24h Post-irradiation Research Articles

Superior Drug Delivery Performance of Multifunctional Bilosomes: Innovative Strategy to Kill Skin Cancer Cells for Nanomedicine Application.

Numerous failures in melanoma treatment as a highly aggressive form of skin cancer with an unfavorable prognosis and excessive resistance to conventional therapies are prompting an urgent search for more effective therapeutic tools. Consequently, to increase the treatment efficiency and to reduce the side effects of traditional administration ways, herein, it has become crucial to combine photodynamic therapy as a promising therapeutic approach with the selectivity and biocompatibility of a novel colloidal transdermal nanoplatform for effective delivery of hybrid cargo with synergistic effects on melanoma cells. The self-assembled bilosomes, co-stabilized with L-α-phosphatidylcholine, sodium cholate, Pluronic® P123, and cholesterol, were designated, and the stability of colloidal vesicles was studied using dynamic and electrophoretic light scattering, also provided in cell culture medium (Dulbecco's Modified Eagle's Medium). The hybrid compounds - a classical photosensitizer (Methylene Blue) along with a complementary natural polyphenolic agent (curcumin), were successfully co-loaded, as confirmed by UV-Vis, ATR-FTIR, and fluorescent spectroscopies. The biocompatibility and usefulness of the polymer functionalized bilosome with loaded double cargo were demonstrated in vitro cyto- and phototoxicity experiments using normal keratinocytes and melanoma cancer cells. Thein vitrobioimaging and immunofluorescence study upon human skin epithelial (A375) and malignant (Me45) melanoma cell lines established the protective effect of the PEGylated bilosome surface. This effect was confirmed in cytotoxicity experiments, also determined on human cutaneous (HaCaT) keratinocytes. The flow cytometry experiments indicated the enhanced uptake of the encapsulated hybrid cargo compared to the non-loaded MB and CUR molecules, as well as a selectivity of the obtained nanocarriers upon tumor cell lines. The phyto-photodynamic action provided 24h-post irradiation revealed a more significant influence of the nanoplatform on Me45 cells in contrast to the A375 cell line, causing the cell viability rate below 20% of the control. As a result, we established an innovative and effective strategy for potential metastatic melanoma treatment through the synergism of phyto-photodynamic therapy and novel bilosomal-origin nanophotosensitizers.

Pediatric Burkitt Leukemia with Underlying MSH2, MUTYH and POT1 Germline Variants: The Complexities and Relevance of Understanding Cancer Predisposition

Monoallelic germline variants in POT1 can lead to POT1-tumor predisposition (POT1-TPD), an autosomal dominant disorder leading to a spectrum of neoplasia, predominantly, melanoma, glioma, angiosarcoma and chronic lymphocytic leukemia (CLL). Recently, germline POT1 variants have been described to be associated with familial clonal hematopoiesis syndrome.The POT1 protein forms a part of shelterin complex, which has been implicated in DNA damage protection, telomere length regulation and chromosomal stability. The proposed mechanism of oncogenesis by POT1 variants is presumed to be secondary to increased telomere length causing genomic instability and cellular immortality. While POT1-TPD can cause CLL in adulthood, childhood leukemias have not been commonly observed. Here, we present a case of a previously healthy 12-year-old male diagnosed with Burkitt leukemia. He presented with leukocytosis and tumor lysis syndrome. Bone marrow evaluation revealed hypercellular marrow involved by Burkitt lymphoma, comprising over 90% marrow cellularity. Flow cytometry showed an abnormal lambda monotypic B-lymphoid population positive for CD10, CD19, CD20, CD22 and CD45. FISH studies demonstrated low-level MYC rearrangement (9%); negative for BCL2 and BCL6 rearrangement. EBV staining was negative. Acute leukemia NGS panel (63 genes) revealed a Tier 1 somatic variant in PHF6. RNA sequencing did not detect additional fusions. His chemotherapy course was complicated by significant toxicities including prolonged count recovery, severe peripheral neuropathy, neurotoxicity, kidney injury and several infectious complications. Given extensive family history of cancers on the maternal side including colorectal, duodenal, brain and skin cancers, a germline cancer predisposition NGS panel was sent from cultured skin fibroblast and identified two heterozygous pathogenic variants in MSH2 (c.942+3A>T) and MUTYH (c.1187G>A) and a heterozygous likely pathogenic variant in POT1 (c.233T>C). To delineate potential roles of these germline variants in oncogenesis, additional testing was performed. Immunohistochemical staining of bone marrow revealed intact nuclear expression of MLH1, PMS2, MSH2,and MSH6,suggesting microsatellite stability. Extended tumor profiling through whole exome sequencing did not identify additional somatic variants or copy loss of heterozygosity in MSH2, MUTYH and POT1 but additional somatic loss of function variants were seen in CCND3, CASP8, ARID1, and PTPRD.To assess potential functional consequences of the POT1 germline variant on DNA double-strand break (DSB) damage repair, the patient lymphocytes were exposed to 2 Gy ionizing radiation and mobilization and kinetics of DNA DSB repair was assessed at 1h and 24h post-irradiation (IR) using phosphorylation (p) of ATM, SMC1 and H2AX (gamma), and cell death and apoptosis was also assessed at 24h post-irradiation by flow cytometry. There was normal mobilization and repair of DNA DSBs in T, B and NK cells although there was a subset of CD19dim B cells that showed constitutive DNA DSBs at1h with partial repair at 24h. It is not evident that this defect can be attributed to the germline defects as they are also observed in several other clinical contexts, including post-chemotherapy. Telomere length assay from peripheral blood demonstrated telomere length at 50 th percentile in both lymphocytes and granulocytes. Children in families with adult-onset cancer predisposition syndromes may present with cancers but the potential contribution of these germline variants to these childhood cancers is unknown. Our molecular and functional studies on both germline and tumor tissues did not suggest direct contribution of these germline variants on lymphomagenesis, however there might be other mechanism of inactivation not identified from this study. This case highlights the complexities of understanding genetic susceptibility in childhood cancers. We propose a multifactorial oligogenic inheritance of cancer susceptibility in this patient through several mechanisms of genomic instability.

