Characterization of Natural Killer cells infiltrating irradiated murine tumors through flow cytometry.

The radiation-induced immune response is increasingly well documented. However, some aspects remain unclear, notably the role of Natural Killer (NK) cells, a subgroup of innate lymphoid cells involved in the antitumor response, in the response to RT. It therefore seems necessary to better characterize NK cells infiltrating irradiated tumors in order to better understand the mechanisms of action of RT, enabling its subsequent optimization and combination with other immunomodulatory treatments. A key technology for studying intratumoral immune cells is flow cytometry, which can simultaneously quantify and analyze the phenotype of numerous cells. Here, we propose a method for phenotyping intratumoral NK cells through flow cytometry in mice bearing colorectal tumors treated with radiotherapy. This procedure can also be used to study the radiation-induced NK cell response in a wide range of solid tumors.

Benefits of High-Dose Vitamin D in Managing Cutaneous Adverse Events Induced by Chemotherapy and Radiation Therapy.

The use of chemotherapy and radiation for cancer treatment can result in cutaneous adverse events (AEs) such as toxic erythema of chemotherapy (TEC) and radiation-induced dermatitis. High-dose vitamin D supplementation has been suggested to potentially improve and shorten recovery for these AEs, primarily based on data from case reports and case series. In this article, we discuss the role of vitamin D in the most prevalent cancers (breast and colorectal cancer) and changes in vitamin D levels after chemotherapy or radiation treatments. We also summarize reports on high-dose vitamin D supplementation for treating chemotherapy-induced and radiation-induced skin toxicity. Larger studies and randomized controlled trials are essential to clarify the roles of vitamin D in malignancy and in cutaneous AEs associated with cancer treatment. The existing studies we reviewed lack standardized dosing regimens and exhibited heterogeneity across study populations, making it challenging to draw generalizable conclusions.

Non-homogenous intratumor ionizing radiation doses synergize with PD1 and CXCR2 blockade.

The efficacy and side effects of radiotherapy (RT) depend on parameters like dose and the volume of irradiated tissue. RT induces modulations of the tumor immune microenvironment (TIME) that are dependent on the dose. Low dose RT (LDRT, i.e., single doses of 0.5-2 Gy) has been shown to promote immune infiltration into the tumor. Here we hypothesize that partial tumor irradiation combining the immunostimulatory/non-lethal properties of LDRT with cell killing/shrinkage properties of high dose RT (HDRT) within the same tumor mass could enhance anti-tumor responses when combined with immunomodulators. In models of colorectal and breast cancer in immunocompetent female mice, partial irradiation (PI) with millimetric precision to deliver LDRT (2 Gy) and HDRT (16 Gy) within the same tumor induces substantial tumor control when combined with anti-PD1. Using flow cytometry, cytokine profiling and single-cell RNA sequencing, we identify a crosstalk between the TIME of the differentially irradiated tumor volumes. PI reshapes tumor-infiltrating CD8+ T cells into more cytotoxic and interferon-activated phenotypes but also increases the infiltration of pro-tumor neutrophils driven by CXCR2. The combination of the CXCR2 antagonist SB225002 with PD1 blockade and PI improves tumor control and mouse survival. Our results suggest a strategy to reduce RT toxicity and improve the therapeutic index of RT and immune checkpoint combinations.

miR-1226-5p is involved in radioresistance of colorectal cancer by activating M2 macrophages through suppressing IRF1.

