Dying cell clearance and its impact on the outcome of tumor radiotherapy

The induction of tumor cell death is one of the major goals of radiotherapy and has been considered to be the central determinant of its therapeutic outcome for a long time. However, accumulating evidence suggests that the success of radiotherapy does not only derive from direct cytotoxic effects on the tumor cells alone, but instead might also depend – at least in part – on innate as well as adaptive immune responses, which can particularly target tumor cells that survive local irradiation. The clearance of dying tumor cells by phagocytic cells of the innate immune system represents a crucial step in this scenario. Dendritic cells and macrophages, which engulf, process and present dying tumor cell material to adaptive immune cells, can trigger, skew, or inhibit adaptive immune responses, respectively. In this review we summarize the current knowledge of different forms of cell death induced by ionizing radiation, the multi-step process of dying cell clearance, and its immunological consequences with special regard toward the potential exploitation of these mechanisms for the improvement of tumor radiotherapy

Transcriptomic analyses of the radiation response in head and neck squamous cell carcinoma subclones with different radiation sensitivity: time-course gene expression profiles and gene association networks

Background: Acquired and inherent radioresistance of tumor cells is related to tumor relapse and poor prognosis not only in head and neck squamous cell carcinoma (HNSCC). The underlying molecular mechanisms are largely unknown. Therefore, systemic in-depth analyses are needed to identify key regulators of radioresistance. In the present study, subclones of the CAL-33 HNSCC cell line with different radiosensitivity were analyzed to identify signaling pathways related to the different phenotypes. Methods: Subclones with altered radiosensitivity were generated by fractionated irradiation of the parental CAL-33 cells. Differences in radiosensitivity were confirmed in colony formation assays. Selected subclones were characterized at the genomic and transcriptomic level by SKY, array CGH, and mRNA-microarray analyses. Time-course gene expression analyses upon irradiation using a natural cubic spline regression model identified temporally differentially expressed genes. Moreover, early and late responding genes were identified. Gene association networks were reconstructed using partial correlation. The Reactome pathway database was employed to conduct pathway enrichment analyses. Results: The characterization of two subclones with enhanced radiation resistance (RP) and enhanced radiosensitivity (SP) revealed distinct genomic and transcriptomic changes compared to the parental cells. Differentially expressed genes after irradiation shared by both subclones pointed to important pathways of the early and late radiation response, including senescence, apoptosis, DNA repair, Wnt, PI3K/AKT, and Rho GTPase signaling. The analysis of the most important nodes of the gene association networks revealed pathways specific to the radiation response in different phenotypes of radiosensitivity. Exemplarily, for the RP subclone the senescence-associated secretory phenotype (SASP) together with GPCR ligand binding were considered as crucial. Also, the expression of endogenous retrovirus ERV3-1in response to irradiation has been observed, and the related gene association networks have been identified. Conclusions: Our study presents comprehensive gene expression data of CAL-33 subclones with different radiation sensitivity. The resulting networks and pathways associated with the resistant phenotype are of special interest and include the SASP. The radiation-associated expression of ERV3-1 also appears highly attractive for further studies of the molecular mechanisms underlying acquired radioresistance. The identified pathways may represent key players of radioresistance, which could serve as potential targets for molecularly designed, therapeutical intervention

Release of monocyte migration signals by breast cancer cell lines after ablative and fractionated gamma-irradiation

Background: Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells. Methods: Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards.-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry. Results: Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to.-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39. Conclusions: Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches

Fabrication and characterization of a multimodal 3D printed mouse phantom for ionoacoustic quality assurance in image-guided pre-clinical proton radiation research

Objective. Image guidance and precise irradiation are fundamental to ensure the reliability of small animal oncology studies. Accurate positioning of the animal and the in-beam monitoring of the delivered radio-therapeutic treatment necessitate several imaging modalities. In the particular context of proton therapy with a pulsed beam, information on the delivered dose can be retrieved by monitoring the thermoacoustic waves resulting from the brief and local energy deposition induced by a proton beam (ionoacoustics). The objective of this work was to fabricate a multimodal phantom (x-ray, proton, ultrasound, and ionoacoustics) allowing for sufficient imaging contrast for all the modalities. Approach. The phantom anatomical parts were extracted from mouse computed tomography scans and printed using polylactic acid (organs) and a granite/polylactic acid composite (skeleton). The anatomical pieces were encapsulated in silicone rubber to ensure long term stability. The phantom was imaged using x-ray cone-beam computed tomography, proton radiography, ultrasound imaging, and monitoring of a 20 MeV pulsed proton beam using ionoacoustics. Main results. The anatomical parts could be visualized in all the imaging modalities validating the phantom capability to be used for multimodal imaging. Ultrasound images were simulated from the x-ray cone-beam computed tomography and co-registered with ultrasound images obtained before the phantom irradiation and low-resolution ultrasound images of the mouse phantom in the irradiation position, co-registered with ionoacoustic measurements. The latter confirmed the irradiation of a tumor surrogate for which the reconstructed range was found to be in reasonable agreement with the expectation. Significance. This study reports on a realistic small animal phantom which can be used to investigate ionoacoustic range (or dose) verification together with ultrasound, x-ray, and proton imaging. The co-registration between ionoacoustic reconstructions of the impinging proton beam and x-ray imaging is assessed for the first time in a pre-clinical scenario

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI

Plasminogen Activator Inhibitor-1 Promotes Neutrophil Infiltration and Tissue Injury on Ischemia–Reperfusion

Objective Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure. Approach and Results Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of 2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis. Conclusions Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury