Female Athymic Nude Mice Research Articles

Single-Frequency Birdcage Coils for Deep Tissue Perfluorocarbon Magnetic Resonance Imaging in Mice.

Fluorine-19 (19F) MRI has become an established tool for invivo cell tracking following exvivo or invivo labelling of various cell types with 19F perfluorocarbons (PFCs). Here, we developed and evaluated novel mouse-specific radiofrequency (RF) hardware for improved dual 1H anatomical imaging and deep tissue 19F MR detection of PFCs. Three linearly polarized birdcage RF coils were constructed-a dual-frequency 1H/19F coil, and a pair of single-frequency 1H and 19F coils, designed to be used sequentially. RF coil quality factors (Q values), signal homogeneity and sensitivity were benchmarked against a commercially constructed dual-frequency 1H/19F surface coil. RF homogeneity was assessed using a phantom designed to mimic PFC localization at depth in a mouse. The single-frequency birdcage coils (1H and 19F) displayed more uniform coverage and enhanced signal-to-noise ratios (SNRs) compared to both the birdcage and surface dual-frequency coils for 19F detection. Bilateral injection of a perfluoropolyether nanoemulsion into the footpads of female athymic nude mice, resulting in drainage to various lymph nodes and subsequent accumulation in lymph node macrophages, provided a platform to assess differences in SNRs and contrast-to-noise ratios (CNR) between both coil configurations as a function of depth and location. The single-frequency 1H coil provided significantly increased CNR in anatomical images (p < 0.001) with increased anatomical coverage compared to the dual-frequency surface coil. The single-frequency 19F birdcage coil offered increased PFC detectability with significantly higher SNR in renal, lumbar, sciatic and popliteal lymph nodes (p < 0.01) compared to the dual-frequency surface coil. Interestingly, the percentage difference between SNR measurements in lymph nodes between the single-frequency 19F coil and the 1H/19F surface coil had a linear relationship with increasing distance from the surface coil (R2 = 0.6352; p < 0.0001), indicating a potential disagreement for imaging experiments that rely on 19F spin quantification at increasing depth within the mouse using surface RF coils.

Intraperitoneal delivery of cannabidiol (CBD) and Δ9-tetrahydocannabinol (THC) promotes papillomavirus infections in athymic nude mice.

We used our mouse papillomavirus (MmuPV1) model to test the hypothesis that two primary psychoactive ingredients of marijuana, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), promote papillomavirus persistence in the oral mucosa of infected mice. We conducted intraperitoneal (ip) injections of a moderate dose (3 mg/kg) of either CBD and/or THC in both male and female athymic nude mice and followed the mice up to 20 weeks post-infection. These doses are comparable to what is estimated for human conventional cannabis consumption. All mice were infected with MmuPV1 in the oral cavity at week 4 post-ip delivery of CBD, THC, or a combination of THC and CBD (T+C). THC and CBD were detected in the blood of treated mice for up to 72 hours after ip injection. Significantly higher levels of viral DNA were detected in males from both CBD and T+C-treated groups compared to those in the control group at 9- 10-and 12-weeks post infection. A marginally increased viral RNA was also detected in the infected tongues of males in all tested groups compared to that in males in the vehicle control group; the opposite was observed in females. We detected significantly higher levels of dermal dendritic cells (CD205+CD11c+), granulocytes (Ly6G+), but macrophages (F4-80+) recruited to the infected tongues of CBD-treated females. Our findings suggest that CBD may play a role in promoting MmuPV1 persistence in the oral cavity.

PAR1 Is a Candidate Target for the Treatment of Peritoneal Dissemination in Gastric Cancer.

Peritoneal dissemination (PD) is a frequent cause of death in gastric cancer (GC), and there is evidence of an association between protease-activated receptor-1 (PAR1) and the development of PD. This study hypothesized that PD in GC might be influenced by PAR1. The cytotoxic effect of paclitaxel (PTX) on PAR1-transfected MKN45 (MKN45/PAR1) cells was analyzed using the MTT assay, and IC50 values were determined. In female athymic nude mice, MKN45/PAR1 cells were suspended in 0.05 ml phosphate-buffered saline (PBS) medium and inoculated into the stomach mid-wall. In each group, intraperitoneal injections of PBS, PTX, SCH79797 (PAR1-antagonist), or PTX plus SCH79797 were administered on days 8, 15, and 22 following tumor inoculation. At 56 days after tumor inoculation, mice were examined for both abdominal tumor nodule status and size and weight of the tumors. The IC50 of PTX for MKN45/PAR1 cells was 0.0697 μM and that of SCH79797 was 0.0145 μM. Mean survival of the MKN45/PAR1 mice in the PBS group was 28.75 days, whereas survival times for the mice treated with SCH79797, PTX, or a combination of PTX and SCH79797 were 31.2, 49.2, and 48.5 days, respectively. Tumor weight was smaller in the group receiving PTX and SCH79797 intraperitoneally compared with that in the PBS group (1,086±127.2 mg vs. 33.2±19.9 mg; p<0.001). The PAR1 antagonist was found to inhibit PD in a PAR1-expressing GC cell line. PAR1 may serve as a promising therapeutic target for managing PD in gastric cancer, as it plays a crucial role in its progression.

PET Imaging of Differentiated Thyroid Cancer with TSHR-Targeted [89Zr]Zr-TR1402.

Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [89Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [89Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89Zr (t1/2 = 78.4 h, β+ = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [89Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [89Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T (P < 0.0001) and FTC133 (P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T (P < 0.0001) and TSHR+ FTC133 (P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [89Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference (P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference (P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [89Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

Antitumor effect of Iso-mukaadial acetate on MCF-7 breast cancer mice xenograft model

Antitumor drugs used today have shown significant efficacy and are derived from natural products such as plants. Iso-mukaadial acetate (IMA) has previously been shown to possess anticancer properties by inducing apoptosis. The purpose of this study was to investigate the therapeutic effect of IMA in the breast cancer xenograft mice model. Female athymic nude mice were used and inoculated with breast cancer cells subcutaneously. Untreated group one served as a negative control and positive control group two (cisplatin) was administered intravenously. IMA was administered orally to group three (100 mg/kg) and group four (300 mg/kg). Blood was collected (70 μL) from the tail vein on day zero, day one and day three. Tumor regression was measured every second day and body mass was recorded each day. Estimation of serum parameters for renal indices was examined using a creatinine assay. Histopathological analysis was conducted to evaluate morphological changes of liver, kidney, and spleen tissues before and after compound administration under a fluorescence light microscope. Histopathological analysis of tumors was conducted before and after compound administration. Apoptotic analysis using the TUNEL system was conducted on liver, kidney, and spleen tissues. Tumor shrinkage and reduction in body mass were observed after treatment with IMA. Serum creatinine was slightly elevated after treatment with IMA at a dosage of 100 and 300 mg/kg. Histopathological results of the liver exhibited no changes before and after IMA while the kidney and spleen tissues showed changes in the cellular structure. IMA showed no cytotoxic effect on the tumor cells, and cell proliferation was observed. Apoptotic assay stain with TUNEL showed apoptotic cells in spleen tissue and kidney but no apoptotic cells were observed in liver tissue section treated with IMA. IMA showed clinical toxic signs that resulted in the suffering and death of the mice immediately after IMA administration. Histopathology of tumor cells showed that IMA did not inhibit cell proliferation and no cellular damage was observed. Therefore, based on the results obtained, we cannot make any definitive conclusion on the complete effect of IMA in vivo. IMA is toxic, poorly soluble, and not safe to use in animal studies. The objective of the study was not achieved, and the hypothesis was rejected.

Abstract PO1-26-03: Activity of alpelisib in a panel of breast XPDX models harboring hotspot and uncommon PIK3CA mutations

Abstract Background: Therapies targeting mutated phosphoinositide 3-kinase (PI3K) p110α catalytic subunit (PIK3CA) cancers remain an active field of research. Alpelisib was approved in combination with fulvestrant in ER+/HER2- breast cancer patients harboring PIK3CA mutations on hotspot amino acids E542, E545 Q546 or H1047. While mutation at these sites have been widely studied, they only comprise ~25% of identified variants in this protein. Recent studies have reported other mutations, including those at the c-terminus, which can lead to constitutive activation of PIK3CA; however, whether these variants are sensitive to agents like alpelisib is unclear. To better understand these variants and their role in PIK3CA-mediated breast cancer, we established a panel of PIK3CA-mutated ER+ and ER- breast XPDX models and evaluated each in vivo with single agent alpelisib. Methods: Ninety previously developed XPDX models representing PIK3CA-mutated ER+ and ER- breast cancer were evaluated in this study. Models were grown subcutaneously in female athymic nude mice supplemented with estradiol in drinking water when necessary and ER expression confirmed at multiple passes; PIK3A variants were determined by WES and RNAseq. For in vivo studies, alpelisib was administered once daily by oral gavage at 35 mg/kg through study completion. Endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C&amp;lt; 0%) versus Day 0 tumor volume was also reported. Results: 60% of PIK3CA-mutated models were found ER+ with 40% also HER2+ while 20% of ER- PIK3CA-mutated models were also HER2+. H1047X mutations accounted for 40% of all PIK3CA variants, 75% of which were single or dual PIK3CAH1047R. Several hotspot-mutated models were sensitive to alpelisib some with regressions including ST986 (E542K), ST1097 (E454K) and STF040 (H1047R), as well as ST1245C (R88Q/N1044H), ST2076 (102-103ins/R357Q) and ST4176 (E726K/H1047R). HER2+ models were less sensitive to alpelisib although STM148D (H1047L) reported tumor regressions. Interestingly, ST1799/PBR (E542K/H1065L), a palbociclib resistant clone of the ER+ parent ST1799 model was resistant to alpelisib while the parent was found sensitive. Conclusion: We have benchmarked a panel of PIK3CA-mutated breast XPDX models with alpelisib and compared activity of models harboring hotspot versus uncommon PIK3CA mutations with ER and HER2 expression. This data is a valuable tool in further developing PIK3CA inhibitors and identifying new variant targets in breast cancer. Citation Format: Mercedes Castaneda, Alyssa Simonson, Johnnie Flores, Maci DeBoer, Jim Lund, Tahmi Rouzbahan, Kyriakos P. Papadopoulos, Amy Lang, Gladys Rodriguez, Maryam Elmi, Arthur Rosenthal, Drew Rasco, Victor Moreno, Amita Patnaik, Tatiana Hernandez, Emiliano Calvo, Lon Smith, Manish Sharma, Muralidhar Beeram, Michael Wick. Activity of alpelisib in a panel of breast XPDX models harboring hotspot and uncommon PIK3CA mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-03.

Characterization of AST-001 non-clinical pharmacokinetics: A novel selective AKR1C3-activated prodrug in mice, rats, and cynomolgus monkeys.

