In this webinar, Katie will discuss the role hypoxia plays in disease progression and treatment response, specifically in cancer. She will also dive into the various molecular imaging technologies that can be used to visualize and assess hypoxia in preclinical cancer models. Some modalities that will be covered include magnetic resonance imaging (MRI), positron emission tomography (PET), and optical imaging.  Topics to be covered: What is hypoxia? Is there a link between hypoxia and cancer? What imaging modalities can be used to visualize hypoxia in vivo? What are the advantages and limitations of each technique? What are some applications of hypoxia imaging? Hypoxia has been shown to influence many facets of cancer including tumor growth, treatment response, and metastatic potential. Thus, the ability to noninvasively visualize hypoxia in vivo may be critical to understanding the underlying tumor biology, guiding treatment plans, and determining prognosis in the clinic. Many different modalities have been used for preclinical hypoxia imaging. While some techniques have been around for decades and have extensive data behind them, others are emerging technologies that aim to overcome existing limitations in the field. Choosing the right modality can be challenging and is dependent on experimental conditions including tumor model, animal strain, and the desired measurement, as each technique will target a different aspect of hypoxia. In this webinar, we will discuss some molecular imaging techniques that can be used to visualize and characterize tumor hypoxia including MRI, PET, optical, and PAI. We will compare the various options, discuss the advantages and limitations of each approach, and show some examples of how scientists are using these techniques within their research. References Rebecca A. D’Alonzo, Suki Gill, Pejman Rowshanfarzad, Synat Keam, Kelly M. MacKinnon, Alistair M. Cook & Martin A. Ebert (2021) In vivo noninvasive preclinical tumor hypoxia imaging methods: a review, International Journal of Radiation Biology, 97:5, 593-631, DOI: 10.1080/09553002.2021.1900943

1. Imaging Hypoxia
Katie Parkins, PhD
Scintica Instrumentation Inc.
Head of Scientific Integration
kparkins@scintica.com

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Topics of discussion
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What is hypoxia?
The relationship between hypoxia and cancer
Monitoring hypoxia in preclinical cancer models
What are the advantages and limitations of each technique?
The clinical relevance of hypoxia

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What is hypoxia?
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What is Hypoxia?
When O2 in the cell or organ drops below
physiologically normal levels
What is the physiologically normal O2 level?
It depends on specific cell or organ but is <20.9%
Physiologic Median O2 Levels in Organs and Tissues
Image from Novus Biologicals
*Oxygen deficiency affects cellular functions and
disrupts various biological processes

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Hypoxia and cancer…
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Tumour
vasculature
Hypoxia associated with:
• Increased aggressiveness
• Increased metastasis
• Resistance to therapy
• Poor patient prognosis
Cancer Normal tissue
<2.5 mmHg 20-40 mmHg
Common feature of most solid malignancies, resulting from an imbalance between oxygen delivery and consumption.

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The clinical relevance of hypoxia
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• Patients with hypoxic tumours have worse prognosis and reduced
chance of survival
• Preclinical and clinical evidence showing hypoxic tumours are
therapy resistant
- Radioresistance
- Chemoresistance
Hypoxia targeted therapies
Telarovic, I., Wenger, R.H. & Pruschy, M. J Exp Clin Cancer Res 40, 197 (2021).

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Monitoring hypoxia
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• Identifying patients that will benefit from hypoxia
targeted therapies
• Monitor changes in hypoxia following targeted therapy
There is a need for robust, specific and
reproducible biomarkers of hypoxia
Suwa, T., Kobayashi, M., Nam, JM. et al. Exp Mol Med 53, 1029–1035 (2021).

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Methods to detect hypoxia
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Invasive techniques
• Oxygen polarographic electrodes
• Fiber optic probes
• Immunohistochemical detection
Limitations
• Not reflective of the whole tumour
• Tumour needs to be accessible
• Does not capture the dynamic aspect of hypoxia changes

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Methods to detect hypoxia
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Noninvasive techniques
• Positron emission tomography (PET)
- Hypoxia tracers
• Magnetic resonance imaging (MRI)
- DCE-MRI
- OE-MRI
• Optical imaging
- Reporter gene systems
• Live cell imaging
- Visualize cells while maintaining desired O2 levels

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Positron emission tomography (PET)
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Courtesy of Dr. M. Desco & J.J. Vaquero, UMCE Hospital
Gregorio Marañón HGUGM (Madrid, Spain)
James ML, Gambhir SS. Physiological reviews. 2012

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Hypoxia specific PET radiotracers
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• Retained in hypoxic regions only and not in normoxic or necrotic areas
• Fast clearance from well oxygenated regions
• Not affected by perfusion or pH
• Robust and reproducible across tumour types
Carlin et al. JNMR. Mar 2014, 55 (3) 515-521.

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18F-Fluoromisonidazole (18F-FMISO)
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Zhao, S., Yu, W., Ukon, N. et al. EJNMMI Res 9, 51 (2019).
• First generation tracer
• Highly lipophilic
• High specificity

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18F-Fluoromisonidazole (18F-FMISO)
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Postema, Ernst J., et al. European journal of nuclear medicine and molecular imaging 36.10 (2009): 1565-1573.
• Hypoxic/normoxia vs. tumour growth
• Guiding hypoxia targeted therapy
• Monitoring changes following treatment

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18F-Fluoroazomycin arabinoside (18F-FAZA)
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• Second generation tracer
• More hydrophilic (faster uptake in hypoxic
tissues)
• Improved specificity
Busk et al. Eur J Nucl Med Mol Imaging (2013) 40:186–197

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Limitations of hypoxia imaging with PET
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• Slow clearance from normoxic regions can delay imaging time
• Low spatial resolution
• Large voxel size may confound interpretation of hypoxic volume
• Relatively expensive technology to adapt
• Need access to cyclotron
Sean Carlin and John L. Humm. JNM. August 2012, 53 (8) 1171-1174.

