Putative Cell Surface Research Articles

A High-Affinity Monoclonal Antibody Against the Pancreatic Ductal Adenocarcinoma Target, Anterior Gradient-2 (AGR2/PDIA17).

Anterior Gradient-2 (AGR2/PDIA17) is a member of the protein disulfide isomerase (PDI) family of oxidoreductases. AGR2 is up-regulated in several solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Given the dire need for new therapeutic options for PDAC patients, we investigated the expression and function of AGR2 in PDAC and developed a novel series of affinity-matured AGR2-specific single-chain variable fragments (scFvs) and monoclonal antibodies. We found that AGR2 was expressed in approximately 90% of PDAC but not normal pancreas biopsies, and the level of AGR2 expression correlated with increasing disease stage. AGR2 expression was inversely related to SMAD4 status in PDAC and colorectal cancer cell models and was secreted from cells into their media. In normal tissues, a high density of AGR2 was detected in the epithelium of cells in the digestive tract but was lacking in most other normal tissue systems. The addition of recombinant AGR2 to cell culture and genetic overexpression of AGR2 increased the adhesion, motility, and invasiveness of both human and mouse PDAC cells. Human phage display library screening led to the discovery of multiple AGR2-specific scFv clones that were affinity-matured to produce monoclonal antibody (MAb) clones with low picomolar binding affinity (S31R/A53F/Y). These high-affinity MAbs inhibited AGR2-mediated cell adhesion, migration, and binding to LYPD3, which is a putative cell surface binding partner of AGR2. Our study provides novel, high-affinity, fully human, anti-AGR2 MAbs that neutralize the pro-tumor effects of extracellular AGR2 in PDAC.

Helicobacter pylori Chronic Infection Selects for Effective Colonizers of Metaplastic Glands.

Chronic gastric infection with Helicobacter pylori can lead to progressive tissue changes that culminate in cancer, but how H. pylori adapts to the changing tissue environment during disease development is not fully understood. In a transgenic mouse gastric metaplasia model, we found that strains from unrelated individuals differed in their ability to infect the stomach, to colonize metaplastic glands, and to alter the expression of the metaplasia-associated protein TFF3. H. pylori isolates from different stages of disease from a single individual had differential ability to colonize healthy and metaplastic gastric glands. Exposure to the metaplastic environment selected for high gastric colonization by one of these strains. Complete genome sequencing revealed a unique alteration in the frequency of a variant allele of the putative adhesin sabB, arising from a recombination event with the related sialic acid binding adhesin (SabA) gene. Mutation of sabB in multiple H. pylori strain backgrounds strongly reduced adherence to both normal and metaplastic gastric tissue, and highly attenuated stomach colonization in mice. Thus, the changing gastric environment during disease development promotes bacterial adhesin gene variation associated with enhanced gastric colonization. IMPORTANCE Chronic infection with Helicobacter pylori is the primary risk factor for developing stomach cancer. As disease progresses H. pylori must adapt to a changing host tissue environment that includes induction of new cell fates in the cells that line the stomach. We tested representative H. pylori isolates collected from the same patient during early and later stages of disease in a mouse model where we can rapidly induce disease-associated tissue changes. Only the later-stage H. pylori strains could robustly colonize the diseased stomach environment. We also found that the ability to colonize the diseased stomach was associated with genetic variation in a putative cell surface adhesin gene called sabB. Additional experiments revealed that SabB promotes binding to stomach tissue and is critical for stomach colonization by the late-stage strains. Thus, H. pylori diversifies its genome during disease progression and these genomic changes highlight critical factors for bacterial persistence.

LilrB3 is a putative cell surface receptor of APOE4.

The three isoforms of apolipoprotein E (APOE2, APOE3, and APOE4) only differ in two amino acid positions but exert quite different immunomodulatory effects. The underlying mechanism of such APOE isoform dependence remains enigmatic. Here we demonstrate that APOE4, but not APOE2, specifically interacts with the leukocyte immunoglobulin-like receptor B3 (LilrB3). Two discrete immunoglobin-like domains of the LilrB3 extracellular domain (ECD) recognize a positively charged surface patch on the N-terminal domain (NTD) of APOE4. The atomic structure reveals how two APOE4 molecules specifically engage two LilrB3 molecules, bringing their intracellular signaling motifs into close proximity through formation of a hetero-tetrameric complex. Consistent with our biochemical and structural analyses, APOE4, but not APOE2, activates human microglia cells (HMC3) into a pro-inflammatory state in a LilrB3-dependent manner. Together, our study identifies LilrB3 as a putative immune cell surface receptor for APOE4, but not APOE2, and may have implications for understanding the biological functions as well as disease relevance of the APOE isoforms.

