Affinity-based enrichment of circulating tumor cells

Background

Circulating Tumor Cells (CTCs) are tumor cells found in the peripheral blood of cancer patients, and the presence of these cells are considered the “real culprits” of metastatic disease that accounts for 90% of all cancer-related deaths. Several studies were conducted in various types of cancers (breast, lung, colon, prostate, melanoma, etc.), presenting CTCs have proven prognosis value in these cancer types [1–3]. CTCs have also been shown to be valuable to predict drug-targeted therapy response in lung cancer patients [4]. As a result, there is increased interest in CTC research. In order to study CTCs these cells must be isolated and this can be challenging. In this chapter, we highlight one of the most widely used CTC-isolation techniques—affinity-based CTC capture. We discuss the principles behind the techniques, as well as explore emerging technologies employing these techniques. Lastly, we discuss the value and limitations of these various techniques.

Affinity-based enrichment of CTCs

As a rare population of cells in circulation, CTCs are difficult to study without the use of powerful technology that sets these cells apart from the surrounding and overwhelming population of leukocytes, red blood cells and other blood components. Among the various CTC enumeration technologies currently available, the use of the affinity-based isolation technique is the most widely adopted. These technologies utilize a unique antigen expression pattern on CTCs. The rationale behind the use of this type of CTC enumeration technology is based on unique antigen(s) expressed by CTC depending on their tissue of origin that are not shared with other normal components circulating in the blood. One of the most common examples among these is the use of the Epithelial Cell Adhesion Molecule (EpCAM) for CTC capture in epithelial cancer types. EpCAM is a transmembrane glycoprotein that is being expressed in cancers of epithelial origin, and not in normal components in circulation. Thus, antibodies against EpCAM can be used to selectively capture epithelial CTCs from peripheral blood. Here, we discuss several types of affinity-based technologies utilizing EpCAM or other tumor specific antigens in the capture of CTC cells (CellSearch and MagSweeper), as well as technologies that combine affinity-based capture with other physical parameters such as cell size (CTC-Chip and GEDI).

CellSearch

To date, CellSearch is the gold standard in the CTC field. It is the first and still the only technology that is cleared by US Food and Drug Administration (FDA) for clinical CTC testing in metastatic prostate, breast, and colorectal cancers. Utilizing the principle of magnetic field-based cell sorting, CellSearch captures CTCs by labeling them with ferrofluid nanoparticles functionalized with anti-EpCAM antibodies. These cells are then isolated via a magnetic field. Brie?y, a blood sample is mixed with buffer and centrifuged, followed by aspiration of the plasma and buffer. Ferrofluid nanoparticles are then added to enrich CTCs with EpCAM expression. Post CTC enumeration, cells are labeled with Cytokeratin (Cytokeratin 8,18,19-phycoerythrin), which are epithelial markers to identify CTC; CD45 (CD45allophycocyanin), which is a leukocyte marker for negative identification and DAPI, which labels nucleated cells. Results of harvested cells are then reviewed and CTCs are identified based on their staining profile (Cytokeratin positive, DAPI positive and CD45 negative) and morphology features [5].

The clinical utility of the CellSearch platform has been validated in various studies. The first milestone study in metastatic breast cancer patients indicates that, based on a cutoff of 5 CTCs detected by CellSearch system per 7.5 mL blood sample, metastatic breast cancer patients can be strati?ed to predict their disease outcome (Progression Survival and Overall Survival) [1]. This has also been validated in studies of other disease types such as prostate and colorectal cancer with a cutoff set at 5 and 3 CTCs respectively [2, 3].

MagSweeper

MagSweeper is an automatic immunomagnetic separation technology for CTC enumeration. Blood samples are pre-diluted with a buffer and then incubated with magnetic-bead coated with anti-EpCAM antibodies to mark epithelial CTCs. Samples are then subjected to MagSweeper enumeration, during which magnetic rods covered with a plastic sheath are placed to sweep the sample well. After CTCs are captured on the rods, they are washed with a buffer and collected by external magnets that are located at the bottom of the well [6].

CTC-Chip

CTC-Chip technology utilizes microposts functionalized with anti-EpCAM antibodies to capture CTCs. On the first generation of the CTC-Chip, 78,000 microposts were distributed on a 970-mm2 surface and were functionalized with anti-EpCAM antibodies. Blood samples were processed through the CTC-Chip at a flow rate of 1–2 mL/h and captured CTCs were then identified via Cytokeratin positive, DAPI positive and CD45 negative staining. Although CTC-Chip technology is effective in capturing and analyzing CTCs, the volume that can be processed is limited to 2–3 mL [7]. This could be a major drawback if applied in clinical settings, as the standard blood sample collected is 7.5 mL. Reducing the sample volume could affect the sensitivity of the CTC capture. Thus, a second generation of the CTC-Chip, HB-Chip, was designed with a Herringbone structure to disrupt the flow of blood and increase CTC collision with antibody-functionalized walls on the chip. This new design not only increases the volume of blood samples that can be processed using the chip, but also enables the capture of CTC clusters [8] that have shown to be clinically significant when analyzed by other CTC enrichment platforms [9]. The third generation of the CTC-Chip, CTC-iChip, utilizes a combination of hydrodynamic size-based cell sorting to deplete small cells in blood such as red blood cells and platelets. The inertial focusing microfluidics positions the cells into a single cell line and then either enriches the affinity-based labeled EpCAM positive CTCs or depletes the CD45 positive leukocytes with a magnetic field. This third generation of CTC-iChip not only allows for positive affinity-based enumeration of CTCs by magnetic activated cell sorting, but also allows for negative depletion of leucocytes for antigen agnostic CTC enumeration [10].

The clinical utility of the CTC-Chip still awaits large-scale clinical trial examination. A pilot study has indicated that by employing the CTC-Chip to analyze EGFR mutation in CTCs harvested from lung cancer patients, the responses for targeted therapies can be potentially predicted [4].

GEDI

Geometrically enhanced differential immunocapture (GEDI) is a technology that captures prostate circulating tumor cells (PCTCs) based on their expression of prostate membrane-specific antigen (PSMA). The geometry of the device is designed to allow large prostate CTC’s to collide onto obstacles, while smaller, non-target cells are displaced onto streamlines that do not collide with the microposts. This combination of affinity and size-based enrichment results in better capture efficiency and purity [11].

Worth noting, by switching the antibody used to functionalize the GEDI device, it is possible to achieve capture of CTCs from breast and gastric cancers using antiHER2 antibody [12], pancreatic CTCs using anti-EpCAM and MUC1 antibodies [13], and mouse epidermal stem cells using anti-CD34 antibody [14].

Emerging technologies for affinity-based enrichment of CTCs

As a fast expanding community, CTC research has gained more and more attention in recent years. Thus, as previously described affinity-based CTC capture platforms are improving and a boom of fast-developing technologies are emerging. A few examples of these are discussed below:

NanoVelcro

NanoVelcro is a chip to capture CTCs based on their EpCAM expression. It is a device that is comprised of two parts; a patterned silicon nanowire substrate coated with anti-EpCAM antibodies and a polydimethylsiloxane (PDMS) chaotic mixer to generate flows that increase the collision between CTCs and capture substrates. Post CTC capture processes, CK/CD45/DAPI staining are executed to identify CTCs on the chip. A small, pilot cohort study of prostate cancer patients, utilizing the NanoVelcro chip, showed promising results for clinical applications [15]. Additionally, by grafting the NanoVelcro Chip with poly(N-isopropylacrylamide) (PIPAAm), a thermoresponsive polymer, captured tumor cells can be released via a temperature switch from 37 to 4 °C and cultured as demonstrated in model systems [16].

Graphene oxide nanosheets

This technology utilizes flower-shaped gold patterns to absorb graphene oxide (GO) and then functionalize the GO nanosheets with anti-EpCAM antibodies to capture CTCs in a PDMS chamber, and identify CTCs stained with CK/CD45/DAPI. The chamber-like platform also favors on-chip cell culture, tested with model systems where cultured tumor cells are spiked into blood, retrieved by GO nanosheets and cultured on chip [17].

VerIFAST

VerIFAST is a technology based on immiscible phase filtration (IPF) in which target cells are incubated with paramagnetic particles (PMPs), coated with EpCAM antibodies for capture and pulled with magnetic force into sequential chambers to achieve staining for EpCAM, Ki67, and Hoechst nuclear staining. The sieve chamber design in this technology can allow for an on-chip capture and staining workflow that minimizes substantial cell loss [18].

Immuno-microbubbles

This technology uses perfluorocarbon gas ?lled microbubbles conjugated with antiEpCAM antibodies to capture CTCs. After CTCs are captured by the microbubbles, the layer of bubbles can be separated by centrifugation. The captured cell population is then subject to pan-CK/CD45/Hoechst staining for CTC identification [19].

GILUPI CellCollector

This emerging technology addresses the increasing need for in vivo CTC collection. This device collects CTCs by a functionalized and structured medical wire with anti-EpCAM antibodies that is inserted into the cubital vein of cancer patients for 30 min. This in vivo collection process, via this device, can analyze CTCs from 1.5 to 3 L of blood as compared with 7.5 mL blood analyzed by other ex vivo CTC assays [20].

Molecular and functional analysis of circulating tumor cells downstream of affinity-based CTC Enrichment

There has been a recent surge in the CTC capturing technology industry. Molecular and functional characterization of CTCs is becoming “hot-beds” for researchers seeking better utilization of CTCs in the clinical setting. Although the identification and quantification of CTCs alone can be effective in prediction of disease outcome [1–3], molecular and functional characterizations of CTCs are likely to increase the specificity of the CTC assay, leading to personalized targeted therapies.

