Technical Advance
Abstract
Reliable diagnostic strategies for individuals with cancer demand practical methods for highly sensitive and specific detection of tumor cells. Amplification of genomic regions that include putative oncogenes is common in tumor cells of various types. Genomic array platforms offer the opportunity to identify and precisely map amplified genomic regions (ampGRs). The stable existence of these tumor cell–specific genomic aberrations during and after therapy, in theory, make ampGRs optimal targets for cancer diagnostics. In this study, we mapped ampGRs around the proto-oncogene MYCN of human neuroblastomas using a high-resolution tiling array (HR-TA). Based on the HR-TA data, we were able to precisely describe the telomeric and centromeric borders of the ampGRs and deduce virtual fusion sites of the joined ampGRs (amplicon fusion sites [AFSs]). These AFSs served as blueprints for the subsequent design of AFS bridging PCR assays (AFS-PCRs). Strikingly, these assays were absolutely tumor cell specific and capable of detecting 1 tumor cell in 1 × 106 to 8 × 106 control cells. We successfully proved the in vivo practicability of AFS-PCR by detecting and quantifying the specific AFS DNA of human MYCN-amplified neuroblastomas in the patients’ corresponding peripheral blood and bone marrow samples. Thus, we believe AFS-PCR could become a powerful and nevertheless feasible personalized diagnostic tool applicable to a large number of cancer patients, including children with MYCN-amplified neuroblastomas.
Authors
Axel Weber, Sylvia Taube, Sven Starke, Eckhard Bergmann, Nina Merete Christiansen, Holger Christiansen
Abstract
The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.
Authors
Fernanda I. Staquicini, Michael G. Ozawa, Catherine A. Moya, Wouter H.P. Driessen, E. Magda Barbu, Hiroyuki Nishimori, Suren Soghomonyan, Leo G. Flores 2nd, Xiaowen Liang, Vincenzo Paolillo, Mian M. Alauddin, James P. Basilion, Frank B. Furnari, Oliver Bogler, Frederick F. Lang, Kenneth D. Aldape, Gregory N. Fuller, Magnus Höök, Juri G. Gelovani, Richard L. Sidman, Webster K. Cavenee, Renata Pasqualini, Wadih Arap
Abstract
Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease.
Authors
Pradyot Dash, Jennifer L. McClaren, Thomas H. Oguin III, William Rothwell, Brandon Todd, Melissa Y. Morris, Jared Becksfort, Cory Reynolds, Scott A. Brown, Peter C. Doherty, Paul G. Thomas
Abstract
Wilms tumor (WT) is a genetically heterogeneous childhood kidney tumor. Several genetic alterations have been identified in WT patients, including inactivating mutations in WT1 and loss of heterozygosity or loss of imprinting at 11p15, which results in biallelic expression of IGF2. However, the mechanisms by which one or a combination of genetic alterations results in tumorigenesis has remained challenging to determine, given the lack of a mouse model of WT. Here, we engineered mice to sustain mosaic, somatic ablation of Wt1 and constitutional Igf2 upregulation, mimicking a subset of human tumors. Mice with this combination of genetic alterations developed tumors at an early age. Mechanistically, Wt1 ablation blocked mesenchyme differentiation, and increased Igf2 expression upregulated ERK1/2 phosphorylation. Importantly, a subset of human tumors similarly displayed upregulation of ERK1/2 phosphorylation, which suggests ERK signaling might contribute to WT development. Thus, we have generated a biologically relevant mouse model of WT and defined one combination of driver alterations for WT. This mouse model will provide a powerful tool to study the biology of WT initiation and progression and to investigate therapeutic strategies for cancers with IGF pathway dysregulation.
Authors
Qianghua Hu, Fei Gao, Weihua Tian, E. Cristy Ruteshouser, Yaqing Wang, Alexander Lazar, John Stewart, Louise C. Strong, Richard R. Behringer, Vicki Huff
Abstract
Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.
Authors
Xingshen Sun, Hongshu Sui, John T. Fisher, Ziying Yan, Xiaoming Liu, Hyung-Ju Cho, Nam Soo Joo, Yulong Zhang, Weihong Zhou, Yaling Yi, Joann M. Kinyon, Diana C. Lei-Butters, Michelle A. Griffin, Paul Naumann, Meihui Luo, Jill Ascher, Kai Wang, Timothy Frana, Jeffrey J. Wine, David K. Meyerholz, John F. Engelhardt
Abstract
Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (α1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded α1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor–mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.
Authors
S. Tamir Rashid, Sebastien Corbineau, Nick Hannan, Stefan J. Marciniak, Elena Miranda, Graeme Alexander, Isabel Huang-Doran, Julian Griffin, Lars Ahrlund-Richter, Jeremy Skepper, Robert Semple, Anne Weber, David A. Lomas, Ludovic Vallier
Abstract
Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.
Authors
Richard J. Stanton, Katarina Baluchova, Derrick J. Dargan, Charles Cunningham, Orla Sheehy, Sepehr Seirafian, Brian P. McSharry, M. Lynne Neale, James A. Davies, Peter Tomasec, Andrew J. Davison, Gavin W.G. Wilkinson
Abstract
Pulmonary metastasis remains the leading ca use of death for cancer patients. Opportunities to improve treatment outcomes for patients require new methods to study and view the biology of metastatic progression. Here, we describe an ex vivo pulmonary metastasis assay (PuMA) in which the metastatic progression of GFP-expressing cancer cells, from a single cell to the formation of multicellular colonies, in the mouse lung microenvironment was assessed in real time for up to 21 days. The biological validity of this assay was confirmed by its prediction of the in vivo behavior of a variety of high- and low-metastatic human and mouse cancer cell lines and the discrimination of tumor microenvironments in the lung that were most permissive to metastasis. Using this approach, we provide what we believe to be new insights into the importance of tumor cell interactions with the stromal components of the lung microenvironment. Finally, the translational utility of this assay was demonstrated through its use in the evaluation of therapeutics at discrete time points during metastatic progression. We believe that this assay system is uniquely capable of advancing our understanding of both metastasis biology and therapeutic strategies.
Authors
Arnulfo Mendoza, Sung-Hyeok Hong, Tanasa Osborne, Mohammed A. Khan, Kirk Campbell, Joseph Briggs, Ananth Eleswarapu, Lauren Buquo, Ling Ren, Stephen M. Hewitt, El-H. Dakir, Susan Garfield, Renard Walker, Glenn Merlino, Jeffrey E. Green, Kent W. Hunter, Lalage M. Wakefield, Chand Khanna
Abstract
Preterm infants are highly susceptible to life-threatening infections that are clinically difficult to detect, such as late-onset septicemia and necrotizing enterocolitis (NEC). Here, we used a proteomic approach to identify biomarkers for diagnosis of these devastating conditions. In a case-control study comprising 77 sepsis/NEC and 77 nonsepsis cases (10 in each group being monitored longitudinally), plasma samples collected at clinical presentation were assessed in the biomarker discovery and independent validation phases. We validated the discovered biomarkers in a prospective cohort study with 104 consecutively suspected sepsis/NEC episodes. Proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid A (SAA) were identified as the most promising biomarkers. The ApoSAA score computed from plasma apoC2 and SAA concentrations was effective in identifying sepsis/NEC cases in the case-control and cohort studies. Stratification of infants into different risk categories by the ApoSAA score enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of nonsepsis infants. The negative predictive value of this antibiotic policy was 100%. The ApoSAA score could potentially allow early and accurate diagnosis of sepsis/NEC. Upon confirmation by further multicenter trials, the score would facilitate rational prescription of antibiotics and target infants who require urgent treatment.
Authors
Pak Cheung Ng, Irene Ling Ang, Rossa Wai Kwun Chiu, Karen Li, Hugh Simon Lam, Raymond Pui On Wong, Kit Man Chui, Hon Ming Cheung, Eddy Wing Yin Ng, Tai Fai Fok, Joseph Jao Yiu Sung, Yuk Ming Dennis Lo, Terence Chuen Wai Poon
Abstract
Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6Chi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6Clo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6Chi monocytes in allografts prompted us to screen for a Ly-6Chi monocyte–associated imaging marker. Low-density array data revealed that Ly-6Chi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6Clo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6Chi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored.
Authors
Filip K. Swirski, Moritz Wildgruber, Takuya Ueno, Jose-Luiz Figueiredo, Peter Panizzi, Yoshiko Iwamoto, Elizabeth Zhang, James R. Stone, Elisenda Rodriguez, John W. Chen, Mikael J. Pittet, Ralph Weissleder, Matthias Nahrendorf
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)