TY - JOUR T1 - Albumin as a probe for clathrin-independent endocytosis and transcytosis into experimental brain metastases of breast cancer JF - bioRxiv DO - 10.1101/2022.08.09.503253 SP - 2022.08.09.503253 AU - Imran Khan AU - Brunilde Gril AU - Anurag Paranjape AU - Christina Burks AU - Christina Robinson AU - Simone Difilippantonio AU - Wojciech Biernat AU - Michał Bieńkowski AU - Rafał Pęksa AU - Renata Duchnowska AU - Jacek Jassem AU - Priscilla K. Brastianos AU - Philippe Metellus AU - Emilie Bialecki AU - Carolyn C. Woodroofe AU - Haitao Wu AU - Rolf Swenson AU - Patricia S. Steeg Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/08/11/2022.08.09.503253.abstract N2 - Background Improvements in drug delivery to brain metastases are needed. We hypothesized that albumin transcytosis would demonstrate widespread distribution in experimental brain metastases of breast cancer.Methods The metastatic and uninvolved brain localization of five transcytotic pathways was determined in two hematogenous models of breast cancer brain metastasis. Albumin was compared to distribution of a Biocytin-TMR, which uses paracellular permeability. An in vitro model for transcytosis of albumin across a blood-tumor barrier (BTB) was developed and characterized for modes of endocytosis. Human craniotomy specimens were stained for endocytic proteins.Results Albumin transcytosis occurred in virtually all metastases, including micrometastases; distribution was greater than, and independent of paracellular permeability. Albumin uptake into the normal brain parenchyma was negligible. Using an in vitro assay that permits albumin transcytosis through a BTB, albumin used a form of macropinocytosis with features of clathrin-independent endocytosis (CIE), involving the neonatal Fc receptor (FcRn), Galectin-3 and membrane sphingolipids. Human craniotomy specimens demonstrated FcRn and Galectin-3 staining.Conclusions The data identify CIE as a pathway with optimal features for drug delivery into brain metastases.Key PointsAlbumin transcytosis occurred in virtually all experimental brain metastases in two model systems.Albumin transcytosis occurred independently of paracellular permeability.Albumin transcytosis uses a clathrin-independent endocytosis pathway.Importance of the Study Improvements in drug delivery to brain metastases are needed to enhance therapeutic efficacy. We report that labeled albumin has a distribution pattern in two models of experimental brain metastases of breast cancer that may be optimal for translational development: Albumin transcytosis occurred in nearly all lesions, including micrometastases, and was independent of paracellular permeability. Using an in vitro model of the blood-tumor barrier, we found that albumin uses a clathrin-independent endocytic pathway requiring FcRn and galectin-3. Human craniotomy specimens stained for both proteins, demonstrating that the pathway can be operative in the human. A CIE pathway may represent an advantageous mechanism for linkage to drugs for brain metastasis therapy.Competing Interest StatementThe authors have declared no competing interest. ER -