Nanotherapy for Breast Cancer Targeting Tumor Macrophages

Institution: The Burnham Institute for Medical Research
Investigator(s): Gaurav  Sharma , Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14FB-0107 Award: $90,000
Award Type: Postdoctoral Fellowship
Research Priorities
Innovative Treatments>Immune therapy: mobilizing the body's defenses



Initial Award Abstract (2008)

Macrophages are versatile, plastic cells that are a key component of the body’s immune system. They have a variety of biological roles which include antigen “presentation”, a crucial role in initiating an immune response, scavenging debris, tissue remodeling and killing target cells such as bacteria etc. Macrophages are often prominent in tumor tissues, comprising up to 80% of the cell mass in breast carcinoma. Evidence currently available suggests that these tumor associated macrophages (TAMs) are reprogrammed by cancer cells and have little cytotoxicity for tumor cells. In fact, TAMs actually promote tumor cell proliferation and metastasis by secreting a wide range of chemicals. Due to their high concentration in breast tumor tissues, TAMs provide an ideal target for anti-breast cancer therapies, but selectively delivering drugs to kill TAMs with minimal side-effects is a major hurdle.

The goal of this project is to work at the interface of nanotechnology and medicine to design “smart” nanoparticles that will selectively target and kill TAMs, thereby suppressing tumor progression and metastasis. Recent advances in nanoparticle design, when properly integrated with the evolving knowledge of tumor microenvironment biology, can provide an ideal platform to overcome the challenges associated with conventional breast cancer therapies. Recently it was shown for the first time that legumain, a member of the endopeptidase family is highly overexpressed by TAMs in murine and human breast tumor tissues and hence provides an ideal starting point for breast tumor targeting. First, we will use a novel strategy to change the shape of nanoparticles that will endow them with the “stealth” to remain in circulation for longer times by evading the body’s reticuloendothelial system. This approach should increase the ability of TAM-directed nanoparticle to “home” in on target tumor sites. Next, to validate TAM targeting, we will use well defined macrophage cell lines and TAMs isolated from mouse breast tumor tissues. The goal is to identify specific TAM molecular “biomarkers” that can be targeted. Finally, the “stealth” nanoparticles will be made functional with targeting moieties, such as antibodies to legumain or other biomarkers identified in the course of these studies.

The proposed study has two innovative elements: (1) Instead of directly targeting and killing cancer cells, an approach that has met with limited success so far because of inefficient targeting and risk of side-effects due to high toxicity of anti-cancer drugs, we seek to alter the tumor microenvironment by targeting and killing TAMs that support angiogenesis and metastasis; and (2) to make the drug carrying nanoparticles more potent for anti-cancer therapy I propose to change their morphology to endow them with the “stealth” and give them an additional level of selectivity for tumor sites.




Final Report (2010)

Tumor associated macrophages (TAMs) are a prominent tissue in breast tumors comprising up to 80% of the cell mass in breast carcinoma. TAMs have been known to promote tumor cell proliferation, angiogenesis and metastasis by secreting a wide range of growth and pro-angiogenic factors. Therefore, targeting TAMs can be a validated cancer therapy and may complement more conventional breast cancer treatment regimens.

In this project I worked at the interface of nanotechnology and biology and successfully developed a "two pronged" strategy to target and deliver drugs to TAMs for breast cancer therapy. In the first step I developed a nanoparticulate formulation that encapsulates a drug which kills macrophages and have decorated these nanoparticles with a molecule that can bind to TAMs. In the second step I used a novel technique to change nanoparticle shape to further boost their internalization by macrophages and hence drug-delivery to TAMs.

The therapeutic nanoparticles that I developed are fabricated from polylactic-co-glycolic acid (PLGA) polymer that is approved by the FDA for a variety of drug delivery applications. These PLGA nanoparticles encapsulate clodronate (a bisphosphonate "bone drug" originally used in the clinic for the treatment of osteoporosis) as an anti-macrophage drug. I then "decorated" these nanoparticles with a peptide known as Lyp-1 which has been shown to selectively target TAMs. Using mouse model of breast cancer I first showed that LyP-1-functionalized nanoparticles have a 4-fold increase in tumor targeting compared to control particles. The clodronate-loaded functionalized NPs when injected in breast tumor bearing mice slowed tumor growth by 33%.

