Identification and characterization of mesenchymal-epithelial progenitor-like cells in normal and injured rat liver.

In normal rat liver, thymocyte antigen 1 (Thy1) is expressed in fibroblasts/myofibroblasts and in some blood progenitor cells. Thy1-expressing cells also accumulate in the liver during impaired liver regeneration. The origin and nature of these cells are not well understood. By using RT-PCR analysis and immunofluorescence microscopy, we describe the presence of rare Thy1(+) cells in the liver lobule of normal animals, occasionally forming small collections of up to 20 cells. These cells constitute a small portion (1.7% to 1.8%) of nonparenchymal cells and reveal a mixed mesenchymal-epithelial phenotype, expressing E-cadherin, cytokeratin 18, and desmin. The most potent mitogens for mesenchymal-epithelial Thy1(+) cells in vitro are the inflammatory cytokines interferon γ, IL-1, and platelet-derived growth factor-BB, which are not produced by Thy1(+) cells. Thy1(+) cells express all typical mesenchymal stem cell and hepatic progenitor cell markers and produce growth factor and cytokine mRNA (Hgf, Il6, Tgfa, and Tweak) for proteins that maintain oval cell growth and differentiation. Under appropriate conditions, mesenchymal-epithelial cells differentiate in vitro into hepatocyte-like cells. In this study, we show that the adult rat liver harbors a small pool of endogenous mesenchymal-epithelial cells not recognized previously. In the quiescent state, these cells express both mesenchymal and epithelial cell markers. They behave like hepatic stem cells/progenitors with dual phenotype, exhibiting high plasticity and long-lasting proliferative activity.

Combined immunotherapy with Listeria monocytogenes-based PSA vaccine and radiation therapy leads to a therapeutic response in a murine model of prostate cancer.

Radiation therapy (RT) is an integral part of prostate cancer treatment across all stages and risk groups. Immunotherapy using a live, attenuated, Listeria monocytogenes-based vaccines have been shown previously to be highly efficient in stimulating anti-tumor responses to impact on the growth of established tumors in different tumor models. Here, we evaluated the combination of RT and immunotherapy using Listeria monocytogenes-based vaccine (ADXS31-142) in a mouse model of prostate cancer. Mice bearing PSA-expressing TPSA23 tumor were divided to 5 groups receiving no treatment, ADXS31-142, RT (10 Gy), control Listeria vector and combination of ADXS31-142 and RT. Tumor growth curve was generated by measuring the tumor volume biweekly. Tumor tissue, spleen, and sera were harvested from each group for IFN-γ ELISpot, intracellular cytokine assay, tetramer analysis, and immunofluorescence staining. There was a significant tumor growth delay in mice that received combined ADXS31-142 and RT treatment as compared with mice of other cohorts and this combined treatment causes complete regression of their established tumors in 60 % of the mice. ELISpot and immunohistochemistry of CD8+ cytotoxic T Lymphocytes (CTL) showed a significant increase in IFN-γ production in mice with combined treatment. Tetramer analysis showed a fourfold and a greater than 16-fold increase in PSA-specific CTLs in animals receiving ADXS31-142 alone and combination treatment, respectively. A similar increase in infiltration of CTLs was observed in the tumor tissues. Combination therapy with RT and Listeria PSA vaccine causes significant tumor regression by augmenting PSA-specific immune response and it could serve as a potential treatment regimen for prostate cancer.

Nitric oxide-releasing nanoparticles accelerate wound healing in NOD-SCID mice.

