Genomic Data Heterogeneity across Molecular Diagnostic Laboratories: A Real-World Connect Myeloid Disease Registry Perspective on Variabilities in Genomic Assay Methodology and Reporting.

Genomic data variability from laboratory reports can impact clinical decisions and population-level analyses; however, the extent of this variability and the impact on the data's value are not well characterized. This pilot study used anonymized genetic and genomic test reports from the Connect Myeloid Disease Registry (NCT01688011), a multicenter, prospective, observational cohort study of patients with newly diagnosed myelodysplastic syndromes, acute myeloid leukemia, or idiopathic cytopenia of undetermined significance, to analyze laboratory test variabilities and limitations. Results for 56 randomly selected patients enrolled in the Registry were independently extracted and evaluated (data cutoff, January 2020). Ninety-five reports describing 113 assay results from these 56 patients were analyzed for discrepancies. Almost all assay results [101 (89%)] identified the sequencing technology applied, and 94 (83%) described the test limitations; 95 (84%) described the limits of detection, but none described the limit of blank for detecting false positives. RNA transcript identifiers were not provided for 20 (43%) variants analyzed by next-generation sequencing and reported by the same laboratory. Of 42 variants with variant allele frequencies ≥30%, 16 (38%) of the variants did not have report text indicating that the variants might be germline. Variabilities and lack of standardization present challenges for incorporating this information into clinical care and render data collation ineffective and unreliable for large-scale use in centralized databases for therapeutic discovery.

Radiotherapy alters expression of molecular targets in prostate cancer in a fractionation- and time-dependent manner.

The efficacy of molecular targeted therapy depends on expression and enzymatic activity of the target molecules. As radiotherapy modulates gene expression and protein phosphorylation dependent on dose and fractionation, we analyzed the long-term effects of irradiation on the post-radiation efficacy of molecular targeted drugs. We irradiated prostate cancer cells either with a single dose (SD) of 10 Gy x-ray or a multifractionated (MF) regimen with 10 fractions of 1 Gy. Whole genome arrays and reverse phase protein microarrays were used to determine gene expression and protein phosphorylation. Additionally, we evaluated radiation-induced pathway activation with the Ingenuity Pathway Analysis software. To measure cell survival and sensitivity to clinically used molecular targeted drugs, we performed colony formation assays. We found increased activation of several pathways regulating important cell functions such as cell migration and cell survival at 24 h after MF irradiation or at 2 months after SD irradiation. Further, cells which survived a SD of 10 Gy showed a long-term upregulation and increased activity of multiple molecular targets including AKT, IGF-1R, VEGFR2, or MET, while HDAC expression was decreased. In line with this, 10 Gy SD cells were more sensitive to target inhibition with Capivasertib or Ipatasertib (AKTi), BMS-754807 (IGF-1Ri), or Foretinib (VEGFR2/METi), but less sensitive to Panobinostat or Vorinostat (HDACi). In summary, understanding the molecular short- and long-term changes after irradiation can aid in optimizing the efficacy of multimodal radiation oncology in combination with post-irradiation molecularly-targeted drug treatment and improving the outcome of prostate cancer patients.

Radiation-induced Adaptive Response: New Potential for Cancer Treatment.

Radiotherapy is highly effective due to its ability to physically focus the treatment to target the tumor while sparing normal tissue and its ability to be combined with systemic therapy. This systemic therapy can be utilized before radiotherapy as an adjuvant or induction treatment, during radiotherapy as a radiation "sensitizer," or following radiotherapy as a part of combined modality therapy. As part of a unique concept of using radiation as "focused biology," we investigated how tumors and normal tissues adapt to clinically relevant multifraction (MF) and single-dose (SD) radiation to observe whether the adaptations can induce susceptibility to cell killing by available drugs or by immune enhancement. We identified an adaptation occurring after MF (3 × 2 Gy) that induced cell killing when AKT-mTOR inhibitors were delivered following cessation of radiotherapy. In addition, we identified inducible changes in integrin expression 2 months following cessation of radiotherapy that differ between MF (1 Gy × 10) and SD (10 Gy) that remain targetable compared with preradiotherapy. Adaptation is reflected across different "omics" studies, and thus the range of possible molecular targets is not only broad but also time, dose, and schedule dependent. While much remains to be studied about the radiation adaptive response, radiation should be characterized by its molecular perturbations in addition to physical dose. Consideration of the adaptive effects should result in the design of a tailored radiotherapy treatment plan that accounts for specific molecular changes to be targeted as part of precision multimodality cancer treatment.

