Abstract:The purpose of the study was to investigate vascularization, oxygenation, and the effect of sunitinib treatment in pancreatic ductal adenocarcinoma (PDAC). BxPC-3 and Capan-2 xenografts grown in dorsal window chambers were used as preclinical models of human PDAC. Tumor angiogenesis and the morphology and function of tumor vascular networks were studied by intravital microscopy, and tumor hypoxia was assessed by immunohistochemistry. The PDAC models differed in vessel distribution and oxygenation, and the diff… Show more
“…Fig. 6b-c and e was modified from Gaustad et al [ 47 ] Fig. 8a A T 1 -weighted magnetic resonance (MR) image of calibration phantoms (P) and a mouse bearing a window chamber (WC) with an A07-GFP melanoma xenograft (T), and a color-coded K trans image of the A07-GFP tumor calculated from dynamic contrast-enhanced MR data by using Tofts generalized pharmacokinetic model.…”
Purpose
The purpose of the study was to demonstrate the performance and possible applications of an intravital microscopy assay using a standard fluorescence microscope.
Methods
Melanoma and pancreatic ductal adenocarcinoma xenografts were initiated in dorsal window chambers and subjected to repeated intravital microscopy. The entire tumor vasculature as well as the normal tissue surrounding the tumor was imaged simultaneously with high spatial and temporal resolution. Vascular morphology images were recorded by using transillumination, and vascular masks were produced to quantify vessel density, vessel diameter, vessel segment length, and vessel tortuosity. First-pass imaging movies were recorded after an intravenous injection of a fluorescent marker and were used to investigate vascular function. Lymphatics were visualized by intradermal injections of a fluorescent marker.
Results
The intravital microscopy assay was used to study tumor growth and vascularization, tumor vessel morphology and function, tumor-associated lymphatics, and vascular effects of acute cyclic hypoxia and antiangiogenic treatment. The assay was sensitive to tumor-line differences in vascular morphology and function and detected tumor-induced lymphatic dilation. Acute cyclic hypoxia induced angiogenesis and increased the density of small diameter vessels and blood supply times, whereas antiangiogenic treatment selectively removed small-diameter vessels, reduced blood supply times, and induced hypoxia. Moreover, the window chamber was compatible with magnetic resonance imaging (MRI), and parametric images derived by dynamic contrast-enhanced MRI were shown to reflect vascular morphology and function.
Conclusions
The presented assay represents a useful and affordable alternative to intravital microscopy assays using confocal and multi-photon microscopes.
“…Fig. 6b-c and e was modified from Gaustad et al [ 47 ] Fig. 8a A T 1 -weighted magnetic resonance (MR) image of calibration phantoms (P) and a mouse bearing a window chamber (WC) with an A07-GFP melanoma xenograft (T), and a color-coded K trans image of the A07-GFP tumor calculated from dynamic contrast-enhanced MR data by using Tofts generalized pharmacokinetic model.…”
Purpose
The purpose of the study was to demonstrate the performance and possible applications of an intravital microscopy assay using a standard fluorescence microscope.
Methods
Melanoma and pancreatic ductal adenocarcinoma xenografts were initiated in dorsal window chambers and subjected to repeated intravital microscopy. The entire tumor vasculature as well as the normal tissue surrounding the tumor was imaged simultaneously with high spatial and temporal resolution. Vascular morphology images were recorded by using transillumination, and vascular masks were produced to quantify vessel density, vessel diameter, vessel segment length, and vessel tortuosity. First-pass imaging movies were recorded after an intravenous injection of a fluorescent marker and were used to investigate vascular function. Lymphatics were visualized by intradermal injections of a fluorescent marker.
