Payel Bhanja

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 β-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 β-catenin from the cytosol to nucleus and upregulation of β-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-β-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.

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.

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.

Macrophage-derived extracellular vesicle-packaged WNTs rescue intestinal stem cells and enhance survival after radiation injury.

WNT/β-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation.

Serum microRNAs are early indicators of survival after radiation-induced hematopoietic injury.

Serum miRNAs can predict long-term radiation-induced hematopoietic injury immediately after radiation and thereby facilitate timely medical intervention and improve overall survival of exposed individuals. Spotting radiation injury with serum microRNAs Three Mile Island, Chernobyl, and Fukushima were catastrophic nuclear power plant accidents in the United States, Ukraine, and Japan, respectively. The radiation from these accidents took terrible tolls on human lives, not only at the time of the accident, but also long-term, as individuals suffer from unpredictable cancer, gut damage, and infections. Predicting such toxicity is imperfect, and current methods do not account for latent damage to organs and systems. In a crucial step toward better indicators of radiation injury, Acharya and colleagues investigated microRNA profiles and hematopoietic damage in mice exposed to various doses of total body irradiation (TBI). Mice received between 0 and 8 Gy TBI. Serum miRNA profiles distinguished animals receiving different doses of radiation, whereas bone marrow mononuclear cell counts did not. Such miRNA signatures may be useful in distinguishing humans with mild radiation-related injury from those with more severe (often nonrecoverable) bone marrow damage—even if all were exposed to sublethal doses of TBI. Importantly, animals receiving radiation mitigation in the form of amifostine, a radioprotectant, or stem cell transplant, demonstrated serum miRNA profiles that changed to match 0-Gy controls, indicating that miRNAs reflect impact of radiation (hematopoietic function) rather than dose. Similar results were obtained in “humanized” mice, suggesting the translatability of this miRNA-based approach to predicting radiation toxicity in people. Future studies with human samples will allow for validation of such indicators, as well as further investigations into novel intervention measures, to improve care of patients and enhance survival after radiation exposure. Accidental radiation exposure is a threat to human health that necessitates effective clinical planning and diagnosis. Minimally invasive biomarkers that can predict long-term radiation injury are urgently needed for optimal management after a radiation accident. We have identified serum microRNA (miRNA) signatures that indicate long-term impact of total body irradiation (TBI) in mice when measured within 24 hours of exposure. Impact of TBI on the hematopoietic system was systematically assessed to determine a correlation of residual hematopoietic stem cells (HSCs) with increasing doses of radiation. Serum miRNA signatures distinguished untreated mice from animals exposed to radiation and correlated with the impact of radiation on HSCs. Mice exposed to sublethal (6.5 Gy) and lethal (8 Gy) doses of radiation were indistinguishable for 3 to 4 weeks after exposure. A serum miRNA signature detectable 24 hours after radiation exposure consistently segregated these two cohorts. Furthermore, using either a radioprotective agent before, or radiation mitigation after, lethal radiation, we determined that the serum miRNA signature correlated with the impact of radiation on animal health rather than the radiation dose. Last, using humanized mice that had been engrafted with human CD34+ HSCs, we determined that the serum miRNA signature indicated radiation-induced injury to the human bone marrow cells. Our data suggest that serum miRNAs can serve as functional dosimeters of radiation, representing a potential breakthrough in early assessment of radiation-induced hematopoietic damage and timely use of medical countermeasures to mitigate the long-term impact of radiation.

Stressed Out - Therapeutic Implications of ER Stress Related Cancer Research

The unfolded protein response (UPR) is an established and well-studied cellular response to the stress and serves to relieve the stress and reinstate cellular homeostasis. It occurs in the endoplasmic reticulum (ER), responsible of properly folding and processing of secretory and transmembrane proteins. It is extremely sensitive to alteration in homeostasis caused by various internal or external stressors which leads to accumulation of misfolded or unfolded proteins in the ER lumen. The UPR works by restoring protein homeostasis in the ER, either through the boosting of protein-folding and degradation capability or by assuaging the demands for such effects, and can cause the activation of cell death if unable to do so. Cancer cells have adapted to gain advantage from the UPR and keeping the cell away from apoptosis and promoting survival, including survival of the cancer stem cells and evading the immune system. Several components of the UPR are overexpressed in a malignant cell and are responsible for resistance from various chemotherapy options and radiotherapy, which are also responsible for causing ER stress and activating the UPR. In this review, we discuss the various ways in which UPR can aid different cancers to survive and evade therapy and highlight recent research, which exploits the UPR to confer sensitivity to these cancer cells against various drugs and radiation.

