A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Atorvastatin and simvastatin, but not pravastatin, up-regulate LPS-induced MMP-9 expression in macrophages by regulating phosphorylation of ERK and CREB.

Statins suppress expression of pro-inflammatory cytokines in endothelial cells, whereas they enhance it in immune cells. Pro-inflammatory cytokines and lipopolysaccharide (LPS) induce matrix metalloproteinase (MMP)-9 gene expression in macrophages, which has been linked to progress of various inflammatory diseases. The aim of this study was to identify effects of various statins on LPS-induced MMP-9 gene expression in macrophages and microglia. MMP-9 expression was analyzed by real-time PCR or zymography. Effect of statins on activation of signaling pathways was analyzed by time-dependent phosphorylation of signaling molecules. Atorvastatin and simvastatin, but not pravastatin, up-regulated LPS-induced MMP-9 expression in murine RAW 264.7 macrophages and BV2 microglia. The phosphorylation duration of extracellular signal regulated kinases was extended by simvastatin, but not by atorvastatin or pravastatin. The up-regulation of LPS-induced MMP-9 gene expression by the statins was dependent on extracellular calcium ions and mediated by enhancing phosphorylation of cAMP-responsive element binding protein. Geranylgeranyl pyrophosphate, a precursor for cholesterol synthesis, could suppress up-regulation of LPS-mediated MMP-9 gene expression by atorvastatin and simvastatin. Atorvastatin and simvastatin-mediated up-regulation of LPS-induced MMP-9 gene expression in macrophages and microglia in vitro raises an important concern about use of the widely-prescribed statins in certain inflammatory conditions that are mediated by LPS.

Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.

Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.

Effect of Antigen Retrieval on Genomic DNA From Immunodissected Samples.

Immunohistochemical (IHC) staining is an established technique for visualizing proteins in tissue sections for research studies and clinical applications. IHC is increasingly used as a targeting strategy for procurement of labeled cells via tissue microdissection, including immunodissection, computer-aided laser dissection (CALD), expression microdissection (xMD), and other techniques. The initial antigen retrieval (AR) process increases epitope availability and improves staining characteristics; however, the procedure can damage DNA. To better understand the effects of AR on DNA quality and quantity in immunodissected samples, both clinical specimens (KRAS gene mutation positive cases) and model system samples (lung cancer patient-derived xenograft tissue) were subjected to commonly employed AR methods (heat induced epitope retrieval [HIER], protease digestion) and the effects on DNA were assessed by Qubit, fragment analysis, quantitative PCR, digital droplet PCR (ddPCR), library preparation, and targeted sequencing. The data showed that HIER resulted in optimal IHC staining characteristics, but induced significant damage to DNA, producing extensive fragmentation and decreased overall yields. However, neither of the AR methods combined with IHC prevented ddPCR amplification of small amplicons and gene mutations were successfully identified from immunodissected clinical samples. The results indicate for the first time that DNA recovered from immunostained slides after standard AR and IHC processing can be successfully employed for genomic mutation analysis via ddPCR and next-generation sequencing (NGS) short-read methods.

An advanced molecular medicine case report of a rare human tumor using genomics, pathomics, and radiomics.

Background: Pulmonary Sclerosing Pneumocytoma (PSP) is a rare tumor of the lung with a low malignant potential that primarily affects females. Initial studies of PSP focused primarily on analyzing features uncovered using conventional X-ray or CT imaging. In recent years, because of the widespread use of next-generation sequencing (NGS), the study of PSP at the molecular-level has emerged. Methods: Analytical approaches involving genomics, radiomics, and pathomics were performed. Genomics studies involved both DNA and RNA analyses. DNA analyses included the patient's tumor and germline tissues and involved targeted panel sequencing and copy number analyses. RNA analyses included tumor and adjacent normal tissues and involved studies covering expressed mutations, differential gene expression, gene fusions and molecular pathways. Radiomics approaches were utilized on clinical imaging studies and pathomics techniques were applied to tumor whole slide images. Results: A comprehensive molecular profiling endeavor involving over 50 genomic analyses corresponding to 16 sequencing datasets of this rare neoplasm of the lung were generated along with detailed radiomic and pathomic analyses to reveal insights into the etiology and molecular behavior of the patient's tumor. Driving mutations (AKT1) and compromised tumor suppression pathways (TP53) were revealed. To ensure the accuracy and reproducibility of this study, a software infrastructure and methodology known as NPARS, which encapsulates NGS and associated data, open-source software libraries and tools including versions, and reporting features for large and complex genomic studies was used. Conclusion: Moving beyond descriptive analyses towards more functional understandings of tumor etiology, behavior, and improved therapeutic predictability requires a spectrum of quantitative molecular medicine approaches and integrations. To-date this is the most comprehensive study of a patient with PSP, which is a rare tumor of the lung. Detailed radiomic, pathomic and genomic molecular profiling approaches were performed to reveal insights regarding the etiology and molecular behavior. In the event of recurrence, a rational therapy plan is proposed based on the uncovered molecular findings.

