Molecular Imaging of Metastatic Lymph Nodes in Breast Cancer

Institution: University of California, San Francisco
Investigator(s): Ella  Jones , Ph.D. -
Award Cycle: 2007 (Cycle 13) Grant #: 13IB-0061 Award: $149,600
Award Type: IDEA
Research Priorities
Imaging, Biomarkers, & Molecular Pathology>Biomarkers and novel screening approaches: unmasking the hidden signs



Initial Award Abstract (2007)
Accurate staging of lymph nodes is a critical component in determining whether the tumor is likely to have metastasized, which dictates the therapeutic options for cancer patients. Currently, the most common practice of assessing lymph nodes is excision (lymphadenectomy) followed by microscopic (histological) evaluation to detect cancer cells. However, this is an invasive procedure that causes tissue damage and leads to lymphedema. Sentinel node biopsy is an improvement, such that many patients can be spared from the need for more extensive lymph node staging.

Some current approaches being explored to improve lymph node imaging include: a) lymphography to evaluate the internal nodal architecture; b) cross-sectional imaging using CT and MRI to assess the primary tumor and lymph nodes along the drainage path, and c) optical fluorescence imaging. Unfortunately none of these techniques can accurately detect malignancy. Recently, PET/CT imaging showed promise, but false-positives can occur due to non-cancer inflammation and infections. Therefore, a non-invasive imaging technique is needed to not only to visualize lymph nodes, but also to characterize the metastatic status of tumor cells that might be present.

In this proposal, we plan to develop a novel molecular imaging probe for assessing the metastatic status of lymph nodes in breast cancer. Cathepsin B, a lysosomal cysteine protease, is a known biomarker responsible for cancer invasion and metastasis. It is highly expressed in aggressive human breast cancer cells. Our goal is to exploit the Cathepsin B “molecular signature” and use these properties to determine the degree of tumor cell infiltration into the lymphatic system. Our strategy is to use a “dendrimer”-based probe with fluorogenic properties (i.e., detected by near-infrared light) that contains peptides that are cleaved by Cathepsin B. We plan to test this approach in mouse models of breast cancer and use newly-developed optical detection instruments to differentiate tumor vs. normal lymph nodes. The dendrimer- Cathepsin B probes should remain optically “silent” until cleaved by Cathepsin B at sites of tumor cell infiltration. My collaborator, Francis Szoka at UCSF, is a leader in the development and application of dendrimers (uniform populations of repeatedly branched, synthetic molecules, like tiny snowflakes) in the field of nanotechnology.

This project aims to develop a non-invasive imaging probe to characterize lymph nodes and breast cancer metastases at molecular level. This molecular imaging probe not only will offer a non-invasive method to visualize lymph nodes, it will potentially provide quantifiable information of tumor invasion. Because the procedure will be performed without surgical intervention, common side effects, such as lymphedema, can be avoided.


Final Report (2010)

Background: The goal of our project was to develop a novel “nanotechnology” agent that would allow external optical detection of tumor cells in the lymph nodes of patients as a prognostic biomarker of metastatic potential. Cathepsin B is a digestive protease that exists in the lysosome in the cell. In malignant tumors, however, Cathepsin B is produced errantly in large quantities onto the cell surface. The overexpression of Cathepsin B facilitates the degradation of the cell basement membranes and allow cancer cells to spread (metastasis) into the lymphatic system. To investigate Cathespin B in breast cancer metastatic lymph nodes, a molecular probe specific to Cathepsin B has been developed.

Methods: A multi-functional polyacid dendrimer (i.e., repeatedly branched, roughly spherical large molecules) was used as a backbone to incorporate multiple copies of a fluorescent Cathepsin B selective substrate. To accomplish this, first a multivalent dendrimer platform was built using a polyacid denrimer (MW = 4100 Da, 36 surface acid group) that was activated by carbodiimidazole. Next, the dendrimer surface was subsequently linked with bifunctional methyletherpolyethylene glycol (mPEG) chains and coupled with a fluorescent Cathespin B specific peptide substrate (NH2-GK(5-FAM)QVSGFRFGC(DACM)G-CONH2) via standard conjugation chemistry. Under normal conditions (i.e., in the absence of Cathepsin B), the quencher (DACM) extinguishes the fluorescence of 5-FAM, making the probe optically silent. However, upon digestion by Cathepsin B, the quencher is removed, and the “molecular beacon” is turned on. The detection of this fluorescence indicates the presence of Cathepsin B.

