Enhancing radiotherapy response via intratumoral injection of a TLR9 agonist in autochthonous murine sarcomas.

Radiation therapy (RT) is frequently used to treat cancers, including soft-tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to RT in transplanted tumors, but the mechanisms of this enhancement remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft-tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and 2 doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to CpG+RT, we performed bulk RNA-Seq, single-cell RNA-Seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and IFN-γ. CpG+RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG+RT, TCR clonality analysis suggests an increase in clonal T cell dominance. Collectively, these findings demonstrate that CpG+RT significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft-tissue sarcoma.

The COMPASS complex maintains the metastatic capacity imparted by a subpopulation of cells in UPS.

Intratumoral heterogeneity is common in cancer, particularly in sarcomas like undifferentiated pleomorphic sarcoma (UPS), where individual cells demonstrate a high degree of cytogenic diversity. Previous studies showed that a small subset of cells within UPS, known as the metastatic clone (MC), as responsible for metastasis. Using a CRISPR-based genomic screen in-vivo, we identified the COMPASS complex member Setd1a as a key regulator maintaining the metastatic phenotype of the MC in murine UPS. Depletion of Setd1a inhibited metastasis development in the MC. Transcriptome and chromatin sequencing revealed COMPASS complex target genes in UPS, such as Cxcl10, downregulated in the MC. Deleting Cxcl10 in non-MC cells increased their metastatic potential. Treating mice with human UPS xenografts with a COMPASS complex inhibitor suppressed metastasis without affecting tumor growth in the primary tumor. Our data identified an epigenetic program in a subpopulation of sarcoma cells that maintains metastatic potential.

NRG Oncology White Paper on the Relative Biological Effectiveness in Proton Therapy.

This position paper, led by the NRG Oncology Particle Therapy Work Group, focuses on the concept of relative biologic effect (RBE) in clinical proton therapy (PT), with the goal of providing recommendations for the next-generation clinical trials with PT on the best practice of investigating and using RBE, which could deviate from the current standard proton RBE value of 1.1 relative to photons. In part 1, current clinical utilization and practice are reviewed, giving the context and history of RBE. Evidence for variation in RBE is presented along with the concept of linear energy transfer (LET). The intertwined nature of tumor radiobiology, normal tissue constraints, and treatment planning with LET and RBE considerations is then reviewed. Part 2 summarizes current and past clinical data and then suggests the next steps to explore and employ tools for improved dynamic models for RBE. In part 3, approaches and methods for the next generation of prospective clinical trials are explored, with the goal of optimizing RBE to be both more reflective of clinical reality and also deployable in trials to allow clinical validation and interpatient comparisons. These concepts provide the foundation for personalized biologic treatments reviewed in part 4. Finally, we conclude with a summary including short- and long-term scientific focus points for clinical PT. The practicalities and capacity to use RBE in treatment planning are reviewed and considered with more biological data in hand. The intermediate step of LET optimization is summarized and proposed as a potential bridge to the ultimate goal of case-specific RBE planning that can be achieved as a hypothesis-generating tool in near-term proton trials.

MR histology reveals tissue features beneath heterogeneous MRI signal in genetically engineered mouse models of sarcoma.

Purpose: To identify significant relationships between quantitative cytometric tissue features and quantitative MR (qMRI) intratumorally in preclinical undifferentiated pleomorphic sarcomas (UPS). Materials and methods: In a prospective study of genetically engineered mouse models of UPS, we registered imaging libraries consisting of matched multi-contrast in vivo MRI, three-dimensional (3D) multi-contrast high-resolution ex vivo MR histology (MRH), and two-dimensional (2D) tissue slides. From digitized histology we generated quantitative cytometric feature maps from whole-slide automated nuclear segmentation. We automatically segmented intratumoral regions of distinct qMRI values and measured corresponding cytometric features. Linear regression analysis was performed to compare intratumoral qMRI and tissue cytometric features, and results were corrected for multiple comparisons. Linear correlations between qMRI and cytometric features with p values of <0.05 after correction for multiple comparisons were considered significant. Results: Three features correlated with ex vivo apparent diffusion coefficient (ADC), and no features correlated with in vivo ADC. Six features demonstrated significant linear relationships with ex vivo T2*, and fifteen features correlated significantly with in vivo T2*. In both cases, nuclear Haralick texture features were the most prevalent type of feature correlated with T2*. A small group of nuclear topology features also correlated with one or both T2* contrasts, and positive trends were seen between T2* and nuclear size metrics. Conclusion: Registered multi-parametric imaging datasets can identify quantitative tissue features which contribute to UPS MR signal. T2* may provide quantitative information about nuclear morphology and pleomorphism, adding histological insights to radiological interpretation of UPS.

