Changes In Tumor Oxygenation Research Articles

Combined Oxygen-Enhanced MRI and Perfusion Imaging Detect Hypoxia Modification from Banoxantrone and Atovaquone and Track Their Differential Mechanisms of Action.

Oxygen-enhanced MRI (OE-MRI) has shown promise for quantifying and spatially mapping tumor hypoxia, either alone or in combination with perfusion imaging. Previous studies have validated the technique in mouse models and in patients with cancer. Here, we report the first evidence that OE-MRI can track change in tumor oxygenation induced by two drugs designed to modify hypoxia. Mechanism of action of banoxantrone and atovaquone were confirmed using in vitro experiments. Next, in vivo OE-MRI studies were performed in Calu6 and U87 xenograft tumor models, alongside fluorine-18-fluoroazomycin arabinoside PET and immunohistochemistry assays of hypoxia. Neither drug altered tumor size. Banoxantrone reduced OE-MRI hypoxic fraction in Calu6 tumors by 52.5% ± 12.0% (P = 0.008) and in U87 tumors by 29.0% ± 15.8% (P = 0.004) after 3 days treatment. Atovaquone reduced OE-MRI hypoxic fraction in Calu6 tumors by 53.4% ± 15.3% (P = 0.002) after 7 days therapy. PET and immunohistochemistry provided independent validation of the MRI findings. Finally, combined OE-MRI and perfusion imaging showed that hypoxic tissue was converted into necrotic tissue when treated by the hypoxia-activated cytotoxic prodrug banoxantrone, whereas hypoxic tissue became normoxic when treated by atovaquone, an inhibitor of mitochondrial complex III of the electron transport chain. OE-MRI detected and quantified hypoxia reduction induced by two hypoxia-modifying therapies and could distinguish between their differential mechanisms of action. These data support clinical translation of OE-MRI biomarkers in clinical trials of hypoxia-modifying agents to identify patients demonstrating biological response and to optimize treatment timing and scheduling. Significance: For the first time, we show that hypoxic fraction measured by oxygen-enhanced MRI (OE-MRI) detected changes in tumor oxygenation induced by two drugs designed specifically to modify hypoxia. Furthermore, when combined with perfusion imaging, OE-MRI hypoxic volume distinguished the two drug mechanisms of action. This imaging technology has potential to facilitate drug development, enrich clinical trial design, and accelerate clinical translation of novel therapeutics into clinical use.

Influence of Phase Change Droplet Activation and Microbubble Cavitation on the Microenvironment of Hepatocellular Carcinoma

ObjectiveBoth microbubble ultrasound contrast agents and acoustic phase change droplets (APCD) have been explored in hepatocellular carcinoma (HCC). This work aimed to evaluate changes to the HCC microenvironment following either microbubble or APCD destruction in a syngeneic pre-clinical model. MethodsMouse RIL-175 HCC tumors were grown in the right flank of 64 immunocompetent mice. Pre-treatment, photoacoustic volumetric tumor oxygenation, and power Doppler measurements were obtained using a Vevo 3100 system (VisualSonics, Toronto, Canada). The experimental groups received a 0.1 mL bolus injection of either Definity ultrasound contrast agent (Lantheus Medical Imaging) or APCD fabricated by condensing Definity. Following injection, ultrasound destruction was performed using flash-replenishment sequences on a Sequoia with a 10L4 probe (Siemens) for the duration of enhancement. Tumor oxygenation and power Doppler measurements were then repeated immediately post-ultrasound treatment. Twenty-four hours post-treatment, animals were euthanized, and tumors were harvested and stained for CD31, Cleaved Caspase 3 and CD45. ResultsImaging biomarkers demonstrated a significant reduction in percent vascularity following either microbubble or APCD destruction in the tumor microenvironment ( p < 0.022) but no significant changes in tumor oxygenation (p = 0.12). Similarly, immunohistochemistry data demonstrated a significant decrease in CD31 expression (p < 0.042) and an increase in apoptosis (p < 0.014) in tumors treated with destroyed microbubbles or APCD relative to controls. Finally, a significant increase in CD45 expression was observed in tumors treated with APCD (p = 0.046), indicating an increase in tumor immune response. ConclusionUltrasound-triggered destruction of both microbubbles and APCD reduces vascularity, increases apoptosis, and may also increase immune response in this HCC model.

