Effects of Ultra-high doserate FLASH Irradiation on the Tumor Microenvironment in Lewis Lung Carcinoma: Role of Myosin Light Chain.

PURPOSE: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR. METHODS AND MATERIALS: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed. RESULTS: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors. CONCLUSIONS: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR.

Tumor-Associated Macrophages Enhance Tumor Hypoxia and Aerobic Glycolysis.

Tumor hypoxia and aerobic glycolysis are well-known resistance factors for anticancer therapies. Here, we demonstrate that tumor-associated macrophages (TAM) enhance tumor hypoxia and aerobic glycolysis in mice subcutaneous tumors and in patients with non-small cell lung cancer (NSCLC). We found a strong correlation between CD68 TAM immunostaining and PET 18fluoro-deoxyglucose (FDG) uptake in 98 matched tumors of patients with NSCLC. We also observed a significant correlation between CD68 and glycolytic gene signatures in 513 patients with NSCLC from The Cancer Genome Atlas database. TAM secreted TNFα to promote tumor cell glycolysis, whereas increased AMP-activated protein kinase and peroxisome proliferator-activated receptor gamma coactivator 1-alpha in TAM facilitated tumor hypoxia. Depletion of TAM by clodronate was sufficient to abrogate aerobic glycolysis and tumor hypoxia, thereby improving tumor response to anticancer therapies. TAM depletion led to a significant increase in programmed death-ligand 1 (PD-L1) expression in aerobic cancer cells as well as T-cell infiltration in tumors, resulting in antitumor efficacy by PD-L1 antibodies, which were otherwise completely ineffective. These data suggest that TAM can significantly alter tumor metabolism, further complicating tumor response to anticancer therapies, including immunotherapy. SIGNIFICANCE: These findings show that tumor-associated macrophages can significantly modulate tumor metabolism, hindering the efficacy of anticancer therapies, including anti-PD-L1 immunotherapy.

Tumor hypoxia and reoxygenation: the yin and yang for radiotherapy.

Tumor hypoxia, a common feature occurring in nearly all human solid tumors is a major contributing factor for failures of anticancer therapies. Because ionizing radiation depends heavily on the presence of molecular oxygen to produce cytotoxic effect, the negative impact of tumor hypoxia had long been recognized. In this review, we will highlight some of the past attempts to overcome tumor hypoxia including hypoxic radiosensitizers and hypoxia-selective cytotoxin. Although they were (still are) a very clever idea, they lacked clinical efficacy largely because of 'reoxygenation' phenomenon occurring in the conventional low dose hyperfractionation radiotherapy prevented proper activation of these compounds. Recent meta-analysis and imaging studies do however indicate that there may be a significant clinical benefit in lowering the locoregional failures by using these compounds. Latest technological advancement in radiotherapy has allowed to deliver high doses of radiation conformally to the tumor volume. Although this technology has brought superb clinical responses for many types of cancer, recent modeling studies have predicted that tumor hypoxia is even more serious because 'reoxygenation' is low thereby leaving a large portion of hypoxic tumor cells behind. Wouldn't it be then reasonable to combine hypoxic radiosensitizers and/or hypoxia-selective cytotoxin with the latest radiotherapy? We will provide some preclinical and clinical evidence to support this idea hoping to revamp an enthusiasm for hypoxic radiosensitizers or hypoxia-selective cytotoxins as an adjunct therapy for radiotherapy.

Radiation-induced immune responses: mechanisms and therapeutic perspectives.

Recent advancement in the radiotherapy technology has allowed conformal delivery of high doses of ionizing radiation precisely to the tumors while sparing large volume of the normal tissues, which have led to better clinical responses. Despite this technological advancement many advanced tumors often recur and they do so within the previously irradiated regions. How could tumors recur after receiving such high ablative doses of radiation? In this review, we outlined how radiation can elicit anti-tumor responses by introducing some of the cytokines that can be induced by ionizing radiation. We then discuss how tumor hypoxia, a major limiting factor responsible for failure of radiotherapy, may also negatively impact the anti-tumor responses. In addition, we highlight how there may be other populations of immune cells including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) that can be recruited to tumors interfering with the anti-tumor immunity. Finally, the impact of irradiation on tumor hypoxia and the immune responses according to different radiotherapy regimen is also delineated. It is indeed an exciting time to see that radiotherapy is being combined with immunotherapy in the clinic and we hope that this review can add an excitement to the field.

Hyaluronate-Death Receptor 5 Antibody Conjugates for Targeted Treatment of Liver Metastasis.

The liver is the most frequent site of metastasis with a 5-year survival rate of only 20-40%. In this work, hyaluronate (HA)-death receptor 5 antibody (DR5 Ab) conjugate was synthesized as a dual targeting therapeutic agent to treat liver metastasis. Dual targeting was achieved by DR5 Ab, a humanized agonistic monoclonal antibody binding to DR5 frequently overexpressed in many kinds of cancer cells, and by HA, a natural polysaccharide binding to HA receptors highly expressed in both the liver and cancer cells. Thiol end-modified HA was site-specifically conjugated to N-glycan on Fc region of oxidized DR5 Ab using a heterobifunctional linker of 3-(2-pyridyldithio)propionyl hydrazide (PDPH). The successful synthesis of HA-DR5 Ab conjugate was confirmed by (1)H NMR, purpald assay, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). In vitro analysis of HA-DR5 Ab conjugate revealed that the conjugation of HA to DR5 Ab did not affect the binding affinity and anticancer efficacy of DR5 Ab. Remarkably, according to in vivo bioimaging study, HA-DR5 Ab conjugate appeared to be highly accumulated in the liver and dramatically effective in inhibiting the tumor growth in liver metastasis model mice.

