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Recent preclinical studies have shown that interstitial fluid pressure (IFP) within tumors can be heterogeneous Andersen et al. (2019). In that study tumors of two xenograft models, respectively, HL-16 cervical carcinoma and Panc-1 pancreatic carcinoma, were investigated. Significant heterogeneity in IFP was reported and it was proposed that this was associated with division of tissue into compartments separated by thick connective tissue bands for the HL-16 tumors and with dense collagen-rich extracellular matrix for the Panc-1 tumors. The purpose of the current work is to explore these experimental observations by using in silico generated tumor models. We consider a mathematical multiphase model which accounts for tumor cells, fibroblasts and interstitial fluid. The model has been trained to comply with experimental in vitro results reported in Shieh et al. (2011) which has identified autologous chemotaxis, ECM remodeling, and cell-fibroblast interaction as drivers for invasive tumor cell behavior. The in silico model is informed with parameters that characterize the leaky intratumoral vascular network, the peritumoral lymphatics which collect the fluid, and the density of ECM as represented through the hydraulic conductivity of the interstitial space. Heterogeneous distribution of solid stress may result in heterogeneous compression of blood vessels and, thus, heterogeneous vascular density inside the tumor. To mimic this we expose the in silico tumor to an intratumoral vasculature whose net effect of density of blood vesssels and vessel wall conductivity is varied through a 2D Gaussian variogram constrained such that the resulting IFPs lie within the range as reported from the preclinical study. The in silico cervical carcinoma model illustrates that sparse ECM was associated with uniform intratumoral IFP in spite of heterogeneous microvascular network, whereas compartment structures resulted in more heterogeneous IFP. Similarly, the in silico pancreatic model shows that heterogeneity in the microvascular network combined with dense ECM structure prevents IFP to even out and gives rise to heterogeneous IFP. The computer model illustrates how a heterogeneous invasive front might form where groups of tumor cells detach from the primary tumor and form isolated islands, a behavior which is natural to associate with metastatic propensity. However, unlike experimental studies, the current version of the in silico model does not show an association between metastatic propensity and elevated IFP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Intratumor heterogeneity is associated with aggressive disease and poor survival rates in several types of cancer. A novel method for assessing intratumor heterogeneity in medical images, named the spatial gradient method, has been developed in our laboratory. In this study, we measure intratumor heterogeneity in K trans maps derived by dynamic contrast-enhanced magnetic resonance imaging using the spatial gradient method, and we compare the performance of the novel method with that of histogram analyses and texture analyses using the Haralick method. K trans maps of 58 untreated and sunitinib-treated pancreatic ductal adenocaricoma (PDAC) xenografts from two PDAC models were investigated. Intratumor heterogeneity parameters derived by the spatial gradient method were sensitive to tumor line differences as well as sunitinib-induced changes in intratumor heterogeneity. Furthermore, the parameters provided additional information to the median value and were not severely affected by imaging noise. The parameters derived by histogram analyses were insensitive to spatial heterogeneity and were strongly correlated to the median value, and the Haralick features were severely influenced by imaging noise and did not differentiate between untreated and sunitinib-treated tumors. The spatial gradient method was superior to histogram analyses and Haralick features for assessing intratumor heterogeneity in K trans maps of untreated and sunitinib-treated PDAC xenografts, and can possibly be used to assess intratumor heterogeneity in other medical images and to evaluate effects of other treatments as well., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gaustad and Rofstad.)

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., (© 2021. The Author(s).)

Tumor hypoxia is a major cause of treatment resistance and poor survival in locally-advanced cervical carcinoma (LACC). It has been suggested that K trans and v e maps derived by dynamic contrast-enhanced magnetic resonance imaging can provide information on the oxygen supply and oxygen consumption of tumors, but it is not clear whether and how these maps can be combined to identify tumor hypoxia. The aim of the current study was to find the optimal strategy for calculating hypoxic fraction and predicting survival from K trans and v e maps in cervical carcinoma. K trans and v e maps of 98 tumors of four patient-derived xenograft models of cervical carcinoma as well as 80 patients with LACC were investigated. Hypoxic fraction calculated by using K trans maps correlated strongly ( P < 0.0001) to hypoxic fraction assessed with immunohistochemistry using pimonidazole as a hypoxia marker and was associated with disease-free and overall survival in LACC patients. Maps of v e did not provide information on hypoxic fraction and patient outcome, and combinations of K trans and v e were not superior to K trans alone for calculating hypoxic fraction. These observations imply that K trans maps reflect oxygen supply and may be used to identify hypoxia and predict outcome in cervical carcinoma, whereas v e is a poor parameter of oxygen consumption and does not provide information on tumor oxygenation status., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gaustad and Rofstad.)

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 ( K trans ) and the extravascular extracellular volume fraction ( v e ) have been derived from DCE-MRI data by using Tofts standard pharmacokinetic model and a population-based arterial input function. K trans 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. K trans 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.

Background: Melanoma patients with metastatic growth in the meninges have poor prognosis and few treatment options. Although treatment with BRAF inhibitors or immune checkpoint inhibitors has provided promising results, most patients with advanced melanoma are resistant to these treatments and develop severe side effects. Novel treatment strategies are needed for patients with meningeal melanoma metastases, and the potential of antiangiogenic therapy was investigated in this preclinical study., Methods: Two GFP-transfected melanoma models (A-07 and D-12) differing substantially in VEGF-A expression were included in the study, and the anti-VEGF-A antibody bevacizumab was used as therapeutic agent. Meningeal metastases were initiated in BALB/c nu/nu mice by intracranial inoculation of melanoma cells, and bevacizumab treatment was given twice a week in i.p. doses of 10 mg/kg until the mice became moribund. Therapeutic effects were evaluated by determining tumor host survival time, assessing tumor growth and angiogenic activity by quantitative analyses of histological preparations, and measuring the expression of angiogenesis-related genes by quantitative PCR., Results: Meningeal A-07 melanomas showed higher expression of VEGF-A than meningeal D-12 melanomas, whereas the expression of ANGPT2 and IL8, two important angiogenesis drivers in melanoma, was much higher in D-12 than in A-07 tumors. Bevacizumab treatment inhibited tumor angiogenesis and prolonged host survival in mice with A-07 tumors but not in mice with D-12 tumors. Meningeal A-07 tumors in bevacizumab-treated mice compensated for the reduced VEGF-A activity by up-regulating a large number of angiogenesis-related genes, including ANGPT2 and its receptors TIE1 and TIE2. Melanoma cells migrated from meningeal tumors into the cerebrum, where they initiated metastatic growth by vessel co-option. In the A-07 model, the density of cerebral micrometastases was higher in bevacizumab-treated than in untreated mice, either because bevacizumab treatment increased mouse survival or induced increased tumor gene expression., Conclusions: The development of antiangiogenic strategies for the treatment of meningeal melanoma metastases is a challenging task because the outcome of treatment will depend on the angiogenic signature of the tumor tissue, treatment-induced alterations of the angiogenic signature, and the treatment sensitivity of metastatic lesions in other intracranial sites.

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 differences were induced by the initial tumor angiogenesis. In both models, sunitinib treatment reduced intratumor and peritumor vessel densities by selectively removing small-diameter vessels. Sunitinb treatment resulted in a general decrease in vessel density and scattered hypoxia in BxPC-3 tumors, and depleted most vessels and induced massive hypoxia in central parts of Capan-2 tumors. The study demonstrates that PDAC xenografts can differ in vascularization, and the differences can impact oxygenation and effects of treatment. Neoadjuvant sunitinib treatment is inappropriate in combination with conventional therapy for human PDACs resembling the PDAC xenografts used here, because sunitinib-induced hypoxia can impair the effect of most conventional therapies.

