Single-Cell Analysis

Automated Circulating Tumor Cell (CTC) Isolation Overcomes Limitations of otherLiquid Biopsy Methods

The Genesis System Enables Sensitive Isolation of CTCs for Enrichment or Enumeration Applications
Liquid biopsies are rapidly gaining traction and becoming a standard of care for cancer. They are an attractive alternative to invasive,
painful, costly tissue biopsies and can facilitate real-time monitoring while providing insight into the disease state, even for tissues that are
difficult to access, such as non-small cell lung cancer (NSCLC). These assays target circulating tumor cells (CTCs) or circulating tumor
DNA (ctDNA).
Limitations of ctDNA as a Marker for Disease Progression
While CTCs have been known to researchers for more thana century, ctDNA has received more attention in recent years
due to advances surrounding Next Generation Sequencing(NGS) technologies. However, ctDNA is found in plasma in low
concentrations and NGS analysis is complicated by the low
frequency of driver mutations. Additionally, plasma frequently
contains contamination from cellular debris and genomic DNA that
may not represent the current tumor state. As more sophisticatedcell isolation technologies have been developed, there is renewedinterest in CTCs as biomarkers.

Fig. 1. Circulating tumor cells are shed from the primary tumor and carried through the
body in the blood. Tumor cells ( ); RBCs ( ); WBCs ( ).

Circulating Tumor Cells

CTC enumeration can be used to detect the risk for early
development of (micro)metastases, assess therapeutic responses
Circulating
Tumor
Cells
shedforfromof advanced disease,andselectaretreatment
various types
CTC Enumeration: A Simple and Predictive Biomarker
of cancer fromprimary
to metastaticstates. Detection theof fiveprimarytumor
andcarried throughout
The implementation of molecular and genomic characterization
or more7.5 mL of blood in subjects with metastatic
body
inCTCsthe perblood
7of CTCs can contribute to improving diagnosis and personalizing
breast cancer (MBC) is associated with disease progression.
1
treatment selection. CTCs are shed by the primary tumor throughMounting clinical evidence has prompted international experts tothe bloodstream and lymphatic systems (Figure 1). They are a
recommend the use of CTC enumeration for staging metastatic
2–3
main source of metastases but cannot be detected by CT or
breast cancer and disease stratification in prospective clinical
PET scans. The enumeration of CTCs is a simple and predictive
trials.8 The American Society of Clinical Oncology biomarker
biomarker with many applications in cancer research for monitoring
guidelines note that CTCs may be used to monitor metatastic
4–6
and prognosis.
tumors during and after treatment. For example, a decrease in
CTC count after one cycle of chemotherapy is indicative of tumor
A unique advantage of CTCs compared to other blood-based
response, increased progression-free survival, and increased
biomarkers is they represent a cancer-derived cell population,
9overall survival in stage III–IV cancer.
providing researchers a powerful tool to study tumor heterogeneityand progression of the disease at various stages.

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Automated Circulating Tumor Cell (CTC) Isolation Overcomes Limitations of other Liquid Biopsy Methods

Addressing Limitations of Legacy CTC Isolation Systems

The Genesis System supports Celselect SlidesTM, which features
patented microfluidics paired with 56,400 microchambers (Figure3) to capture and isolate CTCs based on their size (>8 µm). White
blood cells (WBCs) captured can be quickly identified by using
the WBC-specific marker CD45 and excluded from analysis.
Advantages to using the Genesis System compared to legacy
systems are found in Table 1.