AST-001 is a chemically synthesized inactive nitrogen mustard prodrug that is selectively cleaved to a cytotoxic aziridine (AST-2660) via aldo-keto reductase family 1 member C3 (AKR1C3). The purpose of this study was to investigate the pharmacokinetics and tissue distribution of the prodrug, AST-001, and its active metabolite, AST-2660, in mice, rats, and monkeys. After single and once daily intravenous bolus doses of 1.5, 4.5, and 13.5mg/kg AST-001 to Sprague-Dawley rats and once daily 1 h intravenous infusions of 0.5, 1.5, and 4.5mg/kg AST-001 to cynomolgus monkeys, AST-001 exhibited dose-dependent pharmacokinetics and reached peak plasma levels at the end of the infusion. No significant accumulation and gender differences were observed after 7days of repeated dosing. In rats, the half-life of AST-001 was dose independent and ranged from 4.89 to 5.75h. In cynomolgus monkeys, the half-life of AST-001 was from 1.66 to 5.56h and increased with dose. In tissue distribution studies conducted in Sprague-Dawley rats and in liver cancer PDX models in female athymic nude mice implanted with LI6643 or LI6280 HepG2-GFP tumor fragments, AST-001 was extensively distributed to selected tissues. Following a single intravenous dose, AST-001 was not excreted primarily as the prodrug, AST-001 or the metabolite AST-2660 in the urine, feces, and bile. A comprehensive analysis of the preclinical data and inter-species allometric scaling were used to estimate the pharmacokinetic parameters of AST-001 in humans and led to the recommendation of a starting dose of 5mg/m2 in the first-in-human dose escalation study.

Abstract B045: In vivo ovarian cancer detection using folate receptor-α targeted iron oxide nanoparticles

Abstract Ovarian cancer is the second most common and most deadly gynecological cancer in women. Early and accurate detection is crucial in improving survival rate and quality of life of patients. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used in a variety of cancer detection/imaging applications, including Magnetic Resonant Imaging (MRI), Magnetic Particle Imaging (MPI) as well as Superparamagnetic Relaxometry (SPMR) detection currently being developed in-house. SPMR is a highly sensitive detection technology that can differentiate the magnetic signature of nanoparticles bound to tumor cells from unbound nanoparticles (NP). Folate receptor alpha (FRα) is a folate transporter overexpressed in approximately 90% of ovarian cancer, which makes it a suitable molecular target to use in developing tumor imaging methods. Our earlier study using xenograft model with KB (FRα +ve) and A549 (FRα -ve) cells implanted subcutaneously on the flank region of female athymic nude mice demonstrated higher level tumor accumulation of folate-NPs in KB tumor compared to A549 tumor, detectable by SPMR and MRI. To translate such results into a potential clinical application, we recently generated two orthotopic mice ovarian cancer models by implanting KB cells surgically into ovary as well intraperitoneally (IP) into mice abdominal cavity (metastasis model). Implanted tumor cells were allowed to grow for about 2 weeks. Folate-NPs (20mg Fe/kg) were delivered via tail vein or IP injections. PEG-NPs were also delivered as controls. Mice were euthanized 24-hour post-dosing. Mesenteric tumor tissue and reproductive organs with tumors were resected and measured on SPMR. Same tissues from mice without NP injection or with PEG-NP injection were also measured. Our results demonstrated that Folate-NPs reached the tumor sites in both orthotopic models and accumulated specifically at high level detectable by SPMR ex vivo. The amount of NP accumulation in mice with orthotopically implanted tumor was 3x-10x more than those of the xenograft subcutaneous flank implanted tumor model, indicating efficient NP delivery and binding of NP with tumor cells in its natural tissues/organs. The repeat studies of these orthotopic mice models used in the SPMR experiment are currently being conducted using MRI as an alternative in vivo detection method with images taken pre- and post- dosing of the NPs. We anticipate that folate-NPs presence in the tumors will be easily observable on MRI images as it produces T2 contrast along with additional anatomical information. Favorable orthotopic mice model results presented here will lay the groundwork for IND-enabling development of these targeted NP as T2 contrast agent for early ovarian cancer detection using MRI. This type of targeted imaging technology will improve the accuracy of cancer detection in vivo without the use of radioisotopes or radiation, therefore, minimize the needs of invasive biopsies/surgery, with further potential of monitoring therapy response or recurrence. Citation Format: Marie Zhang, Yamitha Perera, Kathirvel Kandasamy, Dan Inglese. In vivo ovarian cancer detection using folate receptor-α targeted iron oxide nanoparticles [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B045.

Radiolabelled 177 Lu-Bispidine-Trastuzumab for Targeting Human Epidermal Growth Factor Receptor 2 Positive Cancers.

Radioimmunotherapy (RIT) is a promising alternative to conventional treatment options. Here, we present experimental work on the synthesis, radiochemistry, and in vivo performance of a lanthanoid-selective nonadentate bispidine ligand suitable for 177 Lu3+ ion complexation. The ligand (bisp,1) was derivatised with a photoactivatable aryl azide (ArN3 ) group as a bioconjugation handle for light-induced labelling of proteins. Quantitative radiosynthesis of [177 Lu]Lu-1+ was accomplished in 10 minutes at 40 °C. Subsequent incubation of [177 Lu]Lu-1+ with trastuzumab, followed by irradiation with light at 365 nm for 15 min, at room temperature and pH 8.0-8.3, gave the radiolabelled mAb, [177 Lu]Lu-1-azepin-trastuzumab ([177 Lu]Lu-1-mAb) in a decay-corrected radiochemical yield of 14 %, and radiochemical purity (RCP)>90 %. Stability studies and cellular binding assays in vitro using the SK-OV-3 human ovarian cancer cells confirmed that [177 Lu]Lu-1-mAb remained biological active and displayed specific binding to HER2/neu. Experiments in immunocompromised female athymic nude mice bearing subcutaneous xenograft models of SK-OV-3 tumours revealed significantly higher tumour uptake in the normal group compared with the control block group (29.8±11.4 %ID g-1 vs. 14.8±6.1 %ID g-1 , respectively; P-value=0.037). The data indicate that bispidine-based ligand systems are suitable starting points for constructing novel, high-denticity chelators for specific complexation of larger radiotheranostic metal ion nuclides.

Non-Contrast-Enhanced Multiparametric MRI of the Hypoxic Tumor Microenvironment Allows Molecular Subtyping of Breast Cancer: A Pilot Study.

