Intravital microscopy of tumor vessel morphology and function using a standard fluorescence microscope.

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 intervenous 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.

DCE-MRI of Tumor Hypoxia and Hypoxia-Associated Aggressiveness.

Tumor hypoxia is associated with resistance to treatment, aggressive growth, metastatic dissemination, and poor clinical outcome in many cancer types. The potential of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to assess the extent of hypoxia in tumors has been investigated in several studies in our laboratory. Cervical carcinoma, melanoma, and pancreatic ductal adenocarcinoma (PDAC) xenografts have been used as models of human cancer, and the transfer rate constant (Ktrans) and the extravascular extracellular volume fraction (ve) have been derived from DCE-MRI data by using Tofts standard pharmacokinetic model and a population-based arterial input function. Ktrans was found to reflect naturally occurring and treatment-induced hypoxia when hypoxia was caused by low blood perfusion, radiation responsiveness when radiation resistance was due to hypoxia, and metastatic potential when metastasis was hypoxia-induced. Ktrans was also associated with outcome for patients with locally-advanced cervical carcinoma treated with cisplatin-based chemoradiotherapy. Together, the studies imply that DCE-MRI can provide valuable information on the hypoxic status of cervical carcinoma, melanoma, and PDAC. In this communication, we review and discuss the studies and provide some recommendations as to how DCE-MRI data can be analyzed and interpreted to assess tumor hypoxia.

Intratumor Heterogeneity in Interstitial Fluid Pressure in Cervical and Pancreatic Carcinoma Xenografts.

Preclinical studies have suggested that interstitial fluid pressure (IFP) is uniformly elevated in the central region of tumors, whereas clinical studies have revealed that IFP may vary among different measurement sites in the tumor center. IFP measurements are technically difficult, and it has been claimed that the intratumor heterogeneity in IFP reported for human tumors is due to technical problems. The main purpose of this study was to determine conclusively whether IFP may be heterogeneously elevated in the central tumor region, and if so, to reveal possible mechanisms and possible consequences. Tumors of two xenograft models were included in the study: HL-16 cervical carcinoma and Panc-1 pancreatic carcinoma. IFP was measured with Millar SPC 320 catheters in two positions in each tumor and related to tumor histology or the metastatic status of the host mouse. Some tumors of both models showed significant intratumor heterogeneity in IFP, and this heterogeneity was associated with a compartmentalized histological appearance (i.e., the tissue was divided into compartments separated by thick connective tissue bands) in HL-16 tumors and with a dense collagen-I-rich extracellular matrix in Panc-1 tumors, suggesting that these connective tissue structures prevented efficient interstitial convection. Furthermore, some tumors of both models developed lymph node metastases, and of the two IFP values measured in each tumor, only the higher value was significantly higher in metastatic than in non-metastatic tumors, suggesting that metastatic propensity was determined by the tumor region having the highest IFP.