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The major goal of radiotherapy is the induction of tumor cell death. Additionally, radiotherapy can function as in situ cancer vaccination by exposing tumor antigens and providing adjuvants for anti-tumor immune priming. In this regard, the mode of tumor cell death and the repertoire of released damage-associated molecular patterns (DAMPs) are crucial. However, optimal dosing and fractionation of radiotherapy remain controversial. Here, we examined the initial steps of anti-tumor immune priming by different radiation regimens (20 Gy, 4 × 2 Gy, 2 Gy, 0 Gy) with cell lines of triple-negative breast cancer in vitro and in vivo. Previously, we have shown that especially high single doses (20 Gy) induce a delayed type of primary necrosis with characteristics of mitotic catastrophe and plasma membrane disintegration. Now, we provide evidence that protein DAMPs released by these dying cells stimulate sequential recruitment of neutrophils and monocytes in vivo. Key players in this regard appear to be endothelial cells revealing a distinct state of activation upon exposure to supernatants of irradiated tumor cells as characterized by high surface expression of adhesion molecules and production of a discrete cytokine/chemokine pattern. Furthermore, irradiated tumor cell-derived protein DAMPs enforced differentiation and maturation of dendritic cells as hallmarked by upregulation of co-stimulatory molecules and improved T cell-priming. Consistently, a recurring pattern was observed: The strongest effects were detected with 20 Gy-irradiated cells. Obviously, the initial steps of radiotherapy-induced anti-tumor immune priming are preferentially triggered by high single doses – at least in models of triple-negative breast cancer.

Purpose Target delineation in lung cancer radiotherapy using CT and/or PET-CT is affected by large variability. MRI has excellent soft tissue visualization and better spatial resolution than PET-CT. The main purpose of this study is to analyze delineation variability for lung cancer using MRI. Methods and materials Seven physicians delineated the tumor volumes of ten patients for the following scenarios: (1) CT only; (2) PET-CT fusion images registered to CT (“clinical standard”); and (3) post-contrast T1-weighted MRI registered with diffusion-weighted MRI. To compute interobserver variability, the median surface was generated from all observers’ contours and used as the reference surface. A physician labeled the interface types (tumor to lung, atelectasis (collapsed lung), hilum, mediastinum, or chest wall) on the median surface. Contoured volumes and bidirectional local distances (BLDs) between individual observers’ contours and the reference contour were analyzed. Results CT- and MRI-based tumor volumes normalized relative to PET-CT-based volumes were31.62±0.76 (mean±SD) and 1.38±0.44, respectively. Volume differences between the imaging modalities were not significant. Between observers, the mean normalized volumes per patient averaged over all patients varied significantly by a factor of 1.6 (MRI) and 2.0 (CT and PET-CT) (p=4.10×10−5 – 3.82×10−9). The tumor-atelectasis interface had a significantly higher variability than other interfaces for all modalities combined (p=0.0006). The interfaces with the smallest uncertainties were tumor-lung (on CT) and tumor-mediastinum (on PET-CT and MRI). Conclusions While MRI-based contouring showed overall larger variability than PET-CT, contouring variability depended on the interface type and was not significantly different between modalities despite of the limited observer experience with MRI. Multimodality imaging and combining different imaging characteristics might be the best approach to define the tumor volume most accurately.

ObjectivesTo investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry.Materials and MethodsHuman colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n = 21 (n = 11 therapy group; n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1rnu). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings.ResultsCEUS perfusion parameter WiAUC decreased significantly (116,989 ± 77,048 a.u. to 30,076 ± 27,095a.u.; p = 0.005) under therapy with no significant changes (133,932 ± 65,960 a.u. to 84,316 ± 74,144 a.u.; p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 ± 191 vs. 802 ± 460 a.u.; p = 0.006); SI10min: 226 ± 149 vs. 645 ± 461 a.u.; p = 0.009). PF and PV decreased significantly (PF: 147 ± 58 mL/100 mL/min to 71 ± 15 mL/100 mL/min; p = 0.003; PV: 13 ± 3% to 9 ± 4%; p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 ± 1.8 vs. 17.8 ± 4.6; p < 0.001), CD31 (8.1 ± 3.0 vs. 20.8 ± 5.7; p < 0.001) and Ki-67 (318.7 ± 94.0 vs. 468.0 ± 133.8; p = 0.004) and significantly more TUNEL (672.7 ± 194.0 vs. 357.6 ± 192.0; p = 0.003) positive cells in the therapy group. CEUS parameters showed significant (p < 0.05) correlations to DCE-MRI parameters and immunohistochemistry.ConclusionsCEUS with VEGFR2-targeted microbubbles allowed for monitoring regorafenib functional and molecular therapy effects on experimental colorectal adenocarcinomas with a significant decline of CEUS and DCE-MRI perfusion parameters as well as a significant reduction of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2.

In order to provide an authentic standard and to generate pure material for biological testing, an efficient synthetic route to 1 was developed. This represents the first total synthesis of a major bioactive diynone from E. pallida. Keywordsdiynones; selective alkyne reduction; coupling reaction; selective desilylation Echinacea extracts are used by millions of people as botanical dietary supplements. 1 While they are purchased primarily to stimulate the immune system, there are many bioactive compounds in the supplements. It is important to have a good understanding of the biological profile of key components of the supplements so that unfavorable drug interactions can be avoided. Most commercial botanical dietary supplements contain mixtures of three of the nine species of Echinacea: E. pallida, E. angustifolia and E. purpurea. The chemical fingerprint of E. angustifolia has diacetylenic isobutylamides as the diagnostic hydrophobic constituents. In contrast, the chemical fingerprint of E. purpurea has tri-and tetraenic isobutyl amides as the most abundant hydrophobic constituents, while E. pallida contains acetylenic ketones as the diagnostic hydrophobic constituents. Ketones 1 and 2 represent the most abundant acetylenic ketones of E. pallida (Figure 1). Hydroxy ketone 2 is derived from ketone 1 by reaction with molecular oxygen. 2 Extracts from E. pallida also exhibit antiviral activity. Ketones 1 and 2 have been shown to be potent antifungal agents; 3 however, the full range of biological activity of these novel compounds has not yet been determined, partly due to the difficulty in obtaining pure 1 from plant extracts containing many other compounds of similar polarity.As part of an interdisciplinary team of plant scientists, food scientists and chemists whose goal is to identify key bioactive constituents of Echinacea, 4 we describe herein the first total synthesis of ketone 1. The synthetic route is illustrated below in Scheme 1. Our synthetic route began with the known acetylenic alcohol 3. 5 Alcohol 3 was protected as the tetrahydropyranyl (THP) ether 4 in 90% yield (dihydropyran, PPTS, CH 2 Cl 2 , 0 °C). The protected alcohol was converted into 5 by hydroxymethylation using ethylmagnesium bromide and formaldehyde, 6 followed by conversion of the alcohol into the iodide using triphenylphosphine and iodine. 7 The overall yield of 5 from acetylene 4 was 61%. The reaction of iodide 5 with the anion of trimethylsilylacetylene, generated from potassium carbonate and copper iodide, provided diacetylene 6 in 45% isolated yield. 8 Iodide 5 reacted very slowly with the anion of trimethylsilylacetylene. Yields of 72% for this reaction required potassium carbonate that was dried over phosphorus pentoxide. Surprisingly, the reaction of iodide 5 with the lithium salt of trimethylsilylacetylene, generated via the reaction of the acetylene with n-butyllithium, afforded only recovered starting material.