NCI workshop on ctDNA in cancer treatment and clinical care.

Detection of cell-free circulating tumor DNA (ctDNA) from solid tumors is a fast-evolving field with significant potential for improving patient treatment outcomes. The spectrum of applications for ctDNA assays is broad and includes very diverse intended uses that will require different strategies to demonstrate utility. On September 14-15, 2023, the National Cancer Institute held an in-person workshop in Rockville, MD titled "ctDNA in Cancer Treatment and Clinical Care.

Evaluation of Tumor Development Using Hemoglobin Saturation Profile on Rodent Dorsal Window Chamber.

Tumor development can be indirectly evaluated using features of the tumor microenvironment (TME), such as hemoglobin saturation (HbSat), blood vessel dilation, and formation of new vessels. High values of HbSat and other features of the TME could indicate high metabolic activity and could precede the formation of angiogenic tumors; therefore, changes in HbSat profile can be used as a biomarker for tumor progression. One methodology to evaluate HbSat profile over time, and correlate it with tumor development in vivo in a preclinical model, is through a dorsal skin-fold window chamber.

Exogenous sickle erythrocytes combined with vascular disruption trigger disseminated tumor vaso-occlusion and lung tumor regression.

Hypoxic tumor niches are chief causes of treatment resistance and tumor recurrence. Sickle erythrocytes' (SSRBCs') intrinsic oxygen-sensing functionality empowers them to access such hypoxic niches wherein they form microaggregates that induce focal vessel closure. In search of measures to augment the scale of SSRBC-mediated tumor vaso-occlusion, we turned to the vascular disrupting agent, combretastatin A-4 (CA-4).

Circulating Tumor DNA Assays in Clinical Cancer Research.

The importance of circulating free DNA (cfDNA) in cancer clinical research was recognized in 1994 when a mutated RAS gene fragment was detected in a patient's blood sample. Up to 1% of the total circulating DNA in patients with cancer is circulating tumor DNA (ctDNA) that originates from tumor cells. As ctDNA is rapidly cleared from the blood stream and can be obtained by minimally invasive methods, it can be used as a dynamic cancer biomarker for cancer early detection, diagnosis, and treatment monitoring.

The National Institutes of Health Affordable Cancer Technologies Program: Improving Access to Resource-Appropriate Technologies for Cancer Detection, Diagnosis, Monitoring, and Treatment in Low- and Middle-Income Countries.

Point-of-care (POC) technologies have proved valuable in cancer detection, diagnosis, monitoring, and treatment in the developed world, and have shown promise in low-and-middle-income countries (LMIC) as well. Despite this promise, the unique design constraints presented in low-resource settings, coupled with the variety of country-specific regulatory and institutional dynamics, have made it difficult for investigators to translate successful POC cancer interventions to the LMIC markets. In response to this need, the National Cancer Institute has partnered with the National Institute of Biomedical Imaging and Bioengineering to create the National Institutes of Health Affordable Cancer Technologies (ACTs) program.

Rapid prototyping of biomimetic vascular phantoms for hyperspectral reflectance imaging.

The emerging technique of rapid prototyping with three-dimensional (3-D) printers provides a simple yet revolutionary method for fabricating objects with arbitrary geometry. The use of 3-D printing for generating morphologically biomimetic tissue phantoms based on medical images represents a potentially major advance over existing phantom approaches. Toward the goal of image-defined phantoms, we converted a segmented fundus image of the human retina into a matrix format and edited it to achieve a geometry suitable for printing.

In vivo microscopy of microvessel oxygenation and network connections.

Abnormal or compromised microvascular function is a key component of various diseases. In vivo microscopy of microvessel function in preclinical models can be useful for the study of a disease state and effects of new treatments. Wide-field imaging of microvascular oxygenation via hemoglobin (Hb) saturation measurements has been applied in various applications alone and in combination with other measures of microvessel function, such as blood flow.

Limitations of the dorsal skinfold window chamber model in evaluating anti-angiogenic therapy during early phase of angiogenesis.

Background: Angiogenesis is an essential process during tumor development and growth. The murine dorsal skinfold window chamber model has been used for the study of both tumor microvasculature and other vascular diseases, including the study of anti-angiogenic agents in cancer therapy. Hyperspectral imaging of oxygen status of the microvasculature has not been widely used to evaluate response to inhibition of angiogenesis in early tumor cell induced vascular development.