For clinical adoption, analysis of CTCs must overcome a numberof challenges, including low abundance in blood, contamination
with leukocytes, and the loss of cell viability during isolation
processes. Early techniques frequently relied on some type of preprocessing like density gradients or emulsion based separation.
These approaches added extra processing time and were
frequently accompanied by cell loss and lower capture efficiencies.
In addition, many of the legacy CTC platforms rely on immune
markers to detect CTCs. Unfortunately, no single perfect marker
can identify all CTC types, due to the inherent heterogeneity andgenetic instability of cancer. The flexibility to use multiple markers
or customize markers for the detection of specific CTC types
enhances the accuracy and utility of CTC enumeration.
The Genesis System: Automated CTC Isolation and Analysis
Technology
The lack of reproducibility and sensitivity with existing technologies
has limited the clinical adoption of CTCs. The Genesis System(Figure 2) was developed to address these shortcomings, including
CTC isolation and enumeration. This system offers a robust
solution that can accelerate clinical research and the acceptanceof CTCs as a routine biomarker.

Fig. 3. Size-based enrichment of CTCs. A schematic of the microfluidic chambers used tocapture CTCs ( ) based on their size.

Table 1. Advantages of the Genesis System over legacy CTC detection
technologies.
Legacy CTC Detection Limitations

The Genesis System Advantage

Legacy systems have low capture
efficiency

High capture efficiency, reliably detects
10
1 in 107 cells per mL of blood

Emulsion technologies suffer WBC
contamination

WBCs are easily excluded from analysis
by immunostaining with CD45

Harsh processing methods negatively
affect cell viability

Viable cell isolation

Labor intensive with limited automation

Fully-automated cell capture andstaining

Solely dependent upon EpCAM-staining
and only detect epithelial CTCs

Customizable staining to detectepithelial and mesenchymal CTCs for
improved specificity

Designed only for enumeration of CTCs

Automated workflows for CTC
enrichment and enumeration

Fig. 2. The Genesis System.

© 2022 Bio-Rad Laboratories, Inc.

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Automated Circulating Tumor Cell (CTC) Isolation Overcomes Limitations of other Liquid Biopsy Methods

Prepare Liquid Biopsy Sample

Isolate and Retrieve Cells

Celselect Slides enable rapid analysis without compromising cellcapture efficiency. They have been validated with several human
11
cancer cell lines: MCF7 (breast), SKBR3 (breast), LnCAP (prostate),
PC3 (prostate) and HT29 (colorectal), and capture efficiency was
found to be greater than 80%. The slides captured both epithelial
cancer cells, MCF7 and SKBR3, and mesenchymal cells, MDA12
MB-231. Any liquid biopsy including peripheral blood, urine, pleural
fluid, or cerebral spinal fluid can be processed on the Genesis
System.

Downstream Analyses

Automated

Add buffer to liquid biopsy
sample and add to sample
inlet.

Genesis System automatesisolation and retrievalof cells.

Cells can be characterizedwith methods such as
sequencing (bulk or singlecell), Droplet Digital™ PCR,FISH, Flow Cytometry or cells
cultured for further in vitro andin vivo analysis.

Fig. 4. Cell Enrichment Workflow. Liquid biopsy sample is prepared with the Dilution Buffer
and added to the sample inlet. CTCs are isolated automatically in the Celselect Slide while
RBCs and WBCs pass through. Cells are retrieved in a single tube for downstream analyses.

Prepare Liquid Biopsy Sample

The general workflow for CTC enrichment is demonstrated in
Figure 4. After loading the sample onto the Genesis System, theremainder of the enrichment, purification, and staining procedures
are automated in the Celselect Slides. During enrichment,
cells are retrieved in a tube for downstream anlayses such as
immunohistochemistry, Droplet Digital PCR, flow cytometry,
12 Cells
PrepareLiquidhybridization
Biopsy Sample
Isolate (Figure
Immunostaining
fluorescencein situ(FISH)
5), sequencing(Figure 6), or development of cell cultures. If the cells are to be
counted after staining for CTC enumeration (Figure 7) slides
are compatible with imaging using many automated scanning
Automated
microscopes.

Isolate Cells

Immunostaining

Image

Automated

Add Fixing Reagent to liquidbiopsy sample and add tosample inlet. Sample andstaining reagents are prepped
according to predefined
protocols.

Genesis System automates isolation andimmunostaining on Celselect Slides.

