Development of patient derived organoids for cancer drug screening applications

Cancer is a disease characterised by abnormal cell growth that can invade or spread to other regions of the body. Organoids are three-dimensional ex vivo tissue cultures made from embryonic stem cells, induced pluripotent stem cells, progenitor cells or tissue that serve as a physiological model for cancer research. These are designed to recapitulate the in vivo properties of tumours. Importantly, effective recapitulation of the structure of tissues and function is believed to predict patient response, allowing for the creation of personalised therapy in a timely manner that may be used in the clinic. This Review discusses the pre-clinical model and different types of human organoids as models for the development of high throughput drug screening and also aims to highlight how organoids are shaping the future of cancer research.     

White matter damage precedes that in gray matter despite similar magnetic resonance imaging changes following cerebral hypoxia-ischemia in neonatal rats

We hypothesized that the cerebral injury produced by hypoxia-ischemia (HI) in neonatal rats would differ in white compared with gray matter as detected histologically or with magnetic resonance (MR) imaging methods. Maps of T₂ and the apparent diffusion coefficient (ADC) of water were acquired in 1-week-old rats at times prior to cerebral HI (right carotid artery occlusion plus 1.5 h of hypoxia), within the last 5–10 min of HI, and 1 h or 24 h after HI. Near the end of HI, ADC decreased and T₂ increased in both cortical gray and subcortical white matter within the cingulum of the HI hemisphere. One hour after HI, ADC partially recovered, but T₂ remained increased and then increased further by 24 h post-HI. In contrast to the similar MR responses in white and gray matter, histological evidence for irreversible cell damage occurred in white matter earlier than in gray matter within the HI hemisphere. At 1 h post-HI, rarefied or disrupted nerve fibers and an increase in TUNEL-positive cells were observed within white matter in the cingulum, whereas neurons within the cortical gray matter appeared normal. By 24 h post-HI, damage was apparent in both white and gray matter. Thus, MR imaging detected acute tissue edema following cerebral HI in both gray and white matter but did not distinguish between the early irreversible tissue injury detected histologically in white but not gray matter in this rather severe model of neonatal encephalopathy.     

急性颅脑损伤早期血浆CNP和脑脊液乳酸测定的临床意义

目的 :探讨了血浆C -型利钠多肽 (CNP)和脑脊液乳酸水平在急性颅脑损伤早期的病理、生理作用。方法 :分别应用放射免疫分析血浆CNP水平和生化法测定脑脊液乳酸水平。结果 :在治疗前颅脑损伤患者血浆CNP水平非常显著地低于正常人组 (p <0 0 1 ) ,脑脊液乳酸水平明显高于正常人水平 (p <0 0 1 ) ,治疗 1 5天后与正常人比较无显著差异 (p >0 0 5 )。 结论 :观察血浆CNP和脑脊液乳酸水平的变化对研究急性颅脑损伤早期的病理生理变化、判断疗效及预后观察具有十分重要的临床意义     

Expression of vascular endothelial growth factor receptor 3 in blood and lymphatic vessels of lung adenocarcinoma

Vascular endothelial growth factor receptor 3 (VEGFR-3) has been proposed as a marker for lymphatic endothelial cells. This study investigated the expression of VEGFR-3 in the tumour vessels of lung adenocarcinoma and evaluated whether VEGFR-3 staining was useful for identifying lymphatic vessels within the tumour stroma. It also explored whether active growth of lymphatic vessels occurred in lung adenocarcinoma. Formalin-fixed, paraffin-embedded specimens obtained from 60 cases of lung adenocarcinoma, including five cases of pure bronchiolo-alveolar carcinoma (BAC) without stromal, vascular, and pleural invasion, were examined. No VEGFR-3-positive vessels were observed in pure BAC, but varying numbers of VEGFR-3-positive vessels were found in 39 of 55 (70.9%) invasive adenocarcinomas. A comparison of serial sections stained for VEGFR-3, CD31, and laminin-1 showed that most of the VEGFR-3-positive vessels appeared to be blood vessels (CD31-positive, laminin-1-positive), but some had the characteristics of lymphatic vessels (variable staining for CD31, little or no staining for laminin-1). VEGFR-3 staining highlighted lymphatic invasion by cancer cells; this invasion could not be detected by CD31 or haematoxylin and eosin (H&E) staining. Active growth of lymphatic vessels (as indicated by nuclear Ki-67 labelling of the endothelium) was observed in five tumours, four of which showed a high level of lymphatic invasion by cancer cells. It was concluded that VEGFR-3 immunostaining did not discriminate clearly between vascular and lymphatic endothelial cells, since expression of VEGFR-3 can be up-regulated in tumour blood vessels. However, VEGFR-3 staining combined with laminin-1 and CD31 staining would be useful for identifying lymphatic vessels and their invasion by tumour cells in a more objective way. Finally, proliferation of lymphatic endothelial cells may occur in association with lymphatic invasion by cancer cells.     