VEGF neutralization can prevent and normalize arteriovenous malformations in an animal model for hereditary hemorrhagic telangiectasia 2.

Arteriovenous malformation (AVM) refers to a vascular anomaly where arteries and veins are directly connected through a complex, tangled web of abnormal AV fistulae without a normal capillary network. Hereditary hemorrhagic telangiectasia (HHT) types 1 and 2 arise from heterozygous mutations in endoglin (ENG) and activin receptor-like kinase 1 (ALK1), respectively. HHT patients possess AVMs in various organs, and telangiectases (small AVMs) along the mucocutaneous surface.

In vivo spectral and fluorescence microscopy comparison of microvascular function after treatment with OXi4503, Sunitinib and their combination in Caki-2 tumors.

Vascular targeting agents on their own have been shown to be insufficient for complete treatment of solid tumors, emphasizing the importance of studying the vascular effects of these drugs for their use with conventional therapies in the clinic. First-pass fluorescence imaging combined with hyperspectral imaging of hemoglobin saturation of microvessels in the murine dorsal window chamber model provides an easily implementable, low cost method to analyze tumor vascular response to these agents in real-time. In this study, the authors utilized these methods to spectroscopically demonstrate distinct vessel structure, blood flow and oxygenation changes in human Caki-2 renal cell carcinoma following treatment with OXi4503 alone, Sunitinib alone and both drugs together.

Forecasting new development of tumor areas using spatial and temporal distribution profiles of hemoglobin saturation in a mouse model.

Features of the tumor microenvironment (TME), such as hemoglobin saturation (HbSat), can provide valuable information on early development and progression of tumors. HbSat correlates with high metabolism and precedes the formation of angiogenic tumors; therefore, changes in HbSat profile can be used as a biomarker for early cancer detection. In this project, we develop a methodology to evaluate HbSat for forecasting early tumor development in a mouse model.

Drug-loaded sickle cells programmed ex vivo for delayed hemolysis target hypoxic tumor microvessels and augment tumor drug delivery.

Selective drug delivery to hypoxic tumor niches remains a significant therapeutic challenge that calls for new conceptual approaches. Sickle red blood cells (SSRBCs) have shown an ability to target such hypoxic niches and induce tumoricidal effects when used together with exogenous pro-oxidants. Here we determine whether the delivery of a model therapeutic encapsulated in murine SSRBCs can be enhanced by ex vivo photosensitization under conditions that delay autohemolysis to a time that coincides with maximal localization of SSRBCs in a hypoxic tumor.

Combination of spectral and fluorescence imaging microscopy for wide-field in vivo analysis of microvessel blood supply and oxygenation.

Hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence imaging of blood transit time were combined to analyze the oxygenation of and blood flow through microvessel networks. The combination imaging technique was demonstrated in a mouse dorsal window chamber model of a growing Caki-2 human renal cell carcinoma over time. Data from Hb saturation and blood supply time maps show the formation of arteriovenous malformations and shunting of blood directly from arteries to the tumor core and into veins in the periphery of the tumor.

Comparative evaluation of differential laser-induced perturbation spectroscopy as a technique to discriminate emerging skin pathology.

Fluorescence spectroscopy has been widely investigated as a technique for identifying pathological tissue; however, unrelated subject-to-subject variations in spectra complicate data analysis and interpretation. We describe and evaluate a new biosensing technique, differential laser-induced perturbation spectroscopy (DLIPS), based on deep ultraviolet (UV) photochemical perturbation in combination with difference spectroscopy. This technique combines sequential fluorescence probing (pre- and post-perturbation) with sub-ablative UV perturbation and difference spectroscopy to provide a new spectral dimension, facilitating two improvements over fluorescence spectroscopy.

Wide-field functional imaging of blood flow and hemoglobin oxygen saturation in the rodent dorsal window chamber.

The rodent dorsal window chamber is a widely used in vivo model of the microvasculature. The model consists of a 1cm region of exposed microvasculature in the rodent dorsal skin that is immobilized by surgically implanted titanium frames, allowing the skin microvasculature to be visualized. We describe a detailed protocol for surgical implantation of the dorsal window chamber which enables researchers to perform the window chamber implantation surgery.

3D in vivo optical coherence tomography based on a low-voltage, large-scan-range 2D MEMS mirror.

