Establishing the Impact of Vascular Damage on Tumor Response to High-Dose Radiation Therapy.

Approximately half of all patients with cancer receive radiotherapy, which is conventionally delivered in relatively small doses (1.8-2 Gy) per daily fraction over one to two months. Stereotactic body radiation therapy (SBRT), in which a high daily radiation dose is delivered in 1 to 5 fractions, has improved local control rates for several cancers. However, despite the widespread adoption of SBRT in the clinic, controversy surrounds the mechanism by which SBRT enhances local control. Some studies suggest that high doses of radiation (≥10 Gy) trigger tumor endothelial cell death, resulting in indirect killing of tumor cells through nutrient depletion. On the other hand, mathematical models predict that the high radiation dose per fraction used in SBRT increases direct tumor cell killing, suggesting that disruption of the tumor vasculature is not a critical mediator of tumor cure. Here, we review the application of genetically engineered mouse models to radiosensitize tumor cells or endothelial cells to dissect the role of these cellular targets in mediating the response of primary tumors to high-dose radiotherapy in vivo These studies demonstrate a role for endothelial cell death in mediating tumor growth delay, but not local control following SBRT.

Deletion of Atm in Tumor but not Endothelial Cells Improves Radiation Response in a Primary Mouse Model of Lung Adenocarcinoma.

Stereotactic body radiotherapy is utilized to treat lung cancer. The mechanism of tumor response to high-dose radiotherapy (HDRT) is controversial, with competing hypotheses of increased direct tumor cell killing versus indirect effects on stroma including endothelial cells. Here we used dual recombinase technology in a primary murine lung cancer model to test whether tumor cells or endothelial cells are critical HDRT targets. Lenti-Cre deleted one or two copies of ataxia-telangiectasia mutated gene (Atm; KPAFL/+ or KPAFL/FL), whereas adeno-FlpO-infected mice expressed Cre in endothelial cells to delete one or both copies of Atm (KPVAFL/+ or KPVAFL/FL) to modify tumor cell or endothelial cell radiosensitivity, respectively. Deletion of Atm in either tumor cells or endothelial cells had no impact on tumor growth in the absence of radiation. Despite increased endothelial cell death in KPVAFL/FL mice following irradiation, tumor growth delay was not significantly increased. In contrast, a prolonged tumor growth delay was apparent in KPAFL/FL mice. Primary tumor cell lines lacking Atm expression also demonstrated enhanced radiosensitivity as determined via a clonogenic survival assay. These findings indicate that tumor cells, rather than endothelial cells, are critical targets of HDRT in primary murine lung cancer. SIGNIFICANCE: These findings establish radiosensitizing tumor cells rather than endothelial cells as the primary mechanism of tumor response to high-dose radiotherapy, supporting efforts to maximize local control by radiosensitizing tumors cells.See related commentary by Hallahan, p. 704.

Mice Lacking RIP3 Kinase are not Protected from Acute Radiation Syndrome.

Exposure to high doses of ionizing radiation can cause lethal injury to normal tissue, thus inducing acute radiation syndrome. Acute radiation syndrome is caused by depletion of bone marrow cells (hematopoietic syndrome) and irreparable damage to the epithelial cells in the gastrointestinal tract (gastrointestinal syndrome). Although radiation initiates apoptosis in the hematopoietic and gastrointestinal compartments within the first few hours after exposure, alternative mechanisms of cell death may contribute to injury in these radiosensitive tissues. In this study, we utilized mice lacking a critical regulator of necroptosis, receptor interacting protein 3 (RIP3) kinase, to characterize the role of RIP3 in normal tissue toxicity after irradiation. Our results suggest that RIP3-mediated signaling is not a critical driver of acute radiation syndrome.

Dual-Energy CT Imaging of Tumor Liposome Delivery After Gold Nanoparticle-Augmented Radiation Therapy.

