Abstract 2823: Development of an <i>in vitro</i> blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system
Authors: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines
Journal: Cancer Research (2024)

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological malignancy, is characterized by abnormal thymocyte proliferation, frequently with mutations of the Notch-1 gene and has an overall 10-year survival rate of ~27%. A marker of poor prognosis is the presence of T-ALL within the central nervous system (CNS), which is treated with intrathecal chemotherapy and cranial irradiation; two toxic treatments with high morbidities. Gene-expression profiling of Notch-1-induced adhesion regulators showed that C-C chemokine receptor 7 (CCR7) was highly expressed and was associated with leukemic T-cell invasion of the CNS. Normal functions of CCR7 include lymph node homing and immune cell trafficking in response to CCR7 ligands, CC-chemokine ligand 19 (CCL19) or CCL21. In several studies CCL19 has been linked to T-cell invasion of the CNS across the blood-brain barrier (BBB). We hypothesize that antagonizing CCR7 binding to CCL19 will block migration of T-ALL into the CNS and thereby alleviate the need for toxic therapies. To test our hypothesis, we are using receptor internalization assays, calcium mobilization assays, transwell migration assays and an in vitro model of an intact human BBB. We have used flow cytometry-based CCR7 internalization assays ± CCR7 antagonist and found that we can completely block CCL19-induced receptor internalization in human CCR7-expressing CCRF-CEM, and HuT-78 T-ALL cells, and chemotaxis via β1 integrins across a fibronectin-coated transwell membrane. Our BBB employs HBEC-5i, human cerebral microvascular endothelial cells, in transwell chambers coated with gelatin and fibronectin for the adhesion of endothelial cells. HBEC-5i cells are a suitable cell line to make a 3D model of the BBB, as they form an endothelial monolayer with tight junctions (TJ), which is crucial to maintaining the integrity and inhibiting permeability of the BBB. We are establishing a transwell migration assay to assess the migration of CCRF-CEM T-ALL cells across the HBEC-5i cell monolayer. The integrity of the HBEC-5i cell monolayer is assessed by trans-epithelial electrical resistance (TEER), which measures the electrical resistance across the HBEC-5i monolayer, a measure of TJ integrity. Using TEER measurements, we observe that HBEC-5i form a monolayer that reaches an intact monolayer functioning with TJs over 72-96 hours. We are using this model system to investigate the migration of CCR7-expressing CCRF-CEM cells through the BBB ± CCL19. Subsequently, we will add the CCL19 antagonist (CCL197-77) to determine if inhibiting CCR7 is sufficient to eliminate migration of the CCRF-CEM cells through the BBB. If successful, this model will allow us to test the contribution of CCR7 signaling to integrins, the contribution of other immune cell types and the overall molecular mechanisms that mediate invasion of the CNS in a 3-D model of the BBB. Citation Format: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines. Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2823.