Hypoxia promotes lymph node metastasis in human melanoma xenografts by up-regulating the urokinase-type plasminogen activator receptor

Clinical studies have shown that metastatic spread is associated with hypoxia in the primary tumor. The mechanism behind this association has not been identified and, in fact, it has not been established whether hypoxia induces metastasis or whether the most metastatic cell phenotypes develop the most hypoxic tumors. The present study demonstrates that hypoxia promotes spontaneous lymph node metastasis in R-18 human melanoma xenografts by up-regulating the urokinase-type plasminogen activator receptor (uPAR). Pimonidazole was used as a hypoxia marker, and hypoxia and uPAR expression were detected by immunohistochemistry. R-18 cells were capable of up-regulating uPAR under hypoxic conditions in vitro, as revealed by Western and Northern blot analyses, and uPAR-positive regions showed a high degree of colocalization with hypoxic regions in R-18 tumors. There was a strong correlation between uPAR-positive fraction and hypoxic fraction in individual tumors (P < 0.00001). Incidence of metastases, hypoxic fraction, and uPAR-positive fraction increased with the size of the primary tumor with similar kinetics. Metastatic tumors showed approximately 1.5-fold higher hypoxic fraction (P = 0.00004) and approximately 1.4-fold higher uPAR-positive fraction (P = 0.0003) than nonmetastatic tumors of the same size. Moreover, treatment with neutralizing antibody against uPAR prevented metastasis almost completely. Only 1 of 30 treated mice developed metastases, whereas 14 of 30 control mice were metastasis positive, suggesting that functional uPAR is a prerequisite for lymph node metastasis in R-18 tumors. The study reported here suggests that metastatic spread may be promoted by hypoxia in the primary tumor and identifies the plasminogen activation system as an important target for the treatment of malignant melanoma.     

Increased metastatic dissemination in human melanoma xenografts after subcurative radiation treatment: radiation-induced increase in fraction of hypoxic cells and hypoxia-induced up-regulation of urokinase-type plasminogen activator receptor

Cancer patients showing local failure after radiation treatment have increased probability for developing metastatic disease. The mechanisms behind this observation have not been identified. In the present work, metastatic spread after inadequate radiation therapy was studied by using R-18 human melanoma xenografts as models of cancer in humans. Pimonidazole was used as a hypoxia marker, and hypoxia and urokinase-type plasminogen activator receptor (uPAR) expression were detected by immunohistochemistry. R-18 tumors regrowing after subcurative irradiation showed a higher frequency of lymph node metastasis than unirradiated tumors. The expression of uPAR was up-regulated in hypoxic tumor regions, and the fractions of hypoxic and uPAR-positive cells were approximately 2-fold higher in regrowing irradiated tumors than in untreated tumors. Treatment with anti-uPAR antibody blocked metastasis almost completely in irradiated as well as unirradiated tumors. The metastatic frequency was higher in tumors regrowing after irradiation than in unirradiated tumors because the irradiation induced tumor hypoxia, and tumor hypoxia induced up-regulation of uPAR.     

Fluctuating and diffusion-limited hypoxia in hypoxia-induced metastasis.

PURPOSE: Most tumors develop regions with hypoxic cells during growth, owing to permanent limitations in oxygen diffusion (chronic or diffusion-limited hypoxia) and/or transient limitations in blood perfusion (acute or fluctuating hypoxia). The aim of this study was to investigate the relative significance of chronic and acute hypoxia in the development of metastatic disease. EXPERIMENTAL DESIGN: D-12 and R-18 human melanoma xenografts were used as models of human cancer. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Fraction of radiobiologically hypoxic cells (HF(Rad)) was measured in individual primary tumors by using a radiobiological assay based on the paired survival curve method. Fraction of immunohistochemically hypoxic cells (HF(Imm)) was assessed in the same tumors by using a pimonidazole-based immunohistochemical assay optimized with respect to achieving selective staining of chronically hypoxic cells. HF(Imm) and the difference between HF(Rad) and HF(Imm), HF(Rad) - HF(Imm), were verified to be adequate variables for fraction of chronically hypoxic cells and fraction of acutely hypoxic cells, respectively. RESULTS: Chronic as well as acute hypoxia were found to promote spontaneous metastasis of D-12 and R-18 tumors. Acute hypoxia influenced metastasis to a greater extent than chronic hypoxia, partly because the fraction of acutely hypoxic cells was larger than the fraction of chronically hypoxic cells in most tumors and partly because acutely hypoxic cells showed a higher metastatic potential than chronically hypoxic cells. CONCLUSIONS: It may be beneficial to focus on fluctuating hypoxia rather than diffusion-limited hypoxia when searching for hypoxia-related prognostic variables and predictive assays.     