Long-term Tumor Adaptation after Radiotherapy: Therapeutic Implications for Targeting Integrins in Prostate Cancer.

Adaptation of tumor cells to radiotherapy induces changes that are actionable by molecular targeted agents and immunotherapy. This report demonstrates that radiation-induced changes in integrin expression can be targeted 2 months later. Integrins are transmembrane cell adhesion molecules that are essential for cancer cell survival and proliferation. To analyze the short- and long-term effects of radiation on the integrin expression, prostate cancer cells (DU145, PC3, and LNCaP) were cultured in a 3D extracellular matrix and irradiated with either a single dose of radiation (2-10 Gy) or a multifractionated regimen (2-10 fractions of 1 Gy). Whole human genome microarrays, immunoblotting, immunoprecipitation assays, and immunofluorescence staining of integrins were performed. The results were confirmed in a prostate cancer xenograft model system. Interestingly, ß1 and ß4 integrins (ITGB1 and ITGB4) were upregulated after radiation in vitro and in vivo. This overexpression lasted for more than 2 months and was dose dependent. Moreover, radiation-induced upregulation of ß1 and ß4 integrin resulted in significantly increased tumor cell death after treatment with inhibitory antibodies. Combined, these findings indicate that long-term tumor adaptation to radiation can result in an increased susceptibility of surviving cancer cells to molecular targeted therapy due to a radiation-induced overexpression of the target. IMPLICATIONS: Radiation induces dose- and schedule-dependent adaptive changes that are targetable for an extended time; thus suggesting radiotherapy as a unique strategy to orchestrate molecular processes, thereby providing new radiation-drug treatment options within precision cancer medicine.

Exploiting Radiation-Induced Signaling to Increase the Susceptibility of Resistant Cancer Cells to Targeted Drugs: AKT and mTOR Inhibitors as an Example.

Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of "The Cancer Genome Atlas" database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional-cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein-protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355-67. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Exploiting Gene Expression Kinetics in Conventional Radiotherapy, Hyperfractionation, and Hypofractionation for Targeted Therapy.

The dramatic changes in the technological delivery of radiation therapy, the repertoire of molecular targets for which pathway inhibitors are available, and the cellular and immunologic responses that can alter long-term clinical outcome provide a potentially unique role for using the radiation-inducible changes as therapeutic targets. Various mathematical models of dose and fractionation are extraordinarily useful in guiding treatment regimens. However, although the model may fit the clinical outcome, a deeper understanding of the molecular and cellular effect of the individual dose size and the adaptation to repeated exposure, called multifraction (MF) adaptation, may provide new therapeutic targets for use in combined modality treatments using radiochemotherapy and radioimmunotherapy. We discuss the potential of using different radiation doses and MF adaptation for targeting transcription factors, immune and inflammatory response, and cell "stemness." Given the complex genetic composition of tumors before treatment and their adaptation to drug treatment, innovative combinations using both the pretreatment molecular data and also the MF-adaptive response to radiation may provide an important role for focused radiation therapy as an integral part of precision medicine and immunotherapy.

Comprehensive molecular tumor profiling in radiation oncology: How it could be used for precision medicine.

New technologies enabling the analysis of various molecules, including DNA, RNA, proteins and small metabolites, can aid in understanding the complex molecular processes in cancer cells. In particular, for the use of novel targeted therapeutics, elucidation of the mechanisms leading to cell death or survival is crucial to eliminate tumor resistance and optimize therapeutic efficacy. While some techniques, such as genomic analysis for identifying specific gene mutations or epigenetic testing of promoter methylation, are already in clinical use, other "omics-based" assays are still evolving. Here, we provide an overview of the current status of molecular profiling methods, including promising research strategies, as well as possible challenges, and their emerging role in radiation oncology.

