Evaluations of Oxygenation and Vascular Perfusion in Pre-Clinical Models of Cancer and Wound Healing Using OS-DCE OAI

Summary

Here, we present a protocol for performing Oxygen Sensitive – Dynamic Contrast Enhanced Optoacoustic Imaging (OS-DCE OAI) for evaluating pre-clinical models of cancer and wound healing. OS OAI can image oxy‑ and deoxyhemoglobin, mapping tissue oxygenation. DCE-OAI can image the pharmacokinetics of an exogenous contrast agent, thereby mapping vascular perfusion.

Abstract

Oxygen-sensitive optoacoustic imaging (OS OAI) and dynamic contrast–enhanced optoacoustic imaging (DCE OAI) provide complementary, noninvasive readouts of tissue physiology. OS OAI leverages multispectral detection of endogenous oxyhemoglobin and deoxyhemoglobin to generate maps of blood oxygen saturation (%sO₂), while DCE OAI tracks the in vivo pharmacokinetics of a near-infrared absorber to quantify vascular perfusion and permeability. The goal of this protocol is to present a unified OS-DCE OAI workflow that enables simultaneous assessment of oxygenation and perfusion in two widely used pre-clinical settings: orthotopic breast cancer tumors and full-thickness cutaneous wounds.

In this protocol, we demonstrated OAI of mouse models of breast cancer or a laceration wound. For OS OAI, multi-slice multispectral scans are acquired at 700–875 nm to allow spectral unmixing and estimation of %sO₂. For DCE OAI, repeated acquisitions at ≤5-s temporal resolution are performed before and after intravenous bolus injection of indocyanine green (ICG), using either multispectral sampling at 700-875 nm or single-wavelength sampling at 800 nm to generate time versus OA signal amplitude curves. The development of animal models, preparation for OA imaging, OS OAI and DCE OAI acquisitions, and analyses to measure characteristics of oxygenation and vascular perfusion are detailed, along with key troubleshooting guidance for motion artifacts, superficial absorbers, and failed injections.

Together, this OS-DCE OAI protocol yields an integrated functional portrait of hypoxia and vascular transport that can detect physiological changes in tumors and wounds earlier than gross anatomical measures. OS-DCE OAI is readily adaptable to other disease models.

Introduction

Optoacoustic imaging (OAI), also referred to as photoacoustic imaging or multispectral optoacoustic tomography, is a relatively new modality that couples optical excitation with ultrasound (US) detection to enable deep tissue imaging^1,2. In OAI, brief nanosecond laser pulses excite endogenous chromophores such as oxy- and deoxyhemoglobin, producing localized thermoelastic expansion and wideband acoustic waves that are detected by US transducers. These signals enable OAI to yield images with optical contrast at ultrasound-like spatial resolution while achieving centimeter-scale depth of view in vivo. W....

Protocol

Conduct all procedures under the approval of the Institutional Animal Care and Use Committee (IACUC) of your institution. All procedures in the protocol below were approved by the IACUC of our institution.

1. Anesthesia (for subsequent procedures)

1. Induce anesthesia. Initially induce and maintain anesthesia with 5% isoflurane in 100% oxygen carrier gas until the mouse is no longer awake.
2. Maintain anesthesia. Continue to anesthetize the mouse with 1.5–2% isoflurane in 100% oxygen carrier gas. Verify the absence of pedal reflex.
3. Maintain normothermia. Make sure to maintain animals on a war....

Results

OAI signal can generate maps of oxygen saturation in tumors and wounds
Following acquisition and reconstruction of multispectral OAI datasets, regions of interest (ROIs) can be defined within tumors or wound beds to quantify signal intensity at different wavelengths. Spectral unmixing algorithms are then applied to separate the contributions of oxyhemoglobin (HbO₂) and deoxyhemoglobin (Hb) from the composite optoacoustic signal. This processing enables the generation of quantitative maps o.......

Discussion

DCE OAI PK curves may be affected by a variety of artifacts that reduce the reliability of PK analysis. One common pitfall is the presence of superficial wounds, scabs, or dried blood within the imaging field, which can introduce strong, non-specific absorption at the skin surface and obscure deeper signals (Figure 8). When drawing ROIs within the tumor, care should be taken to avoid including these superficial structures to ensure that the extracted curves reflect intratumoral contrast dyna.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20 G Needle	Exel INT	26417	Hypodermic needle; 20G x 1", used for preparing heparin solutions
4T1 cells	ATCC	CRL-2539	Triple negative breast cancer cell line
Anti-Anti (100x)	Gibco	15240-062	Antibiotic cocktail
Biopsy punch	Integra	33-36	6 mm disposable biopsy punch
Cell Culture Grade Water	Corning	25-055-CV	500 mL; tested to USP sterile sater for inection specifications
Collagen Matrix Solution	Corning	354262	Collagen matrix for tumor fomation and injection
Ethanol	Deacon Laboratory	2701	General purpose antiseptic
Eye Lube	Optixcare	50-218-8442	20 g/.70 oz; for animal use only
Gauze	Fisherbrand	22-415-468	Cotton gauze pad; 3" x 3" (7.6 x 7.6 cm); 12-Ply
Gloves	VWR	76457-130	Nitrile examination gloves
Heparin	Meitheal	NDC71288-402-10	10,000 USP units per 10 mL, 1000 USP units per mL; 10 mL Multi-Dose Vial
Indocyanine green	TCI	I0535	Optoacoustic Contrast Agent
Isoflurane	Isospire	17033-091-25	A nonflammable, nonexplosive inhalation anesthetic for use in horses and dogs; 250 mL
Laboratory Film	Parafilm	PM-992	2" x 250'; 1" diameter core
Nair body bream Hair remover	Nair	LL4038	Depiliatory Cream; rich cocoa butter & vitamin E
Needle	Terumo	NN2732R	27G x 1 1/4" (6.40 mm x 32 mm); R.W. Reg. Bevel
Optoacoustic Imaging Machine	iTheraMedical	MSOT inVision 512	Optoacoustic scanner
PBS, 1x	Corning	21-040-CV	500 mL; Phosphate-buffered saline without calcium and magnesium
RPMI	Corning	10-040-CV	Cell culture media
Saline	Hospira	NDC0409-4888-02	10 mL single dose; 0.9% sodium chloride injection, USP
Stopwatch	iTheraMedical	4135333	Used to record minutes and seconds
Syringes	Cardinal Health	1188128012	1 mL Insulin Syringe; U-100 Insulin only; 27G x 1/2" (0.35 x 12.7 mm)
Transparent Wound Dressing	Tegaderm	1624W	transparent film dressing frame style; 2 3/8" x 2 3/4" (6 cm x 7 cm)
Tweezers	Termo Electron	7626	Used to pull skin back when injecting cells into mice
Ultrasound gel	EcoVue	283	32 g packet, prpylene glycol free, water soluble, paraben and dye free
Undyed Sutures	Ethicon	VR493	18" cutting; P-3 3/8 13 mm; undyed and braided, synthetic absorbable suture, non-USP
Warming pad	Repti Therm UTH	120VAC 60Hz 8W	Used to warm animals while under anesthetic;
Would Splints	Grace Bio-Labs	476684	Clear Silicone, 14 mm OD x 10 mm ID 0.5 thickness with suture sites
Wound clips	Reflex 7	203-1000	Reflex skin closure system, 7 mm, stainless stee