Gold nanoparticles (AuNPs) are emerging as promising agents for both cancer therapy and computed tomography (CT) imaging. AuNPs absorb x-rays and subsequently release low-energy, short-range photoelectrons during external beam radiation therapy (RT), increasing the local radiation dose. When AuNPs are near tumor vasculature, the additional radiation dose can lead to increased vascular permeability. This work focuses on understanding how tumor vascular permeability is influenced by AuNP-augmented RT, and how this effect can be used to improve the delivery of nanoparticle chemotherapeutics. Methods: Dual-energy CT was used to quantify the accumulation of both liposomal iodine and AuNPs in tumors following AuNP-augmented RT in a mouse model of primary soft tissue sarcoma. Mice were injected with non-targeted AuNPs, RGD-functionalized AuNPs (vascular targeting), or no AuNPs, after which they were treated with varying doses of RT. The mice were injected with either liposomal iodine (for the imaging study) or liposomal doxorubicin (for the treatment study) 24 hours after RT. Increased tumor liposome accumulation was assessed by dual-energy CT (iodine) or by tracking tumor treatment response (doxorubicin). Results: A significant increase in vascular permeability was observed for all groups after 20 Gy RT, for the targeted and non-targeted AuNP groups after 10 Gy RT, and for the vascular-targeted AuNP group after 5 Gy RT. Combining targeted AuNPs with 5 Gy RT and liposomal doxorubicin led to a significant tumor growth delay (tumor doubling time ~ 8 days) compared to AuNP-augmented RT or chemotherapy alone (tumor doubling time ~3-4 days). Conclusions: The addition of vascular-targeted AuNPs significantly improved the treatment effect of liposomal doxorubicin after RT, consistent with the increased liposome accumulation observed in tumors in the imaging study. Using this approach with a liposomal drug delivery system can increase specific tumor delivery of chemotherapeutics, which has the potential to significantly improve tumor response and reduce the side effects of both RT and chemotherapy.

Characterizing the Potency and Impact of Carbon Ion Therapy in a Primary Mouse Model of Soft Tissue Sarcoma.

Carbon ion therapy (CIT) offers several potential advantages for treating cancers compared with X-ray and proton radiotherapy, including increased biological efficacy and more conformal dosimetry. However, CIT potency has not been characterized in primary tumor animal models. Here, we calculate the relative biological effectiveness (RBE) of carbon ions compared with X-rays in an autochthonous mouse model of soft tissue sarcoma. We used Cre/loxP technology to generate primary sarcomas in KrasLSL-G12D/+; p53fl/fl mice. Primary tumors were irradiated with a single fraction of carbon ions (10 Gy), X-rays (20 Gy, 25 Gy, or 30 Gy), or observed as controls. The RBE was calculated by determining the dose of X-rays that resulted in similar time to posttreatment tumor volume quintupling and exponential growth rate as 10 Gy carbon ions. The median tumor volume quintupling time and exponential growth rate of sarcomas treated with 10 Gy carbon ions and 30 Gy X-rays were similar: 27.3 and 28.1 days and 0.060 and 0.059 mm3/day, respectively. Tumors treated with lower doses of X-rays had faster regrowth. Thus, the RBE of carbon ions in this primary tumor model is 3. When isoeffective treatments of carbon ions and X-rays were compared, we observed significant differences in tumor growth kinetics, proliferative indices, and immune infiltrates. We found that carbon ions were three times as potent as X-rays in this aggressive tumor model and identified unanticipated differences in radiation response that may have clinical implications. Mol Cancer Ther; 17(4); 858-68. ©2018 AACR.

Genetically engineered mouse models for studying radiation biology.

Genetically engineered mouse models (GEMMs) are valuable research tools that have transformed our understanding of cancer. The first GEMMs generated in the 1980s and 1990s were knock-in and knock-out models of single oncogenes or tumor suppressors. The advances that made these models possible catalyzed both technological and conceptual shifts in the way cancer research was conducted. As a result, dozens of mouse models of cancer exist today, covering nearly every tissue type. The advantages inherent to GEMMs compared to in vitro and in vivo transplant models are compounded in preclinical radiobiology research for several reasons. First, they accurately and robustly recapitulate primary cancers anatomically, histopathologically, and genetically. Reliable models are a prerequisite for predictive preclinical studies. Second, they preserve the tumor microenvironment, including the immune, vascular, and stromal compartments, which enables the study of radiobiology at a systems biology level. Third, they provide exquisite control over the genetics and kinetics of tumor initiation, which enables the study of specific gene mutations on radiation response and functional genomics in vivo. Taken together, these facets allow researchers to utilize GEMMs for rigorous and reproducible preclinical research. In the three decades since the generation of the first GEMMs of cancer, advancements in modeling approaches have rapidly progressed and expanded the mouse modeling toolbox with techniques such as in vivo short hairpin RNA (shRNA) knockdown, inducible gene expression, site-specific recombinases, and dual recombinase systems. Our lab and many others have utilized these tools to study cancer and radiobiology. Recent advances in genome engineering with CRISPR/Cas9 technology have made GEMMs even more accessible to researchers. Here, we review current and future approaches to mouse modeling with a focus on applications in preclinical radiobiology research.

