Lymph node metastasis and the physicochemical micro-environment of pancreatic ductal adenocarcinoma xenografts.

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.

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.

Functional intratumoral lymphatics in patient-derived xenograft models of squamous cell carcinoma of the uterine cervix: implications for lymph node metastasis.

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.

Patient-derived xenograft models of squamous cell carcinoma of the uterine cervix.

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.

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.

Tumor interstitial fluid pressure-a link between tumor hypoxia, microvascular density, and lymph node metastasis.

High microvascular density (MVD) in the primary tumor has been shown to be associated with increased incidence of lymph node metastases and poor clinical outcome. Other investigations have revealed that a large fraction of hypoxic tissue in the primary tumor is associated with metastatic disease and impaired survival. These data are apparently incompatible because tumor hypoxia is primarily a consequence of poor oxygen supply caused by an inadequate vasculature with increased intervessel distances. Here, we provide an explanation of these observations. Human melanoma xenografts were used as preclinical cancer models. Tumors that metastasized to lymph nodes showed higher interstitial fluid pressure (IFP) than those that did not metastasize, and compared with tumors with low IFP, tumors with high IFP showed large hypoxic fractions centrally, high MVD in the periphery, high peritumoral density of lymphatics, and elevated expression of vascular endothelial growth factor A (VEGF-A) and VEGF-C. Significant correlations were found between peripheral MVD and central hypoxia, and lymph node metastasis was associated with high values of both parameters. These findings suggest that the outcome of cancer may be associated with both high MVD and extensive hypoxia in the primary tumor. We propose that proangiogenic factors are upregulated in the tumor center and that the outward interstitial fluid flow caused by the elevated IFP transports these factors to the tumor surface where they evoke hemangiogenesis and lymphangiogenesis, and consequently, that the IFP serves as a link between tumor hypoxia, peripheral tumor hemangiogenesis, peritumoral lymphangiogenesis, and lymph node metastasis.

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.

DCE-MRI of the hypoxic fraction, radioresponsiveness, and metastatic propensity of cervical carcinoma xenografts.

BACKGROUND AND PURPOSE: Locoregional treatment failure and poor survival rates are associated with extensive hypoxia in the primary tumor in advanced cervical carcinoma. The potential of gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the hypoxic fraction, radioresponsiveness, and metastatic propensity of cervical carcinomas was investigated in this preclinical study. MATERIALS AND METHODS: CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K(trans) and v(e) were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a hypoxia marker. Tumor radioresponsiveness was determined by irradiating tumors with five fractions of 4 Gy in 48 h and measuring cell survival in vitro. Metastatic propensity was determined by examining host mice for tumor growth in lymph nodes. RESULTS: Low values of K(trans) were associated with extensive hypoxia and radiation resistance in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between ve and hypoxia, radioresponsiveness, or metastatic propensity were not found in any of the tumor lines. CONCLUSION: K(trans) is a potentially useful biomarker of tumor hypoxia, radiation resistance, and metastatic growth in advanced cervical carcinoma.

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.

Dynamic contrast-enhanced magnetic resonance imaging of the metastatic potential of melanoma xenografts.

PURPOSE: Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested as a useful noninvasive method for characterizing the physiologic microenvironment of tumors. In the present study, we investigated whether Gd-DTPA-based DCE-MRI has the potential to provide biomarkers for hypoxia-associated metastatic dissemination. METHODS AND MATERIALS: C-10 and D-12 melanoma xenografts were used as experimental tumor models. Pimonidazole was used as a hypoxia marker. A total of 60 tumors were imaged, and parametric images of K(trans) (volume transfer constant of Gd-DTPA) and v(e) (fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. The host mice were killed immediately after DCE-MRI, and the primary tumor and the lungs were resected and prepared for histologic assessment of the fraction of pimonidazole-positive hypoxic tissue and the presence of lung metastases, respectively. RESULTS: Metastases were found in 11 of 26 mice with C-10 tumors and 14 of 34 mice with D-12 tumors. The primary tumors of the metastatic-positive mice had a greater fraction of hypoxic tissue (p = 0.00031, C-10; p < 0.00001, D-12), a lower median K(trans) (p = 0.0011, C-10; p < 0.00001, D-12), and a lower median v(e) (p = 0.014, C-10; p = 0.016, D-12) than the primary tumors of the metastatic-negative mice. CONCLUSIONS: These findings support the clinical attempts to establish DCE-MRI as a method for providing biomarkers for tumor aggressiveness and suggests that primary tumors characterized by low K(trans) and low v(e) values could have a high probability of hypoxia-associated metastatic spread.

pO2 fluctuation pattern and cycling hypoxia in human cervical carcinoma and melanoma xenografts.

