Combined radiotherapy with doxorubicin in preclinical soft tissue sarcoma and endothelial cell models: evaluation of the variable relative biological effectifness
Year: 2025
Type: dissertation
Abstract: Summary Multimodal approaches in cancer treatment include combinations of surgery, chemotherapy (CTX), and radiotherapy (RT). Sarcomas are highly aggressive mesenchymal tumours that represent a heterogenous group of rare cancers with high genomic complexity. Sarcoma can occur throughout the body and at all ages and can be divided into bone sarcoma and soft tissue sarcoma (STS). STS is treated according to subtype in expert reference centres. Doxorubicin (Dox) is a first-line drug for STS which inhibits deoxyribonucleic acid (DNA) transcription as an alkylating agent. STS treatment with systemic CTX such as Dox targets tumour tissue but causes side effects in normal tissues. Locoregional RT affects normal tissue around the tumour in a manner dependent on tumour localisation. The overall goal of cancer treatment is to kill the tumour cells while limiting damage to normal tissue. In general, RT uses ionising radiation (IR) that can cause direct DNA damage or indirect damage of cellular structures via generation of reactive oxygen species (ROS). DNA damage is detected by cells, and repair can be initiated via different pathways. If cells re-enter the cell cycle without sufficient DNA damage repair, cell death is induced. The principle of dose fractionation relies on less-efficiently repaired DNA damage in rapidly proliferating tumour cells relative to slowly proliferating normal tissue cells, resulting in higher tumour cell death rates as normal tissue recovers. Thus, the “therapeutic window” describes dose–volume constraints balancing tumour control and toxicity to normal tissue. New therapeutic options seek to widen the therapeutic window. While photons (X) deliver their maximal dose in the beginning of their range, protons (H) are characterised by an inverse depth dose profile, sparing normal tissue before and especially behind the tumour. In general, treatment planning for proton beam therapy (PBT) assumes 10%-higher relative biological effectiveness (RBE) of H compared to X. In the concept of a variable RBE, several factors, including linear energy transfer (LET), cell type, experimental endpoint, or position in the treatment field are important, although they are not currently considered in treatment planning. Due to rising LET at the end of the range, H induce more complex and clustered DNA damage relative to X. Paediatric patients or patients with tumours near critical normal tissue structures can benefit from lower normal tissue doses of H. This thesis project addressed four overall aims: (I) Non-inferiority of combined radiochemotherapy with H and Dox were tested in comparison to X and Dox in STS 1 Summary 7 models. (II) The potential impact of sequence of Dox and radiation were evaluated in neoadjuvant, adjuvant, and concurrent settings. (III) Tumour and normal tissue effects were compared. (IV) Finally, measured effects were evaluated in a LET-dependent manner within a proton Bragg peak (BP) curve to extract RBE values. Tumour and normal tissue cells were irradiated with X or H at mid-spread-out Bragg peak (SOBP), and Dox was applied 3 h before irradiation (DoxA, neoadjuvant), 3 h before irradiation and post irradiation until the end of the experiment (DoxB, concurrent), or only postirradiation until the end of the experiment (DoxC, adjuvant). The first contributed study (13) investigated the radiation responses of the following three preclinical STS cell lines with different TP53 statuses: (a) HT-1080 fibrosarcoma cells (TP53 wildtype), (b) GCT undifferentiated pleomorphic sarcoma cells (containing two heterozygous TP53 mutations), and (c) RD embryonal rhabdomyosarcoma cells (containing a single, homozygous TP53 mutation). Clonogenic survival, cell cycle distribution, apoptotic cell death, proliferation, metabolic viability, morphology, and motility were investigated. RD cells were most radiosensitive among the STS cell panel; in addition, GCT cells showed a chemo-resistant phenotype, whereas HT-1080 cells were radioresistant but sensitive to Dox treatment. In sensitive STS cell lines, prolonged Dox treatment was the most effective sequence. For most endpoints, the combination of H irradiation with Dox produced similar effects compared with X irradiation. Additive effects occurred more frequently with X than with H (13). In the second contributed study (15), the radiation response of HMEC-1 human microvascular endothelial cells was evaluated in the same experimental configuration as the STS cells regarding irradiation, Dox schedule, and endpoints. The results showed that Dox monotreatment produced minor effects on all investigated endpoints in endothelial cells (ECs). However, in combination with irradiation, additive effects for longer Dox schedules were found, for example regarding reduced proliferation. Increased viability of HMEC-1 cells following X irradiation alone or in combination with Dox point towards the onset of senescence. Cellular effects emphasised differential processing of endothelial damage dependent on radiation quality (15). The third contributed study (14) examined LET-dependent RBE changes in STS and microvascular ECs in an experimental configuration with a multi-step range shifter (MSRS). Cells were irradiated with monoenergetic BP curves to represent dose and LET combinations encountered in a treatment field (entrance region, BP, and distal fall-off). Following irradiation with the MSRS, the proliferation and viability of STS cells 2 Zusammenfassung 8 demonstrated a stepwise alteration mimicking an inverse proton depth dose profile. RBE values were extracted from viability and proliferation data of fibrosarcoma and HMEC-1 cells. The results showed an increase of RBE at the BP distal fall-off, with proliferation being the more robust endpoint for these specific experimental conditions (14). In conclusion, this thesis demonstrates (I) that H irradiation at mid-SOBP in combination with Dox treatment exhibited non-inferiority relative to X irradiation in all STS models and that cell-line-specific effects dependent on TP53 mutational status could be detected. Moreover, (II) the sequence of Dox treatment and irradiation impacted the radiation response of STS and ECs, with Dox treatment pre- and postirradiation (DoxB, concurrent) being the most effective. In general, (III) H irradiation in combination with Dox treatment exhibited non-inferiority relative to X irradiation in an EC model, but some indications of different mechanisms of induction of endothelial damage dependent on radiation quality were detected. Finally, (IV) increasing LET along the BP curve enhanced the biological effectiveness of H on viability and proliferation of STS cells in a combined biological assay, presenting two alternative endpoints for RBE calculation relative to clonogenic survival.
Author: Bernardo, Teresa
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W6906544460
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