Identification and Targeting of Micro-RNAs Involved in Breast Cancer Metastasis

Patient Tumor-Derived Orthotopic Breast Cancer Models in Mice with Spontaneous Metastases

Investigator:
Geoffrey Greene

Co-Investigators:
Huiping Liu
Michael Clarke
Ralph Weichselbaum

To explore effective strategies that target metastasis, including poly-metastasis versus oligometastasis, a reliable, more representative and predictive model of human metastatic disease is needed. The commonly used models to study metastases, including those involving established human cancer cell line explants in mouse tumor models, metastatic tumor models via bloodstream injections, or murine tumor models, do not fully recapitulate human disease.  To overcome these limitations, we have generated more representative human-in-mouse tumor models by implanting dissected patient tumors, or tumor cells derived from pleural effusions, directly into NOD/SCID mouse mammary fat pads. We established 8 orthotopic breast tumor xenograft models, two derived from ER+ breast cancer specimens, two from Her2+ specimens and four (M1-M4) from ER-PR-Her2- triple negative breast cancer specimens. M1-M4 developed spontaneous lung metastases.  In addition, we used lentiviral vectors to transduce breast cancer stem cells (BCSCs) with firefly luciferase-eGFP or –tdTomato (luc-GFP or luc-Tom), thereby establishing a noninvasive bioluminescence imaging approach to monitor the metastatic progression of BCSC-initiated tumors. These tagged, patient-derived tumors will be extremely useful for monitoring treatment response in these mouse models. Also, we are using these models for functional validation studies of candidate genes, microRNAs, and/or signaling pathways to determine their roles in polymetastasis versus oligometastasis.

Identification of miRNAs that Regulate Lung Metastases

Investigator:
Geoffrey Greene

Co-Investigators:
Huiping Liu
Michael Clarke
Ralph Weichselbaum

Using miRNA real-time PCR primers (Taqman assay probes from AB), we have begun to examine miRNA expression profiles in human breast cancer xenografts and their derivative models from lung metastases, which grow in the orthotopic mammary fat pads at a slower rate and in a less aggressive manner than the parental tumors. A short list of miRNAs that are significantly down regulated in parental tumor models has been identified and includes miR-30c.  We hypothesize that differentially expressed miRNAs play a regulatory role in lung metastases of human breast cancer models. Future plans include functional validation of these miRNAs in breast cancer cells line in vitro, primary tumor cells in vitro and human breast cancers in vivo. Experimental parameter readouts will include tumor growth and invasion as well as bioluminescent imaging of tumor growth and metastasis in vivo.  In addition, we use 3D cultures of primary breast cancer cells for validation studies.  These models will help define the importance and roles of miRNAs in breast cancer polymetastasis versus oligometastasis in the lung and other metastatic sites. In addition, the information gained can be linked directly back to the patient from which each tumor line was derived.

Development of Cancer Cell Targeted Nanoparticles for Imaging and Therapy

Investigator:
Geoffrey Greene

Co-Investigators:
Huiping Liu
Matthew Tirrel
Seungpyo Hong

In conjunction with our microRNA work, we are developing two different types of nanoparticle formulations for microRNA delivery. One involves dendron-based polymers. A second approach uses self-assembling micelles, formed electrostatically between negatively charged mi-RNA mimetics and peptide amphiphiles or positively charged peptides, Our preliminary work shows that both nanoparticle formulations can assemble with DNA nucleotide mimetics of microRNAs.

To specifically target tumor cells or cancer stem cells, we are using anti-CD47 mAb to generate an F(ab’)2 that can be coupled to different nanoparticle formulations. We are currently optimizing the coupling of anti-CD47 to Dendron polymer nanoparticles and electrostatically driven micelles. These particles will be then be tested for efficacy in cell-based studies.