An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays.

The analysis of antigen-specific T-cell responses has become routine in many laboratories. Functional T-cell assays like enzyme-linked-immuno-spot (ELISPOT), which depend on antigen-specific stimulation, increasingly use peptides to represent the antigen of interest. Besides single peptides, mixtures of peptides (peptide pools) are very frequently applied. Such peptide pools may, for example, represent entire proteins (with overlapping peptides covering a protein sequence) or include noncontiguous peptides such as a collection of T-cell-stimulating peptides. The optimum specification of single peptides or peptide pools for T-cell stimulation assays will depend on the purpose of the test, the target T-cell population, the availability of sample, requirements regarding reproducibility, and, last but not least, the available budget, to mention only the most important factors. Because of the way peptides are produced, they will always contain certain amounts of impurities such as peptides with deletions or truncated peptides, and there may be additional by-products of peptide synthesis. Optimized synthesis protocols as well as purification help reduce impurities that might otherwise cause false-positive assay results. However, specific requirements with respect to purity will vary depending on the purpose of an assay. Finally, storage conditions significantly affect the shelf life of peptides, which is relevant especially for longitudinal studies. The present book chapter addresses all of these aspects in detail. It should provide the researcher with all necessary background knowledge for making the right decisions when it comes to choosing, using, and storing peptides for ELISPOT and other T-cell stimulation assays.

Peptide microarray-based analysis of antibody responses to SARS-CoV-2 identifies unique epitopes with potential for diagnostic test development.

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n = 24 patient samples and n = 12 control samples were screened for antibodies against the entire SARS-CoV-2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, OC43, and 229E. While widespread cross-reactivity was revealed across several immunodominant regions of S and N, IgG binding to several SARS-CoV-2-derived peptides provided statistically significant discrimination between COVID-19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID-19-specific diagnostic antibody tests.

Expression of programmed death ligand (PD-L1) in different tumors. Comparison of several current available antibody clones and antibody profiling.

PD-L1 is a surface molecule which is expressed on different types of cells, including antigen presenting cells, vascular endothelial cells and other cells of human tissues. Expression of PD-L1 is also found on human tumor cells. PD-L1 as the ligand to PD1 receptor molecule of CD8+ T cells inhibits its cytotoxic effect on the tumor cell. The modern target therapy uses this interaction to inhibit the PD-1 molecule of T cells to stimulate tumor necrosis. To compare expression differences, twelve frequent types of malignant tumors with ten patients per group were selected. Immunohistochemical stains with different antibodies for PD-L1 (DAKO, Spring Bioscience, Ventana, Cell Signaling, Biocare Medical, Abcam, Zeta Corporation) were performed, analyzed and compared. To summarize, we detected variable expression pattern of PD-L1 with general higher mean value of expression of tumor cells with clone SP263 in most tumor groups. In the comparison of selected cases of lung cancer, therapy relevant differences of PD-L1 expression on tumor cells with different antibodies were observed. Additionally, the profiling study of several PD-L1-antibody clones (28-8 Abcam and 28-8 DAKO, SP142, SP263) with Signal-to-Amino Acid Residue Plots was performed with interesting findings of cross-activity of SP142 with two peptides from PD-1, which can explain why clone SP142 stains immune cells more intensively, as previously published.

Isolation of circulating tumor cells from pancreatic cancer by automated filtration.

It is now widely recognized that the isolation of circulating tumor cells based on cell surface markers might be hindered by variability in their protein expression. Especially in pancreatic cancer, isolation based only on EpCAM expression has produced very diverse results. Methods that are independent of surface markers and therefore independent of phenotypical changes in the circulating cells might increase CTC recovery also in pancreatic cancer. We compared an EpCAM-dependent (IsoFlux) and a size-dependent (automated Siemens Healthineers filtration device) isolation method for the enrichment of pancreatic cancer CTCs. The recovery rate of the filtration based approach is dramatically superior to the EpCAM-dependent approach especially for cells with low EpCAM-expression (filtration: 52%, EpCAM-dependent: 1%). As storage and shipment of clinical samples is important for centralized analyses, we also evaluated the use of frozen diagnostic leukapheresis (DLA) as source for isolating CTCs and subsequent genetic analysis such as KRAS mutation detection analysis. Using frozen DLA samples of pancreatic cancer patients we detected CTCs in 42% of the samples by automated filtration.