Wound healing is a complex process, coordinated by various biological factors. In immunocompromised states wound healing can be interrupted as a result of decreased numbers of immune cells, impairing the production of effector molecules such as nitric oxide (NO). Therefore, topical NO-releasing platforms, such as diethylenetriamine (DETA NONOate), have been investigated to enhance wound healing. Recently, we demonstrated a nanoparticle platform that releases NO (NO-NPs) in a sustained manner, accelerating wound healing in both uninfected and infected murine wound models. Here, NO-NPs were investigated and compared to DETA NONOate in an immunocompromised wound model using non-obese, diabetic, severe combined immunodeficiency mice. NO-NP treatment accelerated wound closure as compared to controls and DETA NONOate treatment. In addition, histological assessment revealed that wounds treated with NO-NPs had less inflammation, more collagen deposition, and more blood vessel formation as compared to other groups, consistent with our previous data in immunocompetent animals. These data suggest that NO-NPs may serve as a novel wound-healing therapy in the setting of immunocompromised states associated with impaired wound healing. FROM THE CLINICAL EDITOR: Wound healing in an immunocompromised host is often incomplete and is a source of major concern in such conditions. This work demonstrates in a murine model that in these settings NO releasing nanoparticles significantly enhance wound healing.

Snai2 Maintains Bone Marrow Niche Cells by Repressing Osteopontin Expression.

Bone marrow (BM) mesenchymal stem and progenitor cells (MSPCs) are a critical constituent of the hematopoietic stem cell (HSC) niche. Previous studies have suggested that the zinc-finger epithelial-mesenchymal transition transcription factor Snai2 (also known as Slug) regulated HSCs autonomously. Here, we show that Snai2 expression in the BM is restricted to the BM stromal compartment where it regulates the HSC niche. Germline or MSPC-selective Snai2 deletion reduces the functional MSPC pool and their mesenchymal lineage output and impairs HSC niche function during homeostasis and after stress. RNA sequencing analysis revealed that Spp1 (osteopontin) expression is markedly upregulated in Snai2-deficient MSPCs. Genetic deletion of Spp1 in Snai2-deficient mice rescues MSPCs' functions. Thus, SNAI2 is a critical regulator of the transcriptional network maintaining MSPCs by the suppression of osteopontin expression.

Aspiration risk and postoperative radiation for head and neck cancer.

The aim of the study was to assess the aspiration risk following postoperative radiation for head and neck cancer. Thirty-seven patients had Modified Barium Swallow before and following treatment. Dysphagia severity was graded from 1 to 7. Before treatment there were sixteen grade 1, seventeen grade 2, three grade 3 and one grade 5. Following postoperative radiation, two patients had grade 1, eleven patients had grade 2, thirteen patients had grade 3, four patients had grade 4, four patients had grade 5, one patients had grade 6, and two patients had grade 7. Nineteen percent (7/37) of the patients developed aspiration (grade 5-7). Aspiration is life-threatening and may develop for all tumor sites and stages.

Localized hyperthermia combined with intratumoral dendritic cells induces systemic antitumor immunity.

Prostate adenocarcinoma, treated with localized tumor hyperthermia (LTH), can potentially serve as a source of tumor antigen, where dying apoptotic/necrotic cells release tumor peptides slowly over time. In addition, LTH-treated cells can release heat shock proteins that can chaperone antigenic peptides to antigen-presenting cells, such as dendritic cells. We attempted to discern whether sequential LTH and intratumoral dendritic cell and/or systemic granulocyte macrophage colony-stimulating factor (GM-CSF) would activate antitumor immune response in a syngeneic murine model of prostate cancer (RM-1). Palpable RM-1 tumors, grown in the distal appendage of C57BL/6 male mice, were subjected to LTH (43.7 degrees C for 1 h) x 2, separated by 5 days. Following the second LTH treatment, animals received either PBS or dendritic cells (2 x 10(6)) intratumorally (every 3 days for three injections). Separate cohorts also received i.v. injection of recombinant adenovirus-expressing murine GM-CSF (AdGMCSF), 1 day after LTH. Control animals received AdenoLacZ or AdenoGFP. Intratumoral dendritic cell injection induced tumor-specific T-helper cell activity (IFNgamma ELISPOTS) and CTL activity, which was further augmented by AdGMCSF, indicating amplification of tumor-specific TH1 immunity. The combination of LTH, AdGMCSF, and intratumoral dendritic cell injection resulted in significant tumor growth delays when compared with animal cohorts that received LTH alone. These results support an in situ autovaccination strategy where systemic administration of GM-CSF and/or intratumoral injection of autologous dendritic cells, when combined with LTH, could be an effective treatment for local and systemic recurrence of prostate cancer.

Tumor biology-guided radiotherapy treatment planning: gross tumor volume versus functional tumor volume.

This issue of Seminars in Nuclear Medicine deals with a watershed event in cancer treatment -- the combined use of functional and anatomical information to guide therapeutic interventions. The use of positron emission tomography/computed tomography (PET/CT) in radiation treatment planning and tumor response evaluation brings a paradigm change in the development of image-guided therapies into routine clinical practice. The implications, as seen in the following articles, are not only promising but also groundbreaking. And, as in every new scientific breakthrough, each step forward generates a myriad of additional important clinical and research questions. Functional imaging takes advantage of the subtle differences between normal and malignant tissues at the cellular level to reveal in vivo unique functional characteristics of neoplasms. The ultimate goal of the partnership between nuclear medicine physicians and radiation oncologists is to use this information with absolute clarity in target definition for radiation treatment planning and therapy, as well as response evaluation. Functional imaging can provide metabolic information and behavioral correlation along with the anatomical imaging for correlative target delineation. Additionally, as a purely diagnostic instrument, PET/CT provides a tool for oncologists to make critical decisions regarding radiation treatment planning modifications secondary to changes in tumor staging (up or down), treatment field modifications, localized control, sites of residual and/or metastatic disease and post therapy response evaluation. The articles in this issue of the seminars provide insights into the current state-of-the-art of functional imaging techniques, mostly centered on the use of (18)F-fluorodeoxyglucose PET/CT in image guided oncologic therapies. Because it is a novel science, the future of image-guided functional treatment planning is bright with technologic and biologic innovations, translational research and new clinical applications.

Sustained release nitric oxide releasing nanoparticles: characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites.

A new platform using biocompatible materials is presented for generating powders comprised of nanoparticles that release therapeutic levels of nitric oxide (NO) in a controlled and sustained manner. The capacity of these particles to retain and gradually release NO arises from their having combined features of both glassy matrices and hydrogels. This feature allows both for the generation of NO through the thermal reduction of added nitrite by glucose and for the retention of the generated NO within the dry particles. Exposure of these robust biocompatible nanoparticles to moisture initiates the sustained release of the trapped NO over extended time periods as determined both fluorimetrically and amperometrically. The slow sustained release is in contrast to the much faster release pattern associated with the hydration-initialed NO release in powders derived from glassy matrices. These glasses are prepared using trehalose and sucrose doped with either glucose or tagatose as the source of thermal electrons needed to convert nitrite to gNO. Significantly, the release profiles for the NO in the hydrogel/glass composite materials are found to be an easily tuned parameter that is modulated through the specific additives used in preparing the hydrogel/glass composites. The presented data raise the prospect that these new NO releasing nanoparticles can be easily formulated for use under a wide range of therapeutic circumstances.

Nanoparticle-Encapsulated Doxorubicin Demonstrates Superior Tumor Cell Kill in Triple Negative Breast Cancer Subtypes Intrinsically Resistant to Doxorubicin.

The effect of size and release kinetics of doxorubicin-nanoparticles on anti-tumor efficacy was evaluated in a panel of human cancer cell lines, including triple-negative breast cancer (TNBC) cells that frequently demonstrate resistance to doxorubicin. Different nano-formulations of sol-gel-based Doxorubicin containing nanoparticles were synthesized. Increased cell kill in chemoreffactory triple-negative breast cancer cells was associated with the smallest size of nanoparticles and the slowest release of Dox. Modeling of dose-response parameters in Dox-sensitive versus Dox-resistant lines demonstrated increased EMax and area under the curve in Dox-resistant mesenchymal TNBC cells, implying potentially favorable activity in this molecular subtype of breast cancer. Mesenchymal TNBC cells demonstrated a high rate of fluorescent bead uptake suggestive of increased endocytosis, which may partially account for the enhanced efficacy of Dox-np in this subtype. Thus, manipulation of size and release kinetics of this nanoparticle platform is associated with enhanced dose-response metrics and tumor cell kill in therapeutically recalcitrant TNBC cell models. This platform is easily customizable and warrants further exploration.

