Abstract 3067: Constitutive macropinocytosis in BRAFV600E thyroid cancer promotes response to a serum-albumin conjugate of monomethyl auristatin E
Authors: Hu Huiyu , Ng Thomas S. , Kang Mikyung , Scott Ella , Li Ran , Quintana Jeremy M. , Matvey Dylan , Vantaku Venkata R. , Weissleder Ralph , Parangi Sareh , Miller Miles A. 
Published: April 2023

Abstract
Background: The non-specific uptake of extracellular materials through macropinocytosis can fuel tumor growth and survival, and is especially studied in cancer-types that frequently harbor RAS or PTEN mutations. However, relatively little data describe macropinocytosis in follicular, papillary, and anaplastic thyroid cancers (FTC, PTC, and ATC, respectively) despite the abundance of prominent MAPK and PI3K pathway mutations in these malignancies. We hypothesized that thyroid cancers exhibit elevated macropinocytosis and efficiently accumulate chemotherapy covalently conjugated to serum albumin, a common macropinocytosis substrate.
Methods: We assessed the impact of ectopic BRAFV600E expression or targeted kinase inhibition on macropinocytosis in vitro across a panel of thyroid cancer cell lines with varied BRAF mutation status. Cellular uptake of dextran and serum albumin was quantified across ≥ 246 individual cells using fluorescence microscopy in triplicate experiments. The in vivo uptake of serum albumin by both ATC cells and tumor infiltrating immune cells, including tumor-associated macrophages, was assessed across n = 4 replicates of an immunocompetent orthotopic model of BrafV600E p53-/- ATC in B6129SF1/J mice. The same mouse model was used to measure the effects of an albumin-drug conjugate based on the microtubule-destabilizing monomethyl auristatin E (MMAE) linked to serum albumin via a cathepsin-cleavable peptide (Alb-vc-MMAE) on tumor volume using n ≥ 10 tumors per group. Groups were compared by two-tailed Wilcoxon rank-sum tests.
Results: FTC and ATC cells exhibited greater macropinocytosis than PTC and immortalized follicular thyroid cells. Fluorescence microscopy of thyroid cancer cells revealed punctate subcellular co-localization between dextran and serum albumin. Treatment with the Na+/H+ exchanger (NHE) inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced cellular uptake by roughly 50%, consistent with macropinocytosis (P &lt; 0.01). Ectopic BRAFV600E enhanced macropinocytosis by 2-fold, while BRAF and MEK kinase inhibition decreased uptake by 50% (P &lt; 0.01). In vivo, ATC tumors accumulated serum albumin at 8.5%ID/g (% injected dose per gram tissue). The albumin-drug-conjugate Alb-vc-MMAE, but not MMAE alone, blocked more than 90% of ATC tumor growth (P &lt; 0.01).
Conclusions: ATC exhibits high constitutive macropinocytosis driven by elevated oncogenic signaling, including via MAPK/ERK activity stimulated by BRAFV600E mutation. Macropinocytosis in ATC, therefore, may be a target for manipulating tumor metabolism and has implications for the delivery of some albumin-bound and other drugs that are affected by macropinocytic cell uptake.
Citation Format: Huiyu Hu, Thomas S. Ng, Mikyung Kang, Ella Scott, Ran Li, Jeremy M. Quintana, Dylan Matvey, Venkata R. Vantaku, Ralph Weissleder, Sareh Parangi, Miles A. Miller. Constitutive macropinocytosis in BRAFV600E thyroid cancer promotes response to a serum-albumin conjugate of monomethyl auristatin E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3067.