Engineering Lymphatic Vasculature Models for Cancer Surveillance
Jamie
1
Hernandez ,
Harini
3
Kantamneni ,
Margot
2
Zevon ,
Vidya
2
Ganapathy ,
Prabhas V.
2,3
Moghe
1Department of Biomedical Engineering, University of Arizona, Tucson, AZ
2Department
of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ
3Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ
Background and Significance
Results
A
C
B
ANC
A
B
HER2- Cell
Crosslinker
(Glutaraldehyde)
Figure 4: HER2+ cells
(HCC 1954), grown in
culture seen with
epithelial morphology
[A],as compared to
HER2- cells (SCP2-TR)
seen with mesenchymal
morphology [B].
Qualitative observations
based on morphologies
of cells grown in coculture with a 1:1 ratio
[C], and 2:1 ratio of
HER2+/HER2- [D]
determined 2:1 to be the
optimal growth ratio.
D
HER2- Cell
HER2+ Cell
Functionalize
(AMD3100)
REs
Figure 2: Coacervation process for forming ANCs with RE metal cores
HER2+ Cell
+ ANC
+ AMD3100
Morphology of HER2+/- Cells in Co-Culture
Phenotype Differentiation
ANC Synthesis
HER2- cells
- ANC
-AMD3100
Figure 3: 3D printed
model made with
mouse, rat, human
dimensions [A].
Fluidics tested
through PDMS
human model with
trypan blue [B] and
in rat Model with
ANCs (imaged in
green) injected
through the channel
[C].
Materials and Methods
To differentiate between phenotypes, cells with and
without HER2 receptor were specifically studied.
HER2+ cells (HCC1954) were targeted using the
HER2 antibody. To confirm this discriminated
between cell types, immunocytochemistry (ICC)
was performed. To track HER2- cells, chemokine
receptor 4(CXCR4) was targeted using ANCs
functionalized with the antagonist, AMD3100.
Because CXCR4 is present in both cell lines, this
receptor was blocked in HER2+ cells in culture.
(Figure 1A)
HER2+ cells
Lymph Vessel Model Design and Fluidics
C
Solvent
(Ethanol)
HER2+ cells
+ ANC
- AMD3100
Over a quarter million women are diagnosed with breast cancer each year. To
prevent the spread of tumors to healthy tissues, it is important to detect metastasis
early. For breast cancer, tumors typically metastasize through the lymphatic
system, specifically the axillary lymph vessel and the sentinel lymph node.
Currently sentinel lymph node biopsy is used as a diagnostic tool to determine
whether a tumor has metastasized from the breast. This leads to high false
negative rates possibly leading to unnecessary surgeries and a compromised
immune system. Our approach is to use bioactive nanoprobes made of ceramic
nanoparticles doped with rare earth (RE) cations encapsulated in human serum
albumin (REANCs).The REs can be imaged in the shortwave infrared spectrum
(SWIR), so that they can be separated from the auto fluorescence spectrum that
native tissues exhibit. The albumin coating of these nanocomposites make these
particles biocompatible, and make it possible to decorate with drugs and ligands
that can be used to target cells. REANCs were able to detect tumor cells in
previous in vivo experiments in mice, but signal was not able to be detected in the
tumor infiltrated lymph nodes. The aim of this project is to engineer a model
that mimics lymphatic vasculature to test the particles’ ability in cancer
surveillance by molecular discrimination of different tumor cell populations
in this model and also size exclusion principle of REANCs. Determination of
cell phenotype of heterogeneous tumor populations is especially critical in order to
optimize detection of the tumor, and sequentially tailor effective cancer treatments.