Real-time Tumor Oxygenation Changes After Single High-dose Radiation Therapy in Orthotopic and Subcutaneous Lung Cancer in Mice: Clinical Implication for Stereotactic Ablative Radiation Therapy Schedule Optimization.

PURPOSE: To investigate the serial changes of tumor hypoxia in response to single high-dose irradiation by various clinical and preclinical methods to propose an optimal fractionation schedule for stereotactic ablative radiation therapy. METHODS AND MATERIALS: Syngeneic Lewis lung carcinomas were grown either orthotopically or subcutaneously in C57BL/6 mice and irradiated with a single dose of 15 Gy to mimic stereotactic ablative radiation therapy used in the clinic. Serial [(18)F]-misonidazole (F-MISO) positron emission tomography (PET) imaging, pimonidazole fluorescence-activated cell sorting analyses, hypoxia-responsive element-driven bioluminescence, and Hoechst 33342 perfusion were performed before irradiation (day -1), at 6 hours (day 0), and 2 (day 2) and 6 (day 6) days after irradiation for both subcutaneous and orthotopic lung tumors. For F-MISO, the tumor/brain ratio was analyzed. RESULTS: Hypoxic signals were too low to quantitate for orthotopic tumors using F-MISO PET or hypoxia-responsive element-driven bioluminescence imaging. In subcutaneous tumors, the maximum tumor/brain ratio was 2.87 ± 0.483 at day -1, 1.67 ± 0.116 at day 0, 2.92 ± 0.334 at day 2, and 2.13 ± 0.385 at day 6, indicating that tumor hypoxia was decreased immediately after irradiation and had returned to the pretreatment levels at day 2, followed by a slight decrease by day 6 after radiation. Pimonidazole analysis also revealed similar patterns. Using Hoechst 33342 vascular perfusion dye, CD31, and cleaved caspase 3 co-immunostaining, we found a rapid and transient vascular collapse, which might have resulted in poor intratumor perfusion of F-MISO PET tracer or pimonidazole delivered at day 0, leading to decreased hypoxic signals at day 0 by PET or pimonidazole analyses. CONCLUSIONS: We found tumor hypoxia levels decreased immediately after delivery of a single dose of 15 Gy and had returned to the pretreatment levels 2 days after irradiation and had decreased slightly by day 6. Our results indicate that single high-dose irradiation can produce a rapid, but reversible, vascular collapse in tumors.

Radiation-Induced Esophagitis In Vivo and In Vitro Reveals That Epidermal Growth Factor Is a Potential Candidate for Therapeutic Intervention Strategy.

PURPOSE: To establish and characterize radiation-induced esophagitis (RIE) in vivo and in vitro. METHODS AND MATERIALS: Fractionated thoracic irradiation at 0, 8, 12, or 15 Gy was given daily for 5 days to Balb/c or C57Bl/6 mice. Changes in body weight gain and daily food intake were assessed. At the end of the study, we removed the esophagus and examined histology by hematoxylin and eosin staining, immune cell infiltration and apoptosis by fluorescence-activated cell sorting, and gene expression changes by quantitative real-time polymerase chain reaction. Het-1A human esophageal epithelial cells were irradiated at 6 Gy, treated with recombinant human growth factors, and examined for gene expression changes, apoptosis, proliferation, and signal transduction pathways. RESULTS: We observed that irradiation at 12 Gy or 15 Gy per fraction produced significant reduction in body weight and decreased food intake in Balb/c mice but not as much in C57Bl/6 mice. Further analyses of Balb/c mice irradiated at 12 Gy/fraction revealed attenuated epithelium, inflamed mucosa, and increased numbers of infiltrating CD4+ helper T cells and apoptotic cells. Moreover, we found that expression of tissue inhibitor for metalloproteinase-1, plasminogen activator inhibitor-1, granulocyte macrophage-colony stimulating factor, vascular endothelial growth factor, and stromal-derived factor-1 were increased, whereas epidermal growth factor (EGF) was decreased. Irradiated Het-1A cells similarly showed a significant decrease in expression of EGF and connective tissue growth factor (CTGF). Treatment of EGF but not CTGF partially protected Het-1A cells from radiation-induced apoptosis and revealed phosphorylation of EGFR, AKT, and ERK signaling pathways. CONCLUSIONS: We established a mouse model of RIE in Balb/c mice with 12 Gy × 5 fractions, which showed reduced body weight gain, food intake, and histopathologic features similar to those of human esophagitis. Decreased EGF expression in the irradiated esophagus suggests that EGF may be a potential therapeutic intervention strategy to treat RIE.

In vivo imaging of activated microglia in a mouse model of focal cerebral ischemia by two-photon microscopy.

Microglia are brain resident macrophages rapidly responding to various stimuli to exert appropriate inflammatory responses. Although they have recently been exploited as an attractive candidate for imaging neuroinflammation, it is still difficult to visualize them at the cellular and molecular levels. Here we imaged activated microglia by establishing intracranial window chamber (ICW) in a mouse model of focal cerebral ischemia by using two-photon microscopy (TPM), in vivo. Intravenous injection of fluorescent antibodies allowed us to detect significantly elevated levels of Iba-1 and CD68 positive activated microglia in the ipsilateral compared to the contralateral side of the infarct. We further observed that indomethacin, a non-steroidal anti-inflammatory drug significantly attenuated CD68-positive microglial activation in ICW, which was further confirmed by qRT-PCR biochemical analyses. In conclusion, we believe that in vivo TPM imaging of ICW would be a useful tool to screen for therapeutic interventions lowering microglial activation hence neuroinflammation.

Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8.

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.