3D in vivo optical imaging on a mouse has been obtained using a 2D MEMS mirror for lateral scanning in a time-domain optical coherence tomography (OCT) system. The MEMS mirror aperture size is 1 x 1 mm(2), and the device footprint is 2 x 2 mm(2). The MEMS mirror scans +/- 30 degrees optical angles about both x and y-axis at only 5.

Intravital microscopy imaging of macrophage localization to immunogenic particles and co-localized tissue oxygen saturation.

Well-designed biomaterial polymer particle-based vaccines will optimally promote immune cell antigen-presenting behavior while minimizing adverse inflammatory responses to the particles and encapsulated drugs or adjuvants. It is important in the design of particle-based vaccines to consider possible harmful effects of immune response on tissue at the vaccination site. Intravital microscopy with rodent dorsal skin window chambers enables in vivo serial observations in the same animal, and such models which have been used to study angiogenesis and macrophage response to implanted biomaterials may also be useful for the development of particle-based vaccines.

Spectral imaging reveals microvessel physiology and function from anastomoses to thromboses.

Abnormal microvascular physiology and function is common in many diseases. Numerous pathologies include hypervascularity, aberrant angiogenesis, or abnormal vascular remodeling among the characteristic features of the disease, and quantitative imaging and measurement of microvessel function can be important to increase understanding of these diseases. Several optical techniques are useful for direct imaging of microvascular function.

In vivo functional differences in microvascular response of 4T1 and Caki-1 tumors after treatment with OXi4503.

4T1 mouse mammary adenocarcinomas and Caki-1 human renal cell carcinomas grown in mouse dorsal window chambers were serially treated with the vascular disrupting agent (VDA) OXi4503 and their responses compared. The real-time in vivo response was assessed using spectral imaging of microvascular hemoglobin saturation. To our knowledge this is the first use of spectral imaging technology for investigation of vascular disrupting agents.

ACE2 activation promotes antithrombotic activity.

The aim of the present study was to test the hypothesis that the activation of the angiotensin-converting enzyme (ACE)2/angiotensin-(1-7)/Mas receptor axis by use of a novel ACE2 activator (XNT) would protect against thrombosis. Thrombi were induced in the vena cava of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats, and ACE2 and ACE activity in the thrombus was determined. Real-time thrombus formation was viewed through intravital microscopy of vessels in nude mice.

Real-time imaging of de novo arteriovenous malformation in a mouse model of hereditary hemorrhagic telangiectasia.

Arteriovenous malformations (AVMs) are vascular anomalies where arteries and veins are directly connected through a complex, tangled web of abnormal arteries and veins instead of a normal capillary network. AVMs in the brain, lung, and visceral organs, including the liver and gastrointestinal tract, result in considerable morbidity and mortality. AVMs are the underlying cause of three major clinical symptoms of a genetic vascular dysplasia termed hereditary hemorrhagic telangiectasia (HHT), which is characterized by recurrent nosebleeds, mucocutaneous telangiectases, and visceral AVMs and caused by mutations in one of several genes, including activin receptor-like kinase 1 (ALK1).

Novel imaging provides new insights into mechanisms of oxygen transport in tumors.

Hypoxia is a common feature of solid tumors, and abnormal tumor oxygen transport is a key factor in the imbalance between tumor oxygen supply and demand. Novel advanced imaging techniques can enable new insights into the complexities of tumor oxygen transport and hypoxia that were not previously known or fully appreciated. In this paper, we document new insights into tumor oxygen transport enabled by spectral imaging of microvascular hemoglobin saturation.

Multimodal optical imaging of microvessel network convective oxygen transport dynamics.

Convective oxygen transport by microvessels depends on several parameters, including red blood cell flux and oxygen saturation. We demonstrate the use of intravital microscopy techniques to measure hemoglobin saturations, red blood cell fluxes and velocities, and microvessel cross-sectional areas in regions of microvascular networks containing multiple vessels. With these methods, data can be obtained at high spatial and temporal resolution and correlations between oxygen transport and hemodynamic parameters can be assessed.

Spectral imaging facilitates visualization and measurements of unstable and abnormal microvascular oxygen transport in tumors.

Abnormal microvasculature contributes to the pathophysiologic microenvironment of tumors. Understanding microvascular tumor oxygen transport is necessary to comprehend the factors that influence tumor biology, physiology, and therapy. Previously, we described an in vivo spectral imaging microscopy system for measurements of microvessel hemoglobin saturation (HbSat).

Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity.

In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing.