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RESEARCH APPROACH AND EXPECTED OUTCOMES

Tissue from both the primary and the metastatic tumor have been cultured in humanized mice and will soon be passaged to a second generation of mice, called patient-derived xenografts (PDX). Growing the tissue in mice allows for the best simulation of growth in the body, and provides tissue for future studies.

In addition, we have genetic analyses of the original tumor, and tissue to employ for RNA sequencing and protein level determination in the original and metastatic tumors. This will allow us to characterize the original tissues, the xenografts, and eventually cells derived from the xenografts by analysis of a majority (possibly >60%) of gene products expressed. Thus, we will be able to tell which cells are similar to the original tumors, and study how they change during xenograft or in vitro culture.

Although cell lines have been derived from TNBC patients in the past, these are usually after neoadjuvant therapy (and thus changed by chemotherapy), and often not from the original tumors. Such detailed analysis of gene expression, while attempted in some studies, has not been consistently carrier out in original tumor, metastatic tumor, xenografts, and in vitro cultures. The result is that some of the classic TNBC cell lines may not be the best models for research; for instance, one such line was found to be TNBC, while the original tumor was not, and a second line was eventually found to be a melanoma line, obviating some of the relevance of the more than 200 studies performed with these cells over the years.

We hope to establish a well-documented set of xenograft mouse lines and in vitro cell lines that can reveal some of the secrets of this cancer without some of these complications. Though this resource may not be absolutely unique in any one sense, this is, to our knowledge, the first time anyone has attempted model development at all these levels with such a complete characterization using highly aggressive TNBC tumors, with an unusual genetic profile and without neoadjuvant therapy.

Once the xenografts and cell lines are derived, careful gene expression analyses by RNA sequencing, confirmation by immunofluorescence microscopy and proteomics, and coordinated genomic sequencing will allow us to understand the changes in genes (mutations), gene expression, gene amplification, and signaling that are associated with aspects of the cancer, and whether and how these change with culture conditions, whether the tumors are homogeneous or not, etc.

From these data, we can make specific hypotheses about which pathways directly underlie tumor phenotypes by studies in the cell culture and xenograft models. These results can be compared to databases of gene expression analyses that have been done elsewhere for TNBC and other cancers. Genes and pathways that may be in common to specific subtypes of TNBC and which are known to be related to cancer phenotype in our models can then be verified in other TNBC models, including cell culture and PDX mice.

Potential therapeutic targets identified may be tested in the PDX mice (KK and others) to determine efficacy and toxicological aspects of potential new therapies, or new combinations of old therapies. In this way we hope to turn the unusual KK resource into an aspect of a much larger research program and link to similar programs across the country to the benefit of TNBC patients worldwide. Further, we expect that what is learned will contribute to delineation of new therapeutic targets and pathways that can be used in other breast cancers as well.


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