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Classical markers of tumor-initiating cells (TIC), aldehyde dehydrogenase 1 (ALDH1) activity and the cell-surface markers, CD44pos/CD24low/neg, show insufficient cell type specificity to identify TIC definitively, nor do they allow robust enrichment by FACS. As a consequence, analyses of TIC function and gene expression to date are not definitive. Identification and characterization of the TIC subpopulation is critically important in breast cancer since these cells are thought to mediate treatment resistance, as well as local and distant recurrence. This is particularly critical for “triple negative” breast cancer (TNBC), since currently there are no targeted therapies available for this clinical subtype. Lentiviral signaling reporter vectors allow purification of enriched TIC subpopulations thereby facilitating single cell analyses to identify distinct sets of cells with TIC features. We used a collection of fluorescent lentiviral reporters that enable visualization and flow cytometric enrichment of cells undergoing STAT3-, GLI-, WNT-, or OCT4/SOX2-mediated transcriptional activity. Our collection of patient derived xenograft (PDX) models of TNBC were infected with the lentiviral reporters, re-transplanted, and screened for reporter activity upon regrowth. TIC function of reporter-expressing cells relative to negative cells was assayed by mammosphere formation and limiting dilution transplantation. Reporter expressing cells with enriched TIC function were then analyzed by single cell RNAseq for expression of classical markers to identify the subset of cells most likely to represent the TIC subpopulation. Our results indicate that STAT3 and GLI transcriptional activity can serve as uniquely PDX-dependent indicators for TIC function for a subpopulation of cancer cells. Also, multiple potential TIC signaling pathways, including STAT3, GLI, WNT, and OCT4/SOX2, can be active in a single PDX model, but do not necessarily associate with TIC function, indicating TIC diversity in TNBC. These data in conjunction with treatment response and single-cell RNA-seq sheds light on the molecular diversity of TIC populations in TNBC, identifies candidate targets for therapeutic intervention, and will indicate whether TIC diversity is associated with differential response to different chemotherapeutic agents. Citation Format: John Landua, Ping Gong, Michael T. Lewis. Heterogeneity of tumor-initiating cell related signaling revealed by lentiviral signaling reporters in breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1689. |