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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: This study had a dual purpose: to investigate (1) whether bevacizumab can change the microvasculature and oxygenation of cervical carcinomas and (2) whether any changes can be detected with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)., Methods and Materials: Two patient-derived xenograft models of cervical cancer (BK-12 and HL-16) were included in the study. Immunostained histologic preparations from untreated and bevacizumab-treated tumors were analyzed with respect to microvascular density, vessel pericyte coverage, and tumor hypoxia using CD31, α-SMA, and pimonidazole as markers, respectively. DCE-MRI was performed at 7.05 T, and parametric images of K trans and v e were derived from the data using the Tofts pharmacokinetic model., Results: The tumors of both models showed decreased microvascular density, increased vessel pericyte coverage, and increased vessel maturation after bevacizumab treatment. Bevacizumab-treated tumors were more hypoxic and had lower K trans values than untreated tumors in the BK-12 model, whereas bevacizumab-treated and untreated HL-16 tumors had similar hypoxic fractions and similar K trans values. Significant correlations were found between median K trans and hypoxic fraction, and the data for untreated and bevacizumab-treated tumors were well fitted by the same curve in both tumor models., Conclusions: Bevacizumab-treated tumors show less abnormal microvessels than untreated tumors do, but because of treatment-induced vessel pruning, the overall function of the microvasculature might be impaired after bevacizumab treatment, resulting in increased tumor hypoxia. DCE-MRI has great potential for monitoring bevacizumab-induced changes in tumor hypoxia in cervical carcinoma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Antiangiogenic treatment (AAT) used in combination with radiation therapy or chemotherapy is a promising strategy for the treatment of several cancer diseases. The vascularity and oxygenation of tumors may be changed significantly by AAT, and consequently, a noninvasive method for monitoring AAT-induced changes in these microenvironmental parameters is needed. The purpose of this study was to evaluate the potential usefulness of diffusion-weighted magnetic resonance imaging (DW-MRI). DW-MRI was conducted with a Bruker Biospec 7.05-T scanner using four diffusion weightings and diffusion sensitization gradients in three orthogonal directions. Maps of the apparent diffusion coefficient (ADC) were calculated by using a monoexponential diffusion model. Two cervical carcinoma xenograft models (BK-12, HL-16) were treated with bevacizumab, and two pancreatic carcinoma xenograft models (BxPC-3, Panc-1) were treated with sunitinib. Pimonidazole and CD31 were used as markers of hypoxia and blood vessels, respectively, and fraction of hypoxic tissue (HF Pim ) and microvascular density (MVD) were quantified by analyzing immunohistochemical preparations. MVD decreased significantly after AAT in BK-12, HL-16, and BxPC-3 tumors, and this decrease was sufficiently large to cause a significant increase in HF Pim in BK-12 and BxPC-3 tumors. The ADC maps of treated tumors and untreated control tumors were not significantly different in any of these three tumor models, suggesting that the AAT-induced microenvironmental changes were not detectable by DW-MRI. DW-MRI is insensitive to changes in tumor vascularity and oxygenation induced by bevacizumab or sunitinib treatment., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was dual: to investigate (a) whether sunitinib may induce changes in tumor microvasculature and hypoxia in pancreatic ductal adenocarcinoma (PDAC) and (b) whether any changes can be detected by DCE-MRI. Sunitinib-treated and untreated control tumors of two PDAC xenograft models (BxPC-3 and Panc-1) were subjected to DCE-MRI before the imaged tumors were prepared for quantitative analysis of immunohistochemical preparations. Pimonidazole was used as a hypoxia marker, and fraction of hypoxic tissue (HF Pim ), density of CD31-positive microvessels (MVD CD31 ), and density of αSMA-positive microvessels (MVD αSMA ) were measured. Parametric images of K trans and v e were derived from the DCE-MRI data by using the Tofts pharmacokinetic model. BxPC-3 tumors showed increased HF Pim , decreased MVD CD31 , unchanged MVD αSMA , and increased vessel maturation index (VMI = MVD αSMA /MVD CD31 ) after sunitinib treatment. The increase in VMI was seen because sunitinib induced selective pruning rather than maturation of αSMA-negative microvessels. Even though the microvessels in sunitinib-treated tumors were less abnormal than those in untreated tumors, this microvessel normalization did not improve the function of the microvascular network or normalize the tumor microenvironment. In Panc-1 tumors, HF Pim , MVD CD31 , MVD αSMA , and VMI were unchanged after sunitinib treatment. Median K trans increased with increasing MVD CD31 and decreased with increasing HF Pim , and the correlations were similar for treated and untreated BXPC-3 and Panc-1 tumors. These observations suggest that sunitinib may induce significant changes in the microenvironment of PDACs, and furthermore, that K trans may be an adequate measure of tumor vascular density and hypoxia in untreated as well as sunitinib-treated PDACs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Four patient-derived xenograft (PDX) models (BK-12, ED-15, HL-16, LA-19) of carcinoma of the uterine cervix have been developed in our laboratory, and their stability during serial transplantation in vivo was investigated in this study. Two frozen cell stocks were established, one from xenografted tumors in passage 2 (early generation) and the other from xenografted tumors transplanted serially in mice for approximately two years (late generation), and the biology of late generation tumors was compared with that of early generation tumors. Late generation tumors showed higher incidence of lymph node metastases than early generation tumors in three models (ED-15, HL-16, LA-19), and the increased metastatic propensity was associated with increased tumor growth rate, increased microvascular density, and increased expression of angiogenesis-related and cancer stem cell-related genes. Furthermore, late generation tumors showed decreased fraction of pimonidazole-positive tissue ( i.e. , decreased fraction of hypoxic tissue) in two models (HL-16, LA-19) and decreased fraction of collagen-I-positive tissue (i.e., less extensive extracellular matrix) in two models (ED-15, HL-16). This study showed that serially transplanted PDXs may not necessarily mirror the donor patients' diseases, and consequently, proper use of serially transplanted PDX models in translational cancer research requires careful molecular monitoring of the models., Competing Interests: CONFLICTS OF INTEREST The authors have no potential conflicts of interest to disclose.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor outcome. Resistance to treatment is associated with impaired vascularity, extensive hypoxia, and interstitial hypertension. In this study, the potential of dynamic contrast-enhanced (DCE)-MRI as a method for assessing the microvascular density (MVD), the fraction of hypoxic tissue, and the interstitial fluid pressure (IFP) of PDACs was investigated., Material and Methods: Intramuscular BxPC-3, Capan-2, MIAPaCa-2, and Panc-1 PDAC xenografts were used as preclinical models of human PDACs. DCE-MRI with Gd-DOTA as contrast agent was conducted with a 7.05-T scanner, and the DCE-MRI series were analyzed voxelwise by using the Tofts pharmacokinetic model. Tumor MVD and hypoxia were measured in histological preparations by using pimonidazole as a hypoxia marker and CD31 as a marker of endothelial cells. IFP was measured with a Millar catheter., Results: K trans (the volume transfer constant of Gd-DOTA) increased with increasing MVD and decreased with increasing hypoxic fraction, but was not associated with IFP. Any association between v e (the fractional distribution volume of Gd-DOTA) and MVD, hypoxic fraction, or IFP could not be detected., Conclusions: This study shows that DCE-MRI is a useful modality for assessing important features of the microenvironment of PDAC xenografts and thus provides the basis for future preclinical and clinical DCE-MRI investigations of PDAC.

