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.

In silico investigations of intratumoral heterogeneous interstitial fluid pressure.

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.

Antifibrotic therapy to normalize the tumor microenvironment.

Most tumors develop abnormal fibrotic regions consisting of fibroblasts, immune cells, and a dense extracellular matrix (ECM) immersed in a viscous interstitial fluid, and an abundant fibrotic tumor microenvironment (TME) is associated with poor outcome of treatment. It has been hypothesized that the treatment of cancer may be improved by interventions aiming to normalize this TME. The approaches used in attempts to normalize the fibrotic TME can be categorized into three strategies of targeted antifibrotic therapy: targeting of components of the ECM, targeting of the producers of the ECM components-the activated cancer-associated fibroblasts (CAFs), and targeting of the signaling pathways activating CAFs. To target the ECM, enzymes against components of the ECM have been used, including collagenase, relaxin, hyaluronidase, and lyxyl oxidase. Targeting of CAFs have been investigated by using agents aiming to eliminate or reprogram CAFs. CAFs are activated primarily by transforming growth factor-ß (TGF-ß), hedgehog, or focal adhesion kinase signaling, and several agents have been used to target these signaling pathways, including angiotensin II receptor I blockers (e.g., losartan) to inhibit the TGF-ß pathway. Taken together, these studies have revealed that antifibrotic therapy is a two-edged sword: while some studies suggest enhanced response to treatment after antifibrotic therapy, others suggest that antifibrotic therapy may lead to increased tumor growth, metastasis, and impaired outcome of treatment. There are several possible explanations of these conflicting observations. Most importantly, tumors contain different subpopulations of CAFs, and while some subpopulations may promote tumor growth and metastasis, others may inhibit malignant progression. Furthermore, the outcome of antifibrotic therapy may depend on stage of disease, duration of treatment, treatment-induced activation of alternative profibrotic signaling pathways, and treatment-induced recruitment of tumor-supporting immune cells. Nevertheless, losartan-induced suppression of TGF-ß signaling appears to be a particularly promising strategy. Losartan is a widely prescribed antihypertensive drug and highly advantageous therapeutic effects have been observed after losartan treatment of pancreatic cancer. However, improved understanding of the mechanisms governing the development of fibrosis in tumors is needed before safe antifibrotic treatments can be established.

Bevacizumab treatment of meningeal melanoma metastases.

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.

Pharmacokinetic analysis of DCE-MRI data of locally advanced cervical carcinoma with the Brix model.

Background: There is significant evidence that DCE-MRI may have the potential to provide clinically useful biomarkers of the outcome of locally advanced cervical carcinoma. However, there is no consensus on how to analyze DCE-MRI data to arrive at the most powerful biomarkers. The purpose of this study was to analyze DCE-MRI data of cervical cancer patients by using the Brix pharmacokinetic model and to compare the biomarkers derived from the Brix analysis with biomarkers determined by non-model-based analysis [i.e., low-enhancing tumor volume (LETV) and tumor volume with increasing signal (TVIS)] of the same patient cohort. Material and methods: DCE-MRI recordings of 80 patients (FIGO stage IB-IVA) treated with concurrent cisplatin-based chemoradiotherapy were analyzed voxel-by-voxel, and frequency distributions of the three parameters of the Brix model (ABrix, kep, and kel) were determined. Moreover, risk volumes were calculated from the Brix parameters and termed RV-ABrix, RV-kep, and RV-kel, where the RVs represent the tumor volume with voxel values below a threshold value determined by ROC analysis. Disease-free survival (DFS) and overall survival (OS) were used as measures of treatment outcome. Results: Significant associations between the median value or any other percentile value of ABrix, kep, or kel and treatment outcome were not found. However, RV-ABrix, RV-kep, and RV-kel correlated with DFS and OS. Multivariate analysis revealed that the prognostic power of RV-ABrix, RV-kep, and RV-kel was independent of well-established clinical prognostic factors. RV-ABrix, RV-kep, and RV-kel correlated with each other as well as with LETV and TVIS. Conclusion: Strong biomarkers of the outcome of locally advanced cervical carcinoma can be provided by subjecting DCE-MRI series to pharmacokinetic analysis using the Brix model. The prognostic power of these biomarkers is not necessarily superior to that of biomarkers identified by non-model-based analyses.

Metastatic pathway and the microvascular and physicochemical microenvironments of human melanoma xenografts.