Fraction of radiobiologically hypoxic cells in human melanoma xenografts measured by using single-cell survival, tumour growth delay and local tumour control as end points.

Four human melanoma xenograft lines (A-07, D-12, R-18, U-25) grown orthotopically in Balb/c nu/nu mice were characterized with respect to the fraction of radiobiologically hypoxic cells. The purpose of the study was to establish a firm radiobiological basis for future use of the lines in the development and evaluation of non-invasive assays of tumour hypoxia. The hypoxic fractions were assessed using three different assays, the single cell survival assay, the tumour growth delay assay and the local tumour control assay, and the means +/- s.e. were found to be 6 +/- 3%, 3 +/- 1% and 5 +/- 2% respectively (A-07), 26 +/- 5%, 25 +/- 6% and 22 +/- 6% respectively (D-12), 55 +/- 9%, 65 +/- 8% and 48 +/- 7% respectively (R-18) and 52 +/- 8%, 59 +/- 7% and 47 +/- 7% respectively (U-25). The three assays gave numerical values for the hypoxic fraction that were not significantly different for any of the lines. The hypoxic fraction differed significantly among the lines; the R-18 and U-25 lines showed higher hypoxic fractions than the D-12 line (P < 0.05), which in turn showed a higher hypoxic fraction than the A-07 line (P < 0.05), regardless of the assay. The wide range of the hypoxic fractions and the significant differences among the lines suggest that A-07, D-12. R-18 and U-25 tumours should be useful models in future studies attempting to develop non-invasive assays of tumour hypoxia.     

Identification of Metastasis-Associated Metabolic Profiles of Tumors by 1H-HR-MAS-MRS

Tumors develop an abnormal microenvironment during growth, and similar to the metastatic phenotype, the metabolic phenotype of cancer cells is tightly linked to characteristics of the tumor microenvironment (TME). In this study, we explored relationships between metabolic profile, metastatic propensity, and hypoxia in experimental tumors in an attempt to identify metastasis-associated metabolic profiles. Two human melanoma xenograft lines (A-07, R-18) showing different TMEs were used as cancer models. Metabolic profile was assessed by proton high resolution magic angle spinning magnetic resonance spectroscopy (¹H-HR-MAS-MRS). Tumor hypoxia was detected in immunostained histological preparations by using pimonidazole as a hypoxia marker. Twenty-four samples from 10 A-07 tumors and 28 samples from 10 R-18 tumors were analyzed. Metastasis was associated with hypoxia in both A-07 and R-18 tumors, and ¹H-HR-MAS-MRS discriminated between tissue samples with and tissue samples without hypoxic regions in both models, primarily because hypoxia was associated with high lactate resonance peaks in A-07 tumors and with low lactate resonance peaks in R-18 tumors. Similarly, metastatic and non-metastatic R-18 tumors showed significantly different metabolic profiles, but not metastatic and non-metastatic A-07 tumors, probably because some samples from the metastatic A-07 tumors were derived from tumor regions without hypoxic tissue. This study suggests that ¹H-HR-MAS-MRS may be a valuable tool for evaluating the role of hypoxia and lactate in tumor metastasis as well as for identification of metastasis-associated metabolic profiles.     

Hypoxia-induced angiogenesis and vascular endothelial growth factor secretion in human melanoma.

