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Purpose: To investigate the effects of short-term administration of dexamethasone (DEX) on radiation-induced responses in the mouse lung, focusing on expression of pro-inflammatory cytokine and related genes. Methods and Materials: At indicated times after thoracic irradiation and/or drug treatment, mRNA expression levels of cytokines (mTNF-α, mIL-1α, mIL-1β, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFN-γ) and related genes in the lungs of C3H/HeN mice were measured by RNase protection assay. Results: Radiation-induced pro-inflammatory cytokine mRNA expression levels in lung peak at 6 h after thoracic irradiation. DEX (5 mg/kg) suppresses both basal cytokine mRNA levels and this early response when given immediately after irradiation. However, by 24 h, in mice treated with DEX alone or DEX plus radiation, there was a strong rebound effect that lasted up to 3 days. Modification of the early radiation-induced response by DEX did not change the second wave of cytokine gene expression in the lung that occurs at 1 to 2 weeks, suggesting that early cytokine gene induction might not determine subsequent molecular events. A single dose of DEX attenuated, but did not completely suppress, increases in cytokine mRNA levels induced by lipopolysaccharide (2.5 mg/kg) treatment, but, unlike with radiation, no significant rebound effect was seen. Five days of dexamethasone treatment in the pneumonitic phase also inhibited pro-inflammatory cytokine gene expression and, again, there was a rebound effect after withdrawal of the drug. Conclusions: Our findings suggest that short-term use of dexamethasone can temporarily suppress radiation-induced pro-inflammatory cytokine gene expression, but there may be a rebound after drug withdrawal and the drug does little to change the essence and course of the pneumonitic process.

IL-3 gene expression within tumors leads to host-cell infiltration, particularly by macrophages, slower tumor growth, and enhanced immunogenicity. Surprisingly, tumor-associated macrophages (TAMs) from within FSAN-JmIL3 tumors had decreased expression of TNF-α and iNOS. On short-term culture, TAMs from FSAN-JmIL3 tumors regained their capacity to produce TNF-α and NO, indicating that they were primedin vivo. In vitro experiments were unable to demonstrate differences between FSAN-JmIL3 and FSAN tumor cells in their ability to stimulate TNF-α production by TAMs. In the absence of evidence that TAM activation was responsible for the slower growth of FSAN-JmIL3 tumors, the response of tumor cells to these effector molecules was studied. TNF-α and NO were cytotoxic for FSAN-JmIL3 cells but growth stimulatory for FSAN. These tumor-related phenotypic changes may contribute as much if not more than functional changes in host infiltrating cells to the slower growth of FSAN-JmIL3 tumorsin vivo.

The goal of this study was to explore immunological strategies to increase local and systemic tumor control in patients receiving radiation therapy. In previous studies, interleukin-3 (IL-3) gene expression within murine tumors was shown to increase their response to irradiation through immune mechanisms. In this study, the efficacy of systemically administered IL-3 gene-transduced irradiated tumor cell vaccines was tested for their ability to augment radiation responses against established immunogenic (FSAR) and nonimmunogenic (FSAN) tumors. Vaccines of irradiated FSAR/FSAN or FSAN-JmIL-3/FSAR-JmIL-3 cells were given intraperitoneally just before and after local irradiation of parental tumors with diameters of 8 mm, as well as in two booster doses. The IL-3 gene-transduced tumor cell vaccines were more effective than the parental vaccines at delaying tumor growth after irradiation, although no complete cures resulted. Responses were largely specific to the tumor type, indicating that tumor-specific immunity was enhanced by IL-3 vaccine administration. When the experiment was repeated in the C3H/HeJ mice, which are deficient in tumor necrosis factor-a production, the vaccines were still effective, but less so than in C3H/HeN mice. Systemic IL-3 vaccine treatment increased intratumoral levels of intercellular adhesion molecule-1, Mac-1, EB22/5.3, tumor necrosis factor-a, and IL-1 mRNA in irradiated tumors, indicating that cellular infiltration was part of the response. The study demonstrates that local radiation therapy can enhance the efficacy of genetically altered vaccine-based immunotherapy for cancer by decreasing tumor burden. At the same time, tumor cell vaccines may improve the cure rate of local radiation therapy by eliminating residual cancer cells. Although less effective than intratumoral gene expression, administration of IL-3 gene-transduced tumor cell vaccines is clinically a more feasible strategy that may be useful in situations in which the tumor load is small. Cancer Gene Therapy (2000) 7, 1172‐1178

Background: Being able to predict the response of tumors to radiation therapy would improve the decision-making process involved in choosing treatment options for cancer. Expression of certain oncogenes and/or inactivation of tumor suppressor genes has been shown to alter cellular radiation responses; however, it is still not clear what marker or combination of markers would best indicate a radioresistant tumor, or whether such screening would be clinically useful. Current choices of markers are derived mainly from in vitro studies on cell survival after irradiation. In general, expression of transforming oncogenes increases cellular radioresistance. This was also demonstrated in this study for v-abl, bcr abl, v-Ha-ras, v-mos, and v-fes expressed in rat-1 cells. There are, however, conflicting data. Some of the discrepancies may in part be due to interactions between the oncogene-activated signals and other intrinsic or activated pathways. One downstream pathway that is required for oncogene-induced transformation involves c-myc. There is evidence that in some systems myc expression can potentiate ras-induced radiation resistance. Myc may therefore play an important role in determining tumor radioresistancy in the context of other oncogenes. Methods and Results: In this study, the role of c-myc in modulating intrinsic and oncogene-induced cellular radiation responses was investigated in more detail. Retroviral vectors were used to express c-myc and dominant negative mutant c-myc genes in rat 1 cells, with and without ca-transfection of v-abl as measured by clonogenic assay, rat 1. Cells infected with c-myc or v-abl were more resistant to irradiation than neo-transfected cells or control cells; however, cells doubly infected were not resistant, even though they had an increased transformation index. This indicates that transformation-related events per se do not necessarily lead to radiation resistance. It also suggests that the effects of c-myc on radioresistance may depend on what other pathways are activated. This conclusion was strengthened by the finding that expression of a dominant negative c-myc (dn-myc) mutant gene blocked v-abl-induced radiation resistance, but on its own made rat-1 cells more resistant to radiation. Conclusions: The apparently contradictory effects of c-myc in either enhancing or reducing radioresponsiveness may be explained by the dualistic roles of c-myc in promoting signal transduction pathways resulting in either cell proliferation or death, depending on what other pathways are activated. The studies indicate that it will be difficult to predict tumor response to radiation purely by examining expression of transforming oncogenes and it is likely that a number of markers will need to be examined to derive a reliable indication of tumor radiation response.

In order to better understand the actions of proinflammatory cytokines in the mammalian CNS, a transgenic approach was employed in which the expression of IL-6, IL-3 or TNF-alpha was targeted to astrocytes in the intact CNS of mice. Transgenic mice exhibited distinct chronic-progressive neurological disorders with neurodegeneration and cognitive decline due to IL-6 expression, macrophage/microglial-mediated primary demyelination with motor impairment due to IL-3 expression and lymphocytic meningoencephalomyelitis with paralysis induced by TNF-alpha expression. Thus, expression of specific cytokines alone in the intact CNS results in unique neuropathological alterations and functional impairments, thereby directly implicating these mediators in the pathogenesis of CNS disease.

