 12-mer colocalisation with full length SIDT1 and the SIDT1 ectodomain was confirmed using controlled epifluorescence microscopy. Levels of SIDT1 protein expression correlate with the presence of perineural invasion in human resection specimens (P<0.001). Discussion Using a multimodality screening approach we have identified novel SIDT1 binding partners. The role of SIDT1 and its interacting partners in heterotypic cellular interactions, e.g. with peritumoral neurons, warrants further investigation. Citation Format: Mohamed O. Elhassan, Jennifer Christie, Marlieke Molendijk, Mark Duxbury. Multimodality interrogation of systemic RNA interference-defective-1 transmembrane family member 1 (SIDT1) identifies axon guidance protein interactions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5272. doi:10.1158/1538-7445.AM2013-5272

Protease-activated receptor-2 regulates cyclooxygenase-2 expression in human bile duct cancer via the pathways of mitogen-activated protein kinases and nuclear factor kappa B

Recent studies have suggested that protease-activated receptor-2 (PAR-2) activity correlates with cell proliferation and tumor growth, and its activation induces expression of cyclooxygenase-2 (COX-2). However, no previous reports have investigated PAR-2 signaling pathways in bile duct cancer. The aim of this study was to determine whether PAR-2 activation can regulate COX-2 expression via mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) in human bile duct cancer cells. We immunohistochemically examined PAR-2 and COX-2 expression in 104 resected human specimens of extrahepatic bile duct cancer. We then determined how inhibitors of MAPKs and NF-κB signaling pathways influence COX-2 expression under PAR-2 activation in HuCCT1 and TKKK, human bile duct cancer cell lines. PAR-2 and COX-2 proteins were immunohistochemically recognized in 63 and 57% of specimens and were significantly correlated. PAR-2 agonist peptide activated mRNA and protein expression of COX-2 in HuCCT1 and TKKK. Pharmacologic blockade of p44/42 or p38 MAPK significantly inhibited PAR-2-activated mRNA and protein expression of COX-2 in both cells. COX-2 protein expression was also inhibited by the blocker of NF-κB pathway in both cells. PAR-2 may regulate COX-2 expression in human bile duct cancer via the MAPKs and NF-κB pathways.

412 Neuroplastic Changes in a Mouse Model of Pancreatic Ductal Adenocarcinoma (PDAC)

Perineural tumor invasion of intrapancreatic nerves and extrapancreatic nerve plexuses is a key feature of pancreatic malignancies and is thought to play an important role in pancreatic cancer-related pain and cancer spread. Here we provide a preliminary description of changes in pancreatic innervation in a mouse model of PDAC that will allow us to examine the mechanisms underlying cancer-induced pathophysiology of the peripheral nervous system. Several neurotrophic factors, including nerve growth factor (NGF) and members of the glial cell line-derived neurotrophic factor (GDNF) family of growth factors, and their receptors can be found in the pancreas and are increased in human pancreatic tumors. These growth factors are capable of inducing neuronal outgrowth and may contribute to sprouting and hypertrophy of pancreatic nerves, which is one of the hallmarks of human PDAC. We hypothesized that artemin, a member of the GDNF family of growth factors, and NGF play a central role in this cross-talk between cancer cells and pancreatic nerves. Experiments were performed with transgenic mice with pancreas-specific expression of the mutated Kras oncogene and heterozygous deletion of the p53 tumor suppressor (Kras). Animals were sacrificed at time points ranging from 13.9-17.4weeks and tissuewas retrieved for histological analysis and assessment of growth factor expression using real time PCR. All Kras mice had developed multifocal pancreatic cancer (by week 13) as evidenced by large masses, obstruction of the biliary tree (by week 17) and a strong desmoplastic reaction. Normal pancreatic innervation was altered with hypertrophied nerves running along blood vessels and extending into the pancreatic parenchyma and tumor nodules, but not into the fibrous tissue. Immunohistochemical experiments showed prominent expression of tyrosine hydroxylase and the artemin receptor GFRα3 on nerve fibers. Compared to wildtype mice, the expression of NGF and its receptor trkA was increased 3.22-fold and 8.85-fold, respectively, in cancer animals. While not statistically significant, a 1.99-fold increase in artemin expression was measured in the Kras animals. Finally, a 2.27-fold decrease in the expression of GFRα3 in cancer animals was observed. These data indicate significant neuroplastic changes occur in the Kras model of pancreatic cancer and these changes correlate with altered expression of growth factors, mimicking data obtained in human resection specimens. Thus, this animal model will enable us to systemically study the impact of changes in trophic factor signaling on cancer growth and animal behavior.

Clinical versatility of porcine hepatocytes in the light of interspecies differences in cytochrome P450 regulation and expression

In fulminant hepatic failure, the clinical use of bioartifical liver support with porcine hepatocytes is the subject of a controversial debate. Cytochrome P450 (CYP) metabolic functions have relevant implications for drug metabolism and detoxification. In this study, we investigate interspecies differences in CYP gene expression between human and porcine primary hepatocytes and the impact of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) exposition mimicking cytokine release in fulminant hepatic failure. Primary hepatocyte cultures were isolated from human resection specimens and from German landrace pigs. Cell cultures (single and co-cultures) were exposed to porcine vs. human IL-6 and TNF-alpha, respectively. Changes in quantitative CYP gene expression were investigated by semi-quantitative RT-PCR. Significant differences in species-specific CYP gene expression by human and porcine hepatocytes were found after exposure to species-identical IL-6 (10 ng/ml) for CYP 1A1, CYP 2C, CYP 3A (P = 0.002, 0.022, 0.017, respectively) or species-identical TNF-alpha (30 ng/ml) for CYP 1A2 and CYP 2A (P = 0.037, 0.023, respectively). In single vs. co-culture, human hepatocytes demonstrated stronger repression of CYP 1A1, 2C8 and 3A4 expression after dosage with human IL-6 (10 ng/ml) (P = 0.022, 0.031, 0.014, respectively). Our findings demonstrate significant species-specific differences in CYP gene expression and regulation when high doses of IL-6 and TNF-alpha are used (10 and 30 ng/ml, respectively). These findings may point to species-specific physiological incompatibilities of porcine hepatocytes and thus limit their clinical versatility.

Morphological Observation and In Vitro Angiogenesis Assay of Endothelial Cells Isolated From Human Cerebral Cavernous Malformations

Little is known about the role of endothelial cells (ECs) in the pathogenesis of cerebral cavernous malformation because of the difficulties to obtain highly pure ECs. Thus, this study attempted to establish a reliable procedure to isolate and culture ECs from human cerebral cavernous malformation lesions. The biological features and the angiogenic potential of the cultured ECs were also investigated. A modified protocol was developed to isolate and culture cerebral cavernous malformation endothelial cells (CECs)from surgically resected human specimens. The biological features of CECs were investigated by electron microscope, immunostaining, real-time polymerase chain reaction, fluorescence-activated cell sorter, and Western blotting. The tube formation by CECs was examined in an in vitro angiogenesis model with or without the addition of vascular endothelial growth factor. CECs from the specimens unaffected by the intraoperative bipolar coagulation were cultivated successfully with higher than 95% purity. Comparing to the ECs from control brain tissue, CECs presented primitive nucleus in ultrathin section, expressed higher levels of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2, and spontaneously formed tube structures in a 3-dimensional collagen matrix. The tube formation by CECs was significantly promoted by vascular endothelial growth factor treatment. A modified protocol for the attainment of purified CECs and the first in vitro angiogenesis model of CECs were successfully established. We provided initial evidence that CECs had enhanced angiogenic potential and showed increased responsiveness to vascular endothelial growth factor.

Physiological incompatibilities of porcine hepatocytes for clinical liver support

In fulminant hepatic failure, the use of bioartificial liver support (BAL) with porcine hepatocytes is the subject of a current and controversial debate.1 Specifically, the issue of cross-species physiological incompatibilities has not been addressed so far. We therefore investigated the effects of species-specific cytokines in single and cocultures on hepatocyte function. Hepatocyte cultures were isolated from human resection specimens and from Landrace pigs. Single and cocultures were exposed to porcine and human interleukin (IL)-6 or tumor necrosis factor (TNF)-alpha. Changes in expression of C-reactive protein (CRP), albumin, CCAAT enhancer binding protein (C/EBP)-alpha and C/EBP-beta and metabolic competence of cultured cells was studied by measuring testosterone metabolite production. After human or porcine IL-6 dosing, CRP was induced up to 100-fold in human hepatocyte cultures, while porcine hepatocytes responded marginally (2- to 5-fold). Treatment with human or porcine IL-6 or TNF-alpha resulted in reduced albumin production, albeit at different levels when human and porcine hepatocytes were compared (P = 0.001). Unlike human, porcine hepatocytes produced less of 6alpha-hydroxytestosterone (6alpha-HT) (P < 0.001) and 7alpha-HT (P < 0.001) after human or porcine IL-6 dosing and treatment with species-specific TNF-alpha induced (human hepatocytes) or decreased (porcine hepatocytes) 6beta-HT production (P = 0.021). In coculture with free exchange of metabolites, porcine hepatocytes produced less 6alpha-HT (P = 0.048) and 16alpha-HT (P = 0.033), whereas after treatment with human IL-6 reduced CRP gene and protein expression was observed with human hepatocytes (P = 0.013). In conclusion, species-specific responses of hepatocytes to cytokines and interactions with xenobiotic metabolites may limit the clinical effectiveness of porcine hepatocytes in BAL.

