Fatty acid oxidation fuels glioblastoma radioresistance with CD47-mediated immune evasion.

Glioblastoma multiforme (GBM) remains the top challenge to radiotherapy with only 25% one-year survival after diagnosis. Here, we reveal that co-enhancement of mitochondrial fatty acid oxidation (FAO) enzymes (CPT1A, CPT2 and ACAD9) and immune checkpoint CD47 is dominant in recurrent GBM patients with poor prognosis. A glycolysis-to-FAO metabolic rewiring is associated with CD47 anti-phagocytosis in radioresistant GBM cells and regrown GBM after radiation in syngeneic mice. Inhibition of FAO by CPT1 inhibitor etomoxir or CRISPR-generated CPT1A-/-, CPT2-/-, ACAD9-/- cells demonstrate that FAO-derived acetyl-CoA upregulates CD47 transcription via NF-κB/RelA acetylation. Blocking FAO impairs tumor growth and reduces CD47 anti-phagocytosis. Etomoxir combined with anti-CD47 antibody synergizes radiation control of regrown tumors with boosted macrophage phagocytosis. These results demonstrate that enhanced fat acid metabolism promotes aggressive growth of GBM with CD47-mediated immune evasion. The FAO-CD47 axis may be targeted to improve GBM control by eliminating the radioresistant phagocytosis-proofing tumor cells in GBM radioimmunotherapy.

Dual blockage of STAT3 and ERK1/2 eliminates radioresistant GBM cells.

Radiotherapy (RT) is the major modality for control of glioblastoma multiforme (GBM), the most aggressive brain tumor in adults with poor prognosis and low patient survival rate. To improve the RT efficacy on GBM, the mechanism causing tumor adaptive radioresistance which leads to the failure of tumor control and lethal progression needs to be further elucidated. Here, we conducted a comparative analysis of RT-treated recurrent tumors versus primary counterparts in GBM patients, RT-treated orthotopic GBM tumors xenografts versus untreated tumors and radioresistant GBM cells versus wild type cells. The results reveal that activation of STAT3, a well-defined redox-sensitive transcriptional factor, is causally linked with GBM adaptive radioresistance. Database analysis also agrees with the worse prognosis in GBM patients due to the STAT3 expression-associated low RT responsiveness. However, although the radioresistant GBM cells can be resensitized by inhibition of STAT3, a fraction of radioresistant cells can still survive the RT combined with STAT3 inhibition or CRISPR/Cas9-mediated STAT3 knockout. A complementally enhanced activation of ERK1/2 by STAT3 inhibition is identified responsible for the survival of the remaining resistant tumor cells. Dual inhibition of ERK1/2 and STAT3 remarkably eliminates resistant GBM cells and inhibits tumor regrowth. These findings demonstrate a previously unknown feature ofSTAT3-mediated ERK1/2 regulation and an effective combination of two targets in resensitizing GBM to RT.

Outcomes of Preoperative Versus Postoperative Radiation for Heterotopic Ossification Prevention in Children With Neuromuscular Hip Dysplasia Undergoing Proximal Femoral Resection.