Trial in Progress: Tagraxofusp-Based Therapy to Eradicate Measurable Residual Disease of Acute Myelogenous Leukemia Prior to Allogeneic Hematopoietic Cell Transplantation

Background Numerous studies convincingly demonstrate that measurable residual disease (MRD) in AML at time of allogeneic cell transplantation (alloHCT) is a powerful, independent predictor of subsequent relapse. Attempts to eradicate AML MRD pre-transplant with consolidation chemotherapy have been unsuccessful, with reported MRD conversion rates as low as 20% (Appelbaum et al., Blood 2019). Tagraxofusp is an IL-3 truncated diphtheria toxin recombinant protein that binds CD123 with high affinity. Preliminary data using tagraxofusp as consolidation therapy in AML patients has demonstrated good tolerability (Lane et al., Blood 2017). Pathways of resistance to tagraxofusp have been identified preclinically as loss of the intracellular target for diphtheria toxin, diphthamide, through methylation of an enzyme involved in diphthamide synthesis. Treatment with a hypomethylating agent has the potential to restore expression of diphthamide and increase tagraxofusp sensitivity in AML cells (Togami et al., J Clin Invest 2019). Design This is a single-arm, open-label, multi-center phase Ib/II clinical trial to evaluate the safety and effectiveness of tagraxofusp +/- azacitidine in AML patients who are in first or second remission with detectable MRD and are planned to undergo alloHCT. MRD will be measured by multi-dimensional flow cytometry with sensitivity of 0.02% (Hematologics). Patients will be enrolled in a modified 3+3 dose escalation design with two cohorts: cohort A) tagraxofusp 12 mcg/kg IV qday on days 1-5; and cohort B) azacitidine 75 mg/m2 subq qday on days 1-5 plus tagraxofusp 12 mcg/kg qday on days 8-12. Patients achieving MRD-negativity after cycle 1 will proceed to alloHCT. If a patient remains MRD-positive, an additional cycle of investigational therapy will be administered. All patients will proceed to alloHCT after no more than 2 cycles of investigational therapy. Both cohorts contain a dose escalation and a dose expansion phase. Cohort B will open if cohort A is not efficacious. Up to 44 participants will be enrolled in this trial at University of California Los Angeles and University of California San Francisco (two constituent sites of the University of California Hematologic Malignancies Consortium). Objectives The primary objective is to estimate the rate of conversion from MRD-positive pre-investigational therapy to MRD-negative after 1 or 2 cycles of investigational therapy. Key secondary objectives include to determine the safety of tagraxofusp +/- azacitidine in this patient population and to evaluate if adverse effects of this investigational therapy lead to delays in alloHCT. Exploratory objectives will involve use of single cell RNA sequencing to investigate if response to investigational therapy is predicted by leukemia-specific biomarkers and to evaluate if putative cell surface markers (inferred from RNA detection) are associated with MRD persistence. Main Outcomes The primary endpoint is MRD status after 1 or 2 cycles of investigational therapy. Key secondary endpoints include dose-limiting toxicities (any grade 4 or 5 non-hematologic toxicity or any grade 3 or greater hematologic toxicity at day 42) and number of days between investigational therapy and alloHCT. Conclusions There is an unmet need to improve outcomes in AML with detectable MRD pre-transplant. The strategy in this study is to employ tagraxofusp as an alternative mechanism of action to eradicate chemo-resistant AML cells responsible for subsequent relapse. In order to mitigate any potential resistance the residual leukemia population may have against tagraxofusp, azacitidine will be added to optimize the likelihood for MRD negativity and the possibility for improved outcomes after alloHCT. This study was sponsored by the ASH CRTI. References Appelbaum J, Loeb A, Gardner K, et al. Additional Cytotoxic Chemotherapy Is Unlikely to Eliminate Measurable Residual Acute Myeloid Leukemia (AML). Blood. 2019;134(Supplement_1):260-260. Lane AA, Sweet KL, Wang ES, et al. Results from Ongoing Phase 1/2 Trial of SL-401 As Consolidation Therapy in Patients with Acute Myeloid Leukemia (AML) in Remission with Minimal Residual Disease (MRD). Blood. 2017;130(Supplement 1):2583-2583. Togami K, Pastika T, Stephansky J, et al. DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance. J Clin Invest. 2019;129(11):5005-5019. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-cell transcriptional profiling reveals cellular and molecular divergence in human maternal–fetal interface

