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Triple negative breast cancer (TNBC) is a heterogeneous and a highly aggressive type of breast cancer. Standard of care for TNBC patients includes surgery, radio-, chemo- and immunotherapy, depending on the stage of the disease. Immunotherapy is ineffective as monotherapy but can be enhanced with taxane chemotherapy or radiotherapy. Radiation can stimulate the immune system by activating the type I interferon (IFN-I) response through cGAS-STING signaling, which recognizes cytosolic double-stranded DNA (dsDNA). Cytosolic dsDNA can be cleared by autophagy, thereby preventing activation of cGAS-STING signaling. Autophagy inhibition was therefore proposed to potentiate the immunostimulatory effects of radiation. Here we show that different molecular features of TNBC cell lines influence the effect of X-ray and carbon ion (C-ion) irradiation and autophagy inhibition on immunogenic signaling. MDA-MB-468, with low basal autophagy and high cytosolic dsDNA, activates the IFN-I response after radiation. In contrast, MDA-MB-231, characterized by high autophagy rates and low cytosolic dsDNA, induces NF-κB signaling and CXCL10 expression upon autophagy inhibition with the VPS34 inhibitor SAR405. Autophagy inhibition in TNBC cells triggers a stronger activation of innate immune cells (monocytes, natural killer cells and dendritic cells) compared to radiation. In BRCA1-mutated MDA-MB-436 cells, C-ion irradiation was more potent compared to X-rays in inducing the NF-κB-driven immunogenic response but failed to activate immune cells. Upregulation of PD-L1 by X-rays, and especially C-ions, may contribute to reduced immune cell activation, underscoring the need for combination strategies with immune checkpoint blockade. Collectively, our study highlights the NF-κB-driven immunostimulatory effects of autophagy inhibition and the importance of understanding the molecular heterogeneity in TNBC with regard to autophagy rates, IFN-I and NF-κB signaling when designing effective treatments that target these pathways.

Microdosimetry is increasingly adopted in the characterization of proton and carbon ion beams used in cancer therapy. Spectra and mean values of lineal energy calculated in frequency and dose are seen by many as the tools which, by complementing dosimetric measurements, allow for the most complete characterization of the therapeutic radiation fields. The urgency is now to consolidate the experience and converge to commonly accepted methodologies. In this context, the purpose of this work is to study the effects of the energy-loss straggling and the delta-ray escape, considering slab-sensitive volumes; these are, in fact, the typical shapes of solid-state microdosimeters, which are widely used in investigating light ion therapy beams. The method considers the energy distribution of delta rays resulting from the collision of the impinging ion and, taking into account the escape, convolutes it with itself as many times as the expected number of collisions in the sensitive volume thickness. The resulting distribution is compared to the experimental microdosimetric spectrum showing a substantially good agreement. The extension of the methodology to a wider range of ion energy and detector characteristics is instrumental for a detector-independent microdosimetric assessment of the radiation fields.