BACKGROUND: Although the representative treatment for colorectal cancer (CRC) is radiotherapy, cancer cells survive due to inherent radioresistance or resistance acquired after radiation treatment, accelerating tumor malignancy and causing local recurrence and metastasis. However, the detailed mechanisms of malignancy induced after radiotherapy are not well understood. To develop more effective and improved radiotherapy and diagnostic methods, it is necessary to clearly identify the mechanisms of radioresistance and discover related biomarkers. METHODS: To analyze the expression pattern of miRNAs in radioresistant CRC, sequence analysis was performed in radioresistant HCT116 cells using Gene Expression Omnibus, and then miR-1226-5p, which had the highest expression in resistant cells compared to parental cells, was selected. To confirm the effect of miR-1226-5 on tumorigenicity, Western blot, qRT-PCR, transwell migration, and invasion assays were performed to confirm the expression of EMT factors, cell mobility and invasiveness. Additionally, the tumorigenic ability of miR-1226-5p was confirmed in organoids derived from colorectal cancer patients. In CRC cells, IRF1, a target gene of miR-1226-5p, and circSLC43A1, which acts as a sponge for miR-1226-5p, were discovered and the mechanism was analyzed by confirming the tumorigenic phenotype. To analyze the effect of tumor-derived miR-1226-5p on macrophages, the expression of M2 marker in co-cultured cells and CRC patient tissues were confirmed by qRT-PCR and immunohistochemical (IHC) staining analyses. RESULTS: This study found that overexpressed miR-1226-5p in radioresistant CRC dramatically promoted epithelial-mesenchymal transition (EMT), migration, invasion, and tumor growth by suppressing the expression of its target gene, IRF1. Additionally, we discovered circSLC43A1, a factor that acts as a sponge for miR-1226-5p and suppresses its expression, and verified that EMT, migration, invasion, and tumor growth are suppressed by circSLC43A1 in radioresistant CRC cells. Resistant CRC cells-derived miR-1226-5p was transferred to macrophages and contributed to tumorigenicity by inducing M2 polarization and secretion of TGF-ß. CONCLUSIONS: This study showed that the circSLC43A1/miR-1226-5p/IRF1 axis is involved in radioresistance and cancer aggressiveness in CRC. It was suggested that the discovered signaling factors could be used as potential biomarkers for diagnosis and treatment of radioresistant CRC.

Molecular Mechanism of Radioresponsiveness in Colorectal Cancer: A Systematic Review.

Background/Objectives: Colorectal cancer (CRC) is the third most diagnosed cancer globally. Radiotherapy is a common treatment strategy for patients but factors such as gene expressions and molecular mechanism effects may affect tumor radioresponse. The aim of this review is to systematically identify genes suggested to have molecular mechanism effects on the radioresponsiveness of CRC patients. Methods: By following the PRISMA guidelines, a comprehensive literature search was conducted on Pubmed, EMBASE and Cochrane Library. After exclusion and inclusion criteria sorting and critical appraisal for study quality, data were extracted from seven studies. A gene set analysis was conducted on reported genes. Results: From the seven studies, 56 genes were found to have an effect on CRC radioresponsiveness. Gene set analysis show that out of these 56 genes, 24 genes have roles in pathways which could affect cancer radioresponse. These are AKT1, APC, ATM, BRAF, CDKN2A, CTNNB1, EGFR, ERBB2, FLT3, KRAS, MET, mTOR, MYC, NFKB1, KRAS, PDGFRA, PIK3CA, PTEN, PTGS1, PTGS2, RAF1, RET, SMAD4 and TP53. The current project was conducted between the period May 2024 to August 2024. Conclusions: The current review systematically presented 56 genes which have been reported to be related to RT or CRT treatment effectiveness in rectal cancer patients. Gene set analysis shows that nearly half of the genes were involved in apoptosis, DNA damage response and repair, inflammation and cancer metabolism molecular pathways that could affect cancer radioresponse. The gene cohort identified in this study may be used as a foundation for future works focusing on the molecular mechanism of specific pathways contributing to the radioresponse of CRC.

Relative biological effectiveness of clinically relevant photon energies for the survival of human colorectal, cervical, and prostate cancer cell lines.

Objective.Relative biological effectiveness (RBE) differs between radiation qualities. However, an RBE of 1.0 has been established for photons regardless of the wide range of photon energies used clinically, the lack of reproducibility in radiobiological studies, and outdated reference energies used in the experimental literature. Moreover, due to intrinsic radiosensitivity, different cancer types have different responses to radiation. This study aimed to characterize the RBE of clinically relevant high and low photon energiesin vitrofor three human cancer cell lines: HCT116 (colon), HeLa (cervix), and PC3 (prostate).Approach.Experiments were conducted following dosimetry protocols provided by the American Association of Physicists in Medicine. Cells were irradiated with 6 MV x-rays, an192Ir brachytherapy source, 225 kVp and 50 kVp x-rays. Cell survival post-irradiation was assessed using the clonogenic assay. Survival fractions were fitted using the linear quadratic model, and survival curves were generated for RBE calculations.Main results.Cell killing was more efficient with decreasing photon energy. Using 225 kVp x-rays as the reference, the HCT116 RBESF0.1for 6 MV x-rays,192Ir, and 50 kVp x-rays were 0.89 ± 0.03, 0.95 ± 0.03, and 1.24 ± 0.04; the HeLa RBESF0.1were 0.95 ± 0.04, 0.97 ± 0.05, and 1.09 ± 0.03, and the PC3 RBESF0.1were 0.84 ± 0.01, 0.84 ± 0.01, and 1.13 ± 0.02, respectively. HeLa and PC3 cells had varying radiosensitivity when irradiated with 225 and 50 kVp x-rays.Significance.This difference supports the notion that RBE may not be 1.0 for all photons through experimental investigations that employed precise dosimetry. It highlights that different cancer types may not have identical responses to the same irradiation quality. Additionally, the RBE of clinically relevant photons was updated to the reference energy of 225 kVp x-rays.