Tumor neoangiogenesis is an important hallmark of cancer progression, triggered by alternating selective pressures from the hypoxic tumor microenvironment. Non-invasive, non-contrast-enhanced multiparametric MRI combining blood-oxygen-level-dependent (BOLD) MRI, which depicts blood oxygen saturation, and intravoxel-incoherent-motion (IVIM) MRI, which captures intravascular and extravascular diffusion, can provide insights into tumor oxygenation and neovascularization simultaneously. Our objective was to identify imaging markers that can predict hypoxia-induced angiogenesis and to validate our findings using multiplexed immunohistochemical analyses. We present an in vivo study involving 36 female athymic nude mice inoculated with luminal A, Her2+, and triple-negative breast cancer cells. We used a high-field 9.4-tesla MRI system for imaging and subsequently analyzed the tumors using multiplex immunohistochemistry for CD-31, PDGFR-β, and Hif1-α. We found that the hyperoxic-BOLD-MRI-derived parameter ΔR2* discriminated luminal A from Her2+ and triple-negative breast cancers, while the IVIM-derived parameter fIVIM discriminated luminal A and Her2+ from triple-negative breast cancers. A comprehensive analysis using principal-component analysis of both multiparametric MRI- and mpIHC-derived data highlighted the differences between triple-negative and luminal A breast cancers. We conclude that multiparametric MRI combining hyperoxic BOLD MRI and IVIM MRI, without the need for contrast agents, offers promising non-invasive markers for evaluating hypoxia-induced angiogenesis.

CLX-155: A Novel, Oral 5-FU Prodrug Displaying Antitumor Activity in Human Colon Cancer Xenograft Model in Nude Mice

Introduction: Capecitabine is an oral prodrug of 5-FU, which interpatient pharmacokinetic (PK) variability related to liver function and severe adverse events (e.g., hand-foot syndrome, myelosuppression, and neurotoxicity) limits. CLX-155 is a novel oral 5’-DFCR prodrug involving 5’-DFCR as an intermediate for generating 5-FU, unlike capecitabine, which the liver does not metabolize. This study addresses the following research question: what is the activity of CLX-155 in a human colon cancer xenograft model in nude mice? Methods: This study involved 50 Foxn1 athymic nude female mice implanted with the human colon cancer cell line HCT116 (5 million cells per site). Investigators randomized animals into five treatment groups (N = 10): vehicle control, CLX-155 at doses of 125, 250, and 500 mg/kg/day, or capecitabine 1000 mg/kg/day. Animals received oral treatment once daily for five days a week with two days off for a total of three consecutive weeks. Investigators evaluated treatment toxicity based on body weight loss. Calculations for tumor growth inhibition involved comparing changes in tumor volume on a given day to tumor volumes on Day 1. Results: CLX-155 demonstrated statistically significant, dose-dependent tumor growth inhibition at all doses compared to vehicle control (p&lt;0.0001). Tumor growth inhibition at Day 15 for CLX-155 treatment groups of 125, 250, and 500 mg/kg/day was 57.8%, 70.4%, and 90.6% respectively. Two animals in the CLX-155 500 mg/kg/day treatment group experienced complete tumor regression, and all animals in the CLX-155 treatment groups survived. Two animals in the CLX-155 250 and 500 mg/kg/day dosing groups experienced a decrease in body weight. In contrast, two mice in the capecitabine group exhibited clinical signs of hunchback and scaly skin, progressive weight loss, and eventual death. Conclusion: CLX-155 demonstrated comparable tumor growth inhibition to capecitabine but at a lower dose, suggesting increased potency. In addition, CLX-155 exhibited improved tolerability and fewer adverse effects. These promising results support further investigation in Phase 1 clinical trials for managing colon cancer.

Abstract C002: Identification of biomarkers predictive of sensitivity to the CHK1/2 inhibitor ACR-368 using high-resolution phosphoproteomics and development of an ACR-368-tailored patient responder identification 3-marker test, ACR-368 OncoSignature