The CD24hi smooth muscle subpopulation is the predominant fraction in uterine fibroids.

Uterine fibroids are the most common gynecological tumors affecting women in their reproductive age. Despite this high incidence the pathogenesis of fibroids is widely unsolved. Whereas formerly only imbalances in hormonal levels were considered to account for tumor development, the identification of genetic changes likely to affect myometrial stem cell reservoirs provided a novel approach to fibroid genesis. Here, we identified a certain subset of cells by the surface marker CD24 with increased abundance in fibroids compared with myometrial tissue. Fibroid cells expressing CD24 shared certain features of immature or progenitor-like cells such as quiescence, reduced expression of smooth muscle differentiation markers and elevated expression of genes involved in the wingless-type (WNT)-pathway such as beta-catenin. In addition, a positive correlation between CD24 and wingless-type family member 4 (WNT4) expression was observed in uterine fibroids with mediator subcomplex 12 gene (MED12) mutations. Our findings suggest that cells highly expressing CD24 represent a type of immature smooth muscle progenitor cells. Their accumulation might be driven by disturbed differentiation processes caused by genetic changes possibly involving MED12 mutations or high mobility group AT-hook (HMGA)2 rearrangements.

A novel mouse model that closely mimics human uterine leiomyomas.

OBJECTIVE: To develop a predictive mouse model for uterine fibroids. DESIGN: Human fibroid cells xenografted to immunodeficient mice. SETTING: University and industrial research center. ANIMAL(S): Immunodeficient scid/beige mice. INTERVENTION(S): Subcutaneous and intrauterine injection of fibroid-derived cells, SV40 transformation of primary cells by lentiviral transduction, proliferation determined by immunohistochemistry, FISH. MAIN OUTCOME MEASURE(S): Characterization of primary and immortalized cells by Western blot and soft agar assay, determination of in vivo tumorigenicity, comparative histology and immunohistochemistry, fluorescence in situ hybridization. RESULT(S): Tumorigenicity of primary myoma cells disappears upon in vitro culture. Transformation and immortalization does not restore or conserve the in vivo growth potential of cultured cells. Injection of primary cells into myometrium of mice leads to xenografts with a leiomyoma-like histology. CONCLUSION(S): Primary myoma cells are suited to generate fibroid-like xenografts for studying pathogenesis without genetic modifications. In contrast, in vitro culture abolishes transplantability, and neither transformation nor immortalization is sufficient to restore tumorigenic capacity.

Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition.

BACKGROUND: Circulating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay. METHODS: We used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available "Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array". RESULTS: Although the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT). CONCLUSIONS: The use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.

Evaluation of activity and combination strategies with the microtubule-targeting drug sagopilone in breast cancer cell lines.

Sagopilone, a fully synthetic epothilone, is a microtubule-stabilizing agent optimized for high in vitro and in vivo activity against a broad range of tumor models, including those resistant to paclitaxel and other systemic treatments. Sagopilone development is accompanied by translational research studies to evaluate the molecular mode of action, to recognize mechanisms leading to resistance, to identify predictive response biomarkers, and to establish a rationale for combination with different therapies. Here, we profiled sagopilone activity in breast cancer cell lines. To analyze the mechanisms of mitotic arrest and apoptosis and to identify additional targets and biomarkers, an siRNA-based RNAi drug modifier screen interrogating 300 genes was performed in four cancer cell lines. Defects of the spindle assembly checkpoint (SAC) were identified to cause resistance against sagopilone-induced mitotic arrest and apoptosis. Potential biomarkers for resistance could therefore be functional defects like polymorphisms or mutations in the SAC, particularly in the central SAC kinase BUB1B. Moreover, chromosomal heterogeneity and polyploidy are also potential biomarkers of sagopilone resistance since they imply an increased tolerance for aberrant mitosis. RNAi screening further demonstrated that the sagopilone-induced mitotic arrest can be enhanced by concomitant inhibition of mitotic kinesins, thus suggesting a potential combination therapy of sagopilone with a KIF2C (MCAK) kinesin inhibitor. However, the combination of sagopilone and inhibition of the prophase kinesin KIF11 (EG5) is antagonistic, indicating that the kinesin inhibitor has to be highly specific to bring about the required therapeutic benefit.