Background: Studies investigating the oxygenation status and the development of hypoxia in microscopic tumors are sparse. The purpose of this study was to measure the extent of hypoxia in microscopic melanoma xenografts and to search for possible mechanisms leading to the development of hypoxia in these tumors., Methods: A-07, D-12, R-18, and U-25 human melanoma xenografts grown in dorsal window chambers or as flank tumors were used as preclinical tumor models. Morphologic and functional parameters of vascular networks were assessed with intravital microscopy, and the expression of angiogenesis-related genes was assessed with quantitative PCR. Microvessels, pericytes, and the extent of hypoxia were assessed by immunohistochemistry in microscopic tumors by using CD31, αSMA, and pimonidazole as markers, and the extent of radiobiological hypoxia was assessed in macroscopic flank tumors., Results: Macroscopic R-18 and U-25 tumors showed extensive hypoxia, whereas macroscopic A-07 and D-12 tumors were less hypoxic. R-18 and U-25 tumors developed hypoxic regions before they reached a size of 2-3 mm in diameter, whereas A-07 and D-12 tumors of similar size did not show hypoxic regions. The development of hypoxic regions was not caused by low vessel density, but was rather a result of inadequate vascular function. Inadequate vascular function was not caused by low vessel diameters or long vessel segments, but was associated with poor vascular pericyte coverage. Poor pericyte coverage was associated with the expression of eight angiogenesis-related genes., Conclusions: Two of the four investigated melanoma models developed hypoxic regions in microscopic tumors, and the development of hypoxia was associated with poor vascular pericyte coverage and inadequate vascular function.

Apparent diffusion coefficient (ADC) values derived from diffusion-weighted magnetic resonance imaging (DW-MRI) are known to reflect the cellular environment of biological tissues. However, emerging evidence accentuates the influence of stromal elements on ADC values. The current study sought to elucidate whether a correlation exists between ADC and the fraction of collagen I-positive tissue across different tumor models of uterine cervical cancer. Early and late generation tumors of four patient-derived xenograft (PDX) models of squamous cell carcinoma (BK-12, ED-15, HL-16, and LA-19) were included. DW-MRI was performed with diffusion encoding constants ( b ) of 200, 400, 700, and 1000 s/mm 2 and diffusion gradient sensitization in three orthogonal directions. The fraction of collagen I-positive connective tissue was determined by immunohistochemistry. Mono-exponential decay curves, from which the ADC value of tumor voxels was calculated, yielded good fits to the diffusion data. A significant inverse correlation was detected between median tumor ADC and collagen I fraction across the four PDX models, indicating that collagen fibers in the extracellular space have the ability to inhibit the movement of water molecules in these xenografts. The results encourage further exploration of DW-MRI as a non-invasive imaging method for characterizing the stromal microenvironment of tumors., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to declare.

Background: Abnormalities in the tumor microenvironment are associated with resistance to treatment, aggressive growth, and poor clinical outcome in patients with advanced cervical cancer. The potential of dynamic contrast-enhanced (DCE) MRI to assess the microvascular density (MVD), interstitial fluid pressure (IFP), and hypoxic fraction of patient-derived cervical cancer xenografts was investigated in the present study., Methods: Four patient-derived xenograft (PDX) models of squamous cell carcinoma of the uterine cervix (BK-12, ED-15, HL-16, and LA-19) were subjected to Gd-DOTA-based DCE-MRI using a 7.05 T preclinical scanner. Parametric images of the volume transfer constant (K trans ) and the fractional distribution volume (v e ) of the contrast agent were produced by pharmacokinetic analyses utilizing the standard Tofts model. Whole tumor median values of the DCE-MRI parameters were compared with MVD and the fraction of hypoxic tumor tissue, as determined histologically, and IFP, as measured with a Millar catheter., Results: Both on the PDX model level and the single tumor level, a significant inverse correlation was found between K trans and hypoxic fraction. The extent of hypoxia was also associated with the fraction of voxels with unphysiological v e values (v e  > 1.0). None of the DCE-MRI parameters were related to MVD or IFP., Conclusions: DCE-MRI may provide valuable information on the hypoxic fraction of squamous cell carcinoma of the uterine cervix, and thereby facilitate individualized patient management.

Pancreatic ductal adenocarcinoma (PDAC) patients develop lymph node metastases early and have a particularly poor prognosis. The poor prognosis has been shown to be associated with the physicochemical microenvironment of the tumor tissue, which is characterized by desmoplasia, abnormal microvasculature, extensive hypoxia, and highly elevated interstitial fluid pressure (IFP). In this study, we searched for associations between lymph node metastasis and features of the physicochemical microenvironment in an attempt to identify mechanisms leading to metastatic dissemination and growth. BxPC-3 and Capan-2 PDAC xenografts were used as preclinical models of human PDAC. In both models, lymph node metastasis was associated with high IFP rather than high fraction of hypoxic tissue or high microvascular density. Seven angiogenesis-related genes associated with high IFP-associated lymph node metastasis were detected by quantitative PCR in each of the models, and these genes were all up-regulated in high IFP/highly metastatic tumors. Three genes were mutual for the BxPC-3 and Capan-2 models: transforming growth factor beta, angiogenin, and insulin-like growth factor 1. Further comprehensive studies are needed to determine whether there is a causal relationship between the up-regulation of these genes and high IFP and/or high propensity for lymph node metastasis in PDAC.

Background: Studies comparing the effect of antiangiogenic agents targeting different angiogenic pathways are sparse. The purpose of this study was to compare the effect of properdistatin and sunitinib treatment in a preclinical model of malignant melanoma. Properdistatin is a small peptide derived from the thrombospondin-1 domain of the plasma protein properdin, and sunitinib is a tyrosine kinase inhibitor targeting several receptors including the vascular endothelial growth factor receptors., Methods: R-18 human melanoma xenografts growing in dorsal window chambers were treated with properdistatin, sunitinib, or vehicle. Parameters describing the morphology of tumor vasculature were assessed from high-resolution transillumination images, and BST (blood supply time; the time needed for arterial blood to flow from the main supplying artery to downstream microvessels) was assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by using pimonidazole as a hypoxia marker., Results: Properdistatin treatment selectively removed small-diameter vessels and reduced BST, whereas sunitinib treatment reduced the density of small- and large-diameter vessel similarly and did not change BST. These observations imply that properdistatin treatment reduced geometric resistance to blood flow and improved vascular function, whereas sunitinib treatment did not affect vascular function. Accordingly, sunitinib-treated tumors showed higher hypoxic fractions than properdistatin-treated tumors., Conclusions: Properdistatin and sunitinib both inhibited angiogenesis, but had distinctly different effects on vascular morphology, vascular function, and extent of hypoxia in R-18 human melanoma xenografts.