Placenta plays essential role in successful pregnancy, as the most important organ connecting and interplaying between mother and fetus. However, the cellular characteristics and molecular interaction of cell populations within the fetomaternal interface is still poorly understood. Here, we surveyed the single-cell transcriptomic landscape of human full-term placenta and revealed the heterogeneity of cytotrophoblast cell (CTB) and stromal cell (STR) with the fetal/maternal origin consecutively localized from fetal section (FS), middle section (Mid_S) to maternal section (Mat_S) of maternal–fetal interface. Then, we highlighted a subpopulation of CTB, named trophoblast progenitor-like cells (TPLCs) existed in the full-term placenta and mainly distributed in Mid_S, with high expression of a pool of putative cell surface markers. Further, we revealed the putative key transcription factor PRDM6 that might promote the differentiation of endovascular extravillous trophoblast cells (enEVT) by inhibiting cell proliferation, and down-regulation of PRDM6 might lead to an abnormal enEVT differentiation process in PE. Together, our study offers important resources for better understanding of human placenta and stem cell-based therapy, and provides new insights on the study of tissue heterogeneity, the clinical prevention and control of PE as well as the maternal–fetal interface.

Transcriptional Control of Gene Expression and the Heterogeneous Cellular Identity of Erythroblastic Island Macrophages.

During definitive erythropoiesis, maturation of erythroid progenitors into enucleated reticulocytes requires the erythroblastic island (EBI) niche comprising a central macrophage attached to differentiating erythroid progenitors. Normally, the macrophage provides a nurturing environment for maturation of erythroid cells. Its critical physiologic importance entails aiding in recovery from anemic insults, such as systemic stress or acquired disease. Considerable interest in characterizing the central macrophage of the island niche led to the identification of putative cell surface markers enriched in island macrophages, enabling isolation and characterization. Recent studies focus on bulk and single cell transcriptomics of the island macrophage during adult steady-state erythropoiesis and embryonic erythropoiesis. They reveal that the island macrophage is a distinct cell type but with widespread cellular heterogeneity, likely suggesting distinct developmental origins and biological function. These studies have also uncovered transcriptional programs that drive gene expression in the island macrophage. Strikingly, the master erythroid regulator EKLF/Klf1 seems to also play a major role in specifying gene expression in island macrophages, including a putative EKLF/Klf1-dependent transcription circuit. Our present review and analysis of mouse single cell genetic patterns suggest novel expression characteristics that will enable a clear enrichment of EBI subtypes and resolution of island macrophage heterogeneity. Specifically, the discovery of markers such as Epor, and specific features for EKLF/Klf1-expressing island macrophages such as Sptb and Add2, or for SpiC-expressing island macrophage such as Timd4, or for Maf/Nr1h3-expressing island macrophage such as Vcam1, opens exciting possibilities for further characterization of these unique macrophage cell types in the context of their critical developmental function.

Longitudinal Large-Scale Semiquantitative Proteomic Data Stability Across Multiple Instrument Platforms.