Defining molecular signature of pro-immunogenic radiotherapy targets in human prostate cancer cells.

To understand the impact of clinically relevant radiation therapy (RT) on tumor immune gene expression and to utilize the changes that occur during treatment to improve cancer treatment outcome, we examined how immune response genes are modulated in prostate cancer cells of varying p53 status. LNCaP (p53 wild-type), PC3 (p53 null) and DU145 (p53 mutant) cells received a 10 Gy single dose or 1 Gy × 10 multifractionated radiation dose to simulate hypofractionated and conventionally fractionated prostate radiotherapy. Total RNA was isolated 24 h after multifractionated radiation treatment and single-dose treatments and subjected to microarray analysis and later validated by RT-PCR. RT-PCR was utilized to identify total-dose inflection points for significantly upregulated genes in response to multifractionated radiation therapy. Radiation-induced damage-associated molecular pattern molecules (DAMPs) and cytokine analyses were performed using bioluminescence and ELISA. Multifractionated doses activated immune response genes more robustly than single-dose treatment, with a relatively larger number of immune genes upregulated in PC3 compared to DU145 and LNCaP cells. The inflection point of multifractionated radiation-induced immune genes in PC3 cells was observed in the range of 8-10 Gy total radiation dose. Although both multifractionated and single-dose radiation-induced proinflammatory DAMPs and positively modulated the cytokine environment, the changes were of higher magnitude with multifractionated therapy. The findings of this study together with the gene expression data suggest that cells subjected to multifractionated radiation treatment would promote productive immune cell-tumor cell interactions.

Differential expression of stress and immune response pathway transcripts and miRNAs in normal human endothelial cells subjected to fractionated or single-dose radiation.

UNLABELLED: Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2 Gy × 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed. IMPLICATIONS: Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy.

mRNA Expression Profiles for Prostate Cancer following Fractionated Irradiation Are Influenced by p53 Status.

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy x 10, 1 Gy x 10, and 2 Gy x 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 x 10(-73), 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 x 10(-30)) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.

Radiation survivors: understanding and exploiting the phenotype following fractionated radiation therapy.

Radiation oncology modalities such as intensity-modulated and image-guided radiation therapy can reduce the high dose to normal tissue and deliver a heterogeneous dose to tumors, focusing on areas deemed at highest risk for tumor persistence. Clinical radiation oncology produces daily doses ranging from 1 to 20 Gy, with tissues being exposed to 30 or more daily fractions. Hypothesizing the cells that survive fractionated radiation therapy have a substantially different phenotype than the untreated cells, which might be exploitable for targeting with molecular therapeutics or immunotherapy, three prostate cancer cell lines (PC3, DU145, and LNCaP) and normal endothelial cells were studied to understand the biology of differential effects of multifraction (MF) radiation of 0.5, 1, and/or 2 Gy fraction to 10 Gy total dose, and a single dose of 5 and 10 Gy. The resulting changes in mRNA, miRNA, and phosphoproteome were analyzed. Significant differences were observed in the MF radiation exposures including those from the 0.5 Gy MF that produces little cell killing. As expected, p53 function played a major role in response. Pathways modified by MF include immune response, DNA damage, cell-cycle arrest, TGF-ß, survival, and apoptotic signal transduction. The radiation-induced stress response will set forth a unique platform for exploiting the effects of radiation therapy as "focused biology" for cancer treatment in conjunction with molecular targeted or immunologically directed therapy. Given that more normal tissue is treated, albeit to lower doses with these newer techniques, the response of the normal tissue may also influence long-term treatment outcome.

Space-relevant radiation modifies cytokine profiles, signaling proteins and Foxp3+ T cells.

