Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

Comparability of Computer-Supported Concurrent Voice Analysis.

OBJECTIVE: The assessment of vocal ability parameters can be made easier and more objective by computer-supported analysis systems. For this, multiple commercially available systems exist. But how strongly do the measured results of one method differ from the results of another and is the evaluation result of one method replaceable by or directly comparable with another? PATIENTS AND METHODS: 15 women aged 20-55 years and 15 men aged 18-56 years took part in this prospective study. The programs lingWAVES and rpSzene® were compared for the results of jitter, shimmer and glottal-to-noise excitation ratio. Correlation coefficients, Youden plots and Bland-Altman plots were calculated to make the comparison. RESULTS: For both systems, none of the values were in accordance with a normal distribution. The results of the two programs did not produce satisfactory consistency. CONCLUSIONS: In this study, absolute equivalency in the recording was ensured by the simultaneous recording of the voice signal and subsequent analysis with two different voice analysis methods. Nevertheless, the results varied so much that they cannot be compared properly. To reach a comparability of voice analyses, it is necessary to develop perturbation measurements which guarantee good clinical applicability and comparability between different analysis systems.

In Vitro Modeling of Reoxygenation Effects on mRNA and Protein Levels in Hypoxic Tumor Cells upon Entry into the Bloodstream.

BACKGROUND: Solid epithelial tumors like breast cancer are the most frequent malignancy in women. Circulating tumor cells (CTCs) are frequently released from hypoxic areas into the blood, where CTCs face elevated oxygen concentrations. This reoxygenation might challenge the use of CTCs for liquid biopsy. METHODS: We modeled this situation in vitro using the breast cancer cell lines-MCF-7, MDA-MB-468, MDA-MB-231-and the cell line BC-M1 established from DTCs in the bone marrow. Cells were cultured under hypoxia, followed by a reoxygenation pulse for 4 h, reflecting the circulation time of CTCs. Analyzed were gene products like EGFR, ErbB-2, EpCAM, PD-L1 on mRNA and protein level. RESULTS: mRNAs of erbb2 or pdl1 and protein levels of PD-L1 displayed significant changes, whereas ErbB-2 protein levels remained constant. The strongest discrepancy between protein and mRNA levels under hypoxia was observed for EGFR, supporting the idea of cap-independent translation of egfr mRNA. Analyses of the phosphorylation of AKT, Erk 1/2, and Stat3 revealed strong alterations after reoxygenation. CONCLUSIONS: CTCs reaching secondary sites faster than reoxygenation could alter the mRNA and protein levels in the cells. CTC and DTC with high PD-L1 levels might become quiescent under hypoxia but were easily reactivated by reoxygenation.

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+ ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

Small protein sequences can induce cellular uptake of complex nanohybrids.

In this letter, we report on the ability of functional fusion proteins presenting a lytic gamma peptide, to promote interactions with HeLa cells and delivery of large hybrid nanostructures.

Compensation for chronic oxidative stress in ALADIN null mice.

Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response.

A novel nanobody-based target module for retargeting of T lymphocytes to EGFR-expressing cancer cells via the modular UniCAR platform.

Recent treatments of leukemias with chimeric antigen receptor (CAR) expressing T cells underline their impressive therapeutic potential. However, once adoptively transferred into patients, there is little scope left to shut them down after elimination of tumor cells or in case adverse side effects occur. This becomes of special relevance if they are directed against commonly expressed tumor associated antigens (TAAs) such as receptors of the ErbB family. To overcome this limitation, we recently established a modular CAR platform technology termed UniCAR. UniCARs are not directed against TAAs but instead against a unique peptide epitope on engineered recombinant targeting modules (TMs), which guide them to the target. In the absence of a TM UniCAR T cells are inactive. Thus an interruption of any UniCAR activity requires an elimination of unbound TM and the TM complexed with UniCAR T cells. Elimination of the latter one requires a disassembly of the UniCAR-TM complexes. Here, we describe a first nanobody (nb)-based TM directed against EGFR. The novel TM efficiently retargets UniCAR T cells to EGFR positive tumors and mediates highly efficient target-specific and target-dependent tumor cell lysis both in vitro and in vivo. After radiolabeling of the novel TM with 64Cu and 68Ga, we analyzed its biodistribution and clearance as well as the stability of the UniCAR-TM complexes. As expected unbound TM is rapidly eliminated while the elimination of the TM complexed with UniCAR T cells is delayed. Nonetheless, we show that UniCAR-TM complexes dissociate in vitro and in vivo in a concentration-dependent manner in line with the concept of a repeated stop and go retargeting of tumor cells via the UniCAR technology.

Retargeting of T lymphocytes to PSCA- or PSMA positive prostate cancer cells using the novel modular chimeric antigen receptor platform technology "UniCAR".

New treatment options especially of solid tumors including for metastasized prostate cancer (PCa) are urgently needed. Recent treatments of leukemias with chimeric antigen receptors (CARs) underline their impressive therapeutic potential. However CARs currently applied in the clinics cannot be repeatedly turned on and off potentially leading to severe life threatening side effects. To overcome these problems, we recently described a modular CAR technology termed UniCAR: UniCAR T cells are inert but can be turned on by application of one or multiple target modules (TMs). Here we present preclinical data summarizing the retargeting of UniCAR T cells to PCa cells using TMs directed to prostate stem cell- (PSCA) or/and prostate specific membrane antigen (PSMA). In the presence of the respective TM(s), we see a highly efficient target-specific and target-dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and PCa cell lysis both in vitro and experimental mice.