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Author(s): Alexander, Roger P; Giraldez, MD; Spengler, RM; Etheridge, A; Godoy, PM; Barczak, AJ; Srinivasan, S; De Hoff, PL; Tanriverdi, K; Courtright, A; Lu, S; Khoory, J; Rubio, R; Baxter, D; Driedonks, TAP; Buermans, HPJ; Nolte-t Hoen, ENM; Jiang, H; Wang, K; Ghiran, I; Wang, Y; Van Keuren-Jensen, K; Freedman, JE; Woodruff, PG; Laurent, LC; Erle, DJ; Galas, DJ; Tewari, M

Author(s): Bouchareychas, L; Duong, P; Covarrubias, S; Alsop, E; Q Phu, TA; Chung, A; Gomes, M; Wong, D; Meechoovet, B; Capili, A; Yamamoto, R; Nakauchi, H; Mcmanus, M; Carpenter, S; Van Keuren-Jensen, K; Raffai, RL

The choroid plexus (CP) is a highly vascularized structure located in the ventricles that forms the blood-CSF barrier (BCSFB) and separates the blood from the cerebrospinal fluid (CSF). In addition to its role as a physical barrier, the CP functions in CSF secretion, transport of nutrients into the central nervous system (CNS) and a gated point of entry of circulating immune cells into the CNS. Aging and neurodegeneration have been reported to affect CP morphology and function and increase protein leakage from blood to the CSF. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with both upper and lower motor neuron loss, as well as altered proteomic and metabolomic signatures in the CSF. The role of the BCSFB and the CP in ALS is unknown. Here we describe a transcriptomic and ultrastructural analysis of BCSFB and CP alterations in human postmortem tissues from ALS and non-neurologic disease controls. ALS-CP exhibited widespread disruptions in tight junctional components of the CP epithelial layer and vascular integrity. In addition, we detected loss of pericytes around ALS blood vessels, accompanied by activation of platelet aggregation markers vWF and Fibrinogen, reminiscent of vascular injury. To investigate the immune component of ALS-CP, we conducted a comprehensive analysis of cytokines and chemokine panels in CP lysates and found a significant down-regulation of M-CSF and V-CAM1 in ALS, as well as up-regulation of VEGF-A protein. This phenotype was accompanied by an infiltration of MERTK positive macrophages into the parenchyma of the ALS-CP when compared to controls. Taken together, we demonstrate widespread structural and functional disruptions of the BCSFB in human ALS increasing our understanding of the disease pathology and identifying potential new targets for ALS therapeutic development.

Additional file 1: Figure S1. ZO-1 immunofluorescence was performed on control (n = 4) and ALS CP (n = 5) and staining was imaged under 20x using confocal microscopy. DAPI denotes nuclei and DIC (differential interference contrast) images were taken to show tissue morphology. Scale bar: 50um. Red arrowheads point to areas of loss of ZO-1 staining in between CP epithelial cells. Figure S2. A) Immunohistochemistry for Claudin 5 was performed on control (n = 3) and ALS CP (n = 5). Pictures were taken at 40X magnification. Scale bar: 20 μm. Black Arrows point to staining in endothelial cells and red arrowheads point to staining within the lumen of blood vessels. B) Western blot analysis was performed on 9 controls and 18 SALS CP lysates and membranes were probed for Claudin 3 and 5, as well as Occludin. GAPDH was used as a loading control. Figure S3. A) Quantification of immunofluorescence signal intensity for CD13 and PDGFRbeta on n = 3 control and n = 5 ALS CP samples was performed using Imaris. Image stacks were taken at 63x magnification and ten different Z-stack fields per sample were captured and quantified. Tissues boundaries were delineated and signal intensity was normalized to section volume. The asterisk denotes significance with a p-value of 0.0095. Values for PDGFRbeta were not found to be significant. B) CD13/ANPEP immunohistochemistry in ALS (n = 5) and control (n = 4) CP. Black arrows point to CD13 stain around blood vessels on controls and red arrowheads denote vascular areas of CD13 loss in ALS. Figure S4 A) CD3 staining in ALS-CP tissues. Scale bar: 20 μm. B) MERTK immunohistochemistry in ALS and Control CP. Pictures were taken at low magnification to show extent of MERTK expression. Scale bar 100 μm C) p-TDP43 (S409/410) staining in CP from ALS and Control postmortem tissues. Only two ALS cases showed any inclusions, and these were located in the choroid stroma. Scale bar: 20 μm. Figure S5 Heatmap showing Z-scores across all samples for all gene targets that had reliable MSD signal. Samples are presented in their respective groups (control vs ALS), ranked by cumulative sum of all analytes measured. Figure S6 All cytokines and chemokines analyzed by MSD immunoassays in n = 22 controls and n = 44 ALS-CP lysates. None of the shown analytes showed significant differences between the two groups through a logistic regression analysis that accounts for patient age (under 65 vs above 65), as well as the plate on which the sample was run to avoid batch effect bias. Both IFN-γ and TNF-α had low signal to background ratio. Supplemental Table 1. ToppGene Analysis showing the top 30 enriched Biological Processes. Supplemental Table 2. MSD immunoassay analysis of CP lysates from ALS and non-neurological disease controls. Supplemental Table 3. Demographics for post-mortem tissue samples. Supplemental Table 4. CP samples used for RNA-seq. Supplemental Table 5. List of antibodies used in analysis of CP samples.

Triple-negative breast cancer (TNBC) is an aggressive subtype accounting for nearly 20% of all breast cancer (BC) cases with disproportionally poor prognosis. Treatment options are limited by the lack of expression of common BC receptors targeted by FDA-approved small molecule inhibitors, resulting in an unmet need for efficient and cost effective treatments. Immunotherapies are emerging as a powerful alternative to standard chemo- and radiation therapy. Our objectives are to generate vaccines targeting TNBC and to identify circulating biomarkers capable of monitoring their therapeutic efficacy in a human transgenic mouse model (C57BL/6) compatible with EO771.LMB, a murine cell line of TNBC with a reported basal-like phenotype. Our current study characterized the cancer-related proteomic and metabolic signatures derived from extracellular vesicles isolated from EO771.LMB. It has been shown that extracellular vesicles (EV) shed by tumors carry complex cargos comprised of proteins, metabolites and nucleic acids and may exhibit cancer-specific signatures with a potential as prognostic markers or predictors of therapy response. EVs released by TNBC tumors into circulation might provide non-invasive and highly actionable insight into the tumor biology of this aggressive cancer. To further our understanding of cancer-specific EV proteomes, we used mass-spectrometry based proteomics and identified a total of 2265 unique proteins from EVs isolated from conditioned media from EO771.LMB. Our survey across the EV proteome, the expression proteome (5096) and phosphoproteome (2728) identified 6461 unique proteins overall. Within EV sub-fractions, microvesicles and apoptotic bodies were characterized by proteomic cargos different from exosomes. Over 90% of proteins identified in EVs shed by cultured EO771.LMB mapped with proteins curated in ExoCarta. Of interest, exploration of the EV proteome identified 47 GPI-anchored surface proteins. These candidate membrane proteins included Glypican-1 and CD109, previously reported as highly specific to cancer, as well as potential TNBC-specific markers. Our multipronged strategy for deep proteomic profiling of EO771.LMB enhanced identification by ˜25% compared to global whole cell proteomics alone. We also identified ˜3150 metabolites corresponding to lysophosphatidyl choline (35%), fatty-acids, -esters, -amides, -alcohols (30%), glycerophospholipids and sphingolipids (16%), sugar (1%), amino acids and biogenic amines (4%), organic acids and derivatives, cyclic alcohols, aromatic compounds and steroids (13%). Of these, 1450 metabolites were shared between cell line and EVs. A proteogenomic approach expanded identification to onco-proteoforms and tumor-specific peptides not represented in canonical databases. Custom, cell-specific protein databases derived from whole transcriptome sequencing of EO771.LMB elucidated novel protein variants and mutations. Pan-cancer markers vs. TNBC-specific markers will be verified in other murine cell lines, across multiple cancer types and fibroblast controls. Optimal membrane markers for downstream immune-affinity purification from plasma will be proposed, and their ability to diagnose disease or monitor tumor load during treatment will be discussed. Citation Format: Igyártó B, Sharma R, Garcia K, David-Dirgo V, Pathak K, Kroll M, Terraf P, Van Keuren-Jensen K, Pirrotte P. An integrative approach to the discovery of triple-negative breast cancer markers derived from extracellular vesicles [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-21.

Author(s): Alexander, Roger P; Giraldez, MD; Spengler, RM; Etheridge, A; Godoy, PM; Barczak, AJ; Srinivasan, S; De Hoff, PL; Tanriverdi, K; Courtright, A; Lu, S; Khoory, J; Rubio, R; Baxter, D; Driedonks, TAP; Buermans, HPJ; Nolte-t Hoen, ENM; Jiang, H; Wang, K; Ghiran, I; Wang, Y; Van Keuren-Jensen, K; Freedman, JE; Woodruff, PG; Laurent, LC; Erle, DJ; Galas, DJ; Tewari, M

Author(s): Giraldez, MD; Spengler, RM; Etheridge, A; Godoy, PM; Barczak, AJ; Srinivasan, S; De Hoff, PL; Tanriverdi, K; Courtright, A; Lu, S; Khoory, J; Rubio, R; Baxter, D; Driedonks, TAP; Buermans, HPJ; Nolte-‘t Hoen, ENM; Jiang, H; Wang, K; Ghiran, I; Wang, Y; Van Keuren-Jensen, K; Freedman, JE; Woodruff, PG; Laurent, LC; Erle, DJ; Galas, DJ; Tewari, M | Abstract: Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.