EPR Monitoring of Oxygenation Levels in Tumors After Chlorophyllide-Based Photodynamic Therapy May Allow for Early Prediction of Treatment Outcome

PurposeMolecular oxygen, besides a photosensitizer and light of appropriate wavelength, is one of the three factors necessary for photodynamic therapy (PDT). In tumor tissue, PDT leads to the killing of tumor cells, destruction of endothelial cells and vasculature collapse, and the induction of strong immune responses. All these effects may influence the oxygenation levels, but it is the vasculature changes that have the main impact on pO2. The purpose of our study was to monitor changes in tumor oxygenation after PDT and explore its significance for predicting long-term treatment response.ProceduresElectron paramagnetic resonance (EPR) spectroscopy enables direct, quantitative, and sequential measurements of partial pressure of oxygen (pO2) in the same animal. The levels of chlorophyll derived photosensitizers in tumor tissue were determined by transdermal emission measurements.ResultsThe noninvasive monitoring of pO2 in the tumor tissue after PDT showed that the higher ΔpO2 (pO2 after PDT minus pO2 before PDT), the greater the inhibition of tumor growth. ΔpO2 also correlated with higher levels of the photosensitizers in the tumor and with the occurrence of a severe edema/erythema after PDT.ConclusionMonitoring of PDT-induced changes in tumor oxygenation is a valuable prognostic factor and could be also used to identify potentially resistant tumors, which is important in predicting long-term treatment response.

Sensitive simultaneous measurements of oxygenation and extracellular pH by EPR using a stable monophosphonated trityl radical and lithium phthalocyanine

The monitoring of acidosis and hypoxia is crucial because both factors promote cancer progression and impact the efficacy of anti-cancer treatments. A phosphonated tetrathiatriarylmethyl (pTAM) has been previously described to monitor both parameters simultaneously, but the sensitivity to tackle subtle changes in oxygenation was limited. Here, we describe an innovative approach combining the pTAM radical and lithium phthalocyanine (LiPc) crystals to provide sensitive simultaneous measurements of extracellular pH (pHe) and pO2. Both parameters can be measured simultaneously as both EPR spectra do not overlap, with a gain in sensitivity to pO2 variations by a factor of 10. This procedure was applied to characterize the impact of carbogen breathing in a breast cancer 4T1 model as a proof-of-concept. No significant change in pHe and pO2 was observed using pTAM alone, while LiPc detected a significant increase in tumor oxygenation. Interestingly, we observed that pTAM systematically overestimated the pO2 compared to LiPc. In addition, we analyzed the impact of an inhibitor (UK-5099) of the mitochondrial pyruvate carrier (MPC) on the tumor microenvironment. In vitro, the exposure of 4T1 cells to UK-5099 for 24 h induced a decrease in pHe and oxygen consumption rate (OCR). In vivo, a significant decrease in tumor pHe was observed in UK-5099-treated mice, while there was no change for mice treated with the vehicle. Despite the change observed in OCR, no significant change in tumor oxygenation was observed after the UK-5099 treatment. This approach is promising for assessing in vivo the effect of treatments targeting tumor metabolism.

Radiosensitizing oxygenation changes in murine tumors treated with VEGF-ablation therapy are measurable using oxygen enhanced-MRI (OE-MRI)

PurposeThere is a significant need for a widely available, translatable, sensitive and non-invasive imaging biomarker for tumor hypoxia in radiation oncology. Treatment-induced changes in tumor tissue oxygenation can alter the sensitivity of cancer tissues to radiation, but the relative difficulty in monitoring the tumor microenvironment results in scarce clinical and research data. Oxygen-Enhanced MRI (OE-MRI) uses inhaled oxygen as a contrast agent to measure tissue oxygenation. Here we investigate the utility of dOE-MRI, a previously validated imaging approach employing a cycling gas challenge and independent component analysis (ICA), to detect VEGF-ablation treatment-induced changes in tumor oxygenation that result in radiosensitization. MethodsMurine squamous cell carcinoma (SCCVII) tumor-bearing mice were treated with 5 mg/kg anti-VEGF murine antibody B20 (B20-4.1.1, Genentech) 2–7 days prior to radiation treatment, tissue collection or MR imaging using a 7 T scanner. dOE-MRI scans were acquired for a total of three repeated cycles of air (2 min) and 100% oxygen (2 min) with responding voxels indicating tissue oxygenation. DCE-MRI scans were acquired using a high molecular weight (MW) contrast agent (Gd-DOTA based hyperbranched polygylcerol; HPG-GdF, 500 kDa) to obtain fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters derived from the MR concentration–time curves. Changes to the tumor microenvironment were evaluated histologically, with cryosections stained and imaged for hypoxia, DNA damage, vasculature and perfusion. Radiosensitizing effects of B20-mediated increases in oxygenation were evaluated by clonogenic survival assays and by staining for DNA damage marker γH2AX. ResultsTumors from mice treated with B20 exhibit changes to their vasculature that are consistent with a vascular normalization response, and result in a temporary period of reduced hypoxia. DCE-MRI using injectable contrast agent HPG-GDF measured decreased vessel permeability in treated tumors, while dOE-MRI using inhaled oxygen as a contrast agent showed greater tissue oxygenation. These treatment-induced changes to the tumor microenvironment result in significantly increased radiation sensitivity, illustrating the utility of dOE-MRI as a non-invasive biomarker of treatment response and tumor sensitivity during cancer interventions. ConclusionsVEGF-ablation therapy-mediated changes to tumor vascular function measurable using DCE-MRI techniques may be monitored using the less invasive approach of dOE-MRI, an effective biomarker of tissue oxygenation that can monitor treatment response and predict radiation sensitivity.

