Back Jan 7th

There will be no posts over winter break, but I will start again 3 weeks from today, on January 7th.

Roundup: 3 Interesting CTC articles published in the last 30 Days

The circulating tumor cell literature is expanding rapidly, with over 30 publications in the last month alone! So I have decided to do a roundup of articles I found interesting and impactful about once per month. If there are other papers you found interesting, I encourage you to share them.

Clinical applications of CTC analysis

IvanovIvanov et al. Chip-Based Nanostructured Sensors Enable Accurate Identification and Classification of Circulating Tumor Cells in Prostate Cancer Patient Blood Samples. Analytical Chemistry 2012

This is a nice paper describing the development of an on-chip biosensor to electrochemically to measure PSA and detect TMPRSS2:ERG gene fusion in prostate cancer patients.

 CTC capture tech development

GascoyneGascoyne et al. Correlations between the dielectric properties and exterior morphology of cells revealed by dielectrophoretic field-flow fractionation. Electrophoresis 2012

Gascoyne et al. perform really extensive dielectric characterization of NCI-60 cancer cell lines from the Cancer Genome Project. They produced some nice correlatives between cancer cell’s dielectric response and their morphological properties.

 Proof-of-principle experiments

TormoenTormoen et al. Development of Coagulation Factor Probes for the Identification of Procoagulant Circulating Tumor Cells. Frontiers in Oncology 2012; 2:110. *Open Access

I’m cheating because this was published in September, but I think the results are really interesting. Tormoen et al. showed that under certain conditions, metastatic cancer cell lines can induce blood coagulation. There is increasing interest in CTC clusters or microemboli—where CTCs are attached to WBCs and other blood components—and this points to a potential mechanism of their formation.

CTC genetic heterogeneity, a window into the metastatic process

x-posted to Kirby Lab Student Blog

Keywords

BCa: Breast Cancer
CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Epithelial cell surface marker: A protein or receptor sticking out of the cell membrane of epithelial cells.
EMT: Epithelial-to-mesenchymal transition, where cells lose biomarkers associated with their organ of origin and become more stem cell-like.
SNR: Signal-to-noise ratio.

Tumor genetic heterogeneity has emerged as an effective biomarker of malignant processes1-4. However, limited access to tissue in solid tumors makes repeated sampling and tracking of tumor mutations infeasible. CTCs can serve as a “liquid biopsy”, allowing researchers to study genetic progression in real time. The paper I’m reviewing today, “Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines” by Powell et al., demonstrates the utility of single-CTC genetic profiling5. The article is Open Access and available on PLoS ONE.

Immunocapture-Based CTC isolation

The technology used in this study is called the MagSweeper, developed by the Jeffrey Lab at Stanford. Magnetic beads were coated with an antibody targeting epithelial cell surface markers. These antibody-coated  (i.e. immunomagnetic) beads were mixed into blood samples, resulting in cancer cells covered in beads, as shown in figure D. The blood samples were diluted with saline solution, and cancer cells were extracted using a magnetic source. Captured cells were washed while attached to the magnet, and then released when the applied field was removed, as shown in figure B. Cell gene expression and viability were shown to be unaffected by this capture process.

Powell_Magsweeperv2

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Evaluating CTC isolation device performance

Keywords

CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Leukocyte: White blood cell.
Erythrocyte: Red blood cell.

I’ve previously discussed how to sort CTCs, and the standards used to characterize device performance.  Today, I’ll explain what some of the most common evaluation metrics are, and place them in context of eventual clinical/industrial application.

What are common performance criteria?

Capture Efficiency is number of target cells captured divided by total number of target cells introduced into the cell isolation system. This metric can only be assessed using cancer cell lines spiked into solution at known concentrations, as the number of actual CTCs in a patient sample is always unknown.

Enrichment is closely related to capture efficiency. It is the factor increase of the target cells per unit volume, post cell-isolation/sorting. As in the case of capture efficiency, this can only be assessed using model CTCs at controlled concentrations.

Capture Purity is the number of target cells captured divided by the total number of nucleated cells captured by the device. As I’ve mentioned before, the major contaminating cell type is usually leukocytes. Nucleated blood cells are an issue for genetic analyses, where their DNA can swamp out the signal from CTCs. Non-nucleated blood cells (e.g. erythrocytes), do not carry any DNA, and are less of a concern.

Here’s simple example to understand how these criteria are calculated: presume you have a 1mL blood sample that contains 4 cancer cells and 1e9 leukocytes. After processing the blood in your device of choice, you have captured 2 cancer cells and 100 leukocytes. Then the performance stats would be: 5e6-fold enrichment, 50% capture efficiency and 2% capture purity.

