Cancer Cell Lines & CTCs: Benchmarking versus Application

x-posted at Erica Pratt’s 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.

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…

How to Sort Circulating Tumor Cells Part IV: Electrokinetic Separation

x-posted at Erica Pratt’s Blog

Keywords

Anion:a negatively charged ion.asophil, 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:

How to Sort Circulating Tumor Cells Part III: Microscopic Characterization

x-posted at Erica Pratt’s Blog

Keywords

CTC: Circulating tumor cell. Read about what they are and why they’re important here.
Cell Fixation: Chemical preservation of a cell. Post-fixation, a cell is no longer alive.
Cell Staining: Using different markers to visualize cells, or components of cells.
Fluorophore: A fluorescent molecule that is excited at one wavelength of light (excitation), and emits light at another wavelength (emission).
Leukocyte: White blood cell (WBC).
Pathology Slide: A fixed section of unhealthy tissue that can be analyzed with various cell visualization markers.
Phenotype: Observable characteristics of a cell.
RBC: Red blood cell or erythrocyte
Systemic disease: Disease that has spread throughout the body.

Why use microscopic characterization to identify CTCs?

CTCs, which are shed from tumors into the vasculature, are considered to be key players in metastasis, and ultimately cancer patient death. Therefore, the goal of many CTC isolation systems is to separate these abnormal (cancer) cells from normal (blood) cells. Post-capture, analyses can be performed to analyze morphological differences between individual CTCs. Additionally, more researchers are investigating an underpinning assumption in CTC isolation: are the blood cells of a patient with systemic disease normal? One method used to answer that question is high-resolution, microscopic characterization of blood samples.

This technique is unique when compared to size– or immunocapture– based sorting I described previously. Microscopic identification of CTCs does not rely on physically separating them from native blood cells; instead, it uses imaging in combination with rapid scanning to look at almost all cells present in a blood sample. Many devices use microscopy to identify CTCs post-capture, but view other blood cells as contaminants to be identified so as not to confound results. Microscopic characterization aims to look at CTCs and other blood cells to further understand the pathology of the disease^1-6. This method requires extensive image processing, and cell categorization algorithms.

What platforms are used for microscopic CTC characterization?

Most techniques focused on blood cell and CTC characterization have an initial stage to remove RBCs and small WBCs, either through lysis or filtration. The remaining cells are fixed (killed), and analyzed for a number of distinguishing characteristics. I will divide techniques by the substrate used, either non-porous surfaces (i.e. glass slides), or polymer porous surfaces (i.e. microfilters).

Non-porous Surfaces, such as modified glass slides^1,2,3, are used to deposit and fix blood samples after minimal pre-processing (usually RBC lysis). Multiple slides can be produced from one 10mL blood sample³, enabling researchers and clinicians to perform multiple assays on one blood draw with slides left over for storage. The example to the left is an x-y plane image of stained pancreatic CTCs and a 3-D reconstruction using multiple x-y plane images at different depths in the sample (z).

Porous Surfaces use devices like microfilters^4,5,6 to eliminate RBCs and small leukocytes prior to cell fixation and analysis. Cells are captured at regular intervals along the filter, making it simple to create a registry system to store unique information about each isolated cell. Multiple filters are used for one blood draw⁵, enabling the same analyses and storage as in the non-porous surface case.

Both of these techniques allow for morphological analysis of different types of captured cells, while also producing images very similar to standard pathology slides, making them attractive for clinical implementation.

How to Sort Circulating Tumor Cells Part II: Immunocapture

x-posted at Erica Pratt’s blog

Keywords

Antibody: A Y-shaped protein that binds to unique cellular targets (i.e. antigens). Read more about antibody function here.
Aptamer: A peptide, RNA or DNA that binds to unique cellular targets and is analogous to antibodies. Aptamers are easier to produce synthetically and are generally more stable than antibodies binding to the same target. Read more about antibodies vs. aptamers here.
CTC:Circulating tumor cell. Read about what they are and why they’re important here.
Downregulation: Suppression of a cellular component, e.g. protein or receptor.
Erythrocyte: Red blood cell.
Leukocyte: White blood cell.

What is immunocapture?

Immunocapture is analogous micro-scale affinity chromatography based on some unique cell characteristic. For CTC capture, this is usually a cell surface marker (e.g. protein or receptor) that is sticking out of the cell membrane, as seen in my cartoon below. If you can manufacture an antibody or aptamer that is specific only for that cell surface marker, you can chemically link these antibodies/aptamers to device surfaces (a process commonly called immunochemistry) and start pulling CTCs out of blood samples.