Aspiration occurence during chemoradiation for head and neck cancer.

AIM: To assess the risk of developing aspiration during chemoradiation for head and neck cancer. PATIENTS AND METHODS: A retrospective review of 114 patients who underwent concurrent chemoradiation for locally advanced head and neck cancer was undertaken. Patients were determined as having aspiration if they had pneumonia on chest-X-ray (CXR) and/or had documented aspiration on the modified barium swallow (MBS) during their treatment. RESULTS: Fifteen patients (13%) developed aspiration during chemoradiation. Twelve patients (10%) had aspiration demonstrated on CXR alone (9 patients) or combined with MBS (3 patients). Three patients (3%) had aspiration on MBS alone. Three of the six patients with aspiration observed on MBS had normal swallowing on their pretreatment MBS. All 15 patients had severe mucositis and neutropenia at the time of the aspiration. Despite broad-spectrum antibiotics and supportive care, six patients (5%) died. CONCLUSION: Aspiration may develop during chemoradiation for head and neck cancer because of radiation-induced altered swallow.

Aspiration rate following chemoradiation for head and neck cancer: an underreported occurrence.

BACKGROUND AND PURPOSE: We would like to assess the prevalence of aspiration before and following chemoradiation for head and neck cancer. PATIENTS AND METHODS: We reviewed retrospectively the Modified Barium Swallow (MBS) in 63 patients who underwent concurrent chemotherapy and radiation for head and neck cancer. MBS was performed prior to treatment to determine the need for immediate gastrostomy tube placement. MBS was repeated following treatment to assess the safety of oral feeding prior to removal of tube feeding. All patients were cancer free at the time of the swallowing study. No patient had surgery. Dysphagia severity was graded on a scale of 1-7. Tube feedings were continued if patients were diagnosed to have severe aspiration (grade 6-7) or continued weight loss. Patients with abnormal swallow (grade 3-7) received swallowing therapy following MBS. RESULTS: Before treatment, there were 18 grade 1, 18 grade 2, 9 grade 3, 8 grade 4, 3 grade 5, 3 grade 6, and 4 grade 7. Following chemoradiation, at a median follow-up of 2 months (1-10 months), one patient had grade 1, eight patients had grade 2, nine patients had grade 3, eight patients had grade 4, 13 patients had grade 5, seven patients had grade 6, and 11 patients had grade 7. Six patients died from aspiration pneumonia (one before, three during, and two post-treatment), and did not have the second MBS. Overall, 37/63 (59%) patients developed aspiration, six of them (9%) fatal. If we excluded the 10 patients who had severe aspiration at diagnosis and the six patients who died from pneumonia, the prevalence of severe aspiration was 33% (21/63). CONCLUSIONS: Aspiration remained a significant morbidity following chemoradiation for head and neck cancer. Its prevalence is underreported in the literature because of its often silent nature. Diagnostic studies such as MBS should be part of future head and neck cancer prospective studies to assess the prevalence of aspiration, and for rehabilitation.

Safety and effectiveness of prophylactic gastrostomy tubes for head and neck cancer patients undergoing chemoradiation.

BACKGROUND: We would like to assess the safety and effectiveness of prophylactic percutaneous endoscopic gastrostomy (PEG) tube feedings during concurrent chemoradiation for head and neck cancer. METHODS: Patients who underwent chemotherapy and radiation for head and neck malignancies were evaluated for their ability to resume oral feeding following treatment. All patients underwent PEG tube placement prior to the treatment because of the expected mucositis. Gastrostomy tubes were removed following treatment when the patients were able to resume oral feedings without aspiration. RESULTS: Between March 1999 and 2006, 104 patients with locally advanced head and neck cancer underwent concurrent chemotherapy and radiation. One patient declined placement of gastrostomy tube. Ninety patients (86%) developed grade 3-4 mucositis during chemoradiation. Five patients died during treatment from aspiration pneumonia and sepsis. One hundred two patients lost weight during treatment. The mean and median weight loss during concurrent therapy was, respectively, 8.5 and 8 kg (1-23.5 kg). Following treatment, tube feedings were continued 1-41 months (mean: 8 months; median: 5 months) because of continued weight loss, chronic dysphagia, or aspiration. At a median follow-up of 19 months (1-62 months), no patient developed serious complications from tube feedings. CONCLUSION: Dysphagia resulting from the severe mucositis produced severe weight loss, despite tube feedings. Gastrostomy tube feedings are safe. Gastrostomy tubes should be placed prophylactically for patients undergoing chemoradiation for head and neck cancer.

