(692f) 3D Bio-Printed Model of Brain Tumor Microenvironment with Vasculatures

Introduction:

Glioblastoma multiform (GBM) is a
malignant brain tumor. GBMs have a heterogeneous population with cancer stem
cells. The GBM stem cells often develop therapeutic resistance against
chemotherapy or radiotherapy, followed by tumor recurrences. The tumor cells
are also highly invasive and frequently exploit microvessels
as guides for migration. These hallmark behaviors of GBM make it even harder to
completely cure GBM, as residual cells from the surgical resection of tumor
core aggressively invade and cause tumor recurrences.

Tumor microenvironment consisting
of various extracellular matrices, vasculatures, mural cells,
chemical/mechanical stimulations, etc., plays a crucial role in tumor growth.
Proper microenvironments are required to maintain the GBM stemness
and to better mimic tumor cell behaviors in the human body. Current gold standard
model for GBM studies is in vivo
animal model, which has limitations on reproducibility, lethality, and
discrepancy with human physiology. Current in
vitro models such as 2D monolayer culture, transwell
culture, 3D hydrogel culture, and spheroid/organoid models provide more
reproducible and confined microenvironments. However, many models are lack of
sufficient area for tumor invasion/migration, thus have limitations on
investigating GBM invasive behaviors under various conditions/periods. The lack
of functional vasculatures in current in
vitro models also restricts studying perivascular invasion/migration of
GBM.

In this study, we created various
types of brain tumor-vascular microenvironments using patient-derived GBM cells
with stemness (GSC: glioblastoma stem cells),
vasculature endothelial cells (ECs), supporting cells, and hydrogel. 3D
bioprinting technology has been utilized to create complex tissues with multiple
cell types and matrices with perfusable vasculatures. Three different 3D in vitro models developed in the study
maintained GBM stemness and recapitulated cell
type-specific tumor invasion patterns and drug responses. The results show the
potential of the models in studying cellular/molecular interactions between GBM
and tumor microenvironment.

Material
and Methods:

Bioprinted models were fabricated
as previously described [1, 2], using gelatin as a sacrificial material and
collagen/fibrin as main scaffold materials. Three different types of GBM cells
were tested in the model: SD02 and SD03 are patient-derived GSCs; U87MG is
commercially available GBM cell line. For capillary formation (microvessel formation), endothelial cells (human umbilical
vein endothelial cells (HUVECs) or brain microvascular endothelial cells) and
supporting cells (fibroblasts, pericytes, astrocytes) were mixed within fibrin hydrogel. The cells in
3D environment spontaneously create a capillary network through cell
self-assembly. To show the drug test application, Model A was cultured for 26
days until GBM cells invaded into surrounding matrices over 1 millimeter, then 10-100µM
of Temozolomide was injected through the large
vessels in Model A for next 31 days.

Results
and Discussion:

Develop
Three Brain Tumor-Vascular Niche Models

Model A consists of large vasculatures
and a GBM spheroid (Figure 1A). This model is designed to investigate the
interactions between GBM and extracellular matrices and to measure the
diffusion pattern from the large fluidic vessels. Model B consists of capillary
network and GBM spheroid (Figure 1B) to mimic microenvironment of GBM tumor
core. The microvessels in the model can provide a more
physiological environment as well as invasion/migration trails for GBM cells. Model
C consists of capillary network and scattering GBM single cells (Figure 1C).
The model is designed to mimic tumor microenvironments after surgical resection
of tumor core. Since GBM cells are highly invasive, the residual tumor cells
left from the surgical resection often invade, proliferate, and cause
recurrences. The three models had been successfully fabricated, culture over
3-8 weeks, and utilized for various applications.

Model
A: Drug Treatment Application

SD02 spheroid, cultured in the
setting of Model A, aggressively invaded into the surrounding matrices for 26
days of pre-treatment culture (Figure 1 Aii-Aiii).
During the first 3-4 weeks of Temozolomide drug
treatment, the invaded cells were gradually regressed and the tumor core was
shrinking (Figure 1 Aiv).
However, some SD02 cells survived from the treatment and resumed aggressive
invasion even with the continuing treatment (Figure 1 Av). The long-term drug treatment of Model A recapitulates the development
of therapeutic resistance under chemotherapy.

