Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Summary

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Abstract

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (µmBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model µmBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Introduction

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type^1,2. A principal question that arises when studying cancer metastasis is how sub clones migrate from the humoral environment of the bloodstream into an organ such as the brain^3,4. This question has led to many variations of migration, invasion, and extravasation assays. All these methods share the critical step of counting or measuring properties of cells that move from one locatio....

Protocol

1. Prepare the blood brain barrier niche mold

NOTE: The culturing device used in this platform is a PDMS based scaffold that we build a cellular blood brain barrier niche upon. It is made of two parts separated by a porous membrane. To prepare the blood brain barrier niche two SU-8 molds made using photolithography are necessary^26,27. The protocol will be described for the 100 µm thick mold first and then notes will be given for the 200 µm thick mold.

1. To prepare the mold, clean a 4” silicon wafer using acetone with a squeeze bottle and then dry it with a ....

Results

Using this technique, we analyzed cell types labeled with different fluorescent proteins or dyes. We demonstrate the use of this approach with a µmBBN chip formulated with hCMEC/D3-DsRed and non-fluorescent astrocytes. The brain microvascular endothelial cells were seeded onto a porous membrane (5 µm track etched pores) and placed in an incubator³⁴ at 37 °C under 5% CO₂. After three days the confluency of the endothelial layer was confirmed via microscopy and then cancer cells were plac.......

Discussion

We have developed and presented a new method that adapts tools often utilized in clinical imaging analyses for measurement of extravasation and migration of cancer cells through an endothelial barrier into brain tissue. We pose this approach can be useful for both in vivo and in vitro measurements; we have demonstrated its use on a 3D microfluidic system recapitulating brain vasculature. Cancer cell measurements including distance extravasated, percent extravasated by volume, sphericity, and volume are quantified using t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA with phenol red	Thermo Fisher Scientific	25200056
1.5 mm biopsy punch with plunger	Integra LifeSciences Corporation	33-31A-P/25
10x MEM	Thermo Fisher Scientific	11430030
150 mm petri dishes	Fisher Scientific	FB0875714
1x DPBS, without Ca and Mg	Thermo Fisher Scientific	14190144
200uL pipette tip	Fisher Scientific	02-707-411
4 inch silicon wafer	University Wafer	452
48 mm wide packing tape	Fisher Scientific	19-072-097
50 x 75 mm glass slide	Fisher Scientific	12-550C
A1 confocal microscope	Nikon
acetone	Fisher Scientific	A9-20
antibiotic/antimycotic (penicillin/streptomycin/amphotericin)	Gibco	15240062
box cutter blade	Fisher Scientific	NC1721575
dissection scissors	Fisher Scientific	08-951-5
DMEM with 4.5 g/L glucose	Thermo Fisher Scientific	11960-044
double sided tape	Fisher Scientific	NC0879005
EGM-2	Lonza	CC-3162
Fetal Bovine Serum, Heat inactivated	Corning	MT35011CV
Fiji software	ImageJ
glass vial	Fisher Scientific	03-341-25D
glutamax	Thermo Fisher Scientific	35050061
hCMEC/D3	EMD Millipore	SCC066
Jupyter notebook	Anaconda
L-glutamine	Thermo Fisher Scientific	25030081
Matrigel - growth factor reduced with phenol red	Corning	CB-40230A
MDA-MB-231	ATCC	HTB-26
MDA-MB-231-BR-GFP	Dr. Patricia Steeg, NIH
N-2 growth supplement	Thermo Fisher Scientific	17502048
normal human astrocytes (NHA)	Lonza	CC-2565
Orange software	University of Ljubljana
Pasteur pipette	Fisher Scientific	13-711-9AM
Photolithography masks	Photosciences Incorporated
pLL3.7-dsRed	University of Michigan Vector Core
pLL-EV-GFP	University of Michigan Vector Core
pLOX-TERT-iresTK	Addgene	12245
pMD2.G	Addgene	12259
polycarbonate membrane, 5um pore size	Millipore	TMTP04700
psPAX2	Addgene	12260
PureCol, 3 mg/mL	Advanced Biomatrix	5005	Type I bovine collagen
sodium bicarbonate	Thermo Fisher Scientific	25080094
sodium pyruvate	Thermo Fisher Scientific	11360070
Solo cup	Fisher Scientific	NC1416545
SU-8 2075	MicroChem Corporation	Y111074 0500L1GL
SU8 developer	MicroChem Corporation	Y020100 4000L1PE
Sylgard 184	Ellsworth Adhesive Company	NC0162601
Toluene	Sigma-Aldrich	179965-1L
Tricholoro perfluoro octyl silane	Sigma-Aldrich	448931-10G