Tumour cells exposed to hypoxia in vitro can show increased expression of several selected genes, including the gene encoding the vascular endothelial growth factor (VEGF), suggesting that hypoxia followed by reoxygenation might promote the malignant progression of tumours. An in vitro/in vivo study was conducted to investigate whether hypoxia can increase the angiogenic potential of tumour cells through increased VEGF secretion. Four human melanoma cell lines (A-07, D-12, R-18, U-25) were included in the study. Cell cultures were exposed to hypoxia (oxygen concentration <10 p.p.m.) in vitro using the steel chamber method. Rate of VEGF secretion was measured in vitro in aerobic and hypoxic cell cultures by ELISA. Angiogenesis was assessed in vivo using the intradermal angiogenesis assay. Aliquots of cells harvested from aerobic cultures or cultures exposed to hypoxia for 24 h were inoculated intradermally in the flanks of adult female BALB/c-nu/nu mice. Tumours developed and angiogenesis was quantified by scoring the number of capillaries in the dermis oriented towards the tumours. The number of tumour-oriented capillaries did not differ significantly between tumours from hypoxic and aerobic cultures for A-07 and U-25, whereas tumours from hypoxic cultures showed a larger number of tumour-oriented capillaries than tumours from aerobic cultures for D-12 and R-18. The VEGF secretion under aerobic conditions and the absolute increase in VEGF secretion induced by hypoxia were lower for D-12 and R-18 than for A-07 and U-25, whereas the relative increase in VEGF secretion induced by hypoxia was higher for D-12 and R-18 than for A-07 and U-25. VEGF is not a limiting factor in the angiogenesis of some tumours under normoxic conditions. Hypoxia can increase the angiogenic potential of tumour cells by increasing the secretion of VEGF, but only of tumour cells showing low VEGF secretion under normoxia. Transient hypoxia might promote the malignant progression of tumours by temporarily increasing the angiogenic potential of tumour cells showing low VEGF expression under normoxic conditions.     

Effects of hypoxia on radiation-responsive stress-activated protein kinase, p53, and caspase 3 signals in TK6 human lymphoblastoid cells

Despite significant evidence of a role of hypoxia in cellular resistance to ionizing radiation-induced toxicity, the underlying molecular mechanisms remain unclear. This study focused on the influence of hypoxia on radiation-induced signals in TK6 human lymphoblastoid cells. Hypoxic (<10 ppm oxygen) and aerobic cells were exposed to equilethal doses of ionizing radiation, radiation dose ratio, 3:1 (hypoxia:air). Hypoxia alone or radiation treatment under aerobic or hypoxic conditions led to increased levels of phospho-p44/42 mitogen-activated protein kinase. Levels of phospho-p38 mitogen-activated protein kinase did not change as a result of either hypoxia or irradiation. Hypoxia alone had no effect on expression of phospho-stress-activated protein kinase (SAPK), wild-type p53, or cleaved caspase 3. Irradiation under aerobic conditions resulted in an increase in the phospho-SAPK signal, whereas hypoxia suppressed the irradiation-induced increase in the level of phospho-SAPK. Both hypoxic and aerobic cells showed increases in p53 levels in response to radiation. Hypoxia blocked radiation-induced cleavage of caspase 3 and poly-ADP-ribose polymerase. Irradiation of aerobic and hypoxic TK6 cells using 6 and 18 Gy, respectively, resulted in a similar and significant increase in fraction of apoptotic cells within 24 hours postirradiation. In contrast, basal levels of apoptosis were observed at 24 hours postirradiation in aerobic and hypoxic NH32 cells, a p53 null derivative of TK6 cells. These results suggest that radiation-induced apoptosis under hypoxia occurs independent of phospho-SAPK and caspase 3, and the p53 response is an obligatory apoptotic signal in TK6 cells.     

Reoxygenation in a rat rhabdomyosarcoma tumor following X-irradiation

The paired survival curve technique was used to characterize the rate at which the fraction of hypoxic cells in rat rhabdomyosarcoma R-1 tumors returns to the preirradiation value of 37% following a single dose of 225-kVp X rays. Tumors were administered a conditioning x-ray dose of 15-Gy, followed at 0, 3, 6, 12, 24, or 48 hr by a 5-Gy, 10-Gy, or 15-Gy dose of X rays under air-breathing conditions or under hypoxic conditions produced by nitrogen-gas asphyxiation 5 min prior to irradiation. Cellular surviving fractions were determined by the tumor excision assay following in vivo irradiation. From the ratio of the survival fractions measured for tumor cells from air-breathing and hypoxic animals, the fraction of hypoxic cells was determined as a function of time postirradiation. These results indicated that immediately following a 15-Gy dose of X rays, essentially 100% of the viable cells remaining were hypoxic. The tumors reoxygenated rapidly, returning to the preirradiation level of 37% during the first 6 hr postirradiation.     

Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

The aim of this study was to investigate whether tumour hypoxia and/or vascular hot spots promote the development of metastatic disease. The D-12 human melanoma xenograft line was used as a tumour model. Hypoxia and vascular hot spots were detected by immunohistochemistry using pimonidazole as a hypoxia marker and anti-CD31 antibody to visualize endothelial cells. Vascular hot spots were found to be induced in hypoxic foci, owing to hypoxia-induced up-regulation of angiogenesis stimulatory factors. This effect was mediated by interleukin 8 and possibly also by vascular endothelial growth factor. Interleukin 8 positive foci showed a high degree of co-localization with hypoxic foci, as revealed by immunohistochemistry. The incidence of spontaneous pulmonary metastases was associated with the density of hypoxic foci, the density of interleukin 8 positive foci and the density of vascular hot spots in the primary tumour. Treatment with neutralizing antibody against interleukin 8 and/or vascular endothelial growth factor resulted in hypoxia-induced necrosis rather than hypoxia-induced vascular hot spots and inhibited metastasis. Our study suggests a cause-effect relationship between hypoxia and metastasis in cancer and hence an elevated probability of metastatic disease in patients having primary tumours characterized by high densities of hypoxic foci and vascular hot spots.     

Tumor bed effect-induced reduction of tumor radiocurability through the increase in hypoxic cell fraction

Two murine tumors, designated FSA and SA-NH, that exhibit strong tumor bed effect (TBE), were found to be less radiocurable if they grew in the preirradiated s.c. tissue. Tumors were transplanted into the right thighs irradiated with 30 Gy one day earlier, and were irradiated when they grew to 6 mm. The TBE-caused reduction in tumor radiocurability was manifested by the increase in TCD50 values. Tumor irradiation under hypoxic conditions increased TCD50 values less, and the hypoxic cell radiosensitizer misonidazole reduced TCD50 values more, when tumors grew in preirradiated than when they grew in unirradiated legs. This implies that TBE-induced increase in TCD50 was due to increase in hypoxic fraction of tumor cells. For 6 mm SA-NH tumor the estimated increase in hypoxic cell fraction was from the control value of 3% to 12%. Thus, TBE causes the reduction in tumor radiocurability through the increase in hypoxic fraction of tumor cells.     

Antiangiogenic treatment with thrombospondin-1 enhances primary tumor radiation response and prevents growth of dormant pulmonary micrometastases after curative radiation therapy in human melanoma xenografts

Thrombospondin-1 (TSP-1) is a potent antiangiogenic factor that has been shown to inhibit tumor growth by preventing endothelial cells from responding to a wide variety of angiogenic stimulators. We have demonstrated previously that D-12 primary tumors (human melanoma xenografts) suppress the growth of their spontaneous pulmonary micrometastases by secreting TSP-1 into the blood circulation. The same tumor model was used in the present work to study antitumor effects of combined radiation therapy and antiangiogenic treatment with TSP-1. Curative radiation treatment of D-12 primary tumors resulted in rapid growth of previously dormant micrometastases. Growth of dormant micrometastases could be prevented by treating the host mice with exogenous TSP-1 after the radiation treatment. Treatment with exogenous TSP-1 after subcurative radiation treatment reduced the growth rate of recurrent primary tumors in addition to suppressing metastatic growth. TSP-1 suppressed tumor growth at both primary and metastatic sites by inducing apoptosis in tumor-associated microvascular endothelial cells. Treatment with exogenous TSP-1 before radiation treatment enhanced the antitumor effect of the radiation treatment. The radiopotentiation by TSP-1 involved at least two distinctly different mechanisms, i.e., TSP-1 reduced the fraction of radiobiologically hypoxic parenchymal tumor cells and increased the radiation sensitivity of the tumor microvasculature by promoting radiation-induced endothelial cell apoptosis. In conclusion, the present preclinical study showed that TSP-1 has antiangiogenic, antimetastatic, and radiopotentiating properties that merit additional investigation in clinical studies.     