Image

Fig. 7. Cell Enumeration
Workflow. Liquid biopsy
sample is prepared with
Fixing Reagent and added tothe sample inlet. CTCs are
isolated automatically in the
Celselect Slide while RBCs
and WBCs pass through. The
Celselect Slide is imaged on
an automated microscope
and files can be imported forreporting.

Create Report

Image Celselect Slide on an
Automated Microscope.

Import file from microscope to
CTC Analysis Software, choose
cells to include and generatereport.

CTC Enumeration Sensitivity
A clinical study conducted at the Sidney Kimmel Cancer Center at
Thomas Jefferson University revealed CTC detection in prostatecancer is more sensitive with the Genesis System than with the
12
CellSearch System (Menarini Silicon Biosystems). Analysis of
18 blood samples from patients with metastatic prostate cancer
demonstrated the Genesis System detected CTCs in 17/18
samples (94%) whereas the CellSearch System detected CTCs in
only 11/18 samples (61%). CTC counts were typically higher using
the Genesis System, implying greater sensitivity for CTC detection(Figure 8).

Fig. 5. Fluorescence in situ hybridization (FISH). A, DNA FISH performed on CTCs isolated
from metastatic breast cancer; B, RNA FISH performed on CTCs isolated from metastatic
breast cancer.

100
90

CTCs/7.5 mL

80

Celselect
CellSearch

70
60
50
40
30
20
0

Celselect

5

6

7

14

15

16

17

22 86 23 120 8

1

0

60 15 79 11 375 11

19 30

4

4

11

4

0

5

0

0

0

3

0

CellSearch 3

Fig. 6. Sanger sequencing of the PCR amplicons from PC3 (A) and LNCaP (B) that were
captured using Celselect Slides.

2

12

3

6

4

7

0

8

0

9

10

11

23 29 47

12

1

13

7

18

Fig. 8. Comparison of CTC counts using Celselect Slide Technology versus CellSearchTechnology. Number of CTCs determined using either the Genesis System or the CellSearch
13
System were normalized to CTC-count per 7.5 mL of blood.

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Automated Circulating Tumor Cell (CTC) Isolation Overcomes Limitations of other Liquid Biopsy Methods

Futhermore, this study found that Celselect Slide Technology
captured low numbers of leukocytes, and these cells were easily
discriminated from CTCs with differential immunostaining using cell
type-specific antibodies.

50
45

Number of Cells Captured

The cells were further analyzed and found to be prostate-specificantigen (PSA)-positive and nucleated, demonstrating they wereCTCs from the prostate tumor. Not only was the Genesis Systemable to capture and detect more CTCs than the CellSearch
System, it was able to detect subpopulations of CTCs missed byother technologies, making it a more reliable tool for monitoring.

35
30
25
20
15
10
5
0

CTC and Expression of Cancer and Immune Markers

A study conducted at Juntendo University School of Medicine
evaluated the use of CTC enumeration to predict eribulin treatment
13
efficacy in MBC patients. Previous CTC enumeration systems
have relied solely on EpCAM staining of CTCs to detect epithelial

Logrankp=0.0013

mCTC

1.0

20

0.8

0.6

0.4

0.4

0.4

0.2

0.2

0.2

10

20

30

40

50

0.0

0

10

Time (weeks)
Number at risk
Low
7
7
13
10
High
Label

6
2

2
0

total CTC Category Median PFS

2
0

11
9
Label

11
16

60

20

30

40

50

0.0

p=0.6800

0

10

20

Time (weeks)
2
0

50

eCTC

0.8

p=0.0125

0.6

0

40

1.0

0.6

0.0

30

CTCs (eCTCs), but not all CTCs are EpCAM positive. A subpopulation of CTCs with decreased levels of epithelial markers
escape EpCAM-based detection. Mesenchymal CTCs (mCTCs) arenot EpCAM positive as these cells are frequently going through theEpithelial to Mesenchymal transition. To evaluate how CTCs could
be used to predict eribulin efficacy, three populations of CTCs wereanalyzed: eCTCs, mCTCs and total CTCs. The ability to customize
stains on the Genesis System to detect mCTCs made this study
possible.

