Background:Clinical linear accelerators are an accessible platform for preclinical research on the biological effects of ultra-rapid electron irradiation (FLASH). However, they are not inherently designed for the accurate pulse control required for experiments using a small number of relatively high-dose pulses, and available methods for beam control such as respiratory gating can be error-prone owing to system latency. Purpose:Here we experimentally characterize the temporal latency of the respiratory gating system for controlling beam-on and beam-off at the individual linac pulse level. We use this information to develop accurate pulse delivery methods for preclinical FLASH research. Methods and Materials:We used programmable controller boards and a relay circuit to monitor and control delivery of specific numbers of pulses through the built-in monitor chamber and respiratory gating system of a Varian Trilogy linac. We modeled system response latency as a normally distributed random variable and experimentally recorded the probability of successful pulse delivery and inhibition relative to the time of beam-on and beam-off request signals to derive the mean and standard deviation of latency times at different pulse repetition frequencies. We implemented two methods - an adaptive method using only the delivered-pulse signal, and a synchronization method additionally using the linac's internal pulse-timing signal - and characterized their performance for standard and customized pulse sequences. Results:The mean and standard deviation values of the respiratory gating latency at 60, 90 and 180 Hz pulse repetition frequency were respectively 2.0±0.8 ms, 2.1±2.8 ms, and 2.4±1.9 ms for beam-on and 1.3±0.9 ms, 1.9±2.9 ms, and 1.8±2.1 ms for beam-off. Beam-on and beam-off latencies were similar to each other, and similar across pulse repetition frequencies. Characterizing the latency parameters permitted choosing optimal timing parameters that maximized the rate of successfully delivering the desired number of pulses using both adaptive and synchronization methods, exceeding 99% at 90 Hz for both methods, and reaching 95% (adaptive) and 80% (synchronization) at 180 Hz. This also enabled successful implementation of custom pulse sequences not natively available on the system. Conclusions:We demonstrated that accounting for latency and/or using the ability to read the prior information on expected pulse timing can provide high accuracy in delivering specified numbers of pulses. This reliability is critical for accurate dose delivery in preclinical FLASH research of single fraction and especially fractionated dosing regimens. The ability to generate custom pulse sequences enables more detailed exploration of the temporal dependence of biological FLASH effects.

Radiation therapy (RT) is a central component of cancer treatment, including breast cancer, and primarily damages DNA in cancer cells, causing cell death. However, secondary effects of RT that may influence tumor and normal tissue both within and beyond the site of irradiation are not well characterized, particularly at the level of the circulating blood proteome. In this study, isotopic acrylamide labeling of proteins and high-resolution LC-MS/MS were used to analyze plasma proteins from breast cancer patients before, during, and after RT. Clustering analysis was used to group proteins according to how their levels changed over time. A total of 31 proteins were identified whose levels were altered during RT but returned to baseline following the conclusion of treatment, representing key parts of the immune response to RT. Another set of 31 proteins related to the extracellular matrix and other functions was altered during RT and remained altered after RT. Specific time points were also examined, and acute changes were identified at the start of RT in the levels of 63 proteins involved in coagulation, cell-cell adhesion, and carbohydrate metabolism. After the end of RT, the circulating levels of 23 extracellular matrix proteins remained altered. These LC-MS/MS findings were validated orthogonally by ELISAs of three proteins in a larger group of breast cancer patients undergoing RT. Overall, in this study, temporal changes in the circulating blood proteome of breast cancer patients undergoing RT were characterized, and these altered protein levels were linked to potential alterations in biological pathways.

This study demonstrates that FLASH radiotherapy reduces radiation-induced oral mucositis and tongue ulceration compared to conventional irradiation in both wild-type and Fanca-deficient mice. FLASH also decreased persistent gH2AX DNA damage within the tongue's basal epithelium, indicating that its normal tissue-sparing effects remain preserved despite impaired Fanconi anemia DNA repair pathways.

