Biomarker Imaging for Preclinical Cancer Research

Biomarker Imaging for Preclinical Cancer Research

Scientific article by Nina Culum, MSc (Inside Scientific)

Despite advances in diagnostics and treatment, cancer remains a leading cause of death worldwide [1]. Many cancers are already metastatic at presentation due to limited options for early cancer screening, many of which involve invasive physical exams, tissue biopsies, or radiation for imaging [2]. A major challenge in effective cancer treatment is therefore early disease diagnosis, which would greatly improve patient survival. Current and emerging molecular biomarkers for cancer detection and progression monitoring are vital to cancer management, but the challenge remains to identify those with adequate sensitivity and specificity for accurate diagnosis and prognosis [3].

Preclinical and Clinical Applications of Cancer Biomarker Imaging

Biospecimen-derived and imaging biomarkers are both widely used in oncology research and practice. In healthcare settings, biomarker imaging applications include diagnosing and staging cancer, targeting treatments, and predicting and monitoring therapeutic efficacy and/or toxicity, while research applications include guiding drug development and tracking drug efficacy and resistance [4]. Although biomarkers are useful for clinical practice, those that do not cross the translational gap can still further research and drug discovery. For example, left ventricular ejection fraction, a safety biomarker, can guide recruitment and continuation in many clinical trials through scintigraphy or ultrasound imaging [5]. In this article, we review several modalities commonly used for cancer biomarker imaging at preclinical stages, as well as recent technological advances.

Common Techniques for Cancer Biomarker Imaging in Preclinical Research

Noninvasive preclinical cancer imaging enables the study of proliferation, metabolism, apoptosis, angiogenesis, and gene expression, and can be used to evaluate the therapeutic effects of drug candidates and guide surgical approaches [6]. The choice of imaging technique depends on device availability, accessibility, and contrast that allows for best tumor imaging [7]. Common biomarker imaging methods include fluorescence, bioluminescence, ultrasound, photoacoustics, positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI), though this article will focus on optical and acoustic preclinical imaging techniques.

Fluorescence and Bioluminescence Biomarker Imaging

Fluorescence imaging requires an external light source to excite a fluorophore which, on decay, releases low energy light [7]. Although much of fluorescence imaging is done in 2D, 3D imaging is possible with fluorescence molecular tomography, which can visualize tissue with a penetration depth of several centimeters in the red to near-infrared (NIR) region [8]. Traditional fluorescence relies on fluorophores excited by light in the 650-950 nm region (NIR-I), but this intrinsically suffers from strong light attenuation in tissues as well as high autofluorescence that leads to shallow tissue penetration depth, low sensitivity, and high background signals [9]. Imaging at the 1000-1700 nm range (NIR-II), however, minimizes photon scattering and tissue autofluorescence, thereby increasing tissue penetration depth, sensitivity, and resolution [9].

A great number of novel probes for improved fluorescence imaging have been reported in recent years, such as enzyme-activated NIR-II probes [10, 11] and plasmonic-fluorescent nanoparticles [12]. A smart nanoprobe consisting of a self-assembling cyclopeptide-dye has been investigated, which responds to pH decreases in the tumor microenvironment by aggregating from small (80 nm) to large (500 nm) nanoparticles, allowing for high contrast and resolution tumor imaging in mice [13]. Rare earth-doped nanoparticles have also allowed for the visualization of tiny (2 mm) metastatic lesions in mice [14], while targeted heptamethine cyanine-based fluorophores have been used to target a diverse range of tumors [15]. First described in 2022, neodymium (3+)-coordinated black phosphorus quantum dots have been used for efficient glioblastoma imaging in mice as well as synergistic x-ray-induced photodynamic chemotherapy [16], while gadolinium-based virus-like nanoparticles have been shown to intraoperatively recognize residual tumors in a mouse model of breast cancer (Figure 1) [17].

Bioluminescence imaging relies on the oxidation of a substrate (i.e., luciferin) by an enzyme (i.e., luciferase) to produce light, and has been used to monitor expression systems, visualize tumor growth, detect metastasis, and assess viral and gene therapies [7, 18]. However, genetic engineering is required to express luciferase in mammalian cells. Current research efforts are focused on developing luciferin-luciferase systems that are shifted towards the NIR region for brighter and more sustained signals compared to naturally occurring systems [18]. For example, red-shifted luciferins based on synthetic coelenterazine analogs and corresponding NanoLuc mutants have recently been used to develop a bioluminescence resonance energy-based Antares reporter for increased signal intensity and tissue penetration in vivo [19].

