Biophysical technologies for understanding circulating tumor cell biology and metastasis

Abstract

An understanding of cancer evolution in lung cancer with its associated resistance to therapy can only be achieved with repeated sampling and analysis of the cancer. Given the high risks and costs associated with repeat physical biopsy, alternative technologies must be applied. Several modalities exist for analysis and re-analysis of cancer biology. Among them are the CellSearch platform, the CTC chip, and the high-definition CTC platform. While the former is primarily able to provide prognosticating information in the form of CTC enumeration, the latter two have the advantage of serving as a platform to study tumor biology. Techniques for analysis of single cell genomics, as well as protein expression on a single cell basis provide scientists with the capacity to understand cancer cell populations as a collection of individual cells, rather than as an average of all cells. A multimodal combination of circulating tumor DNAs (ctDNAs), CTCs, proteomics, and CTC-derived xenografts (CDXs) to create computational models useful in diagnosis, prognostication, and predictiveness to treatment is likely the future of tailoring individualized cancer care.

Keywords: CellSearch; Circulating tumor cells (CTCs); lung cancer.

Figure 1

CellSearch Platform for CTC enumeration. Blood is drawn into the CellSave Preservative Tube with EDTA and a cellular preservative (A); 7.5 mL of blood is transferred into a separate tube and centrifuged to separate solid blood components and plasma (B); the sample is placed into the CELLTRACKS^® AUTOPREP^® System. Plasma is aspirated, and the sample is resuspended in buffer (C); ferrofluid nanoparticles coated with EpCAM antibodies are added and bind to EpCAM positive cells, hence “enriching” CTCs of epithelial origin. These ferrofluid bound cells are then magnetically separated from other cells (D); CTCs are stained with CK8, CK18, and CK19 antibodies. CD45 positive staining cells are considered leukocytes and are excluded from analysis (E); DAPI stain is applied to stain the nuclei of cells (F); a magnetic force is applied to separate and pull ferrofluid bound EpCAM positive cells to a single focal depth (G); cells are scanned to identify CK positive, DAPI positive, and CD45 negative cells for review (H). EDTA, ethylenediaminetetraacetic acid; EpCAM, epithelial cell adhesion molecule; DAPI, 4’-6’-diamino-2-phenylindole; CK, cytokeratin.

Figure 2

CTC chip and Herringbone chip. Panel I: blood is collected from a patient with lung cancer (A); whole blood (B) is pushed through the surface of the CTC chip pneumatically. Each post is coated with EpCAM antibodies (C); captured cells are stained for CK, CD45, and DAPI (D); CTCs can be enumerated or analyzed for mutations (e.g., EGFR T790M mutation) (E); Panel II: whole blood is collected from a patient with lung cancer in EDTA tubes (A); then pushed pneumatically through an EpCAM antibody bound chip etched with herringbone design, with a single inlet and outlet (B); captured cells are stained for CK, CD45, and DAPI (C); CTCs can be enumerated, stained for other markers like AR or PSMA, or also undergo molecular testing (e.g., EGFR T790M mutation) (D). EGFR, epithelial growth factor receptor; PSA, prostate specific antigen; PSMA, prostate specific membrane antigen; AR, androgen receptor.

Figure 3

HD-CTC platform. Peripheral blood from patients with lung cancer is drawn in anticoagulated blood tubes (A); subjected to erythrocyte lysis with ammonium chloride solution, then centrifuged (B); about 0.5 mL of nucleated cells (~3 million nucleated cells) are attached as a monolayer to glass slides with proprietary coating to allow maximal retention of live cells (C); slides are frozen and thawed just before further analysis. Slides are incubated with and cells stained for CK, CD45, and DAPI. Other characterization antibodies may also be added (D); a custom fluorescent scanning microscope scans images of and analyzes cells using a custom digital pathology software based on stringent criteria for HD-CTC (E); HD-CTCs are saved and presented to pathologist for confirmation. Images are saved and digitally catalogued for future re-analysis (F); genomic analysis such as FISH or NGS can be completed (e.g., ALK rearrangement) (G). ALK, anaplastic lymphoma kinase; FISH, fluorescent in situ hybridization; NGS, next generation sequencing.

Figure 4

Blood is drawn from patients with lung cancer (A), processed (B), and injected directly into mouse models (C). As the tumor grows, further studies to understand tumor behavior and mechanisms of drug resistance can be undertaken, including assessing response to chemotherapy (E), assessing CDX tumor characteristics and genomic testing (F,G), and identify novel targeted therapies (H).