With the rapid developments in mass spectrometry (MS)-based proteomics methods, label-free semiquantitative proteomics has become an increasingly popular tool for profiling global protein abundances in an unbiased manner. However, the reproducibility of these data across time and LC-MS platforms is not well characterized. Here, we evaluate the performance of three LC-MS platforms (Orbitrap Elite, Q Exactive HF, and Orbitrap Fusion) in label-free semiquantitative analysis of cell surface proteins over a six-year period. Sucrose gradient ultracentrifugation was used for surfaceome enrichment, following gel separation for in-depth protein identification. With our established workflow, we consistently detected and reproducibly quantified >2300 putative cell surface proteins in a human acute myeloid leukemia (AML) cell line on all three platforms. To our knowledge this is the first study reporting highly reproducible semiquantitative proteomic data collection of biological replicates across multiple years and LC-MS platforms. These data provide experimental justification for semiquantitative proteomic study designs that are executed over multiyear time intervals and on different platforms. Multiyear and multiplatform experimental designs will likely enable larger scale proteomic studies and facilitate longitudinal proteomic studies by investigators lacking access to high throughput MS facilities. Data are available via ProteomeXchange with identifier PXD022721.

A Subpopulation of Microglia Generated in the Adult Mouse Brain Originates from Prominin-1-Expressing Progenitors.

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro To determine whether Prom1-expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion.SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

Real-time PCR method for qualitative and quantitative detection of Lactobacillus sakei group species targeting novel markers based on bioinformatics analysis

Latilactobacillus sakei group comprises four closely related species, making it difficult to accurately distinguish them with standard markers such as the 16S rRNA gene. The objective of our study was to mine novel markers for PCR detection and discrimination of L. sakei group species and L. sakei subspecies by comparative pan-genomic analysis. A total of 63 genome sequences of L. sakei group species consisted of 119,899 coding genes, yielding 5741 pan-genomes, 831 core-genomes, 3347 accessory-genomes, and 1563 unique-genomes. The accessory-genome was compared to extract unique candidate genes common only to genomes of the same species. The candidate genes were then aligned with the other bacterial genomes to select marker genes present in all genomes of a given species, but not in the genomes of other species. We identified the arginine/ornithine antiporter, putative cell surface protein precursor, sodium:solute symporter, PRD domain protein, PTS sugar transporter subunit IIC, and phosphoenolpyruvate-dependent sugar phosphotransferase system EIIC as marker genes for L. sakei, L. sakei subsp. sakei, L. sakei subsp. carnosus, L. curvatus, L. graminis, and L. fuchuensis, respectively. Primer pairs were designed for each marker and showed 100% specificity for 48 lactic acid bacterial reference strains. The PCR method developed in this study was used to evaluate 106 strains isolated from fermented foods to demonstrate that the marker genes provided a viable alternative to the 16S rRNA gene. We also applied the method to the monitoring of kimchi samples to quantify L. sakei group species or subspecies. Our PCR method based on novel markers can rapidly identify L. sakei group with high accuracy and high throughput.

Abstract 5120: Surveying the AML surfaceome for novel immunotherapeutic targets

Abstract Acute myeloid leukemia (AML) therapy requires very intensive chemotherapy/stem cell transplant that cures only 60% of children and 25% of adults with AML. The development of chimeric antigen receptor (CAR) T cells for AML has been hampered by the lack of optimal surface antigens. We hypothesized that unbiased identification of AML cell surface proteins will enable creation of novel CAR T cell immunotherapies and that integration of AML surfaceome data with AML RNA and DNA sequencing data will facilitate identification of new therapeutic targets. We also hypothesized that pediatric and adult surfaceomes are overlapping but distinguishable. Sucrose gradient ultracentrifugation was used for surfaceome enrichment of patient leukemia and normal bone marrow samples without chemical labeling or affinity purification followed by GeLC-MS. The MOLM-14 human AML cell line served as internal control. Mass spectrometry data were processed with MaxQuant, and 9 GO terms were used to define putative cell surface proteins. Samples with at least 6 of 12 known AML surface markers detected were further analyzed. RNA and DNA were extracted for a subset of samples and sequenced. To date, we have surfaceome data for 25 pediatric AML, 12 pediatric normal bone marrow, 9 adult AML, and 9 MOLM-14 samples. A median of 2050 ± 319 surface proteins were detected in the MOLM-14 samples. The mean Pearson correlation coefficient for these replicates was 0.82, confirming the robustness of our workflow. In addition, we found a high representation of known AML cell surface proteins among the captured surface proteins, such as CD33, CD123 and FLT3. A median 1269 ± 474 and 1994 ± 480 surface proteins were found in the pediatric primary AML and normal control samples, respectively and 1790 ± 553 proteins were identified in adult primary AML samples. The average correlation coefficients for the pediatric AML, pediatric control, and adult AML samples were 0.63, 0.75, and 0.63, respectively. Comparison of the pediatric AML and control samples revealed 208 unique and 19 differentially expressed (>2-fold, p<0.05) surface proteins in the patient samples. Similarly, comparison of pediatric and adult AML proteomes identified 210 unique and 23 differentially expressed surface proteins in the pediatric samples. RNA and DNA sequencing has also been performed on a subset of the pediatric and adult primary AML samples, and proteogenomic integration of the different datasets is in progress. We demonstrate that the surfaceome of primary pediatric and adult AML patient samples can be surveyed at depth for the discovery of new potential immunotherapeutic targets. The presence of unique or differentially expressed surface proteins increases our optimism that targeting AML with potent immunotherapies may become clinically feasible. A proteogenomic integration of these AML surfaceome and RNA and DNA sequencing datasets may illuminate AML biology as well as present novel therapeutic targets. Citation Format: Tina Glisovic-Aplenc, Kevin Nestler, Congcong Lu, Lusha Cao, Joseph Cesare, Hyoungjoo Lee, Mitchelle Matesva, Terzah Horton, Sarah Tasian, John Shern, Saar Gill, Benjamin Garcia, Richard Aplenc. Surveying the AML surfaceome for novel immunotherapeutic targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5120.