Molecular discrimination of HER2+/- cells
CXCR4 blocked
CXCR4 not blocked
CXCR4 not blocked
HER2 Ab
HER2 Ab
HER2 Ab
Figure 6: HER2+ (HCC 1954) and HER2- (SCP2-TR) cells grown in culture and stained with a primary HER2 antibody, followed
by the secondary antibody Alexa Fluor 647. Cells were cultured without ANCs, with ANCs, and with ANCs functionalized with
AMD3100. HER2 (imaged in red) is labeled in HER2+ cells, as the signal is not observed in HER2- cells. ANC signal,
measured by the autoflourescence of the albumin and glutaraldehyde (FITC), is not observed in the HER2+ cells with blocked
CXCR4 receptors. Functionalized ANCs are seen clearly around the cells without CXCR4 blocking. Trace signal in the ANC
channel is also observed with the treatment of unfunctionalized particles to a lesser degree.
HER2 Tracking
A
B
Discussion and Conclusions
C
Nucleus
Nucleus
HER2+ cell
HER2
Figure 1: The project is focused on
differentiating between cell phenotypes
[A] and the development of a lymph
vessel model to analyze the behavior of
the particles through the lymphatic
system [B]. Long term aims for the project
bring together the elements of tumor cell
tracking within a lymphatic model [C].
Albumin nanocomposites were formed with pH
controlled coacervation of human serum albumin (HSA). The albumin cage formed
within the ethanol is crosslinked with glutaraldehyde to increase the rigidity of the
particles. Size of the particles were assessed using a zetasizer. Ideal particles
(approximately 90 nm) were functionalized with AMD3100 and used for phenotype
differentiation.
Lymph Vessel Model
The model was designed in AutoCAD 2015 to have basic channels with the
dimensions of mouse (144 µm), rat (200 µm), and human (300 µm) axillary lymph
vessels. This model was 3D printed using a printer with a 16 µm resolution
(Stratasys Connex 350). Forms were made using polydimethylsiloxane (PDMS)
from this 3D printed mold to form a basic channel. ANCs were injected into this
channel for a basic test size exclusion (Figure 3B).
Figure 5: HER2+ cells [A], HER2- cells [B], and 2:1 ratio of HER2+/- cells [C] stained with the a
primary HER2 antibody and the secondary antibody Alexa Fluor 488 (FITC). Nuclei of the cells
imaged in blue, and HER2 is seen in green. Signal in the FITC channel is absent in the HER2- cell
image.
ANC Synthesis, Characterization, and Functionalization
Sample
1
2
3
4
5
ANC
fANC
Average Size (nm)
92.63
92.94
109.1
103.7
97.4
85.25
113.7
Average PDI
0.068
0.065
0.194
0.144
0.223
0.06
0.294
Standard Deviation
1.009
0
2.566
1.358
4.632
0.007
8.218
Table 1: Zetasizer results for average size (nm), polydispersity index (PDI), and standard
deviation of synthesized ANC samples. Average percent yield was calculated to be
60.32%±6.52. Sizing for functionalized particles listed, compared to pre-functionalized
particle size. AMD3100 functionalization appears to have no significant impact on size,
with minimal addition to the diameter of the particles. These particles were determined to
be suitable for HER2- cell tracking in future in vitro experiments
Results from these experiments demonstrated:
 Molecular discrimination of Her2+/- populations in a co-culture model using a HER2
specific antibody
 ANC functionalization with AMD 3100, the antagonist of the cancer cell marker CXCR4
 The development of a basic 3D model of lymph vessel that mimics mouse, rat and
human dimensions
 Successful passage of ANCs through a basic PDMS microfluidic channel with
anatomical dimensions of a rat
Future Directions
Future research to differentiate cell phenotypes will be focused on optimizing the particles
ability to mark the tumor cells. To develop a model that realistically models a murine lymph
vessel, the basic model will be expanded to focus on the interactions of the vessel materials
and the particles. Research will continue to develop:
 Collagen coating to mimic the extracellular matrix of the lymph vessel
 Endothelial cell lining
Phenotype discrimination methods will be incorporated into this intricate model for
application for in vitro particle tracking as well as cancer drug screening within the lymphatic
system
Acknowledgements
This research was supported by RiSE at Rutgers, REU in Cellular Bioengineering: From Biomaterials to
Stem Cells - NSF EEC 1262924, NIH T34 GM0871, and NIH NIBIB EB018378-01 and EB015169-02