NPARS-A Novel Approach to Address Accuracy and Reproducibility in Genomic Data Science.

Background: Accuracy and reproducibility are vital in science and presents a significant challenge in the emerging discipline of data science, especially when the data are scientifically complex and massive in size. Further complicating matters, in the field of genomic-based science high-throughput sequencing technologies generate considerable amounts of data that needs to be stored, manipulated, and analyzed using a plethora of software tools. Researchers are rarely able to reproduce published genomic studies. Results: Presented is a novel approach which facilitates accuracy and reproducibility for large genomic research data sets. All data needed is loaded into a portable local database, which serves as an interface for well-known software frameworks. These include python-based Jupyter Notebooks and the use of RStudio projects and R markdown. All software is encapsulated using Docker containers and managed by Git, simplifying software configuration management. Conclusion: Accuracy and reproducibility in science is of a paramount importance. For the biomedical sciences, advances in high throughput technologies, molecular biology and quantitative methods are providing unprecedented insights into disease mechanisms. With these insights come the associated challenge of scientific data that is complex and massive in size. This makes collaboration, verification, validation, and reproducibility of findings difficult. To address these challenges the NGS post-pipeline accuracy and reproducibility system (NPARS) was developed. NPARS is a robust software infrastructure and methodology that can encapsulate data, code, and reporting for large genomic studies. This paper demonstrates the successful use of NPARS on large and complex genomic data sets across different computational platforms.

MAGI1 as a link between endothelial activation and ER stress drives atherosclerosis.

The possible association between the membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) and inflammation has been suggested, but the molecular mechanisms underlying this link, especially during atherogenesis, remain unclear. In endothelial cells (ECs) exposed to disturbed flow (d-flow), p90 ribosomal S6 kinase (p90RSK) bound to MAGI1, causing MAGI1-S741 phosphorylation and sentrin/SUMO-specific protease 2 T368 phosphorylation-mediated MAGI1-K931 deSUMOylation. MAGI1-S741 phosphorylation upregulated EC activation via activating Rap1. MAGI1-K931 deSUMOylation induced both nuclear translocation of p90RSK-MAGI1 and ATF-6-MAGI1 complexes, which accelerated EC activation and apoptosis, respectively. Microarray screening revealed key roles for MAGI1 in the endoplasmic reticulum (ER) stress response. In this context, MAGI1 associated with activating transcription factor 6 (ATF-6). MAGI1 expression was upregulated in ECs and macrophages found in atherosclerotic-prone regions of mouse aortas as well as in the colonic epithelia and ECs of patients with inflammatory bowel disease. Further, reduced MAGI1 expression in Magi1-/+ mice inhibited d-flow-induced atherogenesis. In sum, EC activation and ER stress-mediated apoptosis are regulated in concert by two different types of MAGI1 posttranslational modifications, elucidating attractive drug targets for chronic inflammatory disease, particularly atherosclerosis.

Surgical thyroparathyroidectomy prevents progression of 5TGM1 murine multiple myeloma in vivo.

The 5TGM1 multiple myeloma transplanted C57BL6/KaLwRij model recapitulates many disease features including monoclonal paraprotein production as well as the development of osteolytic bone lesions. Since a significant association between serum parathyroid hormone PTH variations, bone anabolism and myeloma progression in patients receiving proteasome inhibitors exists, this study investigated the effect of the PTH axis on murine myeloma development in vivo. C57BL6/KaLwRij myeloma-bearing mice underwent thyroparathyroidectomy (TPTX) before and after 5TGM1 cell transplantation. TPTX significantly and permanently inhibited 5TGM1 myeloma cell engraftment and prevented multiple myeloma growth and progression. These data support the hypothesis that the PTH axis is an important mediator of myeloma bone disease.

