While targeted therapy increases overall survival rates in HER2-positive breast cancer patients, most patients will experience recurrence due to resistance to initially successful therapy (trastuzumab-based). Accordingly, there is an unmet, patient-driven need to understand and circumvent breast cancer resistance, to even targeted therapies. At the cellular level, cell-to-cell variation (heterogeneity) is a hallmark of cancer. Perhaps surprisingly, molecular heterogeneity is also a hallmark of HER2+ breast cancer. Spanning the outer membrane of a cancer cell, the large protein HER2 displays an extracellular domain, which is the target of ‘targeted therapies’ (vs. chemo- or radiation therapies). These HER2+ breast cancer targeted therapies include the landmark drug Herceptin® (trastuzumab). But the HER2 protein manifests as a family of proteins (called protein isoforms), not just a single molecular form. Regrettably, numerous of these HER2 isoforms lack the extracellular domain of the full-length HER2 protein, making the cell non-responsive otherwise powerful anti- HER2 targeted therapies. These smaller HER2 isoforms are known as ‘truncated isoforms’, with a 95 kDa form ‘P95HER2’ being especially potent in drug resistance. Until our previous R01 research, the ability to discern full-length HER2 from the truncated P95HER2 isoform was not readily possible with same-cell resolution. Consequently, to advance our knowledge of resistance to anti-HER2 targeted therapies, we propose to build on our team’s capacity to precisely distinguish P95HER2 from other HER2 protein forms to scrutinize the role of P95HER2 in: (1) the potent, signal-activating HER2 dimers that reside on the surface of each breast cancer cell and (2) potentially ultra-resistant breast cancer cell subpopulations that exhibit both the P95HER2 protein isoform and the resistance-driving DNA mutation (PIK3CA). These are two cellular ‘modes’ (P95HER2 homo/heterodimers; co-expression of P95HER2 and PIK3CA mutation) that no other tools can directly and with high-specificity concurrently measure in minute tissue samples, down to single-cell resolution. Our clinical, biostatistics, and bioengineering team will conduct research to yield tools that can perform these isoform- involved multimodal assays in tissues and cells from HER2-positive breast cancer patient biopsies (Stanford Breast Tissue Bank), after performing early development on well-characterized breast cancer cell lines. The ability to directly measure the truncated HER2 isoforms and interaction modes in sparingly available breast biopsy tissues and with single-cell resolution should yield a tremendous advantage for understanding and then assessing the potential for drug resistance. These studies will allow us to profile the cellular and molecular heterogeneity of HER2 to advance understanding of persistent breast cancer resistance to anti-HER2 targeted treatment and, ultimately, to identify approaches to reduce or eliminate recurrence.