Deciphering colorectal cancer radioresistance and immune microrenvironment: unraveling the role of EIF5A through single-cell RNA sequencing and machine learning.

Background: Radiotherapy (RT) is a critical component of treatment for locally advanced rectal cancer (LARC), though patient response varies significantly. The variability in treatment outcomes is partly due to the resistance conferred by cancer stem cells (CSCs) and tumor immune microenvironment (TiME). This study investigates the role of EIF5A in radiotherapy response and its impact on the CSCs and TiME. Methods: Predictive models for preoperative radiotherapy (preRT) response were developed using machine learning, identifying EIF5A as a key gene associated with radioresistance. EIF5A expression was analyzed via bulk RNA-seq and single-cell RNA-seq (scRNA-seq). Functional assays and in vivo experiments validated EIF5A's role in radioresistance and TiME modulation. Results: EIF5A was significantly upregulated in radioresistant colorectal cancer (CRC) tissues. EIF5A knockdown in CRC cell lines reduced cell viability, migration, and invasion after radiation, and increased radiation-induced apoptosis. Mechanistically, EIF5A promoted cancer stem cell (CSC) characteristics through the Hedgehog signaling pathway. Analysis of the TiME revealed that the radiation-resistant group had an immune-desert phenotype, characterized by low immune cell infiltration. In vivo experiments showed that EIF5A knockdown led to increased infiltration of CD8+ T cells and M1 macrophages, and decreased M2 macrophages and Tregs following radiation therapy, thereby enhancing the radiotherapy response. Conclusion: EIF5A contributes to CRC radioresistance by promoting CSC traits via the Hedgehog pathway and modulating the TiME to an immune-suppressive state. Targeting EIF5A could enhance radiation sensitivity and improve immune responses, offering a potential therapeutic strategy to optimize radiotherapy outcomes in CRC patients.

Theranostic GPA33-Pretargeted Radioimmunotherapy of Human Colorectal Carcinoma with a Bivalent 177Lu-Labeled Radiohapten.

Radiolabeled small-molecule DOTA-haptens can be combined with antitumor/anti-DOTA bispecific antibodies (BsAbs) for pretargeted radioimmunotherapy (PRIT). For optimized delivery of the theranostic γ- and ß-emitting isotope 177Lu with DOTA-based PRIT (DOTA-PRIT), bivalent Gemini (DOTA-Bn-thiourea-PEG4-thiourea-Bn-DOTA, aka (3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(DOTA-benzyl thiourea)) was developed. Methods: Gemini was synthesized by linking 2 S-2-(4-isothiocyanatobenzyl)-DOTA molecules together via a 1,14-diamino-PEG4 linker. [177Lu]Lu-Gemini was prepared with no-carrier-added 177LuCl3 to a molar-specific activity of 123 GBq/µmol and radiochemical purity of more than 99%. The specificity of BsAb-177Lu-Gemini was verified in vitro. Subsequently, we evaluated biodistribution and whole-body clearance for [177Lu]Lu-Gemini and, for comparison, our gold-standard monovalent [177Lu]Lu-S-2-(4-aminobenzyl)-DOTA ([177Lu]Lu-DOTA-Bn) in naïve (tumor-free) athymic nude mice. For our proof-of-concept system, a 3-step pretargeting approach was performed with an established DOTA-PRIT regimen (anti-GPA33/anti-DOTA IgG-scFv BsAb, a clearing agent, and [177Lu]Lu-Gemini) in mouse models. Results: Initial in vivo studies showed that [177Lu]Lu-Gemini behaved similarly to [177Lu]Lu-DOTA-Bn, with almost identical blood and whole-body clearance kinetics, as well as biodistribution and mouse kidney dosimetry. Pretargeting [177Lu]Lu-Gemini to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]Lu-Gemini for blood, tumor, liver, spleen, and kidneys of 3.99, 455, 6.93, 5.36, and 14.0 cGy/MBq, respectively. Tumor-to-normal tissue absorbed-dose ratios (i.e., therapeutic indices [TIs]) for the blood and kidneys were 114 and 33, respectively. In addition, we demonstrate that the use of bivalent [177Lu]Lu-Gemini in DOTA-PRIT leads to improved TIs and augmented [177Lu]Lu-Gemini tumor uptake and retention in comparison to monovalent [177Lu]Lu-DOTA-Bn. Finally, we established efficacy in SW1222 tumor-bearing mice, demonstrating that a single injection of anti-GPA33 DOTA-PRIT with 44 MBq (1.2 mCi) of [177Lu]Lu-Gemini (estimated tumor-absorbed dose, 200 Gy) induced complete responses in 5 of 5 animals and a histologic cure in 2 of 5 (40%) animals. Moreover, a significant increase in survival compared with nontreated controls was noted (maximum tolerated dose not reached). Conclusion: We have developed a bivalent DOTA-radiohapten, [177Lu]Lu-Gemini, that showed improved radiopharmacology for DOTA-PRIT application. The use of bivalent [177Lu]Lu-Gemini in DOTA-PRIT, as opposed to monovalent [177Lu]Lu-DOTA-Bn, allows curative treatments with considerably less administered 177Lu activity while still achieving high TIs for both the blood (>100) and the kidneys (>30).

Polymorphism in the Hsa-miR-4274 seed region influences the expression of PEX5 and enhances radiotherapy resistance in colorectal cancer.

Identifying biomarkers for predicting radiotherapy efficacy is crucial for optimizing personalized treatments. We previously reported that rs1553867776 in the miR-4274 seed region can predict survival in patients with rectal cancer receiving postoperative chemoradiation therapy. Hence, to investigate the molecular mechanism of the genetic variation and its impact on the radiosensitivity of colorectal cancer (CRC), in this study, bioinformatics analysis is combined with functional experiments to confirm peroxisomal biogenesis factor 5 (PEX5) as a direct target of miR-4274. The miR-4274 rs1553867776 variant influences the binding of miR-4274 and PEX5 mRNA, which subsequently regulates PEX5 protein expression. The interaction between PEX5 and Ku70 was verified by co-immunoprecipitation and immunofluorescence. A xenograft tumor model was established to validate the effects of miR-4274 and PEX5 on CRC progression and radiosensitivity in vivo. The overexpression of PEX5 enhances radiosensitivity by preventing Ku70 from entering the nucleus and reducing the repair of ionizing radiation (IR)-induced DNA damage by the Ku70/Ku80 complex in the nucleus. In addition, the enhanced expression of PEX5 is associated with increased IR-induced ferroptosis. Thus, targeting this mechanism might effectively increase the radiosensitivity of CRC. These findings offer novel insights into the mechanism of cancer radioresistance and have important implications for clinical radiotherapy.

Multi-institutional analysis of extracranial oligometastatic colorectal cancer patients treated with stereotactic body radiation therapy: TROD 02-008 study.

PURPOSE: To investigate the treatment outcomes of extracranial oligometastatic colorectal cancer (CRC) patients treated with stereotactic body radiotherapy (SBRT). MATERIALS AND METHODS: The clinical data of 388 extra-cranial oligometastatic CRC (≤ 5 lesions) patients and 463 lesions treated with SBRT at 19 cancer institutions were retrospectively analyzed. The prognostic factors predicting overall survival (OS), progression-free survival (PFS), and local control (LC) were assessed in uni- and multivariable analyses. RESULTS: The median age was 62 years (range, 29-92 years). The majority of the patients (90.5%) received surgery and systemic treatment for their primary tumor, had ≤ 2 metastasis (83.3%), had single organ involvement (90.3%), and staged using flouro-deoxyglucose positron emission tomography (FDG-PET/CT) (76%). The median fraction and total radiation doses were 10 Gy (range: 6-34 Gy) and 50 Gy (range: 8-64 Gy), respectively, delivered in a median of 4 fractions (range: 1-8). The median follow-up time for the entire cohort was 30.7 months (interquartile range: 27.0-34.3 months). The 3­year OS, PFS, and LC rates were 64.0%, 42.3%, and 72.7%, respectively. The 3­year LC rate was significantly higher in patients receiving BED10 ≥ 100 Gy than those receiving BED10 < 100 Gy (76.0% vs. 67.3%; p = 0.04). The 3­year PFS and OS rates were higher in patients receiving BED10 ≥ 100 Gy than those receiving BED10 < 100 Gy (33.2% vs. 25.2%; p = 0.03; 53.7% vs.  44.8%; p = 0.02). Single metastasis and complete response after SBRT were independent prognostic factors for survival in multivariable analysis. CONCLUSIONS: In this multi-center study, we demonstrated that SBRT is an effective treatment option of metastatic lesions in oligometastatic CRC patients by providing promising LC rates. Higher SBRT doses beyond BED10 ≥ 100 Gy were associated with improved LC and survival. LC of treated lesion and lower tumor burden after SBRT were associated with better outcomes.