KDEL receptor is a cell surface receptor that cycles between the plasma membrane and the Golgi via clathrin-mediated transport carriers.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

An expanded role for dipeptidyl peptidase 4 in cell regulation.

Dipeptidyl peptidase 4 (DPP4) is a serine protease with diverse regulatory functions in healthy and diseased cells. Much remains unknown about the mechanisms and targets of DPP4. Here we discuss new studies exploring DPP4-mediated cellular regulation, provide an updated list of potential targets of DPP4, and discuss clinical implications of each. Recent studies have sought enhanced efficacy of targeting DPP4's role in regulating hematopoietic stem and progenitor cells for improved clinical application. Further studies have identified DPP4 functions in different cellular compartments and have proposed ways to target this protein in malignancy. These findings, together with an expanded list of putative extracellular, cell surface, and intracellular DPP4 targets, provide insight into new DPP4-mediated cell regulation. DPP4 posttranslationally modifies proteins and peptides with essential roles in hematopoietic cell regulation, stem cell transplantation, and malignancy. Targets include secreted signaling factors and may include membrane proteins and transcription factors critical for different hematopoietic functions. Knowing these targets and functions can provide insight into new regulatory roles for DPP4 that may be targeted to enhance transplantation, treat disease, and better understand different regulatory pathways of hematopoiesis.

Distinct Contributions of CD18 Integrins for Binding and Phagocytic Internalization of Pseudomonas aeruginosa

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133high and CD133low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

Characterization of two putative Dichelobacter nodosus footrot vaccine antigens identifies the first lysozyme inhibitor in the genus

The Gram-negative anaerobic bacterium Dichelobacter nodosus (Dn) causes footrot in ruminants, a debilitating and highly contagious disease that results in necrotic hooves and significant economic losses in agriculture. Vaccination with crude whole-cell vaccine mixed with multiple recombinant fimbrial proteins can provide protection during species-specific outbreaks, but subunit vaccines containing broadly cross-protective antigens are desirable. We have investigated two D. nodosus candidate vaccine antigens. Macrophage Infectivity Potentiator Dn-MIP (DNO_0012, DNO_RS00050) and Adhesin Complex Protein Dn-ACP (DNO_0725, DNO_RS06795) are highly conserved amongst ~170 D. nodosus isolates in the https://pubmlst.org/dnodosus/ database. We describe the presence of two homologous ACP domains in Dn-ACP with potent C-type lysozyme inhibitor function, and homology of Dn-MIP to other putative cell-surface and membrane-anchored MIP virulence factors. Immunization of mice with recombinant proteins with a variety of adjuvants induced antibodies that recognised both proteins in D. nodosus. Notably, immunization with fimbrial-whole-cell Footvax vaccine induced anti-Dn-ACP and anti-Dn-MIP antibodies. Although all adjuvants induced high titre antibody responses, only antisera to rDn-ACP-QuilA and rDn-ACP-Al(OH)3 significantly prevented rDn-ACP protein from inhibiting lysozyme activity in vitro. Therefore, a vaccine incorporating rDn-ACP in particular could contribute to protection by enabling normal innate immune lysozyme function to aid bacterial clearance.

