Methods to assess radiation-induced fibrosis in mice.

Therapeutic radiation is used to treat a variety of cancers in organs and tissues throughout the body. Exposure of benign normal tissue to radiation can result in late injury in a subset of patients. Radiation induced fibrosis is one chronic, progressive late toxicity of radiation exposure that can occur in many organs and tissues, including skin and lung. Radiation fibrosis has few effective treatments. The process of radiation fibrosis is known to involve many mitogenic and immunomodulatory cytokines, inflammatory programs, and processes such as stem cell senescence. Murine models of radiation fibrosis can be used to evaluate agents that may prevent, mitigate, or treat this injury. Here, we provide a detailed protocol for the development of radiation induced dermal and pulmonary fibrosis in mice and describe protocols for the measurement of this injury in treated tissue.

Natural variation in macrophage polarization and function impact pneumocyte senescence and susceptibility to fibrosis.

Radiation-induced pulmonary fibrosis (RIPF), a late adverse event of radiation therapy, is characterized by infiltration of inflammatory cells, progressive loss of alveolar structure, secondary to the loss of pneumocytes and accumulation of collagenous extracellular matrix, and senescence of alveolar stem cells. Differential susceptibility to lung injury from radiation and other toxic insults across mouse strains is well described but poorly understood. The accumulation of alternatively activated macrophages (M2) has previously been implicated in the progression of lung fibrosis. Using fibrosis prone strain (C57L), a fibrosis-resistant strain (C3H/HeN), and a strain with intermediate susceptibility (C57BL6/J), we demonstrate that the accumulation of M2 macrophages correlates with the manifestation of fibrosis. A comparison of primary macrophages derived from each strain identified phenotypic and functional differences, including differential expression of NADPH Oxidase 2 and production of superoxide in response to M2 polarization and activation. Further, the sensitivity of primary AECII to senescence after coculture with M2 macrophages was strain dependent and correlated to observations of sensitivity to fibrosis and senescence in vivo. Taken together, these data support that the relative susceptibility of different strains to RIPF is closely related to distinct senescence responses induced through pulmonary M2 macrophages after thoracic irradiation.

Senescence-associated tumor growth is promoted by 12-Lipoxygenase.

Radiation therapy is a commonly used treatment modality for cancer. Although effective in providing local tumor control, radiation causes oxidative stress, inflammation, immunomodulatory and mitogenic cytokine production, extracellular matrix production, and premature senescence in lung parenchyma. The senescence associated secretory phenotype (SASP) can promote inflammation and stimulate alterations in the surrounding tissue. Therefore, we hypothesized that radiation-induced senescent parenchymal cells in irradiated lung would enhance tumor growth. Using a murine syngeneic tumor model of melanoma and non-small cell lung cancer lung metastasis, we demonstrate that radiation causes a significant increase in markers of premature senescence in lung parenchyma within 4 to 8 weeks. Further, injection of B16F0 (melanoma) or Lewis Lung carcinoma (epidermoid lung cancer) cells at these time points after radiation results in an increase in the number and size of pulmonary tumor nodules relative to unirradiated mice. Treatment of irradiated mice with a senolytic agent (ABT-737) or agents that prevent senescence (rapamycin, INK-128) was sufficient to reduce radiation-induced lung parenchymal senescence and to mitigate radiation-enhanced tumor growth. These agents abrogated radiation-induced expression of 12-Lipoxygenase (12-LOX), a molecule implicated in several deleterious effects of senescence. Deficiency of 12-LOX prevented radiation-enhanced tumor growth. Together, these data demonstrate the pro-tumorigenic role of radiation-induced senescence, introduces the dual TORC inhibitor INK-128 as an effective agent for prevention of radiation-induced normal tissue senescence, and identifies senescence-associated 12-LOX activity as an important component of the pro-tumorigenic irradiated tissue microenvironment. These studies suggest that combining senotherapeutic agents with radiotherapy may decrease post-therapy tumor growth.

