generating-imaging-mouse-human-epithelial-organoids-from-normal-tumor

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

University of Texas Southwestern Medical Center

To investigate the frequency of xenotropic murine leukemia virus (MLV) presence in human cell lines established from mouse xenografts and to search for the evidence of horizontal viral spread to other cell lines.

Frequent detection of infectious xenotropic murine leukemia virus (XMLV) in human cultures established from mouse xenografts.

In an effort to identify novel biallelically inactivated tumor suppressor genes (TSGs) in sporadic invasive and preinvasive non-small-cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multiple 'omics' approach to investigate patient-matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes and in the earliest stages of lung cancer. We found that decreased EYA4 expression is not only associated with poor survival in sporadic lung cancers but also that EYA4 single-nucleotide polymorphisms are associated with increased familial cancer risk, consistent with EYA4s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we found that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross-examination of EYA4 expression across multiple tumor types suggests a cell-type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC. 

EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk.

The human secretin gene (SCT) encodes secretin, a hormone with limited tissue distribution. Analysis of the 450k methylation array data in The Cancer Genome Atlas (TCGA) indicated that the SCT promoter region is differentially hypermethylated in lung cancer. Our purpose was to validate SCT methylation as a potential biomarker for lung cancer.

Validation of SCT Methylation as a Hallmark Biomarker for Lung Cancers.

Despite recent advances in cancer therapy, the overall 5-year survival rate of patients with lung cancer remains low. The aim of our study was to search for novel markers for early diagnosis in patients with lung cancer.

ITPKA Gene Body Methylation Regulates Gene Expression and Serves as an Early Diagnostic Marker in Lung and Other Cancers.

Diffuse gliomas, grades II and III, hereafter called lower-grade gliomas (LGG), have variable, difficult to predict clinical courses, resulting in multiple studies to identify prognostic biomarkers. The purpose of this study was to assess expression or methylation of the homeobox family gene SHOX2 as independent markers for LGG survival.

SHOX2 is a Potent Independent Biomarker to Predict Survival of WHO Grade II-III Diffuse Gliomas.

Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth.

Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes.

Cell-free secretory products (secretome) of human induced pluripotent stem cells (iPSCs) have been shown to attenuate tissue injury and facilitate repair and recovery. To examine whether iPSC secretome facilitates mechanically induced compensatory responses following unilateral pneumonectomy (PNX), litter-matched young adult female hounds underwent right PNX (removing 55%-58% of lung units), followed by inhalational delivery of either the nebulized-conditioned media containing induced pluripotent stem cell secretome (iPSC CM) or control cell-free media (CFM); inhalation was repeated every 5 days for 10 treatments. Lung function was measured under anesthesia pre-PNX and 10 days after the last treatment (8 wk post-PNX); detailed quantitative analysis of lung ultrastructure was performed postmortem. Pre-PNX lung function was similar between groups. Compared with CFM control, treatment with iPSC CM attenuated the post-PNX decline in lung diffusing capacity for carbon monoxide and membrane diffusing capacity, accompanied by a 24% larger postmortem lobar volume and distal air spaces. Alveolar double-capillary profiles were 39% more prevalent consistent with enhanced intussusceptive angiogenesis. Frequency distribution of the harmonic mean thickness of alveolar blood-gas barrier shifted toward the lowest values, whereas alveolar septal tissue volume and arithmetic septal thickness were similar, indicating septal remodeling and reduced diffusive resistance of the blood-gas barrier. Thus, repetitive inhalational delivery of iPSC secretome enhanced post-PNX alveolar angiogenesis and septal remodeling that are associated with improved gas exchange compensation. Results highlight the plasticity of the remaining lung units following major loss of lung mass that are responsive to broad-based modulation provided by the iPSC secretome. To examine whether the secreted products of human induced pluripotent stem cells (iPSCs) facilitate innate adaptive responses following loss of lung tissue, adult dogs underwent surgical removal of one lung, then received repeated administration of iPSC secretory products via inhalational delivery compared with control treatment. Inhalation of iPSC secretory products enhanced capillary formation and beneficial structural remodeling in the remaining lung, leading to improved lung function.

Inhalational delivery of induced pluripotent stem cell secretome improves postpneumonectomy lung structure and function.

Aberrant DNA methylation has long been implicated in cancers. In this work we present a highly discriminative DNA methylation biomarker for non-small cell lung cancers and fourteen other cancers. Based on 69 NSCLC cell lines and 257 cancer-free lung tissues we identified a CpG island in SCT gene promoter which was verified by qMSP experiment in 15 NSCLC cell lines and 3 immortalized human respiratory epithelium cells. In addition, we found that SCT promoter was methylated in 23 cancer cell lines involving 10 cancer types profiled by ENCODE. We found that SCT promoter is hyper-methylated in primary tumors from TCGA lung cancer cohort. Additionally, we found that SCT promoter is methylated at high frequencies in fifteen malignancies and is not methylated in~1000 non-cancerous tissues across 30 organ types. Our study indicates that SCT promoter methylation is a highly discriminative biomarker for lung and many other cancers.

SCT Promoter Methylation is a Highly Discriminative Biomarker for Lung and Many Other Cancers.

Normal lungs do not express α-Klotho (Klotho) protein but derive cytoprotection from circulating soluble Klotho. It is unclear whether chronic supranormal Klotho levels confer additional benefit. To address this, we tested the age-related effects of modest Klotho overexpression on acute lung injury (ALI) and recovery. Transgenic Klotho-overexpressing () and wild-type () mice (2 and 6 mo old) were exposed to hyperoxia (95% O; 72 h; injury; Hx) then returned to normoxia (21% O; 24 h; recovery; Hx-R). Control mice were kept in normoxia. Renal and serum Klotho, lung histology, and bronchoalveolar lavage fluid oxidative damage markers were assessed. Effects of hyperoxia on Klotho release were tested in human embryonic kidney cells stably expressing Klotho. A549 lung epithelial cells transfected with  cDNA or vector were exposed to cigarette smoke; lactate dehydrogenase and double-strand DNA breaks were measured. Serum Klotho decreased with age. Hyperoxia suppressed renal Klotho at both ages and serum Klotho at 2 mo of age.  mice at both ages and 2-mo-old  mice survived Hx-R; 6-mo-old  mice showed lower lung damage than age-matched  mice. Hyperoxia directly inhibited Klotho expression and release in vitro;  transfection attenuated cigarette smoke-induced cytotoxicity and DNA double-strand breaks in lung epithelial cells. Young animals with chronic high baseline Klotho expression were more resistant to ALI. Chronic constitutive Klotho overexpression in older  animals attenuated hyperoxia-induced lung damage and improves survival and short-term recovery despite an acute reduction in serum Klotho during injury. We conclude that chronic enhancement of Klotho expression increases resilience to ALI.

Article	Year
Frequent detection of infectious xenotropic murine leukemia virus (XMLV) in human cultures established from mouse xenografts. Cancer biology & therapy\| PubMed ID: 21750403	2011
EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk. Oncogene\| PubMed ID: 24096489	2014
Validation of SCT Methylation as a Hallmark Biomarker for Lung Cancers. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer\| PubMed ID: 26725182	2016
ITPKA Gene Body Methylation Regulates Gene Expression and Serves as an Early Diagnostic Marker in Lung and Other Cancers. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer\| PubMed ID: 27234602	2016
SHOX2 is a Potent Independent Biomarker to Predict Survival of WHO Grade II-III Diffuse Gliomas. EBioMedicine\| PubMed ID: 27840009	2016
Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes. Translational lung cancer research\| PubMed ID: 29535911	2018
Inhalational delivery of induced pluripotent stem cell secretome improves postpneumonectomy lung structure and function. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 32909918	2020
SCT Promoter Methylation is a Highly Discriminative Biomarker for Lung and Many Other Cancers. IEEE life sciences letters\| PubMed ID: 33758771	2015
Constitutive transgenic α-Klotho overexpression enhances resilience to and recovery from murine acute lung injury. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 34346778	2021

Yu-An Zhang

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