BACKGROUND: Malignant melanoma of the skin can metastasize through blood vessels and lymphatics. The primary tumor develops a vascular microenvironment characterized by abnormal blood vessels and lymphatics and a physicochemical microenvironment characterized by low oxygen tension, regions with hypoxic tissue, and high interstitial fluid pressure (IFP). This study aimed at identifying relationships between the metastatic route of melanomas and characteristic features of the microvascular and physicochemical microenvironments of the primary tumor. METHODS: Two patient-derived xenograft (PDX) models (E-13, N-15) and four cell line-derived xenografts (CDX) models (C-10, D-12, R-18, T-22) of human melanoma were included in the study. Tumors were transplanted to an orthotopic site in BALB/c-nu/nu mice, and when the tumors had grown to a volume of 500-600 mm3, the IFP of the primary tumor was measured and the hypoxia marker pimonidazole was administered before the host mouse was euthanized. The primary tumor, lungs, and six pairs of lymph nodes were evaluated by examining hematoxylin/eosin-stained and immunostained histological preparations. The expression of angiogenesis-related genes was assessed by quantitative PCR. RESULTS: C-10, D-12, and E-13 tumors disseminated primarily by the hematogenous route and developed pulmonary metastases. These tumors showed high angiogenic activity and high expression of the F3 gene as well as ANGPT2 and TIE1, genes encoding proteins of the angiopoietin-tie system. N-15, R-18, and T-22 tumors disseminated mainly by the lymphogenous route and developed metastases in draining lymph nodes. These tumors had highly elevated IFP and showed high expression of NRP2, a gene encoding neuropilin-2. CONCLUSION: The primary metastatic route of orthotopic human melanoma xenografts and the development of lung and lymph node metastases are influenced significantly by the microvascular and physicochemical microenvironments of the primary tumor.

Vascular abnormalities and development of hypoxia in microscopic melanoma xenografts.

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.

DCE-MRI of patient-derived xenograft models of uterine cervix carcinoma: associations with parameters of the tumor microenvironment.

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.

Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts.

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.

Pretreatment late-phase DCE-MRI predicts outcome in locally advanced cervix cancer.

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may provide prognostic biomarkers for cervix carcinoma. We have shown previously that the early phase of the signal intensity-versus-time curve (SITC) may have significant prognostic power. The purpose of the present investigation was to explore the prognostic value of the late phase of the SITC. MATERIAL AND METHODS: DCE-MRI data of 80 patients (FIGO stage IB-IVA) treated with concurrent chemoradiotherapy were examined. Four parameters were calculated from the late-phase SITC: tumor volume with decreasing signal, tumor fraction with decreasing signal, tumor volume with increasing signal (TVIS), and tumor fraction with increasing signal. RESULTS: Multivariate analysis involving clinical parameters and late-phase SITC parameters suggested that TVIS is a strong independent prognostic factor for both disease-free and overall survival. When early-phase SITC parameters were included in the multivariate analysis, the early-phase SITC, but not the late-phase SITC, was found to have independent prognostic value. CONCLUSION: The late-phase SITC can provide prognostic factors for the outcome of cervix carcinoma, that is, a large tumor volume with increasing late-phase SITCs is associated with poor outcome. However, the prognostic power of the late-phase SITC is not as strong as that of the early-phase SITC.

Dynamic contrast-enhanced MRI of the microenvironment of pancreatic adenocarcinoma xenografts.

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: Ktrans (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 ve (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.

Diffusion-weighted and dynamic contrast-enhanced MRI of pancreatic adenocarcinoma xenografts: associations with tumor differentiation and collagen content.

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.

Thrombospondin-1 domain-containing peptide properdistatin improves vascular function in human melanoma xenografts.

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.

Hypoxia biomarkers in squamous cell carcinoma of the uterine cervix.

BACKGROUND: There is significant evidence that severe tumor hypoxia may cause resistance to chemoradiotherapy and promote metastatic spread in locally advanced carcinoma of the uterine cervix. Some clinical investigations have suggested that high expression of hypoxia-inducible factor-1α (HIF-1α) and/or its target gene carbonic anhydrase IX (CAIX) may be useful biomarkers of tumor hypoxia and poor outcome in cervical cancer. Here, we challenged this view by investigating possible associations between HIF-1α expression, CAIX expression, fraction of hypoxic tissue, and lymph node metastasis in experimental human tumors. METHODS: Tumors of two cervical carcinoma xenograft lines (CK-160 and TS-415) were included in the study. Pimonidazole was used as a hypoxia marker, and tumor hypoxia, HIF-1α expression, and CAIX expression were detected by immunohistochemistry. Metastatic status was assessed by examining external lymph nodes in the inguinal, axillary, interscapular, and submandibular regions and internal lymph nodes in the abdomen and mediastinum. RESULTS: Tissue regions staining positive for pimonidazole, HIF-1α, or CAIX were poorly colocalized, both in CK-160 and TS-415 tumors. The expression of HIF-1α or CAIX did not correlate with the fraction of hypoxic tissue in any of the two tumor lines. Furthermore, clinically relevant associations between HIF-1α or CAIX expression and lymph node metastasis were not found. CONCLUSION: Because significant associations between HIF-1α expression, CAIX expression, fraction of hypoxic tissue, and incidence of lymph node metastases could not be detected in any of two preclinical models of human cervical cancer, it is not realistic to believe that high expression of HIF-1α or CAIX can be useful biomarkers of tumor hypoxia and poor outcome in a highly heterogeneous disease like cervical carcinoma.

Early effects of low dose bevacizumab treatment assessed by magnetic resonance imaging.

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.

DW-MRI in assessment of the hypoxic fraction, interstitial fluid pressure, and metastatic propensity of melanoma xenografts.