Previously, a large volume of papers reports that gold nanorods (Au NRs) are able to effectively kill cancer cells upon high laser doses (usually 808 nm, 1-48 W/cm²) irradiation, leading to hyperthermia-induced destruction of cancer cells, i.e, photothermal therapy (PTT) effects. Combination of Au NRs-mediated PTT and organic photosensitizers-mediated photodynamic therapy (PDT) were also reported to achieve synergistic PTT and PDT effects on killing cancer cells. Herein, we demonstrate for the first time that Au NRs alone can sensitize formation of singlet oxygen (¹O₂) and exert dramatic PDT effects on complete destrcution of tumors in mice under very low LED/laser doses of single photon NIR (915 nm

Radiation therapy (RT) remains the front-line treatment for high-grade gliomas; however, tumor recurrence remains the main obstacle for the clinical success of RT. Using a murine astrocytoma tumor cell line, ALTS1C1, the present study demonstrates that whole brain irradiation prolonged the survival of tumor-bearing mice, although the mice eventually died associated with increased tumor infiltration. Immunohistochemical (IHC) analysis indicated that RT decreased the microvascular density (MVD) of the primary tumor core, but increased the MVD of the tumor invasion front. RT also increased the number of tumor-associated macrophages (TAMs) and the expression of stromal cell-derived factor-1 (SDF-1) and hypoxia-inducible factor-1 (HIF-1) at the tumor invasion front. SDF-1 expression suppressed by siRNA (SDFkd tumors) showed a decrease in RT-enhanced tumor invasiveness, leading to prolonged survival of mice bearing these tumors. The invasion front in SDFkd tumors showed a lower MVD and TAM density than that in the islands of the control or irradiated ALTS1C1 tumors. Our results indicate that tumor-secreted SDF-1 is one key factor in RT-induced tumor invasiveness, and that it exerts its effect likely through macrophage mobilization and tumor revascularization.

Simian virus 40 (SV40) transforms cells through the suppression of tumor-suppressive responses by large T and small t antigens; studies on the effects of these two oncoproteins have greatly improved our knowledge of tumorigenesis. Large T antigen promotes cellular transformation by binding and inactivating p53 and pRb tumor suppressor proteins. Previous studies have shown that not all of the tumor-suppressive responses were inactivated in SV40-transformed cells; however, the underlying cause is not fully studied. In this study, we investigated the UVB-responsive transcriptome of an SV40-transformed fibroblast (MRC5CVI) and that of its untransformed counterpart (MRC-5). We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes. MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not. Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation. Our study provides a further understanding of the effects of SV40 transformation on cellular stress responses, and emphasizes the value of SV40-transformed cells in the researches of sensitizing neoplastic cells to radiations.

Activated macrophage/microglia may mediate tissue injury in a variety of CNS disorders. To examine this, transgenic mice were developed in which the expression of a macrophage/microglia activation cytokine, interleukin-3 (IL-3), was targeted to astrocytes using a murine glial fibrillary acidic protein fusion gene. Transgenic mice with low levels of IL-3 expression developed from 5 mo of age, a progressive motor disorder characterized at onset by impaired rota-rod performance. In symptomatic transgenic mice, multi-focal, plaque-like white matter lesions were present in cerebellum and brain stem. Lesions showed extensive primary demyelination and remyelination in association with the accumulation of large numbers of proliferating and activated foamy macrophage/microglial cells. Many of these cells also contained intracisternal crystalline pole-like inclusions similar to those seen in human patients with multiple sclerosis. Mast cells were also identified while lymphocytes were rarely, if at all present. Thus, chronic CNS production of low levels of IL-3 promotes the recruitment, proliferation and activation of macrophage/microglial cells in white matter regions with consequent primary demyelination and motor disease. This transgenic model exhibits many of the features of human inflammatory demyelinating diseases including multiple sclerosis and HIV leukoencephalopathy.

Tumor heterogeneity is a major obstacle to therapy, and thus, how to achieve the maximal therapeutic gain in tumor suppression is an important issue. To accomplish this goal, assessing changes in tumor behaviors before treatment is helpful for physicians to adjust treatment schedules. In this study, the authors longitudinally and spatially investigated tumor perfusion and vascular density by power Doppler imaging and immunohistochemical analysis, respectively. Moreover, the authors developed a method to describe quantitatively the spatial distribution of the vasculature within the central and peripheral regions of tumors.Tumor perfusion was estimated by power Doppler images at an operating frequency of 25 MHz. To avoid the attenuation effect of such high-frequency ultrasound, murine tumors were subcutaneously transplanted into the thighs of mice and then monitored for 11 days. The tumors were removed at various time intervals for immunohistochemical analysis of their vascular density using CD31 staining. The spatial characteristics of the tumor vasculature were quantified by a γ value, which characterizes the rate at which vascular signals increase with the fractional sizes of the peripheral area within the tumor.During tumor progression, the volume of tumor perfusion in the power Doppler images was strongly correlated with the vascular density determined by immunohistochemical analysis. In addition, the γ value significantly decreased with increased tumor size in the power Doppler images but not in the immunohistochemical analysis.Although the tumor perfusion and vascular density estimates showed good temporal correlations during tumor progression, they did not show good spatial correlations due to tumor perfusion patterns changing from homogeneous to heterogeneous. In contrast to the perfusion patterns, the vascular density of the tumor remained uniformly distributed. In the present study, no necrosis regions were found in the tumor experiments. Furthermore, the measurement of γ value is a simple method for assessing the vasculatures of spatial distribution within tumors.

Purpose To investigate vascular responses during fractionated radiation therapy (F-RT) and the effects of targeting pericytes or bone marrow-derived cells (BMDCs) on the efficacy of F-RT. Methods and Materials Murine prostate TRAMP-C1 tumors were grown in control mice or mice transplanted with green fluorescent protein-tagged bone marrow (GFP-BM), and irradiated with 60 Gy in 15 fractions. Mice were also treated with gefitinib (an epidermal growth factor receptor inhibitor) or AMD3100 (a CXCR4 antagonist) to examine the effects of combination treatment. The responses of tumor vasculatures to these treatments and changes of tumor microenvironment were assessed. Results After F-RT, the tumor microvascular density (MVD) was reduced; however, the surviving vessels were dilated, incorporated with GFP-positive cells, tightly adhered to pericytes, and well perfused with Hoechst 33342, suggesting a more mature structure formed primarily via vasculogenesis. Although the gefitinib+F-RT combination affected the vascular structure by dissociating pericytes from the vascular wall, it did not further delay tumor growth. These tumors had higher MVD and better vascular perfusion function, leading to less hypoxia and tumor necrosis. By contrast, the AMD3100+F-RT combination significantly enhanced tumor growth delay more than F-RT alone, and these tumors had lower MVD and poorer vascular perfusion function, resulting in increased hypoxia. These tumor vessels were rarely covered by pericytes and free of GFP-positive cells. Conclusions Vasculogenesis is a major mechanism for tumor vessel survival during F-RT. Complex interactions occur between vessel-targeting agents and F-RT, and a synergistic effect may not always exist. To enhance F-RT, using CXCR4 inhibitor to block BM cell influx and the vasculogenesis process is a better strategy than targeting pericytes by epidermal growth factor receptor inhibitor.