Results
The intravital microscopy assay was used to study tumor growth and vascularization, tumor vessel morphology and function, tumor-associated lymphatics, and vascular effects of acute cyclic hypoxia and antiangiogenic treatment. The assay was sensitive to tumor-line differences in vascular morphology and function and detected tumor-induced lymphatic dilation. Acute cyclic hypoxia induced angiogenesis and increased the density of small diameter vessels and blood supply times, whereas antiangiogenic treatment selectively removed small-diameter vessels, reduced blood supply times, and induced hypoxia. Moreover, the window chamber was compatible with magnetic resonance imaging (MRI), and parametric images derived by dynamic contrast-enhanced MRI were shown to reflect vascular morphology and function.
Conclusions
The presented assay represents a useful and affordable alternative to intravital microscopy assays using confocal and multi-photon microscopes.
“…A functional vascular network is essential to supply oxygen to the pancreatic TME 70 . Pancreatic tumors with a high microvascular density (MVD) tend to be less hypoxic than those with low MVD 71 . Indeed, we have demonstrated this relationship using our intravital animal model in a previous study 35 .…”
Despite decades of improvements in cytotoxic therapy, the current standard of care for locally advanced pancreatic cancer (LAPC) provides, on average, only a few months of survival benefit. Stereotactic Body Radiation Therapy (SBRT), a technique that accurately delivers high doses of radiation to tumors in fewer fractions, has emerged as a promising therapy to improve local control of LAPC; however, its effects on the tumor microenvironment and hypoxia remain poorly understood. To explore how SBRT affects pancreatic tumors, we combined an orthotopic mouse model of pancreatic cancer with an intravital microscopy platform to visualize changes to the in vivo tumor microenvironment in real-time. Mice received SBRT (5×8Gy) or were left untreated and were imaged before and 1, 4, 7, and 14 days after treatment (n = 7–10/group). A fluorescent human pancreatic cancer cell line (BxPC3-DsRed) engineered to express GFP under hypoxic conditions (driven by hypoxia-inducible factor, HIF) was used to monitor tumor hypoxia. Immunohistochemical staining was also performed on tissues to validate in vivo data. Our findings demonstrate a persistent decrease in pancreatic tumor hypoxia as early as one day after SBRT. This coincided with a decrease in both tumor cell proliferation and cell density in the SBRT group. Reduced demand for oxygen after SBRT (due to cell death and growth arrest from treatment) significantly contributed to reoxygenation of the pancreatic TME. Understanding how this reoxygenation phenomenon occurs in a dose-dependent manner will help improve dosing and fractionation schemes for clinical SBRT.
“…This is particularly challenging to accomplish for the pancreas, given its delicate composition, deep anatomical location and proximity to the diaphragm. To circumvent this challenge, a number of laboratories have investigated pancreatic processes ex vivo [37], with the pancreas being exteriorized from the body cavity [38], or using ectopic transplantation of tissues into sites more conveniently imaged, such as the eye [39], kidney [8] or subcutaneous tissue [40,41]. While recent advances have enabled intravital imaging of the pancreas, existing designs are still limited in either stability for long-term time-lapse imaging on the minutes time scale, or their ability to accommodate pancreata of a variety of sizes (e.g.…”
Pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are grave illnesses with high levels of morbidity and mortality. Intravital imaging (IVI) is a powerful technique for visualizing physiological processes in both health and disease. However, the application of IVI to the murine pancreas presents significant challenges, as it is a deep, compliant, visceral organ that is difficult to access, easily damaged and susceptible to motion artefacts. Existing imaging windows for stabilizing the pancreas during IVI have unfortunately shown poor stability for time-lapsed imaging on the minutes to hours scale, or are unable to accommodate both the healthy and tumour-bearing pancreata. To address these issues, we developed an improved stabilized window for intravital imaging of the pancreas (SWIP), which can be applied to not only the healthy pancreas but also to solid tumours like PDAC. Here, we validate the SWIP and use it to visualize a variety of processes for the first time, including (1) single-cell dynamics within the healthy pancreas, (2) transformation from healthy pancreas to acute pancreatitis induced by cerulein, and (3) the physiology of PDAC in both autochthonous and orthotopically injected models. SWIP can not only improve the imaging stability but also expand the application of IVI in both benign and malignant pancreas diseases.
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