肺癌患者化疗前后血清Hcy、IGF-Ⅱ和TSGF检测的临床意义

目的：探讨肺癌患者化疗前后血清Hcy、IGF-Ⅱ和TSGF水平的变化及临床意义。方法：应用免疫法和放射免疫分析对35例肺癌患者进行了血清Hcy、IGF-Ⅱ和TSGF含量检测,并与30名正常健康人作比较。结果：肺癌患者在化疗前血清Hcy、IGF-Ⅱ和TSGF水平均非常显著地高于正常人组（P〈0.01）,化疗后6个月在未复发的27例中明显下降接近于正常人组,而复发的8例,其数值又回升至化疗前水平（P〈0.05）。结论：检测肺癌患者血清Hcy、IGF-Ⅱ和TSGF水平的变化可作为诊断和疗效观察的参考。     

甘氨酸对缺氧/复氧离体大鼠心脏功能的作用

目的：观察甘氨酸(glycine, GLY)对缺氧/复氧离体心脏功能的影响，探讨甘氨酸对心肌缺血-再灌注 (ischemia/reperfusion, I/R)损伤的防治作用及其机制。方法：利用Langendorff灌流装置复制心肌缺氧/复氧(hypoxia/reoxygenation, H/R)模型，观察不同浓度GLY处理后心脏左室收缩压（left ventricular systolic pressure, LVSP）、左室舒张末压（left ventricular end diastolic pressure, LVEDP）、左室发展压 (left ventricular developed pressure, LVDP=LVSP-LVEDP)、左室收缩压最大上升/下降速率(the maximum rising and dropping rates of left ventricular pressure, dp/dtmax and dp/dtmin)，并在相应的时点分别测定冠脉流出液中的超氧化物歧化酶(superoxide dismutase, SOD)活性和丙二醛(malondialdehyde, MDA)的水平。结果：H/R后各时点大鼠心功能各指标均低于缺氧前；GLY处理组复氧后心功能各指标均高于H/R组，并拮抗损伤导致的SOD减少和MDA升高。结论：一定浓度的GLY能显著改善缺氧/复氧心肌的舒缩功能，其机制可能与其提高SOD活性抑制脂质过氧化反应有关。     

长期慢性脑外伤大鼠脑线粒体功能的变化

研究长期慢性轻度脑外伤对大鼠脑线粒体功能的影响。大鼠连续1、5、10、15、20、25、30d轻度闭合性颅脑撞击后分离脑线粒体,测定线粒体肿胀度、膜流动性、膜磷脂含量、呼吸功能、线粒体呼吸酶、超氧化物歧化酶(SOD)、丙二醛(MDA)和Ca2 等指标以显示线粒体功能、抗氧化能力的变化。结果显示,第15、20、25、30d大鼠脑线粒体明显肿胀,膜磷脂降解,膜流动性下降,呼吸功能衰减,呼吸酶、SOD活性降低,Ca2 、MDA含量升高。由此认为,经常性头部撞击可造成大鼠脑线粒体功能受损,其机制可能与脑线粒体膜损伤后继发的自由基生成增加、脑线粒体能量代谢障碍有关。     

FK506 (tacrolimus) improves lung injury through inhibition of Fas-mediated inflammation