Immunotherapy such as anti-PD-1/PD-L1 is effective in several types of tumors, but in general the response rate is less than 30%. Systemic administration of cytokine such as IL-12 could create anti-tumor immunity but has unacceptable side effects. This study is to test our hypothesis that combination of high-dose local radiotherapy (RT) and local production of IL-12 inside tumors within short period could have potent anti-tumor effects but low systemic toxicity. TRAMP-C1 tumors derived from a murine prostate cancer were either no treatment, or irradiated with 25 Gy, or intra-tumorally injected with Ad-sc-IL12 virus (1×10sup8 pfu), or treated with both modalities at the tumor size of 4mm. RT alone and Ad-sc-IL12 alone could not shrink tumors and only caused tumor growth delay around 7 and 12 days, respectively. The combination of RT with Ad-sc-IL12 virus could shrink the tumors to the sizes of < 2mm and further extended the tumor growth delay more than 20 days. The percentage of lymphocytes and IFN-γproduction were evaluated by flow cytometry and ELISA. The percentages of CD4 cells in tumors were too low to be reliably measured in all groups, but the percentages of CD8 cells were increased in Ad-sc-IL12 virus-treated groups and even much higher in those combined with RT. For mice treated with Ad-sc-IL12 virus injection, the serum IL-12 levels were around 4.5 times increase at day7, but those combined with with RT had significantly lower levels than those treated with virus alone. The serum IFN-γ(Th1 response) levels were around 12 times higher in Ad-sc-IL12 groups at day7 as compared to control or RT only group, but had no significant differences between those treated with combined modality and with Ad-sc-IL12 virus alone. Liver damage is an index for IL-12 toxicity and the levels of GOT and GTP were also lower in those treated IL-12 combined with RT than those treated with IL-12 alone. This study supported our hypothesis and showed that combination of local IL-12 production and local RT are effective in increasing CD8 cells, exerting Th1 response, and enhancing tumor growth delay. The most important is that this combination has less systemic toxicity than local IL-12 production alone. This study provides evidence to develop new strategy for producing a strong anti-tumor and low toxicity for radioresistant tumors by combining RT and IL-12 immunotherapy. Citation Format: Ji-Hong Hong, Ching-Fang Yu, Fang-Hsin Chen, Chi-Shiun Chiang. Combination of high-dose irradiation and local interleukin-12 treatment enhance tumor killing and have less toxicities than either treatment alone. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4014.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that leads to the production of GM-CSF, G-CSF, IL-1, IL-6, TNF-α. It can be secreted by various cells, including T cells, NKT cells, fibroblasts, and even tumor cells. In addition, reports have shown that IL-17 could also promote tumor growth by recruiting and expanding granulocytic myeloid suppressor cells (G-MDSCs). In our preliminary study, we found that Tramp-C1, a murine prostate cancer cell line, had remarkable IL-17 level when it grew in pre-irradiated (PreIR) stroma. In contrast, B16F0, a murine melanoma cell line, had much less IL-17 and stronger tumor bed effect, in terms of tumor growth delay, but lung metastasis were formed gradually as B16F0 developed. When the recombinant IL-17 was directly injected into the B16F0 tumors growing in PreIR stroma, we found that the probability of lung metastasis was decreased despite a slight increase of tumor growth at primary site. This indicates that IL-17 may have different effects on irradiated tumor bed and distant metastatic site. The influence of IL-17 on tumor microenvironments of distant metastatic sites and irradiated tumor bed is currently under investigation. In conclusion, we propose a new therapeutic approach of reducing distant metastasis following radiation therapy.. This work was kindly supported by MOST-104-2314-B-182 -024 grant to Fang-Hsin Chen. Citation Format: Yi-Nan Li, Fang-Hsin Chen, Ji-Hong Hong, Chi-Shiun Chiang. Intratumoral injection of interleukin-17 inhibits distant metastasis of radiation-induced recurrent tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1575.

Recent studies indicate that sunitinib, a tyrosine kinase inhibitor, could decease accumulation of myeloid-derived suppressor cells (MDSC) in tumors and also in peripheral blood. This study aims to investigate the behavior and dynamic profile of myeloid-derived suppressor cells in tumor tissues and peripheral blood of sunitinib-treated mice bearing prostate adenocarcinoma TRAMP-C1 tumors. To this end, 3×106 TRAMP-C1 tumor cells were inoculated at shank muscle of C57BL6/J mouse and sunitinib was administered by intraperitoneal injection of a daily dose of 20mg/kg into mice bearing 4 mm diameter tumor. Comparing to tumors in untreated group, sunitinib administration slightly delayed tumor growth delay for 2 days, but significantly decreased micro-vascular density and induced chronic hypoxia in tumors, resulting in the specific accumulation of CD11b+Gr-1+ MDSCs at the tumor necrotic region within CA-IX positive chronic hypoxic area. Flow cytometry was used to analyze the change of CD11b+ myeloid cells within tumor tissues and peripheral blood. Results showed that sunitinib treatment decreased the percentage of CD11b+Ly6G-LyC- tumor-associated macrophages (TAMs), but increased the percentage of CD11b+Ly6G-Ly6C+ monocytic MDSCs (M-MDSCs) while had no effect on CD11b+Ly6G+Ly6C+ neutrophilic MDSCs (N-MDSCs) within tumor tissues. In contrast, both the percentage of N-MDSCs and M-MDSC were expanded in peripheral blood of sunitinib-treated mice. Multiplex immunoassay showed significant increase of CCL2, CCL3, CCL5, CXCL5, IL-17a, GM-CSF, G-CSF and VEGF-A in tumor tissues, but only CXCL5, IL-á, IL-6 and G-CSF in the peripheral blood of sunitinib-treated mice were affected. In conclusion, this study shows that sunitinib has anti-vascular effect and could disrupt the recruitment of CD11b+Ly6G-Ly6C- TAMs into tumors. We also propose that sunitinib might exert different effects on tumor microenvironment and peripheral blood. Citation Format: Fang-Hsin Chen, Sheng-Yung Fu, Chun-Chieh Wang, Chi-Shiun Chiang, Ji-Hong Hong. Sunitinib has opposite roles to regulate the myeloid-derived suppressor cells in tumors and peripheral blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 759.

The herpes simplex virus thymidine kinase/ganciclovir (HSV-sr39tk/GCV) system is a well-established prodrug system used in cancer gene therapy. However, this system is currently not effective enough to eradicate malignant tumors completely. This study aimed to evaluate whether co-expression of interleukin-3 (IL-3) could enhance the anti-tumor activity of HSV-sr39tk/GCV prodrug gene therapy using a murine TRAMP-C1 prostate tumor model. In vitro results demonstrated that HSV-sr39tk-transfected cells exhibited enhanced sensitivity to the GCV prodrug, which was not affected by co-expression of the mIL-3 gene. However, in vivo studies showed that co-expression of the mIL-3 gene significantly increased the HSV-sr39tk/GCV-induced tumor growth delay and even cured the tumor. The TRAMP-C1-specific immune response of spleen lymphocytes from mice bearing HSV-sr39tk- and IL-3-expressing TRAMP-C1 tumors was measured by ELISA. Results showed that IL-3-activated IL-4-dominant lymphocytes became IFN-γ- dominant lymphocytes after combined HSV-sr39tk/GCV therapy. The efficacy of combined therapies on tumor regression was reduced when macrophages populations were depleted by carrageenan or NO production was inhibited by administration of the iNOS inhibitor, L-NAME. These results suggest that utilizing a bicistronic vector to express HSV-sr39tk and the IL-3 gene induced an enhanced macrophage- or NO-dependent anti-tumor effect.