The synthesized probe was fully characterized by gel permeation chromatography (GPC) and standard fluorescence assays. Further evaluation was conducted using purified Cathepsin B, and we used breast tumor cells that were known to overexpress Cathespin B.

Results: The polyacid dendrimer probe with multi-functionality for Cathespin B targeting and reporting has been successfully synthesized and characterized. In vitro evaluation using purifed Cathepsin B shows a complete digestion at 37C after 24 h. Tumor cell lines MCF-7 and DU4475 with high expressions of surface-bound Cathepsin B also demonstrate an effective activation after 24 h of incubation at 37C.

Conclusions: All synthetic challenges to solve the probe chemistry and in vitro evaluations have been overcome. The preliminary in vitro data collected here show great promises of the utility of this probe for non-invasive assessment of the breast cancer Cathepsin B biomarker. We hope to use this probe to study tumor spread to the lymphatic nodes near the breast as an aid to evaluate (i.e., a prognostic marker) the metastatic potential of breast cancer in the clinical setting.




Symposium Abstract (2010)

Background: Metastatic breast cancer primary route of proliferation is through the lymphatic system. Accurate assessment of lymph nodes is critical for staging the disease progression and determination of therapeutic options for patients. Lymphadenectomy is the current clinical method for lymph nodes assessment, which involves microscopical examination of surgically removed lymph nodes. This painful and invasive procedure causes nodal destruction and swelling and is not always analytically reliable. Molecular imaging affords non-invasive options for visualizing lymph nodes. However, available imaging techniques cannot accurately ascertain the malignancy of the infected lymph nodes, because they show only anatomical structures without revealing the bioactivities associated with disease progression. Cathepsin-B (Cat-B), a lysosomal protease, is an established biomarker for metastatic breast cancer and is implicated in the pathway for invasion of cancerous cells into the lymph nodes through the destruction of the basement membranes.

Method: We have developed an fluorescence imaging probe with the potential to highlight diseased lymph nodes by being selectively turned on in the presence of Cat-B. The developed molecular probe is consisted of three components:
(1) a dendritic polyester platform that allows for tumor homing,
(2) Cat-B specific peptidic substrates at the periphery,
(3) molecular weight modifier mPEG surface groups.
Natively, the probe is in the off state, because the fluorescence quencher moiety is placed adjacent to the fluorescence dye on the peptidic substrate. Fluorescence is activated when Cat-B proteolytically removes the fluorescence quencher moieties from the probe. In vivo, this ‘light switch’ design will highlight the bioactivity of Cat-B, affording a viable method for non-invasive assessment of lymph nodes.

Results: The synthesized peptidic reporter NH2-GK(5-FAM)QVSGFRFGC(DACM)G-CONH2 have been assayed with Cat-B, and shown to have comparable specificity with known Cat-B substrates (Table 1). We have further demonstrated that the dendritic probe is efficiently activated by breast cancer cell lines, MCF-7 and DU-4475, that overexpressed Cathepsin-Bin in vitro studies.

Conclusion: The demonstrated Cat-B specificity of the peptidic reporter warrants further in vivo investigations of this probe. The in vitro results has highlight the promising utility of this probe in non-invasive assessment of metastatic breast cancer in the lymph nodes.

Table 1: Proteolysis Data of Cathepsin-B Substrates

Cathepsin B Substrates

Km

(mM)

kcat

(s-1)

kcat / Km

(M-1•s-1)

Cbz-FR-AMC  (@ pH 6.0)

0.6

33.8

54.6

NH2-GK(5-FAM)QVSGFRFGC(DACM)G-CONH2  (@ pH 5.5)

1.0

45.5

47.8

NH2-GK(5-FAM)QVSGFRFGC(DACM)G-CONH2  (@ pH 7.4)

2.8

40.8

14.6