Breast Cancer Studies in a 3-D Cell Culture System

Institution: The Burnham Institute for Medical Research
Investigator(s): Kristiina Vuori, M.D., Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0132 Award: $188,602
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)
Monolayer cell cultures have long served as mainstay models for studies of breast cancer biology studies. However, two-dimensional (2-D) culture conditions fail to replicate tumor architecture, and therefore cannot effectively model the “outside-to- inside” cellular gradients of oxygen and nutrients found in non-vascular regions of the tumor. Persistent metabolic stress provides a breeding ground for malignant progression and for the outgrowth of therapeutically refractory cells. 3-D cultures of breast cancer cells are potentially useful model systems for studies of the roles of metabolic stress in tumor growth and therapeutic responsiveness.

We plan to use 3-D breast cancer cell cultures to test the integrated hypotheses that spatially-localized hypoxia and nutrient deprivation significantly alters the cellular response to radiotherapy, and that metabolic stress in growing tumors can be exploited for the development of novel therapies that selectively eliminate oxygen and nutrient-starved breast tumor cells. During the project period, we will pursue the following specific aims: 1) to develop and characterize a panel of 3-D culture systems for detailed studies of the impact of hypoxia and nutrient starvation on breast cancer cell growth and viability. 2) to determine whether the disruption of cellular bioenergetics by the mTOR inhibitor, rapamycin, selectively enhances the killing of hypoxic breast cancer cells by low-dose radiation.

Our initial efforts will focus on the optimization of protocols for the preparation of 3-D cultures of established human breast cancer cell lines. We will use these 3-D cultures in conjunction with molecular biochemical, and imaging technologies to comparatively examine the survival, growth, and death pathways that govern the behaviors of cells residing in the outer versus inner zones of the tumor.

The outcomes of this project will provide the breast cancer research community with improved model systems for studies of the signaling events and environmental interactions that contribute to the progression of breast cancer, and variations in the clinical response to cancer therapy. Studies will also address fundamental questions related to the mechanisms of tumor cell killing by conventional radio- and chemo-therapy. Finally, we believe that stress-signaling pathways represent fertile ground for cancer drug discovery. We propose that studies with 3-D model systems will ultimately lead to the discovery of novel drugs for breast cancer therapy.


Final Report (2007)
Introduction: Solid tumors, such as breast cancer, contain groups of tumor cells at varying distances from the blood vessels that supply oxygen and nutrients. Therefore, some cells within the tumor are subjected to the stress that occurs under low oxygen and nutrient conditions. It is believed that these “stressed” cells are the most resistant to chemo- and radio-therapies. Thus, a pressing need exists for improved model systems, which allow scientists to probe the inner workings of breast cancer cells, such as variations in the supplies of oxygen and nutrients needed for tumor growth, and to develop therapeutic strategies targeting this highly resistant group of tumor cells.

Topic Addressed: Currently, nearly all of the laboratory research performed with human cancer cells employs technologies involving cells cultured as single-cell layers on plastic dishes. We hope to be able to change this paradigm by demonstrating the relevance and utility of 3-D culture systems for detailed dissections of tumor biology, and to prove that these systems offer unparalleled opportunities for the development of novel agents for cancer therapy. Thus, our goal is to develop a 3-dimensional culture system using human cells to better mimic the complex nature of a real breast tumor, including the cell-cell interactions, nutrient and oxygen gradients, and 3-dimensional architecture of a breast tumor. This model will then be used to answer basic biology questions about how cancer cells die following combination therapy with rapamycin and low dose irradiation (an exciting new therapy in phase II clinical trials for multiple kinds of cancers). Future studies are aimed at understanding the mechanism of cell death in 3-dimensional models treated with rapamycin and radiotherapy, and to specifically impair this mode of cell death to find new targets to promote tumor cell killing.

Accomplished Under Specific Aims:
In Aim 1, we evaluated the utility of different human cell types under 3-D culture conditions as a model for the solid tumor tissues found in human breast cancer. Our findings indicate that several human breast cancer cell lines are amenable to 3-D culture conditions in the presence of fibroblasts or Matrigel, but that the T47D cell line can form spheroids without additional factors. We then used these 3-D cultures to develop the technology to image the live and dead cells within the intact spheroid under confocal microscopy, and to separate out hypoxic subpopulations of cells from within the 3-D structure with the Hypoxyprobe stain during FACS analysis. Thus, the successful completion of studies in Aim 1 now provides the tools and expertise to us to tackle Aim 2 of the application, and beyond.

In Aim 2, we found that the T47D breast cancer cells exhibit differential sensitivity to rapamycin and fractionated radiotherapy when cultured as a monolayer or as a 3-D spheroid. In 3-D culture, rapamycin acts as a radiosensitizing agent and causes significantly more cell death, as assessed by a spheroid re-growth assay, than either treatment alone. Neither PARP cleavage, nor LC3 cleavage were specifically detected in the T47D spheroids treated with irradiation and rapamycin, suggesting that the induction of apoptosis or autophagy are not playing major roles in the cell death caused by the combination treatment. Thus, our original biological hypothesis that was to be tested in this Aim turns out to be correct, while future work is needed to uncover the molecular mechanisms of cell death taking place in “stressed” cancer cells upon combined rapamycin and radiotherapy treatment.

Future direction and impact: The outcomes of this project will be improved model systems for studies of the signaling events and environmental interactions that contribute to the pathobiology of breast cancer, and the clinical response to cancer therapy. Studies will also address fundamental questions related to the mechanisms of tumor cell killing by conventional radio- and chemo-therapy. Finally, we believe that stress-signaling pathways represent fertile ground for cancer drug discovery. We propose that studies with 3-D model systems will ultimately lead to the discovery of novel drugs for breast cancer therapy.


Symposium Abstract (2007)
How a particular breast cancer will respond to a given therapy is influenced by a number of factors. One such important factor is the tumor microenvironment. Breast tumors exist in a complex environment where cells are growing, dividing, and invading other tissues. As a result of these changes, the cancer cells are subjected to stresses, such as limiting amounts of oxygen and nutrients. These so-called metabolic stresses affect how the cells communicate with each other and how they respond to signals from the environment.

The growth of human cancer cells as spheroids better recapitulate the metabolic stresses seen in living tissues than traditional cell culture in a 2-D monolayer. One of our goals was to establish a 3-D system using human breast cancer cells. We have grown the human breast cancer cell line, T47D, as multicellular spheroids and used this as a model system to address how breast cancer cells, exhibiting cell-cell interactions and under metabolic stress, respond to radio- and chemo-therapies.

We have examined how T47D spheroids respond to a combination therapy of low-dose radiation and rapamycin treatment (currently in clinical trials for several cancer types). The combination of rapamycin and radiation caused a 15-day delay in spheroid growth following the cessation of treatment, compared to 0 days or 3 days following sole radiation or rapamycin treatment, respectively. This suggested that the combination treatment caused significantly more cell death than either treatment alone. Our subsequent experiments address the mechanism by which the cells die, and whether this cell death occurs in the nutrient and oxygen-stressed cells in the center of the spheroids. The outcome of this research provides further insight into how cells within a tumor respond to radio- or chemo-therapy, which may lead to better predictions of clinical response.