Metabolic signatures associated with a NAD synthesis inhibitor-induced tumor apoptosis identified by 1H-decoupled-31P magnetic resonance spectroscopy.

PURPOSE: Attempts to selectively initiate tumor cell death through inducible apoptotic pathways are increasingly being exploited as a potential anticancer strategy. Inhibition of NAD+ synthesis by a novel agent FK866 has been recently reported to induce apoptosis in human leukemia, hepatocarcinoma cells in vitro, and various types of tumor xenografts in vivo. In the present study, we used 1H-decoupled phosphorus (31P) magnetic resonance spectroscopy (MRS) to examine the metabolic changes associated with FK866 induced tumor cell death in a mouse mammary carcinoma. EXPERIMENTAL DESIGN: Induction of apoptosis in FK866-treated tumors was confirmed by histology and cytofluorometric analysis. FK866-induced changes in mammary carcinoma tumor metabolism in vivo were investigated using 1H-decoupled 31P MRS. To discern further the changes in metabolic profiles of tumors observed in vivo, high-resolution in vitro 1H-decoupled 31P MRS studies were carried out with perchloric acid extracts of mammary carcinoma tumors excised after similar treatments. In addition, the effects of FK866 on mammary carcinoma tumor growth and radiation sensitivity were studied. RESULTS: Treatment with FK866 induced a tumor growth delay and enhanced radiation sensitivity in mammary carcinoma tumors that was associated with significant increases in the 31P MR signal in the phosphomonoester region and a decrease in NAD+ levels, pH, and bioenergetic status. The 31P MRS of perchloric acid extracts of treated tumors identified the large unresolved signal in the phosphomonoester region as the resultant of resonances originating from intermediates of tumor glycolysis and guanylate synthesis in addition to alterations in pyridine nucleotide pools and phospholipid metabolism. CONCLUSION: The present results suggest that FK866 interferes with multiple biochemical pathways that contribute to the increased cell death (apoptosis) and subsequent radiation sensitivity observed in the mammary carcinoma that could be serially monitored by 31P MRS.

Identifying Urinary and Serum Exosome Biomarkers for Radiation Exposure Using a Data Dependent Acquisition and SWATH-MS Combined Workflow.

PURPOSE: Early and accurate assessment of radiation injury by radiation-responsive biomarkers is critical for triage and early intervention. Biofluids such as urine and serum are convenient for such analysis. Recent research has also suggested that exosomes are a reliable source of biomarkers in disease progression. In the present study, we analyzed total urine proteome and exosomes isolated from urine or serum for potential biomarkers of acute and persistent radiation injury in mice exposed to lethal whole body irradiation (WBI). METHODS AND MATERIALS: For feasibility studies, the mice were irradiated at 10.4 Gy WBI, and urine and serum samples were collected 24 and 72 hours after irradiation. Exosomes were isolated and analyzed using liquid chromatography mass spectrometry/mass spectrometry-based workflow for radiation exposure signatures. A data dependent acquisition and SWATH-MS combined workflow approach was used to identify significantly exosome biomarkers indicative of acute or persistent radiation-induced responses. For the validation studies, mice were exposed to 3, 6, 8, or 10 Gy WBI, and samples were analyzed for comparison. RESULTS: A comparison between total urine proteomics and urine exosome proteomics demonstrated that exosome proteomic analysis was superior in identifying radiation signatures. Feasibility studies identified 23 biomarkers from urine and 24 biomarkers from serum exosomes after WBI. Urinary exosome signatures identified different physiological parameters than the ones obtained in serum exosomes. Exosome signatures from urine indicated injury to the liver, gastrointestinal, and genitourinary tracts. In contrast, serum showed vascular injuries and acute inflammation in response to radiation. Selected urinary exosomal biomarkers also showed changes at lower radiation doses in validation studies. CONCLUSIONS: Exosome proteomics revealed radiation- and time-dependent protein signatures after WBI. A total of 47 differentially secreted proteins were identified in urinary and serum exosomes. Together, these data showed the feasibility of defining biomarkers that could elucidate tissue-associated and systemic response caused by high-dose ionizing radiation. This is the first report using an exosome proteomics approach to identify radiation signatures.