The effect of antiangiogenic agents targeting the vascular endothelial growth factor A (VEGF-A) pathway has been reported to vary substantially in preclinical studies. The purpose of this study was to investigate the effect of sunitinib treatment on tumor vasculature and oxygenation in melanoma xenografts with different angiogenic profiles. A-07, U-25, D-12, or R-18 melanoma xenografts were grown in dorsal window chambers and given daily treatments of sunitinib (40 mg/kg) or vehicle. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply times (BSTs) were assessed from first-pass imaging movies. Tumor hypoxia was assessed with immunohistochemistry by using pimonidazole as hypoxia marker, and the gene expression and the protein secretion rate of angiogenic factors were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The melanoma lines differed substantially in the expression of VEGF-A, VEGF-C, and platelet-derived growth factor A. Sunitinib treatment reduced vessel densities and induced hypoxia in all melanoma lines, and the magnitude of the effect was associated with the gene expression and protein secretion rate of VEGF-A. Sunitinib treatment also increased vessel segment lengths, reduced the number of small-diameter vessels, and inhibited growth-induced increases in the diameter of surviving vessels but did not change BST. In conclusion, sunitinib treatment did not improve vascular function but reduced vessel density and induced hypoxia in human melanoma xenografts. The magnitude of the treatment-induced effect was associated with the VEGF-A expression of the melanoma lines., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In this study, the effect of properdistatin, a novel peptide derived from the thrombospondin 1 (TSP-1) domain of properdin, was investigated in three melanoma xenograft models with different TSP-1 expression. The tumors were grown in dorsal window chambers and were treated with 80 mg/kg/day properdistatin or vehicle. Morphological parameters of the tumor vasculature were assessed from high resolution transillumination images. Blood supply time (i.e., the time required for arterial blood to flow from a supplying artery to downstream microvessels) and plasma velocities were assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Gene and protein expression of TSP-1 were assessed with quantitative PCR and immunohistochemistry, respectively. Properdistatin treatment inhibited angiogenesis in low TSP-1 expressing tumors but did not alter the vasculature in high TSP-1 expressing tumors. In low TSP-1 expressing tumors, properdistatin selectively removed small-diameter capillaries, but did not change the morphology of tumor arterioles or tumor venules. Properdistatin also reduced blood supply times and increased plasma velocities, implying that the treatment reduced the geometric resistance to blood flow and improved vascular function.

Studies of cell line-derived human tumor xenografts have suggested that the lymphatics seen in immunohistochemical preparations from non-peripheral regions of tumors are nonfunctional. In this investigation, lymphangiogenesis, hemangiogenesis, and lymph node metastasis were studied in patient-derived xenograft (PDX) models of carcinoma of the uterine cervix. Lymph vessel density (LVD) and blood vessel density (BVD) were measured in immunohistochemical preparations. The expression of angiogenesis-related genes was investigated by quantitative PCR. Lymphatic functionality was assessed with the ferritin assay, and tumor interstitial fluid pressure (IFP) was measured with a Millar catheter. The PDX models mirrored the angiogenesis and aggressiveness of the donor patients' tumors, and two highly aggressive models developed functional lymphatics within the tumor mass. Tumors with functional intratumoral lymphatics showed low IFP, high LVD, high BVD, high expression of a large number of angiogenesis-related genes, and high incidence of lymph node metastases. LVD correlated with BVD, and lymph node metastasis was associated with high LVD and high BVD. Nine angiogenesis-related genes associated with the development of functional intratumoral lymhatics were identified. High expression of these genes, high LVD, and high BVD may be important biomarkers for poor outcome in cervix carcinoma., Competing Interests: The authors have no potential conflicts of interest to disclose.

Purpose: The aggressiveness of pancreatic ductal adenocarcinoma (PDAC) is highly dependent on the level of differentiation and the composition of the stroma. In this preclinical study, we investigated the potential of diffusion-weighted magnetic resonance imaging (DW-MRI) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) as noninvasive methods for providing information on the differentiation and the stroma of PDACs., Methods: Xenografted tumors initiated from four PDAC cell lines (BxPC-3, Capan-2, MIAPaCa-2, and Panc-1) were included in the study. DW-MRI and DCE-MRI were carried out on a 7.05-T MR scanner, and tumor images of ADC (the apparent diffusion coefficient), K (trans) (the volume transfer constant of Gd-DOTA), and v e (the fractional distribution volume of Gd-DOTA) were produced. The level of differentiation and the amount and structure of collagen I and collagen IV were determined by examining histological preparations., Results: Differentiated tumors showed lower levels of collagen I and collagen IV than non-differentiated tumors. Significant correlations were found between ADC and v e, and both parameters differentiated clearly between collagen-rich non-differentiated tumors and differentiated tumors containing less collagen., Conclusion: Differentiated PDAC xenografts show higher ADC values and higher v e values than their non-differentiated counterparts. This observation supports the application of parametric MR images as tumor biomarkers in PDAC. Patients showing low values of ADC and v e most likely have non-differentiated tumors with extensive stroma and, hence, poor prognosis.

Introduction: A majority of patients with melanoma brain metastases develop multiple lesions, and these patients show particularly poor prognosis. To develop improved treatment strategies, detailed insights into the biology of melanoma brain metastases, and particularly the development of multiple lesions, are needed. The purpose of this preclinical investigation was to study melanoma cell migration within the brain after cell injection into a well-defined intracerebral site., Methods: A-07, D-12, R-18, and U-25 human melanoma cells transfected with green fluorescent protein were injected stereotactically into the right cerebral hemisphere of nude mice. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or histological examination., Results: Intracerebral inoculation of melanoma cells produced multiple lesions involving all regions of the brain, suggesting that the cells were able to migrate over substantial distances within the brain. Multiple modes of transport were identified, and all transport modes were observed in all four melanoma lines. Thus, the melanoma cells were passively transported via the flow of cerebrospinal fluid in the meninges and ventricles, they migrated actively along leptomeningeal and brain parenchymal blood vessels, and they migrated actively along the surfaces separating different brain compartments., Conclusion: Migration of melanoma cells after initial arrest, extravasation, and growth at a single location within the brain may contribute significantly to the development of multiple melanoma brain metastases., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Patient-derived xenograft (PDX) models of cancer are considered to reflect the biology and treatment response of human tumors to a larger extent than xenograft models initiated from established cell lines. The characterization of a panel of four novel PDX models of cervical carcinoma of the uterine cervix is described in this communication. The outcome of treatment differed substantially among the donor patients, and the PDX models were found to mirror the histology, aggressiveness, and metastatic propensity of the donor patients' tumors. Two of the models (BK-12 and LA-19) were highly metastatic, one model (ED-15) was poorly metastatic, and one model (HL-16) was non-metastatic. The primary tumors of the two highly metastatic models showed high density of intratumoral lymphatics, whereas the other two models did not develop intratumoral lymphatics. The potential of the models to metastasize to lymph nodes was associated with high expression of both angiogenesis-related genes and cancer stem cell-related genes. The models may be highly valuable for studying mechanisms linking lymph node metastasis to lymphangiogenesis, hemangiogenesis, and the presence of cancer stem cells., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Patients diagnosed with melanoma brain metastases have few treatment options and poor prognosis, and improved treatment strategies for these patients require detailed understanding of the underlying pathobiology. In this investigation we studied the vascularity and invasiveness of artificial brain metastases established from four human melanoma cell lines., Methods: A-07, D-12, R-18, and U-25 cells transfected with GFP were injected intracerebrally and intra-arterially in nude mice. Moribund mice were killed and autopsied, and the brain was evaluated by fluorescence imaging or by histological examination. Expression and secretion of factors involved in angiogenesis and invasion were assessed by quantitative PCR, ELISA, and immunohistochemistry., Results: The melanoma cells grew preferentially in the meninges and ventricles after intracerebral and intra-arterial injection. Intertumor heterogeneity in the aggressiveness of meningeal tumors reflected differences in angiogenic activity and expression of vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL-8). In contrast, growth and invasion of the brain parenchyma relied primarily on vascular co-option. The cell lines showed different patterns of invasion from meninges to the scull and from meninges to the brain parenchyma, and these differences were associated with differences in expression of the matrix metalloproteinases MMP-2 and MMP-9. Furthermore, the melanoma cells produced multiple brain lesions after intracerebral implantation by using the meningeal linings of the brain as transport routes., Conclusions: The melanoma cell lines showed different growth patterns in the brain, and these differences were associated with differences in expression of the angiogenic factors VEGF-A and IL-8 and the matrix metalloproteinases MMP-2 and MMP-9.

