Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference

Spatiotemporal characterization of ionizing radiation induced DNA damage foci and their relation to chromatin organization

DNA damage sensing proteins have been shown to localize to the sites of DNA double strand breaks (DSB) within seconds to minutes following ionizing radiation (IR) exposure, resulting in the formation of microscopically visible nuclear domains referred to as radiation-induced foci (RIF). This review characterizes the spatiotemporal properties of RIF at physiological doses, minutes to hours following exposure to ionizing radiation, and it proposes a model describing RIF formation and resolution as a function of radiation quality and chromatin territories. Discussion is limited to RIF formed by three interrelated proteins ATM (Ataxia telangiectasia mutated), 53BP1 (p53 binding protein 1) and γH2AX (phosphorylated variant histone H2AX), with an emphasis on the later. This review discusses the importance of not equating RIF with DSB in all situations and shows how dose and time dependence of RIF frequency is inconsistent with a one to one equivalence. Instead, we propose that RIF mark regions of the chromatin that would serve as scaffolds rigid enough to keep broken DNA from diffusing away, but open enough to allow the repair machinery to access the damage site. We review data indicating clear kinetic and physical differences between RIF emerging from dense and uncondensed regions of the nucleus. We suggest that persistent RIF observed days following exposure to ionizing radiation are nuclear marks of permanent rearrangement of the chromatin architecture. Such chromatin alterations may not always lead to growth arrest as cells have been shown to replicate these in progeny. Thus, heritable persistent RIF spanning over tens of Mbp may reflect persistent changes in the transcriptome of a large progeny of cells. Such model opens the door to a “non-DNA-centric view” of radiation-induced phenotypes

BioSig: A bioinformatic system for studying the mechanism of intra-cell signaling

Mapping inter-cell signaling pathways requires an integrated view of experimental and informatic protocols. BioSig provides the foundation of cataloging inter-cell responses as a function of particular conditioning, treatment, staining, etc. for either in vivo or in vitro experiments. This paper outlines the system architecture, a functional data model for representing experimental protocols, algorithms for image analysis, and the requried statistical analysis. The architecture provides remote shared operation of an inverted optical microscope, and couples instrument operation with images acquisition and annotation. The information is stored in an object-oriented database. The algorithms extract structural informaiton such as morphology and organization, and map it to functional information such as inter-cellular responses. An example of usage of this system is included

BioSig: A bioinformatic system for studying the mechanism of intra-cell signaling

Mapping inter-cell signaling pathways requires an integrated view of experimental and informatic protocols. BioSig provides the foundation of cataloging inter-cell responses as a function of particular conditioning, treatment, staining, etc. for either in vivo or in vitro experiments. This paper outlines the system architecture, a functional data model for representing experimental protocols, algorithms for image analysis, and the requried statistical analysis. The architecture provides remote shared operation of an inverted optical microscope, and couples instrument operation with images acquisition and annotation. The information is stored in an object-oriented database. The algorithms extract structural informaiton such as morphology and organization, and map it to functional information such as inter-cellular responses. An example of usage of this system is included

Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts

BACKGROUND: We previously used a three-dimensional (3D) reconstituted basement membrane (rBM) assay to demonstrate that tumorigenic HMT-3522 T4–2 human breast cells can be induced to form morphologically normal structures (“reversion”) by treatment with inhibitors of β1 integrin, the epidermal growth factor receptor (EGFR), or mitogen-activated protein kinase (MAPK). We have now used this assay to identify reversion and/or death requirements of several more aggressive human breast cancer cell lines. METHODS: Breast tumor cell lines MCF7, Hs578T, and MDA-MB-231 were cultured in 3D rBM and treated with inhibitors of β1 integrin, MAPK, or phosphatidylinositol 3-kinase (PI3K). MDA-MB-231 cells, which lack E-cadherin, were transfected with an E-cadherin cDNA. The extent of reversion was assessed by changes in morphology and polarity, growth in 3D rBM or soft agar, level of invasiveness, and tumor formation in nude mice. RESULTS: All three cell lines showed partial reversion (MCF7 the greatest and Hs578T the least) of tumorigenic properties treated with a single β1 integrin, MAPK, or PI3K inhibitor. Combined inhibition of β1 integrin and either PI3K or MAPK resulted in nearly complete phenotypic reversion (MDA-MB-231, MCF7) or in cell death (Hs578T). E-cadherin-transfected MDA-MB-231 cells showed partial reversion, but exposure of the transfectants to an inhibitor of β1 integrin, PI3K, or MAPK led to nearly complete reversion. CONCLUSION: The 3D rBM assay can be used to identify signaling pathways that, when manipulated in concert, can lead to the restoration of morphologically normal breast structures or to death of the tumor cells, even highly metastatic cells. This approach may be useful to design therapeutic intervention strategies for aggressive breast cancers