Visualizing collagen proteolysis by peptide hybridization: From 3D cell culture to in vivo imaging.

Degradation of the extracellular matrix (ECM) is one of the fundamental factors contributing to a variety of life-threatening or disabling pathological conditions. However, a thorough understanding of the degradation mechanism and development of new ECM-targeting diagnostics are severely hindered by a lack of technologies for direct interrogation of the ECM structures at the molecular level. Previously we demonstrated that the collagen hybridizing peptide [CHP, sequence: (GPO)9, O: hydroxyproline] can specifically recognize the degraded and unfolded collagen chains through triple helix formation. Here we show that fluorescently labeled CHP robustly visualizes the pericellular matrix turnover caused by proteolytic migration of cancer cells within 3D collagen culture, without the use of synthetic fluorogenic matrices or genetically modified cells. To facilitate in vivo imaging, we modified the CHP sequence by replacing each proline with a (2S,4S)-4-fluoroproline (f) residue which interferes with the peptide's inherent propensity to self-assemble into homo-triple helices. We show that the new CHP, (GfO)9, tagged with a near-infrared fluorophore, enables in vivo imaging and semi-quantitative assessment of osteolytic bone lesions in mouse models of multiple myeloma. Compared to conventional techniques (e.g., micro-CT), CHP-based imaging is simple and versatile in vitro and in vivo. Therefore, we envision CHP's applications in broad biomedical contexts ranging from studies of ECM biology and drug efficiency to development of clinical molecular imaging.

Counteracting the activation of pAkt by inhibition of MEK/Erk inhibition reduces actin disruption-mediated apoptosis in PTEN-null PC3M prostate cancer cell lines.

The actin cytoskeleton is important in the maintenance of cellular homeostasis and in signal transduction pathways leading to cell growth and apoptotic cell death in eukaryotic cells. Disruption of actin dynamics is associated with morphological changes in cancer cells. Deletion of phosphatase and tensin homolog (PTEN), a tumor suppressor gene involved in the regulation of the cell cycle and apoptosis, leads to cytoskeleton disruption and double-strand breaks (DSBs). To study the mechanism(s) of actin disruption-mediated apoptosis and its potential application for anticancer therapy, PTEN-null PC3M prostate cancer cells were treated with latrunculin B (LB). LB induced destabilization of the actin microfilament and apoptosis in a dose-dependent manner, as demonstrated by morphological changes and nuclear condensation in the PC3M cells. In addition, it resulted in an increase in the levels of γH2AX recruitment, implicating the induction of DNA damage, including DSBs. Induction of Bax, with little effect on Bcl-2 expression, indicated that actin disruption causes apoptosis through activation of Bax signaling in PC3M cells. Treatment with U20126, a mitogen-activated protein kinase kinase (MEK) inhibitor, resulted in attenuated induction of DSBs and apoptosis through activation of protein kinase B (Akt), suggesting that LB-mediated actin dysfunction induces DSBs via the MEK/extracellular signal-regulated kinase (Erk) pathway in cells. Therefore, counteracting activation of phosphorylated Akt stemming from the inhibition of MEK/Erk resulted in attenuation of actin disruption-induced apoptotic events in the PC3M cells. The results of this study provide information not only for use in delineation of the molecular association between actin disruption and tumorigenesis, but also for the development of a strategy for actin-based anticancer chemotherapy against highly metastatic prostate cancer.

Hypoxia antagonizes glucose deprivation on interleukin 6 expression in an Akt dependent, but HIF-1/2α independent manner.

Although both glucose deprivation and hypoxia have been reported to promote cascades of biological alterations that lead to induction of inflammatory mediators, we hypothesized that glucose deprivation and hypoxia might show neutral, synergistic or antagonistic effects to each other on gene expression of inflammatory mediators depending on the regulatory components in their promoters. Gene expression of interleukin 6 (IL-6) was analyzed by real-time PCR, ELISA, or Western blot. Effects of glucose deprivation and/or hypoxia on activation of signaling pathways were analyzed by time-dependent phosphorylation patterns of signaling molecules. We demonstrate that hypoxia antagonized the effects of glucose deprivation on induction of IL-6 gene expression in microglia, macrophages, and monocytes. Hypoxia also antagonized thapsigargin-induced IL-6 gene expression. Hypoxia enhanced phosphorylation of Akt, and inhibition of Akt was able to reverse the effects of hypoxia on IL-6 gene expression. However, inhibition of HIF-1/2α did not reverse the effects of hypoxia on IL-6 gene expression. In addition, phosphorylation of p38, but not JNK, was responsible for the effects of glucose deprivation on IL-6 gene expression.