Results from a recent clinical trial, SWOG 0500, indicates that chemotherapy treatment decisions based on elevated CTC numbers after a first cycle of chemotherapy does not benefit metastatic breast cancer patients [21]. Although the clinical utility of CTC numbers alone in determining treatment options needs further validation, these results exemplify the importance of, and demand for, CTC characterization. Here, we briefly discuss the characterization of CTCs enabled by the technologies we discussed above.

Gene expression analysis to characterize CTC post affinity-based enrichment

Gene expression analysis is important in the prognosis and treatment of cancer as validated in primary tumor gene expression profiling experiments [22–24]. It is very probable that CTC gene expression analysis will more than likely provide critically necessary information for the management of diseases. It will also provide a critical research tool that will enhance the study of metastatic processes, tumor dormancy, tumor tropism, etc. Current attempts are trying to interrogate CTC gene expression profile at different levels.

Gene expression analysis at the mRNA level can be achieved on chip via RNA in situ hybridization (RNA ISH) as demonstrated by HB-CTC Chip to analyze human pancreatic CTC expression of Wnt2 [25].

Off-chip mRNA analysis for products of tumor-specific genes can be analyzed by qRT-PCR or RNA-seq methods from pooled CTCs as demonstrated by data obtained from CTC enrichment methods such as GEDI, GO nanosheets, and HB-CTC Chip [11, 17, 25]. However, due to contaminant leucocytes and other blood components captured nonspecifically by these affinity-based CTC enrichment methods, performing high-throughput mRNA analysis on both tumor and non-tumorspecific genes requires further isolation of pure population of CTCs in the post-capture process. This can be achieved by either the micropipetting of single cells (feasible by CTC-iChip technology) [10] or by microfluidics enabled by a single-cell analysis platform like Fluidigm (feasible by using MagSweeper technology) [26]. These single-cell analysis assays can be implemented to address issues raised regarding CTC heterogeneity that could be critical in the prognosis and treatment of cancer.

CTC gene expression profiles can also be interrogated at the protein level. This interrogation is already seen in most affinity-based CTC capture techniques. As an example, immunofluorescence (IF) staining is being employed to help distinguish CTCs from the background of leukocytes and other blood components. By adding an extra antibody or additional marker(s), it can be helpful in the quali?cation/quantification of protein marker expression levels when enabled by multi-spectrum fluorescence microscopy. CellSearch is a good example of technology that provides a user-defined channel tool that can be used in the characterization of CTCs [27]. There are several ongoing clinical trials are currently utilizing this feature to characterize CTCs for their Her2/Ki-67 expression and using the findings to determine the endocrine therapy used in metastatic breast cancer patients [28]. Multiplex immunofluorescence staining can also be used in the detection of fusion protein products such as TMPRSS2ERG fusion protein found in Castration-Resistant Prostate Cancer patients as demonstrated by the GEDI platform [11].

Genomic analysis of CTC post affinity-based enrichment

CTC genomics is another important aspect in the management of cancer. Analyzing mutations and chromosome rearrangements, as well as lineage tracing using Next Gen Sequencing (NGS) in CTCs, can provide valuable information in treatment selection and personalized therapy. It can also provide important biological-based information regarding the metastasis. DNA fluorescence in situ hybridization (FISH) is a common methodology utilized to analyze DNA point mutation and chromosomal rearrangement on chip. As demonstrated by HB-CTC Chip, FISH for Androgen Receptor (AR) gene locus can be performed on CTCs for copy number analysis [8]. Another efficient method for DNA mutation detection is PCR. This has been shown to be very successful in the detection of EGFR mutation from pooled CTCs isolated with CTC-Chip in lung cancer patients as described above [4]. DNA mutation detection by PCR has also proven to be an effective in the detection of KRAS and other gene mutations from CTC isolated by GILUPI CellCollector [20]. DNA mutation and chromosome translocations can also be detected on CTCs by RNA-ISH/IF techniques that can detect the mRNA or protein product of the mutation/fusion DNA [8, 11].

Next-Gen Sequencing is also being applied to analyze CTCs isolated by affinitybased methods. With a broader spectrum of CTC genomics, it can provide valuable information in treatment options, as well as insight in tumor evolution and the resistance development process. This is exemplified in a publication authored by Jens et al. addressing whole-exome sequencing of CTCs isolated from metastatic prostate cancer patients. Using a combination of automated cell picking with MagSweeper CTC isolation technology, they were able to achieve single cell whole-exome sequencing [29]. Xiaohui Ni et al. also achieved whole-exome sequencing using the CellSearch Platform to isolate CTCs from lung cancer adenoma patients. In this study, whole-exome sequencing was performed to analyze the copy number variation (CNV), and the patterns observed in the CTCs were found to be representative of metastatic tumors [30].

Functional characterization of CTC enabled by affinitybased enrichment

An emerging topic in CTC research is functional characterization of CTCs. This requires techniques that maintain cell viability in samples during the CTC capture process. To achieve optimal CTC viability, additional requirements for the handling, preserving, labeling, and shear pressure control should be implemented. It is worth noting that the CellSearch platform does not allow for viable CTC capture under harsh conditions. Some affinity-based platforms that allow for viable CTC capture are the CTC-iChip [10], GEDI [11], NanoVelcro chip [16], and Graphene Oxide nanosheets [17]. As a result, technologies that allow for viable CTC capture are driving CTC culture technology development and ex vivo drug treatment assays [11, 31] (Fig. 2.1).

Fig. 2.1. Summary of molecular and functional analysis of circulating tumor cells downstream of affinity-based CTC enrichment

Gene expression analysis Genomic analysis Functional characterization
CellSearch Third marker immunofluorescence [28] Copy Number Variation Analysis [31]
CTC-Chip PCR [4]
HB-Chip RNA ISH/Pooled Cells RNA-Seq [25] FISH/Fusion transcript by RT-PCR [7]
CTC-iChip Single Cell qRT-PCR [9] CTC Culture/

Ex-vivo drug treatment [32]

MagSweeper Single Cell qRT-PCR [27] Whole Exome Sequencing [30]
GEDI Pooled Cells RNA-seq [11] Fusion protein by immunofluorescence [11] Viable CTC Capture/

Ex-vivo drug treatment [11]

NanoVelcro Viable CTC capture/ Culture in model system [16]
GO

nanosheets

Pooled Cells qRT-PCR [17] Viable CTC capture/ Culture in model system [17]
GILIPI

CellCollector

PCR [20] Viable CTC capture [20]

Limitations and emerging applications of affinity-based enrichment of CTCs

Limitations

Although affinity-based enrichment of CTCs has shown to be versatile and effective, the fact remains, that limitations related to this type of technology still exist. A major concern for affinity-based capture of CTCs is its utilization of antigen expression. Although EpCAM has shown to be an effective marker for CTC capture and is being used extensively in affinity-based CTC capture platforms, an increasing number of studies are also revealing its limitations. These limitations have been identified that CTCs with no or low EpCAM expression will be overlooked when EpCAM is used as the capture target. This was the case in a study featuring characterization of CTCs with potential for creating brain metastasis, this sub-group of CTCs were found to be EpCAM negative [32]. Another study conducted in metastatic breast cancer patients demonstrated how by combining of Cytokeratin and EpCAM expression for CTC detection, the sensitivity of CTC detection can be increased as compared with using solely EpCAM expression for CTC detection [33]. This could be due to the heterogeneity of CTC populations and the hypothesis that tumor cells undergo Epithelial–Mesenchymal Transition (EMT) and shed epithelial phenotypes such as EpCAM expression in order to increase invasion and metastatic capabilities [34]. It is important to note that the quality of the antibody and the position of the epitope that an antibody recognizes can also affect capture efficiency [35].

Emerging applications for affinity-based enrichment of CTCs

Different strategies were attempted in order to overcome the potential bias introduced by EpCAM-based CTC capture. One strategy sought to increase the sensitivity of affinity-based CTC enrichment by using comprehensive panels to capture CTCs like using Cytokeratin in combination with EpCAM [33] or using a combination panel of EGFR, HPSE, ALDH1 [29]. Another strategy called for the negative depletion of non-tumor cells using an affinity-based isolation method instead of positively captures cells expressing certain markers. The negCTC-iChip is a notable example for this type of technology. By combining depletion of non-tumor cells using anti-CD45 and anti-CD15 antibodies with hydrodynamic cell-sorting based on cell size, the negCTC-iChip mode is able to isolate CTCs without biasing CTC capture with their antigen expression profiles.

Summary

Affinity-based enrichment serves as the first and most widely accepted principle employed in the isolation of CTCs. CellSearch technology, which utilizes this principle, is considered the gold standard in CTC research. Novel and evolving technologies seeking to increase capture efficiency are introducing state-of-the-art microfluidic designs. Numerous application potentials are being investigated to characterize CTCs captured with affinity-based principles at DNA, RNA and protein levels, as well as functional characterization of CTCs. Nevertheless, affinity-based CTC capture also faces some limitations with potential loss of sensitivity as a result of CTC heterogeneity, EMT process, and capture antibody efficiency. To overcome these limitations, technologies have been exploring various approaches to capture CTC that include the combination of panels of targets for CTC capture, combination of affinity-based capture with other principles such as size-based capture, and switching from positive capture to negative depletion of non-tumor cells in the sample.

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