Background: Antiangiogenic treatments have been shown to increase blood perfusion and oxygenation in some experimental tumors, and to reduce blood perfusion and induce hypoxia in others. The purpose of this preclinical study was to investigate the potential of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and diffusion weighted MRI (DW-MRI) in assessing early effects of low dose bevacizumab treatment, and to investigate intratumor heterogeneity in this effect., Methods: A-07 and R-18 human melanoma xenografts, showing high and low expression of VEGF-A, respectively, were used as tumor models. Untreated and bevacizumab-treated tumors were subjected to DCE-MRI and DW-MRI before treatment, and twice during a 7-days treatment period. Tumor images of Ktrans (the volume transfer constant of Gd-DOTA) and ve (the fractional distribution volume of Gd-DOTA) were produced by pharmacokinetic analysis of the DCE-MRI data, and tumor images of ADC (the apparent diffusion coefficient) were produced from DW-MRI data., Results: Untreated A-07 tumors showed higher Ktrans, v e, and ADC values than untreated R-18 tumors. Untreated tumors showed radial heterogeneity in Ktrans, i.e., Ktrans was low in central tumor regions and increased gradually towards the tumor periphery. After the treatment, bevacizumab-treated A-07 tumors showed lower Ktrans values than untreated A-07 tumors. Peripherial tumor regions showed substantial reductions in Ktrans, whereas little or no effect was seen in central regions. Consequently, the treatment altered the radial heterogeneity in Ktrans. In R-18 tumors, significant changes in Ktrans were not observed. Treatment induced changes in tumor size, v e, and ADC were not seen in any of the tumor lines., Conclusions: Early effects of low dose bevacizumab treatment may be highly heterogeneous within tumors and can be detected with DCE-MRI.

Properdistatin is a novel peptide derived from the thrombospondin-1 domain of the plasma protein properdin. The purpose of this study was to investigate the effect of properdistatin treatment on the morphology and function of tumor vasculature. A-07 human melanoma xenografts grown in dorsal window chambers were used as preclinical model. Tumors were treated with 80 mg/kg/day properdistatin or vehicle for 4 days. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time and plasma velocities were assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Properdistatin-treated tumors showed reduced density of small-diameter vessels, reduced blood supply time, and increased plasma velocities. In conclusion, properdistatin treatment inhibited angiogenesis and improved vascular function in A-07 tumors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Antiangiogenic treatment may change the tumor microenvironment and hence influence the effect of conventional therapies. The potential of diffusion weighted magnetic resonance imaging (DW-MRI) and dynamic contrast enhanced MRI (DCE-MRI) in assessing microenvironmental effects of sunitinib treatment was investigated in this preclinical study., Methods: Sunitinib-treated and untreated A-07 tumors were subjected to DW-MRI and DCE-MRI, and parametric images of ADC and Ktrans were produced. Microvascular density, hypoxic fraction, and necrotic fraction were assessed from immunohistochemical preparations, and tumor interstitial fluid pressure (IFP) was assessed with probe measurement., Results: Sunitinib-treated tumors showed reduced microvascular density, increased hypoxic fraction, increased necrotic fraction, increased ADC, and reduced Ktrans, but did not differ from untreated tumors in growth rate and IFP., Conclusions: Sunitinib treatment affected the tumor microenvironment without affecting tumor size. DW-MRI and DCE-MRI were sensitive to the sunitinib-induced changes in the tumor microenvironment.

The efficacy of most cancer treatments is strongly influenced by the tumor blood supply. The results of experimental studies using xenografted tumors to evaluate novel cancer treatments may therefore vary considerably depending on the blood supply of the specific tumor model being used. Mechanisms underlying intertumor heterogeneity in the blood supply of xenografted tumors derived from same tumor line are poorly understood, and were investigated here by using intravital microscopy to assess tumor blood supply and vascular morphology in human melanomas growing in dorsal window chambers in BALB/c nu/nu mice. Two melanoma lines, A-07 and R-18, were included in the study. These lines differed substantially in angiogenic profiles. Thus, when the expression of 84 angiogenesis-related genes was investigated with a quantitative PCR array, 25% of these genes showed more than a 10-fold difference in expression. Furthermore, A-07 tumors showed higher vascular density, higher vessel tortuosity, higher vessel diameters, shorter vessel segments, and more chaotic vascular architecture than R-18 tumors. Both lines showed large intertumor heterogeneity in blood supply. In the A-07 line, tumors with low microvascular density, long vessel segment, and high vessel tortuosity showed poor blood supply, whereas in the R-18 line, poor tumor blood supply was associated with low tumor arteriolar diameters. Thus, tumor-line specific causes of intertumor heterogeneity in blood supply were identified in human melanoma xenografts, and these tumor-line specific mechanisms were possibly a result of tumor-line specific angiogenic profiles., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The purpose of this study was to investigate the effect of acute cyclic hypoxia on tumor vasculature. A-07 human melanoma xenografts growing in dorsal window chambers were used as tumor model. Acute cyclic hypoxia was induced by periodically exposing tumor-bearing mice to a low oxygen atmosphere. The hypoxia treatment consisted of 12 cycles of 10 min of low O(2) (8% O(2) in N(2)) followed by 10 min of air for a total of 4 hr. The treatment started the first day after tumor initiation, and was given daily for 9 days. Vascular morphology was assessed from high-resolution transillumination images, and tumor blood supply was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Hypoxia-treated tumors showed increased vessel density, decreased interstitial distance, and delayed blood supply compared to control tumors. The increase in vessel density was attributed to an increased number of small vessels. In conclusion, acute cyclic hypoxia induced angiogenesis in A-07 tumors resulting in increased density of small-diameter vessels and delayed tumor blood supply., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Purpose: The fraction of hypoxic cells has been shown to differ substantially among human tumors of the same histological type. In this study, a window chamber model was used to identify possible mechanisms leading to the development of highly different hypoxic fractions in A-07 and R-18 human melanoma xenografts., Materials and Methods: Chronic and acute hypoxia was assessed in intradermal tumors using an immunohistochemical and a radiobiological assay. Functional and morphological parameters of the vascular networks of tumors growing in dorsal window chambers were assessed with intravital microscopy., Results: R-18 tumors showed significantly higher hypoxic fractions than A-07 tumors, and the difference was mostly due to acute hypoxia. Compared to A-07 tumors, R-18 tumors showed low vascular densities, low vessel diameters, long vessel segments, low blood flow velocities, frequent fluctuations in blood flow, and a high fraction of narrow vessels with absent or very low and varying flux of red blood cells., Conclusion: The high fraction of acute hypoxia in R-18 tumors was a consequence of frequent fluctuations in blood flow and red blood cell flux combined with low vascular density. The fluctuations were most likely caused by high geometric resistance to blood flow in the tumor microvasculature., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Antiangiogenic agents that disrupt the vascular endothelial growth factor pathway have been demonstrated to normalize tumor vasculature and improve tumor oxygenation in some studies and to induce hypoxia in others. The aim of this preclinical study was to investigate the effect of sunitinib treatment on the morphology and function of tumor vasculature and on tumor oxygenation., Methods: A-07-GFP and R-18-GFP human melanoma xenografts grown in dorsal window chambers were used as preclinical tumor models. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time was assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by use of pimonidazole as a hypoxia marker., Results: Sunitinib treatment reduced vessel densities, increased vessel segment lengths, did not affect blood supply times, and increased hypoxic area fractions., Conclusion: Sunitinib treatment did not improve vascular function but induced hypoxia in A-07-GFP and R-18-GFP tumors.

The purpose of this study was to establish a lymphangiography assay allowing repetitive imaging of tumor-associated lymphatics, and to investigate potential associations between tumor-induced changes in pre-existing lymphatics and tumor angiogenesis. A-07-GFP human melanoma xenografts grown in dorsal window chambers were used as preclinical tumor model. Lymphatics were visualized after multiple intradermal injections of 155 kDa tetramethylrhodamine isothiocyanate dextran outside the window chambers, and tumor vasculature was assessed from high-resolution transillumination images. Lymphangiography was performed thrice in window chambers with or without A-07-GFP tumors, with 3-4 days between repetitions. The lymphangiography assay was highly reproducible. A-07-GFP tumors induced dilation of pre-existing lymphatics after the onset of tumor angiogenesis, and the dilation was correlated with tumor size., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Patients with highly hypoxic primary tumors show increased frequency of locoregional treatment failure and poor survival rates and may benefit from particularly aggressive treatment. The potential of gadolinium diethylene-triamine penta-acetic acid-based dynamic contrast-enhanced-MRI in assessing tumor hypoxia was investigated in this preclinical study. Xenografted tumors of eight human melanoma lines were subjected to dynamic contrast-enhanced-MRI and measurement of the fraction of radiobiologically hypoxic cells and the fraction of pimonidazole-positive hypoxic cells. Tumor images of K(trans) (the volume transfer constant of gadolinium diethylene-triamine penta-acetic acid) and v(e) (the fractional distribution volume of gadolinium diethylene-triamine penta-acetic acid) were produced by pharmacokinetic analysis of the dynamic contrast-enhanced-MRI data, and K(trans) and v(e) frequency distributions of the non-necrotic tumor tissue were established and related to the extent of hypoxia. Tumors showing high K(trans) values and high v(e) values had low fractions of hypoxic cells, whereas tumors showing both low K(trans) values and low v(e) values had high hypoxic fractions. K(trans) differentiated better between tumors with low and high hypoxic fractions than did v(e). This study supports the current attempts to establish dynamic contrast-enhanced-MRI as a method for assessing the extent of hypoxia in human tumors, and it provides guidelines for the clinical development of valid assays., (Copyright © 2011 Wiley Periodicals, Inc.)

Purpose: Interstitial fluid pressure (IFP) is highly elevated in many solid tumors. High IFP has been associated with low radiocurability and high metastatic frequency in human melanoma xenografts and with poor survival after radiation therapy in cervical cancer patients. Abnormalities in tumor vascular networks have been identified as an important cause of elevated tumor IFP. The aim of this study was to investigate the relationship between tumor IFP and the functional and morphological properties of tumor vascular networks., Materials and Methods: A-07-GFP and R-18-GFP human melanomas growing in dorsal window chambers in BALB/c nu/nu mice were used as preclinical tumor models. Functional and morphological parameters of the vascular network were assessed from first-pass imaging movies and vascular maps recorded after intravenous bolus injection of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran. IFP was measured in the center of the tumors using a Millar catheter. Angiogenic profiles of A-07-GFP and R-18-GFP cells were obtained with a quantitative PCR array., Results: High IFP was associated with low growth rate and low vascular density in A-07-GFP tumors, and with high growth rate and high vascular density in R-18-GFP tumors. A-07-GFP tumors showed chaotic and highly disorganized vascular networks, while R-18-GFP tumors showed more organized vascular networks with supplying arterioles in the tumor center and draining venules in the tumor periphery. Furthermore, A-07-GFP and R-18-GFP cells differed substantially in angiogenic profiles. A-07-GFP tumors with high IFP showed high geometric resistance to blood flow due to high vessel tortuosity. R-18-GFP tumors with high IFP showed high geometric resistance to blood flow due to a large number of narrow tumor capillaries., Conclusions: High IFP in A-07-GFP and R-18-GFP human melanoma xenografts was primarily a consequence of high blood flow resistance caused by tumor-line specific vascular abnormalities.

Background: The prognostic and predictive value of magnetic resonance (MR) investigations in clinical oncology may be improved by implementing strategies for discriminating between viable and necrotic tissue in tumors. The purpose of this preclinical study was to investigate whether the extent of necrosis in tumors can be assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and/or T(2)-weighted MR imaging., Material and Methods: Three amelanotic human melanoma xenograft lines differing substantially in tumor necrotic fraction, necrotic pattern, extracellular volume fraction, and blood perfusion were used as experimental models of human cancer. MRI was performed at 1.5 T and a spatial resolution of 0.23 × 0.47 × 2.0 mm(3). Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) was used as contrast agent. Plots of Gd-DTPA concentration versus time were generated for each voxel, and three parameters were calculated for each curve: the extracellular volume fraction (ν(e)), the final slope (a), and the Gd-DTPA concentration at one minute after the contrast administration (C(1min)). Parametric images of ν(e), a, C(1min), and the signal intensity in T(2)-weighted images (SI(T2W)) were compared with the histology of the imaged tissue., Results: The ν(e), a, and C(1min) frequency distributions were significantly different for necrotic and viable tissue in all three tumor lines. By using adequate values of ν(e), a, and C(1min) to discriminate between necrotic and viable tissue, significant correlations were found between the fraction of necrotic tissue assessed by MRI and the fraction of necrotic tissue assessed by image analysis of histological preparations. On the other hand, the SI(T2W) frequency distributions did not differ significantly between necrotic and viable tissue in two of the three tumor lines., Conclusion: Necrotic regions in tumor tissue can be identified in parametric images derived from DCE-MRI series, whereas T(2)-weighted images are unsuitable for detection of tumor necrosis.

Clinical studies have shown that patients with highly hypoxic primary tumors may have poor disease-free and overall survival rates. Studies of experimental tumors have revealed that acutely hypoxic cells may be more metastatic than normoxic or chronically hypoxic cells. In the present work, causal relations between acute cyclic hypoxia and metastasis were studied by periodically exposing BALB/c nu/nu mice bearing A-07 human melanoma xenografts to a low oxygen atmosphere. The hypoxia treatment consisted of 12 cycles of 10 min of 8% O(2) in N(2) followed by 10 min of air for a total of 4 hr, began on the first day after tumor cell inoculation and was given daily until the tumors reached a volume of 100 mm(3). Twenty-four hours after the last hypoxia exposure, the primary tumors were subjected to dynamic contrast-enhanced magnetic resonance imaging for assessment of blood perfusion before being resected and processed for immunohistochemical examinations of microvascular density and expression of proangiogenic factors. Mice exposed to acute cyclic hypoxia showed increased incidence of pulmonary metastases, and the primary tumors of these mice showed increased blood perfusion, microvascular density and vascular endothelial growth factor-A (VEGF-A) expression; whereas, the expression of interleukin-8, platelet-derived endothelial cell growth factor and basic fibroblast growth factor was unchanged. The increased pulmonary metastasis was most likely a consequence of hypoxia-induced VEGF-A upregulation, which resulted in increased angiogenic activity and blood perfusion in the primary tumor and thus facilitated tumor cell intravasation and hematogenous transport into the general circulation.

Purpose: Hypoxic regions have been shown to be a characteristic feature of a wide variety of human primary tumors, whereas the oxygenation status of subclinical micrometastases is in general unknown. The development of hypoxia in a xenograft model of microscopic metastases was investigated in this study., Methods and Materials: U-25-GFP human melanomas growing in dorsal window chamber preparations in BALB/c nu/nu mice were used as a preclinical model of micrometastases. Tumor blood supply time and morphologic parameters of the vascular network were determined from first-pass imaging movies and vascular maps recorded by use of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran as a vascular tracer. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by use of pimonidazole as a hypoxia marker., Results: Nearly half of the tumors had developed hypoxic regions when they reached a diameter of 2 to 3 mm. Tumors with multiple hypoxic foci showed a low growth rate, low blood flow velocity, high vessel tortuosity, high vessel segment length, and high vascular density, whereas tumors with a single hypoxic region showed a high growth rate, high blood flow velocity, low vessel tortuosity, low vessel segment length, and low vascular density. The tumors with hypoxic regions did not differ from those without hypoxia in any single parameter., Conclusions: U-25-GFP xenograft models of vascularized human tumor micrometastases may develop hypoxic regions as a consequence of two distinctly different morphologic abnormalities in the vascular network: high resistance against blood flow (i.e., high vessel tortuosity and high vessel segment length) or low vascular density., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Interstitial fluid pressure (IFP) has been shown to be an independent prognostic parameter for disease-free survival in cervical carcinoma patients treated with radiation therapy. However, the underlying mechanisms are not fully understood. The main aims of this study were to investigate whether tumor radiocurability may be associated with IFP and, if so, to identify possible mechanisms. Human melanoma xenografts transplanted intradermally or in window chamber preparations in BALB/c nu/nu mice were used as preclinical tumor models. Radiation dose resulting in 50% local tumor control was higher by a factor of 1.19 +/- 0.06 in tumors with IFP > or = 9 mm Hg than in tumors with IFP < or = 7 mm Hg. Tumor IFP was positively correlated to vessel segment length and vessel tortuosity and was inversely correlated to vessel density. Compared with tumors with low IFP, tumors with high IFP showed high resistance to blood flow, high frequency of Po(2) fluctuations, and high fractions of acutely hypoxic cells, whereas the fraction of radiobiologically hypoxic cells and the fraction of chronically hypoxic cells did not differ between tumors with high and tumors with low IFP. IFP showed a significant correlation to the fraction of acutely hypoxic cells, probably because both parameters were determined primarily by the microvascular resistance to blood flow. Therefore, the observed association between tumor radiocurability and IFP was most likely an indirect consequence of a strong relationship between IFP and the fraction of acutely hypoxic cells.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a valuable method for characterizing the physiological microenvironment of tumors and thus a promising method for individualizing cancer treatment. The aim of this study was to test the hypothesis that valid parametric images of the tumor microenvironment can be obtained by pharmacokinetic analysis of DCE-MRI series. Cells of four human melanoma xenograft lines (A-07, D-12, R-18 and T-22) were used as preclinical models of human cancer. DCE-MRI was performed at 1.5 T at a spatial resolution of 0.23 x 0.47 x 2.0 mm(3) and a time resolution of 14 s. Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA) was used as contrast agent. The DCE-MRI data were analyzed on a voxel-by-voxel basis by using a pharmacokinetic model recommended for analysis of clinical DCE-MRI series. Parametric DCE-MR images were compared with tumor blood perfusion measured by the (86)Rb uptake method, and fractional volume of the extravascular extracellular space assessed by analysis of histological preparations. Parametric images reflecting tumor blood perfusion and fractional volume of the extravascular extracellular space were obtained. The numerical values of the DCE-MRI-derived parameters were not significantly different from the absolute values of tumor blood perfusion or fractional volume of the extravascular extracellular space in any of the tumor lines. This study shows that DCE-MRI can provide valid quantitative parametric images of the tumor microenvironment in preclinical cancer models and thus supports the suggestion that DCE-MRI may be developed to be a clinically useful method for individualization of microenvironment-based cancer treatment, a possibility that merits increased clinical interest.

Objective: To establish and characterize experimental tumor models of advanced squamous cell carcinoma of the uterine cervix., Methods: Permanent cell lines (CK-160 and TS-415) were established from pelvic lymph node metastases of two cervical carcinoma patients. Xenografted tumors were initiated by inoculating 5 x 10(5) cells into the gastrocnemius muscle of BALB/c nu/nu mice. The tumors were characterized with respect to histological appearance, fraction of necrotic tissue (NF), pimonidazole hypoxic fraction (HF(Pim)), interstitial fluid pressure (IFP), extracellular pH (pH(e)), metastatic propensity, and radiation sensitivity., Results: The xenografted tumors reflected the donor patients' tumors in histological appearance, metastatic propensity, and radiation sensitivity and showed significant intertumor heterogeneity in growth rate, NF, HF(Pim), IFP, and pH(e)., Conclusions: CK-160 and TS-415 xenografts possess properties making them relevant models for studies of the physiological microenvironment of cervical carcinoma and its influence on metastatic dissemination and response to treatment.

Photochemical internalization (PCI) is under development for clinical use in treatment of soft tissue sarcomas and other solid tumors. PCI may release endocytosed bleomycin (BLM) into the cytosol by photochemical rupture of the endocytic vesicles. In this study, the human fibrosarcoma xenograft HT1080 was transplanted into the leg muscle of athymic mice. The photosensitizer disulfonated aluminum phthalocyanine (AlPcS(2a)) and BLM were systemically administrated 48 h and 30 min, respectively, prior to light exposure at 670 nm (30 J cm(-2)). The purposes of this study were to evaluate the treatment response to AlPcS(2a)-photodynamic therapy (PDT) and AlPcS(2a)-PDT in combination with BLM (i.e. PCI of BLM) in an orthotopic, invasive and clinically relevant tumor model and to explore the underlying response mechanisms caused by PDT and PCI of BLM. The treatment response was evaluated by measuring tumor growth, contrast-enhanced magnetic resonance imaging (CE-MRI), histology and fluorescence microscopy. The results show that PCI of BLM is superior to PDT in inducing tumor growth retardation and acts synergistically as compared to the individual treatment modalities. The CE-MRI analyses 2 h after AlPcS(2a)-PDT and PCI of BLM identified a treatment-induced nonperfused central zone of the tumor and a well-perfused peripheral zone. While there were no differences in the vascular response between PDT and PCI, the histological analyses showed that PDT caused necrosis in the tumor center and viable tumor cells were found in the tumor periphery. PCI caused larger necrotic areas and the regrowth in the peripheral zone was almost completely inhibited after PCI. The results indicate that PDT is less efficient in the tumor periphery than in the tumor center and that the treatment effect of PCI is superior to PDT in the tumor periphery.

Tumor blood supply was related to the morphology of the tumor microvasculature and the supplying arteries (SAs) of A-07-GFP and D-12-GFP melanoma xenografts growing in window chamber preparations in BALB/c nu/nu mice. Blood supply and morphologic parameters were determined from first-pass imaging movies and vascular maps recorded after a bolus of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Poorly supplied tumors showed microvascular networks that did not differ from those of well-supplied tumors in vessel tortuosity, diameter, and density. Conversely, the SAs of poorly supplied tumors were more tortuous and had a smaller diameter than those of well-supplied tumors, resulting in lower plasma velocities in the downstream tumor vessels. Consequently, the blood supply of A-07-GFP and D-12-GFP tumors was governed by the geometric resistance of the SAs rather than by the geometric resistance or the vessel density of the tumor microvasculature. The present study suggests that the SAs may represent an important target for physiological interventions of tumors and that it may be beneficial to focus on the tumor SAs rather than the tumor microvasculature when searching for novel therapeutic strategies for modifying tumor blood supply.

Purpose: Patients with advanced cervical cancer and highly hypoxic primary tumors show increased frequency of locoregional treatment failure and poor disease-free and overall survival rates. The potential usefulness of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing tumor hypoxia noninvasively was investigated in the present preclinical study., Methods and Materials: CK-160 and TS-415 human cervical carcinoma xenografts transplanted intramuscularly (i.m.) or subcutaneously (s.c.) in BALB/c nu/nu mice were subjected to DCE-MRI and measurement of fraction of radiobiologically hypoxic cells. Tumor images of K(trans) (the volume transfer constant of Gd-DTPA) and v(e) (the extracellular volume fraction of the imaged tissue) were produced by pharmacokinetic analysis of the DCE-MRI data. Fraction of radiobiologically hypoxic cells was measured by using the paired survival curve method., Results: Fraction of radiobiologically hypoxic cells differed significantly among the four tumor groups. The mean values +/- SE were determined to be 44% +/- 7% (i.m. CK-160), 77% +/- 10% (s.c. CK-160), 23% +/- 5% (i.m. TS-415), and 52% +/- 6% (s.c. TS-415). The four tumor groups differed significantly also in K(trans), and there was an unambiguous inverse relationship between K(trans) and fraction of radiobiologically hypoxic cells. On the other hand, significant differences among the groups in v(e) could not be detected., Conclusions: The study supports the clinical development of DCE-MRI as a method for assessing the extent of hypoxia in carcinoma of the cervix.

Temporal heterogeneities in tumor blood supply were studied by using a recently developed first-pass imaging technique. First-pass imaging movies of A-07-GFP human tumor xenografts growing in window chambers were recorded at a frame rate of approximately 9 fps and a spatial resolution of 10.8 x 10.8 microm(2) after a bolus of 155-kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Each tumor was subjected to imaging thrice, with 20 minutes between each repetition. Highly specific maps of the vascular network and blood supply time (BST) images (i.e., images of the time from when arterial blood enters a tumor through the main supplying artery until it reaches a vessel segment within the tumor) were produced from the movies. The tumors had one to three supplying arterioles and showed substantial temporal heterogeneity in BST. Homogeneous changes in BST in the entire vascular network were seen in tumors supplied by one arteriole. Blood supply time fluctuations in tumor subregions were observed in tumors having two or three supplying arterioles. In addition, individual vessel segments frequently showed significant changes in BST with time. High-magnification transmission microscopy imaging substantiated that BST changes could be a consequence of arterial/arteriolar vasomotor activity, vessel wall compression, varying flow rate, and vascular stasis.

The potential usefulness of gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for assessing hypoxia in tumors with significant necrosis was investigated. Small (100-350 mm(3)) and large (500-1000 mm(3)) D-12 and U-25 tumors were subjected to DCE-MRI, measurement of the fraction of necrotic tissue, and measurement of the fraction of radiobiologically hypoxic cells. Images of E.F (E is the initial extraction fraction of Gd-DTPA and F is perfusion) and lambda (lambda is proportional to extracellular volume fraction) were produced by subjecting the DCE-MRI data to Kety analysis. Necrotic tissue could be identified in lambda images but not in E.F images of the tumors. Most voxels in viable tissue showed lambda values of 0.15-0.70, whereas the lambda values of most voxels in necrotic tissue were either <0.15 or >0.70. The E.F and lambda frequency distributions of the viable tissue, but not the E.F and lambda frequency distributions of the whole tissue, were consistent with the observation that the four groups of tumors showed similar fractions of radiobiologically hypoxic cells. E.F and lambda images may thus provide useful information on the extent of hypoxia in tumors provided that voxels in necrotic tumor regions are identified and excluded from the images.

Gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is considered to be a useful method for characterizing the vascularity of tumors. However, detailed studies of experimental tumors comparing DCE-MRI-derived parametric images with images of the morphology and function of the microvascular network have not been reported. In this communication, we describe a novel MR-compatible mouse dorsal window chamber and report comparative DCE-MRI and intravital microscopy studies of A-07-GFP tumors xenografted to BALB/c nu/nu mice. Blood supply time (BST) images (i.e., images of the time from when arterial blood enters a tumor through the supplying artery until it reaches a vessel segment within the tumor) and morphologic images of the microvascular network were produced by intravital microscopy. Images of E.F (E is the initial extraction fraction of Gd-DTPA and F is perfusion) were produced by subjecting DCE-MRI series to Kety analysis. The E.F images mirrored the morphology (microvascular density) and the function (BST) of the microvascular networks well. Tumor regions showing high E.F values colocalized with tumor regions showing high microvascular density and low BST values. Significant correlations were found between E.F and microvascular density and between E.F and BST, both within and among tumors.

A novel first-pass imaging method for studying blood flow in tumors and normal tissues in mouse dorsal window chamber preparations is reported in this study. A fluorescence-labeled macromolecule is administered i.v. as a bolus dose, and the signal generated by the bolus is detected as the bolus passes through the vascular network. The sensitivity of the method is sufficiently high that the bolus can be detected in any tumor or normal tissue microvessel. The method is particularly useful for measuring the velocity and direction of blood flow in individual vessel segments of normal and malignant tissues and the blood supply time (BST) of individual tumor vessel segments; i.e., the time required for arterial blood to flow from a supplying tumor artery to a downstream microvessel segment. Since all vessels within a window chamber preparation can be studied simultaneously in a single imaging sequence, a significant benefit of the method is that it can be used to produce maps of velocity of blood flow and BST of the entire microvascular network of xenografted tumors.

Purpose: To investigate whether gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may be a useful method for assessing fraction of radiobiologically hypoxic cells in tumors., Materials and Methods: A-07 and R-18 human melanoma xenografts were used as preclinical tumor models. DCE-MRI was performed at a voxel size of 0.23 x 0.47 x 2.0 mm(3). Tumor images of E . F (E is the initial extraction fraction of Gd-DTPA and F is blood perfusion) and lambda (the partition coefficient of Gd-DTPA) were produced by subjecting DCE-MRI series to Kety analysis. Microvascular density and extracellular volume fraction (ECVF) were determined by analysis of histological preparations. The fraction of radiobiologically hypoxic cells was measured by the paired survival curve method., Results: E . F correlated with microvascular density, and lambda correlated with ECVF. The fraction of hypoxic cells was approximately 6.5-fold higher in R-18 tumors than in A-07 tumors, consistent with the observation that A-07 tumors showed higher values for E . F and microvascular density and lower cell density (i.e., higher values for lambda and ECVF) than R-18 tumors., Conclusion: E . F and lambda images obtained by Kety analysis of DCE-MRI series contain information that may be utilized to estimate the extent of radiobiological hypoxia in tumors., ((c) 2007 Wiley-Liss, Inc.)

Purpose: To investigate whether high-resolution images of necrotic regions in tumors can be derived from gadopentetate dimeglumine (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) series., Materials and Methods: E-13 human melanoma xenografts were used as preclinical models of human cancer. DCE-MRI was performed at a voxel size of 0.23 x 0.47 x 2.0 mm3 with the use of spoiled gradient recalled sequences. Tumor images of E . F (E is the initial extraction fraction of Gd-DTPA and F is blood perfusion) and lambda (the partition coefficient of Gd-DTPA, which is proportional to extracellular volume fraction) were produced by subjecting DCE-MRI series to Kety analysis, and these images were compared with histological preparations from the imaged slices., Results: Strong correlations were found between fraction of necrotic tissue and fraction of voxels with lambda > lambdaL for lambdaL values of 0.4 to 0.6. Binary lambda images differentiating between lambda values > lambdaL and lambda values < lambdaL were found to mirror necrotic regions well in tumors with large necroses. However, necrotic foci that were small compared with the voxel size were not detectable., Conclusion: Clinically relevant images of necrotic tumor regions can be obtained for E-13 melanomas by subjecting Gd-DTPA-based DCE-MRI series to Kety analysis., (Copyright 2007 Wiley-Liss, Inc.)