Abstract 1776: Focused ultrasound can act as a chemosensitizer by modulating unfolded protein response with activation of apoptotic pathway

Unfolded protein response (UPR) is a stress response induced in tumor cells because of higher rate of protein synthesis and subsequent misfolding of newly synthesized proteins in the endoplasmic reticulum (ER). To restore normal function of the cell, ER induces the expression of molecular chaperones, such as heat-shock proteins (Hsps) that try to correct protein misfolding. If the correction machinery fails, programmed cell death (apoptosis) could be induced in these cells. Therapeutic focused ultrasound is a promising non-invasive approach to ablate solid tumors. We have demonstrated previously that low intensity focused ultrasound (LOFU) induces mechanical stress in tumor cells and induce UPR. We hypothesized that induction of unfolded/misfolded protein burden in ER by LOFU could sensitize the tumor tissue for chemotherapeutic agents, such as, 17AAG, a HSP90 inhibitor by increasing the ER stress and switching on the apoptotic pathway. We explored whether concomitant application of 17AAG along with LOFU could increase the therapeutic ratio in murine prostate cancer model. RM-1 (murine prostate cancer cells) tumors were subjected to intratumor injection of 17AAG (10mg/kg of body weight, 2/week) and then treated with LOFU (3W at a frequency of 1Mhz, 100% duty cycle) (1/week) for three weeks. Tumor growth was measured twice a week. Three weeks after initiation of LOFU-17AAG treatment cycle, animals were sacrificed and histopathological analysis (HE 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1776. doi:10.1158/1538-7445.AM2011-1776

Abstract 1393: Transplantation of Bone marrow-derived adherent stem cells mitigates radiation-induced gastrointestinal syndrome

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 diarrhea, microbial infection and septic shock. While growth factors, such as, R-spondin1 and KGF can protect mice from RIGS, mitigation is rare, following exposure to lethal doses of irradiation (IR). We hypothesized that IR-induced depletion and injury of intestinal stem cells (ISC) and stromal cells of the ISC niche induces RIGS. Since stromal cells provide critical growth factor/signals for ISC regeneration, we examined whether transplantation of bone marrow (BM)-derived adherent stem cells (BMASC) containing mesenchymal stem cells (MSC), endothelial progenitor cells (EPC) and macrophages would mitigate RIGS. Harvested BM cells, from C57Bl/6 mice, were cultured in MSCBM (Cambrex-Lonza) for 4 days, followed by collection of adherent cells from culture plates as BMASC. C57Bl/6 mice received a single fraction of either whole body irradiation (WBI; 8-12 Gy) or total abdominal irradiation (AIR; 16-20 Gy), followed by transplantation of BMASC (1×10 6 cells/mice) 24 and 72 hours after exposure to IR via tail vein injection. Irradiated controls received MSC culture medium. To evaluate the mitigating effect of myeloid cells, BMASC were fractionated with anti-CD11b-magnetic beads (MACS), followed by transplantation of CD11b+ and CD11b- BMASC cells in irradiated mice as above, respectively. Animals were observed for survival (Kaplan-Meier) and histopathological evaluation (Hematoxylin-eosin, TUNEL and BrdU immunohistochemistry). Xylose absorption test was performed to assess the functional integrity of intestinal mucosal barrier. Flow cytometry demonstrated 48% + 2.1 MSC (CD105+CD45-), 8.1% + 0.53 EPC (CD133+ CD45-) and 17.3% + 1.8 myeloid/macrophages in BMASC donor cells. In contrast to irradiated controls, 100% of the mice that received BMASC transplantation survived a lethal dose of WBI (10.4 Gy) and AIR (18Gy) (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1393.