Targeting glutamine metabolism improves sarcoma response to radiation therapy in vivo.

Diverse tumor metabolic phenotypes are influenced by the environment and genetic lesions. Whether these phenotypes extend to rhabdomyosarcoma (RMS) and how they might be leveraged to design new therapeutic approaches remains an open question. Thus, we utilized a Pax7Cre-ER-T2/+; NrasLSL-G12D/+; p53fl/fl (P7NP) murine model of sarcoma with mutations that most frequently occur in human embryonal RMS. To study metabolism, we infuse 13C-labeled glucose or glutamine into mice with sarcomas and show that sarcomas consume more glucose and glutamine than healthy muscle tissue. However, we reveal a marked shift from glucose consumption to glutamine metabolism after radiation therapy (RT). In addition, we show that inhibiting glutamine, either through genetic deletion of glutaminase (Gls1) or through pharmacological inhibition of glutaminase, leads to significant radiosensitization in vivo. This causes a significant increase in overall survival for mice with Gls1-deficient compared to Gls1-proficient sarcomas. Finally, Gls1-deficient sarcomas post-RT elevate levels of proteins involved in natural killer cell and interferon alpha/gamma responses, suggesting a possible role of innate immunity in the radiosensitization of Gls1-deficient sarcomas. Thus, our results indicate that glutamine contributes to radiation response in a mouse model of RMS.

A Novel Dual ATM/DNA-PK Inhibitor, XRD-0394, Potently Radiosensitizes and Potentiates PARP and Topoisomerase I Inhibitors.

A majority of patients with cancer receive radiotherapy as part of their treatment regimens whether using external beam therapy or locally-delivered radioisotopes. While often effective, some tumors are inadequately controlled with radiation and radiotherapy has significant short-term and long-term toxicities for cancer survivors. Insights into molecular mechanisms involved in cellular responses to DNA breaks introduced by radiation or other cancer therapies have been gained in recent years and approaches to manipulate these responses to enhance tumor cell killing or reduce normal tissue toxicity are of great interest. Here, we report the identification and initial characterization of XRD-0394, a potent and specific dual inhibitor of two DNA damage response kinases, ATM and DNA-PKcs. This orally bioavailable molecule demonstrates significantly enhanced tumor cell kill in the setting of therapeutic ionizing irradiation in vitro and in vivo. XRD-0394 also potentiates the effectiveness of topoisomerase I inhibitors in vitro. In addition, in cells lacking BRCA1/2 XRD-0394 shows single-agent activity and synergy in combination with PARP inhibitors. A phase Ia clinical trial (NCT05002140) with XRD-0394 in combination with radiotherapy has completed. These results provide a rationale for future clinical trials with XRD-0394 in combination with radiotherapy, PARP inhibitors, and targeted delivery of topoisomerase I inhibitors.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

Radiotherapy Combined with Intralesional Immunostimulatory Agents for Soft Tissue Sarcomas.

Immunotherapy has shifted the treatment paradigm for many types of cancer. Unfortunately, the most commonly used immunotherapies, such as immune checkpoint inhibitors (ICI), have yielded limited benefit for most types of soft tissue sarcoma (STS). Radiotherapy (RT) is a mainstay of sarcoma therapy and can induce immune modulatory effects. Combining immunotherapy and RT in STS may be a promising strategy to improve sarcoma response to RT and increase the efficacy of immunotherapy. Most combination strategies have employed immunotherapies, such as ICI, that derepress immune suppressive networks. These have yielded only modest results, possibly due to the limited immune stimulatory effects of RT. Combining RT with immune stimulatory agents has yielded promising preclinical and clinical results but can be limited by the toxic nature of systemic administration of immune stimulants. Using intralesional immune stimulants may generate stronger RT immune modulation and less systemic toxicity, which may be a feasible strategy in accessible tumors such as STS. In this review, we summarize the immune modulatory effects of RT, the mechanism of action of various immune stimulants, including toll-like receptor agonists, and data for combinatorial strategies utilizing these agents.