How to Sort Circulating Tumor Cells Part I: Size

x-posted at Erica Pratt’s blog

Keywords

Leukocyte: White blood cell.
Erythrocyte: Red blood cell.
Thrombocyte: Platelets, important in the formation of blood clots.
Reynolds number: is the ratio of inertial to viscous fluid forces. In pressure-driven flow, this is the ratio of the pressure force applied (to actuate flow) as compared to the resistance of the fluid to deformation or shearing. You can read more about the derivation of the Reynolds number here.

Why sort CTCs based on size?

Many researchers have anecdotally observed that CTCs can be larger than other cells in the blood (e.g. leukocytes, erythrocytes, thrombocytes). A commonly quoted range for CTCs is 12-25 microns¹, which is bigger than 90-95% of the largest blood cell population, leukocytes. For this reason, size-based sorting is an attractive, label-free, isolation method if the ratio between CTC and blood cell size is large, and if CTC biochemical properties are not well understood.

What types of engineering techniques are used?

The three types of size-based sorting of cancer cells I’ll describe today (in order of frequency of use) are centrifugation, microfilters, and hydrodynamic sorting. Centrifugation is the current state of the art^2,3,4, while microfilters^1,5 and hydrodynamic sorting^6,7 are engineering technologies developed more recently.

Engineering Microfluidic Devices for Neural Culture

We recently communicated our efforts toward the development and implementation of a novel neural cell culture device with spatiotemporal solute delivery. Our work, in collaboration with Gary Banker and Cheng Fang at Oregon Health and Sciences University, details the construction and culture of nerve cells within a microfluidic device. We demonstrate that neurons seeded in our device grow predictably and survive for several days within the closed device. We additionally present an analytical framework to engineer solute interactions in nerve cell culture, which can be generalized to other device geometries.

This work is motivated by the need to screen and evaluate solutes for their effects on intracellular transport within the neuron. Several researchers have proposed that the cause of neurodegeneration is due to transport defects within individual nerve cells. Our platform can impose solutes on neurons at specific locations. These solutes can be toxins, meant to stimulate a transport defect within the cell, or a therapeutic agent, to protect against transport failures. Additionally, our device layout is both multiplexed and directional; these design aspects can lead to high inference studies, as a large number of cells can be probed on one device with minimal time spent determining cell polarity/orientation.

Our design approach of the neural cell culture system considers both the effects of geometry on the cells (via shear stress) as well as the solute distribution (via concentration profiles in space and time), in addition to other effects such as materials compatibility. Toward generalizing our approach to different types of cells and solutes, we simplify the output of the governing convection-diffusion equations to parametrized plots and algebraic relations so that designs may easily be evaluated by predicting device performance.

Link to PDF.

Why Study Circulating Tumor Cells?

x-posted at Erica Pratt’s blog

Keywords

Metastasis: The spreading of the initial (primary) tumor to another site in the body (secondary tumor).
Peripheral blood: Blood circulating through arteries, veins, capillaries, etc. Not what’s in your liver, lymphatic system, bone marrow, etc.
PSA: Prostate-specific antigen, a protein secreted by the prostate, and elevated in cancer. Measuring PSA levels is a standard clinical tool when assessing prostate cancer, read about the test here.

What is a circulating tumor cell?

Cancer is public health problem receiving increasing scrutiny, resulting in over 500,000 deaths in 2011 alone¹. However, most cancer deaths are not from intial tumor formation, but from metastases². The diagram to the left gives a simple picture of a metastatic process. Within the primary tumor, a subpopulation of cells are able to break away from the tumor and worm their way into the lymphatic system, or blood circulation. Cells that enter the bloodstream we call circulating tumor cells (CTCs), and a subset of these cells are believed to be capable of secondary tumor (metastasis) formation.

CTCs’ existence has been known since the 1800s, when physician Thomas Ashworth observed abnormal, cancerous-looking, cells in the peripheral blood of a metastatic cancer patient³. However, it’s only in the last ten years or so that devices have been able to isolate CTCs from blood efficiently. Why has it taken so long? Because CTCs are extraordinarily rare, in one milliliter of blood there may be single or tens of CTCs as compared to over one billion blood cells! In a future post, I will discuss the engineering strategies employed to overcome this problem.