IGF-1 Receptor Signaling Regulates Type II Pneumocyte Senescence and Resulting Macrophage Polarization in Lung Fibrosis.

PURPOSE: Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging. METHODS AND MATERIALS: Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63). RESULTS: IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 µg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis. CONCLUSIONS: This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.

12-Lipoxygenase is a Critical Mediator of Type II Pneumocyte Senescence, Macrophage Polarization and Pulmonary Fibrosis after Irradiation.

Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive complication of therapeutic irradiation of the thorax. It has been suggested that senescence of type II pneumocytes (AECIIs), an alveolar stem cell, plays a role in the development of RIPF through loss of replicative reserve and via senescent AECII-driven release of proinflammatory and profibrotic cytokines. Within this context, we hypothesized that arachidonate 12-lipoxygenase (12-LOX) is a critical mediator of AECII senescence and RIPF. Treatment of wild-type AECIIs with 12S-hydroxyeicosateraenoic acid (12S-HETE), a downstream product of 12-LOX, was sufficient to induce senescence in a NADPH oxidase 4 (NOX4)-dependent manner. Mice deficient in 12-LOX exhibited reduced AECII senescence, pulmonary collagen accumulation and accumulation of alternatively activated (M2) macrophages after thoracic irradiation (5 × 6 Gy) compared to wild-type mice. Conditioned media from irradiated or 12S-HETE-treated primary pneumocytes contained elevated levels of IL-4 and IL-13 compared to untreated pneumocytes. Primary macrophages treated with conditioned media from irradiated AECII demonstrated preferential M2 type polarization when AECIIs were derived from wild-type mice compared to 12-LOX-deficient mice. Together, these data identified 12-LOX as a critical component of RIPF and a therapeutic target for radiation-induced lung injury.

Hyperpolarized [1-13C]-Pyruvate Magnetic Resonance Spectroscopic Imaging of Prostate Cancer In Vivo Predicts Efficacy of Targeting the Warburg Effect.

Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect.Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 µg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro LDH activity was assessed in tumor homogenates.Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro LDH activity correlated to FX-11 sensitivity.Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137-48. ©2018 AACR.

Mithramycin A Enhances Tumor Sensitivity to Mitotic Catastrophe Resulting From DNA Damage.

PURPOSE: Specificity protein 1 (SP1) is involved in the transcription of several genes implicated in tumor maintenance. We investigated the effects of mithramycin A (MTA), an inhibitor of SP1 DNA binding, on radiation response. METHODS AND MATERIALS: Clonogenic survival after irradiation was assessed in 2 tumor cell lines (A549, UM-UC-3) and 1 human fibroblast line (BJ) after SP1 knockdown or MTA treatment. DNA damage repair was evaluated using γH2AX foci formation, and mitotic catastrophe was assessed using nuclear morphology. Gene expression was evaluated using polymerase chain reaction arrays. In vivo tumor growth delay was used to evaluate the effects of MTA on radiosensitivity. RESULTS: Targeting of SP1 with small interfering RNA or MTA sensitized A549 and UM-UC-3 to irradiation, with no effect on the BJ radiation response. MTA did not alter γH2AX foci formation after irradiation in tumor cells but did enhance mitotic catastrophe. Treatment with MTA suppressed transcription of genes involved in cell death. MTA administration to mice bearing A549 and UM-UC-3 xenografts enhanced radiation-induced tumor growth delay. CONCLUSIONS: These results support SP1 as a target for radiation sensitization and confirm MTA as a radiation sensitizer in human tumor models.

IL-13 is a therapeutic target in radiation lung injury.