Validation of cDNA Microarray Analysis to Distinguish Tumor Type Ex Vivo

Tissue biopsy alone is diagnostic in less than 10% of cases of metastatic bone disease with an unknown primary tumor. Accordingly, to delineate the neoplastic process in cases of metastatic bone disease, where the primary tumor cannot be determined with conventional modalities, new tools are attractive. Complementary deoxyribonucleic acid cDNA microarrays may be such a tool. Verification and validation of this technology for analysis of specimens ex vivo is mandatory if it is to be used to analyze the complexity of heterogenous biologic tissues. To convey the issues involved in the clinical application of cDNA microarray analysis of tumor specimens ex vivo, a set of experiments analyzing surgically resected human specimens is presented. The specific aims of the analysis were to assess cDNA microarray reproducibility of serial tissue preparations of tumor samples and to confirm that heterogeneity within a given clinical tumor specimen does not preclude this technique as a tool to discern tumor types. Complementary deoxyribonucleic microarray analysis, as it applies to heterogeneous clinical tumor samples in limited numbers, based on preliminary data, seems to be an appropriate but still experimental method for distinguishing tumor types.

Preclinical study of endoscopic ultrasonography with electronic radial scanning echoendoscope.

To evaluate the imaging possibility of a newly designed electronic radial scanning echoendoscope (ER-ES). In the in vivo study of swine, we obtained B-mode endoscopic ultrasonography (EUS) images of the gastric and gallbladder (GB) walls and checked the ability to detect Doppler signals using ER-ES and electronic linear array echoendoscope (EL-ES). Furthermore, in the ex vivo study of swine, B-mode EUS images of fixed gastric and GB wall specimens were obtained using ER-ES, EL-ES and mechanical radial scanning echoendoscope (MR-ES). In the study of resected human specimens, we obtained B-mode EUS images of five resected GB specimens (three normal GB, one cholecystitis and one cancerous) using the three types of echoendoscope. In the in vivo study of swine, ER-ES and EL-ES depicted the gastric walls as five-layered, and the GB walls as single-layered structures. The ability to detect Doppler signals was equal between ER-ES and EL-ES. In the ex vivo study of swine, ER-ES, MR-ES and EL-ES equally delineated the gastric walls as five-layered and GB walls as three-layered structures. In the study of resected human specimens, results demonstrated the normal GB walls as three-layered, the cholecystitis as a combination of outer high-echoic and inner low-echoic layers, and the cancer as a protruded tumor. We conclude that ER-ES has faculties for making B-mode images as well as EL-ES and MR-ES. In addition, in the in vivo study, ER-ES can analyze blood flow information as well as EL-ES.

Studies on the acetylating capacity of human colonic epithelial cells for 5-aminosalicylic acid.

Following oral administration of sulphasalazine, 5-aminosalicylic acid (5-ASA) is present in serum at very low concentrations and is mostly in the acetylated form. Analysis of rectal mucosal biopsies from patients on sulphasalazine maintenance therapy has shown that the predominant metabolite present is N-acetyl ASA (Clin Sci 1987: 72; 79P). The authors have now investigated whether this acetylation takes place in the epithelium. Isolated colonic epithelial cell suspensions were prepared in Krebs-Henseleit saline from fresh human resection specimens by EDTA digestion and mechanical disaggregation. Cell suspensions remained viable, as determined by lactate production, over 3 hours. Aliquots of cell suspension were incubated with 5-ASA, 0.1 mM, and glucose, 5 mM, at 37°C for 1 or 2 hours. Following incubation, the supernatant was analysed by high performance liquid chromatography for the presence of acetyl-ASA. Following washing, the cell pellet was disrupted ultrasonically, and intracellular acetyl-ASA was also measured. Production of acetyl-ASA in 1 hour was 112.5 (+27 SD) nmol/g dry weight (extracellular) and 48 (+15.6 SD) nmol/g dry weight (intracellular). No intracellular 5-ASA was detected, suggesting rapid and complete acetylation. A cytosolic fraction of the epithelial cells was prepared by centrifugation of homogenized cells at 13,000 g. In the presence of 5-ASA and acetyl-CoA, this enzyme solution produced acetyl-ASA linearly at a rate of 37 nmol/minutes/ mg protein. No acetyltransferase activity was found in the brush border. The cytosol was further purified by passing it through a Superose 12 gel filtration column. Maximum acetyltransferase activity was found in the fraction corresponding to a molecular weight of approximately 30,000. Thus, the human colonic epithelial cell is probably the primary site of acetylation of 5-ASA, and the N-acetyltransferase activity is found in the cytosol.