An extra copy of p53 suppresses development of spontaneous Kras-driven but not radiation-induced cancer.

The tumor suppressor p53 blocks tumor progression in multiple tumor types. Radiation-induced cancer following exposure to radiation therapy or space travel may also be regulated by p53 because p53 has been proposed to respond to DNA damage to suppress tumorigenesis. Here, we investigate the role of p53 in lung carcinogenesis and lymphomagenesis in LA-1 KrasG12D mice with wild-type p53 or an extra copy of p53 (super p53) exposed to fractionated total body irradiation with low linear energy transfer (low-LET) X-rays or high-LET iron ions and compared tumor formation in these mice with unirradiated controls. We found that an additional copy of p53 suppressed both Kras-driven lung tumor and lymphoma development in the absence of radiation. However, an additional copy of p53 did not affect lymphoma development following low- or high-LET radiation exposure and was unable to suppress radiation-induced expansion of thymocytes with mutated Kras. Moreover, radiation exposure increased lung tumor size in super p53 but not wild-type p53 mice. These results demonstrate that although p53 suppresses the development of spontaneous tumors expressing KrasG12D, in the context of exposure to ionizing radiation, an extra copy of p53 does not protect against radiation-induced lymphoma and may promote KrasG12D mutant lung cancer.

Acute DNA damage activates the tumour suppressor p53 to promote radiation-induced lymphoma.

Genotoxic cancer therapies, such as chemoradiation, cause haematological toxicity primarily by activating the tumour suppressor p53. While inhibiting p53-mediated cell death during cancer therapy ameliorates haematologic toxicity, whether it also impacts carcinogenesis remains unclear. Here we utilize a mouse model of inducible p53 short hairpin RNA (shRNA) to show that temporarily blocking p53 during total-body irradiation (TBI) not only ameliorates acute toxicity, but also improves long-term survival by preventing lymphoma development. Using Kras(LA1) mice, we show that TBI promotes the expansion of a rare population of thymocytes that express oncogenic Kras(G12D). However, blocking p53 during TBI significantly suppresses the expansion of Kras(G12D)-expressing thymocytes. Mechanistically, bone marrow transplant experiments demonstrate that TBI activates p53 to decrease the ability of bone marrow cells to suppress lymphoma development through a non-cell-autonomous mechanism. Together, our results demonstrate that the p53 response to acute DNA damage promotes the development of radiation-induced lymphoma.

Tumor cells, but not endothelial cells, mediate eradication of primary sarcomas by stereotactic body radiation therapy.

Cancer clinics currently use high-dose stereotactic body radiation therapy as a curative treatment for several kinds of cancers. However, the contribution of vascular endothelial cells to tumor response to radiation remains controversial. Using dual recombinase technology, we generated primary sarcomas in mice with targeted genetic mutations specifically in tumor cells or endothelial cells. We selectively mutated the proapoptotic gene Bax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. Bax deletion from endothelial cells did not affect radiation-induced cell death in tumor endothelial cells or sarcoma response to radiation therapy. Although Atm deletion increased endothelial cell death after radiation therapy, deletion of Atm from endothelial cells failed to enhance sarcoma eradication. In contrast, deletion of Atm from tumor cells increased sarcoma eradication by radiation therapy. These results demonstrate that tumor cells, rather than endothelial cells, are critical targets that regulate sarcoma eradication by radiation therapy. Treatment with BEZ235, a small-molecule protein kinase inhibitor, radiosensitized primary sarcomas more than the heart. These results suggest that inhibiting ATM kinase during radiation therapy is a viable strategy for radiosensitization of some tumors.

Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium.

Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

Inhibiting glycogen synthase kinase-3 mitigates the hematopoietic acute radiation syndrome in mice.

Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.