BACKGROUND: Few studies exist to inform the extrapolated practice of irradiating children for heterotopic ossification (HO) prevention. We report the incidence of HO formation following prophylactic preoperative compared with postoperative radiation therapy (RT) in children with neuromuscular hip dysplasia (NHD) following proximal femoral resection (PFR). METHODS: A retrospective, 2-institution chart review was performed. Eligibility was limited to patients with at least 1 year of follow-up. Evaluation included radiographic HO grading by a combined severity scale, assessment of synchronous symptoms of pain or decreased range of motion, and stratification by preoperative versus postoperative reception of RT. A control cohort included 4 nonirradiated hips with NHD after PFR. RESULTS: Twenty-five hips in 20 children met eligibility criteria. Eleven hips were irradiated preoperatively and 14 postoperatively. Radiographic evidence of post-RT development of radiographic evidence of heterotopic ossification (rHO) was found in all 25 hips and earlier in patients irradiated preoperatively (median time to rHO was 4.0 vs. 15.7 mo, P=0.03, 95% confidence interval, 0.24-21.5). There was no statistically significant difference in the development of symptomatic HO (P=0.62) between the preoperative (45.5%) and postoperative (35.7%) groups, nor in HO grade (P=0.34). Seven (28%) of the 25 hips (5 preoperative and 2 postoperative) had documentation of rHO-free intervals after surgery, with an average duration of 5.6 months, while the remaining presented with rHO at first follow-up visit. All eligible control hips (100%) developed rHO and symptomatic heterotopic ossification. CONCLUSIONS: Perioperative RT did not prevent the formation of HO in any child with NHD after PFR. Extrapolation of evidence of the efficacy of RT for HO prevention in ambulatory adults after traumatic hip injury to a population of children with central nervous system injury and NHD may be premature. Additional studies are needed to clarify optimal prevention of HO in this population. LEVEL OF EVIDENCE: Level III-therapeutic retrospective comparative study.

Label-free fluorescence lifetime spectroscopy detects radiation-induced necrotic changes in live brain in real-time.

Current clinical imaging modalities do not reliably identify brain tissue regions with necrosis following radiotherapy. This creates challenges for stereotaxic biopsies and surgical-decision making. Time-resolved fluorescence spectroscopy (TRFS) provides a means to rapidly identify necrotic tissue by its distinct autofluorescence signature resulting from tissue breakdown and altered metabolic profiles in regions with radiation damage. Studies conducted in a live animal model of radiation necrosis demonstrated that necrotic tissue is characterized by respective increases of 27% and 108% in average lifetime and redox ratio, when compared with healthy tissue. Moreover, radiation-damaged tissue not visible by MRI but confirmed by histopathology, was detected by TRFS. Current results demonstrate the ability of TRFS to identify radiation-damaged brain tissue in real-time and indicates its potential to assist with surgical guidance and MRI-guided biopsy procedures.

Converting Treatment Plans From Helical Tomotherapy to L-Shape Linac: Clinical Workflow and Dosimetric Evaluation.

This work evaluated a commercial fallback planning workflow designed to provide cross-platform treatment planning and delivery. A total of 27 helical tomotherapy intensity-modulated radiotherapy plans covering 4 anatomical sites were selected, including 7 brain, 5 unilateral head and neck, 5 bilateral head and neck, 5 pelvis, and 5 prostate cases. All helical tomotherapy plans were converted to 7-field/9-field intensity-modulated radiotherapy and volumetric-modulated radiotherapy plans through fallback dose-mimicking algorithm using a 6-MV beam model. The planning target volume (PTV) coverage ( D1, D99, and homogeneity index) and organs at risk dose constraints were evaluated and compared. Overall, all 3 techniques resulted in relatively inferior target dose coverage compared to helical tomotherapy plans, with higher homogeneity index and maximum dose. The organs at risk dose ratio of fallback to helical tomotherapy plans covered a wide spectrum, from 0.87 to 1.11 on average for all sites, with fallback plans being superior for brain, pelvis, and prostate sites. The quality of fallback plans depends on the delivery technique, field numbers, and angles, as well as user selection of structures for organs at risk. In actual clinical scenario, fallback plans would typically be needed for 1 to 5 fractions of a treatment course in the event of machine breakdown. Our results suggested that <1% dose variance can be introduced in target coverage and/or organs at risk from fallback plans. The presented clinical workflow showed that the fallback plan generation typically takes 10 to 20 minutes per case. Fallback planning provides an expeditious and effective strategy for transferring patients cross platforms, and minimizing the untold risk of a patient missing treatment(s).

Stereotactic Ablative Radiation Therapy Induces Systemic Differences in Peripheral Blood Immunophenotype Dependent on Irradiated Site.