Macrophages display different phenotypes with distinct functions and can rapidly respond to environmental changes. Previous studies on TRAMP-C1 tumor model have shown that irradiation has a strong impact on tumor microenvironments. The major changes include the decrease of microvascular density (MVD), the increase of avascular hypoxia, and the aggregation of tumor associated macrophages (TAMs) in avascular hypoxic regions. Similar changes were observed no matter the irradiation was given to tissue bed before tumor implantation (pre-IR tumors), or to established tumors (IR tumors). Recent results on three murine tumors, TRAMP-C1 prostate adenocarcinoma, ALTS1C1 astrocytoma, and GL261 glioma, further demonstrate that different phenotypes of inflammatory cells are spatially distributed into different microenvironments in both IR and pre-IR tumors. Regions with avascular hypoxia and central necrosis have CD11bhigh/Gr-1+ neutrophils in the center of the necrotic area. Next to them are CD11blow/F4/80+ macrophages that sit at the junctions between central necrotic and surrounding hypoxic regions. The majority of cells in the hypoxic regions are CD11blow/CD68+ macrophages. These inflammatory cell populations express different levels of Arg I. This distribution pattern, except for neutrophils, is not observed in tumors receiving chemotherapy or an anti-angiogenesis agent which also lead to avascular hypoxia. This unique distribution pattern of inflammatory cells in IR tumor sites is interfered with by targeting the expression of a chemokine protein, SDF-1, by tumor cells, and this also increases radiation-induced tumor growth delay. This indicates that irradiated-hypoxia tissues have distinct tumor microenvironments that favor the development of M2 macrophages and that is affected by the levels of tumor-secreted SDF-1.

Understanding the cytotoxicity of quantum dots strongly relies upon the development of new analytical techniques to gather information about various aspects of the system. In this study, we demonstrate the in vivo biodistribution and fate of CdSe quantum dots in the murine model by means of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). By comparing the hot zones of each element acquired from LA-ICP-MS with those in fluorescence images, together with hematoxylin and eosin-stained images, we are able to perceive the fate and in vivo interactions between quantum dots and rat tissues. One hour after intravenous injection, we found that all of the quantum dots had been concentrated inside the spleen, liver and kidneys, while no quantum dots were found in other tissues (i.e., muscle, brain, lung, etc.). In the spleen, cadmium-114 signals always appeared in conjunction with iron signals, indicating that the quantum dots had been filtered from main vessels and then accumulated inside splenic red pulp. In the liver, the overlapped hot zones of quantum dots and those of phosphorus, copper, and zinc showed that these quantum dots have been retained inside hepatic cells. Importantly, it was noted that in the kidneys, quantum dots went into the cortical areas of adrenal glands. At the same time, hot zones of copper appeared in proximal tubules of the cortex. This could be a sign that the uptake of quantum dots initiates certain immune responses. Interestingly, the intensity of the selenium signals was not proportional to that of cadmium in all tissues. This could be the result of the decomposition of the quantum dots or matrix interference. In conclusion, the advantage in spatial resolution of LA-ICP-MS is one of the most powerful tools to probe the fate, interactions and biodistribution of quantum dots in vivo.

A distinguishing feature of high-grade gliomas is the infiltration of neoplastic cells into adjacent brain tissues that mark most of these tumors surgically incurable. To study the factors associated with tumor invasion, we established a new murine brain tumor model, ALTS1C1 derived from SV40 large T antigen-transfected astrocytes. This new brain tumor model recapitulates several histopathological features of human high-grade glioma including increased cellularity, prominent cellular pleomorphism, geographic necrosis, active mitosis, and extensive invasion of tumor cells into adjacent brain tissues. More importantly, ALTS1C1 expressed a relatively high level of stromal-derived factor-1 (SDF-1/CXCL12) in vitro and in vivo and higher microvascular density (MVD) in vivo. To define the roles of SDF-1 in this tumor model, the expression of SDF-1 in ALTS1C1 cells was inhibited by specific siRNA. SDF-knockdown ALTS1C1 (SDF(kd)) cells took longer than parental ALTS1C1 cells to form tumors and in contrast to the wild-type tumors they had well-defined regular borders and lacked infiltration tracts. The SDF(kd) tumors were also associated with a lower MVD and more hypoxic areas. In contrast to parental tumors, the density of F4/80-positive tumor-associated macrophages (TAMs) in SDF(kd) tumor was higher in non-hypoxic than in hypoxic regions. SDF-1 production by tumor cells therefore seems critical for the aggregation of TAMs into areas of hypoxia and tumor invasiveness. This study not only provides new insight into the role of SDF-1 in brain tumor invasion and the relationship between TAMs and hypoxia, but also provides a new preclinical brain tumor model for designing new treatment options for invasive cases.

Baculovirus is an insect virus that is non-pathogenic to humans and has emerged as a promising gene therapy vector. Since solid tumor growth/metastasis critically relies on angiogenesis and hEA, a fusion protein comprising human endostatin and angiostatin, exhibits potent antiangiogenic and antitumor efficacy in mouse models; this study aimed to evaluate the feasibility of baculovirus for hEA expression and antiangiogenesis-based cancer gene therapy. Toward this end, we constructed Bac-hEA that mediated transient hEA expression and Bac-ITR-hEA that exploited the adeno-associated virus inverted terminal repeats (ITRs) for prolonged hEA expression. Western blot and ELISA analyses showed that both Bac-hEA and Bac-ITR-hEA expressed hEA in transduced mammalian cells, yet Bac-ITR-hEA only marginally prolonged the hEA expression. In comparison with Bac-hEA, nonetheless, Bac-ITR-hEA significantly enhanced the hEA expression level that concurred with augmented antiangiogenic properties, as demonstrated by cell proliferation, migration and tubule network formation assays. Importantly, intratumoral injection of Bac-ITR-hEA into prostate cancer mouse models, when compared with Bac-hEA, exerted stronger antiangiogenic effects in vivo, more potently inhibited tumor growth and significantly prolonged mouse survival. This study collectively supported the notion that hEA is an effective antiangiogenic protein and proved the potential of baculovirus as a vector for antiangiogenesis-based cancer therapy, which may be combined with chemotherapy, radiotherapy or gene therapies using other vectors.

Honokiol (HNK) is a phenolic compound isolated from the bark of houpu (Magnolia officinalis), a plant widely used in traditional Chinese and Japanese medicine. While substantial evidence indicates that HNK possesses anti-inflammatory activity, its effect on dendritic cells (DCs) during the inflammatory reaction remains unclear. The present study investigates how HNK affects lipopolysaccharide (LPS)-stimulated human monocyte-derived DCs. Our experimental results show that HNK inhibits the inflammatory response of LPS-induced DCs by (1) suppressing the expression of CD11c, CD40, CD80, CD83, CD86, and MHC-II on LPS-activated DCs, (2) reducing the production of TNF-α, IL-1β, IL-6, and IL-12p70 but increasing the production of IL-10 and TGF-β1 by LPS-activated DCs, (3) inhibiting the LPS-induced DC-elicited allogeneic T-cell proliferation, and (4) shifting the LPS-induced DC-driven Th1 response toward a Th2 response. Further, our results show that HNK inhibits the phosphorylation levels of ERK1/2, p38, JNK1/2, IKKα, and IκBα in LPS-activated DCs. Collectively, the findings show that the anti-inflammatory actions of HNK on LPS-induced DCs are associated with the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways.

The goals of this study were to quantify myelin-associated changes in the brain following single doses of radiation and to determine their relationship to the dose limits that this tissue can tolerate. Mice developed a transient loss of balance 1 month after 60 Gy doses 250 kV p X-rays to the brain and 3–4 months after 30–45 Gy radiation, but not after lower doses. The symptoms were transient and lasted ∼ 1 month. The ED50300 for radiation-induced brain death, which occurred largely between 200 and 240 days, was 32.4 Gy (29.1, 35.5 Gy, 95% confidence limit of mean). At the time that animals developed neurological symptoms, 3–4 months after irradiation with doses of 30–45 Gy, biochemical assays of myelin-associated proteins showed decreases in 2′,3′-cyclic nucleotide phosphohydrolase (CNPase) and myelin basic protein (MBP) levels that were not seen with lower radiation doses. By 120–180 days, further dose-dependent decreases in both CNPase and MBP levels were found after 20–45 Gy irradiation that preceded and correlated with death. The correlation of the decrease in CNPase and MBP levels with the incidence of transient neurological malfunction and animal death, together with histological evidence, suggests that demyelination is responsible for these phenomena.

