Circulating tumour cells are cancer cells, which are separated from the primary tumour and travel through the body in the bloodstream. The number of circulating tumour cells in the blood (CTCs – circulating tumour cells) gives an indication of the stage of cancer and permits (limited) conclusions to be made on its further progress. Moreover, the success of a cancer treatment can be checked. However, the concentration of CTCs in the blood is so low (one to ten cells per millilitre of blood) that an investigation is difficult and time consuming and is not part of standard analysis. The detection of CTCs also requires great expertise from the leading experimenter. The only commercially available system for metastatic breast cancer, metastatic colon cancer and metastatic prostate cancer approved by the U.S. Food and Drug Administration – all of these mentioned already being advanced cancers – is Cell Search®. Here the tumour cells in a blood sample are marked by an antibody-antigen interaction involving magnetic beads and then sorted out by applying a magnetic field.
Separation on the basis of mechanical properties
Now physicists at the University of Augsburg have developed a system that can detect not only very small numbers of CTCs reliably and specifically, but could be applied quickly and inexpensively in every doctor’s office without much equipment. The “Non-Inertial Lift Induced Cell Sorting“, or NILICS, is based on a physical separation of the tumour cells from the other blood cells, due to their different mechanical characteristics. “The advantage of this system is that we are able to isolate the tumour cells label-free, i.e. without marking them (for instance with magnetic particles) from diluted whole blood. Signal amplification and enrichment of cells are also eliminated”, explains Prof. Thomas Franke, head of the working group Soft Matter/ Biological Physics at the Institute for Experimental Physics of the University of Augsburg.
Tumour cells are moved by the lift force
A few microlitres of blood are introduced into a very small, microfluidic channel with a diameter of between ten and 100 microns and transported using a certain pressure through the channel. Due to the small size of the channel laminar flow becomes the predominant form of motion, in which no turbulence occurs and all particles and cells flow in parallel. Tumour cells differ from other cells and other blood cells in their elasticity and malleability. “These differing properties are exploited in the system. “Rigid” cells will remain at exactly the position in which they are introduced, whereas elastic cells, by way of a so-called lift force, a force originated in this system, are repelled away from the wall”, explains Prof. Franke. The cells thus move away from the wall of the canal until they reach an equilibrium state due to the decreasing shear forces. After a certain distance the channel divides, in such a way that the cells are collected at various outlets. Using a microscope and a counting plate existing tumour cells can be counted.
Scheme of the experimental set-up: The sample is injected by “sample flow”, which is driven by a syringe pump, into the channel and is subjected to “sheath flow” in a focussed manner. At the end of the channel, the cell populations are separated into two receptacles. © University of Augsburg, Institute of Experimental Physics I
Applicable to each type of cancer
Scientist Thomas Geislinger from the research group under Prof. Franke used for the tests, among others, skin cancer cell line MV3 and the osteosarcoma cell line SAOS 2. “The method is from a theoretical point of view universally applicable to all cancer cell types. The only requirement is the difference in the physical properties, which are given for all currently known tumour cell lines “, explains Geislinger.
The significance of circulating tumour cells
Essentially, cells of all tumour types are able to circulate in the blood. Through this process cell metastasis occurs. In early cancers (primary disease) circulating tumour cells are found in about ten to 20 percent of all patients. Where there is a occurrence of metastases – depending on the method of examination – 50 to 90 percent of CTCs can be detected. In studies it has been shown that in cases of metastatic breast cancer with a count of ≥ 5 CTCs per 7.5 ml of blood, the survival period (overall survival) is significantly shorter than for patients with less than 5 CTCs per 7.5 ml of blood. Such a connection has been shown for other types of cancer as well, as Bao et al. recently published. In advanced cancer, the analysis of CTCs also allows conclusions about the responsiveness or non-responsiveness to chemotherapy or hormone therapy to be made.
Test for CTCs by the family doctor is something conceivable
On the basis of these findings, it might be useful at an early stage and on a regular basis to determine CTC levels. Mr. Geislinger explains: “A test that could be developed on the basis of the method presented would already be usable in the family doctor’s screening process, as no prior knowledge of the nature or severity of any disease would be necessary. A normal blood sample would be sufficient for a first test”. For the installation of such a system the appropriate infrastructure would have to be provided. The researchers envision a test system in the form of a single-use article: “The channels required could be inexpensively manufactured and be operated in a fully automated manner, which would favour its application locally. The fact that the cells do not need to be labelled would make handling easier than the method previously available”. Some companies have already expressed interest in the method.
Diabetes, high blood pressure, malaria: more to come
In earlier experiments NILICS was used to separate red blood cells from platelets. A separation of erythrocytes from leukocytes is also possible. “Other diseases in which the physical properties of the cells get changed, and thus our method might be able to be used, include for example diabetes, high blood pressure, malaria, sickle cell anaemia,” says Mr. Geislinger, expressing the potential of the method.
Further separation methods for CTCs
Other research groups are attempting the task of isolating circulating cancer cells. Thus, the working group Industrial Simulation working under project leader Prof. Thomas Schrefl, his scientific staff colleague Markus Gusenbauer (Dipl.Ing.) at the Fachhochschule St. Pölten in Austria and other various project partners have simulated separation and filtration methods for CTCs on a computer. The main principle is always a combination of size and affinity filtering at microfluidic scale. For example, one simulation using soft magnetic particles which are coated with an antibody in order to produce dynamic filter structures. In the magnetic field the soft magnetic particles form chains, thus producing a comb filter. The spacing between the chains can be adjusted by means of targeted control of the magnetic fields, and thereby adapted to the different tumour cell types. Two other similar approaches have also been simulated and are now being tested on a prototype. With these methods the appropriate antibodies need to be available for each cell type being filtered. This is as yet not the case. This difficulty is being bypassed by the Augsburg researchers in a clever way.