Glioblastoma cells have increased capacity to repair radiation-induced DNA damage after migration to the olfactory bulb.

The invasive nature of GBM combined with the diversity of brain microenvironments creates the potential for a topographic heterogeneity in GBM radioresponse. Investigating the mechanisms responsible for a microenvironment-induced differential GBM response to radiation may provide insights into the molecules and processes mediating GBM radioresistance. Using a model system in which human GBM stem-like cells implanted into the right striatum of nude mice migrate throughout the right hemisphere (RH) to the olfactory bulb (OB), the radiation-induced DNA damage response was evaluated in each location according to γH2AX and 53BP1 foci and cell cycle phase distribution as determined by flow cytometry and immunohistochemistry. RNAseq was used to compare transcriptomes of tumor cells growing in the OB and the RH. Protein expression and neuron-tumor interaction were defined by immunohistochemistry and confocal microscopy. After irradiation, there was a more rapid dispersal of γH2AX and 53BP1 foci in the OB versus in the RH, indicative of increased double strand break repair capacity in the OB and consistent with the OB providing a radioprotective niche. With respect to the cell cycle, by 6h after irradiation there was a significant loss of mitotic tumor cells in both locations suggesting a similar activation of the G2/M checkpoint. However, by 24h post-irradiation there was an accumulation of G2 phase cells in the OB, which continued out to at least 96h. Transcriptome analysis showed that tumor cells in the OB had higher expression levels of DNA repair genes involved in non-homologous end joining and genes related to the spindle assembly checkpoint. Tumor cells in the OB were also found to have an increased frequency of soma-soma contact with neurons. GBM cells that have migrated to the OB have an increased capacity to repair radiation-induced double strand breaks and altered cell cycle regulation. These results correspond to an upregulation of genes involved in DNA damage repair and cell cycle control. Because the murine OB provides a source of radioresistant tumor cells not evident in other experimental systems, it may serve as a model for investigating the mechanisms mediating GBM radioresistance.

Targeted Nanoparticles to Mitigate Radiation‐Induced Blood‐Brain Barrier Disruption and Cognitive Impairment

BackgroundUp to 90% of brain cancer survivors develop radiation‐induced cognitive dysfunction following treatment. Damage to neurovascular endothelium contributes to cognitive decline through increased blood‐brain barrier (BBB) permeability. In our preliminary studies of endothelial cells (ECs) and mice, increased mitochondrial reactive oxygen species (mtROS), inducing heightened mitochondrial DNA (mtDNA) damage and release as cell‐free DNA are detected following irradiation (IR). This cell‐free mtDNA is known to activate Toll‐like receptor 9 (TLR9), triggering an immune response and further ROS. These endothelial responses are prevented with TLR9 inhibition and mtROS scavenging, suggesting a key role for TLR9 in sustained IR‐induced endothelial damage and dysfunction.Here, we posit that specific inhibition of TLR9 in cerebral ECs during radiation treatment will prevent subsequent BBB permeability and cognitive decline. Thus, the objective of this study is to examine the role of TLR9 in IR‐induced cognitive decline using TLR‐specific loaded nanoparticles (NP).Methods and ResultsTo target TLR9 in irradiated ECs, poly (lactic‐co‐glycolic acid)‐based NP coated with cyclic Arg‐Gly‐Asp (cRGD)‐peptide were designed and loaded with TLR9 inhibitor (ODN‐2088, 2.23ug/mg particle). cRGD‐peptide is a ligand for integrin αvβ3, which is highly expressed in ECs post‐IR. cRGD‐NP loaded with coumarin‐6 were used to first evaluate specificity in vitro and in vivo. ECs 24h post‐IR (4 Gy) displayed higher fluorescence than non‐IR, indicating selective uptake. Moreover, in C57Bl6 mice 24h post unilateral cerebral IR (12Gy) with cRGD‐NP, uptake accumulated in endothelium of the IR hemisphere.Mice were administered NP‐ODN‐2088 or empty NP (3mg injection) and subjected to IR as described above. 30 days post‐IR, they were tested for memory and learning ability using the Novel Object Recognition test. IR induced a significant impairment in the memory ability of empty NP treated mice compared to NP‐ODN‐2088. BBB permeability was analyzed in the same mice 30 days post‐IR with immunohistochemistry using anti‐mouse IgG. Significant BBB permeability was detected in the IR hemisphere of empty NP mice compared to the non‐IR hemisphere. However, the IR hemisphere of NP‐ODN‐2088 mice showed attenuated permeability compared to empty NP treated mice.ConclusionInhibition of TLR9 in irradiated ECs was observed to protect against BBB disruption and cognitive decline. These data support a pathway of radiation‐induced endotheliopathy through which TLR9 activation links vascular mitochondrial injury to neuropathology. Currently there are no pharmacological options for treating radiation‐induced normal tissue injury and targeting TLR9 poses a promising strategy.