Abstract Introduction ACR-368 (prexasertib) is a potent and selective CHK1/2 inhibitor with demonstrated durable, single-agent activity in patients with advanced solid tumors. Genomic biomarkers have been unsuccessful in predicting response to ACR-368 due in part to the complex genetic changes in cancer that translate into dysregulated protein signaling pathways. To address this challenge, we sought to identify protein-based, predictive biomarkers that measure the ACR-368-sensitive dysregulated signaling driving tumorigenesis using our proprietary approach, AP3 (Acrivon Predictive Precision Proteomics). Methods ACR-368 anticancer activity was measured in a panel of ovarian cancer cell lines using CellTiter-Glo. Quantitative phosphoproteomics was performed on ACR-368 sensitive and resistant ovarian cancer cell lines using data-independent acquisition mass spectrometry (DIA-MS). Kinase activity inference analysis and signaling pathway analyses were conducted to uncover ACR-368-regulated pathways associated with tumorigenesis. A quantitative, multiplexed immunofluorescent (IF) assay was developed based on three biomarkers, termed ACR-368 OncoSignature. Ovarian PDX studies were conducted in female athymic nude or CB-17 Scid mice. Results ACR-368 demonstrated diverse anti-proliferative activity in ovarian cancer cell lines. Phosphoproteome-profiling analysis from ACR-368 sensitive and resistant cells exposed to ACR-368 yielded &amp;gt;17,000 confidently localized phospho-sites (localization probability &amp;gt; 0.75), with 8272 being significantly regulated by ACR-368 (FC &amp;gt; 1.5, Q-value &amp;lt; 0.05, Limma t-test). Unsupervised hierarchical clustering analysis revealed overrepresentation of ATM/ATR and CDK1/2-associated substrate sites in the upregulated group, while the downregulated group showed overrepresentation of CHK1-associated sites. Pathway enrichment analysis highlighted the differential regulation of the homology-directed repair (HDR) pathway between sensitive and non-sensitive ovarian cancer cells. Through analysis of HDR-related signaling networks, three functionally orthogonal predictive biomarkers were assembled into a quantitative multiplex in situ assay for FFPE tissue that provides a direct readout of a tumor’s dependency on the signaling axis inhibited by ACR-368. Quantitation of these biomarkers in cancer cell lines using the IF-based OncoSignature demonstrated efficient classification based on ACR-368 sensitivity. The assay accurately predicted sensitivity to ACR-368 across ovarian cancer PDX models with an AUC of 0.9 (95% confidence interval: 0.71 to 1; p-value = 0.025). Conclusions Employing our AP3 platform, which combines MS-based phosphoproteomics and quantitative multiplexed-IF staining of drug-tailored biomarkers, we developed a response-predictive test for ACR-368 that enables identification of responders to ACR-368 treatment. A clinical trial (NCT05548296) evaluating the efficacy of ACR-368 based on the OncoSignature test status is currently recruiting in patients with platinum-resistant ovarian, endometrial, and urothelial cancer. Citation Format: Caroline Wigerup, Michail Shipitsin, Ayesha Murshid, Lei Shi, Magnus E. Jakobsson, Dorte Bekker-Jensen, Sibgat Choudry, James Dunyak, David Proia, Jesper V. Olsen, Kristina Masson, Peter Blume-Jensen. Identification of biomarkers predictive of sensitivity to the CHK1/2 inhibitor ACR-368 using high-resolution phosphoproteomics and development of an ACR-368-tailored patient responder identification 3-marker test, ACR-368 OncoSignature [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C002.

Abstract 3586: In vivo targeted detection and imaging of ovarian cancer by SPMR and MRI using anti-folate receptor functionalized iron oxide nanoparticles

Abstract Ovarian cancer is the second most common and most deadly gynecological cancer in women. Early and accurate detection is crucial in improving survival rate and quality of life of patients. Folate receptor alpha (FRα) is a folate transporter that has been reported to be overexpressed in approximately 90% of ovarian cancer, which makes it a suitable molecular target to use in developing tumor imaging methods. Iron oxide nanoparticles (NPs) have been used in a variety of cancer detection/imaging applications. Here we show that anti-FRα targeted superparamagnetic iron oxide nanoparticles (SPION) can be used for in vivo detection by two different imaging methods. Superparamagnetic Relaxometry (SPMR) is a highly sensitive in vivo detection technology that is able to differentiate the magnetic signature of nanoparticles bound to tumor cells from unbound nanoparticles. Same targeted SPIONs accumulated in tumor are able to provide molecular magnetic resonance imaging (MRI) contrast as well. Our results have shown that the anti-FRα NPs can distinguish high and low FRα expression cell lines, such as KB and A549, respectively in an in vitro cell based assay by SPMR, demonstrating good sensitivity, specificity and selectivity. In vivo study using xenograph model with KB and A549 cells implanted subcutaneously on the flank region of female athymic nude mice demonstrated higher level tumor accumulation of anti-FRα NPs in KB tumor compared to A549 tumor, measurable by ex vivo tumor detection using SPMR. Using a PEG NPs (without the target ligand) as control for the mice study have shown that minimum nanoparticle tumor accumulated were observed, demonstrating the specificity of the nanoparticles in vivo. Organ distribution study indicated that anti-FRα NPs mostly accumulated in liver and spleen as these nanoparticles were cleared through these two organs. Other organs such as brain, heart, lung, kidney didn’t show appreciable accumulation. The repeat of same type of xenograph mice model used in the SPMR study is currently under the way using MRI as an alternative in vivo detection method with images taken pre- and post- dosing of the nanoparticles. Based on the specificity and sufficient accumulation of nanoparticles demonstrated in the SPMR study, it is expected that MRI will show similar performance with additional anatomical information.Our anti-FRα NPs provided targeted and specific delivery to cancerous tissue and generated measurable signal by SPMR. Furthermore, demonstration of these anti-FRα NPs generating tumor specific contrast in MRI is currently under the way. These studies lay the groundwork for use of bio-safe magnetic particles as a detection and contrast agent for early ovarian cancer detection with improved accuracy to minimize the needs of invasive biopsies/surgery as well as potential utility for monitoring therapy response or recurrence. Citation Format: Marie Zhang, Kathirvel Kandasamy, Yamitha Perera, Dan Inglese, Eric Smith-Nguyen. In vivo targeted detection and imaging of ovarian cancer by SPMR and MRI using anti-folate receptor functionalized iron oxide nanoparticles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3586.

Abstract 6240: The potent quadruplex-binding compound QN-302 shows anti-tumor activity as a monotherapy in an orthotopic in vivo model of pancreatic cancer

