This poster describes the generation of invasive breast cancer cell lines for in vivo imaging, as presented at AACR 2013. Authors: Jae Beom Kim, Kenneth Wong, and Konnie Urban (Life Sciences & Technology, PerkinElmer Inc., Alameda, CA). To find out more about the PerkinElmer technologies presented in this poster, please use the following links: In Vivo Imaging & Analysis - http://bit.ly/17hNxIE Microscopy Imaging Systems and Software - http://bit.ly/ZuqpmV

1. Generation of invasive breast cancer cell lines for in vivo imaging
Jae Beom Kim, Kenneth Wong, and Konnie Urban. Life Sciences & Technology, PerkinElmer Inc., Alameda, CA
Abstract 1 In vivo monitoring of tumor growth 4 Biomarkers in MDA-MB-231-luc2-LN cells
Whole animal non-invasive imaging contributes significantly to understand tumor behavior. It Day 102 Day 107 Day 115 Day 126 Tumor volume Bioluminescence
A
also plays a critical role in drug discovery and development. Optical imaging is convenient
A B
because it does not require radioactive materials for imaging. Especially in preclinical
MDA-MB-231-luc2
M #3 - Excision Day 126
1,000
applications, optical imaging can be a very useful tool because genetic modification is 800
Tumor Volume
600
(mm^3)
feasible. The most popular optical imaging is using bioluminescence. We introduced various 400
La Very Very Large 200
cancer cell lines that express firefly luciferase. These cells have stable expression of light rg Large 0
0 30 60 90 120 150
emission for prolonged cell culture situation. This enables researchers to implant the cells into e Cultured MDA-MB-231-luc2-LN cells were stained with
Experimental Day
Before Unmixing After Unmixing PathScan signaling nodes multiplex
animals and monitor tumor development and metastasis. In addition, these tumor cells can be immunofluorescence kit (Cell Signaling Technology).
detected using fluorescent agents that target tumor cells. As a consequence, one can co- C D Cells were counter stained with DAPI and fluorescent
register both bioluminescent and fluorescent images. Cells also can be labeled with both B Day 73 Day 93 Day 101 Day 106 Tumor volume Bioluminescence Fluorescence images were taken using Nuance camera. Each signal
was spectrally unmixed using Nuance software. (A)
MDA-MB-231-luc2-tdTomato
bioluminescent and fluorescent markers such as luciferases and fluorescent proteins. Image before unmixing, (B) Spectrally unmixed image,
#M6 - Excision Day 108 1.0E+12 M#6 - Excision Day 108 M #6 - Excision Day 108 (C) Unmixed DAPI image, (D) Unmixed Phospho-
Although there are many different types of cell lines available for different tumor types, 3,000 1.0E+11
10
Erk1/2 image (Alexa Flour 488), (E) Unmixed Phospho
Total Radiant Efficiency
9
2,500 1.0E+10 8
Akt (Alexa Flour 555), (F) Unmixed Phospho-S6
Tumor Volume
studying metastasis can be challenging. That is mainly because most cell lines show delayed 7
(photons/sec)
2,000 1.0E+09
Total Flux
(mm^3)
6
( X 10^8)
1,500 1.0E+08 5 ribosomal protein (Alexa Flour 647).
DAPI Phospho-Erk1/2
4
metastasis when implanted in the animal. To expedite the metastasis, intravenous injection or 1,000
500
1.0E+07
1.0E+06
3
2
1.0E+05 1
intracardiac injection is performed to generate secondary tumors in the animal. However, 0
E F
1.0E+04 0
0 30 60 90 120 0 30 60 90 120 150 0 30 60 90 120 150
these methods do not represent true metastasis from originated organs. One of the most Experimental Day M #6 Experimental Day M #6 Experimental Day
Non-Palpable Tumor (N=3) Non-Palpable Tumor (N=3)
popular breast cancer cell line is MDA-MB-231. When these cells are implanted into Background Background
mammary fat pads of female nude mice, it typically takes more than 90 days to detect
MDA-MB-231-luc2 and MDA-MB-231-luc2-tdTomato cells were implanted into the mammary fat pads of athymic nude mice.
metastasis in the secondary sites. Therefore, to study the tumor behavior or to examine the Tumor growth was monitored using bioluminescent imaging. Tumor volumes were measured using a caliper. Bioluminescent
drug efficacy, one has to wait for a long time to see metastasis with MDA-MB-231 cell line. signals were quantitated using Living Image software. Representative mice were shown. (A) Mouse implanted with MDA-
MB-231-luc2 cells. (B) Mouse implanted with MDA-MB-231-luc2-tdTomato cells. Graphs next to the animals images are Phospho Akt Phospho-S6
Here, we generated tumor cell lines that were derived from MDA-MB-231 originated cells. We quantitation for tumor volumes, bioluminescent signals, and fluorescent signals.