To further boost drug-delivery to TAMs, I used engineering principles to change nanoparticle shape so as to identify a shape that can stimulate internalization by macrophages. Towards this end I fabricated a library of nanoparticle shapes and found that nanoparticles in the shape of oblate ellipsoids are internalized much faster compared to conventional spherical nanoparticles.

Overall, I have successfully developed a novel nanoparticulate formulation to selectively deliver clodronate to TAMs and have shown that these nanoparticles can abrogate TAMs and slow tumor growth. Since these nanoparticles are made from a FDA-approved polymer and encapsulate an FDA-approved drug they will have immediate clinical utility. Furthermore, I have also changed nanoparticle morphology to increase their internalization by macrophages.




Symposium Abstract (2010)

Gaurav Sharma (PI), Priya Karmali, Michael Ramirez, Hui Xie, Erkki Ruoslahti, Jeffrey Smith (mentor)

Macrophages are versatile, plastic cells that are a key component of the body’s immune system. They have a variety of biological roles which include initiating an immune response, scavenging debris, tissue remodeling and killing target cells such as bacteria etc. Macrophages are often prominent in tumor tissues, comprising up to 80% of the cell mass in breast carcinoma. Evidence currently available suggests that these tumor associated macrophages (TAMs) are reprogrammed by cancer cells and have little cytotoxicity for tumor cells. In fact, TAMs actually promote tumor cell proliferation and metastasis by secreting a wide range of chemicals. The pivotal role played by TAMs in tumor growth and metastasis combined with the limitations of conventional cancer therapies highlight the importance of investigating TAMs as a validated therapeutic target for cancer therapy.

The goal of my research project is to work at the interface of nanotechnology and medicine to develop “smart” nanoparticles that will selectively target and kill TAMs but not normal tissue or breast epithelial cells, thereby suppressing tumor progression and metastasis while limiting any side-effects of the anti-cancer therapy.  Towards this end, we have developed a novel anti-macrophage agent that can be selectively targeted to TAMs. This agent consists of a FDA approved drug (clodronate) encapsulated in nanoparticles made from PLGA, a FDA-approved biodegradable and biocompatible copolymer. Clodronate belongs to a class of bisphosphonate (BP) compounds that are used in the clinic to prevent or inhibit osteoporosis. Once delivered inside the cell cytoplasm, BPs reacts with nonhydrolyzable analogue of ATP resulting in apoptosis. Encapsulation of clodronate in a nano-particulate system therefore allows for an efficient delivery of the drug to macrophages as they rapidly internalize particles.

We have fabricated clodronate-loaded particles (clodNPs) in the size range 200 – 300 nm and with high drug encapsulation efficiency of 40 – 60%. We next showed that treatment with clodNPs killed mouse macrophages in a concentration dependent manner while there was no effect on cell viability on cells treated with empty NPs and drug only.  Non-macrophage cells used as control were not affected upon incubation with clodNPs. To test if these particles can be targeted to TAMs we functionalized them with LyP-1, a newly identified tumor targeting peptide. In particular the LyP-1 peptide recognizes a subpopulation of activated macrophages in tumors that express the receptor for this peptide. Our in-vivo results using BALB/c mice bearing 4T1 breast tumors show a preferential accumulation of LyP-1-functionalized NPs in tumors compared to control particles. Our preliminary tumor therapy experiments show a 40% reduction in tumor volume of mice treated with LyP-1-functionalized clodNPs compared to vehicles.

The potential impact of this study is extremely high; my studies will not only provide the proof-of-concept for targeting TAMs but can also lead to a new therapeutic candidate for treating tumors with minimal side-effects. My studies will also provide the framework for a highly promising approach for targeting macrophages in other diseases, where they play an important role, such as atherosclerosis, AIDS, and infectious diseases like leishmania.



Polymer particle shape independently influences binding and internalization by macrophages.
Periodical:Journal of Controlled Release
Index Medicus: J Control Release
Authors: Sharma G, Valenta DT, Altman Y, Harvey S, Xie H, Mitragotri S, Smith JW
Yr: 2010 Vol: 7 Nbr: Abs: Pg:116 "in press"