Tirapazamine administered as a neoadjuvant to radiotherapy reduces metastatic dissemination.

PURPOSE: The level of hypoxia in primary tumors has been linked both clinically and experimentally to the incidence of metastases. This study was designed to address the effect of selectively targeting hypoxic cells in primary tumors on subsequent presentation of metastasis. EXPERIMENTAL DESIGN: The murine KHT model was used as a reproducible temporal and spatial onset of metastases is revealed following treatment of primary ( approximately 400 mm(3)) s.c. tumors with a 25 Gy radiation dose. The bioreductive drugs tirapazamine and RB6145 were administered in multiple doses before radiotherapy. RESULTS: Fractionated treatment with both tirapazamine and RB6145 significantly reduced the hypoxic fraction of the primary tumor, as assessed by pimonidazole binding, and had no effect on the overall growth rate of the primary tumor. Excision assays showed an increased level of cell kill in tirapazamine-treated versus RB6145-treated tumors consistent with tirapazamine targeting hypoxic cells at a broader range of oxygen tensions than RB6145. Tirapazamine treatment significantly reduced the presentation of metastases following radiotherapy (P = 0.003 versus saline controls) whereas RB6145 had no effect. Local control rates increased from 20% to 32% and 50% when radiation was combined with RB6145 and tirapazamine, respectively. CONCLUSIONS: These data provide direct evidence that selective targeting of hypoxic cells in primary tumors is a viable approach in the control of metastatic disease. The enhanced efficacy of tirapazamine versus RB6145 suggests that the radioresistant cells at intermediate oxygen tensions, conducive to targeting with tirapazamine but not with the more stringent bioreductive RB6145, predominate in terms of linking primary tumor hypoxia and metastases.     

Acute hypoxia enhances spontaneous lymph node metastasis in an orthotopic murine model of human cervical carcinoma

An orthotopic mouse model of cervical carcinoma has been used to investigate the relationship between acute (cyclic) hypoxia and spontaneous lymph node metastasis in vivo. The human cervical carcinoma cell line ME-180 was stably transfected to express the fluorescent protein DsRed2, which allowed the in vivo optical monitoring of tumor growth and metastasis by fluorescent microscopy. The surgically implanted primary tumors metastasize initially to local lymph nodes and later to lung, a pattern consistent with the clinical course of the disease. The effect of acute hypoxia on the growth and spread of these tumors was examined by exposing tumor-bearing mice to treatment consisting of exposure to 12 cycles of 10 min 7% O(2) followed by 10 min air (total 4 h) daily during tumor growth. After 21 days, the tumors were excised, lymph node and lung metastases were quantified, and the hypoxic fraction and relative vascular area of the primary tumors were assessed by immunohistochemical staining for the hypoxic marker drug EF5 [2-(2-nitro-1H-imidazole-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] and the vascular marker CD31, respectively. In untreated mice, the primary tumor size was directly correlated with lymph node metastatic burden. The acute hypoxia treatment resulted in a significant decrease in the size of the primary tumors at the time of excision. However, the mice in the acute hypoxia group had an increased number of positive lymph nodes (2-4) as compared with control mice (1-3). Lung metastasis was not affected. The acute hypoxia treatment also decreased the relative vascular area in the primary tumors but did not affect the hypoxic fraction. These results suggest that fluctuating oxygenation in cervical carcinoma tumors may reduce tumor growth rate, but it may also enhance the ability of tumor cells to metastasize to local lymph nodes.     

Risk of cancer revealed by follow-up of families with hereditary non-polyposis colorectal cancer: Reply to Dr. Eluf-Neto