Impact of Neoadjuvant Bevacizumab on Neuroradiographic Response and Histological Findings Related to Tumor Stemness and the Hypoxic Tumor Microenvironment in Glioblastoma: Paired Comparison Between Newly Diagnosed and Recurrent Glioblastomas.

BackgroundPreviously, we reported that bevacizumab (Bev) produces histological and neuroradiographic alterations including changes in tumor oxygenation, induction of an immunosupportive tumor microenvironment, and inhibition of stemness. To confirm how those effects vary during Bev therapy, paired samples from the same patients with newly diagnosed glioblastoma (GBM) who received preoperative neoadjuvant Bev (neoBev) were investigated with immunohistochemistry before and after recurrence.MethodsEighteen samples from nine patients with newly diagnosed GBM who received preoperative neoBev followed by surgery and chemoradiotherapy and then autopsy or salvage surgery after recurrence were investigated. The expression of carbonic anhydrase 9 (CA9), hypoxia-inducible factor-1 alpha (HIF-1α), nestin, and Forkhead box M1 (FOXM1) was evaluated with immunohistochemistry.For comparison between neoBev and recurrent tumors, we divided the present cohort into two groups based on neuroradiographic response: good and poor responders (GR and PR, respectively) to Bev were defined by the tumor regression rate on T1-weighted images with gadolinium enhancement (T1Gd) and fluid-attenuated inversion recovery images. Patterns of recurrence after Bev therapy were classified as cT1 flare-up and T2-diffuse/T2-circumscribed. Furthermore, we explored the possibility of utilizing FOXM1 as a biomarker of survival in this cohort.ResultsA characteristic “pseudo-papillary”-like structure containing round-shaped tumor cells clustered adjacent to blood vessels surrounded by spindle-shaped tumor cells was seen only in recurrent tumors. Tumor cells at the outer part of the “pseudo-papillary” structure were CA9-positive (CA9+)/HIF-1α+, whereas cells at the inner part of this structure were CA9−/HIF-1α+ and nestin+/FOXM1+. CA9 and HIF-1α expression was lower in T1Gd-GR and decreased in the “T2-circumscribed/T2-diffuse” pattern compared with the “T1 flare-up” pattern, suggesting that tumor oxygenation was frequently observed in T1Gd-GR in initial tumors and in the “T2-circumscribed/T2-diffuse” pattern in recurrent tumors. FOXM1 low-expression tumors tended to have a better prognosis than that of FOXM1 high-expression tumors.ConclusionA “pseudo-papillary” structure was seen in recurrent GBM after anti-vascular endothelial growth factor therapy. Bev may contribute to tumor oxygenation, leading to inhibition of stemness and correlation with a neuroimaging response during Bev therapy. FOXM1 may play a role as a biomarker of survival during Bev therapy.