Spontaneous expression of the CIC::DUX4 fusion oncoprotein from a conditional allele potently drives sarcoma formation in genetically engineered mice.

CIC::DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous soft tissue tumors and disseminated disease in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic Cic function and the distance between adjacent loxP sites. Characterization of soft tissue and presumed metastatic tumors showed that they consistently expressed the CIC::DUX4 fusion protein and many downstream markers of the disease credentialing the models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-sequencing maps, validate CIC::DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

The Next Chapter in Immunotherapy and Radiation Combination Therapy: Cancer-Specific Perspectives.

Immunotherapeutic agents have revolutionized cancer treatment over the past decade. However, most patients fail to respond to immunotherapy alone. A growing body of preclinical studies highlights the potential for synergy between radiation therapy and immunotherapy, but the outcomes of clinical studies have been mixed. This review summarizes the current state of immunotherapy and radiation combination therapy across cancers, highlighting existing challenges and promising areas for future investigation.

Enhancing radiotherapy response via intratumoral injection of the TLR9 agonist CpG to stimulate CD8 T cells in an autochthonous mouse model of sarcoma.

Radiation therapy is frequently used to treat cancers including soft tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to radiation therapy (RT) in transplanted tumors, but the mechanism(s) remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and two doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to RT + CpG, we performed bulk RNA-seq, single-cell RNA-seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and interferon-γ. CpG + RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG + RT, TCR clonality analysis suggests an increase in clonal T-cell dominance. Collectively, these findings demonstrate that RT + CpG significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft tissue sarcoma.

Preclinical Evaluation of the ATR Inhibitor BAY 1895344 as a Radiosensitizer for Head and Neck Squamous Cell Carcinoma.

PURPOSE: Despite aggressive multimodal treatment that typically includes definitive or adjuvant radiation therapy (RT), locoregional recurrence rates approach 50% for patients with locally advanced human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). Thus, more effective therapeutics are needed to improve patient outcomes. We evaluated the radiosensitizing effects of ataxia telangiectasia and RAD3-related (ATR) inhibitor (ATRi) BAY 1895344 in preclinical models of HNSCC. METHODS AND MATERIALS: Murine and human HPV-negative HNSCC cells (MOC2, MOC1, JHU-012) were treated with vehicle or ATRi with or without 4 Gy. Checkpoint kinase 1 phosphorylation and DNA damage (γH2AX) were evaluated by Western blot, and ATRi half-maximal inhibitory concentration was determined by MTT assay for HNSCC cells and immortalized murine oral keratinocytes. In vitro radiosensitization was tested by clonogenic assay. Cell cycle distribution and mitotic catastrophe were evaluated by flow cytometry. Mitotic aberrations were quantified by fluorescent microscopy. Tumor growth delay and survival were assessed in mice bearing MOC2 or JHU-012 transplant tumors treated with vehicle, ATRi, RT (10 Gy × 1 or 8 Gy × 3), or combined ATRi + RT. RESULTS: ATRi caused dose-dependent reduction in checkpoint kinase 1 phosphorylation at 1 hour post-RT (4 Gy) and dose-dependent increase in γH2AX at 18 hours post-RT. Addition of RT to ATRi led to decreased BAY 1895344 half-maximal inhibitory concentration in HNSCC cell lines but not in normal tissue surrogate immortalized murine oral keratinocytes. Clonogenic assays demonstrated radiosensitization in the HNSCC cell lines. ATRi abrogated the RT-induced G2/M checkpoint, leading to mitosis with unrepaired DNA damage and increased mitotic aberrations (multinucleated cells, micronuclei, nuclear buds, nucleoplasmic bridges). ATRi and RT significantly delayed tumor growth in MOC2 and JHU-012 in vivo models, with improved overall survival in the MOC2 model. CONCLUSIONS: These findings demonstrated that BAY 1895344 increased in vitro and in vivo radiosensitivity in HPV-negative HNSCC preclinical models, suggesting therapeutic potential warranting evaluation in clinical trials for patients with locally advanced or recurrent HPV-negative HNSCC.