In the past decade, immunotherapies targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death 1 (PD-1), and PD-1 ligand (PD-L1) have been approved for solid tumors. However, some patients demonstrate suboptimal clinical outcomes due to resistance. The tumor microenvironment (TME) significantly affects the efficiency of immunotherapy by mediating interactions between tumor and non-tumor cells, including dendritic cells, T cells, B cells, macrophages, neutrophils, NK cells, and myeloid-derived suppressor cells (MDSCs). These non-tumor cells often exhibit two phenotypes with altered functions, and tumor cells drives their transition towards tumor promotion through tumor-education. Tumor-educated cells (TECs) are cells influenced by tumor cells, which acquire immune-suppressive phenotypes and promote tumor progression through resistance to anti-cancer therapies. These cells undergo modifications in response to signals from the tumor, which can influence their roles in tumor progression. Their dynamic interactions with tumor cells contribute to the reshaping of the TME, facilitating cancer growth and immune modulation. This review summarizes research on TECs in TME, explores mechanisms related to tumor education, and discusses their role in tumor progression and immunotherapy resistance. Additionally, potential therapeutic approaches targeting these cells are also reviewed, which may complement current treatment strategies.

The RNA demethylase enzyme FTO is emerging as an important oncogenic factor and therapeutic target in a variety of solid tumors. However, its effect on cancer metabolism remains largely unknown. Here we show that FTO promotes cysteine metabolism, a hallmark of metabolic rewiring in non-small cell lung cancer (NSCLC) cells. Mechanistically, FTO inhibition reduces the expression of multiple targets in the cysteine metabolism pathway including the cystine transporter SLC7A11 involved in exogenous cystine uptake as well as two key enzymes cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CTH) involved in de novo cysteine synthesis. Functionally, FTO inhibition reduces cystine uptake and NSCLC growth and survival in a homocysteine-dependent manner. As cysteine plays a crucial role in glutathione biosynthesis and maintenance of cellular redox homeostasis, FTO inhibition decreased glutathione levels and increased reactive oxygen species (ROS) levels in NSCLC cells. To therapeutically exploit the increased oxidative stress in NSCLC cells treated with FTO inhibitors, we combined FTO inhibition with radiation therapy. FTO inhibition enhanced the radiosensitivity of NSCLC cells. Our findings reveal a novel role for FTO in NSCLC cysteine metabolism and oxidative stress, highlighting the therapeutic potential of FTO inhibition as a strategy to enhance the efficacy of radiation therapy for the treatment of NSCLC. Nishanth Kuganesan, Stavros Melemenidis, Edward E. Graves, Erinn B. Rankin. Identifying and exploiting a novel role of FTO in promoting cysteine metabolism for NSCLC therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4678.

Despite aggressive chemoradiation treatment, the overall survival rate for patients with HPV- head and neck squamous cell carcinoma (HNSCC) remains poor, highlighting the urgent need for more effective drug-radiotherapy combinations to improve the therapeutic index of radiation therapy (RT). The fat mass and obesity-related gene (FTO) is emerging as a promising cancer therapeutic target; however, its role in the RT response has been underexplored. In our study, we found that both genetic and pharmacologic inhibition of FTO enhanced the efficacy of RT in human and mouse HNSCC tumor xenografts. Mechanistically, inhibition of FTO improved the RT response in HPV- HNSCC cells, which was associated with increased DNA damage, reduced efficiency of homology directed repair, and decreased formation of RAD51 homolog 1 (RAD51) foci. Importantly, pharmacologic inhibition of FTO did not exacerbate radiation-induced oral mucositis, a significant normal-tissue toxicity associated with HNSCC RT. In summary, our results indicate a role for FTO in regulating homologous recombination while identifying FTO as a potential therapeutic target to enhance the therapeutic index of RT in HPV- HNSCC treatment.

Monocytes infiltrating tumors acquire various states that distinctly impact cancer treatment. Here, we show that resistance of tumors to radiotherapy (RT) is controlled by the accumulation of monocyte-derived dendritic cells (moDCs). These moDCs are characterized by the expression of CD301b and have a superior capacity to generate regulatory T cells (Tregs). Accordingly, moDC depletion limits Treg generation and improves the therapeutic outcome of RT. Mechanistically, we demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from radioresistant tumor cells following RT is necessary for the accumulation of moDCs. Our results unravel the immunosuppressive function of moDCs and identify GM-CSF as an immunotherapeutic target during RT.

We report a low-cost protocol and platform for whole-abdomen 3D dynamic contrast-enhanced ultrasound (DCE-US) imaging in mice using a clinical matrix-array transducer. Background/Objectives: This platform addresses common limitations of preclinical ultrasound systems. In particular, these systems often lack real-time volumetric and molecular imaging capabilities. Methods: Using a modified silicone cup and water bath configuration, mice with dual subcutaneous tumors were imaged in vivo on a clinical EPIQ 7 system equipped with an X6-1 transducer. Results: Intravenous administration of targeted microbubbles enabled high-resolution, contrast-mode 3D imaging at multiple time points. Volumetric reconstructions captured both tumors and surrounding anatomy in a single scan, while time–intensity curves and Differential Targeted Enhancement (DTE) analysis revealed greater microbubble uptake in irradiated tumors, consistent with elevated P-selectin expression. Conclusions: This standardized imaging platform enables whole-abdomen molecular DCE-US in preclinical studies, facilitating intra-animal comparisons of vascular and molecular features across lesions or organs.

Configuring clinical linear accelerators (linacs) for ultra-high dose rate (UHDR) electron experiments typically requires invasive hardware manipulation and/or irreversible manufacturer modifications, limiting broader implementation. We present an independently developed UHDR electron configuration of a clinical TrueBeam linac that allows reversible switching between preclinical UHDR and conventional (CONV) modes using only non-invasive software settings. UHDR mode was achieved via service mode software with RF and beam current settings typical of a photon beam, the photon target and monitor chamber retracted, and a clinically unused low-energy scattering foil inserted. An external AC current transformer (ACCT) for beam monitoring, anatomy-specific collimator, and sample holder were mounted on the accessory tray, with external ion chamber in solid water for exit dose monitoring. Percent depth dose (PDD) was measured for UHDR and CONV beams. Dose-per-pulse (DPP) was varied by adjusting gun voltage and quantified with radiochromic film at different source-to-surface distances (SSD). Beam profiles assessed dose uniformity and usable field size. Dose calibration was established between film, ACCT, and ion chamber, and day-to-day reproducibility was tested. PDD confirmed similar energies for UHDR (12.8MeV) and CONV (11.9MeV) beams with matching profiles through mouse thickness. Maximum DPP exceeded 0.5Gy, reaching ~1.5Gy for collimated in vivo setups and ~0.7Gy at extended SSD for tissue culture. Field flatness and symmetry were maintained, supporting organ-specific irradiations and up to 5cm fields for culture. Calibration showed strong linearity across detectors, and output variation was <4%. We demonstrated accurate, reproducible UHDR delivery on a widely available clinical linac with no invasive hardware manipulation, enabling preclinical FLASH research on a clinical treatment machine.

Abstract Despite the use of intensive combined modality therapy such as chemoradiation or surgery and radiation, the overall survival rate for patients with HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. This underscores the unmet clinical need for the development of more effective drug-radiotherapy combinations to enhance the therapeutic index of radiation therapy (RT). The RNA demethylase enzyme FTO has emerged as a promising target for cancer therapy. However, its role as a radiosensitizer in HNSCC remains unknown. Here, we investigated the potential of targeting FTO to augment the effectiveness of RT in HPV-negative HNSCC. We found that genetic and pharmacologic inhibition of FTO enhanced the efficacy of RT in both human and mouse HNSCC tumor xenografts. Importantly, FTO inhibition did not increase radiation-induced oral mucositis. Mechanistically, FTO inhibition radiosensitizes HPV-negative HNSCC cells through amplifying DNA damage while reducing RAD51 foci formation, providing a molecular rationale for exploiting FTO inhibitors as radiation sensitizers in HNSCC. Collectively, our results suggest that therapeutic targeting of FTO may be an effective strategy to enhance the therapeutic index of RT for the treatment of HNSCC. Citation Format: Lu Ji, Leighton Pu, Hongbin Cao, Stavros Melemenidis, Subarna Sinha, Li Guan, Eyiwunmi E. Laseinde, Sara Richter, Rie Eyben, Kerriann M. Casey, Christina Kong, Edward Graves, Quynh-Thu Le, Erinn Rankin. FTO inhibition enhances the therapeutic index of radiation therapy in head and neck cancer. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B021.

