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

Cell culture

Human breast cancer cell line MDA-MB-231 cells were transfected with luciferase 2 cDNA (MDA-MB-231-luc2, PerkinElmer). MDA-MB-231-luc2-tdTomato cells were generated by transfecting tdTomato

cDNA into MDA-MB-231-luc2 cells. Cells were grown in MEM media supplemented with 10% fetal

bovine serum (Hyclone) without antibiotics. Growth curves were generated by seeding 100,000 cells

in a T25 flask. At each time point, cells were trypsinized and counted using an automatic cell counter

(Nexcelom, Lawrence, MA). Total numbers of cells were plotted in a logarithmic scale. Secondary tumor cell lines were established using limited dilution. The luminescence and/or fluorescence

stability were monitored for 4 weeks.

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

tumor cells were tested for the presence of mycoplasma and mouse pathogens. Female athymic

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

nude mice.

In vitro and in vivo fluorescent and bioluminescent imaging

For in vivo imaging, mice were anesthetized with isoflurane. Mice were subjected to in vivo tdTomato

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

metastasized into lymph nodes, secondary tumor was excised from the animals and bioluminescent

images were taken using IVIS Spectrum.

Histology, Immunofluorescence and tissue image processing Tissues were harvested at post-implantation day 7 and embedded in paraffin. Sections were cut at 7

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.

Once the staining was done, cells were mounted using DAPI containing mounting media. Multi-

spectral images were taken with an epifluorescence microscope (Nikon E400) equipped with Nuance camera (PerkinElmer).

Abstract 1

Tumor metastases with MDA-MB-231-luc2 and MDA-MB-luc2-tdTomato cells and establishment of new cell lines

In vivo monitoring of tumor growth

Summary

MDA-MB-231-luc2-LN cells were implanted into the mammary fat pads of athymic nude mice (n=12). Tumor growth was

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

was performed using Living Image software. MDA-MB-231-luc2-tdTomato-LN cells showed similar pattern of metastases (Data not shown).

1. Tumor growth was monitored using MDA-MB-231-luc2 and MDA-MB-

luc2-tdTomato cells in vivo using bioluminescence and/or

fluorescence imaging in real time.

2. Secondary tumors were isolated from metastasized lymph nodes

and new cell lines were established.

3. Newly established cell lines showed different growth patterns from those of parental cell lines.

4. MDA-MB-231-luc2-LN and MDA-MB-231-luc2-tdTomato-LN cell lines

metastasize faster than parental cells.

4 Biomarkers in MDA-MB-231-luc2-LN cells

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.

5 Instruments

IVIS and Nuance camera

Autofluorescence (removed from quantitation)

Spectral Unmixing

Acquisition

RGB

Representatio

n of Spectral

Cube

Label A

Label B

Label C

Label D

RGB

Spectra of labels

Once unmixed, labels can be measured accurately.

Materials & Methods

2

3 Tumor metastases with MDA-MB-231-luc2-LN cells

Large

Very Large

Very Large

MDA-MB-231-luc2 and MDA-MB-231-luc2-tdTomato cells were implanted into the mammary fat pads of athymic nude mice.

Tumor growth was monitored using bioluminescent imaging. Tumor volumes were measured using a caliper. Bioluminescent 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 quantitation for tumor volumes, bioluminescent signals, and fluorescent signals.

MDA-MB-231-luc2 and MDA-MB-231-luc2-tdTomato cells were implanted into the mammary fat pads of athymic nude mice

and tumor metastases were monitored using bioluminescent imaging. Representative mice are shown. Ex vivo images of excised lymph nodes and growth curves are shown. Top Row: MDA-MB-231-luc2 cells. Bottom row: MDA-MB-231-luc2-

tdTomato cells.

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Background

Cultured MDA-MB-231-luc2-LN cells were stained with

PathScan signaling nodes multiplex immunofluorescence kit (Cell Signaling Technology).

Cells were counter stained with DAPI and fluorescent

images were taken using Nuance camera. Each signal was spectrally unmixed using Nuance software. (A)

Image before unmixing, (B) Spectrally unmixed image, (C) Unmixed DAPI image, (D) Unmixed Phospho-

Erk1/2 image (Alexa Flour 488), (E) Unmixed Phospho

Akt (Alexa Flour 555), (F) Unmixed Phospho-S6 ribosomal protein (Alexa Flour 647).

A B

C D

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DAPI Phospho-Erk1/2

Phospho Akt Phospho-S6

Before Unmixing After Unmixing

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MDA-MB-231-luc2-tdTomato vs. MDA-MB-luc2-tdTomato-LN

MDA-MB-231-luc2-tdTomato MDA-MB-231-luc2-tdTomato-LN

Bioluminescence Tumor volume

Bioluminescence Tumor volume Fluorescence

Growth Curve

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Whole animal non-invasive imaging contributes significantly to understand tumor behavior. It

also plays a critical role in drug discovery and development. Optical imaging is convenient

because it does not require radioactive materials for imaging. Especially in preclinical

applications, optical imaging can be a very useful tool because genetic modification is

feasible. The most popular optical imaging is using bioluminescence. We introduced various

cancer cell lines that express firefly luciferase. These cells have stable expression of light

emission for prolonged cell culture situation. This enables researchers to implant the cells into

animals and monitor tumor development and metastasis. In addition, these tumor cells can be

detected using fluorescent agents that target tumor cells. As a consequence, one can co-

register both bioluminescent and fluorescent images. Cells also can be labeled with both

bioluminescent and fluorescent markers such as luciferases and fluorescent proteins.

Although there are many different types of cell lines available for different tumor types,

studying metastasis can be challenging. That is mainly because most cell lines show delayed

metastasis when implanted in the animal. To expedite the metastasis, intravenous injection or

intracardiac injection is performed to generate secondary tumors in the animal. However,

these methods do not represent true metastasis from originated organs. One of the most

popular breast cancer cell line is MDA-MB-231. When these cells are implanted into

mammary fat pads of female nude mice, it typically takes more than 90 days to detect

metastasis in the secondary sites. Therefore, to study the tumor behavior or to examine the

drug efficacy, one has to wait for a long time to see metastasis with MDA-MB-231 cell line.

Here, we generated tumor cell lines that were derived from MDA-MB-231 originated cells. We

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

(MDA-MB-231-luc2-LN and MDA-MB-231-luc2-tdTomato-LN). The growth patterns of these

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

biomarker expression patterns with multiplexing multispectral microscopy. These cells can be

used to study tumor metastasis and drug discovery using non-invasive in vivo imaging.