Model
B&C: Different Tumor Invasion Patterns & Cell-Cell Interactions

In the Models B&C, three GBM
cell types presented different patterns of invasions and vascular interactions
(Figure 1B-C). SD02 cells showed spiky, spreading branch-like invasions (Figure
1 Bii). The
cells also restricted capillary formation in both spheroid and single cell form
(Figure 1 Bii
& Cii).
SD03 spheroid has a more dispersed invasion pattern than SD02 and also presented
a large number of single cell migrations (Figure 1 Biii). SD03 cells showed the most
perivascular interactions. The
cells spread along with capillaries (Figure 1 Biii & Ciii). Some tumor cells formed a
cluster near the capillary branching area (Figure 1 Ciii).  SD03 tumor spheroid did not restrict the
capillary formation (Figure 1 Biii & Ciii). The non-stem GBM cell line U87MG showed a lumpy
invasion pattern and caused the death of neighboring ECs in Model B (Figure 1 Biv). But in
Model C, U87MG cells presented a scattered pattern of proliferation and wrapped
around the capillaries without inducing EC death (Figure 1 Civ).

In Model B, the tumor invasion
patterns of all three cell types are similar to that of in vivo mouse models with injected GBM cells (data not shown). Also,
the patient-derived GSCs culture in Model B maintained its stemness
over three weeks of the culture period. Putting the cell type-specific invasions
from Model B and the long-term drug responses demonstrated in Model A, our
models has a high potential in establishing a personalized therapeutic
strategy.

While Model B is closely
mimicking in vivo models, Model A and
Model C provides unique culture environments that cannot be created in current in vivo models. The models have a promise
for designing and building customized microenvironments for cellular/molecular
interaction studies which are hard to be performed on in vivo models.

By using the three models
presented in this study, we have generated various combinations of culture
conditions with following parameters: 1) with or without microvessels;
2) tumor spheroid vs. single cells; 3) GBM cells with or without stemness; and 4) with or without drug perfusion. In
addition to these factors, we can modify the scaffold hydrogel compositions,
vascular cell types, surrounding cell types, fluidic perfusion through the vasculatures,
etc. Thus, our models have a great expandability in diverse in vitro research fields and can serve
as a valuable tool to model various co-culture conditions and to efficiently
control microenvironment.

Conclusions:

This study demonstrated the
development and several applications of 3D brain tumor microenvironment with vascultures, shifting the research focus to an essential area
of tumor microenvironments. The GBM-vascular models responded differently to
GBM cell types, showing its potential in investigating patient-specific tumor behaviors
under chemo-/radio-therapy conditions and consequentially helping to tailor
patient-specific treatment strategy. In addition, the 3D models can be adapted
to other biological systems and serve as a valuable tool to fabricate
customized microenvironments.

Figure
1. (A) Model A with a tumor
spheroid and large vasculatures. (Ai)
Schematics. (Aii-Av) GBM response to Temozolomide treatment.
(Aii-Aiii) GBM cells invaded into the surrounding
matrices during 26 days of pre-treatment phase. (Aiv) GBM tumor core was regressed
during the first three weeks of drug treatment. (Av) The tumor cells developed therapeutic resistance after long-term
drug treatment then aggressively invaded into the surrounding tissue area. (B) Model B with a tumor spheroid and
capillaries. (Bi) Schematics.
(Bii)
Patient-derived SD02 cells presented spiky, spreading branch-like invasion
patterns with restricted capillary formation around the tumor spheroid. (Biii)
Patient-derived SD03 cells presented more dispersed invasion pattern with a lot
of single cell migrations. SD03 tumor spheroid did not restrict the capillary
formation. (Biv)
Commercially available GBM cell line U87MG showed lumpy invasion pattern. The
cells often caused endothelial cell deaths around the tumor spheroid. (C) Model C with dispersed tumor cells
and capillaries. (Ci)
Schematics. (Cii)
SD02 cells restricted capillary formation and did not present many cell-cell
interactions with endothelial cells. (Ciii) SD03 cells spread along with capillaries. Some tumor
cells formed a cluster near the capillary branching area. (Civ) U87MG cells presented a
scattered pattern of proliferation and wrapped around the capillaries without
inducing EC death.

References:

[1] Lee, V. K., Kim, D. Y., Ngo, H., Lee, Y., Seo, L., Yoo, S. S., ... & Dai, G. (2014). Creating perfused functional
vascular channels using 3D bio-printing technology. Biomaterials, 35(28),
8092-8102.

[2] Lee, V. K., Lanzi, A.
M., Ngo, H., Yoo, S. S., Vincent, P. A., & Dai,
G. (2014). Generation of multi-scale vascular network system
within 3D hydrogel using 3D bio-printing technology. Cellular
and molecular bioengineering, 7(3), 460-472.