Capture efficiency, enrichment, and capture purity are the values you will see reported most commonly in the literature. However, there are two more metrics that are gaining increasing attention as the field moves forward:

Cell Viability is percentage of target cells that are still alive after the cell sorting/isolation process. It can also be defined as percentage of cells alive after a certain number of days in cell culture.  The first definition can only be assessed using model CTCs, while the second can be calculated from actual patient samples. This is important for assays that require live cells to investigate how CTCs respond to different biomechanical and biochemical cues.

Release Efficiency is the percentage of captured target cells that can be successfully removed from a device in a viable state. The material and optical properties of CTC isolation devices are often suboptimal for traditional biological assays, requiring researchers to create new protocols for each new type of CTC isolation device. Releasing viable cells is gaining increasing focus as scientists and engineers try to streamline and standardize this technology for use in hospitals, drug development, and pharma companies.

Criteria are a baseline, not the end goal

Characterizing device performance is important for optimization, as well as for comparison to the industrial and academic state-of-the-art. However, the area of greatest impact is no longer optimizing every performance metric, but what you can do with CTCs once you’ve captured them. CTCs are a platform for a variety of tests, ranging from morphological analyses1 and molecular profiling2 to chemotherapeutic testing3. Depending on the test that will be applied to the CTCs, the performance criteria that are prioritized will be different. For example, if the end-goal is to enable sensitive genetics testing, you might build a more discriminating device and trade capture efficiency (sensitivity) for capture purity (specificity). If the goal is to culture CTCs ex-vivo for other types of assays, then cell viability and release efficiency become paramount.

CTC enumeration is the traditiaonal tool to inform patient prognosis. However, there is increasing work using CTC to infer information on actual tumor state. These analyses range from looking for novel gene fusion correlated with patient outcomes, to using patient CTCs to evaluate chemotherapeutic efficacy, to culturing CTCs outside the body.

CTC enumeration is the traditiaonal tool to inform patient prognosis. However, there is increasing work using CTCs to infer information about the primary tumor or metastasis. These analyses range from [L-R] looking for novel gene fusions correlated with patient outcomes, to using patient CTCs to evaluate chemotherapeutic efficacy, to culturing CTCs outside the body.


At the World CTC Summit, this was termed fit-to-purpose CTC isolation technology. While it would be ideal to have a “magic bullet” tech that has perfect performance metrics and is facile for every time of biological and pharmacological test, in reality most CTC isolation technologies will have to be designed, and selected, based on their end-goal application.

References

1. Marrinucci D., Bethel K., Lazar D., Fisher J., Huynh E., Clark P., Bruce R., Nieva J. & Kuhn P. (2010). Cytomorphology of circulating colorectal tumor cells:a small case series., Journal of oncology, PMID:

2. Stott S.L., Lee R.J., Nagrath S., Yu M., Miyamoto D.T., Ulkus L., Inserra E.J., Ulman M., Springer S. & Nakamura Z. & Isolation and characterization of circulating tumor cells from patients with localized and metastatic prostate cancer., Science translational medicine, PMID:

3. Kirby B.J., Jodari M., Loftus M.S., Gakhar G., Pratt E.D., Chanel-Vos C., Gleghorn J.P., Santana S.M., Liu H. & Smith J.P. & (2012). Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device., PloS one, PMID:

Cancer Cell Lines & CTCs: Benchmarking versus Application

x-posted at Kirby Lab Student Blog

Keywords

Cell Surface Marker: Some protein or receptor sticking out of the cell membrane. I explain this more in depth in my immunocapture post.
CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Microemboli: A small mass of cells or tissue inside the bloodstream.
Platelets: aka thrombocytes, important in the formation of blood clots.
Phenotype: Observable characteristics of a cell.
RBCs: Red blood cells, aka erythrocytes.
Senescence: When a cell hits the Hayflick limit and can no longer divide naturally.
WBCs: White blood cells, aka leukocytes.

Benchmarking is critical for assessing CTC isolation tech performance

In my “How to Sort CTCs”  series, I covered a variety of sorting methodologies used for patient prognosis. However, before clinical implementation, it is important characterize device performance with a series of standards. This is impossible to do with a patient blood sample, because there is an unknown number of CTCs floating around with other blood cells, which can be effected by the cancer treatment process (e.g. radiation patients often have anemia)1. Furthermore, this is all changing dynamically as a function of both time and treatment.

