21:T46c,The primary cilium permits compartmentalization of specific signaling pathways, including elements of the Hedgehog (Hh) pathway. Hh transcriptional activity is thought to be negatively regulated by constitutively high ciliary cAMP maintained by the Gα(s)-coupled GPCR, GPR161. However, cilia also sequester many other Gα(s)-coupled GPCRs with unknown potential to regulate Hh. Here we used biosensors optimized for ciliary cAMP and strategies to isolate signals in the cilium from the cell body and neighboring cells. We found that ciliary cAMP was not elevated relative to cellular cAMP, inconsistent with constitutive cAMP production. Gα(s)-coupled GPCRs (e.g., the 5-HT₆ serotonin and D1R dopamine receptor) had reduced ability to generate cAMP upon trafficking to the ciliary membrane. However, activation of the Hh pathway restored or amplified GPCR function to permit cAMP elevation selectively in the cilium. Hh therefore enables its own local GPCR-dependent cAMP regulatory circuit. Considering that GPCRs comprise much of the druggable genome, these data suggest alternative strategies to modify Hh signaling.22:T4c7,Basal-like breast cancer (BLBC) shows brain metastatic (BM) capability and overexpresses EGFR and death-receptors 4/5 (DR4/5); however, the anatomical location of BM prohibits efficient drug-delivery to these targetable markers. In this study, we developed BLBC-BM mouse models featuring different patterns of BMs and explored the versatility of estem cell (SC)-mediated bi-functional EGFR and DR4/5-targeted treatment in these models. Most BLBC lines demonstrated a high sensitivity to EGFR and DR4/5 bi-targeting therapeutic protein, E_VDR_L [anti-EGFR VHH (E_V) fused to DR ligand (DR_L)]. Functional analyses using inhibitors and CRISPR-Cas9 knockouts revealed that the E_V domain facilitated in augmenting DR4/5-DR_L binding and enhancing DR_L-induced apoptosis. E_VDR_L secreting stem cells alleviated tumor-burden and significantly increased survival in mouse models of residual-tumor after macrometastasis resection, perivascular niche micrometastasis, and leptomeningeal metastasis. This study reports mechanism based simultaneous targeting of EGFR and DR4/5 in BLBC and defines a new treatment paradigm for treatment of BM.

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