Expression of the CIC-DUX4 fusion oncoprotein mimics human CIC-rearranged sarcoma in genetically engineered mouse models.

CIC-DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous tumors and widespread metastasis in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic CIC function and the distance between loxP sites. Characterization of primary and metastatic mouse tumors showed that they consistently expressed the CIC-DUX4 fusion protein as well as other downstream markers of the disease credentialing these models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-seq maps, validate CIC-DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

Loss of function of Atrx leads to activation of alternative lengthening of telomeres in a primary mouse model of sarcoma.

The development of a telomere maintenance mechanism is essential for immortalization in human cancer. While most cancers elongate their telomeres by expression of telomerase, 10-15% of human cancers use a pathway known as alternative lengthening of telomeres (ALT). In this work, we developed a genetically engineered primary mouse model of sarcoma in CAST/EiJ mice which displays multiple molecular features of ALT activation after CRISPR/Cas9 introduction of oncogenic KrasG12D and loss of function mutations of Trp53 and Atrx. In this model, we demonstrate that the loss of Atrx contributes to the development of ALT in an autochthonous tumor, and this process occurs independently of telomerase function by variation of mTR alleles. Furthermore, we find that telomere shortening from the loss of telomerase leads to higher chromosomal instability while loss of Atrx and activation of ALT lead to an increase in telomeric instability, telomere sister chromatid exchange, c-circle production, and formation of ALT-associated promyelocytic leukemia bodies (APBs). The development of this primary mouse model of ALT could enable future investigations into therapeutic vulnerabilities of ALT activation and its mechanism of action.

Temporary Knockdown of p53 During Focal Limb Irradiation Increases the Development of Sarcomas.

Approximately half of patients with cancer receive radiotherapy and, as cancer survivorship increases, the low rate of radiation-associated sarcomas is rising. Pharmacologic inhibition of p53 has been proposed as an approach to ameliorate acute injury of normal tissues from genotoxic therapies, but how this might impact the risk of therapy-induced cancer and normal tissue injuries remains unclear. We utilized mice that express a doxycycline (dox)-inducible p53 short hairpin RNA to reduce Trp53 expression temporarily during irradiation. Mice were placed on a dox diet 10 days prior to receiving 30 or 40 Gy hind limb irradiation in a single fraction and then returned to normal chow. Mice were examined weekly for sarcoma development and scored for radiation-induced normal tissue injuries. Radiation-induced sarcomas were subjected to RNA sequencing. Following single high-dose irradiation, 21% of animals with temporary p53 knockdown during irradiation developed a sarcoma in the radiation field compared with 2% of control animals. Following high-dose irradiation, p53 knockdown preserves muscle stem cells, and increases sarcoma development. Mice with severe acute radiation-induced injuries exhibit an increased risk of developing late persistent wounds, which were associated with sarcomagenesis. RNA sequencing revealed radiation-induced sarcomas upregulate genes related to translation, epithelial-mesenchymal transition (EMT), inflammation, and the cell cycle. Comparison of the transcriptomes of human and mouse sarcomas that arose in irradiated tissues revealed regulation of common gene programs, including elevated EMT pathway gene expression. These results suggest that blocking p53 during radiotherapy could minimize acute toxicity while exacerbating late effects including second cancers. SIGNIFICANCE: Strategies to prevent or mitigate acute radiation toxicities include pharmacologic inhibition of p53 and other cell death pathways. Our data show that temporarily reducing p53 during irradiation increases late effects including sarcomagenesis.

Radiation-induced bone loss in mice is ameliorated by inhibition of HIF-2α in skeletal progenitor cells.