PURPOSE: Blood perfusion in tumors is spatially and temporally heterogeneous, resulting in local fluctuations in tissue oxygen tension (pO(2)) and tissue regions showing cycling hypoxia. In this study, we investigated whether the pO(2) fluctuation pattern and the extent of cycling hypoxia differ between tumor types showing high (e.g., cervical carcinoma xenograft) and low (e.g., melanoma xenograft) fractions of connective tissue-associated blood vessels. METHODS AND MATERIALS: Two cervical carcinoma lines (CK-160 and TS-415) and two melanoma lines (A-07 and R-18) transplanted into BALB/c nu/nu mice were included in the study. Tissue pO(2) was measured simultaneously in two positions in each tumor by using a two-channel OxyLite fiber-optic oxygen-sensing device. The extent of acute and chronic hypoxia was assessed by combining a radiobiological and a pimonidazole-based immunohistochemical assay of tumor hypoxia. RESULTS: The proportion of tumor regions showing pO(2) fluctuations, the pO(2) fluctuation frequency in these regions, and the relative amplitude of the pO(2) fluctuations were significantly higher in the melanoma xenografts than in the cervical carcinoma xenografts. Cervical carcinoma and melanoma xenografts did not differ significantly in the fraction of acutely hypoxic cells or the fraction of chronically hypoxic cells. However, the ratio between fraction of acutely hypoxic cells and fraction of chronically hypoxic cells was significantly higher in melanoma than in cervical carcinoma xenografts. CONCLUSIONS: Temporal heterogeneity in blood flow and tissue pO(2) in tumors may depend on tumor histology. Connective tissue surrounding microvessels may stabilize blood flow and pO(2) and, thus, protect tumor tissue from cycling hypoxia.

Assessment of tumor hypoxia and interstitial fluid pressure by gadomelitol-based dynamic contrast-enhanced magnetic resonance imaging.

BACKGROUND AND PURPOSE: Extensive hypoxia and high interstitial fluid pressure (IFP) in the primary tumor may cause resistance to radiation treatment and promote metastatic spread. The potential of gadomelitol-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the extent of hypoxia and the level of interstitial hypertension in tumors was investigated in this preclinical study. MATERIALS AND METHODS: Twenty-three A-07 tumors were subjected to DCE-MRI and subsequent measurement of IFP and fraction of pimonidazole-positive hypoxic tissue (HF(Pim)). Parametric tumor images of K(trans), v(e), and V(b)(Tofts) (Tofts model) and of K(i) and V(b)(Patlak) (Patlak model) were produced by pharmacokinetic analyses of the DCE-MRI series. RESULTS: There was no correlation between IFP and HF(Pim) in the tumors. K(trans) and K(i) decreased significantly with increasing HF(Pim), whereas V(b)(Tofts) and V(b)(Patlak) increased significantly with increasing IFP. CONCLUSION: Information on both the extent of hypoxia and the level of interstitial hypertension in A-07 tumors can be derived from a single DCE-MRI series by using gadomelitol as contrast agent.

Interstitial fluid pressure and vascularity of intradermal and intramuscular human tumor xenografts.

PURPOSE: High interstitial fluid pressure (IFP) in tumors has been shown to be associated with poor prognosis. Mechanisms underlying the intertumor heterogeneity in IFP were investigated in this study. METHODS AND MATERIALS: A-07 melanoma xenografts were transplanted intradermally or intramuscularly in BALB/c nu/nu mice. IFP was measured in the center of the tumors with a Millar catheter. Tumor blood perfusion and extracellular volume fraction were assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). The necrotic fraction, vascular density, and vessel diameters of the tumors were determined by image analysis of histological preparations. RESULTS: Significant intertumor heterogeneity in IFP, blood perfusion, and microvascular morphology was observed whether the tumors were transplanted intradermally or intramuscularly. High IFP was mainly a consequence of high resistance to blood flow caused by low vessel diameters in either transplantation site. IFP decreased with increasing blood perfusion in intradermal tumors and increased with increasing blood perfusion in intramuscular tumors, mainly because the morphology of the tumor microvasculature differed systematically between the two tumor models. CONCLUSION: The potential of DCE-MRI as a noninvasive method for assessing the IFP of tumors may be limited because any relationship between IFP and blood perfusion may differ with the tumor growth site.

Magnetic resonance imaging of tumor necrosis.

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.

Quantitative assessment of hypoxia in melanoma xenografts by dynamic contrast-enhanced magnetic resonance imaging: intradermal versus intramuscular tumors.

BACKGROUND AND PURPOSE: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested to be a useful method for assessing the extent of hypoxia in tumors. In this study, we investigated whether differences in hypoxic fraction between tumors caused by the site of growth can be detected by DCE-MRI. MATERIALS AND METHODS: Intradermal and intramuscular A-07 tumors were subjected to DCE-MRI, histological analysis of microvascular characteristics, and measurement of hypoxic cell fractions using a radiobiological assay and a pimonidazole-based immunohistochemical assay. Parametric images of E·F (blood perfusion) and v(e) (extracellular volume fraction) were produced by pharmacokinetic analysis of the DCE-MRI series. RESULTS: The intramuscular tumors had 3-4-fold higher hypoxic fractions than the intradermal tumors, owing to a lower microvascular density. This difference in extent of hypoxia was not detectable in the parametric MR images. Most likely, larger vessel diameters compensated for the lower vessel density in the intramuscular tumors, resulting in E·F images that were similar to those of the intradermal tumors. CONCLUSION: Quantitative assessment of hypoxic fractions from parametric MR images may require tumor site-specific translational criteria.

