Mindy I. Davis Ph.D. National Institute of Health

A review of literature where researchers used DNA-barcoded antibody sensing to detect proteins from fine-needle aspirates.

The ability to detect and quantitate proteins on the surface and inside cells accurately has many potential applications in cancer. Most techniques require large numbers of cells or have limitations in the number of proteins that can be simultaneously interrogated. The authors* here sought to develop a methodology that is (1) sensitive down to <100 cells and in some cases even just a single cell, (2) enables a high degree of multiplexing, and (3) can be completed in one day (see Figure).

The ability to assess small numbers of cells allows fine-needle aspirates (FNAs) to be used rather than the more invasive core biopsies to collect cells from a patient. After depleting the aspirates of non-tumor cells, such as leukocytes, the authors used a total of 90 “barcoded” antibodies simultaneously for detection of proteins. The barcoding process consists of attaching a unique DNA ~70mer to the antibody via a photocleavable linker. The tagged antibody is bound to the fixed and permeabilized cells, and after washing, the DNA tags are released by both proteolytic cleavage and photocleavage methods. Using fluorescence hybridization technology, a method that unlike quantitative polymerase chain reaction does not require amplification, the quantity of each tag can be assessed. The antibodies selected included diagnostic markers, housekeeping proteins, control proteins, and proteins that represent the major pathways considered to be hallmarks of cancer.

The method was initially validated using cell lines, and the data were robust in technical replicates on different days for the MDA-MB-231 breast cancer cell line. In comparing the single cell data to the bulk data for A431 cells, an epidermoid carcinoma cell line, there was some heterogeneity, but in general the single cell profile matched the bulk profile. Upon comparing A431 cells treated with gefitinib to gefitinib-treated naïve cells, differences in key markers, consistent with expectations from the literature, were detected. This opens up the possibility to be able to monitor changes in the patient's cells during the course of treatment by collecting sequential FNAs. As the tumor shrinks, there is much less material that can be used for harvesting, so the ability to collect a FNA rather than a core biopsy is a big advantage for this application.

Comparing the treatment of HT1080 cells at five different doses of taxol revealed dose–response curves for several protein markers. Using clinical samples from lung adenocarcinoma patients required additional steps on the front end prior to detection with the antibodies. First, the EpCAM-positive cells were isolated and then single cells were separated from debris via micromanipulation. As might be expected, the profile correlation between a single patient cell to the bulk patient cells had a lower correlation than the same test on cell lines. This technique will allow us to get a better understanding of tumor heterogeneity, and it also reaffirms that cell lines do not adequately represent the complexity of tumor cells. In a proof-of-principle study, blinded data collected for four cancer patients undergoing PI3K inhibitor treatment were able to detect a difference in markers between responders and nonresponders and also a difference between the two responders who had received different doses. This new methodology for rapidly assessing protein levels in a very small number of cells is an important step towards the ultimate goal of personalized medicine.

Figure. Multiplexed protein analysis in single cells. (A) Cells were harvested from cancer patients by FNAs. In this case, a heterogeneous population of EpCAM-positive cancer cells (green) is displayed alongside mesothelial cells (red) with nuclei shown in blue (Hoechst) from an abdominal cancer FNAs. Cancer cells were enriched and isolated via magnetic separation in polydimethylsiloxane (PDMS) microfluidic devices with herringbone channels using both positive (for example, EpCAM+/CK+) and negative (for example, CD45) selection modes. (B) Cells of interest were incubated with a cocktail of DNA-conjugated antibodies containing a photocleavable linker (Fig. S1 in the article’s Supplementary Material) to allow DNA release after exposure to ultraviolet light. (C) DNA-antibody conjugates released from lysed cells (Fig. S2 in the article’s Supplementary Material) were isolated using size separation and IgG pull-down. Released “alien” DNA barcodes were processed with a fluorescent DNA barcoding platform (NanoString). Fluorescent barcodes were hybridized and imaged using a CCD camera. The quantified barcodes were translated to protein expression levels by normalizing to DNA per antibody and housekeeping proteins and subtracting nonspecific binding from control IgGs. A representative profile of SKOV3 ovarian cancer cell lines shows high CD44 and high Her2 expression, characteristic of this cell line.

*Abstract from Science Translation Medicine 2014, Vol. 6: 219ra9

Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps.

After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Science Translational Medicine titled "Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates". Authors of the paper are Ullal AV, Peterson V, Agasti SS, Tuang S, Juric D, Castro CM, and Weissleder R.

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