Abstract 5974: Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse

Abstract [Purpose] In pancreatic ductal adenocarcinoma (PDAC) which is characterized by an intense desmoplastic feature, the extracellular matrix (ECM) can significantly influence the tumor microenvironment (TME). Hyaluronan (HA), a major component of ECM, is associated with elevated tumor pressure, vascular collapse, and poor perfusion in TME, conferring hypoxia. HA expression is also correlated with poor prognosis in the patients with PDAC. PEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan in tumors. The resultant improvement in vascular patency and blood perfusion is expected to increase the delivery of therapeutic molecules. The aim of this study was to investigate the change in physiologic and metabolic profile of the tumor in response to treatment with PEGPH20 using multi-modal imaging techniques. We also investigated the capability of PEGPH20 to enhance treatment effect of radiation. [Methods] Athymic nude mice were inoculated with BxPC3 (human pancreatic adenocarcinoma) tumor cells transduced with hyaluronan synthase 3 (HAS3) to the right tibial periosteum. BxPC3-HAS3 tumor treated with PEGPH20 or control buffer were scanned with Electron paramagnetic Resonance imaging (EPRI), dynamic contrast enhanced (DCE) MRI, ultra-small superparamagnetic iron oxide (USPIO) MRI, Photoacoustic imaging (PAI), and Hyperpolarized 13C-MRI using [1-13C] pyruvate to evaluate intratumor pO2, intratumor perfusion, blood volume, O2 saturation, and glycolysis, respectively. [Results] EPRI showed significantly increased pO2 in PEGPH20 treated group. DCE-MRI and USPIO-MRI showed improved perfusion/permeability and local blood volume, respectively after PEGPH20 treatment, accounting for the increase in tumor oxygenation. PAI provided the evidence of immediate changes in tumor oxygenation after treatment. Hyperpolarized 13C-MRI using [1-13C] pyruvate suggested the decreased glycolytic flux evaluated by lactate/pyruvate ratio after PEGPH20 treatment. Combination of radiotherapy and PEGPH20 synergistically delayed tumor progression and prolonged the survival. [Conclusions] This study examined the effect of PEGPH20 on TME in PDAC xenograft model by using non-invasive multimodal imaging techniques. In summary, the non-invasive imaging modalities were useful in evaluating the changes in hemodynamics and metabolism in TME induced by modulation of ECM such as PEGPH20 treatment. PEGPH20 enhanced treatment effect of radiation therapy. The results validated the utility of the imaging methods to non-invasively monitor the changes in TME and predicted the radiosensitizing effect of hyaluronan depletion. Citation Format: Yu Saida, Tomohiro Seki, Shun Kishimoto, Jeffrey R. Brender, Gadisetti VR. Chandramouli, Yasunori Otowa, Kota Yamashita, Kazutoshi Yamamoto, Nallathamby Devasahayam, Murali C. Krishna. Multimodal molecular imaging detects early reoxygenation induced by hyaluronan depletion in pancreatic cancer model mouse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5974.

Abstract 3058: Molecular imaging of hypoxia to evaluate synergy between HER2 targeted therapy and fractionated radiation in murine models of HER2+ breast cancer

Abstract Introduction: Tumor hypoxia contributes to intra-tumoral heterogeneity and decreases efficacy of cytotoxic therapy. Fractionated radiation is a component of standard-of-care therapy for patients with unresectable human epidermal growth factor receptor 2 (HER2+) breast cancer. This study investigates if [18F]-fluoromisonidazole (FMISO)-PET imaging can quantify anti-HER2 therapy-induced changes in tumor oxygenation and utilize imaging metrics to monitor the synergy of combination targeted and fractionated radiation. Improving treatment synergy in HER2+ breast cancer has potential to increase therapeutic efficacy without increasing cytotoxic therapy. Methods: For in vivo studies, HER2+ cell line (BT474 and MDA-MB-361) and HER2+ patient derived xenograft (BCM 3472) tumor models were engrafted in mice and treated with single agent or combination trastuzumab (4 mg/kg) and fractionated radiation (6/3 Gy). Mice were imaged with FMISO-PET imaging on days 0, 3, and 7 to monitor changes in tumor hypoxia. Hypoxic fraction within the tumor was quantified by a voxel-wise basis with a hypoxic threshold of mean standardized uptake value (SUV) of the muscle + 1 STD. Tumors were either extracted for biological validation of imaging or continued to measure tumor size changes for longitudinal assessment of response. Immunohistochemical analysis included staining for pimonidazole (hypoxia), CD-31 (microvessel), cleaved caspase-3 (apoptosis) and Ki-67 (proliferation). Bliss test of independence and a non-parametric T-test was used to assess for treatment synergy and significance, respectively. Results: In vivo, MDA-MB-361 tumors treated with trastuzumab and fractionated radiation revealed a SUVmean of 0.20 ± 0.09 on day 7, while tumors treated with fractionated radiation had a FMISO SUVmean of 0.31 ± 0.06 (p=0.05). MDA-MB-361 tumors treated with a control or fractionated radiation were 93.7% and 87.7% hypoxic, respectively; whereas, tumors treated with trastuzumab + fractionated radiation were significantly decreased (76.3% hypoxic, p=0.05 and p=0.26, respectively). 30 days after start of therapy, BCM3472 tumors treated with single agent fractionated radiation had a tumor volume of 285.3 ± 59 mm3, whereas tumors treated with trastuzumab and fractionated radiation had a tumor volume of 104 ± 24 mm3 (p&amp;lt;0.05). Treatment synergy started 14 days after start of therapy. Conclusion: HER2+ breast cancer treated with trastuzumab prior to fractionated radiation decreases hypoxia and synergistically increases efficacy of radiotherapy. FMISO-PET imaging has potential to identify in vivo response to combination therapies and better understand changes in the tumor microenvironment to guide combination therapy. Citation Format: Patrick Song, Yun Lu, Sharon Samuel, Katherine Heinzman, Suzanne Lapi, Anna Sorace. Molecular imaging of hypoxia to evaluate synergy between HER2 targeted therapy and fractionated radiation in murine models of HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3058.

