Large scale in vivo micro-RNA loss of function screen identified miR-29a, miR-100 and miR-155 as modulators of radioresistance and tumor-stroma communication.

Micro RNAs (miR) are master regulators of cellular transcriptome. We aimed to investigate the role of miR regulation on tumor radiosensitivity and development of local tumor recurrence by a novel large-scale in vivo loss of function screen. For stable miR silencing, human A431 tumor cells were transduced with lentiviral constructs against 170 validated human miR (miRzip library). Fractionated radiotherapy (5x6Gy) was applied to A431 miRzip library growing s.c. in NCr nude mice. Enrichment of miRZip and miR expression was assessed using multiplexed qRT-PCR. The modulatory effect of miR on tumor and tumor microenvironment response to ionizing radiation was further evaluated by clonogenic survival, apoptosis (Caspase 3/7), DNA double-strand breaks (DSB, nuclear γH2AX foci), tumor microvessel density (MVD), transcriptome and protein analysis. Fractionated irradiation of the A431 miRzip library led to regression of tumors. However, after a latency period, tumors ultimately progressed and formed local recurrences indicating the survival of a subpopulation of miRzip expressing tumor clones. Among the selected miR for subsequent validation studies, loss of miR-29a, miR-100 and miR-155 was found to enhance clonogenic survival, reduce apoptosis and residual γH2AX foci of irradiated tumor cells. Moreover, knockdown of miR increased tumor angiogenesis correlating with elevated VEGF and TGFα expression levels. This phenomenon was most evident after tumor irradiation in vivo suggesting a critical role for tumor-stroma communication in development of the radioresistant phenotype. Engineering radioresistant tumors in vivo by modulating miR expression may lead to identification of critical targets for conquering local therapy failure.

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions.

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database.Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments.In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se Further, our data will have a significant impact on the ongoing debate about patient treatment modalities.

Biosensor Cell-Fit-HD4D for correlation of single-cell fate and microscale energy deposition in complex ion beams.

We present a protocol for the biosensor Cell-Fit-HD4D. It enables long-term monitoring and correlation of single-cell fate with subcellular-deposited energy of ionizing radiation. Cell fate tracking using widefield time-lapse microscopy is uncoupled in time from confocal ion track imaging. Registration of both image acquisition steps allows precise ion track assignment to cells and correlation with cellular readouts. For complete details on the use and execution of this protocol, please refer to Niklas et al. (2022).

Biosensor for deconvolution of individual cell fate in response to ion beam irradiation.

Clonogenic survival assay constitutes the gold standard method for quantifying radiobiological effects. However, it neglects cellular radiation response variability and heterogeneous energy deposition by ion beams on the microscopic scale. We introduce "Cell-Fit-HD4D" a biosensor that enables a deconvolution of individual cell fate in response to the microscopic energy deposition as visualized by optical microscopy. Cell-Fit-HD4D enables single-cell dosimetry in clinically relevant complex radiation fields by correlating microscopic beam parameters with biological endpoints. Decrypting the ion beam's energy deposition and molecular effects at the single-cell level has the potential to improve our understanding of radiobiological dose concepts as well as radiobiological study approaches in general.

Radioresistance and Transcriptional Reprograming of Invasive Glioblastoma Cells.

PURPOSE: Infiltrative growth pattern is a hallmark of glioblastoma (GBM). Radiation therapy aims to eradicate microscopic residual GBM cells after surgical removal of the visible tumor bulk. However, in-field recurrences remain the major pattern of therapy failure. We hypothesized that the radiosensitivity of peripheral invasive tumor cells (peri) may differ from the predominantly investigated tumor bulk. METHODS AND MATERIALS: Invasive GBM populations were generated via debulking of the visible tumor core and serial orthotopic transplantation of peri cells, and sustained proinvasive phenotype of peri cells was confirmed in vitro by scratch assay and time lapse imaging. In parallel, invasive GBM cells were selected by transwell assay and from peri cells of patient-derived 3-dimensional spheroid cultures. Transcriptome analysis deciphered a GBM invasion-associated gene signature, and functional involvement of key pathways was validated by pharmacologic inhibition. RESULTS: Compared with the bulk cells, invasive GBM populations acquired a radioresistant phenotype characterized by increased cell survival, reduced cell apoptosis, and enhanced DNA double-strand break repair proficiency. Transcriptome analysis revealed a reprograming of invasive cells toward augmented activation of epidermal growth factor receptor- and nuclear factor-κB-related pathways, whereas metabolic processes were downregulated. An invasive GBM score derived from this transcriptional fingerprint correlated well with patient outcome. Inhibition of epidermal growth factor receptor and nuclear factor-κB signaling resensitized invasive cells to irradiation. Invasive cells were eradicated with similar efficacy by particle therapy with carbon ions. CONCLUSIONS: Our data indicate that invasive tumor cells constitute a phenotypically distinct and highly radioresistant GBM subpopulation with prognostic impact that may be vulnerable to targeted therapy and carbon ions.

