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Additional file 3. Table S2 Consumables and Reagents.

Additional file 1: Table S1. Detailed patient characteristics.

Additional file 3: Figure S2. Overall survival shown by Kaplan-Meier curves for Musashi (A), Nanog (B), Oct4 (C) and Osteopontin (D) expressed in primary and recurrent tumor. Curves are divided by the median expression rate of the corresponding marker. None of the markers was significantly associated with overall survival.

Additional file 1. Additional figures.

Additional file 4: Figure S3. Overall survival shown by Kaplan-Meier curves for standard molecular markers. Survival was dichotomized by IDH-1/2 mutational status (A), MGMT promotor methylation status (B) and nuclear ATRX expression (C).

Purpose Evaluation of clinical outcome of two-weekly high-dose-rate brachytherapy boost after external beam radiotherapy (EBRT) for localized prostate cancer. Methods 338 patients with localized prostate cancer receiving definitive EBRT followed by a two-weekly high-dose-rate brachytherapy boost (HDR-BT boost) in the period of 2002 to 2019 were analyzed. EBRT, delivered in 46 Gy (DMean) in conventional fractionation, was followed by two fractions HDR-BT boost with 9 Gy (D90%) two and four weeks after EBRT. Androgen deprivation therapy (ADT) was added in 176 (52.1%) patients. Genitourinary (GU)/gastrointestinal (GI) toxicity was evaluated utilizing the Common Toxicity Criteria for Adverse Events (version 5.0) and biochemical failure was defined according to the Phoenix definition. Results Median follow-up was 101.8 months. 15 (4.4%)/115 (34.0%)/208 (61.5%) patients had low-/intermediate-/high-risk cancer according to the D`Amico risk classification. Estimated 5-year and 10-year biochemical relapse-free survival (bRFS) was 84.7% and 75.9% for all patients. The estimated 5-year bRFS was 93.3%, 93.4% and 79.5% for low-, intermediate- and high-risk disease, respectively. The estimated 10-year freedom from distant metastasis (FFM) and overall survival (OS) rates were 86.5% and 70.0%. Cumulative 5-year late GU toxicity and late GI toxicity grade ≥ 2 was observed in 19.3% and 5.0% of the patients, respectively. Cumulative 5-year late grade 3 GU/GI toxicity occurred in 3.6%/0.3%. Conclusions Two-weekly HDR-BT boost after EBRT for localized prostate cancer showed an excellent toxicity profile with low GU/GI toxicity rates and effective long-term biochemical control.

Additional file 1: Supplemental Materials. The primary and secondary antibodies used in this paper. Supplemental Methods.: Comet assay and staining for HDAC5 and DAPI. In order to characterize the tested cell lines, preliminary experiments were carried out via Comet assay. The Comet assay was performed under alkaline conditions following a protocol reported elsewhere [19]. Just before irradiation, cells were embedded in a thin layer of agarose spread on glass microscope slides. The slides were placed on ice, subjected to irradiation and transferred immediately either into ice-cold lysis buffer or in CGM for the indicated times. DNA fragmentation was quantified from the ���Tail Moment��� (TM, given in arbitrary units, a.u.) defined as the product of the percentage of DNA in the comet tail and the tail length. Supplementary Fig. S1 shows the induction and repair of DNA damage measured by the Comet assay in the cells of both cell lines immediately and up to 40 min after irradiation with 5 Gy. As seen in Supplementary Fig. S1, the initial TM was higher in MO59J cells, however, the residual DNA damage was similar in both cell lines, as were the kinetics of DNA damage disappearance. Supplementary Fig. S2 shows the distribution of TM in both cell lines measured immediately after IR. As seen in Supplementary Fig. S2, the histogram of TM values in intrinsically radiosensitive MO59J line was shifted towards higher DNA damage. It is worth mentioning that DNA damage measured by the alkaline Comet assay includes single-strand breaks and alkali-labile sites and base exchanges. Another preliminary test to characterize intrinsically radioresistant MO59K and radiosensitive MO59J cell lines was staining for HDAC5 protein and subsequent HDAC5 foci counting (Supplementary Fig. S3). To this end, cells were cultured on microscope glass slides for at least 24 h and stained with anti-HDAC5 (1:200) primary antibodies essentially as described elsewhere [57] with the exception of primary antibodies. For each experiment or cell line, at least 100 nuclei were examined and HDAC5 foci were scored by eye at a magnification of 1000x. Probes were then quantified by counting the number of foci per nucleus. As seen in Supplementary Fig. S3, HDAC5 foci were much more expressed in radioresistant MO59K cell line. Simultaneously slides were counterstained with DAPI (Supplementary Fig. S4). Fig. S1. DNA damage (Tail Moment, TM) induction and repair measured by the comet assay in human GBM cell lines irradiated with 5 Gy of X-rays in vitro. Immediately after irradiation, the samples were placed at 37 ��C in a 5% CO2 incubator. At the indicated time intervals after X-ray exposure, the cells were lysed and subjected to the alkaline comet assay. Up to 75 cells were analyzed for each slide. Each point (bar) represents the mean value (�� SE) of TM for the respective time point. The curves are best least-square fits of exponential decay function to the datapoints. Fig. S2. DNA damage (Tail Moment) induction measured by the comet assay in human GBM cell lines MO59K and MO59J irradiated with 5 Gy of X-rays in vitro. The cells taken at the indicated time (2 min) after X-ray exposure were lysed and subjected to the alkaline Comet assay. Up to 75 cells were analyzed for each slide. The curves are best least-square fits of normal distribution to the datapoints. Fig. S3. Representative histograms depicting the HDAC5 focus formation in control non-irradiated MO59K and MO59J cells. Cells were analyzed for HDAC5 focus formation 24 h after seeding. About 100 nuclei were counted per each cell line and experiment (n = 3). Fig. S4. Representative histograms depicting the mean DAPI fluorescence intensity per nucleus in control non-irradiated MO59K and MO59J cells. Cells were cultured on the slides and 24 h after seeding stained with DAPI. Fluorescence intensity was quantitated with the ImageJ program in about 100 nuclei counted per each cell line and experiment (n = 3). Fig. S5. Cellular viability measured by an ATP test. Changes of intracellular ATP in 2 tumor cell lines exposed to serial dilutions of PI-103 for 24 h. ATP content was measured by standard luciferase bioluminescence assay. Quadruplicate data derived from at least three independent experiments were averaged, normalized against non-treated controls (DMSO) and analyzed using the standard four-parameter logistic model to generate dose-response curves. Error bars indicate SD values. Fig. S6. Effects of radiation, PI-103 or combined PI-103-radiation treatments on colony-forming ability of MO59K cells. One thousand MO59K cells were plated on Petri dishes for 16 h, treated with radiation (A: 2, 3, and 5 Gy), or with PI-103 (B: 0.5 ��M, 1 ��M, and 2 ��M) or a combination of radiation (C: 3 Gy) and PI-103 (0.5 ��M, 1 ��M and 2 ��M)) added 3 h before irradiation. Twenty four hours post-irradiation the inhibitor was washed out and the cultures were incubated in fresh CGM for the next 12 days, fixed and stained with crystal violet. The numbers of colonies were counted and averaged from three independent experiments. Combination index was determined according Malyarenko et al. (2020) and Chou (2010) and found to be at tested concentrations of PI-103 and radiation dose of 3 Gy

Additional file 2: Supplementary Fig. S12. Uncropped representative Western blots for Figs. 2-6, and Supplementary Figs. S8-S10. The figures show original blots, in some cases the same membrane was used to simultaneously detect different proteins (after cutting) which strongly deviated in molecular weight. All original blots for ��-actin used as loading control for the respective runs are also included.

Figure S1. Western blot analysis of cell-cycle regulatory protein expression in drug-treated DK-MG and SNB19 cells, normalized to β-actin intensity (loading control). Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S2. Western blot analysis of PARP- and cleaved PARP-expression in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios. Figure S3. Western blot analysis of autophagy marker proteins LC3B and p62 in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S4. Western blot analysis of β-actin expression for proteins shown in Fig. 5 and Fig.7 (details in Figs. 5 and 7 legends). Figure S5. Western blot analysis of p-MEK1/2 and p-Erk1/2 in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5). Figure S6. Western blot analysis of Rheb in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S7. Western blot analysis of DNA-repair proteins in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S8. Western blot analysis of β-actin expression for proteins depicted in Fig. S7 (details in Fig. 7 legend). Figure S9. Cumulative plots of radius-normalized fc values (fc·a) vs. external conductivity of DK-MG (A) and SNB19 (B) cells were obtained by contra-rotating-field (CRF) technique. Symbols represent mean fc·a (± SD) values from 20 cells measured at ~ 10, 25 and 40 μS/cm. Lines are best fits of Eq. 2 to the data. The steeper line slopes for DK-MG cells (A) imply smaller Cm values compared to SNB19 cells (B). For Cm and CC values, calculated with Eq. 3, see Additional file 2: Table S4. Figure S10. Impact of drug-treatment on cell radius, Cm, and CC values of DK-MG (A) and SNB19 (B) cells. “*” denotes significant difference at p