Ultrasound and Photoacoustic Biomarker Imaging

Ultrasound imaging relies on a transducer to generate sound pulses that propagate through tissue and are reflected back based on tissue density, and has been shown to closely monitor cancer development and metastasis in animals [7]. Microbubble contrast agents have expanded the utility of preclinical ultrasound imaging to allow for detailed interrogation of the cancer microvasculature, a technique referred to as contrast-enhanced ultrasound (CEUS) imaging [7, 20, 21]. For example, gemcitabine-loaded microbubbles have been used to substantially enhance tumor image quality in a murine pancreatic cancer model [22]. Additionally, microbubbles that target the breast cancer marker B7-H3 have been shown to differentiate normal mammary glands from those containing ductal carcinoma in situ (DCIS) in mice (Figure 2) [23].

Photoacoustic imaging lies at the bridge of optical and acoustic imaging; by detecting optical absorption characteristics of biological tissue with ultrasound resolution, it can visualize molecular functional information in deep tissue better than pure optical imaging techniques [24]. In this method, molecular vibration and small pressure waves caused by light absorption generate thermoelastic expansion, and the resulting acoustic waves are detected by ultrasound transducers [18]. Whole body photoacoustic imaging of small animals has been widely applied in preclinical biomedical research to guide therapies and monitor drug delivery to tumors [24, 25].

Ultrasound and Photoacoustic Biomarker Imaging Solutions by FUJIFILM VisualSonics

FUJIFILM VisualSonics has specialized in ultra-high frequency ultrasound and photoacoustic imaging for more than 20 years. Ultra-high frequency ultrasound (up to 70 MHz) provides the resolution necessary to detect sub-millimeter tumors in preclinical cancer research models [26, 27]. Additionally, researchers can acquire functional data on blood flow, perfusion, and molecular expression with Doppler-based and contrast-enhanced imaging modes. When ultra-high frequency ultrasound is paired with the Vevo LAZR-X for photoacoustic imaging, anatomical and functional information can be co-registered with maps of oxygenation and biomarker distribution. Photoacoustic imaging with Vevo systems has been proven to be a valuable tool in assessing animal models of breast, [23], liver [26], and brain [28] tumors, as well as bacterial infections [29] and many other diseases. This multi-modal imaging allows for tumor detection in a myriad of ways, from tumor volume and vascularization to oxygenation and molecular expression [26, 28].

Visualsonics’ newest flagship platform, the Vevo F2, is the “world’s first” ultra-high to low frequency ultrasound system. The expanded bandwidth of this platform and the addition of new transducers provides increased penetration depth, allowing researchers to image a variety of animal models on the same system. The Vevo F2 features improve signal processing for faster image acquisition and include a triple transducer port for ease of switching between transducers and a new data acquisition mode (called VADA, or Vevo Advanced Data Acquisition), allowing researchers to configure custom image sequences for advanced applications such as ultrafast plane wave imaging. Together, the Vevo F2 and LAZR-X create a multi-modal, hybrid system that can provide researchers with anatomical, functional, and molecular data across different animal models like no other commercially available system.

Several approaches for monitoring drug delivery and treatment response in animals through photoacoustic imaging have been described in recent years. For example, nanocarrier drug release has been monitored in a murine colon cancer model through a paclitaxel-methylene blue conjugate with redox activity; during release, this conjugate spontaneously oxidizes to produce a concentration-dependent photoacoustic signal with up to 649% signal enhancement after 10 hours [30]. Optical-resolution photoacoustic microscopy imaging has also been used to track vascular changes in a mouse model of prostate cancer, and was shown to be a promising method for elucidating drug mechanisms in vivo and for monitoring and guiding cancer therapy [31].

More recently, efforts in developing dual-mode imaging modalities have been published. For instance, a cyanine-based photocage has been described under hypoxic conditions, whose photolysis simultaneously produces fluorescence and photoacoustic signals for dual-mode tumor imaging [32]. The combination of ultrasound and photoacoustic imaging has also been shown to reliably assess differences in tissue oxygenation between control and pancreatic tumor-bearing mice, which holds promise in assessing treatment responses in longitudinal preclinical studies [33].

Conclusions and Future Perspectives

Imaging biomarkers are essential to both routine cancer patient care and in research stages when investigating underlying mechanisms and potential theranostic therapies. Furthermore, noninvasive in vivo approaches for imaging animal models of cancer facilitate longitudinal treatment studies, which can recapitulate therapeutic effects in human cancers [7]. Many preclinical imaging options are available to researchers, each with their own benefits and limitations (Figure 3).

Figure 3: Summary of the preclinical applications, advantages, and disadvantages of each preclinical imaging technique discussed. Created with information from refs 7, 18, 24.

While each imaging modality has its own advantages and disadvantages, the shortcomings of each method can be overcome with the use of multimodal scanners. Dual-mode systems, such as the combination of ultrasound and photoacoustic imaging, could become an integral component of preclinical cancer imaging, enabling the assessment of tumor structure and function, and even facilitate the development of personalized medicine [7]. For more insights into preclinical cancer research and methods, browse our latest webinars here.

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