Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice

Abstract

Introduction: Our goal was to generate xenograft mouse models of human breast cancer based on luciferase-expressing MDA-MB-231 tumor cells that would provide rapid mammary tumor growth; produce metastasis to clinically relevant tissues such as lymph nodes, lung, and bone; and permit sensitive in vivo detection of both primary and secondary tumor sites by bioluminescent imaging.

Method: Two clonal cell sublines of human MDA-MB-231 cells that stably expressed firefly luciferase were isolated following transfection of the parental cells with luciferase cDNA. Each subline was passaged once or twice in vivo to enhance primary tumor growth and to increase metastasis. The resulting luciferase-expressing D3H1 and D3H2LN cells were analyzed for long-term bioluminescent stability, primary tumor growth, and distal metastasis to lymph nodes, lungs, bone and soft tissues by bioluminescent imaging. Cells were injected into the mammary fat pad of nude and nude-beige mice or were delivered systemically via intracardiac injection. Metastasis was also evaluated by ex vivo imaging and histologic analysis postmortem.

Results: The D3H1 and D3H2LN cell lines exhibited long-term stable luciferase expression for up to 4-6 months of accumulative tumor growth time in vivo. Bioluminescent imaging quantified primary mammary fat pad tumor development and detected early spontaneous lymph node metastasis in vivo. Increased frequency of spontaneous lymph node metastasis was observed with D3H2LN tumors as compared with D3H1 tumors. With postmortem ex vivo imaging, we detected additional lung micrometastasis in mice with D3H2LN mammary tumors. Subsequent histologic evaluation of tissue sections from lymph nodes and lung lobes confirmed spontaneous tumor metastasis at these sites. Following intracardiac injection of the MDA-MB-231-luc tumor cells, early metastasis to skeletal tissues, lymph nodes, brain and various visceral organs was detected. Weekly in vivo imaging data permitted longitudinal analysis of metastasis at multiple sites simultaneously. Ex vivo imaging data from sampled tissues verified both skeletal and multiple soft tissue tumor metastasis.

Conclusion: This study characterized two new bioluminescent MDA-MB-231-luc human breast carcinoma cell lines with enhanced tumor growth and widespread metastasis in mice. Their application to current xenograft models of breast cancer offers rapid and highly sensitive detection options for preclinical assessment of anticancer therapies in vivo.

Figure 1

In vitro bioluminescence of MDA-MB-231 sublines D3, D3H1 and D3H2LN. Cells from each subline were serially diluted in duplicate wells from 4000 to 8 cells/well. Luciferin substrate was added to each well and the plate was imaged to obtain photons/s per cell. Wells with media (no cells) or cells alone (no luciferin) were included as controls. The range of bioluminescence for each subline represents data collected from two to three experiments.

Figure 2

Mammary tumor growth is enhanced in D3H1 and D3H2LN bioluminescent sublines of MDA-MB-231 cells. Cells were injected into the mammary fat pad of (a) nude or (b) nude-beige mice, and mean tumor volumes were determined by caliper measurements over time. The tumors from the bioluminescent sublines D3H1 and D3H2LN were also monitored using in vivo imaging, and the correlation between mean photons/s and mean tumor volume are indicated as R² values.

Figure 3

D3H2LN mammary tumors grow rapidly and produce bilateral lymph node metastasis. Bioluminescent D3H2LN cells were injected into nude-beige mice, and tumor growth and metastasis were monitored over time in vivo. Data for a representative mouse are shown. Tumor take rate was high (6/8 mice) and lymph node metastasis to both the right and left thoracic areas was detected by week 9 in all mice with mammary tumors (upper panels). Ex vivo images of excised brachial lymph nodes and lung lobes are shown (middle panels). Histological analysis of the same tissues confirmed perfuse metastasis to the lymph nodes and isolated micrometastasis to the lung lobes (lower panels).

Figure 4

Multiple metastases detected in vivo following intracardiac injection of D3H2LN cells. Ventral (upper panels) and dorsal (lower panels) images taken over time from a representative nude mouse injected with D3H2LN cells are shown. Pseudocolor scale bars were consistent for all images of ventral or dorsal views in order to show relative changes at metastatic sites over time.

Figure 5

Ex vivo and histological data confirm metastases from intracardiac injection of D3H2LN cells. Representative data from selected tissues from two mice that were evaluated by both ex vivo imaging and subsequent histological analysis are shown.