Longitudinal monitoring of tumor response to immune checkpoint inhibitors using noninvasive diffuse reflectance spectroscopy.

Immune checkpoint inhibitors have revolutionized cancer treatment. However, there are currently no methods for noninvasively and nondestructively evaluating tumor response to immune checkpoint inhibitors. We used diffuse reflectance spectroscopy to monitor in vivo tumor microenvironmental changes in response to immune checkpoint inhibitors in a CT26 murine colorectal cancer model. Mice growing CT26 tumor xenografts were treated with either anti-PD-L1, anti-CTLA-4, a combination of both inhibitors, or isotype control on 3 separate days. Monotherapy with either anti-PD-L1 or anti-CTLA-4 led to a large increase in tumor vascular oxygenation within the first 6 days. Reoxygenation in anti-CTLA-4-treated tumors was due to a combination of increased oxygenated hemoglobin and decreased deoxygenated hemoglobin, pointing to a possible change in tumor oxygen consumption following treatment. Within the anti-PD-L1-treated tumors, reoxygenation was primarily due to an increase in oxygenated hemoglobin with the minimal change in deoxygenated hemoglobin, indicative of a likely increase in tumor perfusion. The tumors in the combined treatment group did not show any significant changes in tumor oxygenation following therapy. These studies demonstrate the sensitivity of diffuse reflectance spectroscopy to tumor microenvironmental changes following immunotherapy and the potential of such non-invasive techniques to determine early tumor response to immune checkpoint inhibitors.

Photoacoustic monitoring of oxygenation changes induced by therapeutic ultrasound in murine hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly vascular solid tumor. We have previously shown that ultrasound (US) therapy significantly reduces tumor vascularity. This study monitors US-induced changes in tumor oxygenation on murine HCC by photoacoustic imaging (PAI). Oxygen saturation and total hemoglobin were assessed by PAI before and after US treatments performed at different intensities of continuous wave (CW) bursts and pulsed wave (PW) bursts US. PAI revealed significant reduction both in HCC oxygen saturation and in total hemoglobin, proportional to the US intensity. Both CW bursts US (1.6 W/cm2) and the PW bursts US (0.8 W/cm2) significantly reduced HCC oxygen saturation and total hemoglobin which continued to diminish with time following the US treatment. The effects of US therapy were confirmed by power Doppler and histological examination of the hemorrhage in tumors. By each measure, the changes observed in US-treated HCC were more prevalent than those in sham-treated tumors and were statistically significant. In conclusion, the results show that US is an effective vascular-targeting therapy for HCC. The changes in oxygenation induced by the US treatment can be noninvasively monitored longitudinally by PAI without the use of exogenous image-enhancing agents. The combined use of PAI and the therapeutic US has potential for image-guided vascular therapy for HCC.

Abstract PS10-21: Identifying efficacy of targeted HER2 antibodies in sensitization of HER2 positive breast cancer to fractionated radiation

