Optimization of rVAR2-Based Isolation of Cancer Cells in Blood for Building a Robust Assay for Clinical Detection of Circulating Tumor Cells.

Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. Recombinant malaria protein VAR2CSA (rVAR2) binds a unique chondroitin sulfate modification present on the vast majority of cancers and thereby holds promise as a near-universal tumor cell-targeting reagent to isolate CTCs from complex blood samples. This study describes a technical approach for optimizing the coupling of rVAR2 to magnetic beads and the development of a CTC isolation platform targeting a range of different cancer cell lines. We investigate both direct and indirect approaches for rVAR2-mediated bead retrieval of cancer cells and conclude that an indirect capture approach is most effective for rVAR2-based cancer cell retrieval.

Development of an epitope panel for consistent identification of antigen-specific T-cells in humans.

We aimed to establish a panel of MHC-peptide multimers suitable as a positive control in the detection of HLA A*0201 restricted antigen specific T cells (ASTC) by flow cytometry. MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melanoma targets identified from a literature search and in silico prediction. Peripheral blood mononuclear cells (PBMC) from healthy donors were analysed with the MHC Dextramers using flow cytometry. The best performing epitopes were tested on PBMC from patients undergoing testing for Mycobacterium tuberculosis infection to assess the coverage of this epitope panel. Of 21 candidate epitopes, ASTC could be detected against 12 (57·1%) in at least one of 18 healthy blood donors. Reactivity to two or more epitopes was seen in 17 of the 18 donors (94·4%). We selected the six best-performing epitopes and demonstrated a positive response in 42 (97·7%) of 43 patient samples (healthy, latent and active M. tuberculosis infection). The selected panel of six antigenic epitopes sufficed as a positive control in the detection of ASTC in HLA A*0201. Performance was robust in different stages of latent and active M. tuberculosis infection, indicating reliability also during infection.

Combined microfluidic enrichment and staining workflow for single-cell analysis of circulating tumor cells in metastatic prostate cancer patients.

Circulating tumor cells (CTCs) are precursors of cancer in the blood and provide an attractive source for dynamic monitoring of disease progression and tumor heterogeneity. However, the scarcity of CTCs in the bloodstream has limited their use in clinical practice. In this study, we present a workflow for easy detection of CTCs by cytokeratin staining using the FDA-cleared Parsortix device for size-based microfluidic enrichment. To minimize sample handling, the isolated cells are stained inside the separation cassette and harvested for subsequent single cell isolation and whole genome copy-number analysis. We validated the workflow on a panel of four prostate cancer cell lines spiked into healthy donor blood collected in CellRescue or EDTA tubes, resulting in mean recoveries of 42% (16-69%). Furthermore, we evaluated the clinical utility in a cohort of 12 metastatic prostate cancer patients and found CTCs in 67% of patients ranging from 0 to 1172 CTCs in 10 mL blood. Additionally, we isolated single patient-derived CTCs and identified genomic aberrations associated with treatment response and clinical outcome. Thus, this workflow provides a readily scalable strategy for analysis of single CTCs, applicable for use in monitoring studies to identify genomic variations important for guiding clinical therapy decision.

Tumor-agnostic cancer therapy using antibodies targeting oncofetal chondroitin sulfate.

Molecular similarities between embryonic and malignant cells can be exploited to target tumors through specific signatures absent in healthy adult tissues. One such embryonic signature tumors express is oncofetal chondroitin sulfate (ofCS), which supports disease progression and dissemination in cancer. Here, we report the identification and characterization of phage display-derived antibody fragments recognizing two distinct ofCS epitopes. These antibody fragments show binding affinity to ofCS in the low nanomolar range across a broad selection of solid tumor types in vitro and in vivo with minimal binding to normal, inflamed, or benign tumor tissues. Anti-ofCS antibody drug conjugates and bispecific immune cell engagers based on these targeting moieties disrupt tumor progression in animal models of human and murine cancers. Thus, anti-ofCS antibody fragments hold promise for the development of broadly effective therapeutic and diagnostic applications targeting human malignancies.

The Role of Proteoglycans in Cancer Metastasis and Circulating Tumor Cell Analysis.

Circulating tumor cells (CTCs) are accessible by liquid biopsies via an easy blood draw. They represent not only the primary tumor site, but also potential metastatic lesions, and could thus be an attractive supplement for cancer diagnostics. However, the analysis of rare CTCs in billions of normal blood cells is still technically challenging and novel specific CTC markers are needed. The formation of metastasis is a complex process supported by numerous molecular alterations, and thus novel CTC markers might be found by focusing on this process. One example of this is specific changes in the cancer cell glycocalyx, which is a network on the cell surface composed of carbohydrate structures. Proteoglycans are important glycocalyx components and consist of a protein core and covalently attached long glycosaminoglycan chains. A few CTC assays have already utilized proteoglycans for both enrichment and analysis of CTCs. Nonetheless, the biological function of proteoglycans on clinical CTCs has not been studied in detail so far. Therefore, the present review describes proteoglycan functions during the metastatic cascade to highlight their importance to CTCs. We also outline current approaches for CTC assays based on targeting proteoglycans by their protein cores or their glycosaminoglycan chains. Lastly, we briefly discuss important technical aspects, which should be considered for studying proteoglycans.

Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein.

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.