Positron Emission Tomography (PET) imaging can monitor cancer treatment response by non-invasively detecting apoptosis in vivo. Signal-to-noise (SNR) remains one of the critical barriers to approval for clinical use. We have previously developed a PET tracer [18 F]-C-SNAT4 for imaging capase-3 activity in apoptotic tumors induced by chemo- and immunotherapy. [18 F]-C-SNAT4 is designed to undergo caspase-3 activated intramolecular cyclization. The product then self-assembles in situ into nanoparticles to generate preferential retention of F18 radioactivity in apoptotic cells. This unique mechanism prompted us to investigate if a cold mixture could enhance the probe retention and further augment the sensitivity for imaging radiotherapy. [18 F]-C-SNAT4 and hot/cold mixture [18 F]/[19 F]-C-SNAT4 were used to detect human NSCLC (NCI-H460) apoptosis induced by radiation. Both hot [18 F]-C-SNAT4 and hot/cold mixture [18 F]/[19 F]-C-SNAT4 had significantly increased uptake in radiation treated vs. untreated NCI-H460 cells in vitro. A 1: 80 hot/cold mixture increased signal by 1.6x compared to [18 F]-C-SNAT4 alone. In vivo studies were performed in murine xenograft models in high-dose radiation and low-dose radiation treatment groups. The hot/cold mixture showed an increase in the signal by 2.5x in high-dose radiation treated murine NCI-H460 xenograft models. Low-dose radiation induced apoptosis was only detected with the hot/cold mixture with 2.4x signal compared to hot [18 F]-C-SNAT4. Toxicity and dosimetry safety were evaluated at 250x and 10x respective dosages, then normalized to human dose equivalent. A hot/cold mixture of [18 F]/[19 F]-C-SNAT4 generates significantly more signal compared to hot [18 F]-C-SNAT4, leading to higher sensitivity in detecting treatment response. This may present a solution to low sensitivity in the translation of apoptosis-specific radionuclides to clinical application.

Abstract Radiation therapy (RT) can activate both innate and adaptive immune responses, and the combination of RT and immunotherapy may produce synergistic anti-tumor responses. We found that the “don’t-eat-me” molecule CD47 is highly expressed on the surface of small cell lung cancer (SCLC) cells, a highly metastatic form of lung cancer, and that blockade of CD47 can enhance phagocytosis of SCLC cells by macrophages. In this study, we investigated whether combining CD47 blockade and RT could synergize to inhibit the tumor growth of SCLC as well as other cancer types in preclinical models. We evaluated the immune responses induced by RT and CD47 blockade and their efficacy in controlling tumors in preclinical models of SCLC as well as colon cancer, breast cancer and lymphoma. Cancer cells were engrafted into both flanks of recipient mice and one side was irradiated, with or without CD47 antibodies, to investigate local and systemic effects of RT and CD47 blockade. We also used liver metastases models and endogenous lung tumor models to investigate anti-tumor effects and immune responses in physiological tumor microenvironments. Various radiation doses and fractionation schedules were evaluated to determine the optimal conditions for inducing anti-tumor responses. We found CD47 blockade potently enhances the local anti-tumor effects of RT in all the preclinical models of SCLC we tested. Strikingly, CD47 blockade also stimulates systemic “abscopal” effects inhibiting non-irradiated SCLC tumors in mice receiving RT. These effects are observed in liver metastases, endogenous lung tumors, and subcutaneous tumor models. Surprisingly, these abscopal effects are independent of T cells but require macrophages that migrate into non-irradiated tumor sites in response to inflammatory signals produced by RT and are locally activated by CD47 blockade to phagocytose cancer cells. Similar abscopal anti-tumor effects were observed in other cancer models treated with RT and CD47 blockade. Additionally, we observed that RT increases tumor infiltrating macrophages in human cancer patients. Interestingly, these abscopal anti-tumor effects can be enhanced when combined with PD-1 blockade. Furthermore, we found that a wide range of radiation doses, from 2 Gy to 20 Gy, can induce abscopal responses, with fractionated doses (24 Gy in 3 fractions) inducing stronger abscopal effects than single doses. Our data demonstrate macrophage-mediated abscopal responses following RT when combined with CD47 blockade across multiple cancer models. This systemic activation of anti-tumor macrophages following RT and CD47 blockade may be particularly impactful for cancer patients who suffer from metastatic disease. Given that RT is a standard-of-care treatment and CD47-blocking strategies are undergoing clinical trials, our findings hold significant translational potential for cancer patients. Citation Format: Yoko Nishiga, Edward Graves, Julien Sage.Optimizing macrophage-mediated abscopal effects for enhanced clinical translation: Radiation therapy combined with CD47 blockade.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P016

