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Purpose: To identify differential cellular responses after proton and photon irradiation by comparing transcriptomes of primary fibroblasts irradiated with either radiation type. Methods and Materials: A panel of primary dermal fibroblast cultures was irradiated with low and higher linear energy transfer (LET) proton beams. Cobalt-60 photon irradiation was used as reference. Dose was delivered in 3 fractions of 3.5 Gy (relative biological effectiveness) using a relative biological effectiveness of 1.1 for proton doses. Cells were harvested 2 hours after the final fraction was delivered, and RNA was purified. RNA sequencing was performed using Illumina NextSeq 500 with high-output kit. The edgeR package in R was used for differential gene expression analysis. Results: Pairwise comparisons of the transcriptomes in the 3 treatment groups showed that there were 84 and 56 differentially expressed genes in the low LET group compared with the Cobalt-60 group and the higher LET group, respectively. The higher LET proton group and the Cobalt-60 group had the most distinct transcriptome profiles, with 725 differentially regulated genes. Differentially regulated canonical pathways and various regulatory factors involved in regulation of biological mechanisms such as inflammation, carcinogenesis, and cell cycle control were identified. Conclusions: Inflammatory regulators associated with the development of normal tissue complications and malignant transformation factors seem to be differentially regulated by higher LET proton and Cobalt-60 photon irradiation. The reported transcriptome differences could therefore influence the progression of adverse effects and the risk of developing secondary cancers.

The transcriptional response of cells exposed to proton radiation is not equivalent to the response induced by traditional photon beams. Changes in cellular signalling is most commonly studied using the method Quantitative polymerase chain reaction (qPCR). Stable reference genes must be used to accurately quantify target transcript expression. The study aim was to identify suitable reference genes for normalisation of gene expression levels in normal dermal fibroblasts irradiated with either proton or photon beams. The online tool RefFinder was used to analyse and identify the most stably expressed genes from a panel of 22 gene candidates. To assess the reliability of the identified reference genes, a selection of the most and least stable reference genes was used to normalise target transcripts of interest. Fold change levels varied considerably depending on the used reference gene. The top ranked genes IPO8, PUM1, MRPL19 and PSMC4 produced highly similar target gene expression, while expression using the worst ranked genes, TFRC and HPRT1, was clearly modified due to reference gene instability.

Introduction: Proton beam therapy delivers a more conformal dose distribution than conventional radiotherapy, thus improving normal tissue sparring. Increasing linear energy transfer (LET) along the proton track increases the relative biological effectiveness (RBE) near the distal edge of the Spread-out Bragg peak (SOBP). The severity of normal tissue side effects following photon beam radiotherapy vary considerably between patients. Aim: The dual study aim was to identify gene expression patterns specific to radiation type and proton beam position, and to assess whether individual radiation sensitivity influences gene expression levels in fibroblast cultures irradiated in vitro. Methods: The study includes 30 primary fibroblast cell cultures from patients previously classified as either radiosensitive or radioresistant. Cells were irradiated at three different positions in the proton beam profile: entrance, mid-SOBP and at the SOBP distal edge. Dose was delivered in three fractions × 3.5 Gy(RBE) (RBE 1.1). Cobalt-60 (Co-60) irradiation was used as reference. Real-time qPCR was performed to determine gene expression levels for 17 genes associated with inflammation response, fibrosis and angiogenesis. Results: Differences in median gene expression levels were observed for multiple genes such as IL6, IL8 and CXCL12. Median IL6 expression was 30%, 24% and 47% lower in entrance, mid-SOBP and SOBP distal edge groups than in Co-60 irradiated cells. No genes were found to be oppositely regulated by different radiation qualities. Radiosensitive patient samples had the strongest regulation of gene expression; irrespective of radiation type. Conclusions: Our findings indicate that the increased LET at the SOBP distal edge position did not generally lead to increased transcriptive response in primary fibroblast cultures. Inflammatory factors were generally less extensively upregulated by proton irradiation compared with Co-60 photon irradiation. These effects may possibly infl