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths worldwide with limited treatment options due to extensive radiation and chemotherapy resistance. Monotherapy with immune checkpoint blockade showed no survival benefit. A combination of immunomodulation and radiotherapy may offer new treatment strategies, as demonstrated for non-small cell lung cancer. Radiation-induced anti-tumour immunity is mediated through cytosolic nucleic acid sensing pathways that drive the expression of interferon beta-1 (IFNB1) and proinflammatory cytokines. Human PDAC cell lines (PANC-1, MIA PaCa-2, BxPC-3) were treated with X-rays and protons. Immunogenic cell death was measured based on HMGB1 release. Cytosolic dsDNA and dsRNA were analysed by immunofluorescence microscopy. Cell cycle progression, MHC-I and PD-L1 expression were determined by flow cytometry. Galectin-1 and IFNB1 were measured by ELISA. The expression levels and the phosphorylation status of the cGAS/STING and RIG-I/MAVS signalling pathways were analysed by western blotting, the expression of and proinflammatory cytokines was determined by RT-qPCR and genome-wide by RNA-seq. CRISPR-Cas9 knock-outs and inhibitors were used to elucidate the relevance of STING, MAVS and NF-κB for radiation-induced IFNB1 activation. We demonstrate that a clinically relevant X-ray hypofractionation regimen (3x8 Gy) induces immunogenic cell death and activates IFNB1 and proinflammatory cytokines. Fractionated radiation induces G2/M arrest and accumulation of cytosolic DNA in PDAC cells, which partly originates from mitochondria. RNA-seq analysis shows a global upregulation of type I interferon response and NF-κB signalling in PDAC cells following 3x8 Gy. Radiation-induced immunogenic response is regulated by STING, MAVS and NF-κB. In addition to immunostimulation, radiation also induces immunosuppressive galectin-1. No significant changes in MHC-I or PD-L1 expression were observed. Moreover, PDAC cell lines show similar radiation-induced immune effects when exposed to single-dose protons or photons. Our findings provide a rationale for combinatorial radiation-immunomodulatory treatment approaches in PDAC using conventional photon-based or proton beam radiotherapy.

In order to overcome the resistance to radiotherapy in human chondrosarcoma cells, the prevention from efficient DNA repair with a combined treatment with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) inhibitor AZD7648 was explored for carbon ion (C-ion) as well as reference photon (X-ray) irradiation (IR) using gene expression analysis, flow cytometry, protein phosphorylation, and telomere length shortening. Proliferation markers and cell cycle distribution changed significantly after combined treatment, revealing a prominent G2/M arrest. The expression of the G2/M checkpoint genes cyclin B, CDK1, and WEE1 was significantly reduced by IR alone and the combined treatment. While IR alone showed no effects, additional AZD7648 treatment resulted in a dose-dependent reduction in AKT phosphorylation and an increase in Chk2 phosphorylation. Twenty-four hours after IR, the key genes of DNA repair mechanisms were reduced by the combined treatment, which led to impaired DNA repair and increased radiosensitivity. A time-dependent shortening of telomere length was observed in both cell lines after combined treatment with AZD7648 and 8 Gy X-ray/C-ion IR. Our data suggest that the inhibition of DNA-PKcs may increase sensitivity to X-rays and C-ion IR by impairing its functional role in DNA repair mechanisms and telomere end protection.

Chondrosarcomas are particularly difficult to treat due to their resistance to chemotherapy and radiotherapy. However, particle therapy can enhance local control and patient survival rates. To improve our understanding of the basic cellular radiation response, as a function of dose and linear energy transfer (LET), we developed a novel water phantom-based setup for cell culture experiments and characterized it dosimetrically. In a direct comparison, human chondrosarcoma cell lines were analyzed with regard to their viability, cell proliferation, cell cycle, and DNA repair behavior after irradiation with X-ray, proton, and carbon ions. Our results clearly showed that cell viability and proliferation were inhibited according to the increasing ionization density, i.e., LET, of the irradiation modes. Furthermore, a prominent G2/M arrest was shown. Gene expression profiling proved the upregulation of the senescence genes CDKN1A (p21), CDKN2A (p16NK4a), BMI1, and FOXO4 after particle irradiation. Both proton or C-ion irradiation caused a positive regulation of the repair genes ATM, NBN, ATXR, and XPC, and a highly significant increase in XRCC1/2/3, ERCC1, XPC, and PCNA expression, with C-ions appearing to activate DNA repair mechanisms more effectively. The link between the physical data and the cellular responses is an important contribution to the improvement of the treatment system.