Abstract Introduction: Tumor hypoxia contributes to intra-tumoral heterogeneity and decreases efficacy of cytotoxic therapy. Fractionated radiation therapy has emerged as an alternative to single dose radiation, and is a component of standard-of-care therapy for patients with unresectable human epidermal growth factor receptor 2 (HER2+) breast cancer. Because radiation therapy is dependent on tissue oxygenation, therapies that increase oxygenation could radio-sensitize tumors. The goal of this study is to investigate if molecular imaging with [18F]-fluoromisonidazole (FMISO)-PET can quantify anti-HER2 therapy-induced changes in tumor oxygenation and utilize imaging metrics to enhance the effectiveness of fractionated radiation. Improving treatment synergy in HER2+ breast cancer has potential to increase therapeutic effectiveness without increasing cytotoxic therapy. Methods: For in vitro studies, HER2+ breast cancer cells (BT474, SKBR3, MDA-MB-361 and MDA-MB-453) were treated with various sequencing of combination trastuzumab (1 µg/mL) and fractionated radiation (6/3 Gy) and assessed for cell death. HER2+ cancer cells (BT474 and MDA-MB-361) were also treated with combination trastuzumab and fractionated radiation and analyzed for DNA double strand breaks (DSB) through flow cytometry. For in vivo studies, HER2+ cell line (BT474 and MDA-MB-361) and HER2+ patient derived xenograft (BCM 3472) tumors were engrafted into mice and treated with trastuzumab (4 mg/kg) on days 0 and 3 and fractionated radiation (6/3 Gy) on days 1, 2 and 3 (or single agent control). [18F]-FMISO-PET imaging was conducted on day 0, 3 and 7. At the imaging endpoint, tumors were either extracted for biological validation or continued to measure tumor size changes for longitudinal assessment of response. Bliss test of independence and a non-parametric T-test was used to assess for treatment synergy and significance, respectively. Results: In vitro cell death assay revealed single agent trastuzumab or fractionated radiation treated groups exhibited 15.2% ± 6.5% or 53.6% ± 9% cell death respectively, while trastuzumab prior to fractionated radiation groups exhibited 72.5% ± 2.5% cell death (p = 0.01) on day 7 in MDA-MB-361 cells. Flow cytometry analysis showed MDA-MB-361 cells treated with trastuzumab prior to fractionated radiation exhibited 62.4% ± 8.7% DSB, which significantly increased from single agent trastuzumab (0.74% ± 0.39%) or fractionated radiation groups (37.3% ± 7.3%) (p = 0.01). In vivo, MDA-MB-361 tumors treated with trastuzumab and fractionated radiation had a [18F]-FMISO SUVmean of 0.20 ± 0.09, while tumors treated with fractionated radiation had a [18F]-FMISO SUVmean of 0.31 ± 0.06 on day 7 (p = 0.05). MDA-MB-361 tumors treated with trastuzumab and fractionated radiation experienced a 26.1% ± 16.8% decrease in tumor volume, while tumors treated with single agent fractionated radiation experienced a 10.4% ± 2.8% decrease in tumor volume from day 0 to day 7 (p = 0.11). 30 days after start of therapy, MDA-MB-361 tumors treated with single agent fractionated radiation had a tumor volume of 471.8 ± 120 mm3, whereas tumors treated with combination trastuzumab and fractionated radiation had a tumor volume of 116 ± 38 mm3 (p &amp;lt; 0.01). Bliss test of independence confirmed in vivo treatment synergy of trastuzumab and fractionated radiation starting 14 days after start of therapy. Conclusion: HER2+ breast cancer treated with trastuzumab prior to fractionated radiation synergistically increases efficacy of radiotherapy in vitro and in vivo. [18F]-FMISO-PET imaging has potential to identify in vivo response to combination therapies and better understand changes in the tumor microenvironment to guide combination therapy. Acknowledgements: We thank the American Cancer Society for RSG-18-006-01-CCE and NIH NCI R01 CA240589. Citation Format: Patrick N. Song, Yun Lu, Tiara Napier, Sharon Samuel, Katherine Heinzman, Suzanne E. Lapi, Anna G. Sorace. Identifying efficacy of targeted HER2 antibodies in sensitization of HER2 positive breast cancer to fractionated radiation [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-21.

Combined endogenous MR biomarkers to assess changes in tumor oxygenation induced by an allosteric effector of hemoglobin.

Hypoxia is a crucial factor in cancer therapy, determining prognosis and the effectiveness of treatment. Although efforts are being made to develop methods for assessing tumor hypoxia, no markers of hypoxia are currently used in routine clinical practice. Recently, we showed that the combined endogenous MR biomarkers, R1 and R2*, which are sensitive to [dissolved O2] and [dHb], respectively, were able to detect changes in tumor oxygenation induced by a hyperoxic breathing challenge. In this study, we further validated the ability of the combined MR biomarkers to assess the change in tumor oxygenation induced by an allosteric effector of hemoglobin, myo‐inositol trispyrophosphate (ITPP), on rat tumor models. ITPP induced an increase in tumor pO2, as observed using L‐band electron paramagnetic resonance oximetry, as well as an increase in both R1 and R2* MR parameters. The increase in R1 indicated an increase in [O2], whereas the increase in R2* resulted from an increase in O2 release from blood, inducing an increase in [dHb]. The impact of ITPP was then evaluated on factors that can influence tumor oxygenation, including tumor perfusion, saturation rate of hemoglobin, blood pH and oxygen consumption rate (OCR). ITPP decreased blood [HbO2] and significantly increased blood acidity, which is also a factor that right‐shifts the oxygen dissociation curve. No change in tumor perfusion was observed after ITPP treatment. Interestingly, ITPP decreased OCR in both tumor cell lines. In conclusion, ITPP increased tumor pO2 via a combined mechanism involving a decrease in OCR and an allosteric effect on hemoglobin that was further enhanced by a decrease in blood pH. MR biomarkers could assess the change in tumor oxygenation induced by ITPP. At the intra‐tumoral level, a majority of tumor voxels were responsive to ITPP treatment in both of the models studied.

Quantitative diffuse reflectance spectroscopy of short-term changes in tumor oxygenation after radiation in a matched model of radiation resistance.

There is a critical need to identify patients with radiation-resistant tumors early after treatment commencement. In this study, we use diffuse reflectance spectroscopy (DRS) to investigate changes in vascular oxygenation and total hemoglobin concentration in A549 radiation-sensitive and resistant tumors treated with a clinically relevant dose fraction of 2 Gy. DRS spectra were acquired before, immediately after, 24, and 48 hours after radiation. Our data reveals a significantly higher reoxygenation (sO2) in the radiation-resistant tumors 24 and 48h after treatment, and provides promising evidence that DRS can discern between the reoxygenation trends of radiation-sensitive and resistant tumors.

Photoacoustic imaging to assess pixel-based sO2 distributions in experimental prostate tumors.

A protocol for photoacoustic imaging (PAI) has been developed to assess pixel-based oxygen saturation (sO2) distributions of experimental tumor models. The protocol was applied to evaluate the dependence of PAI results on measurement settings, reproducibility of PAI, and for the characterization of the oxygenation status of experimental prostate tumor sublines (Dunning R3327-H, -HI, -AT1) implanted subcutaneously in male Copenhagen rats. The three-dimensional (3-D) PA data employing two wavelengths were used to estimate sO2 distributions. If the PA signal was sufficiently strong, the distributions were independent from signal gain, threshold, and positioning of animals. Reproducibility of sO2 distributions with respect to shape and median values was demonstrated over several days. The three tumor sublines were characterized by the shapes of their sO2 distributions and their temporal response after external changes of the oxygen supply (100% O2 or air breathing and clamping of tumor-supplying artery). The established protocol showed to be suitable for detecting temporal changes in tumor oxygenation as well as differences in oxygenation between tumor sublines. PA results were in accordance with histology for hypoxia, perfusion, and vasculature. The presented protocol for the assessment of pixel-based sO2 distributions provides more detailed information as compared to conventional region-of-interest-based analysis of PAI, especially with respect to the detection of temporal changes and tumor heterogeneity.

Mathematical Description of Changes in Tumour Oxygenation from Repeated Functional Imaging.

Functional imaging of tumour hypoxia has been suggested as a tool for refining target definition and treatment optimization in radiotherapy. The approach, however, has been slow to be adopted clinically as most of the studies on the topic do not take into account the in-treatment changes of hypoxia. The present study aimed to introduce a function that quantifies the changes of oxygen distributions in repeated PET images taken during treatment. The proposed approach for determining the reoxygenation function was tested for feasibility on patients with head and neck cancer, repeatedly imaged with FMISO PET during radiotherapy. Reoxygenation functions were derived by solving the convolution between functions describing the oxygen distributions of successive images. The method was found to be mathematically feasible. The results indicate that the reoxygenation functions describing the change in oxygenation have distinct shapes prompting the hypothesis that oxygenation changes reflected by them might have predictive power for treatment outcome. Future studies on a larger patient population to search for predictive correlations based on the reoxygenation function are planned.

Photoacoustic signal characterization of cancer treatment response: Correlation with changes in tumor oxygenation

Frequency analysis of the photoacoustic radiofrequency signals and oxygen saturation estimates were used to monitor the in-vivo response of a novel, thermosensitive liposome treatment. The liposome encapsulated doxorubicin (HaT-DOX) releasing it rapidly (<20s) when the tumor was exposed to mild hyperthermia (43°C). Photoacoustic imaging (VevoLAZR, 750/850nm, 40MHz) of EMT-6 breast cancer tumors was performed 30min pre- and post-treatment and up to 7days post-treatment (at 2/5/24h timepoints). HaT-DOX-treatment responders exhibited on average a 22% drop in oxygen saturation 2h post-treatment and a decrease (45% at 750nm and 73% at 850nm) in the slope of the normalized PA frequency spectra. The spectral slope parameter correlated with treatment-induced hemorrhaging which increased the optical absorber effective size via interstitial red blood cell leakage. Combining frequency analysis and oxygen saturation estimates differentiated treatment responders from non-responders/control animals by probing the treatment-induced structural changes of blood vessel.