Rigosertib is a more effective radiosensitizer than cisplatin in concurrent chemoradiation treatment of cervical carcinoma, in vitro and in vivo.

PURPOSE: To compare rigosertib versus cisplatin as an effective radiosensitizing agent for cervical malignancies. METHODS AND MATERIALS: Rigosertib and cisplatin were tested in cervical cancer cell lines, HeLa and C33A. A 24-hour incubation with rigosertib and cisplatin, before irradiation (2-8 Gy), was used for clonogenic survival assays. Cell cycle analysis (propidium iodide staining) and DNA damage (γ-H2AX expression) were evaluated by fluorescence-activated cell sorter cytometry. Rigosertib was also tested in vivo in tumor growth experiments on cervical cancer xenografts. RESULTS: Rigosertib was demonstrated to induce a G2/M block in cancer cells. Survival curve comparison revealed a dose modification factor, as index of radiosensitization effect, of 1.1-1.3 for cisplatin and 1.4-2.2 for rigosertib. With 6-Gy irradiation, an increase in DNA damage of 15%-25% was achieved in both HeLa and C33A cells with cisplatin pretreatment, and a 71-108% increase with rigosertib pretreatment. In vivo tumor growth studies demonstrated higher performance of rigosertib when compared with cisplatin, with 53% longer tumor growth delay. CONCLUSIONS: Rigosertib was more effective than cisplatin when combined with radiation and caused minimal toxicity. These data support the need for clinical trials with rigosertib in combination therapy for patients with cervical carcinoma.

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 1. Flt3 ligand gene transfer increases antitumor effects of a radio-inducible suicide gene therapy in an ectopic tumor model.

Hepatocellular carcinoma (HCC) often presents as a diffuse or multifocal tumor making it difficult to control by surgery or radiation. Radio-inducible herpes simplex virus thymidine kinase (HSV-TK) gene therapy has been shown to enhance local tumor control after radiation therapy (RT), while limiting the expression of the transgene in the irradiated tumor tissues. To prevent liver tumor recurrence and control systemic disease while limiting the potential bystander toxicity of HSV-TK therapy, we proposed to stimulate endogenous dendritic cell (DC) proliferation with systemic adenovirus Flt3 ligand (Adeno-Flt3L) gene therapy, followed by primary tumor radiation therapy combined with a radio-inducible HSV-TK gene therapy. We hypothesized that adenovirus-expressing Flt3L gene therapy will stimulate DC proliferation, allowing the upregulated DCs to locally harness tumor antigens released from HSV-TK/RT-treated HCC cells, thereby converting irradiated tumors to an autologous in situ tumor vaccine in mice with primary liver tumors. To test this hypothesis, an expression vector of HSV-TK was constructed under the control of a radio-inducible promoter early-growth response (Egr-TK) and a recombinant adenovirus-expressing human Flt3L was constructed. The Adeno-Flt3L [10(9) plaque forming units (pfu)] was administered intravenously on days 1 and 8 after radiation therapy. The murine hepatoma cell line (BNL1ME) was stably transfected by Egr-TK or Egr-Null (encoding no therapeutic gene). Palpable tumors in BALB/c mice were treated with a localized dose of 25 Gy of radiation followed by ganciclovir (GCV, 100 mg/kg, 14 days). Four treatment cohorts were compared: Egr-Null/GCV + RT + Adeno-LacZ; Egr-Null/GCV + RT + Adeno-Flt3L; Egr-TK/GCV + RT + Adeno-LacZ; and Egr-TK/GCV + RT + Adeno-Flt3L. There was no primary tumor regression in the Egr-Null tumors after radiation therapy alone. In contrast, Egr-TK tumors had nearly complete tumor regression for 3 weeks after radiation therapy (P < 0.01), however, long-term follow-up demonstrated primary tumor recurrence and death secondary to pulmonary metastasis. Flt3L expression was confirmed by serum bioassay (mean = 88 ng/mL) in these animals and Western blotting of tissue culture medium in Adeno-Flt3L-infected BaF/huFlt3L cells. Radiation therapy with Adeno-Flt3L gene therapy effectively retarded primary tumor growth when compared to radiation therapy alone. The trimodality therapy (Egr-TK/GCV + RT + Adeno-Flt3L) was the most efficacious with 40% complete tumor regression (>100 days) and <20% pulmonary metastases, indicating the development of sustained antitumor immune response. These studies provide a rationale for triple modality therapies with radiation-inducible HSV-TK gene therapy and Adeno-Flt3L when used in combination with primary tumor radiation therapy for improved local and systemic control of HCC.