Simultaneous targeting of TGF-ß/PD-L1 synergizes with radiotherapy by reprogramming the tumor microenvironment to overcome immune evasion.

Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor ß (TGF-ß). We report that a bifunctional fusion protein that simultaneously inhibits TGF-ß and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ T cells. Intriguingly, targeting of the TGF-ß trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.

Carbon ion dosimetry on a fluorescent nuclear track detector using widefield microscopy.

Fluorescent nuclear track detectors (FNTDs) are solid-state dosimeters used in a wide range of dosimetric and biomedical applications in research worldwide. FNTDs are a core but currently underutilized dosimetry tool in the field of radiation biology which are inherently capable of visualizing the tracks of ions used in hadron therapy. The ions that traverse the FNTD deposit their energy according to their linear energy transfer and transform colour centres to form trackspots around their trajectory. These trackspots have fluorescent properties which can be visualized by fluorescence microscopy enabling a well-defined dosimetric readout with a spatial component indicating the trajectory of individual ions. The current method used to analyse the FNTDs is laser scanning confocal microscopy (LSM). LSM enables a precise localization of track spots in x, y and z however due to the scanning of the laser spot across the sample, requires a long time for large samples. This body of work conclusively shows for the first time that the readout of the trackspots present after 0.5 Gy carbon ion irradiation in the FNTD can be captured with a widefield microscope (WF). The WF readout of the FNTD is a factor ∼10 faster, for an area 2.97 times the size making the method nearly a factor 19 faster in track acquisition than LSM. The dramatic decrease in image acquisition time in WF presents an alternative to LSM in FNTD workflows which are limited by time, such as biomedical sensors which combine FNTDs with live cell imaging.

Combined external beam radiotherapy with carbon ions and tumor targeting endoradiotherapy.

External beam radiotherapy (EBRT) with carbon ions and endoradiotherapy using radiolabeled tumor targeting agents are emerging concepts in precision cancer therapy. We report on combination effects of these two promising strategies. Tumor targeting 131I-labelled anti-EGFR-antibody (Cetuximab) was used in the prototypic EGFR-expressing A431 human squamous cell carcinoma xenograft model. A 131I-labelled melanin-binding benzamide derivative was utilized targeting B16F10 melanoma in an orthotopic syngeneic C57bl6 model. Fractionated EBRT was performed using carbon ions in direct comparison with conventional photon irradiation. Tumor uptake of 131I-Cetuximab and 131I-Benzamide was enhanced by fractionated EBRT as determined by biodistribution studies. This effect was independent of radiation quality and significant for the small molecule 131I-Benzamide, i.e., >30% more uptake in irradiated vs. non-irradiated melanoma was found (p<0.05). Compared to each monotherapy, dual combination with 131I-Cetuximab and EBRT was most effective in inhibiting A431 tumor growth. A similar trend was seen for 131I-Benzamide and EBRT in B16F10 melanoma model. Addition of 131I-Benzamide endoradiotherapy to EBRT altered expression of genes related to DNA-repair, cell cycle and cell death. In contrast, immune-response related pathways such as type 1 interferon response genes (ISG15, MX1) were predominantly upregulated after combined 131I-Cetuximab and EBRT. The beneficial effects of combined 131I-Cetuximab and EBRT was further attributed to a reduced microvascular density (CD31) and decreased proliferation index (Ki-67). Fractionated EBRT could be favorably combined with endoradiotherapy. 131I-Benzamide endoradiotherapy accelerated EBRT induced cytotoxic effects. Activation of immune-response by carbon ions markedly enhanced anti-EGFR based endoradiotherapy suggesting further evaluation of this novel and promising radioimmunotherapy concept.