PROJECT SUMMARY Despite advances in surgery, chemotherapy and targeted therapies, survival of metastatic epithelial ovarian cancer remains dismal due in part to tumor heterogeneity and residual microscopic disease undetectable by traditional imaging modalities. This malignancy involves peritoneal carcinomatosis at advanced stages; that is, extensive tumor studding of the pelvic cavity and its resident organs. To address these “invisible” tumors, we introduce a series of molecular-targeted and cell-activatable imaging and therapeutic agents integrated with a newly developed miniaturized microscope for multiplexed molecular imaging in vivo—a cellular-resolution hyperspectral fluorescence microendoscope—that uniquely enable visualization of microscopic deposits of tumor cells deep inside the body. The multiplexed biomarker imaging feature is motivated by the critical need to quantitatively monitor cancer cell phenotypes salient to treatment resistance and escape (e.g., cancer stem cells are defined by multiple cell-surface biomarkers). In parallel, we have also developed near-infrared photocytotoxic immunoconjugates (PICs) that target cell membrane molecules overexpressed by cancer cells, including the epidermal growth factor receptor (EGFR), to create a combined photodynamic and receptor antagonist therapeutic agent for tumor-targeted, activatable photoimmunotherapy (taPIT). The photodynamic and fluorescence components become de-quenched (activated) upon cancer cell binding, cellular internalization and lysosomal antibody proteolysis. This strategy overcomes off-target phototoxicity, including bowel phototoxicity, the major clinical obstacle for photoactivated treatments in complex sites such as the pelvic cavity. Here, we build on our prior work that shows the PIC is activated within ovarian micrometastases with 93% sensitivity and 93% specificity in vivo, in a xenograft mouse model using EGFR overexpressing human epithelial ovarian cancer cells, enabling accurate recognition of tumors as small as 30 μm and selective destruction of disseminated peritoneal micrometastases. We propose to further develop this platform to address tumor heterogeneity and chemoresistant phenotypes lurking within residual tumor deposits, which represents a critical niche in cancer therapy. Current clinical imaging technologies cannot resolve microscopic tumor deposits left behind by surgery and chemotherapy, and there are limited treatment options for patients with recurrent, chemoresistant tumors. We anticipate that this new paradigm for microendoscopy-guided taPIT will complement current treatment modalities for patients with advanced-stage disease and those receiving salvage therapies, opening new avenues for personalized medicine. The new capabilities will be applied to guide dynamically targeted taPIT adaptive to phenotype evolution in response to therapy and for monitoring treatment response of microscopic residual disease.