Objective To investigate whether FK506 (tacrolimus) can inhibit Fas- or A23187-induced interleukin (IL)-8 expression and cell death in A549 human alveolar epithelial cells, plus Fas-mediated acute lung injury in vivo. Methods Assays for IL-8, cell death, and caspase-3 activity were performed. A549 cells were treated with 25 μmol A23187 or 0.2 μg/ml agonistic anti-Fas antibody plus 5 ng/ml interferon-gamma (IFN-γ). Tacrolimus was treated at 0.1–10 ng/ml. For in vivo experiment, agonistic anti-Fas antibody (Jo2) at 2.5 μg/g was intratracheally instilled into C57BL/6 mice. Neutrophils and protein contents in bronchoalveolar lavage (BAL) fluid were measured within 24 h of instillation. Mice were orally treated with 32 mg/kg of tacrolimus 24 h and 1 h prior to instillation. Results Both Fas and A23187 caused significant IL-8 expression and cell death in A549 cells. Tacrolimus inhibited A23187-induced IL-8 expression alone while it protected all Fas-mediated responses. Mice instilled intratracheally with Jo2 at 2.5 μg/g had significant increases in neutrophils, protein contents in BAL fluid and in expression of chemoattractants for neutrophils. These increases were reversed by tacrolimus. Conclusions Tacrolimus serves as a therapeutic option for improving lung injury through inhibition of Fas-mediated inflammation. Received 7 November 2005; returned for revision 28 December 2005; accepted by G. Wallace 2 February 2006     

Presynaptic depolarization in myelinated vagal afferent fibres terminating in the nucleus of the tractus solitarius in the cat

The presynaptic influences that act on terminals of slowly adapting lung stretch receptor afferents and aortic baroreceptor afferents within the nucleus of the solitary tract were assessed using intracellular recording and antidromic stimulation techniques.Central respiratory influences on the axcitability of lung stretch receptor terminals were observed in 29% (4 of 14) of measurements. These were confirmed in intracellular recordings where membrane depolarizations in synchrony with phrenic nerve discharge were seen in 17% (4 of 24) of fibres. In three cases membrane depolarization also occurred synchronously with artificial lung inflation.Neither tests of excitability nor intracellular recording revealed any evidence for equivalent presynaptic influences on 16 myelinated aortic baroreceptor terminals.Stimulation of the superior laryngeal nerve evoked depolarizations in 50% (7 of 14) of lung stretch receptor terminals. These took the form of complex waves of depolarization with both short (3–8 ms) and long latency (27–35 ms) components. The amplitude of the long latency response increased during the period of phrenic nerve discharge, i.e. during central inspiration.These effects are discussed in relation to the central respiratory influences on both respiratory and cardiovascular reflexes.     

Correlation between genetic alterations and histopathological subtypes in bronchiolo-alveolar carcinoma and atypical adenomatous hyperplasia of the lung

Bronchiolo-alveolar carcinoma (BAC) is a type of lung adenocarcinoma characterized by growth along the alveolar wall. It is divided into two subtypes: sclerosing BAC (SBAC), which has central fibrosis, and non-sclerosing BAC (NSBAC), which lacks central fibrosis. We compared the genetic alterations in these two types of BAC with those in atypical adenomatous hyperplasia (AAH). There were 39 cases of SBAC, 19 of NSBAC and 20 of AAH. To detect the loss of heterozygosity (LOH) we used the microsatellite markers D3S1234 and D3S1300 on chromosome 3p, IFNA and D9S144 on 9p, and TP53 on 17p. We also used polymerase chain reaction-SSCP analysis and direct sequencing to examine a point mutation of the p53 gene at exons 5-8. At the TP53 locus, the frequencies of LOH showed a statistical rank-difference correlation among AAH, NSBAC and SBAC. On chromosomes 3p and 9p there were no statistical differences of LOH among AAH, NSBAC and SBAC. We detected a significant statistical rank-difference correlation in the p53 mutation among AAH, NSBAC and SBAC. These findings suggest that a process of multistep carcinogenesis from AAH through NSBAC to SBAC might occur in some cases of adenocarcinoma, and LOH of 3p and 9p might be an early event of carcinogenesis, while the p53 mutation might be a later event.