TLR9 agonist protects mice from radiation-induced gastrointestinal syndrome.

PURPOSE: Radiation-induced gastrointestinal syndrome (RIGS) is due to the clonogenic loss of crypt cells and villi depopulation, resulting in disruption of mucosal barrier, bacterial invasion, inflammation and sepsis. Intestinal macrophages could recognize invading bacterial DNA via TLR9 receptors and transmit regenerative signals to the neighboring crypt. We therefore investigated whether systemic administration of designer TLR9 agonist could ameliorate RIGS by activating TLR9. METHODS AND MATERIALS: Male C57Bl6 mice were distributed in four experimental cohorts, whole body irradiation (WBI) (8.4-10.4 Gy), TLR9 agonist (1 mg/kg s.c.), 1 h pre- or post-WBI and TLR9 agonist+WBI+iMyd88 (pretreatment with inhibitory peptide against Myd88). Animals were observed for survival and intestine was harvested for histological analysis. BALB/c mice with CT26 colon tumors in abdominal wall were irradiated with 14 Gy single dose of whole abdominal irradiation (AIR) for tumor growth study. RESULTS: Mice receiving pre-WBI TLR9 agonist demonstrated improvement of survival after 10.4 Gy (p<0.03), 9.4 Gy (p<0.008) and 8.4 Gy (p<0.002) of WBI, compared to untreated or iMyd88-treated controls. Post-WBI TLR9 agonist mitigates up to 8.4 Gy WBI (p<0.01). Histological analysis and xylose absorption test demonstrated significant structural and functional restitution of the intestine in WBI+TLR9 agonist cohorts. Although, AIR reduced tumor growth, all animals died within 12 days from RIGS. TLR9 agonist improved the survival of mice beyond 28 days post-AIR (p<0.008) with significant reduction of tumor growth (p<0.0001). CONCLUSIONS: TLR9 agonist treatment could serve both as a prophylactic or mitigating agent against acute radiation syndrome and also as an adjuvant therapy to increase the therapeutic ratio of abdominal Radiation Therapy for Gastro Intestinal malignancies.

Bone marrow stromal cell transplantation mitigates radiation-induced gastrointestinal syndrome in mice.

