Identifying Metastatic Breast Cells from Peripheral Blood

Institution: Lawrence Livermore National Laboratory
Investigator(s): Kristen Kulp, Ph.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10IB-0077 Award: $203,223
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2004)
The early and accurate detection of metastatic breast disease would constitute a significant advance in the treatment of breast cancer. Currently, the presence of cancer in the regional lymph nodes remains the most valuable prognostic indicator for metastatic breast cancer; however, as many as 30% of patients considered to be node negative eventually develop recurrent disease. Because breast cancers have been shown to shed tumor cells into the circulation soon after they become invasive, it has been suggested that detection and genetic/protein analysis of circulating tumor cells (CTCs) in the blood would provide an avenue for a simple blood test that could define the risk of metastasis.

The hypothesis of this proposal is that metastatic breast tumor cells circulating in the peripheral blood of breast cancer patients can be positively identified by statistical analysis of molecule-specific images of individual cells. We propose to use "surface imaging mass spectrometry" to measure specific changes in cells that are characteristic of metastases. Metastatic progression results from altered genes, which produce altered protein products that can be measured as changes to the mass spectrum of the cell components. Two cultured mammary adenocarcinoma cell lines, MTLn3 and MTC, will be used to develop methods to identify and differentiate metastatic cells from non-metastatic cells. These cells were originally obtained from the same tumor, so they have a similar genetic background, but they differ in their ability to form metastases. Our aims are, first, to determine the spectral differences of MTLn3 and MTC cells and to develop statistical methods necessary to differentiate the cell mass spectral patterns. Secondly, we plan to develop methods to isolate and identify MTLn3 and MTC cells "spiked" into human blood samples. Centrifugation techniques, specifically designed to enrich for tumor cells, will be compared to immunomagnetic bead separation methods to determine the tumor cell recovery for each method. This aim will establish the limit of detection for epithelial cells per milliliter of sample blood for our method. These preliminary experiments will then be used as a foundation for animal studies that will give us a final "proof-of-principle" to move forward with human studies.

Currently, specific methods of detection are needed to monitor the spread of disease to neighboring tissues, lymph nodes or other parts of the body. We propose to develop a sensitive and reliable method to identify and differentiate metastatic cells circulating in the peripheral blood. Our method will combine the power of "proteomic" analysis with the ability of single cell detection and, if successful, could be used as the basis for a blood test for predicting metastasis of breast cancer.


Final Report (2006)
Introduction: Diagnosing and treating metastases is critical to the survival of breast cancer patients. Currently, the presence of cancer in the regional lymph nodes remains the most valuable predictive indicator for metastatic breast cancer; however, as many as 30% of patients considered to be node negative eventually develop recurrent disease. This makes it clear that better methods are needed to identify breast cancer metastases. Because breast cancers have been shown to shed tumor cells into the circulation soon after they become invasive, it has been proposed that detection of circulating tumor cells in the blood provides an index of the risk for developing malignancies. These cells may also help us detect recurrence of the disease. We are developing a novel mass spectrometry method to detect these circulating metastatic cells. Metastatic progression is caused by altered genes, which produces changes in protein expression and structure that can be detected as differences in the chemical mass spectrum of cellular proteins and small molecules.

Topics Addressed: Although several methods have been employed to analyze circulating tumor cells, none of the methods has the necessary sensitivity and specificity to positively correlate the presence of tumor cells in the blood with metastasis. We are using mass spectrometry to overcome these limitations by measuring unique mass spectra from individual cells. Our aims in this project are as follows:
Aim 1) Determine the differences in mass spectra of rat mammary adenocarcinoma cell lines MTLn3 and MTC and develop statistical methods necessary to differentiate the cell mass spectral patterns. These cells were originally obtained from the same tumor, but differ in their metastatic potential.
Aim 2) Develop methods to isolate and identify MTLn3 and MTC cells spiked into blood samples. Centrifugation techniques, specifically designed to enrich for tumor cells, will be compared to immunomagnetic bead separation methods to determine the epithelial cell recovery for each method. This aim will establish the limit of detection for epithelial cells per milliliter of blood for our method.

Progress toward specific aims: We showed that in our in vitro system using MTLn3 and MTC cells thatwe can differentiate metastatic cells from non-metastatic cells. These breast cancer cells were originally obtained from the same tumor, so they have a closely related genetic background, but they differ in their ability to form metastases. Based on statistical analysis of the spectra acquired from individual cells, we are able to separate these cell lines into 2 distinct visual groups. We have also shown that purifying the cells from the blood does not change the detectable mass spectra of the cells. Because tumor cells are circulating in very low numbers in blood, we are working on refining methods for isolating breast cells from blood. Our method of choice is a commercially-available immunomagnetic bead separation technique in which polystyrene beads are conjugated to a monoclonal antibody that is specific to human epithelial cells. We are able to isolate tumor cells from blood, crush them to expose their contents, and analyze them using ToF-SIMS to identify unique mass spectra of the individual cells.

Future Direction and Impact: In the future we hope to detect circulating cells in blood samples from women known to have metastatic breast cancer, and not find such cells in blood of healthy women. Using mass spectrometry, we will be able to classify circulating tumor cells according to their primary tumor origin and potential for malignancy. Our method will not only have the power of analyzing thousands of cellular components simultaneously with the advantage of single cell detection, but will have thousands of confirming spectra for any given cell. This technique could be used as the basis for a blood test for metastasis. Concern about metastases or recurrence of disease is common among breast cancer survivors. There is a need for objective tests that provide definitive answers. The methods described here could be used as a basis for less expensive, less invasive tests to indicate the presence of circulating metatastic breast cells.