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Magnetic resonance imaging (MRI)
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https://imotions.com
James ML, Gambhir SS. Physiological reviews. 2012

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Dynamic contrast enhanced (DCE-MR) imaging
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Hillestad, Tiril, et al. Cancer Research 80.18 (2020): 3993-4003.
• Evaluates T1 shortening induced by gadolinium-based contrast agents
• Overall goal is to quantify time course of enhancement;
-regional blood flow
-size and number of vessels
-permeability
With pharmacokinetic modeling a number of
regional values can be derived:
1. K-trans (transfer constant)
2. Rate constant
3. Fractional volume of extravascular-extracellular space
4. Fractional volume of the plasma space

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Dynamic contrast enhanced (DCE-MR) imaging
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Wegner, Catherine S., et al. Neoplasia 20.7 (2018): 734-744.
1. Sunitinib may induce significant changes in the microenvironment
2. Ktrans may be an adequate measure of tumor vascular density and hypoxia in PDAC tumors

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Oxygen enhanced MRI (OE-MRI)
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O’Connor et al., 2016
• Uses inhaled molecular oxygen as contrast agent
• Oxygen dissolves within arterial blood and tissue plasma causing
changes in R1
• Readily translatable between animal models and humans

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Oxygen enhanced MRI (OE-MRI)
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O’Connor et al., 2016
• Subcutaneous murine human glioma xenograft
• OE-MRI followed by DCE-MRI and histology
• Area under the curve (AUC) measured for R1 curve (OE-MRI)
• Gadolinium concentration curve (DCE-MRI)
• Tumour regions with low perfusion tended to have positive
correlation with hypoxic regions and negative correlation with
vessel density

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Limitations of hypoxia imaging with MR
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• Oxygen tension measurements affected by other factors:
1. Hemoglobin saturation
2. Vascular geometry
3. Consumption
• May only provide estimates of tissue perfusion and the hypoxic
nature of tumour
*costly and time consuming
Mirabello et al., Front. Chem., 23 February 2018.

21. Poll Question

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Optical (bioluminescence and fluorescence) imaging
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James ML, Gambhir SS. Physiological reviews. 2012

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Reporter gene imaging of hypoxia
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Imaging reporter gene under control
of hypoxia response element (HRE)
Inject hypoxia responsive cell line to
generate cancer model
Only hypoxic cells produce optical
imaging signal

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Reporter gene imaging of hypoxia
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B. Krishnamachary et al. Neoplasia Vol. 22, No. 12, 2020
• Theranostic strategy
• HRE driven expression of luc
• HRE driven expression of yCD
• Image + eliminate hypoxic cells

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Optical imaging of hypoxia
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Zheng, X. et al. Nat Commun 6, 5834 (2015).

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Optical imaging of hypoxia
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Primary tumour- HIF1
Lymph node metastasis-
HIF1
Primary tumour (L) and metastasis (R) - pimonidazole
Zheng, X. et al. Nat Commun 6, 5834 (2015).

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Limitations of optical based hypoxia imaging
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• Hypoxia typically occurs in areas with diminished perfusion
• Bioluminescence imaging relies on substrate delivery
• Some reporters require co-factors to produce
light such as oxygen and ATP
• Depth of penetration

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More recently…live cell imaging of hypoxia
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• Can image cells while maintaining the relevant physiological conditions within a
hypoxia workstation
• Can visualize hypoxia happening in real time (switches) and/or confirm hypoxia
responsive prior to animal injections
• Inexpensive; rapid throughput
• Live cell imaging can be done with compact
brightfield microscopes that fit within incubators (or workstations)
-Basic brightfield and fluorescent imaging

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Monitoring changes to oxygen levels over time
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Danhier, Pierre, et al. Neoplasia 17.12 (2015): 871-881.

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A multimodal example..
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A multimodal example..
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Raman et al. Cancer Res; 66: (20). October 15, 2006.

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A multimodal example..
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Raman et al. Cancer Res; 66: (20). October 15, 2006.

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Choosing a modality
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Gene/transcription based (indirect)
Microscopy
PET
Bioluminescence
MRI
Photoacoustic/Ultrasound
Probe based (direct)
Probe based (direct)
Perfusion based (indirect)
Perfusion based (indirect)
Gilkes, Daniele M., ed. Hypoxia and Cancer Metastasis. Vol. 1136. Springer, 2019.

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Summary
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Noninvasive imaging techniques can be used to longitudinally visualize the degree and
heterogeneous distribution of tumour hypoxia
Non-invasive quantitative imaging for:
• Identification of tumours likely to respond to hypoxia targeted therapies
• Mapping the degree of hypoxia for treatment planning
• Monitoring strategies designed to modify tumour hypoxia for therapeutic gain

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Hypoxia and disease
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Trauma
Multiple organ failure
Stroke
Respiratory diseases
Peripheral artery disease
Heart disease
Cancer
Metabolic diseases
Kidney disease
Reproductive diseases

36. Poll Question

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Scintica Instrumentation
Phone: +1 (519) 857-6199
kparkins@scintica.com
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Thank you for participating!
Katie Parkins, PhD
Head of Scientific Integration