Leukocyte Recruitment, Angiogenesis and Breast Cancer

Institution: La Jolla Institute for Molecular Medicine
Investigator(s): Pragada Sriramarao, Ph.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4JB-0164 Award: $304,797
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1998)
In spite of advances in our current understanding and treatment of breast cancer, approximately 30% of breast cancer patients develop advanced, usually incurable metastatic disease. Unlike solid tumors, the metastatic micro-tumors originating from the breast are resistant to various therapies. Our interest is to study the immunopathology of metastatic breast cancer with the objective of enhancing or modulating the patient's own immune response to combat the disease. Recent studies with immune regulatory factors, such as cytokines and chemokines, have demonstrated their potential to eradicate experimentally induced tumors in animals. The inhibition of tumor growth in these studies was associated with enhanced infiltration of tumor killing lymphocytes. Thus, a better understanding of how immune cells interact with tumor microvessels in breast carcinomas will facilitate the development of strategies to target breast cancer.

Our project will investigate the properties of metastatic micro-tumors, which stimulate local formation of blood vessels through a process called angiogenesis. Our experimental technique is called 'intravital microscopy', which can directly visualize the interaction of immune cells with the tumor microcirculation. For this, we implant a transparent window chamber in the dorsum of a female mouse. Tiny tumor spheroids of human breast carcinoma (e.g., MCF-7 cells) are introduced into the chamber, and this stimulates production of a localized network of blood vessels within 10-14 days. This now becomes our model system where we can introduce immune cells, inhibitory molecules to prevent their attachment to the vessels, and immune-stimulatory compounds, such as cytokines. Our first aim is to test whether leukocyte/T-cell attachment is prevented by (i) inducing the expression of endothelial cell adhesion molecules by stimulation with inflammatory cytokines and/or (ii) blockade of angiogenic factors released by tumors. Next, we will inject mouse T-cells (and CD8+ cells) that have the ability to kill tumor cells, and determine whether vessel stimulation with specific cytokines and chemokines will help in T-cell recruitment into the tumor vessels. Finally, one key step in immune recruitment is the ability of leukocytes to leave the tumor vessels and move into the space where the tumors reside. Using our model of tumor angiogenesis, it will be possible to expose the cytokine-activated tumor vessels to various compounds and examine if the leukocytes or T-cells move from the blood into sites of tumor growth.

In cancer patients, immune cells continuously circulate in close proximity to cancer cells that spread and grow in different parts of the body. However, the ability of immune cells to stem the growth of these tumors appears to be compromised. It is hoped that new information will help in developing strategies to improve immune surveillance as a mechanism to effectively treat metastatic breast cancer.

Final Report (2001)
Successful eradication of metastatic microtumors is dependent upon effective recruitment of immune white blood cells to sites of tumor growth. Studies funded by BCRP have for the first time demonstrated that recruitment of immune cells into the tumor stroma of breast carcinomas is severely restricted/compromised in the angiogenic blood vessels draining into tumors, but not in the surrounding nommal vessels. These results suggest that breast carcinomas (microtumors) have developed a mechanism of evasion of immune surveillance by the host immune cells. Several published studies have demonstrated the potential of cytokines and chemokines in the recruitment of immune effectors and the treatment of experimental tumors in animals. However, the mechanisms by which immune effector cells interact with tumor microvessels/angiogenic blood vessels are not well understood.

As part of this funded proposal we examined whether the microenvironment of metastatic breast carcinomas could be altered by combination treatment with cytokines and chemokines. We investigated if such a combination treatment would induce effective interactions between circulating immune cells and angiogenic blood vessels resulting in the successful recruitment of circulating leukocytes to sites of tumor growth in vivo. We examined the ability of circulating leukocytes to interact in newly formed or angiogenic blood vessels within microtumors (spheroids) of MCF-7 human breast carcinomas implanted in nude mice by intravital microscopy. Our studies revealed that, in absence of local stimulation of blood vessels with cytokines or other inflammatory mediators, immune cells failed to interact with endothelial cells lining the tumor microvessels. However, local treatment of the breast carcinomas with cytokines such as TNF-beta (LT) or IL-4 resulted in augmentation of immune cell interactions in angiogenic blood vessels in a time dependent manner. Leukocyte adhesion was dependent upon the up regulation of vascular P- and E-selectin, while other adhesion molecules failed to appreciably participate in these adhesive interactions. Combination treatment with chemo-attractant resulted in the effective recruitment of circulating blood cells into the core of the tumor stroma. Finally, we have demonstrated that the angiogenic factors that promote tumor growth actually inhibit the expression of adhesion molecules by tumor microvessels, and thus inhibit recruitment of immune cells. Blockade of anti-angiogenic factor release by the breast carcinomas using function-blocking antibodies resulted in the effective rolling and adhesion of circulating leukocytes in tumor microvessels. Our studies suggest that angiogenic growth factors may play an additional role in blocking the recruitment of immune cells to sites of tumor growth.

We anticipate that these novel observations will help in developing strategies for exploitation of immune surveillance as a mechanism to eradicate metastatic breast cancer and eventually have an impact on reducing human/economic costs of breast cancer in California.