Introduction: Radiotherapy is effective for breast cancer treatment but often causes undesirable side effects that impair quality of life. Ultra-high dose rate radiotherapy (FLASH) has shown reduced normal tissue toxicity while achieving comparable tumor growth delay compared to conventional dose rate radiotherapy (CONV). This study evaluated whether FLASH could achieve similar tumor control as CONV with tumor eradication as the primary endpoint, in an orthotopic breast cancer model. Methods: Non-metastatic, orthotopic tumors were generated in the left fourth mammary fat pad using the Py117 mammary tumor cell line in syngeneic C57BL/6J mice. Two sequential irradiation studies were performed using FLASH (93-200 Gy/s) and CONV (0.08 Gy/s) electron beams. Single fractions of 20, 25, or 30 Gy were applied to tumors with varying abdominal wall treatment fields (~3.75 or 2.5 mm treatment margin to tumor). Results: Both FLASH and CONV demonstrated comparable efficacy. Small tumors treated with 30 Gy and larger abdominal wall treatment fields appeared to have complete eradication at 30 days but also exhibited the highest skin toxicity, limiting follow-up and preventing confirmation of eradication. Smaller abdominal wall treatment fields reduced skin toxicity and allowed for extended follow-up, which resulted in 75% tumor-free survival at 48 days. Larger tumors showed growth delay but no eradication. Conclusions: In this preclinical, non-metastatic orthotopic breast cancer model, FLASH and CONV demonstrated equivalent tumor control with single-fraction doses of 20, 25, or 30 Gy. Overall, 30 Gy achieved the highest eradication rate but also resulted in the most pronounced skin toxicity.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are successful in treating many cancers but may cause immune-related adverse events. ICI-mediated myocarditis has a high fatality rate with severe cardiovascular consequences. Targeted therapies for ICI myocarditis are currently limited. METHODS: We used a genetic mouse model of PD1 deletion ( MRL/Pdcd1 −/− ) along with a novel drug-treated ICI myocarditis mouse model to recapitulate the disease phenotype. We performed single-cell RNA-sequencing, single-cell T-cell receptor sequencing, and cellular indexing of transcriptomes and epitopes on immune cells isolated from MRL and MRL/Pdcd1 −/− mice at serial time points. We assessed the impact of macrophage deletion in MRL/Pdcd1 −/− mice, then inhibited CXCR3 (C-X-C motif chemokine receptor 3) in ICI-treated mice to assess the therapeutic effect on myocarditis phenotype. Furthermore, we delineated the functional and mechanistic effects of CXCR3 blockade on T-cell and macrophage interactions. We then correlated the results in human single-cell multiomics data from blood and heart biopsy data from patients with ICI myocarditis. RESULTS: Single-cell multiomics demonstrated expansion of CXCL (C-X-C motif chemokine ligand) 9/10+CCR2+ macrophages and CXCR3hi (C-X-C motif chemokine receptor 3 high-expressing) CD8+ (cluster of differentiation) effector T lymphocytes in the hearts of MRL/Pdcd1 −/− mice correlating with onset of myocarditis development. Both depletion of CXCL9/10+CCR2+ (C-C motif chemokine receptor) macrophages and CXCR3 blockade, respectively, led to decreased CXCR3hi CD8+ T-cell infiltration into the heart and significantly improved survival. Transwell migration assays demonstrated that the selective blockade of CXCR3 and its ligand, CXCL10, reduced CXCR3+CD8+ T-cell migration toward macrophages, implicating this interaction in T-cell cardiotropism toward cardiac macrophages. Furthermore, cardiomyocyte apoptosis was induced by CXCR3hi CD8+ T cells. Cardiac biopsies from patients with confirmed ICI myocarditis demonstrated infiltrating CXCR3+ T cells and CXCL9+/CXCL10+ macrophages. Both mouse cardiac immune cells and patient peripheral blood immune cells revealed expanded TCR s (T-cell receptors) correlating with CXCR3hi CD8+ T cells in ICI myocarditis samples. CONCLUSIONS: These findings bring forth the CXCR3-CXCL9/10 axis as an attractive therapeutic target for ICI myocarditis treatment, and more broadly as a druggable pathway in cardiac inflammation.

