Fig. 8: PEAK1-expressing MSCs promote lapitinib resistance by modulating antiapopototic/DNA damage signaling within a subpopulation of highly plastic HER2-positive breast cancer cells.

a Schematic of the CycIF workflow used for single-cell analysis. b–c t-SNE plots of all cells across biological replicates of cell culture and lapatinib treatment conditions (b) and overlayed with the GFP-positive BT474 HER2-positive breast cancer cells (c, inlay shows GFP gating scheme). d Averaged BT474 GFP-positive breast cancer cell (BCC) number at 48 h post-therapy treatment. e Quantification of ar–––ea under the curve (AUC) for data plotted in d. f Overlay of notable breast cancer cell and MSC subpopulations onto the t-SNE plot from c. g Pseudocoloring of the single-cell antigen intensities of GRP78, MCL1, VIM, p-γH2AX, p65NFκB and p-Akt in the GFP-positive breast cancer cells and αSMA in the GFP-negative MSCs overlayed onto the t-SNE plots for these cell populations. h–m Average integrated signal intensity for indicated tumor cell markers within indicated gated populations of complementary markers across the four cell culture conditions. n–p Histograms representing lapatinib-induced changes of the single-cell antigen expression patterns for p-Akt^high/p-gH2AX^low, MCL1^high/p-gH2AX^low and GRP78^high/VIM^high BT474 cell subpopulations identified in k–m. q Representative microscopy images of nuclear (DAPI), MCL1 and p-gH2AX immunofluorescence across cell culture and lapatinib treatment conditions. IPA-derived canonical pathway (r) or disease/function annotation enrichments (s) for SNAI2, PEAK1, INHBA, CCL4, GDF5, MCL1, AKT1, H2AFX, GRP78 and VIM. t Proposed model of mechanism by which stromal expression of PEAK1 drives tumor growth, metastasis and targeted therapy resistance in neighboring HER2-positive breast cancer cells.