Baicalein Enhances Radiosensitivity in Colorectal Cancer via JAK2/STAT3 Pathway Inhibition.

Radiation resistance is a crucial factor influencing therapeutic outcomes in colorectal cancer (CRC). Baicalein (BE), primarily derived from Scutellaria baicalensis, has demonstrated anti-CRC properties. However, the impact of BE on the radiosensitivity of CRC remains unclear. This study aimed to evaluate the radiosensitization effects of BE and elucidate its mechanism in CRC radiotherapy. We established an in vitro radioresistant cell model (CT26-R) using parental CRC cells (CT26) subjected to ionizing radiation (IR). CT26-R cells were pretreated with or without BE, followed by transfection with pcDNA-NC and pcDNA-JAK2. The proliferation of CT26-R cells treated with BE and IR was assessed using a colony formation assay. A CRC animal model was developed in BALB/c mice via CT26-R cell transplantation. The radiosensitizing effect of BE on CRC was evaluated in vivo. TUNEL assay was conducted to detect apoptosis in tumor tissue. The expression levels of p-STAT3, JAK2, PD-L1, and SOCS3 in vitro and in vivo were measured by western blotting. Our results demonstrated that BE significantly increased radiosensitivity in vitro and in vivo and enhanced apoptosis in tumor tissues. Additionally, BE significantly downregulated the expression of p-STAT3, JAK2, and PD-L1, and significantly upregulated SOCS3 expression. These in vivo effects were reversed by pcDNA-JAK2. In summary, our data suggest that BE enhances CRC radiosensitivity by inhibiting the JAK2/STAT3 pathway.

LncRNA HOTAIR promotes DNA damage repair and radioresistance by targeting ATR in colorectal cancer.

Long non-coding RNAs (lncRNAs) have been implicated in cancer progression and drug resistance development. Moreover, there is evidence that lncRNA HOX transcript antisense intergenic RNA (HOTAIR) is involved in colorectal cancer (CRC) progression. The present study aimed to examine the functional role of lncRNA HOTAIR in conferring radiotherapy resistance in CRC cells, as well as the underlying mechanism. The relative expression levels of HOTAIR were examined in 70 pairs of CRC tumor and para-cancerous tissues, as well as in radiosensitive and radioresistant samples. The correlations between HOTAIR expression levels and clinical features of patients with CRC were assessed using the Chi-square test. Functional assays such as cell proliferation, colony formation and apoptosis assays were conducted to determine the radiosensitivity in CRC cells with HOTAIR silencing after treatment with different doses of radiation. RNA pull-down assay and fluorescence in situ hybridization (FISH) were used to determine the interaction between HOTAIR and DNA damage response mediator ataxia-telangiectasia mutated- and Rad3-related (ATR). HOTAIR was significantly upregulated in CRC tumor tissues, especially in radioresistant tumor samples. The elevated expression of HOTAIR was correlated with more advanced histological grades, distance metastasis and the poor prognosis in patients with CRC. Silencing HOTAIR suppressed the proliferation and promoted apoptosis and radiosensitivity in CRC cells. HOTAIR knockdown also inhibited the tumorigenesis of CRC cells and enhanced the sensitivity to radiotherapy in a mouse xenograft model. Moreover, the data showed that HOTAIR could interact with ATR to regulate the DNA damage repair signaling pathway. Silencing HOTAIR impaired the ATR-ATR interacting protein (ATRIP) complex and signaling in cell cycle progression. Collectively, the present results indicate that lncRNA HOTAIR facilitates the DNA damage response pathway and promotes radioresistance in CRC cells by targeting ATR.

Role and mechanism of KIAA1429 in regulating cellular ferroptosis and radioresistance in colorectal cancer.

