Title: Investigations of intra-tumor heterogeneity in breast cancer
Description: Individual breast tumors are composed of a variety of cell types with different features and behaviors. This "intra-tumor heterogeneity" drives tumor progression, therapeutic resistance, and cancer recurrence. CD44 + CD24 - (but not CD44 +/- CD24 + ) breast cancer cells were previously identified as "cancer stem cells" since they could initiate tumors in mice. Here, we first purified CD44 + CD24 - and CD44 +/- CD24 + breast cancer cells from primary human samples and characterized these two cell types (dubbed "CD44+" and "CD24+" cells, respectively). Based on comprehensive gene expression profiling, CD44+ cells were more stem-cell-like and invasive, and each cell type resembled corresponding normal cells. Activation of the TGF-β pathway in CD44+ cells, possibly due to TGFBR2 hypomethylation, maintained their mesenchymal appearance but was not required for cell growth or survival. CD44+ cell gene signatures correlated with negative breast cancer clinical variables, and CD44+ and CD24+ cells had different drug sensitivities. Markers of these cells were variably expressed among breast tumors, with CD24+ cell markers being higher in metastases, and one mutation was present only in CD24+ cells. Second, we conducted a loss-of-function screen using lentiviral shRNAs targeting 1,576 genes differentially expressed between CD44+ and CD24+ cells, identifying 15 genes specifically required in CD44+ cell-like cell lines, and subsequently investigated related cell-type-specific roles of Stat3. The IL6/JAK2/Stat3 pathway was specifically activated in, and required for the survival of, CD44+ cell-like cell lines and CD44 + CD24 - cells in breast tumors. Additional CD44+ cell-like-cell-line-required pathways regulated Stat3 activation, and a Stat3 activation gene signature correlated with poor breast cancer clinical outcome.

In summary, we found that CD44+ and CD24+ cells are clinically important, and we identified Stat3 as a possible target for killing CD44 + CD24 - breast cancer cells. Our results indicate that cells with properties of cancer stem cells likely act in conjunction with clonal evolution, the continual production and selection of distinct cell populations, to generate and maintain breast cancer intra-tumor heterogeneity and, therefore, that the most effective way to eradicate tumors may be to design therapies that can simultaneously eliminate multiple cancer cell types.