As one of the most intensively studied probes for imaging of the cellular proliferation, [ 18 F]FLT was investigated whether the targeting specificity of thymidine kinase 1 (TK1) dependency could be enhanced through a synergistic effect mediated by herpes simplex type 1 virus (HSV1) tk gene in terms of the TK1 or TK2 expression. 5-[ 123 I]Iodo arabinosyl uridine ([ 123 I]IaraU) was prepared in a radiochemical yield of 8% and specific activity of 21 GBq/μmol, respectively. Inhibition of the cellular uptake of these two tracers was compared by using the arabinosyl uridine analogs such as 5-iodo, 5-fluoro and 5-( E )-iodovinyl arabinosyl uridine along with 2′-fluoro-5-iodo arabinosyl uridine (FIAU). Due to potential instability of the iodo group, accumulation index of 1.6 for [ 123 I]IaraU by HSV1-TK vs. control cells could virtually be achieved at 1.5 h, but dropped to 0.2 compared to 2.0 for [ 18 F]FLT at 5 h. The results from competitive inhibition by these nucleosides against the accumulation of [ 18 F]FLT implied that FLT exerted a mixed TK1- and TK2-dependent inhibition with HSV1- tk gene transfection because of the shifting of thymidine kinase status. Taken together, the combination of [ 18 F]FLT and HSV1-TK provides a synergistic imaging potency.

Purpose To investigate vasculatures and microenvironment in tumors growing from preirradiated tissues (pre-IR tumors) and study the vascular responses of pre-IR tumors to radiation and antiangiogenic therapy. Methods and Materials Transgenic adenocarcinoma of the mouse prostate C1 tumors were implanted into unirradiated or preirradiated tissues and examined for vascularity, hypoxia, and tumor-associated macrophage (TAM) infiltrates by immunohistochemistry. The origin of tumor endothelial cells was studied by green fluorescent protein–tagged bone marrow (GFP-BM) transplantation. The response of tumor endothelial cells to radiation and antiangiogenic agent was evaluated by apoptotic assay. Results The pre-IR tumors had obvious tumor bed effects (TBE), with slower growth rate, lower microvascular density (MVD), and more necrotic and hypoxic fraction compared with control tumors. The vessels were dilated, tightly adhered with pericytes, and incorporated with transplanted GFP-BM cells. In addition, hypoxic regions became aggregated with TAM. As pre-IR tumors developed, the TBE was overcome at the tumor edge where the MVD increased, TAM did not aggregate, and the GFP-BM cells did not incorporate into the vessels. The vessels at tumor edge were more sensitive to the following ionizing radiation and antiangiogenic agent than those in the central low MVD regions. Conclusions This study demonstrates that vasculatures in regions with TBE are mainly formed by vasculogenesis and resistant to radiation and antiangiogenic therapy. Tumor bed effects could be overcome at the edge of larger tumors, but where vasculatures are formed by angiogenesis and sensitive to both treatments. Vasculatures formed by vasculogenesis should be the crucial target for the treatment of recurrent tumors after radiotherapy.

Tumor vasculature is derived angiogenesis within primary tumors from sprouting host preexisting vessels and vasculogenesis within recurrent tumors from bone-marrow. The tumor recurred by means of the pathway of vasculogenesis after the tumor received unsuccessful therapy, and it has lower curability to therapy. Thus, investigating the features of tumor vascular patterns between the two vasculature pathways may provide a method for assisting improving tumor therapy. In this study, we proposed the potential of high-frequency ultrasound 3D Doppler images for characterizing pathways of the tumor vasculatures in recurrent and primary tumors in vivo. Murine tumors were transplanted in non-irradiated and pre-irradiated tissues to imitate the primary and recurrent tumor models, respectively. The 7th days after inoculation, the cineloop of power Doppler-mode images were recorded to reconstruct 3-D tumor vasculature images using 40-MHz Doppler imaging system. Recurrent tumors show the blood vessels were more dilated and continuous than those in primary tumors. Recurrent tumors exhibited higher expression of tumor perfusion and lower the feature of branching pattern in tumor vasculature compared with primary tumors. Tumor volume values had no difference between the two tumor models. The study suggested that tumor perfusion and the feature of branching pattern in tumor vasculature could used to discriminate the angiogenesis and vasculogenesis pathways. It has the potential for exporting the responses of anti-vasculogenesis drug and improving the curability of recurrent tumors in the future.

Objective The blood flow rate in the microcirculation associated with angiogenesis plays an important role in the progression and treatment of cancer. Since the microvascular status of tumor vessels can yield useful clinical information, assessing changes in the tumor microcirculation could be particularly helpful for tumor evaluation and treatment planning. Methods In this study we used a self-developed 25-MHz ultrasound imaging system with a spatial resolution of 150 μm for assessing tumor-microcirculation development and the pattern of the vasculature in three tumor-bearing mice in vivo based on power Doppler images. The total Doppler power (DP) and color pixel density (CPD) revealed the presence of functional vessels distributed throughout a tumor volume. The vasculature distributions in the core and periphery were compared to the regulation of vasculature function, which facilitated determination of when the tumor grew rapidly. Results The data obtained from a quantified analysis of power Doppler images indicated that the tumor vascularity initially increased throughout the tumor. Both DP and CPD increased rapidly in the tumor periphery when the tumor volume exceeded 10 mm 3 . Conclusion Our preclinical findings suggest that power Doppler imaging could be useful for detecting the changes in tumor vascular perfusion and for determining the optimal treatment timing when a tumor begins its rapid volumetric growth.

Cordyceps sinensis (CS), a Chinese tonifying herb, has been widely used for centuries in Asian countries as a medicine and a health supplement. Although ample evidence indicates that CS can modulate immune responses, the functional effect of CS on dendritic cells (DCs) is still unclear. This study examines how CS affects human monocyte-derived DCs in two physiological states: naïve and LPS-induced inflammatory. Our experimental results demonstrate that CS acts as an activator and maturation inducer of immature DCs by stimulating the expression of costimulatory molecules and proinflammatory cytokines by DCs, enhancing the DC-induced, allogeneic T cell proliferation, and reducing the endocytic ability of DCs. In contrast, CS suppresses the LPS-induced, inflammatory response by decreasing the LPS-induced expression of costimulatory molecules and proinflammatory cytokines by DCs. CS also suppresses the LPS-induced, DC-elicited, allogeneic T cell proliferation and shifts the LPS-activated, DC-driven Th1 response toward a Th2 response. These results demonstrate that CS differentially regulates the DC activities according to the presence or absence of the inflammatory signs. Restated, with the lack of an ongoing inflammatory environment, CS primes DCs toward a Th1-type immunity, whereas in a potential inflammatory reaction, CS balances the over-reactivity of elicited Th1 immunity. This investigation illustrates the Yin-Yang balancing effects of CS as a medicine and a health supplement.

Magnetic nanodiamonds were prepared via solid-state microwave arcing of a nanodiamond-ferrocene mixed powder in a focused microwave oven. High-resolution transmission electron microscope (HRTEM) images show that a magnetic nanodiamond is composed of iron nanoparticles encapsulated by graphene layers on the surface of nanodiamonds. Fluorescence property was introduced onto magnetic nanodiamonds by chemical modification of magnetic nanodiamonds via surface grafting of poly(acrylic acids) and fluorescein o-methacrylate. Fluorescent magnetic nanodiamonds are water soluble with a solubility of approximately 2.1 g/L. Cellular-imaging experiments show that fluorescent magnetic nanodiamonds could be ingested by HeLa cells readily in the absence of agonist (i.e., folate) moieties on the surface of nanodiamonds.

This study aimed to evaluate the ability of the health food supplement Cordyceps sinensis (CS) to ameliorate suppressive effects of chemotherapy on bone marrow function as a model for cancer treatment. Mice were treated with Taxol (17 mg/kg body wt) one day before oral administration of a hot-water extract of CS (50 mg/kg daily) that was given daily for 3 weeks. White blood cell counts in peripheral blood of mice receiving Taxol were at 50% of normal levels on day 28 but had recovered completely in mice treated with CS. In vitro assays showed that CS enhanced the colony-forming ability of both granulocyte macrophage colony forming unit (GM-CFU) and osteogenic cells from bone marrow preparations and promoted the differentiation of bone marrow mesenchymal stromal cells into adipocytes, alkaline phosphatase–positive osteoblasts, and bone tissue. This result could be attributed to enhanced expression of Cbfa1 (core binding factor a) and BMP-2 (bone morphogenetic protein) with concurrent suppression of ODF (osteoclast differentiation factor/RANK [receptor activator of NF-κB]) ligand. In summary, CS enhances recovery of mice from leukopenia caused by Taxol treatment. It appears to do so by protecting both hematopoietic progenitor cells directly and the bone marrow stem cell niche through its effects on osteoblast differentiation.

A cell survival assay of the four arabinosyl uridine analogs with functionalities of 5-fluoro, 5-fluorovinyl, 5-iodo, and 5-iodovinyl as potential positron-emitter tagged probe for monitoring cancer gene therapy were performed. Cytotoxicities of 5-fluoro-, 5-iodo-, 5-fluorovinyl, and 5-iodovinyl arabinosyl uridines against SR-39 thymidine kinase transfected murine prostate cancer cells have been evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. None of them showed significant bioactivity. A syn conformation derived from intra-hydrogen bonding was suggested for the unfavorable interaction and diminished bioactivity.

Macrophages display different phenotypes that can switch in response to their micro-environment. In our earlier study (Chiang, C. S., Liu, W. C. and Jung, S. M., 2005. Compartmental responses after thoracic irradiation of mice: strain differences. Int. J. Radiat. Oncol. Biol. Phys. 62:862) on radiation-induced cytokine expression in lung lavage samples, there was a suggestion that the procedures used to harvest lung macrophages affected the profiles they expressed. To further explore this issue, we examined gene expression by cell populations, mainly macrophages, isolated by lavage from lung and peritoneal cavity following either in vivo or in vitro stimulation with LPS, IFN-gamma or irradiation. We found that expression of mRNA for tumor necrosis factor-alpha, IL-1 alpha/beta and IL-6 varied several fold depending on whether the assay was performed on cells immediately after isolation or after in vitro manipulation. The relative level of inducible nitric oxide synthase (iNOS) to arginase I (Arg I), which is frequently used as index of the M1 versus M2 functional macrophage phenotype, also varied. LPS stimulation in vivo was able to change the profile from Arg I expression to one where the iNOS pathway became dominant, but was unable to do this in vitro. This contrasts with the ability of IFN-gamma to generate an iNOS-dominant pathway in vitro, but not in vivo. This study cautions that the expression of inflammatory cytokines and the iNOS to Arg I ratio, which is often used as an index of their functional capacity, varies with the experimental conditions.

The tumor-homing ability of monocytes renders them a potential cellular delivery system for alternative cancer therapies, although their migratory ability can be impaired following reagent uptake. Approaches that enhance monocyte tumor homing and promote their migration will improve the clinical value of these cells as cellular carriers. Previous studies have shown that irradiation (IR) can promote macrophage aggregation in hypoxic regions. To investigate whether IR enhances the infiltration of bone marrow-derived monocytes (BMDMs) into tumors, the infiltration of BMDMs from GFP-transgenic mice in a murine prostate adenocarcinoma TRAMP-C1 model was examined by fluorescence microscopy. IR did not increase the number of BMDMs that infiltrated initially, but did increase monocyte retention within IR-treated tumors for up to 2 weeks. We also showed that BMDMs can take up various imaging and therapeutic agents, although the mobility of BMDMs decreased with increasing load. When BMDMs were differentiated in IR-treated tumor-conditioned medium (IR-CM) in vitro, the nanoparticle load-mediated inhibition of migration was attenuated. These IR-CM-differentiated BMDMs delivered polymer vesicles encapsulating doxorubicin to radiation therapy (RT)-induced hypoxic tumor regions, and enhanced the efficacy of RT. The prolonged retention of monocytes within irradiated tumor tissues and the ability of IR-CM to enhance the migratory ability of cargo-laden BMDMs suggest that monocytes pre-conditioned by IR-CM can potentially act as cellular carriers for targeted therapy following conventional RT.

Radiation therapy (RT) remains the front-line treatment for high-grade gliomas; however, tumor recurrence remains the main obstacle for the clinical success of RT. Using a murine astrocytoma tumor cell line, ALTS1C1, we have previously shown that RT can effectively reduce primary tumor volume and increase mice surviving time, but the mice eventually died associated with increased tumor infiltration. In this study, we explore the role of MMP2 (Matrix metalloproteinase-2), a reported molecule associated with brain tumor invasion, on ALTS1C1 tumor response to RT. In this study, the MMP2 expression was inhibited by specific lentiviral-shRNA construct. Eight Gray of whole brain irradiation could only prolong the median survival days of brain tumor-bearing mice from 25 days to 28 days, but the inhibition of MMP2 expression by siRNA prolonged the median survival days of tumor bearing mice from 25 days to 42 days. The median survival days were further prolonged to 53 days when 8 Gy of radiation and MMP2 inhibition were concurrently applied. Immunohistochemical (IHC) results showed that the synergistic effect as a result of RT-caused vessel density decrease (inadequate vasculature) and the retardation of vessel maturation as a result of MMP2 inhibition-caused the decrease of pericyte-covered vessels (insufficient vessel function). This study provides a new treatment option for treating invasive brain tumors. (This study was supported by NHRI-EX103-10132BI and NSC 102-2314-B-007-003-MY3 grants) Citation Format: Ching-Fang Yu, Ying-Chieh Yang, Ji-Hong Hong, Chi-Shiun Chiang. Inhibition of MMP2 expression enhances the efficacy of radiation therapy for a murine astrocytoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2014-3945

