Metastasis of breast cancer cells to the bone, lung, and lymph nodes promotes resistance to ionizing radiation

Abstract

Background

Metastasis represents the leading cause of breast cancer deaths, necessitating strategies for its treatment. Although radiotherapy is employed for both primary and metastatic breast cancers, the difference in their ionizing radiation response remains incompletely understood. This study is the first to compare the radioresponse of a breast cancer cell line with its metastatic variants and report that such metastatic variants are more radioresistant.

Materials and methods

A luciferase expressing cell line was established from human basal-like breast adenocarcinoma MDA-MB-231 and underwent in vivo selections, whereby a cycle of inoculations into the left cardiac ventricle or the mammary fat pad of athymic nude mice, isolation of metastases to the bone, lung and lymph nodes visualized with bioluminescence imaging, and expansion of obtained cells was repeated twice or three times. The established metastatic cell lines were assessed for cell proliferation, wound healing, invasion, clonogenic survival, and apoptosis.

Results

The established metastatic cell lines possessed an increased proliferative potential in vivo and were more chemotactic, invasive, and resistant to X‑ray-induced clonogenic inactivation and apoptosis in vitro.

Conclusion

Breast cancer metastasis to the bone, lung, and lymph nodes promotes radioresistance.

Zusammenfassung

Hintergrund

Metastasierung ist die Hauptursache für den tödlichen Verlauf von Brustkrebserkrankungen. Darauf müssen spezifische Behandlungsstrategien ausgerichtet werden. Sowohl primäre als auch metastatische Brustkrebsarten können mit einer Strahlentherapie behandelt werden, allerdings sind die Unterschiede in der Reaktion auf ionisierende Strahlung bis heute nicht vollständig verstanden. In dieser Studie wird zum ersten Mal die Strahlenantwort einer Brustkrebszelllinie mit der ihrer metastatischen Varianten verglichen und die erhöhte Strahlenresistenz der metastatischen Varianten gezeigt.

Material und Methoden

Eine Luciferase-exprimierende Zelllinie wurde aus humanen basaloiden Brustadenokarzinomen MDA-MB-231 etabliert und in zwei oder drei Zyklen in vivo selektiert. Dabei wurden die Zellen entweder in den linken Herzventrikel oder in das Fettgewebe der Brust athymischer Nacktmäuse inokuliert. Die durch Biolumineszenz sichtbar gemachten Metastasen wurden aus Knochen, Lunge und Lymphknoten isoliert und expandiert. Die etablierten metastatischen Zelllinien wurden auf Zellproliferation, Wundheilung, Invasion, Klonüberleben und Apoptose getestet.

Ergebnisse

Die etablierten metastatischen Zelllinien wiesen in vivo ein gesteigertes proliferatives Potenzial auf. In vitro zeigten sie erhöhte chemotaktische und invasive Aktivität, zudem besaßen sie eine erhöhte Resistenz gegen röntgenstrahleninduzierte klonogene Inaktivierung und Apoptose.

Schlussfolgerung

Metastasen in Knochen, Lunge und Lymphknoten erhöhen die Strahlenresistenz von Brustkrebs.

Caption Electronic Supplementary Material

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Fig. S1 A flow diagram outlining the isolation of metastases. pGL4.5 cells that stably express luciferase were established from a human basal-like breast cancer cell line MDA-MB-231. pGL4.5 cells were inoculated into the mammary fat pad or the left cardiac ventricle of athymic nude mice. At 20–30 days after inoculation, D‑Luciferin-administered mice underwent bioluminescence imaging. Metastases to the bone, lung and lymph nodes were isolated, and cells were expanded

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Fig. S2 A flow chart schematizing the establishment of metastatic cell lines from pGL4.5 cells by repetitive in vivo selections. pGL4.5 cells underwent injection into the mammary fat pad (MFP) or left ventricular cardiac injection (LVC). Metastases to the bone, lung and lymph nodes were isolated, and cells were expanded. With this first in vivo selection, three, five and four cell lines were obtained from metastases to the lymph nodes, lung and bone (data not shown). Of these, NLN0001, NLU0002 and NBOS0002 cells underwent the second in vivo selection, from which five, three and five cell lines were obtained, respectively (data not shown). From these, NLU0022 and NBOS0022 cells were inoculated into the left cardiac ventricle of five and six mice, respectively, for the third in vivo selection. Cells expanded from isolated lung metastases derived from NLU0022 in five mice were mixed, and named NLU0041 cells. Likewise, cells expanded from isolated bone metastases derived from NBOS0022 in six mice were mixed, and named NBOS0042 cells. For the data in Figs. 1, 2, 3, 4 and 5 and S3, pGL4.5, NLN0001, NLN0104, NLU0041 and NBOS0042 were used (highlighted with boxed characters in the schema)

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Fig. S3 Cell proliferation in vitro tested with cell counting and an Alamar Blue assay. a pGL4.5 (circles), NBOS0042 (squares), NLU0041 (diamonds) and NLN0104 (triangles) cells were counted at every 24–196 h after plating. Growth curves were fitted against the data for the cell numbers at 24–120 h after plating to the exponential equation y = a * exp (b * x), where y, x, a and b are the cell numbers, time in h, intercept and slope, respectively. Doubling time (T _D) in h was calculated as ln (2 / b). b At every 24–120 h after plating, Alamar Blue was added and fluorescence was measured. For each of the pGL4.5 (black), NBOS0042 (medium grey), NLU0041 (grey) and NLN0104 (light grey) cells, the data for 48–120 h time points are presented as fold changes relative to the data for the 24 h time point, and fold changes represent the means and SD of five independent experiments with triplicate measurements

Table S1 Frequency of metastasis in pGL4.5 cells and its metastatic cell lines