Characterization of partial ligation-induced carotid atherosclerosis model using dual-modality molecular imaging in ApoE knock-out mice.

BACKGROUND: Recently, partial ligation of the common carotid artery (CCA) was reported to induce carotid atheromata rapidly in apolipoprotein-E knockout (ApoE(-/-)) mice. We investigated this new atherosclerosis model by using combined matrix-metalloproteinase (MMP) near-infrared fluorescent (NIRF) imaging and macrophage-tracking luciferase imaging. METHODOLOGY AND PRINCIPAL FINDINGS: Partial ligation of the left CCA was performed in 10-week-old ApoE(-/-) mice on a high fat diet (n=33); the internal and external carotid arteries and occipital artery were ligated, while the superior thyroid artery was left intact. Two thirds of the animals were treated with either LiCl or atorvastatin. At 1-week, Raw264.7 macrophages modified to express the enhanced firefly-luciferase reporter gene (10(7) Raw-luc cells) were injected intravenously. At 2-week, NIRF molecular imaging visualized strong MMP-2/9 activity in the ligated area of the left CCA as well as in the aortic arch. Left-to-right ratios of the NIRF signal intensities in the CCA had a decreasing gradient from the highest value in the upper-most ligated area to the lowest value in the lower-most region adjacent to the aortic arch. Luciferase imaging showed that most Raw-luc macrophages were recruited to the ligated area of the CCA rather than to the aortic arch, despite similarly strong MMP-2/9-related NIRF signal intensities in both areas. In addition, LiCl or atorvastatin could reduce MMP-2/9 activity in the aortic arch but not in the ligated area of the CCA. CONCLUSIONS/SIGNIFICANCE: This is the first molecular imaging study to characterize the partial ligation-induced carotid atherosclerosis model. Molecularly divergent types of atherosclerosis were identified: conventional lipogenic atherosclerosis in the aorta vs. flow-related mechanical atherosclerosis in the partially ligated left system.

Actin disruption agents induce phosphorylation of histone H2AX in human breast adenocarcinoma MCF-7 cells.

Modified actin dynamics are a unique feature of transformed cancer cells and thereby promising targets for cancer chemotherapy. While latrunculin B (LB) and pectenotoxin-2 (PTX-2), both derived from natural sources, inhibit actin polymerization, jasplakinolide (JSP) prevents actin depolymerization. The purpose of this study was to examine the detailed molecular action of actin disruption inducing apoptosis via double strand breaks (DSBs). Actin disruption induced phosphorylation of H2AX, a well known DSB marker leading to G2 arrest and consequently resulted in apoptosis on MCF-7 cancer cells. Cells impaired by actin disruption activated Erk (extracellular signal-related kinase) and p53 protein was involved in DNA damage responses, but did not change the levels of p21Cip1/WAF1 protein in MCF-7 cells. To overcome the DSBs by actin disruption, MCF-7 cells set the repair system through the homologous recombination (HR) pathway. These results indicate that actin is involved in the signaling inducing DSBs and HR repair as well as G2 cell cycle arrest in human cancer. Therefore, the results suggest that actin disruption might be a potential candidate for developing anti-cancer therapies in human breast cancer.

Actin disruption inhibits hypoxia inducible factor-1α expression via inactivity of Mdm2-mediated p70S6K.

The intracellular actin cytoskeleton is a central player in tumor cell migration and adhesion, and interacts with the extracellular matrix during the progression to metastasis. Although recent reports on motility events have revealed that the destabilization of actin affects cancer progression and hypoxia inducible factor-1α (HIF-1α) activity, little is known about the responsive activity of HIF-1α following actin disruption. Here, we demonstrate that the inhibition of actin polymerization or depolymerization attenuates HIF-1α expression independently of proteasomal degradation. The disruption of actin dynamics inactivates HIF-1α translational expression through p70S6K translational signaling; this is independent of p53 activation, suggesting that actin dysfunction-mediated HIF-1α destabilization may lead to the development of novel anticancer chemotherapeutic targets.