Supplemental Materials. The primary and secondary antibodies used in this paper. (DOCX 57 kb)

Table S1. Cell cycle-phase distribution in control and irradiated (2 or 8 Gy) SNB19 tumor cells. The cells were fixed either 30 min or 24 h after IR, permeabilized, stained with propidium iodide, and analyzed for their DNA content by flow cytometry. Data are presented as means (± SD) from at least three independent experiments. For detailed description, see legend to Fig. 3. Table S2. Detection of γH2AX as a measure of DNA damage in SNB19 cells by flow cytometry. Mean γH2AX values are normalized to the non-irradiated control (30 min post-irradiation). The data are means ±SE from at least three independent experiments. Table S3. Cloning efficiencies and radiosensitivity parametersa of in vitro irradiated tumor cell lines untreated and pretreated with the MK-2206 and PI-103 either alone or in combination. aMean (± SE) from at least three independent experiments; bCF2 is the colony-forming ability at 2 Gy; cD10 is the radiation dose required to reduce colony-forming ability by 10%; dThe growth inhibition factor IF10 was calculated as (D10 control)/(D10 + inh.). Table S4. Impact of MK-2206, PI-103 either alone or in combination on the area-specific plasma membrane capacitance Cm, the whole-cell capacitance CC* and cell radius. *The data derived from the ROT experiments shown in the Additional file 4: Figure S10 represent the means ± SE of at least 60 cells. (DOCX 63 kb)

Characteristics of healthy individuals and RC patients undergoing chemo-radiotherapy (Summary). Table S2. Patients’ characteristics in regard to chemo-radiation toxicities and alcohol/tobacco consumption. Table S3. DNA damage measured by the histone γ-H2AX in PBMCs isolated from blood of apparently healthy donors (N) and unselected rectal carcinoma (RC) patients after exposure to 0.5 or 2 Gy of X-irradiation in vitro or after 5 clinical radiation fractions. Table S4. DNA damage measured by the 53BP1 foci in PBMCs isolated from blood of apparently healthy donors (N) and unselected rectal carcinoma (RC) patients after exposure to 0.5 or 2 Gy of X-irradiation in vitro or after 5 clinical radiation fractions. Figure S1. DNA damage assessed by the mean number of 53BP1 foci in non-irradiated (A) and irradiated (B-D) PBMCs derived from unselected RC patients (triangles), as compared to cells from apparently healthy donors (circles). For further details, see legend to Fig. 1. Filled squares represent the mean values (± SE) for the respective group. “n.s.” indicates that the difference was not highly significant (p > 0.05). Figure S2. Correlational analysis of mean γ-H2AX and 53BP1 foci counts from 500 nuclei per sample. Non-irradiated (A) and irradiated with 0.5 (B and C) and 2 Gy (D) lymphocytes were fixed 30 min (B) or 24 h (C, D) post-IR. The expression of both proteins was analyzed simultaneously at each time and IR points for n = 48 blood samples derived from unselected RC patients. Figure S3. DNA damage assessed by means of the 53BP1 assay in non-irradiated (A) and irradiated (B-D) PBMCs derived from normally-reacting RC patients (grade 0 and 1, up triangles) and radiation-sensitive (grade 2 and 3, down triangles) cancer patients compared to cells from apparently healthy donors (circles). Filled squares represent the mean values (± SE) for the respective group. For details, see legend to Fig. 2. Figure S4. Correlation between the 53BP1 foci expression and tumor staging (see Additional file 1: Table S2). Peripheral lymphocytes were prepared from the blood samples derived from RC patients. Foci counting for 53BP1 were performed in non-irradiated (A), irradiated in vitro with 0.5 and 2 Gy samples 30 min and 24 h post-IR (B and C) or 72 h after 5 clinical radiation fractions (D). Filled squares represent the mean values (± SE) for the respective group. Figure S5. Comparison of the γ-H2AX foci expression in peripheral lymphocytes of RC patients differing in tumor regression grade (TRG, Additional file 1: Table S2). Foci counting for γ-H2AX were performed in non-irradiated (up triangles and circles) cells or after 5 clinical radiation fractions (down triangles and diamonds). Filled squares represent the mean values (± SE) for the respective group. (DOC 1915 kb)

