Palao, N. | bioRxiv Science

bioRxiv2024

In vitro FLASH irradiation of A549 lung cancer cells and IMR90 healthy human fibroblasts in the synchrocyclotron room of a clinical proton therapy system

PurposeThis study aims to investigate the FLASH irradiation effect on lung tumor (A549) and healthy fibroblast (IMR90) cell lines using an irradiation station installed at the synchrocyclotron room of a clinical proton facility without any permanent beamline modifications. Methods and MaterialsAn irradiation system composed of a lead scatterer and 3D-printed positioning system was designed and fabricated to operate within the beamline gap of the IBA Proteus One proton therapy facility after the extraction of the beam from the synchrocyclotron. A dosimetric analysis of the produced irradiation field was carried out using radiochromic films. FLASH and conventional-rate irradiations were conducted on relevant cell lines for lung cancer at the isocenter of the treatment room. Biological assessments post-irradiation included clonogenic and viability assays for cell survival, immunofluorescence analysis of p21 protein expression, and flow cytometry analysis for cell cycle arrest evaluation. ResultsThe irradiation system successfully delivered homogeneous and repeatable FLASH dose rates (>900 Gy/s) with a positioning accuracy of 1 mm and dose uniformity within 10%. Clonogenic assays revealed no statistically significant differences in survival between FLASH and conventional dose rates for both A549 and IMR90 cell lines, although a trend towards higher viability was observed in IMR90 cells under FLASH conditions. Flow cytometry demonstrated significant differences in cell cycle arrest patterns at doses above 7 Gy, with FLASH-irradiated cells exhibiting a decrease in G2/M phase arrest compared to conventional rates. Immunofluorescence analysis of p21 expression showed no significant differences between irradiation modalities. ConclusionsThe developed irradiation station effectively facilitates FLASH radiotherapy experiments in a clinical proton facility, achieving the necessary dose rates without hardware modification or extra tuning of the facility. Our analysis reported notable alterations in cell cycle dynamics suggesting distinct biological responses between FLASH and conventional rates in both healthy and tumor cells. These findings contribute to the emerging understanding of the FLASH effect and support the potential for its differential impact on cancerous versus healthy tissues.

bioengineering↗

bioRxiv2023

CRISPR/Cas9 screenings unearth protein arginine methyltransferase 7 as a novel driver of metastasis in prostate cancer

Owing to the inefficacy of available treatments, the survival rate of patients with metastatic prostate cancer (mPCa) is severely decreased. Therefore, it is crucial to identify new therapeutic targets to increase their survival. This study aim was to identify the most relevant regulators of mPCa onset by performing two high-throughput CRISPR/Cas9 screenings. Furthermore, some of the top hits were validated using small interfering RNA (siRNA) technology, with protein arginine methyltransferase 7 (PRMT7) being the best candidate. Its inhibition or depletion via CRISPR significantly reduced mPCa cell capacities in vitro. Moreover, PRMT7 ablation reduced mPCa appearance in chicken chorioallantoic membrane and mouse xenograft assays. Molecularly, PRMT7 reprograms the expression of several adhesion molecules through methylation of several transcription factors, such as FoxK1 or NR1H2, which results in primary tumor PCa cell adhesion loss and motility gain. Importantly, PRMT7 is upregulated in advanced stages of Spanish PCa tumor samples and PRMT7 pharmacological inhibition reduces the dissemination of mPCa cells. Thus, here is shown that PRMT7 is a potential therapeutic target and biomarker of mPCa.

cancer biology↗