Abstract The compound QN-302, a tetra-substituted naphthalene diimide derivative has single-digit nM anti-proliferative activity in a panel of human pancreatic cancer (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It has significant anti-tumor activity in the MIA-PACA2 xenograft model for PDAC and in the genetic KPC model. We now report that this compound also shows significant mono-therapy anti-tumor activity in an orthotopic model for PDAC. Female athymic nude mice bearing orthotopically implanted pancreatic BxPC-3 cells were dosed with 1 x weekly IV QN-302 at 0.5 and 1.0 mg/kg, over a period of 21 days. A separate group of mice was treated with Gemcitabine twice weekly IV during this period, at a dosage of 15 mg/kg. Blood samples from animals were collected on a weekly basis and were used to detect and quantify circulating tumor DNA as a method of following treatment efficacy. In line with previous studies QN-302 was well-tolerated at 0.5 and 1.0 mg/kg with no adverse effects on animal health or behavior noted during the study. Circulating tumor DNA levels were significantly less than vehicle controls in gemcitabine-treated animals from day 7 and QN-302 (1.0 mg/kg) treated animals from day 14 of treatment. Survival was significantly increased in gemcitabine and QN-302 (1.0 mg/kg) QW treatment groups. Statistically significant efficacy was observed in gemcitabine and QN-302 (1.0 mg/kg) treated animals. Examination of blood chemistry and biochemistry of treated animals did not indicate any adverse effects due to QN-302. This orthotopic model is the 4th in vivo study in a pancreatic cancer model showing anti-tumor activity for QN-302, further confirming the potential of this compound for human disease. QN-302 is currently in an advanced stage of pre-IND development by Qualigen Inc. Citation Format: Nicole Williams, Danielle Santos, Jenny Worthington, Ahmed Ahmed, Tariq Arshad, Stephen Neidle. The potent quadruplex-binding compound QN-302 shows anti-tumor activity as a monotherapy in an orthotopic in vivo model of pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6240.

Abstract P3-08-05: Correlation of Trop2 expression with in vivo sensitivity to sacituzumab govitecan in a panel of breast XPDX models

Abstract Background: Sacituzumab govitecan (SG) is an antibody-drug conjugate targeting Trop2 with an SN-38 payload recently approved for pretreated patients with locally advanced or metastatic triple-negative breast cancer (TNBC). The XenoSTART Patient-Derived Xenograft (XPDX) breast cancer platform includes over 180 models spanning all subtypes characterized with immunohistochemistry (IHC) including ER, PR, and HER2 protein levels, genomic and transcriptomic sequencing, and in vivo drug sensitivity. To better understand potential benefit of SG in breast cancers other than TNBC and further annotate our platform, Trop2 protein levels were determined in all breast models by IHC. We evaluated tumor growth inhibition by SG in 125 of our XPDX breast models and compared protein expression with agent activity. Methods: 180 breast XPDX models were evaluated for Trop2 expression (AF650, R&amp;D Systems) and 125 were evaluated in vivo against SG; responses were grouped by ER and Trop2 status (+/-). Models were grown subcutaneously in female athymic nude mice and ER+ models supplemented with estradiol. Models were also characterized for PR, HER2, and AR protein expression by IHC and profiled using WES and RNAseq. For in vivo studies, SG was administered by intravenous injection biweekly for two cycles at 1 mg, flat; endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C&amp;lt; 0%) versus Day 0 tumor volume was also reported. Results: 180 breast models were examined by IHC with 75/180 (42%) classified as ER+ and 105/180 (58%) ER-. In ER+ models 38/75 (51%) were Trop2+ and 37/75 (49%) Trop2-, and in ER- models 41/105 (39%) were Trop2+ and 64/105 (61%) Trop2-. In vivo, 20% of ER+/Trop2+ models reported sensitivity to SG, most notably models from patients with acquired resistance to CDK4/6 inhibitors, including STM001 and ST4316B. Interestingly, &amp;gt;70% of ER+/HER2+/Trop2+ models were insensitive to SG, including ST225 and ST340. Of 41 ER-/Trop2+ models, approximately 40% reported some response to SG with 50% of these sensitive to therapy, including ST5954 established from a patient who began treatment with SG following sample collection and is currently in remission. &amp;gt;75% of Trop2- models were insensitive to SG regardless of ER status. Conclusion: We screened 180 models in our XPDX breast cancer platform for Trop2 expression and compared expression with in vivo SG efficacy in 125 models. Analysis is underway to correlate receptor and molecular profiles with SG sensitivity in breast models and we are expanding expression and in vivo testing to additional indications. Citation Format: Alyssa Simonson, Johnnie Flores, Ebony Anderson, Crystal Moreno, George Plasko, Kyriakos P. Papadopoulos, Amita Patnaik, Drew Rasco, Gladys Rodriguez, Amy Lang, Muralidhar Beeram, Luis Rodriguez, Ronald Drengler, Steven Abbate, Hanni Salih, Lon Smith, Maryam Elmi, Brittany DeBerry, Arthur Rosenthal, Tatiana Hernandez, Nehal Lakhani, Manish Sharma, Michael Wick. Correlation of Trop2 expression with in vivo sensitivity to sacituzumab govitecan in a panel of breast XPDX models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-05.

Abstract P1-13-03: Establishment and characterization of two T-DM1-resistant, ER+/HER2+ breast XPDX models developed sequentially from the same patient with differential in vivo sensitivity to trastuzumab deruxtecan (DS-8201a)