Microdosimetry investigates the energy deposition of ionizing radiation at microscopic scales, beyond the assessment capabilities of macroscopic dosimetry. This contributes to an understanding of the biological response in radiobiology, radiation protection and radiotherapy. Microdosimetric pulse height spectra are usually measured using an ionization detector in a pulsed readout mode. This incorporates a charge-sensitive amplifier followed by a shaping network. At high particle rates, the pileup of multiple pulses leads to distortions in the recorded spectra. Especially for gas-based detectors, this is a significant issue, that can be reduced by using solid-state detectors with smaller cross-sectional areas and faster readout speeds. At particle rates typical for ion therapy, however, such devices will also experience pileup. Mitigation techniques often focus on avoiding pileup altogether, while post-processing approaches are rarely investigated. This work explores pileup effects in microdosimetric measurements and presents a stochastic resampling algorithm, allowing for offline simulation and correction of spectra. Initially it was developed for measuring neutron spectra with tissue equivalent proportional counters and is adapted for the use with solid-state microdosimeters in a clinical radiotherapy setting. The algorithm was tested on data acquired with solid-state microdosimeters at the MedAustron ion therapy facility. The successful simulation and reduction of pileup counts is achieved by establishing of a limited number of parameters for a given setup. The presented results illustrate the potential of offline correction methods in situations where a direct pileup-free measurement is currently not practicable.

Microdosimetry investigates the energy deposition of ionizing radiation at microscopic scales, beyond the assessment capabilities of macroscopic dosimetry. This contributes to an understanding of the biological response in radiobiology, radiation protection and radiotherapy. Microdosimetric pulse height spectra are usually measured using an ionization detector in a pulsed readout mode. This incorporates and a charge-sensitive amplifier followed by a shaping network. At high particle rates, the pileup of multiple pulses leads to distortions in the recorded spectra. Especially for gas-based detectors, this is a significant issue, that can be reduced by using solid-state detectors with smaller cross-sectional areas and faster readout speeds. At particle rates typical for ion therapy, however, such devices will also experience pileup. Mitigation techniques often focus on avoiding pileup altogether, while post-processing approaches are rarely investigated. This work explores pileup effects in microdosimetric measurements and presents a stochastic resampling algorithm, allowing for offline simulation and correction of spectra. Initially it was developed for measuring neutron spectra with tissue equivalent proportional counters and is adapted for the use with solid-state microdosimeters in a clinical radiotherapy setting. The algorithm was tested on data acquired with solid-state microdosimeters at the MedAustron ion therapy facility. The successful simulation and reduction of pileup counts is achieved by establishing of a limited number of parameters for a given setup. The presented results illustrate the potential of offline correction methods in situations where a direct pileup-free measurement is currently not practicable.

This paper characterizes the microdosimetric spectra of a single-energy carbon-ion pencil beam at MedAustron using a miniature solid-state silicon microdosimeter to estimate the impact of the lateral distribution of the different fragments on the microdosimetric spectra. The microdosimeter was fixed at one depth and then laterally moved away from the central beam axis in steps of approximately 2 mm. The measurements were taken in both horizontal and vertical direction in a water phantom at different depths. In a position on the distal dose fall-off beyond the Bragg peak, the frequency-mean and the dose-mean lineal energies were derived using either the entire range of y-values, or a sub-range of y values, presumingly corresponding mainly to contributions from primary particles. The measured microdosimetric spectra do not exhibit a significant change up to 4 mm away from the beam central axis. For lateral positions more than 4 mm away from the central axis, the relative contribution of the lower lineal-energy part of the spectrum increases with lateral distance due to the increased partial dose from secondary fragments. The average values yF and yD are almost constant for each partial contribution. However, when all particles are considered together, the average value of yF and yD varies with distance from the axis due to the changing dose fractions of these two components varying by 30 % and 10 % respectively up to the most off axis vertical position. Characteristic features in the microdosimetric spectra providing strong indications of the presence of helium and boron fragments have been observed downstream of the distal part of the Bragg peak. We were able to investigate the radiation quality as function of off-axis position. These measurements emphasize variation of the radiation quality within the beam and this has implications in terms of relative biological effectiveness.