Congenitally athymic nude (nu/nu) mice have been used extensively as hosts to study the metastatic properties of xenografted human tumors. It has not been fully explored to what extent the metastatic behavior of human tumor xenografts reflects the intrinsic properties of the tumor cells and to what extent it is influenced artificially by the host. The purpose of the work reported here was to evaluate the potential usefullness of athymic mice for qualitative and quantitative assessment of the intrinsic metastatic properties of human tumor cells. Four human melanoma cell lines (A-07, D-12, R-18, U-25) were included in the study. Quantitative intradermal and intracranial transplantation assays were used to determine the tumorigenicity and immunogenicity of the cell lines. The metastatic behavior of the cell lines was studied by inoculating cells intra-arterially or intravenously (artificial metastases) or intra-dermally (spontaneous metastases). The influence of the host on the incidence of metastases was assessed by inoculating cells intravenously in immunologically modified athymic mice. Both the intradermal and intracranial tumorigenicity differed substantially between the cell lines. The host immune reactivity against heterotransplantation was significantly lower for D-12 than for A-07, R-18 and U-25 cells. The incidence of metastases was influenced significantly by the host immune reactivity. The cell lines showed organ-specific metastatic patterns in athymic mice. The organs of preference were lungs for A-07 and D-12 cells, lymph nodes for R-18 cells, and brain for U-25 cells. The organ-specific metastatic pattern in athymic mice reflected the pattern of distant metastases in the donor patients. In addition, all cell lines gave rise to metastases in abdominal organ sites, but without organ specificity. The incidence of abdominal metastases was influenced significantly by the tumorigenicity of the cell lines. In conclusion, athymic mice appear to be excellent hosts for assessment of the organ specificity in the metastatic behavior of human tumors. The metastasis frequency of human tumors in athymic mice, however, might be a misleading quantitative measure of the intrinsic metastatic propensity of the tumor cells, owing to the cell-line-dependent host immune reactivity. © 1995 Wiley-Liss, Inc. © 1995 Wiley-Liss, Inc.     

Inhibition of the Kit Ligand/c-Kit Axis Attenuates Metastasis in a Mouse Model Mimicking Local Breast Cancer Relapse after Radiotherapy

PURPOSE: Local breast cancer relapse after breast-saving surgery and radiotherapy is associated with increased risk of distant metastasis formation. The mechanisms involved remain largely elusive. We used the well-characterized 4T1 syngeneic, orthotopic breast cancer model to identify novel mechanisms of postradiation metastasis. EXPERIMENTAL DESIGN: 4T1 cells were injected in 20 Gy preirradiated mammary tissue to mimic postradiation relapses, or in nonirradiated mammary tissue, as control, of immunocompetent BALB/c mice. Molecular, biochemical, cellular, histologic analyses, adoptive cell transfer, genetic, and pharmacologic interventions were carried out. RESULTS: Tumors growing in preirradiated mammary tissue had reduced angiogenesis and were more hypoxic, invasive, and metastatic to lung and lymph nodes compared with control tumors. Increased metastasis involved the mobilization of CD11b(+)c-Kit(+)Ly6G(high)Ly6C(low)(Gr1(+)) myeloid cells through the HIF1-dependent expression of Kit ligand (KitL) by hypoxic tumor cells. KitL-mobilized myeloid cells homed to primary tumors and premetastatic lungs, to give rise to CD11b(+)c-Kit(-) cells. Pharmacologic inhibition of HIF1, silencing of KitL expression in tumor cells, and inhibition of c-Kit with an anti-c-Kit-blocking antibody or with a tyrosine kinase inhibitor prevented the mobilization of CD11b(+)c-Kit(+) cells and attenuated metastasis. C-Kit inhibition was also effective in reducing mobilization of CD11b(+)c-Kit(+) cells and inhibiting lung metastasis after irradiation of established tumors. CONCLUSIONS: Our work defines KitL/c-Kit as a previously unidentified axis critically involved in promoting metastasis of 4T1 tumors growing in preirradiated mammary tissue. Pharmacologic inhibition of this axis represents a potential therapeutic strategy to prevent metastasis in breast cancer patients with local relapses after radiotherapy. Clin Cancer Res; 18(16); 4365-74. ?2012 AACR.     

