https://openworks.mdanderson.org/sumexp22/1035/thumbnail.jp

Chin, Renee L.

Pagel, Mark D.

Robins, Caroline E.

Shin, Ashley K.

English

OpenWorks @ MD Anderson

Introduction: Procedure:Day 0: Prepare and shave 5 mice (6 weeks of age) around the 4th right inguinal nipple 3 days before implantationDay 3: Subcutaneous injection of 4T1 tumor cell line mammary epithelial tumor cells into the mammary fat padDay 12: Baseline MSOT imaging 9 days post-implantation (tumors are around 4-5 mm in diameter)Day 13: MSOT imaging 10 days post-implantation and 3 hours post-injection (ITPP or phosphate buffered saline control)Conclusions:MSOT has shown to accurately determine the oxygenation of in the Su86.86, MIA PaCa-2, Colo357 pancreatic tumor models in mice.MSOT has the potential to determine changes in tumoral oxygenation in response to ITPP.Acknowledgements:I would personally like to thank Renee Chin, Caroline Robins, Marty Pagel, Michelle Zalles,Jorge de la Cerda, Willian Schuler, and other fellow lab mates for support, assistance withexperimentation, and critique. I would like to thank the Small Animal Imaging Facility at the UTMD Anderson Cancer Center. I would also like to express my gratitude for opportunity to be apart of the 2022 MD Anderson Summer Imaging Research Program.Measuring the Effect of ITPP on Tumor Hypoxia with Multispectral Optoacoustic TomographyAshley K. Shin1,2, Caroline E. Robins2,3, Renee L. Chin2, Mark D. Pagel21University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX, USA2Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, USA3Texas Tech University, Lubbock, TX, USAAuthor contact information: Ashley Shin; ashley.k.shin@uth.tmc.eduResults:Figure 1: Hypoxia in tumors leads to poor response to T cell-based immunotherapy. A hypoxic tumor microenvironment promotestumor growth and proliferation. Various factors contribute to hypoxic tumor microenvironment leading to decreased T cell extravasation andincreased T cell apoptosis.1 Abbreviations: CAM, cell adhesion molecules; VEGF, vascular endothelial growth factor; FGF, fibroblast growthfactor; IL, interleukin; CAF, cancer-associated fibroblast; HIF-1α, hypoxia-inducible factor 1α; MDSC, myeloid-derived suppressor cell; TAM,tumor-associated macrophage; Treg, regulatory T cell.Hypoxia within solid tumors creates an environment that promotes tumor growth anddevelopment. Hypoxia induces HIF-1α, which increases tumor angiogenesis. Tumorangiogenesis then generates abnormal vasculature leading to decreased expression ofvascular adhesion molecules, thus decreasing T cell recruitment and extravasation.2 Hypoxiaalso induces an anti-inflammatory, immunosuppressive, M2 phenotype in macrophages thatfurther hinders anti-tumor T cell function and trafficking. In addition, HIF-1α induces T cellapoptosis via Fas to Fas ligand interactions. Because T cells are hindered from entering thetumor environment, the tumors are resistant to T cell-based cancer immunotherapies, thusleading to poor prognoses.2Myo-inositol trispyrophosphate (ITPP) changes oxygen unloading from hemoglobin, thuschanges %sO2.3 %sO! ="#$!"#%"#$!, where HbO2 is oxyhemoglobin and Hb is deoxyhemoglobin.ITPP also activates endothelial Phosphatase-and-Tensin-homologue (PTEN), thus inhibitingtumor cell growth and proliferation.4Multi Spectral Optoacoustic Tomography (MSOT) measures deoxyhemoglobin andoxyhemoglobin at distinct wavelengths.5 MSOT provides a non-invasive way to measure tumorhypoxia to determine response to cancer therapies. Compared to other imaging techniqueslike magnetic resonance imaging, MSOT is quick, patient-friendly and decreases radiationexposure to patients.Pathological angiogenesis Generation of abnormal vasculatureSerpentine-like and leaky vasculature ↑ tumor cell growth↓ CD8+ T cells↓ CAMsExcessive fibrosis↓ O2 deliveryPerfusion-limited hypoxiaHypoxiaDiffusion-limited hypoxia↓ O2 and nutrients for T cells↓ pro-inflammatory cytokinesT cell apoptosis↓ pH (5.8 – 6.5)T cell apoptosisMacrophagesM2 phenotypeAnti-inflammatory, immunosuppressionPancreatic stellate cellsDifferentiate into myofibroblasts↑ collagen deposition ↑ oxidative metabolism of tumor cells↑ diffusion distances↑ tumor cell growth↑ lactate↑ MDSCs, TAMs, Treg↑ VEGF, FGF, IL-8, angiopoetin-2Lipoxygenase enzymesECM remodeling by CAFsHIF-1α↓ T cell recruitment and extravasation↑ O2 consumption by tumor cellsTumorSpinal cordTumorSpinal cordFigure 2: The wavelengths absorbed by various media. MSOT canbe used to detect the oxygenation of tumors because oxyhemoglobin(HbO2) and deoxyhemoglobin (HbR) absorb distinct wavelengths of light.Figure courtesy of iThera Medical, Inc.Figure 3: The use of MSOT to determine the level of oxygenation within the tumor environment. Mouse is given 21% medical grade air. MSOT emits light at different wavelengths (700, 730, 760, 800, 850, and 875 nm). The light isabsorbed by oxyhemoglobin and deoxyhemoglobin, which undergoes thermoelastic expansion, also known as the optoacoustic effect. The thermoelastic expansion results in ultrasound waves. Images are created from the ultrasoundwaves. Spectral unmixing of the tumor image separates the oxyhemoglobin and deoxyhemoglobin absorption. %sO2 is calculated, and the overall oxygenation of the tissue is obtained. The oxygenation levels from the baseline MSOTscan and post-ITPP injection scan are obtained and compared to determine if ITPP improved oxygenation.Top: mouse apparatus to be submerged into MSOTmachine for imaging; Lower Left: MSOT machine for miceand rodents; Lower Middle: laser within MSOT; LowerRight: clinical MSOT machineMethods:Figure 4: Baseline MSOT image (left) of the 4T1 mammary epithelial tumor on the 4th right inguinalnipple before ITPP injection. Post-injection MSOT image (right) of the 4T1 mammary epithelial tumoron the 4th right inguinal nipple 3 hours post-ITPP injection. The tumor was about 4 to 5 millimeters indiameter. The legs and spinal cord were used as landmarks to find the tumor before the scan. Aftercompleting the scans, both sets of the tumor scans (baseline and post-injection) were spectrally unmixedto separate the oxyhemoglobin and deoxyhemoglobin absorption.Future Directions:MSOT will continue to be utilized to determine the %sO2 of tumors. MSOT is currently being tested within the clinical setting on humanpatients. To continue investigating if ITPP improves the effectiveness of checkpoint blockade, further studies will investigate the effect ofITPP on tumor size and the effect of ITPP on immunogenicity. If ITPP shows to reduce tumor size and increase immunogenicity, theITPP injection will be effective in improving responses to checkpoint blockade immunotherapy.Time (min)Figure 5: %sO2 signals from Su86.86 (green), MIAPaCa-2 (blue), and Colo357 (red) tumor models on21% O2 medical grade air. At constant temperature. TheMSOT showed a consistent measures, confirming themouse was stable when breathing 21% O2.Figure 6: The average value and standard deviationof %sO2 of 10 mice with Su86.86, 10 mice with MIAPaCa-2, and 10 mice with Colo357, all breathing 21%O2. Su86.86, MIA PaCa-2, and Colo357 are pancreatictumor model cell lines obtained from patients andconditioned to grow in immunocompromised mice.The study of tumor reoxygenation with ITPP as monitored with MSOT is ongoing (Figure 4). As an example of expected results, Figures5 and 6 demonstrate a related project that shows the classification of normoxic (Su86.86), mildly hypoxic (MIA PaCa-2) and very hypoxic(Colo357) tumors.Responsible Conduct of Research:The MD Anderson PI was responsible for maintaining documents and approvals for all modifications in the protocol. All mice werehandled in compliance with regulations set by MD Anderson's ethics review board (IACUC).References:1. Jayaprakash, P., et al. (2022). Hypoxia reduction sensitizes refractory cancers to immunotherapy. Annual Review of Medicine, 73(1),251–265.2. Jayaprakash, P., et al. (2018). Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer toimmunotherapy. Journal of Clinical Investigation, 128(11), 5137–5149.3. Tran, L.-B.-A., et al. (2018). Impact of myo-inositol trispyrophosphate (ITPP) on tumour oxygenation and response to irradiation inrodent tumour models. Journal of Cellular and Molecular Medicine, 23(3), 1908–1916.4. El Hafny-Rahbi, B., et al. (2021). Tumour angiogenesis normalized by myo-inositol trispyrophosphate alleviates hypoxia in themicroenvironment and promotes antitumor immune response. Journal of Cellular and Molecular Medicine, 25(7), 3284–3299.5. Tomaszewski, M. R., et al. (2018). Oxygen-enhanced and dynamic contrast-enhanced optoacoustic tomography provide surrogatebiomarkers of tumor vascular function, hypoxia, and necrosis. Cancer Research, 78(20), 5980–5991.

Measuring the Effect of ITPP on Tumor Hypoxia with Multispectral Optoacoustic Tomography

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1035&context=sumexp22

Measuring the Effect of ITPP on Tumor Hypoxia with Multispectral Optoacoustic Tomography

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