Purpose: To examine and compare the molecular and cellular processes leading to radiation fibrosis and pneumonitis in C57BL/6J and C3H/HeN mice. Methods and Materials: At indicated times after various doses of thoracic irradiation, the cell populations obtained by bronchoalveolar lavage of C57BL/6J mice were differentially analyzed by cytology and assessed by RNase protection (RPA) assay for levels of cytokines and related genes. The molecular responses in bronchial alveolar lavage (BAL) populations were compared with those in whole lung of C57BL/6J mice and with those of C3H/HeN mice. The former strain develops late radiation fibrosis, whereas the latter develop subacute radiation pneumonitis. Results: In C57BL/6J mice, a decrease in the total number of BAL cells was found 1 week after 6, 12, or 20 Gy thoracic irradiation with a subsequent dose-dependent increase up to 6 months. After 12 and 20 Gy, large, foamy macrophages and multinucleated cells became evident in BAL at 3 weeks, only to disappear at 4 months and reappear at 6 months. This biphasic response was mirrored by changes expression of mRNA for proinflammatory cytokines and the Mac-1 macrophage-associated antigen. As with BAL, whole lung tissue also showed biphasic cytokine and Mac-1 mRNA responses, but there were striking temporal differences between the two compartments, with changes in whole lung tissue correlating better than BAL with the onset of fibrosis in this strain. The radiation-induced proinflammatory mRNA responses had strain-dependent and strain-independent components. Thoracic irradiation of C3H/HeN induced similar increases in tumor necrosis factor (TNF)-α, interleukin (IL)-1α/β, and interferon (IFN)-γ mRNA expression in lung as it did in C57BL/6J mice during the “presymptom” phase at 1–2 months. However, immediately preceding and during the pneumonitic time period at 3–4 months, TNF-α and IL-1α/β mRNAs were highly upregulated in C3H/HeN mice, which develop pneumonitis, but not in C57BL/6J mice, which do not. At the onset of radiation fibrosis in C57BL/6J mice (5–6 months), irradiated lungs had increased levels of IL-1α/β and IFN-γ mRNA expression, but the TNF-α response was, notably, still muted. Conclusions: The major molecular and cellular events in lungs of C57BL/6J and C3H/HeN mice, which develop late fibrosis and subacute pneumonitis after thoracic irradiation respectively, take place within the interstitium and are not reflected within BAL populations. The initial proinflammatory responses are similar in the two strains, but later responses reflect the latent time to lesion development. TNF-α expression at 3–4 months may be important in radiation-induced pneumonitis, and its downregulation is important in avoiding this radiation-induced complication.

Tissue damage caused by exposure to pathogens, chemicals and physical agents such as ionizing radiation triggers production of generic "danger" signals that mobilize the innate and acquired immune system to deal with the intrusion and effect tissue repair with the goal of maintaining the integrity of the tissue and the body. Ionizing radiation appears to do the same, but less is known about the role of "danger" signals in tissue responses to this agent. This review deals with the nature of putative "danger" signals that may be generated by exposure to ionizing radiation and their significance. There are a number of potential consequences of "danger" signaling in response to radiation exposure. "Danger" signals could mediate the pathogenesis of, or recovery from, radiation damage. They could alter intrinsic cellular radiosensitivity or initiate radioadaptive responses to subsequent exposure. They may spread outside the locally damaged site and mediate bystander or "out-of-field" radiation effects. Finally, an important aspect of classical "danger" signals is that they link initial nonspecific immune responses in a pathological site to the development of specific adaptive immunity. Interestingly, in the case of radiation, there is little evidence that "danger" signals efficiently translate radiation-induced tumor cell death into the generation of tumor-specific immunity or normal tissue damage into autoimmunity. The suggestion is that radiation-induced "danger" signals may be inadequate in this respect or that radiation interferes with the generation of specific immunity. There are many issues that need to be resolved regarding "danger" signaling after exposure to ionizing radiation. Evidence of their importance is, in some areas, scant, but the issues are worthy of consideration, if for no other reason than that manipulation of these pathways has the potential to improve the therapeutic benefit of radiation therapy. This article focuses on how normal tissues and tumors sense and respond to danger from ionizing radiation, on the nature of the signals that are sent, and on the impact on the eventual consequences of exposure.

To determine the contribution of intra-alveolar cells as opposed to cells fixed in the interstitium in the development of radiation-induced lung injury.C3H/HeN mice were irradiated to the thorax with various doses of radiation. The cellular composition and cytokine production were assessed in the two sites by histological staining and RNase protection assay.Following thoracic irradiation, there was an initial decrease in the number of bronchial alveolar lavage (BAL) cells that was followed after 2 months by a dose-dependent increase up to 4 months. Foamy Mac-1 positive macrophages were present early in the BAL populations, which also expressed the pro-inflammatory cytokines TNF-alpha, IL-1alpha and IL-1beta, but this response subsided by the time of onset of pneumonitis (3 months). In contrast, in whole lung tissue there was a steady increase in Mac-1 positive cells and increased expression of TNF-alpha, IL-1alpha and IL-1beta mRNAs to maximum levels at 3-4 months.These data indicate distinct temporal and spatial changes in pro-inflammatory cytokine gene expression in different cellular compartments of the irradiated lung. BAL cells became inflammatory early on, but interstitial cells became involved later and were probably more involved in contributing to the pneumonitis.

Radiation therapy for cancer patients works by ionizing damage to nuclear DNA, primarily by creating double-strand breaks (DSB). A major shortcoming of traditional radiation therapy is the set of side effect associated with its long-range interaction with nearby tissues. Low-energy Auger electrons have the advantage of an extremely short effective range, minimizing damage to healthy tissue. Consequently, the isotope 99mTc, an Auger electron source, is currently being studied for its beneficial potential in cancer treatment. We examined the dose effect of a pyrene derivative 99mTc complex on plasmid DNA by using gel electrophoresis in both aqueous and methanol solutions. In aqueous solutions, the average yield per decay for double-strand breaks is 0.011±0.005 at low dose range, decreasing to 0.0005±0.0003 in the presence of 1 M dimethyl sulfoxide (DMSO). The apparent yield per decay for single-strand breaks (SSB) is 0.04±0.02, decreasing to approximately a fifth with 1 M DMSO. In methanol, the average yield per decay of DSB is 0.54±0.06 and drops to undetectable levels in 2 M DMSO. The SSB yield per decay is 7.2±0.2, changing to 0.4±0.2 in the presence of 2 M DMSO. The 95% decrease in the yield of DSB in DMSO indicates that the main mechanism for DSB formation is through indirect effect, possibly by cooperative binding or clustering of intercalators. In the presence of non-radioactive ligands at a near saturation concentration, where radioactive Tc compounds do not form large clusters, the yield of SSB stays the same while the yield of DSB decreases to the value in DMSO. DSBs generated by 99mTc conjugated to intercalators are primarily caused by indirect effects through clustering.

To investigate cytokine gene expression in the lung after single and fractionated doses of radiation, and to investigate the effect of steroids and the genetic background.Expression of cytokine genes (mTNF-alpha, mIL-1alpha, mIL-1beta, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFN-gamma) in the lungs of C3H/HeJ and C57BL/6J mice was measured by RNase protection assay at different times after various doses of radiation. The effects of dexamethasone and fractionated radiation treatment on gene expression were also studied.IL-1beta was the major cytokine induced in the lungs of C3H/HeJ mice within the first day after thoracic irradiation. Radiation doses as low as 1 Gy were effective. Responses to 20 Gy irradiation peaked within 4-8h and subsided by 24 h. With the exception of IL-1alpha and TNF-alpha, the other cytokines that were investigated had undetectable pre-treatment mRNA levels and were not radiation inducible. Similar responses were seen in C57BL/6J mice, although TNF-alpha was induced and there were some quantitative differences. Pre-treatment of C3H/HeJ mice with dexamethasone reduced basal and induced IL-1 levels, but complete inhibition was not achieved. Dexamethasone was also effective if given immediately after irradiation. Fractionated daily doses of radiation (4 Gy/day) helped to maintain cytokine gene expression for a longer period.Inflammatory genes are rapidly induced in the lung by irradiation. This response cannot be readily abolished by steroid pre-treatment. Fractionated treatment schedules help to perpetuate the response.