PURPOSE: The major goal was to evaluate effects of various radiation regimens on leukocyte populations relatively long-term after whole-body irradiation. MATERIALS AND METHODS: C57BL/6 mice were exposed to-low-dose/low-dose rate (LDR) (57)Co γ-rays (0.01 Gy, 0.03 cGy/h), with and without acute 2 Gy proton (1 Gy/min) or γ-ray (0.9 Gy/min) irradiation; analyses were done on days 21 and 56 post-exposure. RESULTS: Numerous radiation-induced changes were noted at one or both time points. Among the most striking differences (P < 0.05) were: (i) High percentage of CD4(+)CD25(+)Foxp3(+) T cells in spleens from the Proton vs. LDR, Gamma and LDR + Proton groups (day 56); (ii) high interleukin-2 (IL-2) in spleen supernatants from the LDR and LDR + Proton groups vs. 0 Gy (day 56), whereas IL-10 was high in the LDR + Gamma group vs. 0 Gy (day 56); (iii) difference in transforming growth factor-ß1 (TGF-ß1) in spleen supernatants from Proton and LDR + Proton groups vs. Gamma and LDR + Gamma groups (both days); (iv) low TGF-ß1 in blood from LDR + Proton vs. LDR + Gamma group (day 21); and (v) high level of activated cJun N-terminal kinase (JNK) in CD4(+) T cells from LDR + Proton vs. LDR + Gamma group (day 21). CONCLUSIONS: The findings demonstrate that at least some immune responses to acute 2 Gy radiation were dependent on radiation quality time of assessment, and pre-exposure to LDR γ-rays.

Fractionated radiation alters oncomir and tumor suppressor miRNAs in human prostate cancer cells.

We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation differentially expressed more genes compared to single-dose radiation. To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA expression in LNCaP, PC3 and DU145 prostate cancer cells treated with single-dose radiation and fractionated radiation by microarray. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. Fractionated radiation significantly altered more miRNAs as compared to single-dose radiation. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with single-dose radiation and fractionated radiation. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using fractionated radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in predicting radiosensitivity.

Gene expression profile of coronary artery cells treated with nonsteroidal anti-inflammatory drugs reveals off-target effects.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have come under scrutiny because of the gastrointestinal, renal, and cardiovascular toxicity associated with prolonged use of these drugs. The purpose of this study was to identify molecular targets for NSAIDs related to cellular toxicity with a view to optimize drug efficacy in the clinic. Coronary artery smooth muscle cells and endothelial cells were treated with low (clinically achievable) and high (typically used in preclinical studies) concentrations of celecoxib, NS398, and ibuprofen for 24 hours. NSAIDs-induced gene expression changes were evaluated by microarray analysis and validated by real-time reverse-transcription polymerase chain reaction and western blotting. The functional significance of differentially expressed genes was evaluated by Ingenuity Pathway Analysis. At high concentrations, NSAIDs altered the expression of genes regulating cell proliferation and cell death. NSAIDs also altered genes associated with cardiovascular functions including inflammation, thrombosis, fibrinolysis, coronary artery disease, and hypertension. The gene expression was most impacted by ibuprofen, celecoxib, and NS398, in that order. This study revealed that NSAIDs altered expression of an array of genes associated with cardiovascular events and emphasizes the potential for fingerprinting drugs in preclinical studies to assess the potential drug toxicity and to optimize the drug efficacy in clinical settings.

Comparison of proton and electron radiation effects on biological responses in liver, spleen and blood.

PURPOSE: To determine whether differences exist between proton and electron radiations on biological responses after total-body exposure. MATERIALS AND METHODS: ICR mice (n=45) were irradiated to 2 Gray (Gy) using fully modulated 70 MeV protons (0.5 Gy/min) and 21 MeV electrons (3 Gy/min). At 36 h post-irradiation liver gene expression, white blood cell (WBC), natural killer (NK) cell and other analyses were performed. RESULTS: Oxidative stress-related gene expression patterns were strikingly different for irradiated groups compared to 0 Gy (P<0.05). Proton radiation up-regulated 15 genes (Ctsb, Dnm2, Gpx5, Il19, Il22, Kif9, Lpo, Nox4, Park7, Prdx4, Prdx6, Rag2, Sod3, Srxn1, Xpa) and down-regulated 2 genes (Apoe, Prdx1). After electron irradiation, 20 genes were up-regulated (Aass, Ctsb, Dnm2, Gpx1, Gpx4, Gpx5, Gpx6, Gstk1, Il22, Kif9, Lpo, Nox4, Park7, Prdx3, Prdx4, Prdx5, Rag2, Sod1, Txnrd3, Xpa) and 1 was down-regulated (Mpp4). Of the modified genes, only 11 were common to both forms of radiation. Comparison between the two irradiated groups showed that electrons significantly up-regulated three genes (Gstk1, Prdx3, Scd1). Numbers of WBC and major leukocyte types were low in the irradiated groups (P<0.001 vs. 0 Gy). Hemoglobin and platelet counts were low in the electron-irradiated group (P<0.05 vs. 0 Gy). However, spleens from electron-irradiated mice had higher WBC and lymphocyte counts, as well as enhanced NK cell cytotoxicity, compared to animals exposed to protons (P<0.05). There were no differences between the two irradiated groups in body mass, organ masses, and other assessed parameters, although some differences were noted compared to 0 Gy. CONCLUSION: Collectively, the data demonstrate that at least some biological effects induced by electrons may not be directly extrapolated to protons.

Low-dose photon and simulated solar particle event proton effects on Foxp3+ T regulatory cells and other leukocytes.

Radiation is a major factor in the spaceflight environment that has carcinogenic potential. Astronauts on missions are continuously exposed to low-dose/low-dose-rate (LDR) radiation and may receive relatively high doses during a solar particle event (SPE) that consists primarily of protons. However, there are very few reports in which LDR photons were combined with protons. In this study, C57BL/6 mice were exposed to 1.7 Gy simulated SPE (sSPE) protons over 36 h, both with and without pre-exposure to 0.01 Gray (Gy) LDR g-rays at 0.018 cGy/h. Apoptosis in skin samples was determined by immunohistochemistry immediately post-irradiation (day 0). Spleen mass relative to body mass, white blood cells (WBC), major leukocyte populations, lymphocyte subsets (T, Th, Tc, B, NK), and CD4(+)CD25(+)Foxp3+ T regulatory (Treg) cells were analyzed on days 4 and 21. Apoptosis in skin samples was evident in all irradiated groups; the LDR+sSPE mice had the greatest expression of activated caspase-3. On day 4 post-irradiation, the sSPE and LDR+sSPE groups had significantly lower WBC counts in blood and spleen compared to non-irradiated controls (p < 0.05 vs. 0 Gy). CD4(+)CD25(+)Foxp3(+) Treg cell numbers in spleen were decreased at day 4, but proportions were increased in the sSPE and LDR+sSPE groups (p < 0.05 vs. 0 Gy). By day 21, lymphocyte counts were still low in blood from the LDR+sSPE mice, especially due to reductions in B, NK, and CD8(+) T cytotoxic cells. The data demonstrate, for the first time, that pre-exposure to LDR photons did not protect against the adverse effects of radiation mimicking a large solar storm. The increased proportion of immunosuppressive CD4+CD25(+) Foxp3(+) Treg and persistent reduction in circulating lymphocytes may adversely impact immune defenses that include removal of sub-lethally damaged cells with carcinogenic potential, at least for a period of time post-irradiation.

A metalloporphyrin antioxidant alters cytokine responses after irradiation in a prostate tumor model.

The goal of this study was to evaluate cytokine secretion capacity in a mouse model of prostate cancer, both with and without metalloporphyrin antioxidant and radiation treatment. C57BL/6 mice with subcutaneous RM-9 tumors were treated daily for 12 days with MnTE-2-PyP(5+) [Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin], beginning 1 day after injection of RM-9 cells; a 10-Gy tumor-localized dose of (60)Co gamma rays was administered in a single fraction on day 7. Spleen, tumors and plasma were collected on day 12. T cells in the spleen were activated with anti-CD3 antibody and supernatants were collected. Twenty-two cytokines were quantified in spleen supernatants, five in tumor homogenates, and three in plasma using multiplex bead array technology and ELISA. The presence of a tumor had significant effects on many of the cytokines quantified (P < 0.05). Tumor-induced depression was evident for eight spleen cytokines (TNF-alpha, G-CSF, GM-CSF, IFN-gamma, IL10, IP-10, MIP-1alpha and mKC), whereas only three were enhanced (IL1beta, IL6 and MCP-1). Radiotherapy resulted in enhanced splenocyte capacity to produce IL4 and IL13 and increased IL4, MCP-1 and VEGF in tumors (P < 0.05). Addition of MnTE-2-PyP(5+) to radiation decreased the concentrations of IL4, IL13 and TGF-beta1 in spleen supernatants and IL4 and VEGF in tumors (P < 0.05 compared to radiation alone). Some differences were also noted in plasma cytokines. Overall, the findings suggest that administration of MnTE-2-PyP(5+) together with radiotherapy may enhance anti-tumor immune responsiveness and decrease the risk for radiation-induced normal tissue toxicities.

Effect of a metalloporphyrin antioxidant (MnTE-2-PyP) on the response of a mouse prostate cancer model to radiation.

BACKGROUND: Metalloporphyrin antioxidants can protect tissues against radiation-induced damage. However, for effective use in radiotherapy as normal tissue radioprotectants, they must not protect the cancer. The major objectives were to evaluate the effects of Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) on tumor response to radiation and to explore mechanisms responsible for the observed effects. MATERIALS AND METHODS: C57BL/6 mice were subcutaneously (s.c.) injected with RM-9 prostate tumor cells on day 0 and grouped according to treatment with MnTE-2-PyP (s.c. 6 mg/kg/day beginning on day 1 for 16 maximum days), 10 Gray (Gy) single fraction radiation on day 7, a combination of both or neither. Subsets per group and non-tumor bearing controls were evaluated for leukocyte populations, red blood cell (RBC) and platelet characteristics and cytokines on day 12; the remaining mice were followed for tumor growth. RESULTS: Although radiation alone significantly slowed tumor growth and the addition of MnTE-2-PyP resulted in slightly slower tumor progression, the difference between radiation and radiation plus drug was not statistically significant. However, the treatment with drug alone significantly elevated T (helper, Th and cyotoxic, Tc) and natural killer (NK) cells in the spleen, B-cells in the blood and spleen, and the capacity to produce interleukin-2. The addition of the drug to radiation did not ameliorate the depression seen in all the major leukocyte types, but did protect against radiation-induced decreases in RBC counts, hemoglobin and hematocrit. Vascular endothelial growth factor (VEGF) increased in the plasma from both the irradiated groups and a trend for increased transforming growth factor-beta1 (TGF-beta1) was noted with radiation alone. CONCLUSION: MnTE-2-PyP did not protect RM-9 prostate tumors against radiation damage and was not toxic under the conditions used. The drug-induced enhancement of certain immune parameters suggests that MnTE-2-PyP may be beneficial not only as a normal tissue radioprotectant, but also as a facilitator of antitumor immunity.

Low dose, low dose rate photon radiation modifies leukocyte distribution and gene expression in CD4(+) T cells.

A better understanding of low dose radiation effects is needed to accurately estimate health risks. In this study, C57BL/6 mice were gamma-irradiated to total doses of 0, 0.01, 0.05, and 0.1 Gy ((57)Co; ~0.02 cGy/h). Subsets per group were euthanized at the end of irradiation (day 0) and on days 4 and 21 thereafter. Relative spleen mass and splenic white blood cell (WBC) counts, major leukocyte populations, and spontaneous DNA synthesis were consistently higher in the irradiated groups on day 0 compared to 0 Gy controls, although significance was not always obtained. In the spleen, all three major leukocyte types were significantly elevated on day 0 (P < 0.05). By day 21 post-irradiation the T, B, and natural killer (NK) cell counts, as well as CD4(+) T cells and CD4:CD8 T cell ratio, were low especially in the 0.01 Gy group. Although blood analyses showed no significant differences in leukocyte counts or red blood cell and platelet characteristics, the total T cells, CD4(+) T cells, and NK cells were increased by day 21 after 0.01 Gy (P < 0.05). Gene analysis of CD4(+) T cells negatively isolated from spleens on day 0 after 0.1 Gy showed significantly enhanced expression of Il27 and Tcfcp2, whereas Inha and Socs5 were down-regulated by 0.01 Gy and 0.1 Gy, respectively (P < 0.05). A trend for enhancement was noted in two additional genes (Il1r1 and Tbx21) in the 0.1 Gy group (P < 0.1). The data show that protracted low dose photons had dose- and time-dependent effects on CD4(+) T cells after whole-body exposure.