Colorectal cancer (CRC) is one of the most common non-cutaneous malignancies, causing significant mortality and a substantial burden. This study aims to explore the role of KIAA1429 (also known as vir-like m6A methyltransferase associated [VIRMA]) protein in the radioresistance of CRC. CRC cells and a radioresistant cell line were cultured, and KIAA1429 expression was detected. After the down-regulation of KIAA1429, its effect on the radioresistance and ferroptosis of cancer cells was analyzed. The role of ferroptosis in radioresistance was verified. The binding relationship among long non-coding RNA endogenous Bornavirus-like nucleoprotein 3, pseudogene (lncRNA EBLN3P), microRNA (miR)-153-3p, and KIAA1429 was analyzed. KIAA1429 and lncRNA EBLN3P were highly expressed in CRC, while miR-153-3p was poorly expressed. KIAA1429 and lncRNA EBLN3P were further increased/decreased in the radioresistant cells. KIAA1429 knockdown decreased the survival rate of the radioresistant cell line after X-ray irradiation and increased gamma H2A histone family member X (γ-H2AX), ferroptosis, and oxidative stress. A ferroptosis inhibitor alleviated the inhibitory effect of KIAA1429 knockdown on radioresistance. KIAA1429-mediated m6A modification up-regulated lncRNA EBLN3P, and lncRNA EBLN3P increased KIAA1429 by competitively binding to miR-153-3p. miR-153-3p silencing or lncRNA EBLN3P overexpression attenuated the promotion of ferroptosis and the inhibition of radioresistance induced by KIAA1429 knockdown. Overall, KIAA1429-mediated m6A modification up-regulated lncRNA EBLN3P expression, and lncRNA EBLN3P increased KIAA1429 expression by competitively binding to miR-153-3p, thus reducing ferroptosis and increasing the radioresistance of CRC.

SNORA28 Promotes Proliferation and Radioresistance in Colorectal Cancer Cells through the STAT3 Pathway by Increasing H3K9 Acetylation in the LIFR Promoter.

Radiotherapy is essential for treating colorectal cancer (CRC), especially in advanced rectal cancer. However, the low radiosensitivity of CRC cells greatly limits radiotherapy efficacy. Small nucleolar RNAs (snoRNAs) are a class of noncoding RNA that primarily direct post-transcriptional modifications of ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs), and other cellular RNAs. While snoRNAs are involved in tumor progression and chemoresistance, their association with radiosensitivity remains largely unknown. Herein, SNORA28 is shown highly expressed in CRC and is positively associated with poor prognosis. Furthermore, SNORA28 overexpression enhances the growth and radioresistance of CRC cells in vitro and in vivo. Mechanistically, SNORA28 acts as a molecular decoy that recruits bromodomain-containing protein 4 (BRD4), which increases the level of H3K9 acetylation at the LIFR promoter region. This stimulates LIFR transcription, which in turn triggers the JAK1/STAT3 pathway, enhancing the proliferation and radioresistance of CRC cells. Overall, these results highlight the ability of snoRNAs to regulate radiosensitivity in tumor cells and affect histone acetylation modification in the promoter region of target genes, thus broadening the current knowledge of snoRNA biological functions and the mechanism underlying target gene regulation.

The Tumor Microbiome Reacts to Hypoxia and Can Influence Response to Radiation Treatment in Colorectal Cancer.

Tumor hypoxia has been shown to predict poor patient outcomes in several cancer types, partially because it reduces radiation's ability to kill cells. We hypothesized that some of the clinical effects of hypoxia could also be due to its impact on the tumor microbiome. Therefore, we examined the RNA sequencing data from the Oncology Research Information Exchange Network database of patients with colorectal cancer treated with radiotherapy. We identified microbial RNAs for each tumor and related them to the hypoxic gene expression scores calculated from host mRNA. Our analysis showed that the hypoxia expression score predicted poor patient outcomes and identified tumors enriched with certain microbes such as Fusobacterium nucleatum. The presence of other microbes, such as Fusobacterium canifelinum, predicted poor patient outcomes, suggesting a potential interaction between hypoxia, the microbiome, and radiation response. To experimentally investigate this concept, we implanted CT26 colorectal cancer cells into immune-competent BALB/c and immune-deficient athymic nude mice. After growth, in which tumors passively acquired microbes from the gastrointestinal tract, we harvested tumors, extracted nucleic acids, and sequenced host and microbial RNAs. We stratified tumors based on their hypoxia score and performed a metatranscriptomic analysis of microbial gene expression. In addition to hypoxia-tropic and -phobic microbial populations, analysis of microbial gene expression at the strain level showed expression differences based on the hypoxia score. Thus, hypoxia gene expression scores seem to associate with different microbial populations and elicit an adaptive transcriptional response in intratumoral microbes, potentially influencing clinical outcomes. SIGNIFICANCE: Tumor hypoxia reduces radiotherapy efficacy. In this study, we explored whether some of the clinical effects of hypoxia could be due to interaction with the tumor microbiome. Hypoxic gene expression scores associated with certain microbes and elicited an adaptive transcriptional response in others that could contribute to poor clinical outcomes.

Transarterial Radioembolization (TARE) Global Practice Patterns: An International Survey by the Cardiovascular and Interventional Radiology Society of Europe (CIRSE).

PURPOSE: An international survey was conducted by the Cardiovascular Interventional Radiological Society of Europe (CIRSE) to evaluate radioembolization practice and capture opinions on real-world clinical and technical aspects of this therapy. MATERIALS AND METHODS: A survey with 32 multiple choice questions was sent as an email to CIRSE members between November and December 2022. CIRSE group member and sister societies promoted the survey to their local members. The dataset was cleaned of duplicates and entries with missing data, and the resulting anonymized dataset was analysed. Data were presented using descriptive statistics. RESULTS: The survey was completed by 133 sites, from 30 countries, spanning 6 continents. Most responses were from European centres (87/133, 65%), followed by centres from the Americas (22/133, 17%). Responding sites had been performing radioembolization for 10 years on average and had completed a total of 20,140 procedures over the last 5 years. Hepatocellular carcinoma treatments constituted 56% of this total, colorectal liver metastasis 17% and cholangiocarcinoma 14%. New sites had opened every year for the past 20 years, indicating the high demand for this therapy. Results showed a trend towards individualized treatment, with 79% of responders reporting use of personalized dosimetry for treatment planning and 97% reporting routine assessment of microsphere distribution post-treatment. Interventional radiologists played an important role in referrals, being present in the referring multi-disciplinary team in 91% of responding centres. CONCLUSION: This survey provides insight into the current state of radioembolization practice globally. The results reveal the increasing significance placed on dosimetry, evolving interventional techniques and increased technology integration.

Inhibition of DNMTs increases neoantigen-reactive T-cell toxicity against microsatellite-stable colorectal cancer in combination with radiotherapy.

The therapeutic efficacy of immunotherapy is limited in the majority of colorectal cancer patients due to the low mutational and neoantigen burdens in this immunogenically "cold" microsatellite stability-colorectal cancer (MSS-CRC) cohort. Here, we showed that DNA methyltransferase (DNMT) inhibition upregulated neoantigen-bearing gene expression in MSS-CRC, resulting in increased neoantigen presentation by MHC class I in tumor cells and leading to increased neoantigen-specific T-cell activation in combination with radiotherapy. The cytotoxicity of neoantigen-reactive T cells (NRTs) to DNMTi-treated cancer cells was highly cytotoxic, and these cells secreted high IFNγ levels targeting MSS-CRC cells after ex vivo expansion of NRTs with DNMTi-treated tumor antigens. Moreover, the therapeutic efficacy of NRTs further increased when NRTs were combined with radiotherapy in vivo. Administration of DNMTi-augmented NRTs and radiotherapy achieved an ∼50 % complete response and extended survival time in an immunocompetent MSS-CRC animal model. Moreover, remarkably, splenocytes from these mice exhibited neoantigen-specific T-cell responses, indicating that radiotherapy in combination with DNMTi-augmented NRTs prolonged and increased neoantigen-specific T-cell toxicity in MSS-CRC patients. In addition, these DNMTi-augmented NRTs markedly increase the therapeutic efficacy of cancer vaccines and immune checkpoint inhibitors (ICIs). These data suggest that a combination of radiotherapy and epi-immunotherapeutic agents improves the function of ex vivo-expanded neoantigen-reactive T cells and increases the tumor-specific cytotoxic effector population to enhance therapeutic efficacy in MSS-CRC.

TOPK Inhibition Enhances the Sensitivity of Colorectal Cancer Cells to Radiotherapy by Reducing the DNA Damage Response.

OBJECTIVE: Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer. However, the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer (CRC) cells is unclear. This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells. METHODS: The expression of TOPK was detected in CRC tissues by immunohistochemistry, and the effect of TOPK knockdown was detected in CRC cells by Western blotting. CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells. Furthermore, proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy. DNA damage was detected by the comet assay. Changes in the DNA damage response signaling pathway were analyzed by Western blotting, and apoptosis was detected by flow cytometry. RESULTS: The expression of TOPK was significantly greater in CRC tissues at grades 2-4 than in those at grade 1. After irradiation, CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins, including phospho-cyclin-dependent kinase 1 (p-CDK1), phospho-ataxia telangiectasia-mutated (p-ATM), poly ADP-ribose polymerase (PARP), and meiotic recombination 11 homolog 1 (MRE11). CONCLUSIONS: TOPK was overexpressed in patients with moderately to poorly differentiated CRC. Moreover, TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.

Phase I trial of single-photon emission computed tomography-guided liver-directed radiotherapy for patients with low functional liver volume.

BACKGROUND: Traditional constraints specify that 700 cc of liver should be spared a hepatotoxic dose when delivering liver-directed radiotherapy to reduce the risk of inducing liver failure. We investigated the role of single-photon emission computed tomography (SPECT) to identify and preferentially avoid functional liver during liver-directed radiation treatment planning in patients with preserved liver function but limited functional liver volume after receiving prior hepatotoxic chemotherapy or surgical resection. METHODS: This phase I trial with a 3 + 3 design evaluated the safety of liver-directed radiotherapy using escalating functional liver radiation dose constraints in patients with liver metastases. Dose-limiting toxicities were assessed 6-8 weeks and 6 months after completing radiotherapy. RESULTS: All 12 patients had colorectal liver metastases and received prior hepatotoxic chemotherapy; 8 patients underwent prior liver resection. Median computed tomography anatomical nontumor liver volume was 1584 cc (range = 764-2699 cc). Median SPECT functional liver volume was 1117 cc (range = 570-1928 cc). Median nontarget computed tomography and SPECT liver volumes below the volumetric dose constraint were 997 cc (range = 544-1576 cc) and 684 cc (range = 429-1244 cc), respectively. The prescription dose was 67.5-75 Gy in 15 fractions or 75-100 Gy in 25 fractions. No dose-limiting toxicities were observed during follow-up. One-year in-field control was 57%. One-year overall survival was 73%. CONCLUSION: Liver-directed radiotherapy can be safely delivered to high doses when incorporating functional SPECT into the radiation treatment planning process, which may enable sparing of lower volumes of liver than traditionally accepted in patients with preserved liver function. TRIAL REGISTRATION: NCT02626312.

Are survival outcomes dependent on the tumour dose threshold of 139 Gy in patients with chemorefractory metastatic colorectal cancer treated with yttrium-90 radioembolization using glass particles? A real-world single-centre study.

OBJECTIVES: To compare the survival and objective response rate (ORR) of the patients receiving estimated tumour absorbed dose (ETAD) <140 Gy versus ETAD ≥140 Gy in patients with advanced chemorefractory colorectal carcinoma liver metastases (CRCLM) treated with yttrium-90 transarterial radioembolization (90Y TARE). METHODS: Between August 2016 and August 2023 adult patients with unresectable, chemorefractory CRCLM treated with 90Y TARE using glass particles, were retrospectively enrolled. Primary outcomes were overall survival (OS) and hepatic progression free survival (hPFS). Secondary outcome was ORR. RESULTS: A total of 40 patients with a mean age of 66.2 ± 7.8 years met the inclusion criteria. Mean ETAD for group 1 (ETAD <140 Gy) and group 2 (ETAD ≥140) were 131.2 ± 17.4 Gy versus 195 ± 45.6 Gy, respectively. The mean OS and hPFS for group 1 versus group 2 were 12 ± 10.3 months and 8.1 ± 9.3 months versus 9.3 ± 3 months and 7.1 ± 8.4 months, respectively and there were no significant differences (P = .181 and P = .366, respectively). ORR did not show significant difference between the groups (P = .432). CONCLUSION: In real-world practice, no significant difference was found in OS, hPFS, and ORR between patients who received ETAD <140 Gy versus ETAD ≥140 Gy in patients with CRCLM, in this series. ADVANCES IN KNOWLEDGE: This study demonstrated that increased tumour absorbed doses in radioembolization may not provide additional significant advantage for OS and hPFS for patients with CRCLM.