Despite the use of intensive combined modality therapy such as chemoradiation or surgery and radiation, the overall survival rate for patients with HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. This underscores the unmet clinical need for the development of more effective drug-radiotherapy combinations to enhance the therapeutic index of radiation therapy (RT). The RNA demethylase enzyme FTO has emerged as a promising target for cancer therapy. However, its role as a radiosensitizer in HNSCC remains unknown. Here, we investigated the potential of targeting FTO to augment the effectiveness of RT in HPV-negative HNSCC. We found that genetic and pharmacologic inhibition of FTO enhanced the efficacy of RT in both human and mouse HNSCC tumor xenografts. Importantly, FTO inhibition did not increase radiation-induced oral mucositis. Mechanistically, FTO inhibition radiosensitizes HPV-negative HNSCC cells through amplifying DNA damage while reducing RAD51 foci formation, providing a molecular rationale for exploiting FTO inhibitors as radiation sensitizers in HNSCC. Collectively, our results suggest that therapeutic targeting of FTO may be an effective strategy to enhance the therapeutic index of RT for the treatment of HNSCC. Lu Ji, Leighton Pu, Hongbin Cao, Stavros Melemenidis, Subarna Sinha, Li Guan, Eyiwunmi E. Laseinde, Sara Richter, Rie Eyben, Kerriann Casey, Christina Kong, Edward Graves, Quynh Le, Erinn Rankin. FTO inhibition enhances the therapeutic index of radiation therapy in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4679.

Our data clearly demonstrate macrophage-mediated abscopal responses of RT when combined with CD47 blockade in a range of cancer models. The systemic activation of antitumor macrophages following radiation and CD47 blockade may be particularly important in cancer patients who suffer from metastatic disease. RT is a part of standard-of-care and CD47-blocking strategies are in clinical trials, therefore our observations may be rapidly translatable to cancer patients.

Purpose/Objective(s) Longitudinal monitoring of tumor volume is a fundamental component of preclinical biomedical research. We explored the use of inexpensive portable ultrasound (US) probes as a viable, cost-and time-efficient alternative to the standard caliper-based volume measurements in laboratory settings, aiming to compare these methods and establish a more precise measurement technique. Materials/Methods 5 BALB/c and 5 C57Bl6/J mice were shaved and injected subcutaneously with 105 colon (CT26) or lung (LLC1) carcinoma cells on the flank. At day 14, tumor length and width (LxW) were measured with either calipers or a portable handheld US probe (frequency: 8-20MHz, maximum depth: 4cm), which provided 2D images across the LxW. Tumors were subsequently excised and imaged with cone-beam computed tomography (CBCT) to obtain a gold-standard volume measurement. The widely-used ellipsoid volume formula was applied to US- and caliper-derived LxW as well as to LxW and the US-derived tumor depth (D) measured from the orthogonal cross-section. In addition to these methods, we developed a novel algorithm designed to estimate tumor volumes from regions of interest (ROIs) defined on two orthogonal US cross sections. Results The CBCT volumes ranged from 30-170mm3, with an average volume of 82.8 ± 49.6mm3. The average of the caliper-derived volume was 206% ± 76% of the reference volume, the average US-derived volume estimated from the caliper formula was 106% ± 41%, and the average volume from the US-derived LxWxD applied to the traditional ellipsoid formula was 90% ± 34%. The average from the ROI-derived volumes from the 2 orthogonal US views was 94% ± 12%. Conclusion Our study demonstrates the feasibility of rapid US-based measurement of tumor volumes that offers improved accuracy relative to standard caliper-based approaches. The adoption of a portable handheld US probe for acquiring width, length, and depth measurements enhances the precision of the average tumor volume estimations by ∼100% when applied to the ellipsoid formula. The calculated volumes from the ROIs using two orthogonal US views closely align with this improved performance and exhibit the least variability across tumors, suggesting that this approach is better suited for tumors with irregular shapes. Although image collection with US was initially two times lengthier than caliper measurements, we anticipate that with increased user proficiency, the time required could decrease further.