Post-irradiation surface viscoelastic integrity of photo-polymerized resin-based composites

ObjectiveA class of ultra-rapid-cure resin-based composites (RBCs) exhibited immediate post-irradiation surface viscoelastic integrity using an indentation-creep/recovery procedure. The aim of this study was to determine whether such behavior is more generally characteristic of a wider range of RBCs. MethodsEight representative RBCs were selected based on different clinical categories: three bulkfills (OBF, Filtek One Bulk Fill; VBF, Venus Bulkfill; EBF, Estelite Bulkfill), three conventional non-flowables (XTE, Filtek Supreme XTE; GSO, GrandioSo; HRZ, Harmonize) and conventional flowables (XTF, Filtek Supreme XTE Flow; GSF, GrandioSo Flow). Stainless steel split molds were used to fabricate cylindrical specimens (4mm (dia)×4mm). These were irradiated (1.2W/cm2) for 20s on the top surface. Post-irradiation specimens (n=3), within their molds, were centrally loaded with a flat-ended 1.5mm diameter indenter under 14MPa stress: either immediately (<2min) or after 24h delayed indentation. Stress was maintained for 2h, then – after removal – recovery measurements continued for a further 2h. Indentation depth (%) versus time was measured continuously to an accuracy of <0.1μm. Data were analyzed by One-way ANOVA and Tukey post-hoc tests (α=0.05). ResultsTime-dependent viscoelastic indentation was observed for all RBCs. For immediate indentation, the maximum indentation range was 1.43–4.92%, versus 0.70–2.22% for 24h delayed indentation. Following 2h recovery, the residual indentation range was 0.86–3.58% after immediate indentation, reducing to 0.22–1.27% for delayed indentation. The greatest immediate indentation was shown by VBF followed by XTF and GSF. OBF, HRZ, XTE and GSO had significantly lower indentations (greater hardness). XTE showed a significantly reduced indentation maximum compared to OBF (p<0.05). Indentations delayed until 24h post-irradiation were reduced (p<0.05) for most materials. SignificanceThe indentation-creep methodology effectively characterized resin-based composites within several categories. Viscoelastic properties evaluated by the indentation-creep method confirmed that highly filled RBCs were more resistant to indentation. Indentations were reduced after 24h post-irradiation due to further matrix-network development.

Conversion kinetics of rapid photo-polymerized resin composites

ObjectiveTo measure the degrees of conversion (DC), conversion kinetics, and the effect of post-irradiation time on rapid photo-polymerized bulk-fill resin composites under conditions equivalent to clinical depths of 1 and 4mm. Methods36 specimens (n=3), based on two resin composites incorporating PowerCure rapid-polymerization technology in two consistencies (PFill; PFlow) and two comparators with matching consistencies (Eceram; EFlow), were investigated from the same manufacturer (Ivoclar AG, Liechtenstein). Specimens were prepared within 4mm diameter cylindrical molds, of either 1mm or 4mm depths respectively, to simulate near-surface and deep locations in a bulk-fill restoration. The independent variables in this study were: materials, thickness and time. Two high irradiance polymerization protocols were utilized for PowerCure materials: 2000 and 3050mW/cm2 for 5 and 3s, respectively. A standard (1200mW/cm2) polymerization protocol was used with control materials. FTIR was utilized to measure DC in real-time for 24h post-irradiation. The data were analyzed using Welch’s-ANOVA, Games-Howell post-hoc test, kinetic dual-exponential sum function and independent sample t-tests (p=0.05). ResultsThe DC of the materials ranged between 44.7–59.0 % after 5min, which increased after 24h reaching 55.7–71.0 % (p<0.05). Specimen thickness did not influence the overall DC. At 5min, the highest DC was shown in EFlow. But PFlow, irradiated for 3s and 5s exhibited comparable results (p>0.05). PFill composite irradiated with the 3s and 5s protocols did not differ from ECeram (p>0.05). Specimen thickness and material viscosity affected polymerization kinetics and rate of polymerization (RPmax). Faster polymerization occurred in 1mm specimens (except PFill-5s and ECeram). PFill and PFlow exhibited faster conversion than the controls. RPmax varied across the specimen groups between 4.3–8.8 %/s with corresponding DC RPmax between 22.2–45.3 %. SignificancePolymerization kinetics and RPmax were influenced by specimen thickness and material viscosity. PFill and PFlow materials produced an overall comparable conversion at 5min and 24h post-irradiation, despite the ultra-short irradiation times, throughout the 4mm specimen thickness.

Effect of time on the post-irradiation curing of six resin-based composites

ObjectivesTo evaluate the effect of time on the Vickers microhardness (VH) at the top and bottom surfaces of six conventional resin-based composites (RBCs) up to twelve weeks after light curing. MethodsFive specimens of Filtek Supreme Ultra, Herculite Ultra, Mosaic Ultra, Tetric EvoCeram, TPH Spectra HV, and Venus Pearl were packed into opaque molds that were 2.3mm in diameter and 2.5mm deep. The uncured RBC specimens were covered by a polyester strip and photo-cured with an Elipar DeepCure-S light-curing unit (LCU) according to the manufacturer's instructions. After irradiation, the polyester strip was removed, and the Vickers microhardness was measured immediately at top and bottom surfaces. The hardness measurements were repeated after 30min, 1h, 2h, 4h, 24h, 1 week, 4 weeks, and 12 weeks. In between, the specimens were stored in dry and dark conditions at 37°C. Two-way ANOVA (α=0.05) followed by Tukey–Kramer post hoc multiple comparison tests were used to determine where statistically significant differences existed. ResultsThe micro-hardness values at the top surface always exceeded those at the bottom surface. A significant logarithmic increase of the micro-hardness due to post-irradiation curing took place between 30min and 24h (p<0.05). There was no significant increase in the VH after 24h. Depending on the RBC, compared to the immediate values the hardness 24h post-irradiation had increased by 11–27% at the top surface and by 21–58% at the bottom. SignificanceEven after 12 weeks, the bottom hardness values never reached the top microhardness values. The results of studies that wait 24h or longer before measuring the properties of RBC specimens will be significantly enhanced by the impact of post-irradiation curing. Especially within the first 4h, the time when specimens are measured is critical information and should be reported.

Pre-heating time and exposure duration: Effects on post-irradiation properties of a thermo-viscous resin-composite

ObjectiveTo evaluate the effects of pre-heating time and exposure duration on the degree of conversion (DC), maximum rate of polymerization (RPmax), polymerization shrinkage strain (PS) and surface micro-hardness (VHN) of Viscalor. MethodsViscalor syringes were pre-heated using a Caps Warmer (VOCO, Germany) in T3 mode (at 68°C) for 30s (T3-30s) and 3min (T3-3min) and then the composite paste was extruded into appropriately sized molds. Light irradiation was applied at zero distance from the upper surface with a LED-LCU of mean irradiance 1200mW/cm2 for either 20 or 40s. The real-time polymerization kinetics and DC at 5min and 24h post-irradiation (DC5min and DC24h) were measured using ATR-FTIR (n=3). PS was obtained with the bonded-disk technique (n=3). Top and bottom Vickers micro-hardness (VHNtop and VHNbottom) were measured at 5min post-irradiation and after 24h dry storage (n=5). Data were analysed using one-way ANOVA, two-way ANOVA, independent t-test and Tukey post hoc tests (p<0.05). ResultsPolymerization kinetic curves of Viscalor from 0 to 15min were similar for different pre-heating times and exposure durations. Pre-heated Viscalor (T3-30s and T3-3min) with 40s exposure had greater VHNtop and VHNbottom than for Viscalor (no heat) (p<0.05). Exposure duration did not significantly affect DC, RPmax and PS (p>0.05). After 24h storage, DC and VHN increased. Pre-heating did not increase the DC24h, relative to no pre-heating (p>0.05). Two-way ANOVA showed that there was no significant interaction between pre-heating time and exposure duration (p>0.05). SignificanceIncreasing irradiation time from 20 to 40s did not affect DC, RPmax or PS, but increased VHNtop. Composite pre-heating had no adverse effect through any premature polymerization. For Viscalor, 3min pre-heating and 20s irradiation were sufficient to provide adequate hardness, without increasing PS or compromising polymerization kinetics.

Flexible film dosimeter for in vivo dosimetry.

The aims of this study were to develop a flexible film dosimeter applicable to the irregular surface of a patient for in vivo dosimetry and to evaluate the device's dosimetric characteristics. A flexible film dosimeter with active layers consisting of radiochromic-sensitive films and flexible silicone materials was constructed. The dose-response, sensitivity, scanning orientation dependence, energy dependence, and dose rate dependence of the flexible film dosimeter were tested. Irradiated dosimeters were scanned 24h post-irradiation, and the region of interest was 5mm×5mm. Biological stability tests ensured the safety of application of the flexible film dosimeter for patients. A preliminary clinical study with the flexible film dosimeter was implemented on four patients. The red channel demonstrated the highest sensitivity among all channels, and the response sensitivity of the dosimeter decreased with the applied dose, which were the same as the characteristics of GAFCHROMIC EBT3 radiochromic films. The flexible film dosimeter showed no significant energy dependence for photon beams of 6MV, 6MV flattening filter-free (FFF), 10MV, and 15MV. The flexible film dosimeter showed no substantial dose rate dependence with 6 or 6MV FFF. In terms of biological stability, the flexible film dosimeter demonstrated no cytotoxicity, no irritation, and no skin sensitization. In the preliminary clinical study, the dose differences between the measurements with the flexible film dosimeter and calculations with the treatment planning system ranged from -0.1% to 1.2% for all patients. The dosimeter developed in this study is a flexible film capable of attachment to a curved skin surface. The biological test results indicate the stability of the flexible film dosimeter. The preliminary clinical study showed that the flexible film dosimeter can be successfully applied as an in vivo dosimeter.

421P - Triggering anti-GBM immune response with EGFR-mediated photoimmunotherapy

BackgroundGlioblastoma (GBM) employs a variety of mechanisms to suppress the tumour immune microenvironment. A therapeutic approach aiming to abolish immunosuppressive cells and activate anti-GBM immunity would provide a powerful treatment strategy against GBM. We investigated whether photoimmunotherapy (PIT) targeting epidermal growth factor receptor (EGFR) can promoteT cell activation, whilst overcoming the immunologically cold status of GBM. MethodsThe phthalocyanine dye (IR700) was conjugated to affibody molecule (ZEGFR:03115). Cell viability, reactive oxygen species (ROS) production and major damage associated molecular patterns (calreticulin (CRT), heat shock protein 70 (Hsp70), high mobility group box 1 (HMGB1), and ATP) were studied in human and murine glioma cell lines post-ZEGFR:03115–IR700 PIT using flow cytometry (FC), Western blot and ELISA. Maturation of dendritic cells (DCs) stimulated by ZEGFR:03115-IR700 PIT was verified by FC. Xenografts and syngeneic murine tumour models (subcutaneous and orthotopic) were used to determine therapeutic and tumour-specific immune response following PIT. Post-treatment tumours were evaluated ex vivo. ResultsCells treated with ZEGFR:03115–IR700 PIT showed a significant decrease in cell viability. Generation of ROS post-PIT resulted in translocation of CRT to the cell membrane and the release of HMGB1, Hsp70 and ATP. FC analysis showed significant increase in the surface expression of CD86 and HLA-DR molecules on DCs stimulated by ZEGFR:03115–IR700 PIT compared to the negative controls. In vivo, PIT led to a significant delay in subcutaneous tumour growth. A therapeutic efficacy of the conjugate was observed in brain tumours as early as 24h post-irradiation. Ki-67, CD31, H&E staining of tumour sections showed a reduced cell proliferation index, distinct differences in vessel density, extensive tumour necrosis and microhaemorrhage on the margins of the treated tumours. In addition, tumour-infiltrating lymphocytes were elevated in the treated mice compared with the controls. ConclusionsEGFR-targeted PIT is an attractive therapeutic strategy that boosts the anti-tumour T cell response which might influence the suppressed immune microenvironment in GBM patients. Legal entity responsible for the studyThe authors. FundingThe Institute of Cancer Research; The National Science Centre (NCNOPUS13#2017/25/B/NZ5/00039); and CRUK Convergence Science Centre Fund. DisclosureAll authors have declared no conflicts of interest.

Ecto-Calreticulin Expression After Particle Irradiation Versus Conventional Photon Irradiation in Human Cancer Cell Lines

Radiotherapy can activate anti-tumor immune responses by triggering immunogenic cell death (ICD) of tumor cells. ICD is characterized by the release of danger-associated molecular patterns (DAMPs). Calreticulin (CRT) is one of the most potent DAMPs. The cell surface translocation of CRT (ecto-CRT) serves as an “eat me” signal to antigen-presenting cells (APCs), which plays a pivotal role in activating anti-tumor immunity. However, there is limited information describing the impact of proton and carbon-ion irradiation on ecto-CRT expression. Thus, we investigated ecto-CRT expression in multiple human cancer cell lines irradiated by proton and carbon-ion in comparison to conventional photon irradiation. This study examined four human cancer cell lines: A549 (lung adenocarcinoma), U251 MG (glioma), Tca8113 (tongue squamous carcinoma) and CNE-2 (nasopharyngeal carcinoma). Cell lines were irradiated to 0,2,4,10Gy with proton, carbon ion, or photon. The expression levels of ecto-CRT were analyzed by flow cytometry at 12h, 24h and 48h post-irradiation. Mean Fluorescence Intensity (MFI) of all samples were calculated by cytometry analysis software. There was a dose-dependent increase of ecto-CRT expression after photon, proton or carbon-ion irradiation in all four cell lines. Higher dose irradiation induced more ecto-CRT expression (p<0.05). The expression levels of ecto-CRT were significantly increased at 48h post-irradiation rather than 12h or 24h post-irradiation (p<0.05). When comparing at the same relative biological effectiveness (RBE) dose, 10GyE, there was no significant difference of ecto-CRT expression changes at 48h post-irradiation with proton and carbon-ion in comparison to photon. The MFI fold changes of 10GyE dose at 48h post-irradiation compared to control group (0Gy) for A549, U251, Tca8113 and CNE-2 were 1.63-1.73, 1.29-1.79,1.54-1.69 and 2.21-2.59, respectively. Proton and carbon-ion as well as photon irradiation can enhance the ecto-CRT expression with a dose-dependent manner in human cancer cell lines.

In vitro anti-tumor effect of low-power laser irradiation (LPLI) on gastroenterological carcinoma cells.

Primary hepatocellular carcinoma (HCC) and colon carcinoma are two of the most common clinical malignancies along with high morbidity and mortality. As low-power laser irradiation (LPLI) can induce cytotoxicity or cell apoptosis on several types of hyperplasia, LPLI may be a potential alternative treatment for gastroenterological cancers. The current in vitro study focused on LPLI-induced apoptosis and mechanism after 532-nm laser irradiation on two different carcinoma cells. Squamous cell carcinoma (VX2) and murine colon carcinoma (CT26) cells were cultured to test the feasibility of LPLI. The applied fluence varied from 0 to 600J/cm2. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide analysis, fluorescence imaging, wound healing assay, and cell apoptosis tests were performed 24h post-irradiation to monitor cellular responses. The current results demonstrated a dose-dependent stimulatory effect of LPLI on the cell viability, migration, and apoptosis of VX2 and CT26 cells. The therapeutic fluence of 600J/cm2 induced statistically significant inhibition in cell viability. Both the wound healing assay and the cell apoptosis tests confirmed that LPLI with high fluences could inhibit cell migration as well as induce cell apoptosis. The current findings demonstrate that LPLI might be a potential treatment for the carcinoma cells. Further studies will be performed to evaluate the feasibility of LPLI in in vivo tumor models.

Abstract 4862: Effect of irradiation and gold nanoparticle on expression dynamics of cell surface markers in MDA-MB 231 breast cancer cells

Abstract Cell surface molecules expressed on cancer cells can be used as diagnostic and therapeutic tools. In breast cancer, CD44 and CD24 were identified as cell surface markers characterizing cancer cell stemness that may correlate with prognosis. Breast cancer cells with high CD44 and low CD24 expression have been shown to exhibit proliferative, invasive and metastatic properties that may relate to drug sensitivity and metastatic risk in patients. In addition to stemness, immune evasion is another hallmark of cancer with CD47 “don’t eat me” and CD274 “don’t find me” molecules playing important roles in antitumor immunity. Although each of these molecules are of interest as therapeutic targets, current therapeutic paradigms involve multimodal approach with radiation therapy often having a central role. Hence, the effect of irradiation, and related radio-sensitizers, on the expression of cancer cells surface markers to be targeted with an adjuvant therapeutic mode must be considered when designing such therapies. The goal of this study was to determine the dynamics of CD44, CD24, CD47 and CD274 expression in radio-sensitized and irradiated MDA-MB 231 breast cancer cells. We have recently reported that gold nanoparticles (AuNPs) sensitized breast cancer cells to irradiation at kV and MV energies. Here, we hypothesize that AuNP may also modulate the irradiation altered expression of cell surface markers. We explored this by using 10 MV energies and 6 Gy radiation dose. MDA-MB-231 cells were incubated for 3 hours with 14nm AuNPs, irradiated and allowed to grow for 24h and 72h after which percenteges of positive cells were determined by flow cytometry. Results are expressed as a percent of control, non-irradiated cells that was set at 100%. At 24h non-irradiated cells pre-incubated with AuNP exhibited a decrease in the percentage of CD24+ and CD44+ cells, while no significant change in the percentage of CD47+ and CD274+ cells was observed. Irradiation with 6Gy at 10 MV induced a decrease in CD24+ and an increase in CD44+ cells and AuNP potentiated this effect. However, 72h post-irradiation, percentages of CD24+ and CD44+ cells significantly increased, compared to non-irradiated controls and this effect was of lesser magnitude in AuNP pretreated cells. Percentage of CD47+ and CD274+ cells also significantly increased 24h post-irradiation, but with less magnitude in AuNP treated cells. At 72h in AuNP treated irradiated cells no change was observed in the percentage of CD274+ cells, while the percentage of CD47+ cells significantly increased in comparison to the irradiated cells not exposed to AuNPs. Preliminary data shown here indicate that expression of molecules important for cancer progression, metastasis and immune evasion undergo changes in response to irradiation, and that these changes are affected by AuNPs. Further studies will shed more light on the mechanisms behind these observed effects. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin Chetty, Benjamin Movsas, Ning Wen. Effect of irradiation and gold nanoparticle on expression dynamics of cell surface markers in MDA-MB 231 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4862.

BYSTANDER WI-38 CELLS MODULATE DNA DOUBLE-STRAND BREAK REPAIR IN MICROBEAM-TARGETED A549 CELLS THROUGH GAP JUNCTION INTERCELLULAR COMMUNICATION.

Bi-directional signaling involved in radiation-induced bystander effect (RIBE) between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to radiation cancer therapy. Using the SPICE-NIRS microbeam, we delivered 500 protons to A549-GFP lung carcinoma cells, stably expressing H2B-GFP, which were co-cultured with normal WI-38 cells. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured up to 24-h post-irradiation in both targeted and bystander cells. As a result, inhibition of gap junction intercellular communication (GJIC) attenuated DSB repair in targeted A549-GFP cells, and suppressed RIBE in bystander WI-38 cells but not in distant A549-GFP cells. This suggests that GJIC plays a two-way role through propagating DNA damage effect between carcinoma to normal cells and reversing the bystander signaling, also called 'rescue effect' from bystander cells to irradiated cells, to enhance the DSB repair in targeted cells.

Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

The distinct role of low-level laser irradiation (LLLI) on endothelial exosome biogenesis remains unclear. We hypothesize that laser irradiation of high dose in human endothelial cells (ECs) contributes to the modulation of exosome biogenesis via Wnt signaling pathway. When human ECs were treated with LLLI at a power density of 80J/cm2, the survival rate reduced. The potential of irradiated cells to release exosomes was increased significantly by expressing genes CD63, Alix, Rab27a, and b. This occurrence coincided with an enhanced acetylcholine esterase activity, pseudopodia formation, and reduced zeta potential value 24h post-irradiation. Western blotting showed the induction of LC3 and reduced level of P62, confirming autophagy response. Flow cytometry and electron microscopy analyses revealed the health status of the mitochondrial function indicated by normal ΔΨ activity without any changes in the transcription level of PINK1 and Optineurin. When cells exposed to high power laser irradiation, p-Akt/Akt ratio and in vitro tubulogenesis capacity were blunted. PCR array and bioinformatics analyses showed the induction of transcription factors promoting Wnt signaling pathways and GTPase activity. Thus, LLLI at high power intensity increased exosome biogenesis by the induction of autophagy and Wnt signaling. LLLI at high power intensity increases exosome biogenesis by engaging the transcription factors related to Wnt signaling and autophagy stimulate.

Tumor heterogeneity determined with a γH2AX foci assay: A study in human head and neck squamous cell carcinoma (hHNSCC) models

PurposeThis study aimed to analyze the intra-tumoral heterogeneity of γH2AX foci in tumor specimens following ex vivo radiation to evaluate the potential of γH2AX foci as predictors for radiosensitivity. Material and methodsγH2AX foci were quantified in tumor specimens of 3hHNSCC tumor models with known differences in radiosensitivity after reoxygenation in culture medium (10h, 24h), single dose exposure (0Gy, 4Gy), and fixation 24h post-irradiation. Multiple, equally treated samples of the same tumor were analyzed for foci, normalized and fitted in a linear mixed-effects model. ResultsThe ex vivo reoxygenation time had no significant effect on γH2AX foci counts. A significant intra model heterogeneity could be shown for FaDu (p=0.033) but not for SKX (p=0.167) and UT-SCC-5 (p=0.082) tumors, respectively. All tumor models showed a significant intra-tumoral heterogeneity between specimens of the same tumor (p<0.01) or among microscopic fields of a particular tumor specimen (p<0.0001). ConclusionSimilar results for ex vivo γH2AX foci between 10h and 24h reoxygenation time support the applicability of the assay in a clinical setting. The high intra-tumoral heterogeneity underlines the necessity of multiple analyzable samples per patient and therewith the need for an automated foci analysis.

Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bi-directional signaling

Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24h post-irradiation in three categories of cells: (1) “targeted”/irradiated A549-GFP cells; (2) “neighboring”/non-irradiated cells directly contacting the “targeted” cells; and (3) “distant”/non-irradiated cells, which were not in direct contact with the “targeted” cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by γ-H2AX levels at 8h post-irradiation, showed a decrease to non-irradiated control level when approaching 24h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that γ-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi-directional signaling had extended a profound influence on cellular sensitivity to radiation as well as the sensitivity to RIBE.

Polymer gel dosimeters for pretreatment radiotherapy verification using the three-dimensional gamma evaluation and pass rate maps

Polymer gel dosimeters (PGDs) have been widely studied for use in the pretreatment verification of clinical radiation therapy. However, the readability of PGDs in three-dimensional (3D) dosimetry remain unclear. In this study, the pretreatment verifications of clinical radiation therapy were performed using an N-isopropyl-acrylamide (NIPAM) PGD, and the results were used to evaluate the performance of the NIPAM PGD on 3D dose measurement. A gel phantom was used to measure the dose distribution of a clinical case of intensity-modulated radiation therapy. Magnetic resonance imaging scans were performed for dose readouts. The measured dose volumes were compared with the planned dose volume. The relative volume histograms showed that relative volumes with a negative percent dose difference decreased as time elapsed. Furthermore, the histograms revealed few changes after 24h postirradiation. For the 3%/3mm and 2%/2mm criteria, the pass rates of the 12- and 24-h dose volumes were higher than 95%, respectively. This study thus concludes that the pass rate map can be used to evaluate the dose-temporal readability of PGDs and that the NIPAM PGD can be used for clinical pretreatment verifications.

Deuteron irradiation of W and WO3 for production of high specific activity 186Re: Challenges associated with thick target preparation

This investigation evaluated target fabrication and beam parameters for scale-up production of high specific activity 186Re using deuteron irradiation of enriched 186W via the 186W(d,2n)186Re reaction. Thick W and WO3 targets were prepared, characterized and evaluated in deuteron irradiations. Full-thickness targets, as determined using SRIM, were prepared by uniaxially pressing powdered natural abundance W and WO3, or 96.86% enriched 186W, into Al target supports. Alternatively, thick targets were prepared by pressing 186W between two layers of graphite powder or by placing pre-sintered (1105°C, 12h) natural abundance WO3 pellets into an Al target support. Assessments of structural integrity were made on each target prepared. Prior to irradiation, material composition analyses were conducted using SEM, XRD, and Raman spectroscopy. Within a minimum of 24h post irradiation, gamma-ray spectroscopy was performed on all targets to assess production yields and radionuclidic byproducts. Problems were encountered with the structural integrity of some pressed W and WO3 pellets before and during irradiation, and target material characterization results could be correlated with the structural integrity of the pressed target pellets. Under the conditions studied, the findings suggest that all WO3 targets prepared and studied were unacceptable. By contrast, 186W metal was found to be a viable target material for 186Re production. Thick targets prepared with powdered 186W pressed between layers of graphite provided a particularly robust target configuration.

SU-G-JeP2-11: Polymer Gels as 3D QA Devices in Magnetic Resonance-Guided Radiation Therapy

Purpose: To demonstrate the potential of polymer gels to serve as 3D QA devices in MR-guided radiotherapy. Methods: Custom-designed polymer gel dosimeters (MGS Research, Madison, CT) were centered on the penumbras of a radiation field, parallel to the magnetic field lines inside a 1.5 T MR scanner integrated with a 7 MV linac (MR-Linac). 15 Gy were delivered perpendicular to the magnetic field. Coronal MR images were acquired with a 2D balanced-Fast Field Echo (b-FFE) sequence in real-time during irradiation and with a 2D spin echo sequence at different times after irradiation. Signal intensities (SI) were measured inside and outside the radiation field on b-FFE images obtained during irradiation. Spin-spin relaxation rate (R2) maps were calculated from images after irradiation and penumbra widths were calculated from these maps. Results: A difference in SI between areas of the dosimeter inside and outside of the radiation field could be measured immediately after the beam was turned on. The difference in SI increased throughout the duration of the exposure. R2 values from immediately after irradiation were 83% of those measured 24h post-irradiation. At 1h after irradiation, the R2 values were 87% of those at 24h indicating that polymerization of the gel was still ongoing. At 24h postirradiation the width of the 80/20 penumbra on the left field edge measured 6.5 mm and on the right 5.6 mm. Conclusion: The MR-Linac allows for MR imaging during treatment and measurements of 3D dose distributions with steep dose gradients without having to transfer dosimeters for read-out. The results showed promise for polymer gels as relative 3D QA devices to verify radiation treatment plans delivered by an MR-Linac. To our knowledge, this was the first time that polymer gels were used for real-time image acquisition. Further investigations into the application of polymer gel dosimetry on MR-Linac are ongoing.