Abstract Background: Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC), consisting of a humanized anti-HER2 (human epidermal growth factor receptor 2) monoclonal antibody linked to a topoisomerase I inhibitor payload using a cleavable tetrapeptide-based linker, approved for the treatment of HER2+ metastatic breast cancer patients refractory to anti-HER2 therapy including T-DM1. While some mechanisms for clinical T-DM1 resistance have been identified, less is known about innate or acquired resistance to DS-8201a. We established two XenoSTART Patient-Derived Xenograft (XPDX) models from tissue samples collected two years apart from a patient with ER+/HER2+ breast cancer before and after HER2 directed therapies. These models designated ST4565 and ST4565C were developed and characterized for receptor expression, genomic alterations, and in vivo drug sensitivities toward multiple chemotherapies and targeted agents, including T-DM1 and DS-8201a. Methods: Models ST4565 and ST4565C were established from breast samples collected from a Caucasian female with ER+/HER2+ metastatic breast cancer; ST4565 was collected at age 35 prior to therapy and ST4564C at age 37 following several treatment regimens including 5-FU/doxorubicin/cyclophosphamide, docetaxel/trastuzumab/pertuzumab, and T-DM1/anastrozole. Both were grown subcutaneously in female athymic nude mice supplemented with exogenous estradiol. The resulting models were passaged, and receptor expression confirmed immunohistochemically; genomic analysis, including WES and RNAseq, was performed to further characterize the models. For in vivo studies, both models were evaluated using several chemotherapy and targeted agents alone and in combination including: trastuzumab, pertuzumab, T-DM1, DS-8201a, neratinib, tucatinib, fulvestrant, alpelisib, sacituzumab, and irinotecan. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C=&amp;lt; 0) versus Day 0 tumor volume was also reported. Results: ST4565 and ST4565C retained comparable receptor expression (ER=2+/HER2=3+) over tested passages with similar histology compared to archival clinical samples. DNA/RNA sequencing identified several conserved variants including loss of CDKN2A/B and MTAP and a CNV=14 for CCND1. In vivo, ST4565 and ST4565C were found resistant to T-DM1 at 3 mg/kg weekly with a T/C of 100% in both models. However, DS-8201a treatment at 3 mg/kg weekly resulted in partial tumor regressions in ST4565 (T/C=-51%) while ST4565C was found resistant (T/C=49%). Both models were found resistant to all tested chemotherapies and all other targeted therapies but reported similar sensitivity to fulvestrant (T/C=~40%). Conclusion: We established two XPDX models representing T-DM1-resistant, ER+/HER2+ breast cancer from breast samples collected two years apart from the same patient that were found differentially responsive to DS-8201a. These models can be utilized as a valuable tool in better understanding innate resistance to T-DM1 and acquired resistance to DS-8201a. Citation Format: George Plasko, Johnnie Flores, Alyssa Simonson, Peter Forofontov, Ashwin Varma, Amy Lang, Gladys Rodriguez, Kyriakos P. Papadopoulos, Drew Rasco, Amita Patnaik, Bruce Conway, Joe Johnston, Michael Wick. Establishment and characterization of two T-DM1-resistant, ER+/HER2+ breast XPDX models developed sequentially from the same patient with differential in vivo sensitivity to trastuzumab deruxtecan (DS-8201a) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-03.

Abstract P1-13-22: Establishment and characterization of two ER+/HER2- XPDX models developed sequentially before and after acquired resistance to the CDK4/6 inhibitor palbociclib from a patient with metastatic breast cancer

Abstract Background: Several CDK4/6 inhibitors have recently been approved in combination with letrozole or fulvestrant in hormone receptor-positive breast cancer. Although this combination therapy has been found effective in some patients, resistance often develops. To aid in developing new therapies for CDK4/6i-resistant breast cancer and better understand potential resistance mechanisms, we established two XenoSTART Patient-Derived Xenograft (XPDX) models representing ER+/HER2- breast cancer from tissue samples collected seventeen months apart from the same patient before and after palbociclib therapy. These models designated ST4887 and ST4887B were developed and characterized for receptor expression, genomic alterations, and in vivo drug sensitivities toward multiple chemotherapies and targeted agents, including CDK4/6i and fulvestrant. Methods: Models ST4887 and ST4887B were established from metastatic samples collected from a Caucasian female with ER+/HER2- metastatic breast cancer; ST4887 was collected at age 38 from a femur mass biopsy following several treatment regimens including paclitaxel/doxorubicin/cyclophosphamide, radiation and tamoxifen. ST4887B was collected at age 39 from a liver biopsy following treatment with palbociclib/letrozole then palbociclib/fulvestrant, and finally ixabepilone/capecitabine. Both were grown subcutaneously in female athymic nude mice supplemented with exogenous estradiol. The resulting models were passaged, and receptor expression confirmed immunohistochemically; genomic analysis, including WES and RNAseq, was performed to further characterize models. For in vivo studies, both models were evaluated using several chemotherapy and targeted agents alone and in combination including cisplatin, docetaxel, CDK4/6i, fulvestrant, letrozole, olaparib, niraparib, and sacituzumab. In vivo study endpoints included tumor volume and time from treatment initiation with %T/C values and tumor regression reported at study completion; a T/C of ≤ 20% versus control was considered sensitive. Tumor regression (%T/C=&amp;lt; 0) versus Day 0 tumor volume was also reported. Results: ST4887 and ST4887B retained comparable receptor expression (ER=3+/HER2=1+) over tested passages with similar histology compared to archival clinical samples. DNA/RNA sequencing identified several conserved variants including a somatic BRCA2 truncation (BRCA2Y2660*); transcriptomic analysis revealed upregulation of several related genes but no notable fusions. In vivo, both models were insensitive to cisplatin or docetaxel, however ST4887 but not ST4887B was sensitive to fulvestrant or CDK4/6i therapies, although abemaciclib demonstrated some activity toward ST4887B. PARP inhibitors were active toward ST4887 and to a lesser extent ST4887B, while sacituzumab did not have a significant effect on either model. Conclusion: We established and characterized two XPDX models from the same patient before and after acquired resistance to the CDK4/6i palbociclib. Both models were found to retain receptor status and drug sensitivities similar to the patient at the time of sample collection. These models can be utilized as a valuable tool in better understanding acquired resistance to palbociclib. Citation Format: Ashwin Varma, Johnnie Flores, Alyssa Simonson, Anna Stackpole, Kyriakos P. Papadopoulos, Amita Patnaik, Drew Rasco, Muralidhar Beeram, Marisa Sandera, Michael Wick. Establishment and characterization of two ER+/HER2- XPDX models developed sequentially before and after acquired resistance to the CDK4/6 inhibitor palbociclib from a patient with metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-22.