CTC Enumeration as a Predictor of Treatment Efficacy

0.8

10

Fig. 9. CTC enumeration and analysis. A, spike in analysis for CTC recovery. NCI-441 cells(PD-L1 positive lung cancer cell line) were spiked into normal blood sample; cell recovery was
reproducible down to 1 CTC in a million cells; B, representative image showing CD45 negative andPD-L1 positive cell; “y” is a variable representing the number cells collected as a function of thenumber of cells spiked in; “R2” is a statistical measure of fit between 0–1 that indicates how muchvariation of a “Cells Captured” is explained by “Cells Spiked.”As part of the study, the lower limits of the reproducible detection(LOD) of the Genesis System were evaluated. As few as 5 PD-L1+
CTCs were reproducibly detected in 4 mL of whole blood. (ThePD-1/PD-L1 pathway is a target for NSCLC immunotherapy.) This
represents a capture rate greater than 1 in 1,000,000 cells (Figure8), and suggested a higher capture rate than the cells captured on
legacy systems.

Total CTC

0

Number of Cells Spiked

In a collaborative study with IncellDx, Cytek Biosciences, and
Qognit, single-cell immune and cancer marker profiling of NSCLC
primary tumor cells were examined to potentially predict the10presence of CTCs in the blood. This screening tool could be
used to select patients for ongoing CTC monitoring for diseaseprogression and response to therapy. A comprehensive study
was performed on tissue with paired blood samples to refine theprediction algorithm.

1.0

y = 0.8006x
R2 = 0.9741

40

2
0

7
1

30

40

50

1
1

0

Time (weeks)
20mCTC Category Median PFS

1

0

9
11
Label

8
9

4
4

1
1

1

eCTC Category Median PFS

<3

Low

25.4 weeks

<2

Low

22.6 weeks

<2

Low

16.1 weeks

>3

High

13.1 weeks

>2

High

13.1 weeks

>2

High

18.0 weeks

Fig. 10. Kaplan–Meier curves of PFS relative to CTC-count. The log-rank test was applied for comparisons of the survival distributions of the groups. Total CTCs including eCTCs and mCTCs was the13
most predictive for PFS.

© 2022 Bio-Rad Laboratories, Inc.

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Automated Circulating Tumor Cell (CTC) Isolation Overcomes Limitations of other Liquid Biopsy Methods

Blood samples were collected from 22 patients before and during
treatment. Progression-free survival (PFS) and CTCs counts weremonitored by CTC type. The results demonstrate the total CTCs
(including eCTCs and mCTCs) was the most predictive of PFS overeCTCs or mCTCs alone (Figure 10). Since legacy systems often
rely on detection of EpCAM positive CTCs (eCTCs), eribulin therapy
monitoring with these systems are not as accurate.

SummaryThis study demonstrated that the Genesis System, with the ability
to customize fluorescent stains, enables the detection of CTC
sub-populations and offers a significant improvement upon legacy
systems.

■

Capture of CTCs with high efficiency from whole blood■

Removal of 99% of RBCs and WBCs■

Automated workflow

The examples described in the four studies above demonstrate
how the Genesis System overcomes the limitations of current CTC
enumeration systems as summarized in Table 1.
The Celselect Slide Technology addresses the challenges of CTC
isolation and analysis by providing the following benefits:■

CTC Enrichment Can Improve AR-V7 Sensitivity
Researchers at the Showa University School of Medicine
demonstrated that testing for the splice variant AR-V7 (the causeof castration-resistant prostate cancer) with polymerase chain
reaction (PCR) can yield positive results in healthy individuals. They
hypothesized that the false positivity was due to contaminationof hematopoietic cells. Testing enriched CTCs were compared towhole blood. Results demonstrated that the AR-V7 false positivity
rate was reduced in the CTC enriched samples. Although ARV7 positivity did not predict therapy effectiveness, AR-V7 wasmore frequently positive than the Extent of Disease (EOD) andis positively correlated with progression of bone metastases
suggesting AR-V7 testing on enriched CTCs is an area of
exploration for prognostic testing.14© 2022 Bio-Rad Laboratories, Inc.

