Radiation-Induced Migration of Breast Cancer Cells

Institution: Stanford University
Investigator(s): Edward Graves, Ph.D. -
Award Cycle: 2013 (Cycle 19) Grant #: 19IB-0106 Award: $234,189
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2013)

Non-technical overview of the research topic and relevance to breast cancer:
Despite advances in early detection and treatment, breast cancer continues to be a major health problem in the United States. Radiation therapy has undergone tremendous advances over the past half century, and is now used to treat more than half of all breast cancer patients. It is also relied upon to control tumors in patients when complete surgical removal of the tumor and its infiltrating cells is not possible. However aggressive tumors, such as triple-negative breast cancers, continue to exhibit limited response to treatment relative to less aggressive cancers. We have recently observed that radiotherapy to a tumor or post-surgical tumor bed may stimulate tumor cells that were circulating in the blood at the time of treatment, to return and re-grow, through a mechanism involving stimulation of granulocyte macrophage colony stimulating factor (GM-CSF). This is of prime interest for breast cancer radiotherapy, because this phenomenon may explain the persistence of local recurrences following radiotherapy in a subset of breast cancer patients. Moreover, the implication of GM-CSF raises concerns over delivery of this agent (trade name Leukine) to stimulate white blood cell production in breast cancer patients during treatment. Understanding this phenomenon may then allow it to be stopped, as well as optimization of a patient’s therapeutic regimen based on the characteristics of their individual disease. The objective of this project is to understand how radiation stimulates breast cancer cell migration, how GM-CSF facilitates this process, and the significance of this phenomenon in human breast cancers.

The question(s) or central hypotheses of the research:
Our main hypothesis is that radiation of tumors and normal tissue stimulates expression of a protein factor that attracts circulating breast cancer cells. A secondary hypothesis of this work is that GM-CSF, the factor we have identified as responsible for this process, is a potential target for drug development that, when inhibited, may slow or stop the recurrence of tumors following radiotherapy.

The general methodology:
We will use a variety of mouse models with metastatic triple-negative breast cancer to characterize this phenomenon, and how different doses of radiation stimulate it. We will use a florescent imaging “label” on the tumor cells that allows us to monitor and count their migration before and after radiation. Model systems of different radiation doses, timing, and surgical interventions will be used to assess how this phenomenon may function in human breast cancer patients undergoing treatment. We will examine the role of GM-CSF in this process both in cells in a petri dish, in animal models, as well as in blood and tissue samples from breast cancer patients. After gaining an understanding of this process at the molecular level, we will test the ability of anti-GM-CSF antibodies to slow or stop this process and improve tumor response to radiation.

Innovative elements of the project:
The proposed project utilizes novel imaging methods, biological models, and preclinical radiotherapy techniques to evaluate a hypothesis that challenges traditional thinking in radiobiology. It is therefore innovative on a number of levels. The concept that tumors re-grow after radiation therapy in part because tumor cells that had entered the circulation and left the primary mass return and proliferate, is a departure from conventional radiobiology. This project will develop and apply a variety of novel techniques and models to answer its research questions. The most notable of these are the multiple mouse tumor models in which tumor cell migration can be monitored by imaging. We will not only elucidate a novel molecular mechanism through which radiation-induced tumor self-seeding operates and establish its clinical significance, but will also produce unique therapeutic approaches towards inhibiting this phenomenon and improving the cure of breast cancers by radiotherapy.




Final Report (2015)

The goal of this project was to evaluate the influence of radiotherapy on the migration of breast cancer cells. It is well known that tumor cells can travel from a primary tumor to a distant site and form metastatic secondary tumors. Recently, it has been shown that these cells can also return to the primary tumor site. We sought to understand whether these returning cells can facilitate tumor regrowth after radiotherapy, and whether radiation affects this trafficking.

Our California Breast Cancer Research Program IDEA award proposed two specific aims. The first, to study breast cancer “self-seeding” and the effects of radiotherapy on this phenomenon, was completed in project year 1. We characterized the effects of radiation on this process using donor-recipient murine tumor models including mouse and human tumors, and evaluated how these trafficking cells lead to tumor recurrence after irradiation. The second specific aim, determining the role of the radiation-inducible cytokine granulocyte macrophage colony stimulating factor (GM-CSF) on this process, was completed using preclinical models in project year 1 and clinical samples collected from human breast cancer patients in project year 2.

We have shown that GM-CSF is necessary to stimulate breast cancer cell migration when expressed after radiation, and that inhibition of this factor is sufficient to thwart radiation-induced tumor cell migration in cell culture and animal models. Clinically, we have examined blood collected before, during, and after radiation therapy of breast cancer patients for increased levels of GM-CSF, but have not been able to detect significant circulating levels of the molecule. At present, we are looking to perform a proteomic analysis of these samples to identify other factors that are stimulated by radiation and that may play a role in this process. We have published a high profile manuscript on this work in Cell Reports in June 2014, and submitted an NIH R01 to continue this work in September 2014. We are awaiting the notice of award for this grant, and look forward to further pursuing the intriguing findings we obtained through the support of this CBCRP IDEA award.

Publications:
Effects of radiation on metastasis and tumor cell migration




Conference Abstract (2016)

Effects of Radiation on Tumor Cell Migration and Metastasis

Marta Vilalta, Marjan Rafat, Jourdan Brune, Meghana Golla, Amato J. Giaccia, Edward E. Graves
Department of Radiation Oncology, Stanford University, Stanford, CA, USA

In our California Breast Cancer Research Program (CBCRP) funded project, we sought to evaluate whether radiotherapy of breast cancers may alter the migration of circulating breast cancer cells. Recently, it has been shown that the process of metastasis, in which tumor cells migrate from their parent tumor to distant sites to form secondary lesions, may function in reverse, resulting in the transit of cells from the circulation and/or secondary tumors back to the primary cancer. We hypothesized that this process of tumor reseeding may limit the efficacy of focal treatments such as radiotherapy, because untreated cells returning to the treated tumor could lead to tumor regrowth. Using in vitro migration assays, we observed that irradiation of breast cancer cells results in the production of the cytokine granulocyte macrophage colony stimulating factor (GM-CSF), which increases the migration of tumor cells. Using conformal animal irradiation techniques and bioluminescent imaging of labeled circulating tumor cells (CTCs), we then demonstrated in vivo that radiotherapy of orthotopic breast cancers attracts CTCs to the site of irradiation and results in tumor recurrence. Radiation-induced secretion of GM-CSF has been found to be a key driver of this process, and inhibition of this factor can counteract CTC migration and tumor regrowth. Furthermore, we have recently observed that irradiation of normal mammary tissues, muscle, and skin can attract CTCs, suggesting that radiation may similarly attract tumor cells to normal tissues and promote metastasis. These novel findings suggest that cancer radiobiology may be driven not solely by cell kill but also by cell migration, and encourage further investigation of these phenomena in other tumor types and in the clinical setting. We are currently evaluating the significance of these phenomena in human breast cancer patients, and developing therapies to inhibit this process so as to further improve the ability of radiation to cure breast cancer.