Dynamic contrast-enhanced magnetic resonance imaging of human cervical carcinoma xenografts: pharmacokinetic analysis and correlation to tumor histomorphology.

BACKGROUND AND PURPOSE: Biomarkers that can predict the outcome of treatment accurately are needed for treatment individualization in advanced carcinoma of the uterine cervix. The potential of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was investigated in the present preclinical study. MATERIALS AND METHODS: CK-160 and TS-415 human cervical carcinoma xenografts were subjected to DCE-MRI at 1.5T using a spatial resolution of 0.23×0.47×2.0mm(3). Parametric images of K(trans) (the volume transfer constant of Gd-DTPA) and v(e) (the extravascular extracellular volume fraction) were produced by pharmacokinetic analysis of the DCE-MRI data and compared with the histomorphology of the imaged tissue. RESULTS: Analysis of small homogeneous tumor regions showed that K(trans), but not v(e), differed significantly between parenchymal tissue, connective tissue, and necrotic tissue, consistent with the vascularity of these compartments. However, strong correlations between K(trans) and the fractional volume of the compartments could not be detected for larger tumor regions, primarily because the majority of the voxels represented a chaotic mixture of parenchymal, connective, and necrotic tissue. CONCLUSION: The potential of DCE-MRI in providing detailed information on the histomorphology of cervical carcinoma is limited, mainly because the tumor tissue shows significant morphological heterogeneity at the subvoxel level.

Tumors exposed to acute cyclic hypoxic stress show enhanced angiogenesis, perfusion and metastatic dissemination.

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.

Associations between radiocurability and interstitial fluid pressure in human tumor xenografts without hypoxic tissue.

PURPOSE: The interstitial fluid pressure (IFP) of the primary tumor is an independent prognostic parameter for cervical cancer patients treated with radiation therapy. The aim of this preclinical study was to investigate whether tumor radiocurability may be associated with IFP through hypoxia-independent mechanisms. EXPERIMENTAL DESIGN: Small A-07 and R-18 melanoma xenografts without hypoxic tissue were used as preclinical tumor models. IFP was measured by using the wick-in-needle method. Radiation dose resulting in 50% local tumor control (TCD(50)), cell density, cell tumorigenicity, plating efficiency in vitro, mitotic index, fraction of Ki67-positive cells, vascular endothelial growth factor-A (VEGF-A) concentration, and radiation-induced endothelial cell apoptosis were assessed in tumors with low and high IFP. RESULTS: TCD(50) was found to be higher for tumors with high IFP than for tumors with low IFP by factors of 1.13 +/- 0.03 (A-07; P < 0.0001) and 1.10 +/- 0.03 (R-18; P < 0.0001). In the A-07 line, tumors with high IFP showed a larger number of clonogenic cells and a higher rate of cell proliferation than tumors with low IFP. In the R-18 line, tumors with high IFP showed a higher concentration of VEGF-A and a lower endothelial cell apoptotic index after irradiation than tumors with low IFP. CONCLUSIONS: The radiation resistance of normoxic tumor tissue with highly elevated IFP may be an indirect consequence of increased tumor cell clonogenicity as well as increased VEGF-A expression, possibly caused by hypertension-induced modifications of signaling pathways regulating cell proliferation, cell survival, and/or angiogenesis.

Radiocurability is associated with interstitial fluid pressure in human tumor xenografts.

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.

Detection of different hypoxic cell subpopulations in human melanoma xenografts by pimonidazole immunohistochemistry.

This study aimed at developing immunohistochemical assays for different subpopulations of hypoxic cells in tumors. BALB/c-nu/nu mice bearing A-07 or R-18 tumors were given a single dose of 90 mg/kg body weight or three doses (3 h apart) of 30 mg/kg body weight of pimonidazole hydrochloride intravenously. The fraction of pimonidazole-labeled cells was assessed in paraffin-embedded and frozen tumor sections and compared with the fraction of radiobiologically hypoxic cells. The staining pattern in paraffin-embedded sections indicated selective staining of chronically hypoxic cells. Frozen sections showed a staining pattern consistent with staining of both chronically and acutely/repetitively hypoxic cells. Fraction of pimonidazole-labeled cells in paraffin-embedded sections was lower than the fraction of radiobiologically hypoxic cells (single-dose and triple-dose experiment). In frozen sections, fraction of pimonidazole-labeled cells was similar to (single-dose experiment) or higher than (triple-dose experiment) fraction of radiobiologically hypoxic cells. Three different subpopulations of hypoxic cells could be quantified by pimonidazole immunohistochemistry: the fraction of cells that are hypoxic because of limitations in oxygen diffusion, the fraction of cells that are hypoxic simultaneously because of fluctuations in blood perfusion, and the fraction of cells that are exposed to one or more periods of hypoxia during their lifetime because of fluctuations in blood perfusion.