Pulmonary fibrosis is a potentially lethal late adverse event of thoracic irradiation. Prior research indicates that unrestrained TGF-ß1 and/or type 2 cytokine-driven immune responses promote fibrosis following radiation injury, but the full spectrum of factors governing this pathology remains unclear. Interleukin 13 (IL-13) is a key factor in fibrotic disease associated with helminth infection, but it is unclear whether it plays a similar role in radiation-induced lung fibrosis. Using a mouse model, we tested the hypothesis that IL-13 drives the progression of radiation-induced pulmonary fibrosis. Irradiated lungs from wild-type c57BL/6NcR mice accumulated alternatively-activated macrophages, displayed elevated levels of IL-13, and extensive fibrosis, whereas IL-13 deficient mice were resistant to these changes. Furthermore, plasma from irradiated wild-type mice showed a transient increase in the IL-13 saturated fraction of the circulating decoy receptor IL-13Rα2. Finally, we determined that therapeutic neutralization of IL-13, during the period of IL-13Rα2 saturation was sufficient to protect mice from lung fibrosis. Taken together, our results demonstrate that IL-13 is a major regulator of radiation-induced lung injury and demonstrates that strategies focusing on IL-13 may be useful in screening for timely delivery of anti-IL-13 therapeutics.

Peptidases released by necrotic cells control CD8+ T cell cross-priming.

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.

Mesenchymal stem cells inhibit cutaneous radiation-induced fibrosis by suppressing chronic inflammation.

Exposure to ionizing radiation (IR) can result in the development of cutaneous fibrosis, for which few therapeutic options exist. We tested the hypothesis that bone marrow-derived mesenchymal stem cells (BMSC) would favorably alter the progression of IR-induced fibrosis. We found that a systemic infusion of BMSC from syngeneic or allogeneic donors reduced skin contracture, thickening, and collagen deposition in a murine model. Transcriptional profiling with a fibrosis-targeted assay demonstrated increased expression of interleukin-10 (IL-10) and decreased expression of IL-1ß in the irradiated skin of mice 14 days after receiving BMSC. Similarly, immunoassay studies demonstrated durable alteration of these and several additional inflammatory mediators. Immunohistochemical studies revealed a reduction in infiltration of proinflammatory classically activated CD80(+) macrophages and increased numbers of anti-inflammatory regulatory CD163(+) macrophages in irradiated skin of BMSC-treated mice. In vitro coculture experiments confirmed that BMSC induce expression of IL-10 by activated macrophages, suggesting polarization toward a regulatory phenotype. Furthermore, we demonstrated that tumor necrosis factor-receptor 2 (TNF-R2) mediates IL-10 production and transition toward a regulatory phenotype during coculture with BMSC. Taken together, these data demonstrate that systemic infusion of BMSC can durably alter the progression of radiation-induced fibrosis by altering macrophage phenotype and suppressing local inflammation in a TNF-R2-dependent fashion.

Inhibition of radiation-induced skin fibrosis with imatinib.

PURPOSE: Dermal fibrosis is a disabling late toxicity of radiotherapy. Several lines of evidence suggest that overactive signaling via the Platelet-derived growth factor receptor-beta (PDGFR-ß) and V-abl Abelson murine leukemia viral oncogene homolog 1 (cAbl) may be etiologic factors in the development of radiation-induced fibrosis. We tested the hypothesis that imatinib, a clinically available inhibitor of PDGFR-ß, Mast/stem cell growth factor receptor (c-kit) and cAbl, would reduce the severity of dermal fibrosis in a murine model. MATERIALS AND METHODS: The right hind legs of female C3H/HeN mice were exposed to 35 Gy of X-rays. Cohorts of mice were maintained on chow formulated with imatinib 0.5 mg/g or control chow for the duration of the experiment. Bilateral hind limb extension was measured serially to assess fibrotic contracture. Immunohistochemistry and biochemical assays were used to evaluate the levels of collagen and cytokines implicated in radiation-induced fibrosis. RESULTS: Imatinib treatment significantly reduced hind limb contracture and dermal thickness after irradiation. Immunohistochemical studies demonstrated a substantial reduction in PDGFR-ß phosphorylation. We also observed reduced Transforming Growth factor-ß (TGF-ß) and collagen expression in irradiated skin of imatinib-treated mice, suggesting that imatinib may suppress the fibrotic process by interrupting cross-talk between these pathways. CONCLUSIONS: Taken together, these results support that imatinib may be a useful agent in the prevention and treatment of radiation-induced dermal fibrosis.