PURPOSE: Despite the strong interest in combining stereotactic ablative radiation therapy (SAR) with immunotherapy, limited data characterizing the systemic immune response after SAR are available. We hypothesized that the systemic immune response to SAR would differ by irradiated site owing to inherent differences in the microenvironment of various organs. METHODS AND MATERIALS: Patients receiving SAR to any organ underwent prospective blood banking before and 1 to 2 weeks after SAR. Peripheral blood mononuclear cells (PBMCs) and serum were isolated. PBMCs were stained with fluorophore-conjugated antibodies against T and natural killer (NK) cell markers. Cells were interrogated by flow cytometry, and the results were analyzed using FlowJo software. Serum cytokine and chemokine levels were measured using Luminex. We analyzed the changes from before to after therapy using paired t tests or 1-way analysis of variance (ANOVA) with Bonferroni's post-test. RESULTS: A total of 31 patients had evaluable PBMCs for flow cytometry and 37 had evaluable serum samples for Luminex analysis. The total number of NK cells and cytotoxic (CD56dimCD16+) NK cells decreased (P = .02) and T-cell immunoglobulin- and mucin domain-containing molecule-3-positive (TIM3+) NK cells increased (P = .04) after SAR to parenchymal sites (lung and liver) but not to bone or brain. The total memory CD4+ T cells, activated inducible co-stimulator-positive and CD25+CD4+ memory T cells, and activated CD25+CD8+ memory T cells increased after SAR to parenchymal sites but not bone or brain. The circulating levels of tumor necrosis factor-α (P = .04) and multiple chemokines, including RANTES (P = .04), decreased after SAR to parenchymal sites but not bone or brain. CONCLUSIONS: Our data suggest SAR to parenchymal sites induces systemic immune changes, including a decrease in total and cytotoxic NK cells, an increase in TIM3+ NK cells, and an increase in activated memory CD4+ and CD8+ T cells. SAR to nonparenchymal sites did not induce these changes. By comparing the immune response after radiation to different organs, our data suggest SAR induces systemic immunologic changes that are dependent on the irradiated site.

Patient-reported quality-of-life outcomes after de-escalated chemoradiation for human papillomavirus-positive oropharyngeal carcinoma: Findings from a phase 2 trial.

BACKGROUND: The current study represents a subset analysis of quality-of-life (QOL) outcomes among patients treated on a phase 2 trial of de-escalated chemoradiation for human papillomavirus (HPV)-associated oropharyngeal cancer. METHODS: Eligibility included newly diagnosed, (American Joint Committee on Cancer, 7th edition) stage III or IV oropharyngeal squamous cell carcinoma, p16 positivity, age ≥ 18 years, and a Zubrod performance status of 0 to 1. Treatment was induction paclitaxel at a dose of 175 mg/m2 and carboplatin at an area under the curve of 6 for 2 cycles followed by response-adapted, dose-reduced radiation of 54 Gy or 60 Gy with weekly concurrent paclitaxel at a dose of 30 mg/m2 . The University of Washington Quality of Life (UW-QOL) and the Functional Assessment of Cancer Therapy-Head and Neck questionnaires were used to assess patient-reported QOL as a secondary endpoint. RESULTS: A total of 45 patients were registered, 40 of whom completed QOL surveys and were evaluable. Nadirs for overall UW-QOL and Functional Assessment of Cancer Therapy-Head and Neck scores were reached at 4 weeks after treatment but returned to baseline at 3 months. Nearly all functional indices returned to baseline levels by 6 to 9 months. The mean overall UW-QOL score was 71.6 at baseline compared with 70.8, 73.0, 83.3, and 81.1, respectively, at 3 months, 6 months, 1 year, and 2 years after therapy. The percentage of patients rating their overall QOL as "very good" or "outstanding" at 6 months, 1 year, and 2 years using the UW-QOL was 50%, 77%, and 84%, respectively. CONCLUSIONS: This de-escalation regimen achieved QOL outcomes that were favorable compared with historical controls. These results serve as powerful evidence that ongoing de-escalation efforts lead to tangible gains in function and QOL. Cancer 2018;124:521-9. © 2017 American Cancer Society.

The effect of radiation dose on the onset and progression of radiation-induced brain necrosis in the rat model.