Abstract 5576: Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening

Abstract Screening and prevention is critical to reducing breast cancer deaths, and mammography is not an ideal tool. Mammography has not reduced invasive breast cancer incidence, it has high numbers of false positives and it detects many non-invasive cancers that are not life threatening. Metastatic breast cancer is the second leading cause of cancer death for women. The development of a new blood test for breast cancer screening could offer significant improvements with the potential to be more accurate, less costly, less intimidating, not subject to age differences, and able to discriminate between cancers subtypes. Detection of breast cancer cells (BCC) in blood samples offers a valuable opportunity for screening. However, BCC in blood are rare and must be identified against a background of millions of other blood cells. Although many technologies have been developed for the isolation and detection of circulating tumor cells using EpCAM and cytokeratins, these tests do not provide levels of sensitivity and specificity required for a cancer screening tool. To address this critical unmet need, we developed a panel of cell surface markers whose expression patterns specifically distinguish breast cancer cells in blood samples. These genes were selected based on a discovery platform tailored to detect early stage breast cancers and include 20 novel putative cell surface molecules that are candidates for breast cancer cell detection and isolation. We prioritized these molecules by specificity of expression in tumor cells including: 1) Ratio >25:1 average breast tumor biopsy expression versus average normal blood expression, 2) High expression relative (>3 fold) in >20% of tumor biopsy samples, 3) High expression (>3 fold) in >40% of breast cancer cell lines, and 4) Expression significantly associated with 10-year outcomes (Kaplan Meier analysis, p<0.05). We combined the top 10 ranking genes in each of these categories, resulting in a list of 20 candidates that represent high priority targets. Antibodies for all 20 genes are commercially available. Expression in micro dissected biopsies was markedly higher than non-breast cancer blood for most. Also, most were expressed at lower levels than EpCAM in normal breast tissue and had background level expression in isolated normal blood cells. The innovation of this strategy is twofold. First, it uses a panel of molecules instead of an individual molecule to detect and isolate breast cancer cells and, second, it relies on molecules that are clinically relevant to breast cancers, rather than relying on generic epithelial cell markers. Current studies include the validation of antibody cocktails with specificity and sensitivity that outperforms the current EpCAM enrichment method and provides a proof of feasibility for screening of breast cancer. Citation Format: Lucas Delmonico, Edward C. Goodwin, Marcia Vasconcellos Fournier. Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5576.

Dental Pulp Cells Isolated from Teeth with Superficial Caries Retain an Inflammatory Phenotype and Display an Enhanced Matrix Mineralization Potential.

We have isolated dental pulp cells (DPCs) from three healthy (hDPCs) and three carious (cDPCs) donors and shown that compared to hDPCs cells isolated from superficial carious lesions show higher clonogenic potential; show an equivalent proportion of cells with putative stem cell surface markers; show enhanced matrix mineralization capability; have enhanced angiogenic marker expression and retain the inflammatory phenotype in vitro characteristic of superficial caries lesions in vivo. Our findings suggest that cDPCs may be used for further investigation of the cross talk between inflammatory, angiogenic and mineralization pathways in repair of carious pulp. In addition cells derived from carious pulps (almost always discarded) may have potential for future applications in mineralized tissue repair and regeneration.

Characterization of Biomarkers of Tumorigenic and Chemoresistant Cancer Stem Cells in Human Gastric Carcinoma.

