A dynamic in vivo-like organotypic blood-brain barrier model to probe metastatic brain tumors

Abstract

The blood-brain barrier (BBB) restricts the uptake of many neuro-therapeutic molecules, presenting a formidable hurdle to drug development in brain diseases. We proposed a new and dynamic in vivo-like three-dimensional microfluidic system that replicates the key structural, functional and mechanical properties of the blood-brain barrier in vivo. Multiple factors in this system work synergistically to accentuate BBB-specific attributes-permitting the analysis of complex organ-level responses in both normal and pathological microenvironments in brain tumors. The complex BBB microenvironment is reproduced in this system via physical cell-cell interaction, vascular mechanical cues and cell migration. This model possesses the unique capability to examine brain metastasis of human lung, breast and melanoma cells and their therapeutic responses to chemotherapy. The results suggest that the interactions between cancer cells and astrocytes in BBB microenvironment might affect the ability of malignant brain tumors to traverse between brain and vascular compartments. Furthermore, quantification of spatially resolved barrier functions exists within a single assay, providing a versatile and valuable platform for pharmaceutical development, drug testing and neuroscientific research.

Figure 1. The integrity and function of blood-brain barrier.

(a) Cellular constituents of the BBB in vivo. The BBB is formed by lined BMECs surrounded by pericytes and astrocytic end-feet. (b) Schematic illustration of BBB function with the expression of several transporters and functional proteins. (c) The design and structure of the integrated BBB device. (i) Device design. It is composed of 16 independent function units connected by a microchannel network (ii). Each unit consists of four uniform BBB regions, one vascular channel, one gas channel, one gas valve and four gel channels. They share the same waste outlet in the middle of the chip. Enlarged view (iii) and sideview (iv) of the barrier regions consisting of BMECs, astrocytes and 3D ECM under flow. (d) Illustration of the procedures to establish the blood-brain barrier under flow conditions. (i) The empty device with gas valve and vascular channels closed. (ii) Collagen gelatin and cell medium infusion with gas valve opened. (iii) Suspension of astrocytes perfused into the vascular channel and attached to the side surface of gelled ECM. (iv) Suspension of BMECs perfused into the vascular channel and attached to the astrocytes. (v) Co-cultures of BMECs and astrocytes in the vascular channels under continuous flow.

Figure 2. Expression and quantification of the barrier-specific functional proteins in BMECs under both static and flow conditions after 48 hours.

(a–c) Expression of adhesive protein VE-cadherin (a), tight junction proteinZO-1 (b) and claudin-5 (c). n = 3. (d,e) Expression of efflux transporter of P-glycoprotein (d) and glucose transporter Glut-1 (e) in BMECs with or without the presence of astrocytes. The flow rate applied to the vascular channel is 1 μL/min, and all scale bars indicate 50 μm. (f) Relative fluorescence intensity statistics of the expression of P-glycoprotein and Glut-1 in different groups. n = 3. Data are presented as mean ± s.e.m. Statistical significance was calculated by Student t-test. *P < 0.05.

Figure 3. Evaluation of the barrier function of the 3D high throughput BBB system.

(a,b) Time-lapse images of permeable sodium fluorescein tracer (NaFl, MW = 376Da, 250 μM) across the BBB into the brain compartment under the static (a) and flow condition (b) over 24 h. Green, NaFl. Scale bar, 25 μm. (c) Quantitative graphs showing the permeability of NaFl across the BBB into the brain compartment in BBB and BMECs alone under the static and flow conditions. (d) TEER measurement of the barrier function in the BBB group and BMECs alone under static and flow conditions. The maximal value of TEER in the BBB group under flow was 1298 ± 86 Ω × cm². n = 3. Data are presented as mean ± s.e.m.

Figure 4. Brain metastasis of malignant cancer cells.

(a) Schematic illustration of the brain metastasis of the common cancer cells into the brain, such as lung cancer, breast cancer and melanoma. Metastases from primary sites spread to the brain through the circulatory system (red arrows) and also to adjacent sites (orange arrows). (b) Schematic of exogenous cancer cells penetrating the brain by crossing the BBB. (c) Time-lapse images of extravasation of different cancer cells across the barrier on this BBB system in lung cancer cells (A549), breast cancer cells (MDA-MB-231), melanoma (M624) and liver cancer cells (BEL-7402). The migration of cancer cells across the BBB was monitored over 72 h. The flow rate is 1 μL/min. The cells were pre-labelled with live-cell staining dyes. Red, BMECs; Blue, astrocytes; Green, cancer cells. Arrows, metastatic cancer cells into the brain compartment by crossing the BBB. (d) Box-and-whiskers plots of cell migration of different cancer cells crossing the BBB. The box represents the 25^th and 75^th percentiles with the median shown by the line bisecting the box. Invasion distance is shown by crosses inside the box. The whiskers represent the 10^th and 90^th percentiles of the data. (e) Time-lapse migration of glioma U87 cells in the brain compartment under vascular flow. U87 cells could not traverse the BBB into the vascular compartment after 72 h. Flow rate, 1 μL/min. Red, BMECs; Blue, astrocytes; Green, U87 cells. Scale bar, 100 μm.

Figure 5. Therapeutic response of glioma cells to clinically relevant pharmaceutical agents on this BBB system.

(a) Characterization of U87 glioma cells in various barrier groups after the addition of eight different chemotherapeutic agents on the vascular compartment under flow. TMZ: lipophilic molecule. CBP, DDP, 5-Fu, NDP and GEM are hydrophilic molecules. FTO and IFO are pro-drugs with hydrophilic properties. The U87 cells were labelled with live/dead staining dyes. Red: dead cells; Green: live cells. (b) Schematic of diffusion of lipophilic and hydrophilic drug compounds across the BBB, and (c) Quantitative assay of live/dead rate of U87 cells triggered by the drugs introduced into the vascular compartment of the BBB. n = 3. (d) The functional response of the BBB to lipophilic TMZ penetration at different concentrations. The U87 cells exhibited dose-dependent responses to the TMZ added in the vascular compartment. Green, live cells; Red, dead cells. Scale bar, 25 μm. (e) Plot of the rate of apoptotic U87 cells as a function of TMZ concentration. n = 3.