PURPOSE: To provide a comprehensive understanding of how the selection of radiation dose affects the temporal and spatial progression of radiation-induced necrosis in the rat model. MATERIALS AND METHODS: Necrosis was induced with a single fraction of radiation exposure, at doses ranging between 20 and 60 Gy, to the right hemisphere of 8-week-old Fischer rats from a linear accelerator. The development and progression of necrosis in the rats was monitored and quantified every other week with T1- and T2-weighted gadolinium contrast-enhanced MRI studies. RESULTS: The time to onset of necrosis was found to be dose-dependent, but after the initial onset, the necrosis progression rate and total volume generated was constant across different doses ranging between 30 and 60 Gy. Radiation doses less than 30 Gy did not develop necrosis within 33 weeks after treatment, indicating a dose threshold existing between 20 and 30 Gy. CONCLUSION: The highest dose used in this study led to the shortest time to onset of radiation-induced necrosis, while producing comparable disease progression dynamics after the onset. Therefore, for the radiation-induced necrosis rat model using a linear accelerator, the most optimum results were generated from a dose of 60 Gy.

NK Cells Preferentially Target Tumor Cells with a Cancer Stem Cell Phenotype.

Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24(+)/CD44(+), CD133(+), and aldehyde dehydrogenase(bright)) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.

Design and development of masked therapeutic antibodies to limit off-target effects: application to anti-EGFR antibodies.

Therapeutic antibodies frequently cause side effects by binding antigen in non-target tissues. Here we demonstrate a novel molecular design of antibodies that addresses this problem by reversibly "masking" antibody complementarity determining regions until they reach diseased tissues containing disease-associated proteases. Specifically, two distinct single-chain Fv (scFv) fragments derived from antibodies against the epidermal growth factor receptor (cetuximab and 425) were fused a protease susceptible linker to their epitopes, which were engineered to encourage intermolecular association. Surface plasmon resonance and flow cytometry were used to confirm that the masked complex poorly interacts with native antigen, whereas protease treatment restores antigen recognition. Minimally, the "masked" scFvs possesses an eight-fold lower association with the epitope compared with the individual scFvs unmasked by proteolytic cleavage. This molecular design may have general utility for targeted release of therapeutic antibodies at disease sites.

A CD8/Lck transgene is able to drive thymocyte differentiation.

Efficient development of thymocytes requires participation of a CD8 or CD4 coreceptor in the TCR:MHC interaction. Both CD8 and CD4 coreceptor cytoplasmic domains associate with Lck. In this study, we attempted to delineate the role of CD8alpha-associated Lck in driving CD8 single positive (SP) thymocyte development. We used a chimeric molecule encoding the extracellular and transmembrane domains of CD8alpha fused to full-length Lck. In mice deficient for CD8alpha and transgenic for 2C, a MHC class I-restricted TCR, robust reconstitution of CD8 SP thymocytes occurred both centrally and peripherally. The reconstituted CD8 SP population was phenotypically and functionally comparable to 2C wild-type counterparts expressing endogenous CD8alpha. A CD8alpha/Lck kinase-dead chimera also resulted in reconstitution of CD8 SP thymocytes. Our results suggest that CD8alpha-associated Lck is sufficient to drive CD8 SP thymocyte development. Furthermore, this CD8 SP development may not necessarily depend on Lck kinase activity.

CD8 T cells are sufficient to mediate allorecognition and allograft rejection.

Different T cell subsets may play different roles in allorecognition and allograft rejection. It has been suggested that CD8 T cells can only initiate rejection with help from CD4 T cells. Since CD8 T cells may have different requirements for allorecognition and for costimulation, it is important to clarify the role of CD8 cells in rejection. We examined the role of CD8 cells in allorecognition using a TCR transgenic mouse transplantation model. In our study, CD8 cells were able to recognize alloantigens and reject allografts in the absence of help from CD4 T cells. Furthermore our study provides a model to study the mechanisms of CD8-mediated allograft rejection. It may be important in the future, to consider that CD8 T cells may need to be targeted independently of CD4 T cells in strategies used to prevent rejection and induce tolerance.