BACKGROUND: Cancer patients with primary tumors showing extensive hypoxia and highly elevated interstitial fluid pressure (IFP) have poor prognosis. The potential of diffusion-weighted magnetic resonance imaging (DW-MRI) in assessing the hypoxic fraction, IFP, and metastatic propensity of tumors was investigated in this study. METHODS: A-07 and R-18 melanoma xenografts were used as general models of human cancer. DW-MRI was performed at 1.5 T, and maps of the apparent diffusion coefficient (ADC) were produced with in-house-made software developed in Matlab. Pimonidazole was used as a hypoxia marker. Tumor cell density and hypoxic fraction were assessed by quantitative analysis of histological sections. IFP was measured with a Millar catheter. Metastatic propensity was determined by examining tumor-bearing mice for pulmonary micrometastases post mortem. RESULTS: ADC decreased with increasing tumor cell density, independent of whether the A-07 and R-18 data were analyzed separately or together. In the A-07 line, ADC decreased with increasing hypoxic fraction and increasing IFP and was lower in metastatic than in nonmetastatic tumors, and in the R-18 line, ADC decreased with increasing hypoxic fraction. There was a strong inverse correlation between ADC and hypoxic fraction as well as between ADC and IFP across the two tumor lines, primarily because low ADC as well as high hypoxic fraction and high IFP were associated with high cell density. CONCLUSION: Low ADC is a potentially useful biomarker of poor prognosis in cancer, since low ADC is mainly a consequence of high cell density, and high cell density may lead to increased hypoxia and interstitial hypertension and, therefore, increased microenvironment-associated metastasis.

Connective tissue of cervical carcinoma xenografts: associations with tumor hypoxia and interstitial fluid pressure and its assessment by DCE-MRI and DW-MRI.

Abstract Background. A high fraction of stroma in malignant tissues is associated with tumor progression, metastasis, and poor prognosis. Possible correlations between the stromal and physiologic microenvironments of tumors and the potential of dynamic contrast-enhanced (DCE) and diffusion-weighted (DW) magnetic resonance imaging (MRI) in quantification of the stromal microenvironment were investigated in this study. Material and methods. CK-160 cervical carcinoma xenografts were used as preclinical tumor model. A total of 43 tumors were included in the study, and of these tumors, 17 were used to search for correlations between the stromal and physiologic microenvironments, 11 were subjected to DCE-MRI, and 15 were subjected to DW-MRI. DCE-MRI and DW-MRI were carried out at 1.5 T with a clinical MR scanner and a slotted tube resonator transceiver coil constructed for mice. Fraction of connective tissue (CTFCol) and fraction of hypoxic tissue (HFPim) were determined by immunohistochemistry. A Millar SPC 320 catheter was used to measure tumor interstitial fluid pressure (IFP). Results. CTFCol showed a positive correlation to IFP and an inverse correlation to HFPim. The apparent diffusion coefficient assessed by DW-MRI was inversely correlated to CTFCol, whereas no correlation was found between DCE-MRI-derived parameters and CTFCol. Conclusion. DW-MRI is a potentially useful method for characterizing the stromal microenvironment of tumors.

Tumor-line specific causes of intertumor heterogeneity in blood supply in human melanoma xenografts.

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.

Tumors exposed to acute cyclic hypoxia show increased vessel density and delayed blood supply.

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.

Assessment of the interstitial fluid pressure of tumors by dynamic contrast-enhanced magnetic resonance imaging with contrast agents of different molecular weights.

BACKGROUND: Cancer patients showing highly elevated interstitial fluid pressure (IFP) in the primary tumor may benefit from particularly aggressive treatment. There is some evidence that gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) may be a useful non-invasive method for providing information on the IFP of tumors. The purpose of this preclinical study was to investigate whether any association between DCE-MRI-derived parametric images and tumor IFP can be strengthened by using MR contrast agents with higher molecular weights than that of Gd-DTPA. MATERIAL AND METHODS: A-07 human melanoma xenografts were used as preclinical models of human cancer. Three contrast agents were compared: Gd-DTPA (0.55 kDa), P846 (3.5 kDa), and gadomelitol (6.5 kDa). A total of 46 tumors were subjected to DCE-MRI and subsequent measurement of IFP. Parametric images of K(trans) (the volume transfer constant of the contrast agent) and v(e) (the fractional distribution volume of the contrast agent) were produced by pharmacokinetic analysis of the DCE-MRI series. RESULTS: Significant inverse correlations were found between median K(trans) and IFP for Gd-DTPA (p = 0.0076; R(2) = 0.46; n = 14) and P846 (p = 0.0042; R(2) = 0.45; n = 16), whereas there was no correlation between median K(trans) and IFP for gadomelitol (p > 0.05; n = 16). Significant correlation between median v(e) and IFP was not found for any of the contrast agents (p > 0.05 for Gd-DTPA, P846, and gadomelitol). CONCLUSION: K(trans) images, but not v(e) images, derived by pharmacokinetic analysis of DCE-MRI data for low-molecular-weight contrast agents may provide information on the IFP of tumors. Any association between K(trans) and IFP cannot be expected to be improved by using contrast agents with higher molecular weights than those of Gd-DTPA and P846.