Abstract 12697: Pharmacological Inhibition of BAG3-HSP70 With the Proposed Breast Cancer Therapeutic JG-98: In-vivo Mice Study

Introduction: The co-chaperone Bcl2-associated athanogene-3 (BAG3), along with its cofactor heat shock protein 70 (HSP70), is fundamental for protein quality control and cell survival in a healthy heart. However, elevated expression of BAG3 is also associated with metastasis in breast and other cancers, and small molecules such as JG-98 that disrupt BAG3-HSP70 binding have been shown to reduce cancer cell proliferation. We previously showed JG-98 is cardiotoxic in neonatal rat ventricular myocytes. Hypothesis: As BAG3-HSP70 is fundamental for autophagic protein turnover, JG-98 will reduce tumor size but have cardiotoxic effects. Methods: BT474 Resistant breast cancer cells were injected bilaterally into mammary fat pads of female athymic nude mice. When tumors reached 25 mm 2 , mice received a baseline echocardiography and were randomized to intraperitoneal injections of either PBS/DMSO vehicle or 3 mg/kg JG-98 twice weekly for 3 weeks (n = 4/group) or 6 weeks (n=10/group). Tumor growth was monitored using calipers and echocardiography was performed prior to sacrifice. Results: After 3 weeks, tumor volume increased by ~44% in Vehicle treated mice but decreased by 17% in JG-98 treated mice, effectively halting tumor growth. However, cardiac echocardiography did not reveal any functional or structural differences between the vehicle and JG-98 treated mice after either 3 or 6 weeks. JG-98 did result in dysregulation of BAG3 and its interactome in the LV. JG-98 slightly increased BAG3 levels in the LV at 3 weeks, but slightly decreased levels at 6 weeks. This agrees with data showing acute stress upregulates BAG3, but chronic stress downregulates it. Similarly, we found altered protein expression of the BAG3 binding partners HSBP8, HSPB5, and HSP70. Conclusions: JG-98 had no overt cardiotoxic effects, suggesting the adult heart may be resistant to disrupted BAG3-HSP70. However, we also observed dysregulated protein levels in the BAG3-mediated autophagy pathways that could precede dysfunction with more chronic treatment. Furthermore, mutations in BAG3 or altered expression levels could sensitize individuals to JG-98 treatment. Further work is necessary to understand the cardiac impact of cancer therapeutics that target BAG3.

Molecular Analysis of Short- versus Long-Term Survivors of High-Grade Serous Ovarian Carcinoma.

Simple SummaryOvarian cancer is commonly associated with poor survival; patients with a diagnosis of high-grade serous ovarian carcinoma (HGSC) have an overall survival of 39% at 5 years. For reasons not well known, about 32% of ovarian cancer patients survive 10 years or more. In this study, our goal was to determine the molecular differences that drive long-term survival (LTS) in patients with HGSC. Indeed, this study shows that patients with LTS have a distinct pattern of gene expression, with TMEM62 being related to LTS. Increased TMEM62 expression led to decreased proliferation of ovarian cancer cells in vitro and decreased tumor burden in vivo.Despite having similar histologic features, patients with high-grade serous ovarian carcinoma (HGSC) often experience highly variable outcomes. The underlying determinants for long-term survival (LTS, ≥10 years) versus short-term survival (STS, <3 years) are largely unknown. The present study sought to identify molecular predictors of LTS for women with HGSC. A cohort of 24 frozen HGSC samples was collected (12 LTS and 12 STS) and analyzed at DNA, RNA, and protein levels. OVCAR5 and OVCAR8 cell lines were used for in vitro validation studies. For in vivo studies, we injected OVCAR8 cells into the peritoneal cavity of female athymic nude mice. From RNAseq analysis, 11 genes were found to be differentially expressed between the STS and LTS groups (fold change > 2; false discovery rate < 0.01). In the subsequent validation cohort, transmembrane protein 62 (TMEM62) was found to be related to LTS. CIBERSORT analysis showed that T cells (follicular helper) were found at higher levels in tumors from LTS than STS groups. In vitro data using OVCAR5 and OVCAR8 cells showed decreased proliferation with TMEM62 overexpression and positive correlation with a longevity-regulating pathway (KEGG HSA04213) at the RNA level. In vivo analysis using the OVCAR8-TMEM62-TetON model showed decreased tumor burden in mice with high- vs. low-expressing TMEM62 tumors. Our results demonstrate that restoring TMEM62 may be a novel approach for treatment of HGSC. These findings may have implications for biomarker and intervention strategies to help improve patient outcomes

Continuous monitoring of postirradiation reoxygenation and cycling hypoxia using electron paramagnetic resonance imaging.

Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO2) in tumor tissues. Human colorectal cancer cell lines, HT29 and HCT116, were used to induce tumor growth in female athymic nude mice. Tumors were irradiated with 3, 10, or 20 Gy using an x‐ray irradiator. Prior to each EPRI scan, magnetic resonance imaging (MRI) was performed to obtain T2‐weighted anatomical images for reference. The differences in the mean pO2 were determined through two‐tailed Student's t‐test and one‐way analysis of variance. The median pO2 60 min after irradiation was found to be lower in HCT116 than in HT29 (9.1 ± 1.5 vs. 14.0 ± 1.0 mmHg, p = 0.045). There was a tendency for delayed and incomplete recovery of pO2 in the HT29 tumor when a higher dose of irradiation (10 and 20 Gy) was applied. Moreover, there was a dose‐dependent increase in the hypoxic areas (pO2 < 10 mmHg) 2 and 24 h after irradiation in all groups. In addition, an area that showed pO2 fluctuation between hypoxia and normoxia (pO2 > 10 mmHg) was also identified surrounding the region with stable hypoxia, and it slightly enlarged after recovery from acute hypoxia. In conclusion, we demonstrated the reoxygenation phenomenon in an in vivo xenograft model study using EPRI. These findings may lead to new knowledge regarding the reoxygenation process and possibilities of a new radiation therapy concept, namely, reoxygenation‐based radiation therapy.