Tumor-line specific pO(2) fluctuations in human melanoma xenografts

PURPOSE: A large number of studies have demonstrated that tumors are heterogeneous in oxygen tension (pO(2)) and may develop regions with chronically or acutely hypoxic cells during growth. In the present study, it was investigated whether experimental tumors of different lines may show characteristic pO(2) fluctuation patterns and hence may differ with respect to the kinetics of acute hypoxia. METHODS AND MATERIALS: A total of 70 xenografted tumors of two human melanoma lines (A-07 and R-18) were included in the study. Tissue pO(2) was measured simultaneously in two positions in each tumor for periods of at least 60 min using a two-channel fiberoptic oxygen-sensing device (OxyLite 2000, Oxford Optronix, Oxford, UK). RESULTS: The mean pO(2) was calculated for each pO(2) trace, and this parameter was significantly greater in A-07 than in R-18 tumors (p <0.000001). Fluctuations in pO(2) around 3, 5, or 10 mm Hg were seen in a large fraction of the tumors of both lines. The pO(2) fluctuation frequency differed among individual traces from 0 to 20/h (A-07) and from 0 to 12/h (R-18) and was significantly greater in A-07 than in R-18 tumors (p = 0.0026). The absolute amplitude of the pO(2) fluctuations ranged from 1 to 16 mm Hg (A-07) and 1 to 33 mm Hg (R-18) and did not differ between the tumor lines. The relative amplitude was significantly higher in R-18 than in A-07 tumors (p <0.000001). The pO(2) values recorded simultaneously in the same tumor were in general not temporally coordinated. CONCLUSION: Experimental tumors of different lines may show individual and characteristic pO(2) fluctuation patterns. The pO(2) fluctuations may result in regions with acutely hypoxic cells. The kinetics of the acute hypoxia may differ among tumors of different lines, individual tumors of the same line, and different regions within the same tumor.     

The constitutive level of vascular endothelial growth factor (VEGF) is more important than hypoxia-induced VEGF up-regulation in the angiogenesis of human melanoma xenografts

Angiogenesis of tumours might develop as a result of environmental conditions, such as hypoxia, and/or as a result of genetic alterations specific for tumour cells. The relative contributions of these mechanisms were investigated by comparing the in vivo expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) to the hypoxic fraction, the angiogenic potential and the vascular density of four human melanoma lines (A-07, D-12, R-18, U-25) grown intradermally in Balb/c nu/nu mice. VEGF expression, bFGF expression and expression of pimonidazole, a marker of hypoxic cells, were investigated by immunohistochemistry. An association between high VEGF and bFGF expression and high angiogenic potential was detected, suggesting an important role for VEGF/bFGF in the angiogenesis of melanomas. High VEGF/bFGF expression was also related to low hypoxic fraction and high vascular density. Thus, the constitutive, genetically determined level of VEGF was probably more important than hypoxia-induced upregulation in the angiogenesis of the melanoma xenografts.     

Hypoxia‐mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression

Tumor hypoxia and high levels of expression of the urokinase‐type plasminogen activator (uPA) receptor (uPAR) represent a poor clinical outcome for patients with various cancers. Here, we examined the effect of hypoxia on in vitro invasion of extracellular matrix and uPAR expression by human carcinoma cells. Compared with culture under 20% O₂, culture for up to 24 hr under 1% or 4% O₂ resulted in increased cell surface uPAR. However, the highest uPAR levels were observed in cells cultured under 1% O₂. Culture of MDA‐MB‐231 breast carcinoma cells under hypoxia also resulted in increased uPAR mRNA levels. Furthermore, incubation with cobalt chloride or with an iron chelator also resulted in elevated uPAR expression, while presence of 30% carbon monoxide in the hypoxic atmosphere reduced the hypoxia‐mediated uPAR mRNA upregulation. Increased uPAR expression was paralleled by higher cell‐associated uPA levels and lower levels of secreted uPA as determined by gel zymography performed on cell extracts and culture‐conditioned media. In addition, the in vitro invasiveness of MDA‐MB‐231 breast carcinoma cells was significantly higher when the invasion assay was performed under hypoxic conditions. This effect of hypoxia on invasion was abrogated by including in the assay a monoclonal, function‐blocking anti‐uPAR antibody or by the presence of 30% carbon monoxide in the hypoxic atmosphere. Our findings indicate that hypoxia stimulates carcinoma cell invasiveness by upregulating uPAR expression on the cell surface through a mechanism that requires a putative heme protein. Through a similar mechanism, hypoxia may stimulate tumor invasion and metastasis in vivo. Int. J. Cancer 80:617–623, 1999. © 1999 Wiley‐Liss, Inc.