BACKGROUND: Nuclear accidents and terrorism presents a serious threat for mass casualty. While bone-marrow transplantation might mitigate hematopoietic syndrome, currently there are no approved medical countermeasures to alleviate radiation-induced gastrointestinal syndrome (RIGS), resulting from direct cytocidal effects on intestinal stem cells (ISC) and crypt stromal cells. We examined whether bone marrow-derived adherent stromal cell transplantation (BMSCT) could restitute irradiated intestinal stem cells niche and mitigate radiation-induced gastrointestinal syndrome. METHODOLOGY/PRINCIPAL FINDINGS: Autologous bone marrow was cultured in mesenchymal basal medium and adherent cells were harvested for transplantation to C57Bl6 mice, 24 and 72 hours after lethal whole body irradiation (10.4 Gy) or abdominal irradiation (16-20 Gy) in a single fraction. Mesenchymal, endothelial and myeloid population were characterized by flow cytometry. Intestinal crypt regeneration and absorptive function was assessed by histopathology and xylose absorption assay, respectively. In contrast to 100% mortality in irradiated controls, BMSCT mitigated RIGS and rescued mice from radiation lethality after 18 Gy of abdominal irradiation or 10.4 Gy whole body irradiation with 100% survival (p<0.0007 and p<0.0009 respectively) beyond 25 days. Transplantation of enriched myeloid and non-myeloid fractions failed to improve survival. BMASCT induced ISC regeneration, restitution of the ISC niche and xylose absorption. Serum levels of intestinal radioprotective factors, such as, R-Spondin1, KGF, PDGF and FGF2, and anti-inflammatory cytokines were elevated, while inflammatory cytokines were down regulated. CONCLUSION/SIGNIFICANCE: Mitigation of lethal intestinal injury, following high doses of irradiation, can be achieved by intravenous transplantation of marrow-derived stromal cells, including mesenchymal, endothelial and macrophage cell population. BMASCT increases blood levels of intestinal growth factors and induces regeneration of the irradiated host ISC niche, thus providing a platform to discover potential radiation mitigators and protectors for acute radiation syndromes and chemo-radiation therapy of abdominal malignancies.

Protective role of R-spondin1, an intestinal stem cell growth factor, against radiation-induced gastrointestinal syndrome in mice.

BACKGROUND: Radiation-induced gastrointestinal syndrome (RIGS) results from a combination of direct cytocidal effects on intestinal crypt and endothelial cells and subsequent loss of the mucosal barrier, resulting in electrolyte imbalance, diarrhea, weight loss, infection and mortality. Because R-spondin1 (Rspo1) acts as a mitogenic factor for intestinal stem cells, we hypothesized that systemic administration of Rspo1 would amplify the intestinal crypt cells and accelerate the regeneration of the irradiated intestine, thereby, ameliorating RIGS. METHODS AND FINDINGS: Male C57Bl/6 mice received recombinant adenovirus expressing human R-spondin1 (AdRspo1) or E.coli Lacz (AdLacz), 1-3 days before whole body irradiation (WBI) or abdominal irradiation (AIR). Post-irradiation survival was assessed by Kaplan Meier analysis. RIGS was assessed by histological examination of intestine after hematoxilin and eosin staining, immunohistochemical staining of BrdU incorporation, Lgr5 and beta-catenin expression and TUNEL staining. The xylose absorption test (XAT) was performed to evaluate the functional integrity of the intestinal mucosal barrier. In order to examine the effect of R-spondin1 on tumor growth, AdRspo1 and AdLacZ was administered in the animals having palpable tumor and then exposed to AIR. There was a significant increase in survival in AdRspo1 cohorts compared to AdLacZ (p<0.003) controls, following WBI (10.4 Gy). Significant delay in tumor growth was observed after AIR in both cohorts AdRspo1 and AdLacZ but AdRspo1 treated animals showed improved survival compared to AdLacZ. Histological analysis and XAT demonstrated significant structural and functional regeneration of the intestine in irradiated animals following AdRspo1 treatment. Immunohistochemical analysis demonstrated an increase in Lgr5+ve crypt cells and the translocation of beta-catenin from the cytosol to nucleus and upregulation of beta-catenin target genes in AdRspo1-treated mice, as compared to AdLacz-treated mice. CONCLUSION: Rspo1 promoted radioprotection against RIGS and improved survival of mice exposed to WBI. The mechanism was likely related to induction of the Wnt-beta-catenin pathway and promotion of intestinal stem cell regeneration. Rspo1 has protective effect only on normal intestinal tissue but not in tumors after AIR and thereby may increase the therapeutic ratio of chemoradiation therapy in patients undergoing abdominal irradiation for GI malignancies.