Purpose: Gastric carcinomas are heterogeneous, and the current therapy remains essentially based on surgery with conventional chemotherapy and radiotherapy. This study aimed to characterize biomarkers allowing the detection of cancer stem cells (CSC) in human gastric carcinoma of different histologic types.Experimental Design: The primary tumors from 37 patients with intestinal- or diffuse-type noncardia gastric carcinoma were studied, and patient-derived tumor xenograft (PDX) models in immunodeficient mice were developed. The expressions of 10 putative cell surface markers of CSCs, as well as aldehyde dehydrogenase (ALDH) activity, were studied, and the tumorigenic properties of cells were evaluated by in vitro tumorsphere assays and in vivo xenografts by limiting dilution assays.Results: We found that a subpopulation of gastric carcinoma cells expressing EPCAM, CD133, CD166, CD44, and a high ALDH activity presented the properties to generate new heterogeneous tumorspheres in vitro and tumors in vivo CD44 and CD166 were coexpressed, representing 6.1% to 37.5% of the cells; ALDH activity was detected in 1.6% to 15.4% of the cells; and the ALDH+ cells represented a core within the CD44+/CD166+ subpopulation that contained the highest frequency of tumorigenic CSCs in vivo The ALDH+ cells possessed drug efflux properties and were more resistant to standard chemotherapy than the ALDH- cells, a process that was partially reversed by verapamil treatment.Conclusions: CD44 and ALDH are the most specific biomarkers to detect and isolate tumorigenic and chemoresistant gastric CSCs in noncardia gastric carcinomas independently of the histologic classification of the tumor. Clin Cancer Res; 23(6); 1586-97. ©2016 AACR.

Transplantation of CD51+ Stem Leydig Cells: A New Strategy for the Treatment of Testosterone Deficiency.

Stem Leydig cell (SLC) transplantation could provide a new strategy for treating the testosterone deficiency. Our previous study demonstrated that CD51 (also called integrin αv) might be a putative cell surface marker for SLCs, but the physiological function and efficacy of CD51+ SLCs treatment remain unclear. Here, we explore the potential therapeutic benefits of CD51+ SLCs transplantation and whether these transplanted cells can be regulated by the hypothalamic-pituitary-gonadal (HPG) axis. CD51+ cells were isolated from the testes of 12-weeks-old C57BL/6 mice, and we showed that such cells expressed SLC markers and that they were capable of self-renewal, extensive proliferation, and differentiation into multiple mesenchymal cell lineages and LCs in vitro. As a specific cytotoxin that eliminates Leydig cells (LCs) in adult rats, ethane dimethanesulfonate (EDS) was used to ablate LCs before the SLC transplantation. After being transplanted into the testes of EDS-treated rats, the CD51+ cells differentiated into mature LCs, and the recipient rats showed a partial recovery of testosterone production and spermatogenesis. Notably, a testosterone analysis revealed a circadian rhythm of testosterone secretion in cell-transplanted rats, and these testosterone secretions could be suppressed by decapeptyl (a luteinizing hormone-releasing hormone agonist), suggesting that the transplanted cells might be regulated by the HPG axis. This study is the first to demonstrate that CD51+ SLCs can restore the neuroendocrine regulation of testicular function by physiologically recovering the expected episodic changes in diurnal testosterone serum levels and that SLC transplantation may provide a new tool for the studies of testosterone deficiency treatment. Stem Cells 2017;35:1222-1232.

Thyroid Progenitors Are Robustly Derived from Embryonic Stem Cells through Transient, Developmental Stage-Specific Overexpression of Nkx2-1.

SummaryThe clinical importance of anterior foregut endoderm (AFE) derivatives, such as thyrocytes, has led to intense research efforts for their derivation through directed differentiation of pluripotent stem cells (PSCs). Here, we identify transient overexpression of the transcription factor (TF) NKX2-1 as a powerful inductive signal for the robust derivation of thyrocyte-like cells from mouse PSC-derived AFE. This effect is highly developmental stage specific and dependent on FOXA2 expression levels and precise modulation of BMP and FGF signaling. The majority of the resulting cells express thyroid TFs (Nkx2-1, Pax8, Foxe1, Hhex) and thyroid hormone synthesis-related genes (Tg, Tpo, Nis, Iyd) at levels similar to adult mouse thyroid and give rise to functional follicle-like epithelial structures in Matrigel culture. Our findings demonstrate that NKX2-1 overexpression converts AFE to thyroid epithelium in a developmental time-sensitive manner and suggest a general methodology for manipulation of cell-fate decisions of developmental intermediates.