Antibody-based Targeting of Breast Cancer Stem Cells

Institution: University of California, Santa Barbara
Investigator(s): Claudia Gottstein, M.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15IB-0049 Award: $150,000
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2009)

Breast cancer is a very heterogeneous disease and different patient subgroups vary drastically in clinical outcomes. While the chances to achieve a complete remission are comparatively good in breast cancer, reoccurrence of the disease remains a major problem. A recently developed hypothesis suggests that cancer stem cells, a subpopulation of tumor cells with the ability to self-renew or differentiate into more mature cancer cells, are the major drivers of tumor propagation and metastasis, and potentially tumor recurrence. A cell surface protein marker, called CD44, has been used as a marker for breast cancer stem cells. While CD44 is not an ideal therapeutic target due to its wide expression on many cell types, it can be used to guide identification of other biomarkers co-expressed on CD44 positive breast cancer cells. However, a selection technique that identifies co-expressed markers on specific cell types has not been developed and is the focus of this research.

We will utilize a novel approach to detect breast cancer stem cell biomarkers using antibodies (Ab). Our initial work will be to screen large combinatorial phage (virus) libraries prepared from patient lymphocyte samples against archived breast tumor tissue samples. Our goal is to isolate Ab-derived protein sequences on the phage which bind to markers co-expressed with CD44 on breast cancer cells. We anticipate isolating a panel of Ab binding to breast cancer stem cells that we can produce in larger quantity using recombinant expression in bacteria (E. coli). We will test these Ab to: (1) further validate the “stem cell hypothesis” for breast cancer, (2) define stem cell “expression signatures” for stratification breast cancers for better targeted treatment therapies, and (3) use as a basis to make additional cancer stem cell specific monoclonal antibodies (mAb) for targeted therapies

Our project is true “high risk-high reward”, and we hope that the Ab generated from our approach will allow to further characterize cancer stem cells and serve as candidates for targeted therapies against breast cancer in patients.




Final Report (2012)

A recently postulated hypothesis states that a subpopulation of cells in a tumor with stem cell-like properties, termed "cancer stem cells" are the major drivers for tumor progression, metastasis and recurrence. These cells are not easily eliminated with conventional therapies, but rather become enriched through therapy. Evidence has now accumulated that cancer stem cells play an important role in breast cancer, but the originally used markers for these cells are insufficient. Antibodies which specifically recognize breast cancer stem cells, particularly those circulating in the blood, would be of tremendous value for diagnosis and treatment, and potentially, long term, for prevention of recurrence.

The goal of the project was to develop tools and methods to generate such antibodies. This has been achieved, although some of the specific aims had to be adjusted due to emerging knowledge and technologies. Some technical barriers had to be overcome that were related to optimizing procedures in order to maximize the amount and quality of material derived from the precious patient samples.

Within this project we have:
i) generated an immune antibody library from 41 breast cancer patients (mostly triple negative receptor status);
ii) developed a new concept in combinatorial design of antibody libraries suited for detection of rare markers or markers on rare cells;
iii) shown first proof-of-concept of this new design;
iv) isolated antibodies binding to breast cancer cells, which are now being further validated.

The work has been presented at two scientific meetings and one meeting for breast cancer patients, and is also scheduled for presentation at the prestigious Gordon conference. A manuscript is in preparation. Pending additional funding, we will follow-up this work by; i) further validating the new concept; ii) supplying other interested researchers in the field with aliquots of the library after full validation and publication; and iii) using the library to isolate antibodies for diagnostic and therapeutic applications, with the long term goal to prevent breast cancer recurrence.




Symposium Abstract (2010)

Iskender Teber1,2, Anne Wallace3, Lincoln Johnson2 and Claudia Gottstein1,2
1 California NanoSystems Institute, University of California Santa Barbara
2 Neuroscience Research Institute, University of California Santa Barbara
3 Department of Surgery, University of California San Diego

Introduction: A recently postulated hypothesis states that a subpopulation of cells in a tumor with stem cell-like properties, termed “tumor initiating cells” or “cancer stem cells” are the major drivers for tumor progression, metastasis and recurrence. These cells are not easily eliminated with conventional therapies, and therefore may be enriched after conventional therapy. If the hypothesis is true, treatment strategies would have to be reconsidered, and relapses of tumors after many years could be explained and potentially specifically treated. A cell surface protein marker, called CD44, has been used as a marker for breast cancer stem cells. While CD44 is not an ideal therapeutic target due to its wide expression on many cell types, it can be used to guide identification of other biomarkers co-expressed on CD44 positive breast cancer cells. The isolation of antibodies that recognize such co-expressed markers from breast cancer patients’ blood leukocytes is the focus of this research. Such antibodies would be invaluable tools to test the tumor stem cell hypothesis and specifically target breast cancer stem cells.

Methods: To derive recombinant antibody pools, blood was obtained from breast cancer patients and antibody producing blood cells were separated by density gradient centrifugation. Ribonucleic acid (RNA) from these cells was isolated and analyzed by microfluidic electrophoresis. The genes encoding for antibodies were amplified from the RNA. In pilot experiments for the selection of such antibodies on breast cancer tissues, control antibodies were cloned and expressed on the surface of a bacteriophage. Sections of human breast cancer tumors grown in mice were incubated with recombinant phage and labeled with fluorescent nanoparticles. Recombinant phage were recovered from the tissue via laser capture microdissection and the recovered antibody genes amplified via PCR. Human primary breast cancer tissues were stained for CD44 with fluorescent nanoparticles to visualize target cells for the selection.

Results: We have obtained high quality RNA (RNA integrity number >8) from human blood lymphocytes and have successfully amplified the DNA coding for antibody variable regions. In pilot experiments on sections of tumors grown in mice, we were able to visualize single tissue binding phage, recover the phage via laser capture microdissection and reamplify the DNA of the antibody coding region. This serves as a proof-of-principle for the proposed methodology. Immunofluorescence staining of primary human breast cancer material revealed positive epithelial staining for CD44 in 8/11 invasive ductal breast carcinomas. These clinical specimens will serve as target for the antibody selections.

Conclusions: We completed several proof-of-principle milestones towards delivering tools for the analysis and targeting of breast cancer stem cells. These tools will serve to test whether treatment strategies have to be redesigned to include the specific elimination of breast cancer stem cells, and also may enable specific targeting of these cells for prognostic or therapeutic applications.