took MDA-MB-231 cells that were labeled with either luciferase (MDA-MB-231-luc2) or
luciferase & tdTomato fluorescent protein (MDA-MB-231-luc2-tdTomato). These cells were
implanted into mammary fat pads of nude mice and secondary tumors were isolated from
lymph nodes. Tissues were dissociated to single cells and clonal cell lines were established 2 Tumor metastases with MDA-MB-231-luc2 and MDA-MB-
(MDA-MB-231-luc2-LN and MDA-MB-231-luc2-tdTomato-LN). The growth patterns of these luc2-tdTomato cells and establishment of new cell lines 5 Instruments
cells were compared to their corresponding parental cells. To find out the metastasis patterns
of these cells, we implanted new cell lines orthotopically into nude mice. Our results showed
that these cell lines showed faster metastasis than parental cell lines. Moreover, we examined Spectral Unmixing
Day 107 Day 115 Day 126 Ex vivo Growth Curve IVIS and Nuance camera
biomarker expression patterns with multiplexing multispectral microscopy. These cells can be
MDA-MB-231-luc2
used to study tumor metastasis and drug discovery using non-invasive in vivo imaging. Acquisition
Materials & Methods
MDA-MB-231-luc2
MDA-MB-231-luc2-tdTomato vs. MDA-MB-luc2- RGB
Cell culture
tdTomato-LN
Spectra of labels
-tdTomato
7.00E+05
6.00E+05
Human breast cancer cell line MDA-MB-231 cells were transfected with luciferase 2 cDNA (MDA-MB- 5.00E+05
4.00E+05
Label A Label
3.00E+05 C
231-luc2, PerkinElmer). MDA-MB-231-luc2-tdTomato cells were generated by transfecting tdTomato 2.00E+05
1.00E+05
cDNA into MDA-MB-231-luc2 cells. Cells were grown in MEM media supplemented with 10% fetal 0.00E+00
0 24
MDA-MB-231-luc2-tdTomato
48 72
MDA-MB-231-luc2-tdTomato-LN
96
bovine serum (Hyclone) without antibiotics. Growth curves were generated by seeding 100,000 cells Label B Label
RGB D
in a T25 flask. At each time point, cells were trypsinized and counted using an automatic cell counter Representatio Once unmixed, labels can
MDA-MB-231-luc2 and MDA-MB-231-luc2-tdTomato cells were implanted into the mammary fat pads of athymic nude mice n of Spectral be measured accurately.
Cube
(Nexcelom, Lawrence, MA). Total numbers of cells were plotted in a logarithmic scale. Secondary and tumor metastases were monitored using bioluminescent imaging. Representative mice are shown. Ex vivo images of
Autofluorescence
tumor cell lines were established using limited dilution. The luminescence and/or fluorescence excised lymph nodes and growth curves are shown. Top Row: MDA-MB-231-luc2 cells. Bottom row: MDA-MB-231-luc2-
(removed from quantitation)
tdTomato cells.
stability were monitored for 4 weeks. IVIS Spectrum and Nuance camera are shown. Whole animal in vivo imaging was done using an IVIS Spectrum. Multispectral
fluorescence microscopic images were taken using a Nuance camera. Spectral unmixing for in vivo and in vitro fluorescence
imaging was done using Living Image and Nuance software respectively.
Tumor implantation
All the procedures for animal care and tumor cell implantation followed the approved animal protocols
and guidelines of the Institutional Animal Care and Use Committee (IACUC). Prior to implantation, all
3 Tumor metastases with MDA-MB-231-luc2-LN cells
tumor cells were tested for the presence of mycoplasma and mouse pathogens. Female athymic
Mouse #2 Mouse #5 Mouse #6 Mouse #8 Mouse #9
nude mice (Charles River) were anesthetized with isoflurane and two million MDA-MB-231-luc2 or
MDA-MB-231-luc2-tdTomato cells were orthotopically implanted into mammary fat pads of athymic Summary
nude mice.
1. Tumor growth was monitored using MDA-MB-231-luc2 and MDA-MB-
MDA-MB-231-luc2-LN
Day 24
In vitro and in vivo fluorescent and bioluminescent imaging luc2-tdTomato cells in vivo using bioluminescence and/or
For in vivo imaging, mice were anesthetized with isoflurane. Mice were subjected to in vivo tdTomato fluorescence imaging in real time.
imaging using the IVIS Spectrum (PerkinElmer). Multispectral images were spectrally unmixed using
Living Image software (PerkinElmer). For MDA-MB-231-luc2 tumor imaging in vivo, mice were
injected with D-luciferin. Bioluminescent images were taken using IVIS Spectrum. Once tumor 2. Secondary tumors were isolated from metastasized lymph nodes
metastasized into lymph nodes, secondary tumor was excised from the animals and bioluminescent and new cell lines were established.
images were taken using IVIS Spectrum.
Day 32
3. Newly established cell lines showed different growth patterns from
Histology, Immunofluorescence and tissue image processing
Tissues were harvested at post-implantation day 7 and embedded in paraffin. Sections were cut at 7 those of parental cell lines.
mm thickness. For MDA-MB-231-luc2-LN cells, PathScan Signaling Nodes Mutiplex IF Kit (Cell
Signaling Technology) was used. Staining procedures followed the manufacture’s recommendation.
MDA-MB-231-luc2-LN cells were implanted into the mammary fat pads of athymic nude mice (n=12). Tumor growth was
4. MDA-MB-231-luc2-LN and MDA-MB-231-luc2-tdTomato-LN cell lines
Once the staining was done, cells were mounted using DAPI containing mounting media. Multi- monitored using bioluminescent imaging. Two animals showed no palpable tumor. Six representative animals are shown at day 24
and 32. For imaging, primary tumors were masked to detect metastases in the lymph nodes. Quantitation of bioluminescence
metastasize faster than parental cells.
spectral images were taken with an epifluorescence microscope (Nikon E400) equipped with Nuance
was performed using Living Image software. MDA-MB-231-luc2-tdTomato-LN cells showed similar pattern of metastases (Data
camera (PerkinElmer). not shown).