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Preclinical evaluation of PEGylated liposomal doxorubicin as an effective radiosensitizer in chemoradiotherapy for lung cancer

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Abstract

Purpose

Development of a safe and effective systemic chemotherapeutic agent for concurrent administration with definitive thoracic radiotherapy remains a major goal of lung cancer management. The synergistic effect of PEGylated liposomal doxorubicin and irradiation was evaluated in lung cancer cell lines both in vitro and in vivo.

Methods

In vitro radiosensitization of A549 and LLC cell lines was evaluated by colony formation assay, γH2AX fluorescent staining and western blot assay, and annexin V staining. A radiosensitization study with healthy human lung-derived cell line BEAS-2B was performed for comparative purposes. In vivo radiosensitization was evaluated by tumor ectopic growth, cell survival, pharmacokinetics, and biodistribution analyses. Cleaved caspase‑3, the marker for apoptosis, was assessed immunohistochemically in A549 xenograft tumors.

Results

Treatment with PEGylated liposomal doxorubicin decreased A549 and LLC cell proliferation in a dose-dependent manner. In vitro studies revealed comparable radiosensitizer advantages of PEGylated liposomal doxorubicin and free doxorubicin, showing equivalent DNA double-strand breaks according to γH2AX fluorescent staining and western blot assays, similar numbers of apoptotic cells in the annexin‑V staining assay, and moderately decreased clonogenic survival. In vivo studies demonstrated markedly slow ectopic tumor growth with prolonged survival following treatment with PEGylated liposomal doxorubicin plus irradiation in both A549 and LLC mouse models, suggesting that PEGylated liposomal doxorubicin is more effective as a radiosensitizer than free doxorubicin in vivo. Pharmacokinetics evaluation showed a longer half-life of approximately 40 h for PEGylated liposomal doxorubicin, confirming that the liposomal carrier achieved controlled release. Biodistribution evaluation of PEGylated liposomal doxorubicin confirmed high accumulation of doxorubicin in tumors, indicating the promising drug delivery attributes of PEGylated liposomal doxorubicin. Although free doxorubicin caused histopathologic myocarditis with the cardiac muscle fibers showing varying degrees of damage, PEGylated liposomal doxorubicin caused no such effects. The immunohistochemical expression of cleaved caspase-3-positive cells was greatest expressed in the irradiation and PEGylated liposomal doxorubicin combined treatment group, indicating prolonged tumoricidal effects.

Conclusions

Our study provides preclinical in vitro and in vivo evidence of the effectiveness of PEGylated liposomal doxorubicin as a radiosensitizer, supporting its potential clinical development as a component of chemoradiotherapy.

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Acknowledgements

We would like to thank the staff of the Core Lab at the Department of Medical Research, National Taiwan University Hospital, for their technical support.

Funding

This work was supported by the National Taiwan University Hospital (grant number NTUH 109-N4547) and the Ministry of Science and Technology (MST, Taiwan, Contract No. 108-2314-B-002-035-MY2 and No. 109-2314-B-002-094-MY2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Radiology, National Taiwan University College of Medicine, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan

    Jenny Ling-Yu Chen M.D., Ph.D., Chun-Kai Pan M.S. & Yu-Sen Huang M.D. Ph.D.

  2. Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan

    Jenny Ling-Yu Chen M.D., Ph.D., Wen-Chi Yang M.D., Feng-Ming Hsu M.D. Ph.D. & Sung-Hsin Kuo M.D. Ph.D.

  3. Cancer Center, College of Medicine, National Taiwan University, No. 57, Lane 155, Section 3, Keelung Road, Taipei, Taiwan

    Jenny Ling-Yu Chen M.D., Ph.D., Wen-Chi Yang M.D., Feng-Ming Hsu M.D. Ph.D. & Sung-Hsin Kuo M.D. Ph.D.

  4. Department of Medical Research, National Taiwan University Hospital, No. 7, Chung-Shan South Road, 100, Taipei, Taiwan

    Chun-Kai Pan M.S., Yu-Li Lin Ph.D. & Ching-Yi Tsai M.S.

  5. Institute of Toxicology, National Taiwan University College of Medicine, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan

    Ching-Yi Tsai M.S.

  6. Department of Medical Imaging, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan

    Yu-Sen Huang M.D. Ph.D.

  7. Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan

    Ming-Jium Shieh M.D. Ph.D.

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  1. Jenny Ling-Yu Chen M.D., Ph.D.
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  2. Chun-Kai Pan M.S.
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  3. Yu-Li Lin Ph.D.
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Contributions

JC, CP, CT, YH, and YL performed the experiments. JC, CP, FH, and WY analyzed and interpreted the data. JC, CP, YH, and YL were major contributors in writing the manuscript. SK and MS supervised the study, reviewed and edited the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yu-Li Lin Ph.D..

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Conflict of interest

J.L.-Y. Chen, C.-K. Pan, Y.-L. Lin, C.-Y. Tsai, Y.-S. Huang, W.-C. Yang, F.-M. Hsu, S.-H. Kuo and M.-J. Shieh declare that they have no competing interests.

Ethical standards

All in vivo experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC 20180464). Humane endpoints were determined according to a clinical scoring system based on that outlined by the IACUC of our institution.

Supplementary Information

66_2021_1835_MOESM1_ESM.pdf

Supplementary Figure 1. Tumor cell viability was evaluated using Cell Counting kit-8 (Sigma-Aldrich, Gillingham, UK). Treatment with pegylated liposomal doxorubicin decreases cell proliferation in a dose-dependent manner.

66_2021_1835_MOESM2_ESM.pdf

Supplementary Figure 2. BALB/c athymic nude mice bearing established subcutaneous A549 cells (mean starting tumor volume = 30 mm3) and C57BL/6 mice bearing established subcutaneous LLC tumors (mean starting tumor volume = 40 mm3) were randomized into five treatment groups and a control group. PEGylated liposomal doxorubicin enhanced tumor suppressive activity and prolonged survivals in both A549 and LLC ectopic mouse models. Mice bearing A549 ectopic tumors were sacrificed on day 45. The immunohistochemically stained cleaved caspase-3 intensity of ectopic tumors was scored using the threshold method in ImageJ and based on three slides per group.

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Chen, J.LY., Pan, CK., Lin, YL. et al. Preclinical evaluation of PEGylated liposomal doxorubicin as an effective radiosensitizer in chemoradiotherapy for lung cancer. Strahlenther Onkol 197, 1131–1142 (2021). https://doi.org/10.1007/s00066-021-01835-9

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