Although radiotherapy is a front line treatment for brain tumors, little is known about the in vivo molecular responses of brain to irradiation. In this study, expression of c-fos, c-jun and junB immediate-early genes were followed in mouse brain after irradiation. C-fos and junB, but not c-jun, mRNA was induced within 15 min in unanesthetized irradiated mice. Induction was transient and lasted4 h. The response was dose-dependent with increases in c-fos and junB mRNA levels after dose ofor = 2 and 7 Gy, respectively. Anesthesia of mice with pentobarbitol delayed the increases in mRNA expression and the response was attenuated. Pre-treatment of mice with dexamethasone, in a schedule which suppressed acute-phase gene expression after brain irradiation, did not significantly change c-fos and junB induction. Our results show that c-fos and junB responses occur in the brain in response to irradiation and that they can be modified by pentobarbital treatment but suggest that there is no direct correlation between the level of mRNA expression and later expression of cytokines or other acute-phase response genes.

The chance of life-threatening complications occurring late after brain irradiation limits the efficacy of this form of cancer therapy. The molecular and cellular events that trigger radiation-induced brain damage are still unknown, but since they have the potential to serve as valuable targets for therapeutic intervention they are worth delineating. In this murine study, the effect of irradiation on the expression of molecules which are known to contribute to brain damage in other model systems was examined. Expression of genes encoding cytokines (TNF-alpha/beta, IL-1 alpha/beta, IL-2, IL-3, IL-4, IL-5, IL-6 and IFN-gamma), cytokine receptors (TNF-Rp55 and p75, IL-1R- p60 and p80, IFN-gamma R, and IL-6R), the cell adhesion molecule (ICAM-1), inducible nitric oxide synthetase (iNOS), anti-chymotrypsin (EB22/5.3), and the gliotic marker (GFAP) was evaluated over a 6-month period using a sensitive RNase protection assay (RPA). We had previously demonstrated that within 24 h of brain irradiation there is an acute transitory molecular response involving TNF-alpha, IL-1, ICAM-1, EB22/5.3 and GFAP. This study shows re-elevation of TNF-alpha, EB22/5.3 and GFAP mRNA levels at 2-3 months, but only TNF-alpha mRNA was overexpressed at 6 months. These time points are when neurological abnormalities are seen after higher doses. The data suggest that TNF-alpha may be involved in late brain responses to irradiation and could contribute to clinical symptoms.

Tumor-associated macrophages (TAMs) are a major non-cancer component in solid tumors and actively participate in remodeling tumor microenvironment after a variety of treatment; however their functions in irradiated tumors are not fully clarified. Our previous studies showed that high-dose irradiation to TRAMP-C1 tumor (a mouse prostate cancer cell line) decreased micro-vascular density (MVD) and induced CD68+ TAMs aggregation at avascular hypoxia region. These findings drive us to examine if these aggregated CD68+ TAMs promote regrowth of irradiated tumors. The TRAMP-C1 tumor cells were i.m. injection into shank muscle and irradiated with single-dose 25Gy at size of 4mm diameter. Sutent, a tyrosine kinase inhibitor, was i.p. injected (20mg/kg per day) to some mice and served as a model for hypoxia induced by anti-angiogenesis treatment. Tumors treated with Sutent alone also had decreased MVD and large amount of avascular hypoxia, but the aggregation of CD68+ TAMs in avascular hypoxia region was only observed in irradiated tumors, not in sutent-treated tumors; this finding suggests this aggregation of CD68+ TAMs in hypoxic region is not a universal finding and could be specific for irradiated tumor microenvironment. The phenotype of TAMs in regions with and without aggregation was studied and the differences existed. Arg-1+iNOS− TAMs were the dominant phenotype outside the avascular hypoxia regions and randomly distributed, but presence and accumulation of Arg-1+iNOS+ TAMs in avascular hypoxia regions was detected. The NOS inhibitor, L-NAME (500mg/L in drinking water), was therefore used to block the effects of nitric oxide produced from specific Arg-1+iNOS+ TAMs accumulated in avascular hypoxia region. The blockade of nitric oxide delayed tumor regrowth in irradiated but not in control tumors. Aggregation of TAMs and presence of Arg-1+ iNOS+ TAMs in avascular hypoxia regions were totally vanished in tumors treated with NOS inhibitor. In addition, the MVD was further decreased and percentages of avascular hypoxia regions were increased in the irradiated tumor treated with NOS inhibitor. These findings suggested that Arg-1+iNOS+TAMs accumulated in avascular hypoxia regions promote tumor regrowth after irradiation, and nitric oxide blockage is a potential strategy to improve the effects of radiotherapy. (This work is supported by grants 101-2314-B-182A-129-MY3 from National Science Council, Taiwan, CMRPG390943 from Chang Gung Memorial Hospital) Citation Format: Ji-Hong Hong, Sheng-Yung Fu, Fang-Hsin Chen, Chun-Chieh Wang, Chi-Shiun Chiang. The role of Arg-1+iNOS+ tumor-associated macrophages and nitric oxide on tumor microenviroment after high-dose irradiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1532. doi:10.1158/1538-7445.AM2013-1532

Singlet oxygen is a very important reactive oxygen species (ROS) involved in peroxidation of olefins and polymers, as well as in clinical photodynamic therapy treatments of tumors. Previously, it was reported that singlet oxygen can be formed via sensitization by spherical metal nanoparticles upon photo-excitation of the surface plasmon resonance (SPR) bands. In this paper, we report that sensitization and formation of singlet O2 is strongly dependent on the morphologies of gold and silver nanostructures. For example, singlet O2 can be generated via photo-irradiation and sensitization of silver decahedrons and silver triangular nanoplates, but not by silver nanocubes and gold decahedrons. The sensitization patterns of silver and gold nanoparticles are the reverse of each other. In the case of gold nanorods, singlet O2 can be generated via photo-excitation at the longitudinal SPR band, but not by excitation at the transverse SPR band. The controlling factors for such a morphology dependent singlet O2 sensitization will be discussed. Furthermore, we also demonstrate in vitro morphology dependent sensitization behaviour of silver nanoparticles in the photodynamic cancer treatment. Our results indicate that metal nanoparticles with certain morphologies are potentially very promising dual functional nanomaterials with capabilities of simultaneously serving as near infrared (NIR) activatable photodynamic therapy and photothermal therapy reagents for cancer treatments.

Numerous studies have documented the altered expression of various cytokines or their genes in the brain in a variety of central nervous system (CNS) pathologic states such as multiple sclerosis (MS), acquired immunodeficiency syndrome (AIDS) encephalopathy, stroke, Alzheimer’s disease, scrapie (spongiform encephalopathy), other infectious diseases and trauma. Whether the presence of cytokines in these neuropathologic states represents a primary or secondary event and the extent to which these factors may contribute to the development and progression of clinicopathologic alterations in these disorders remains largely unknown. We have focused on these issues by utilizing a transgenic approach to direct the constitutive expression of a variety of cytokines to astrocytes in the intact CNS of mice.1–4 Transgenic expression of cytokines in the CNS of mice allows not only the investigation of complex cellular responses at a localized level in the intact brain, but also more closely recapitulates the expression of these mediators as found in pathogenetic states.

Purpose: To investigate the in vivo acute phase molecular response of the brain to ionizing radiation. Methods and Materials: C3Hf/Sed/Kam mice were given midrain or whole-body irradiation. Cerebral expression of interleukins (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6), interferon (IFN-γ), tumor necrosis factors (TNF-α and TNK-β), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), α1-antichymotrypsin (EB22/5.3), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay. The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined. Results: Levels of TNF-α, IL-1β, ICAM-1, EB22/5.3, and to alesser extent IL-1α and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain. Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy. Most responses were rapid, peaking within 4–8, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsides. Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely. Dexamethasone was more inhibitory than pentoxifylline at the doses chosen. Conclusions: The initial response of thebrain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy. This mechanism explains the beneficial effects of the clincial use of steroids in such circumtances.