Radiotherapy remains a common treatment modality for cancer despite skeletal complications. However, there are currently no effective treatments for radiation-induced bone loss, and the consequences of radiotherapy on skeletal progenitor cell (SPC) survival and function remain unclear. After radiation, leptin receptor-expressing cells, which include a population of SPCs, become localized to hypoxic regions of the bone and stabilize the transcription factor hypoxia-inducible factor-2α (HIF-2α), thus suggesting a role for HIF-2α in the skeletal response to radiation. Here, we conditionally knocked out HIF-2α in leptin receptor-expressing cells and their descendants in mice. Radiation therapy in littermate control mice reduced bone mass; however, HIF-2α conditional knockout mice maintained bone mass comparable to nonirradiated control animals. HIF-2α negatively regulated the number of SPCs, bone formation, and bone mineralization. To test whether blocking HIF-2α pharmacologically could reduce bone loss during radiation, we administered a selective HIF-2α inhibitor called PT2399 (a structural analog of which was recently FDA-approved) to wild-type mice before radiation exposure. Pharmacological inhibition of HIF-2α was sufficient to prevent radiation-induced bone loss in a single-limb irradiation mouse model. Given that ~90% of patients who receive a HIF-2α inhibitor develop anemia because of off-target effects, we developed a bone-targeting nanocarrier formulation to deliver the HIF-2α inhibitor to mouse bone, to increase on-target efficacy and reduce off-target toxicities. Nanocarrier-loaded PT2399 prevented radiation-induced bone loss in mice while reducing drug accumulation in the kidney. Targeted inhibition of HIF-2α may represent a therapeutic approach for protecting bone during radiation therapy.

Single-fraction Radiation Treatment Dose Response in a Genetically Engineered Mouse Model of Medulloblastoma.

Medulloblastoma is the most common malignant brain tumor of children. Although standard of care radiotherapy for pediatric medulloblastoma (PM) can lead to long-term remission or cure in many patients, it can also cause life-long cognitive impairment and other adverse effects. The pathophysiological mechanisms involved in radiation-induced cerebral damage are incompletely understood, and their elucidation may lead to interventions that mitigate radiation toxicity. To explore the mechanisms of radiation-induced cerebral damage, transgenic mouse models of PM and non-tumor-bearing controls were exposed to radiation doses that ranged from 0 to 30 Gy. Between 0-20 Gy, a significant dose-dependent reduction in tumor-associated hydrocephalus and increase in overall survival were observed. However, at 30 Gy, hydrocephalus incidence increased and median overall survival fell to near-untreated levels. Immunohistochemistry revealed that both tumor-bearing and non-tumor-bearing mice treated with 30 Gy of radiation had significantly more reactive astrocytes and microvascular damage compared to untreated controls. This effect was persistent across mice that were given 1 and 2 weeks of recovery time after irradiation. Our data suggest that radiation therapy promotes neural death by inducing long-term neuroinflammation in PM, suggesting radiation delivery methods that limit inflammation may be effective at widening the therapeutic window of radiation therapy in PM patients.

Expression of the CIC-DUX4 fusion oncoprotein mimics human CIC-rearranged sarcoma in genetically engineered mouse models.

CIC-DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of tissues and patients. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous tumors and widespread metastasis in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic CIC function and loxP site proximity. Characterization of primary and metastatic mouse tumors showed that they consistently expressed the CIC-DUX4 fusion protein as well as other downstream markers of the disease credentialing these models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-seq maps, validate CIC-DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

Histological assessment of intestinal injury by ionizing radiation.

Given the potential risk of radiological terrorism and disasters, it is essential to develop plans to prepare for such events. In these hazardous scenarios, radiation-induced gastrointestinal (GI) syndrome is one of the many manifestations that may happen after the organism is exposed to a lethal dose of ionizing radiation. Therefore, it is critical to better understand how the intestinal tissues initiate and orchestrate regeneration following severe radiation injury. In this chapter, we aimed to provide several key considerations for researchers who utilize histological assessment to study radiation-induced intestinal injury. Rigor and reproducibility are critical in experimental design and can be achieved by maintaining proper radiation administration, maintaining consistency in sample collection, and selecting and using appropriate controls. We also provided technical details of histological preparation of the intestines with tips on dissecting, cleaning, fixing, and preserving. Step-by-step descriptions of both bundling and Swiss rolling are provided with discussion on how to choose between the two approaches. In the following section, we detailed several histological assessment methods and then provided suggestions on how to use histological assessment to study cellular dynamics in the small intestines. Finally, we touched on some non-histological assessments. We hope that the information provided in this chapter will contribute to the research society of radiation-induced intestinal injury with an ultimate goal of promoting the development of radiation countermeasures against the GI acute radiation syndrome.