Antibodies. List of primary antibodies used in this study. (DOCX 12 kb)

Colony forming assay data. Colony forming efficiencies and radiosensitivity parameters of tumor cells treated with AZD6244 and NVP-BEZ235 derived with the linear quadratic model. (PDF 128 kb)

Representative cell cycle histograms. Representative DNA histograms of SNB19 and A549 cells treated with AZD6244, NVP-BEZ235, a combination of both drugs and IR with 8 Gy. Cells were fixed 30 min, 24 or 48 h after IR, permeabilized, treated with RNase A, stained with PI and analyzed for DNA content. DNA histograms were deconvoluted with the ModFit Software. (PDF 3250 kb)

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo-/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C m = 1.9 µF/cm(2). In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C m values of 3.7-4.0 µF/cm(2), which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.

Although locoregional relapse is frequent after definitive radiotherapy (RT) or multimodal treatments, re-irradiation is only performed in few patients even in palliative settings like e.g. vertebral metastasis. This is most due to concern about potentially severe complications, especially when large volumes are exposed to re-irradiation. With technological advancements in treatment planning the interest in re-irradiation as a local treatment approach has been reinforced. Recently, several studies reported re-irradiation for spinal metastases using SBRT with promising local and symptom control rates and simultaneously low rates of toxicity. These early data consistently indicate that SBRT is a safe and effective treatment modality in this clinical situation, where other treatment alternatives are rare. Similarly, good results have been shown for SBRT in the re-irradiation of head and neck tumors. Despite severe late adverse effects were reported in several studies, especially after single fraction doses >10 Gy, they appear less frequently compared to conventional radiotherapy. Few studies with small patient numbers have been published on SBRT re-irradiation for non-small cell lung cancer (NSCLC). Overall survival (OS) is limited by systemic progression and seems to depend particularly on patient selection. SBRT re-irradiation after primary SBRT should not be practiced in centrally located tumors due to high risk of severe toxicity. Only limited data is available for SBRT re-irradiation of pelvic tumors: feasibility and acceptable toxicity has been described, suggesting SBRT as a complementary treatment modality for local symptom control.

Purpose The purpose of this work is to develop fast deliverable step and shoot IMRT technique. A reduction in the number of segments should theoretically be possible, whilst simultaneously maintaining plan quality, provided that the reduction is accompanied by an increased number of gantry angles. A benefit of this method is that the segment shaping could be performed during gantry motion, thereby reducing the delivery time. The aim was to find classes of such solutions whose plan quality can compete with conventional IMRT. Materials/Methods A planning study was performed. Step and shoot IMRT plans were created using direct machine parameter optimization (DMPO) as a reference. DMPO plans were compared to an IMRT variant having only one segment per angle ("2-Step Fast"). 2-Step Fast is based on a geometrical analysis of the topology of the planning target volume (PTV) and the organs at risk (OAR). A prostate/rectum case, spine metastasis/spinal cord, breast/lung and an artificial PTV/OAR combination of the ESTRO-Quasimodo phantom were used for the study. The composite objective value (COV), a quality score, and plan delivery time were compared. The delivery time for the DMPO reference plan and the 2-Step Fast IMRT technique was measured and calculated for two different linacs, a twelve year old Siemens Primus™ ("old" linac) and two Elekta Synergy™ "S" linacs ("new" linacs). Results 2-Step Fast had comparable or better quality than the reference DMPO plan. The number of segments was smaller than for the reference plan, the number of gantry angles was between 23 and 34. For the modern linac the delivery time was always smaller than that for the reference plan. The calculated (measured) values showed a mean delivery time reduction of 21% (21%) for the new linac, and of 7% (3%) for the old linac compared to the respective DMPO reference plans. For the old linac, the data handling time per beam was the limiting factor for the treatment time reduction. Conclusions 2-Step Fast plans are suited to reduce the delivery time, especially if the data handling time per beam is short. The plan quality can be retained or even increased for fewer segments provided more gantry angles are used.