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Thu, 01.01.1970
FLASH PROTON RADIOTHERAPY IS EQUIPOTENT TO STANDARD RADIATION IN TREATMENT OF MURINE SARCOMAS WHILE REDUCING TOXICITIES TO NORMAL SKIN, MUSCLE AND BONE
Abstract
Background and Aims
Compared to Standard dose rates, the high dose rates of FLASH radiation can reduce radiotherapy toxicities to normal tissues. We examined the potential of FLASH-proton radiotherapy (F-PRT) to treat murine sarcomas and protect normal epithelial and mesenchymal tissues relative to the effects of standard-proton radiotherapy (S-PRT).
Methods
Mice received 30 or 45 Gy of F-PRT (69-124 Gy/sec) or S-PRT (0.39–0.65 Gy/sec) to their hind legs. Skin, muscle and bone injuries were recorded as acute through chronic macroscopic and/or microscopic observations of radiation-induced damage. Murine skin and bone RNAseq analyses were performed to delineate involved mechanisms. Skin stem cell depletion, inflammatory reaction and TGF-β levels were evaluated, and antitumor efficacy of F-PRT was compared to S-PRT in two murine models of sarcoma.
Results
Fewer severe morbidities were induced by F-PRT, with RNAseq revealing S-PRT to upregulate pathways involved in apoptosis signaling and keratinocyte differentiation in skin, and osteoclast differentiation and chondrocyte development in bone. Accordingly, F-PRT reduced skin injury, stem cell depletion and inflammation; mitigated lymphedema; and decreased myofiber atrophy, bone resorption, hair follicle atrophy, and epidermal hyperplasia. Equipotent control of sarcoma growth was achieved by the radiation modalities. Finally, S-PRT produced higher levels of TGF-β1 in murine skin than did F-PRT, and this finding was corroborated in the skin samples of dogs treated on a F-PRT clinical trial.
Conclusions
F-PRT can alleviate radiation-induced damage to both epithelial and mesenchymal tissues without compromise to sarcoma response; continuing investigation will further F-PRT translation to the clinic.
CHARACTERIZATION OF DAMAGE ASSOCIATED MOLECULAR PATTERNS AFTER FLASH RADIOTHERAPY TO AMPLIFY ANTI-TUMOR IMMUNE RESPONSE
Abstract
Background and Aims
Using glioblastoma as model, the aim of the present study was to investigate the role of G2/M arrest in tumor response to FLASH-RT and to characterize Damage Associated Molecular Patterns (DAMPs) that might amplify anti-tumor immunogenic response.
Methods
In vitro, GL261, H454 and PDGC2159 GBM and HaCat normal cells were synchronized (or not) in G2/M phase using a CDK1 (9uM) or PLK1 (25nM) inhibitor 24hours before 20Gy FLASH-RT (2.103Gy/s, 2 pulses of 10Gy, 100Hz) or CONV-RT (~0.1-0.2Gy/s, 10Hz) with eRT6 (Jorge, 2019). Calreticulin, HSPA5, ATP, HMGB1, DNA release, micronuclei formation and cGAS-STING-type I IFN response were investigated. In vivo, murine GL261 and PDGC2159 GBM cells were orthotopically grafted to C57Bl6 and Swiss nude mice. Mice were treated with a single dose of 10Gy delivered Whole-Brain either with FLASH (≥107Gy/s, 1 pulse) or CONV-RT (~0.1-0.2Gy/s). Tumor control, normal brain toxicity, immune response and in situ vaccination were evaluated.
Results
In vitro, the level of micronuclei positive cells was similar after FLASH and CONV (40% vs 0% in non-RT) and HMGB1 mRNA level was enhanced (+1.8fold) in FLASH vs CONV irradiated samples. G2/M blockade significantly increased micronuclei formation (+20%), and cGAS mRNA level (+2.33fold) in FLASH vs CONV irradiated samples. Other markers were not modified. In vivo experiments are ongoing.
Conclusions
These preliminary results support a G2/M-dependent release of DAMPs after FLASH irradiation that might trigger downstream immune response. Experiments are ongoing to characterize this response along with anti-tumor efficacy and normal toxicity in immune-deficient/competent mice.
FLASH SPARING OF MELANOMA CELLS IN VITRO AND IN VIVO
Abstract
Background and Aims
We have previously found that FLASH-irradiation with a pulsed electron beam (average doserate ≥600Gy/s) was less efficient to sterilize cancer cells in-vitro compared with conventional doserate irradiation (CONV, 0.2Gy/s). In the current work we aimed at investigating the effect for a malignant cell line both in vitro and in vivo.
Methods
Radiation response of melanoma cell line B16_F10 was determined in-vitro by clonogenic assays for an absorbed dose in the range 0-9 Gy comparing FLASH to CONV. In-vivo-response was studied in a syngeneic mice model (C57BL/6J) with subcutaneously injected B16_F10-tumors, irradiated to an absorbed dose of 15, 20 or 25Gy (FLASH and CONV). The tumor growth was quantified by using the relative tumor volume, normalized to unity at the time of irradiation, TVrel.
Results
The in-vitro results showed a significantly increased survival after FLASH compared with CONV (F-test: p=0.02). Tumor growth curves in-vivo were similar for CONV and FLASH at 15 and 20Gy, but FLASH was relatively less efficient at 25Gy. Four weeks after irradiation with 25Gy, a relative tumor volume of TVrel<1 was seen in 2/9 mice in the CONV group but in 0/8 mice in the FLASH group. A relative tumor volume of TVrel<4 was seen in 5/9 mice in the CONV group but 0/8 mice in the FLASH group. Severe skin toxicity was observed in 5/9 vs 0/8.
Conclusions
FLASH may be less efficient than CONV to sterilize malignant cells in-vitro as well as in-vivo. Future work will address the differential response between normal tissue and tumors at higher doses.
LONGITUDINAL IN-VIVO ASSESSMENT OF MOUSE SKIN DAMAGE WITH FUNCTIONAL OPTICAL COHERENCE TOMOGRAPHY IN FLASH VERSUS CONVENTIONAL RADIOTHERAPY
Abstract
Background and Aims
Reduced skin toxicity is now shown pre-clinically after >40Gy/s FLASH radiotherapy (RT) as compared to ~0.03Gy/s conventional RT. While this area of study has been advanced significantly, the biological basis of the FLASH sparing effect on skin is yet to be discovered, and diagnostic tools to assess the response biological mechanisms and tools that can be used in the translation of FLASH study in humans are needed. Here we report on direct mechanistic in situ measurements of skin damage, with the aim to quantify and compare microstructural and microvascular changes in skin after RT, while varying dose and supporting the findings with ex vivo histological observations.
Methods
In IACUC approved animal study, right thighs of 54 nude mice (n=6 per dose group) were treated with 0-40Gy single doses of 300Gy/s FLASH and conventional RT. Skin was imaged with functional optical coherence tomography - a non-invasive microscopic 3D imaging technique with light penetration at 1-3 millimeter depths in biological tissues.
Results
Quantification was completed for skin pigmentation, epidermal thickness, remodeling of collagen fibers, alteration of blood and lymphatic networks. These demonstrated inter-connected “passenger versus driver” temporal behaviors of skin tissue components in both RT-treatment types. Response to FLASH RT was characterized by reduced damage to collagen bundles and blood/lymphatic networks together with less desquamation of skin surface (less damage to epidermis) and higher pigmentation.
Conclusions
This study presents first of its kind in-vivo functional longitudinal observations and quantification of the comparative FLASH effects in a mouse model of skin early damage, healing and recovery.
MOUSE ABDOMEN RADIATION USING A 50 MEV PROTON BEAM: FLASH VS. CONVENTIONAL DOSE RATE
Abstract
Background and Aims
The normal tissue toxicity sparing and survival benefits of ultra-high dose rate radiation (FLASH) remain poorly understood. We present preliminary results of mouse abdomen FLASH proton radiation from a low-energy proton system (50 MeV) optimized for small animal radiobiological research.
Methods
We radiated 6-7 week old female C57BL/6 mice with partial abdomen radiation using the plateau region of a continuous (unpulsed) cyclotron-generated 50 MeV preclinical proton beam, transmitting through the abdomen, with 1.5cm width of beam via customized vertical and horizontal collimators. Mice were stratified into 3 groups: 1) control/sham radiation (n=8); 2) conventional dose rate (20Gy at ~1Gy/sec, n=19); and 3) FLASH (20Gy at 48-93Gy/sec, n=22). Mice were observed for survival. Colon tissue was harvested at 1-hour post-radiation. H&E and immunohistochemistry was performed for: yH2aX and cleaved caspase-3. Experiments were repeated in triplicate.
Results
Survival was different between FLASH and conventional groups: FLASH (13 days post radiation, 36.4% survival); conventional (15.6% survival, P = 0.04 ) [Figure 1]. One-hour post radiation, lower cleaved caspase-3 IHC staining was seen in the FLASH group versus conventional group, while yH2aX staining was similar in both groups [Figure 2].
Conclusions
Preliminary results of mouse partial abdomen FLASH proton radiation from a 50 MeV beam suggest FLASH proton radiation leads to better survival than conventional dose rate radiation. More studies are ongoing.
BIOLOGICAL EFFECT OF MURINE VENTRAL SKIN IRRADIATION WITH PULSED FLASH RADIOTHERAPY USING A CLINICAL LINAC
Abstract
Background and Aims
To investigate the biological effect of murine ventral skin irradiation with FLASH radiotherapy compared with conventional irradiation.
Methods
Female FvB mice were randomly assigned to three groups: control, conventional (CONV) and FLASH groups. Mice were irradiated at 9 to 19 Gy of CONV (0.1 Gy/s) or FLASH (38.5-600 Gy/s) irradiation using traditional and modified Elekta Synergy linac (6 MeV), respectively. Doses were verified by Gafchromic films positioned under the body. Body weights were recorded every week 1 to 6 weeks after irradiation. Enzyme linked immunosorbent assay (ELISA) were performed in skin tissue lysis and serum samples of the mice for four inflammatory cytokines: tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and IL-10. Flow cytometry using antibodies for CD3, CD8, CD4 and CD45 in blood were performed pre- and 1-week post irradiation.
Results
A significant increase in weight percentages relative to pre-irradiation were observed in the FLASH group, and the alteration of serum and skin tissue levels of TNF-α, IFN-γ, IL-6 and IL-10 induced by FLASH was mild compared with that of CONV. The CD8+/CD45+ ratio in the blood were higher in the CONV than in FLASH and pre-irradiated ratio. These data indicate that lower inflammatory cytokine levels of serum and skin tissue in FLASH could be the result of minor immune overactivation.
Conclusions
Ultra-high dose rate electron FLASH caused less body weight loss, minor inflammatory cytokine levels of serum and skin tissue, as well as less CD8+/CD45+ ratio in the blood. Thus, electron FLASH irradiation represents a new approach of radiotherapy.