■

On-slide immunostaining or retrieval of viable cells fordownstream analysis
High-sensitivity CTC enumeration to understand tumor
progression and response to therapy

The Genesis System offers an automated, easy-to-use, robust,and precise approach to CTC enumeration that has the potential toaccelerate the clinical adoption of CTCs as a biomarker in cancer
research and potential diagnostics.

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References1.

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2.

Maheswaran S, Haber DA. (2010) Circulating tumor cells: a window into cancer biology and metastasis. Curr Opin Genet Dev 20:
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Hwang WL, Pleskow HM, Miyamoto DT. Molecular analysis of circulating tumors cells: Biomarkers beyond enumeration. Adv Drug
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Jiang Z, Li L, Zhang S, Wang T, Liu Y, Bian L, et al. Predictive value of CTCs biomarkers status in patients with metastatic breast
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Li J, Han X, Yu X, Xu Z, Yang G, Liu B, et al. Clinical applications of liquid biopsy as prognostic and predictive biomarkers inhepatocellular carcinoma: circulating tumor cells and circulating tumor DNA.
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Toss A, Mu Z, Fernandez S, Cristofanilli M. CTC enumeration and characterization: moving toward personalized medicine. Ann Transl
Med. 2014 Nov;2(11):108–108.

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Bidard F-C, Peeters DJ, Fehm T, Nole F, Gisbert-Criado R, Mavroudis D, et al. Clinical validity of circulating tumour cells in patientswith metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncol. 2014 Apr;15(4):406–14.

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Cristofanilli M, Pierga J-Y, Reuben J, Rademaker A, Davis AA, Peeters DJ, et al. The clinical use of circulating tumor cells (CTCs)
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Liberko M, Kolostova K, Bobek V. Essentials of circulating tumor cells for clinical research and practice. Crit Rev Oncol Hematol. 2013
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10. Wang et al (2021) Cell by cell immuno- and cancer marker profiling of non-small cell lung cancer tissue: Checkpoint marker
expression on CD103 + , CD4 + T-cells predicts circulating tumor cells. homeostasis. Translational Oncology 14, 100953
11. Riahi R, Gogoi P, Sepehri S, Zhou Y, Handique K, Godsey J, et al. A novel microchannel-based device to capture and analyze
circulating tumor cells (CTCs) of breast cancer. Int J Oncol. 2014 Jun;44(6):1870–8.
12. Gogoi P, Sepehri S, Zhou Y, Gorin MA, Paolillo C, Capoluongo E, et al. Development of an Automated and Sensitive Microfluidic
Device for Capturing and Characterizing Circulating Tumor Cells (CTCs) from Clinical Blood Samples. PloS One. 2016;11(1):e0147400.
13. Horimoto Y, Tokuda E, Murakami F, Uomori T, Himuro T, Nakai K, et al. Analysis of circulating tumour cell and the epithelial
mesenchymal transition (EMT) status during eribulin-based treatment in 22 patients with metastatic breast cancer: a pilot study. J
Transl Med. 2018 Oct 20;16(1):287.
14. Nakasato T, et. al. A Novel, Circulating Tumor Cell Enrichment Method Reduces ARv7 False Positivity in Patients with CastrationResistant Prostate Cancer. Diagnostics (Basel). 2020 Mar 11;10(3):151. doi: 10.3390/diagnostics10030151. PMID: 32168745; PMCID:
PMC7151149.
BIO-RAD, CELLSELECT SLIDES and DROPLET DIGITAL are trademarks of Bio-Rad Laboratories. All trademarks used herein are the property of their respective owner.
For Research Use Only. Not for use in diagnostics procedures.

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