Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma.

Intratumoral heterogeneity (ITH) has increasingly being described for multiple cancers as the root cause of therapy resistance. Recent studies have started to explore the scope of ITH in glioblastoma (GBM), a highly aggressive and fatal form of brain tumor, to explain its inevitable therapy resistance and disease relapse. In this review, we detail the emerging data that explores the extensive genetic, cellular and functional ITH present in GBM. We discuss current experimental models of human GBM recurrence and suggest harnessing new technologies (CRISPR-Cas9 screening, CyTOF, cellular barcoding, single cell analysis) to delineate GBM ITH and identify treatment-refractory cell populations, thus opening new therapeutic windows. We will also explore why current therapeutics have failed in clinical trials and how ITH can inform us on developing empiric therapies for the treatment of recurrent GBM.

CD133+ brain tumor-initiating cells are dependent on STAT3 signaling to drive medulloblastoma recurrence.

Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.

Autofluorescence spectroscopy augmented by multivariate analysis as a potential noninvasive tool for early diagnosis of oral cavity disorders.

OBJECTIVE: Oral leukoplakia is one of the common potentially malignant lesions encountered worldwide. We report the results of an in vivo clinical evaluation of autofluorescence (AF) spectroscopy for differential diagnosis of oral leukoplakia. Multivariate analysis of spectral data has been incorporated to improve the efficacy of the technique. The results of this noninvasive study are expected to provide potential for extending the technique to other disorders. MATERIALS AND METHODS: A total of 18 patients and 30 normal volunteers participated in this study. AF spectra were acquired from affected sites of patients and from right and left buccal mucosa of normal volunteers. Diagnostic performance was analyzed using spectral intensity ratio (SIR), and principal component analysis followed by linear discriminant analysis (PCA-LDA). RESULTS: AF spectra of leukoplakic patients showed characteristic emissions from flavin adenine dinucleotide (FAD) and porphyrin at 500 and 630 nm, respectively. But the emission from porphyrin is not very prominent in the case of healthy volunteers. Also, significant decrease in spectral intensity is observed for leukoplakia compared with normal volunteers in the unprocessed spectra. Method of SIR yielded 96% sensitivity and 100% specificity and an overall 100% for PCA-LDA respectively for efficient differentiation of the lesions. CONCLUSIONS: The result of this preliminary study shows that PCA-LDA or SIR applied to AF spectroscopy is a useful tool for the differential diagnosis of oral cavity disorders. This has been demonstrated in leukoplakia in a clinical setting, and it is expected that the technique can be extended to other oral cavity disorders as well.

Sonic hedgehog regulates Bmi1 in human medulloblastoma brain tumor-initiating cells.

Bmi1 is a key stem cell regulatory gene implicated in the pathogenesis of many aggressive cancers, including medulloblastoma. Overexpression of Bmi1 promotes cell proliferation and is required for hedgehog (Hh) pathway-driven tumorigenesis. This study aimed to determine if Sonic hedgehog (Shh) modulates the key stem cell regulatory gene Bmi1 in childhood medulloblastoma brain tumor-initiating cells (BTICs). Although current literature suggests that there is a correlation between Shh pathway genes and Bmi1 expression, it is unclear whether there is indeed a direct regulatory mechanism. To address whether Shh induces expression of Bmi1, stem cell-enriched populations from medulloblastoma cell lines and primary samples were treated with Shh ligand and KAAD-cyclopamine (Shh antagonist). Our data indicate that Bmi1 expression positively correlates with increasing Shh ligand concentrations. Chromatin immunoprecipitation reveals that Gli1 preferentially binds to the Bmi1 promoter, and Bmi1 transcript levels are increased and decreased by Gli1 overexpression and downregulation, respectively. Knockdown experiments of Bmi1 in vitro and in vivo demonstrate that Hh signaling not only drives Bmi1 expression, but a feedback mechanism exists wherein downstream effectors of Bmi1 may, in turn, activate Hh pathway genes. These findings implicate Bmi1 and Hh as mutually indispensable pathways in medulloblastoma BTIC maintenance. Recent molecular characterization of medulloblastoma also reveals that Bmi1 is overexpressed across all subgroups of medulloblastoma, particularly in the most aggressive subtypes. Lastly, despite recent identification of BTIC markers, the molecular characterization of these cell populations remains unclear. In this work, we propose that the BTIC marker CD133 may segregate a cell population with a Hh-receptor phenotype, thus demonstrating a cell-cell interaction between the CD133+ Hh receptor cells and the CD133- Hh-secreting cells.

Inhibition of chemically induced carcinogenesis by drugs used in homeopathic medicine.

Homeopathy is considered as one modality for cancer therapy. However, there are only very few clinical reports on the activity of the drugs, as well as in experimental animals. Presently we have evaluated the inhibitory effects of potentized homeopathic preparations against N'-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in rats as well as 3-methylcholanthrene-induced sarcomas in mice. We have used Ruta, Hydrastis, Lycopodium and Thuja, which are commonly employed in homeopathy for treating cancer. Administration of NDEA in rats resulted in tumor induction in the liver and elevated marker enzymes such as gamma-glutamyl transpeptidase, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase and alkaline phosphatase in the serum and in liver. Concomitant administration of homeopathic drugs retarded the tumor growth and significantly reduced the elevated marker enzymes level as revealed by morphological, biochemical and histopathological evaluation. Out of the four drugs studied, Ruta 200c showed maximum inhibition of liver tumor development. Ruta 200c and phosphorus 1M were found to reduce the incidence of 3-methylcholanthrene-induced sarcomas and also increase the life span of mice harboring the tumours. These studies demonstrate that homeopathic drugs, at ultra low doses, may be able to decrease tumor induction by carcinogen administration. At present we do not know the mechanisms of action of these drugs useful against carcinogenesis.

Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin.

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.

Intestinal bioavailability and biotransformation of 3-hydroxybenzo(a)pyrene in an isolated perfused preparation from channel catfish, Ictalurus punctatus.

The intestinal bioavailability and biotransformation of 3-hydroxybenzo(a)pyrene, a major metabolite of benzo(a)pyrene in many animal species, was investigated in an in situ isolated intestinal preparation from the channel catfish, and in vitro with preparations of catfish intestine and blood. 3-Hydroxybenzo(a)pyrene was a good substrate for adenosine 3'-phosphate 5'-phosphosulfate (PAPS)-sulfotransferase and UDP-glucuronosyltransferase in cytosol or microsomes prepared from intestinal mucosa. The benzo(a)pyrene-3-glucuronide and 3-sulfate conjugates were only very slowly hydrolyzed by intestinal beta-glucuronidase and sulfatase. The K(m) values for PAPS-sulfotransferase and UDP-glucuronosyltransferase were 0.4 and 1 microM, respectively, and V(max) were 1.61 +/- 1.08 nmol benzo(a)pyrene-3-sulfate/min/mg of cytosolic protein and 1.08 +/- 0.54 nmol benzo(a)pyrene-3-glucuronide/min/mg of microsomal protein. Hydrolytic enzyme activities were three orders of magnitude slower. In the in situ intestinal preparation, [(3)H]3-hydroxybenzo(a)pyrene was readily metabolized to the glucuronide and sulfate conjugates. After 1 h of incubation of 2 or 20 microM [(3)H]3-hydroxybenzo(a)pyrene in the in situ preparation, the luminal contents contained 3-hydroxybenzo(a)pyrene, benzo(a)pyrene-3,6-dione, benzo(a)pyrene-3-sulfate, and benzo(a)pyrene-3-glucuronide. Mucosal samples contained these components, as well as some unextractable material. The blood contained mainly benzo(a)pyrene-3-sulfate and an as yet unidentified metabolite of 3-hydroxybenzo(a)pyrene bound to hemoglobin. Some, but not all, blood samples contained small amounts of 3-hydroxybenzo(a)pyrene, benzo(a)pyrene-3-glucuronide, and benzo(a)pyrene-3,6-dione. These studies demonstrate the rapid phase 2 conjugation of a phenolic benzo(a)pyrene metabolite in intestinal mucosa, and the transfer of the phase 2 sulfate and glucuronide conjugates to blood.

In vitro responses of equine colonic arterial and venous rings to adenosine triphosphate.

OBJECTIVE: To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. SAMPLE POPULATION: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. PROCEDURES: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine triphosphate (10(-8) to 10(-3) M) was added in a noncumulative manner, and relaxation percentage versus time curves were generated. Areas under the curves (ie, percentage of relaxation time) were calculated. RESULTS: Relaxation response of arterial and venous rings to ATP was dose-dependent. Percentage of relaxation time in response to 10(-4) and 10(-3) MATP was significantly greater, compared with that for rings not treated with ATP Removal of endothelium attenuated but did not eliminate the relaxation response. Addition of L-NAME did not attenuate the relaxation response in arteries. At higher concentrations, the vascular response to ATP was biphasic. CONCLUSIONS AND CLINICAL RELEVANCE: ATP applied to equine colonic arterial and venous rings with and without intact endothelium induced a biphasic response characterized by transient contraction followed by slow, substantial, and sustained relaxation. This ATP-induced response is possibly mediated by a mechanism other than NO. Adenosine triphosphate may be a useful treatment to modulate colonic vasomotor tone in horses with strangulating volvulus of the ascending colon.

Utilization pattern and efficacy of nonsurgical techniques to establish drainage for high biliary obstruction.

PURPOSE: To review the frequency and success of percutaneous and endoscopic techniques in the relief of high biliary obstruction. MATERIALS AND METHODS: A search of the radiologic achieves was performed identifying 70 patients with cholangiographic demonstration of high biliary obstruction defined as proximal to the distal third of the extrahepatic bile duct. Record review determined the frequency and success rates of percutaneous and endoscopic techniques in providing biliary decompression for obstructive jaundice. RESULTS: Endoscopic retrograde cholangiopancreatography was performed in 35 of 70 patients, providing initial endoscopic biliary decompression (EBD) in six patients (two subsequently required percutaneous intervention). Percutaneous biliary drainage (PBD) was attempted in 60 of 70 patients, providing initial decompression in 55 patients. PBD provided decompression after failed endoscopic biliary drainage in 18 of 26 patients. Endoscopic drainage was never attempted after failed percutaneous drainage. Overall EBD success was 23% and overall PBD success was 95%. The complication rate attributed to EBD was 26%; that attributed to PBD was 25%. For those patients who underwent attempts at both EBD and PBD, the complication rate was 16%. CONCLUSION: At an institution with well-developed gastrointestinal medical services and interventional radiologic services, PBD was more successful in providing initial biliary decompression than endoscopic techniques for high biliary obstruction.

Portal venous gas in a patient with Crohn's disease.

Portal venous gas usually occurs in the setting of an acute abdomen. Several causes for benign portal venous gas (PVG) have been reported. We describe the finding of PVG by computed tomography in a febrile patient with Crohn's disease and discuss the clinical implications of such a finding.