Symposium Abstract (2005)
Diagnosing and treating metastases is critical to the survival of breast cancer patients. Currently, the presence of cancer in the regional lymph nodes remains the most valuable predictive indicator for metastatic breast cancer; however, as many as 30% of patients considered to be node negative eventually develop recurrent disease. This makes it clear that better methods are needed to identify breast cancer metastases. Because breast cancers have been shown to shed tumor cells into the circulation soon after they become invasive, it has been proposed that detection of circulating tumor cells in the blood provides an index of the risk for developing malignancies. These cells may also help us detect recurrence of the disease. We are developing a novel mass spectrometry method to detect these circulating metastatic cells. Metastatic progression is caused by altered genes, which produce changes in protein expression and structure that can be detected as differences in the chemical mass spectrum of cellular proteins and small molecules.

Although several methods have been employed to analyze circulating tumor cells, none of the methods has the necessary sensitivity and specificity to positively correlate the presence of tumor cells in the blood with metastasis. We are using mass spectrometry to overcome these limitations by measuring unique mass spectra from individual cells. We show that in our in vitro system, we can differentiate metastatic cells from non-metastatic cells. These breast cancer cells were originally obtained from the same tumor, so they have a closely related genetic background, but they differ in their ability to form metastases. Based on statistical analysis of the spectra acquired from individual cells, we are able to separate these cell lines into 2 distinct visual groups. Because tumor cells are circulating in very low numbers in blood, we are also refining methods for isolating breast cells from blood. Initial experiments show a centrifugation method that isolates cells based on their density works quite well. Another method, that isolates cells based on antigenic response to specific proteins on the cell surface, is being investigated. In the future we hope to detect circulating cells in blood samples from women known to have metastatic breast cancer, and not find such cells in blood of healthy women. Using mass spectrometry, we will be able to classify circulating tumor cells according to their primary tumor origin and potential for malignancy. Our method will not only have the power of analyzing thousands of cellular components simultaneously with the advantage of single cell detection, but will have thousands of confirming spectra for any given cell. This technique could be used as the basis for a blood test for metastasis.

Concern about metastases or recurrence of disease is common among breast cancer survivors. There is a need for objective tests that provide definitive answers. The methods described here could be used as a basis for less expensive, less invasive tests to indicate the presence of circulating metastatic breast cells.

(This work was performed under the auspices of the US DOE by LLNL under contract no. W-7405-Eng-48 and supported by NCI grants CA55861, CBCRP 10IB-0077 and LLNL-LDRD funding 04-ERD-104).


Symposium Abstract (2007)
The survival rate of women diagnosed with breast cancer is highly dependent on the timely diagnosis and treatment of metastases. Since breast tumors have been shown to shed cells from the primary tumor into the circulatory system, it is possible that detecting and characterizing these circulating tumor cells in peripheral blood will reveal whether there is a potential risk for future tumor development. However, detection methods must be improved in order to correlate the presence of circulating tumor cells in the blood with metastasis. The goal of our project is to combine epithelial cell isolation techniques with Time-of-flight Secondary Ion Mass Spectrometry (ToF-SIMS) to analyze and identify circulating breast cells that have the potential cause metastasis.

ToF-SIMS is an imaging mass spectrometry technique that can create chemical images of individual cells. These images contain substantial molecular information, which can be used to predict the cells’ metastatic potential. To fully utilize all of the chemical information that is present in the individual cell images, we use multivariate statistical methods, such as principal component analysis (PCA), to reduce large spectral data sets to a small number of manageable variables and graphically represent the similarities and differences among cells.

Using commercially available methods of cell isolation, such as magnetic bead technology and density gradient centrifugation, we are able to separate epithelial cells from peripheral human blood. The CELLection Kitä is a reliable method that allows us to retrieve tumor cells from whole blood by binding the cells to magnetic beads with antibodies that are specific to epithelial cells. Once bound to the beads, a magnetic chamber is used to isolate the tumor cells from the blood. The second isolation method combines the commercially available OncoQuickä method with flow cytometric techniques. This method separates cells based on size and uses cell sorting to further separate the epithelial cells from the background blood cells. The isolate epithelia cells are attached to silicon substrates, washed and dried using a specifically derived sample preparation protocol, and then individual cells are analyzed using ToF-SIMS. Isolation results show that the CELLectionä method gives us the highest recovery of epithelial cells, but both recovery methods seem to be consistent and reliable techniques. Neither isolation process affects the chemical signature of the cell as determined by ToF-SIMS analysis.

Developing a combined ToF-SIMS and multivariate statistical analysis approach will allow us to classify the isolated cells based on their metastatic potential. This research gives us the necessary tools to develop a method of sensitive and specific metastatic cell detection, which is the foundation for developing a cheaper, less invasive test for breast cancer metastases.

Single cancer cell imaging and cell extract analysis using time-of-flight secondary ion mass spectrometry (ToF-SIMS)
Periodical:American Association for Cancer Research
Index Medicus: AACR
Authors: Kulp KS, Wu KJ, Shattuck L, Wu EJ, Nelson JL, Mongomery JS, Felton JS, and Knize MG
Yr: 2005 Vol: Nbr: Abs: Pg:

Chemical and biological differentiation of three human breast cancer cell types using time-of-flight secondary ion mass spectrometry
Periodical:Analytical Chemistry
Index Medicus: Anal Chem
Authors: Kulp KS, Berman ES, Knize MG, Shattuck DL, Nelson EJ, Wu L, Montgomery JL, Felton JS, Wu K
Yr: 2006 Vol: 78 Nbr: 11 Abs: Pg:3651-8