PatientvsCellLine

For this reason, engineers need an alternative system that can serve as a patient blood model, but it is repeatable and controllable. Immortalized cancer cell lines—derived from cancers of various organs—are commonly used for this purpose. A normal human cell can only divide a set number of times before it undergoes senescence; this is called the Hayflick limit. Immortalized cell lines have been genetically altered to surpass the Hayflick limit and continue dividing indefinitely.

This enables researchers to create standardized systems to benchmark their technology with. A known number of cancer cells can be spiked in varying ratios with different blood components, allowing for measurement of sensitivity and specificity of capture, along with other metrics. For this reason…

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How to Sort Circulating Tumor Cells Part IV: Electrokinetic Separation

x-posted at Kirby Lab Student Blog

Keywords

Anion:a negatively charged ion.
Basophil, Lymphocyte,
Neutrophil:
Different types of white blood cells.
Biofouling:
Fouling of surface with biological material.
Cation: A positively charged ion.
Conductivity
: Measure of a substance’s ability to conduct electricity.
Cytoplasm: Inner contents of the cell, which holds everything outside the nucleus.
CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Erythrocyte: Red blood cell.
PBMCs: Peripheral mononuclear blood cells, aka blood cells that have a nucleus (e.g. white blood cells).  Cartoon of the different kinds of white blood cells here.
MDA-MB-***: Human breast carcinoma  immortalized cell lines.
Phenotype: Observable characteristics of a cell.

This is the last major sorting technique in this series (for now), and I will be using the review paper I co-wrote with Charlie Huang as the framework for my description of this technique1. Charlie works extensively on electrokinetic manipulation of cells, and his half of the review paper lends itself well to explaining how electrokinetics can be used to sort CTCs.

Why use electrokinetic separation?

Most CTC sorting devices target some observed cancer cell phenotype that was determined from studying tumor tissue directly, or from using immortalized cancer cell lines. This means that active sorting techniques, like size-based selection and immunocapture, require some level of a priori knowledge about CTCs before you can engineer a device to capture them. Microscopic characterization is one CTC identification method that circumvents this problem, fixing (killing) the cells, and then using imaging in combination with rapid scanning to look at almost everything present in the blood sample. Electrokinetic separation of cancer cells is another, but enables live cell isolation without knowing its physical or biochemical properties beforehand.

What types of electrokinetic techniques are used?

There are two commonly used types of electrokinetic manipulation for mammalian cells, electrophoresis (EP) and dielectrophoresis (DEP). Electrophoresis involves applying a uniform electric field across a charged particle, causing it to polarize (i.e. free charge aligns with the electric field), inducing a net particle migration. However, if a uniform electric field is applied to an electrically neutral particle, the charges will polarize to form a dipole, but there is no actuation because the force on each anion is cancelled out by the force on its respective cation, and vice versa. To induce actuation, a non-uniform electric field must be applied (Dielectrophoresis), causing the charge on one side of the particle to feel the electrical force more strongly than the other, resulting in particle migration—as shown in the example below (thanks to the Kirby Lab DEP subgroup for the great schematic!).

Electrophoresis is good for moving charged particles around; however, the net charge from cell type-to-cell type is often not distinct enough to sort cells with high resolution. In contrast, dielectrophoresis is excellent for sorting cells because motion is dependent not on net charge, but on cell membrane and cytoplasm electrical properties as well as cell size, as dictated by this equation:

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Thanksgiving Post

No technical post today because of the Thanksgiving holiday. However, in the spirit of the celebration here is a fantastic story about a 15-year-old, self-taught engineer, from Sierra Leone who’s making big waves at MIT’s Media Lab.

“We have not too much electricity,” Doe says in the video. “The lights will come on once in a week and the rest of the month, dark. So, I made my own battery to power lights in people’s houses.”

Doe has scoured trash bins looking for spare parts to build batteries, and later generators and transmitters. He’s also started his own FM radio station, equipped with a self-made music mixer, giving everyone in the community the chance to tune in and get their fix of neighborhood news, according to Sengeh.

The two met at a national high school innovation challenge called Innovate Salone in Sierra Leone, where students are challenged to think about creative ways they can solve some of the most challenging issues within their communities. Prior to the camp, Sengeh says Doe hadn’t left a 10-mile radius of his home. Suddenly, he was becoming the youngest person in history to be invited to the “Visiting Practitioner’s Program,” presenting his technology to undergraduate students from Harvard College and MIT.

Read the rest of the story, and see the video, here.

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