Monitoring Tumor Response to Carbogen Breathing by Oxygen-Sensitive Magnetic Resonance Parameters to Predict the Outcome of Radiation Therapy: A Preclinical Study

In an effort to develop noninvasive invivo methods for mapping tumor oxygenation, magnetic resonance (MR)-derived parameters are being considered, including global R1, water R1, lipids R1, and R2*. R1 is sensitive to dissolved molecular oxygen, whereas R2* is sensitive to blood oxygenation, detecting changes in dHb. This work compares global R1, water R1, lipids R1, and R2* with pO2 assessed by electron paramagnetic resonance (EPR) oximetry, as potential markers of the outcome of radiation therapy (RT). R1, R2*, and EPR were performed on rhabdomyosarcoma and 9L-glioma tumor models, under air and carbogen breathing conditions (95% O2, 5% CO2). Because the models demonstrated different radiosensitivity properties toward carbogen, a growth delay (GD) assay was performed on the rhabdomyosarcoma model and a tumor control dose 50% (TCD50) was performed on the 9L-glioma model. Magnetic resonance imaging oxygen-sensitive parameters detected the positive changes in oxygenation induced by carbogen within tumors. No consistent correlation was seen throughout the study between MR parameters and pO2. Global and lipids R1 were found to be correlated to pO2 in the rhabdomyosarcoma model, whereas R2* was found to be inversely correlated to pO2 in the 9L-glioma model (P=.05 and .03). Carbogen increased the TCD50 of 9L-glioma but did not increase the GD of rhabdomyosarcoma. Only R2* was predictive (P<.05) for the curability of 9L-glioma at 40Gy, a dose that showed a difference in response to RT between carbogen and air-breathing groups. (18)F-FAZA positron emission tomography imaging has been shown to be a predictive marker under the same conditions. This work illustrates the sensitivity of oxygen-sensitive R1 and R2* parameters to changes in tumor oxygenation. However, R1 parameters showed limitations in terms of predicting the outcome of RT in the tumor models studied, whereas R2* was found to be correlated with the outcome in the responsive model.

Determination of real-time tumor oxygenation changes following high-dose radiotherapy in orthotopic and subcutaneous lung cancers in mice

Determination of real-time tumor oxygenation changes following high-dose radiotherapy in orthotopic and subcutaneous lung cancers in mice

Repeatability and sensitivity of T2* measurements in patients with head and neck squamous cell carcinoma at 3T.

PurposeTo determine whether quantitation of T2* is sufficiently repeatable and sensitive to detect clinically relevant oxygenation levels in head and neck squamous cell carcinoma (HNSCC) at 3T.Materials and MethodsTen patients with newly diagnosed locally advanced HNSCC underwent two magnetic resonance imaging (MRI) scans between 24 and 168 hours apart prior to chemoradiotherapy treatment. A multiple gradient echo sequence was used to calculate T2* maps. A quadratic function was used to model the blood transverse relaxation rate as a function of blood oxygenation. A set of published coefficients measured at 3T were incorporated to account for tissue hematocrit levels and used to plot the dependence of fractional blood oxygenation (Y) on T2* values, together with the corresponding repeatability range. Repeatability of T2* using Bland–Altman analysis, and calculation of limits of agreement (LoA), was used to assess the sensitivity, defined as the minimum difference in fractional blood oxygenation that can be confidently detected.Results T2* LoA for 22 outlined tumor volumes were 13%. The T2* dependence of fractional blood oxygenation increases monotonically, resulting in increasing sensitivity of the method with increasing blood oxygenation. For fractional blood oxygenation values above 0.11, changes in T2* were sufficient to detect differences in blood oxygenation greater than 10% (Δ T2* > LoA for ΔY > 0.1).ConclusionQuantitation of T2* at 3T can detect clinically relevant changes in tumor oxygenation within a wide range of blood volumes and oxygen tensions, including levels reported in HNSCC. J. Magn. Reson. Imaging 2016;44:72–80.

Impact of Oxygenation Status on 18F-FDG Uptake in Solid Tumors.

The influence of changes in tumor oxygenation (monitored by EPR oximetry) on the uptake of 18F-FDG tracer was evaluated using micro-PET in two different human tumor models. The 18F-FDG uptake was higher in hypoxic tumors compared to tumors that present a pO2 value larger than 10 mmHg.