Clinical Outcomes and Cost-effectiveness of Primary Prophylaxis of Febrile Neutropenia During Adjuvant Docetaxel and Cyclophosphamide Chemotherapy for Breast Cancer.

Docetaxel and cyclophosphamide (TC) is a widely used breast cancer adjuvant regimen. We sought to compare the rates of febrile neutropenia (FN) between patients receiving no primary prophylaxis (PP) and those receiving PP with either granulocyte-colony stimulating factor (G-CSF) or antibiotics. We also analyzed cost-effectiveness of TC with and without either G-CSF or antibiotics. Charts were reviewed of all 340 patients who received adjuvant TC between January 2008 and December 2012 at two major cancer centers. Rates of FN in the three groups - no PP, PP with G-CSF and PP with antibiotics were compared. A Markov model was constructed comparing cost-effectiveness of PP with G-CSF, PP with antibiotics, and secondary prophylaxis (SP) with G-CSF after an episode of FN in a previous cycle. Costs were based on actual resource utilization and supplemented by the published literature, adjusted to 2012 Canadian dollars. Of the 73 (21%) patients who did not receive any PP, 23 (32%) of patients developed FN. Of the 192 (57%) patients receiving PP with G-CSF alone, only two (1%; p < 0.0001) developed FN; and of the 53 (16%) receiving PP with antibiotics alone, six (11%; p < 0.01) developed FN. From a cost-standpoint, PP with G-CSF was less cost-effective than PP with antibiotics. The rate of FN with TC chemotherapy exceeds 30%, and American Society of Clinical Oncology guidelines recommend PP with G-CSF in this situation. PP with antibiotics is more cost-effective, and is a reasonable option in resource-limited settings or for patients who decline or do not tolerate G-CSF.

Impact of new chemotherapeutic and targeted agents on survival in stage IV non-small cell lung cancer.

PURPOSE: Significant advances in the systemic management of metastatic non-small cell lung cancer (NSCLC) have occurred over the past decade, with options now including multiple lines of chemotherapy, epidermal growth factor receptor inhibitors, and antiangiogenic agents. Improvements in overall survival have been demonstrated in randomized controlled trials comparing these newer agents with best supportive care or standard therapy. This study examined uptake of these therapies in general practice and their impact on survival. METHODS: This retrospective cohort study compared demographic, treatment, and survival data among 987 patients diagnosed with stage IV NSCLC at two institutions in 1998, 2003, and 2008. Cohorts were selected based on intervals when doublet chemotherapy, second-line chemotherapy, and targeted agents were incorporated into the standard treatment regimen. RESULTS: The proportion of patients receiving systemic therapy increased over time (20% in 1998, 42% in 2008). Overall survival improved significantly across cohorts (p < .001), with 2-year survival rates of 0.3% in 1998, 4% in 2003, and 15% in 2008. In a multivariate survival analysis, the 2003 and 2008 cohorts were independently associated with longer survival, as was the use of one or more lines of systemic therapy. Elderly patients (aged ≥70 years) were also more likely to receive systemic therapy over time, with longer overall survival (p < .001). CONCLUSION: Over the past decade, there has been an increasing use of systemic therapy in stage IV NSCLC patients, including the elderly. This has been associated with significantly longer overall survival.

Oncogenes, trousseau syndrome, and cancer-related changes in the coagulome of mice and humans.

Cancer is often associated with venous thrombosis, a phenomenon that was first described by Trousseau in 1865 (Trousseau syndrome). Recent studies have begun to explain how oncogenic events may deregulate the hemostatic system. For instance, activated oncogenes (K-ras, EGFR, PML-RARalpha, and MET) or inactivated tumor suppressors (e.g., 53 or PTEN) may increase the risk of thrombosis by inducing the expression of tissue factor, a potent procoagulant molecule, and plasminogen activator inhibitor-1, a fibrinolysis inhibitor. In a more complex clinical reality, transforming genes may often act in concert with numerous epigenetic factors, including hypoxia, inflammation, anticancer therapy, contact between blood and metastatic cancer cells, and emission of procoagulant vesicles from tumors and their stroma into the circulation. To add to mechanistic insights gained from mouse models, which may not fully phenocopy human Trousseau syndrome, we suggest that valuable clues to progression and thrombosis risk may be obtained by monitoring multiple hemostatic variables in cancer patients ("coagulomics").

Oncogenes and Angiogenesis: down-regulation of thrombospondin-1 in normal fibroblasts exposed to factors from cancer cells harboring mutant ras.

The onset of angiogenesis in cancer often involves down-regulation of endogenous angiogenesis inhibitors, of which thrombospondin-1 (TSP-1) is a paradigm. As this effect is thought to occur under the influence of transforming genetic lesions (e.g., expression of the mutant ras oncogene), its nature is regarded as intrinsic to cancer cells themselves. Here, we show that ras-transformed cancer cells can also induce TSP-1 down-regulation in their adjacent nontransformed stromal fibroblasts, but not in endothelial cells, in a paracrine and distance-dependent manner. Indeed, several H-ras-expressing fibrosarcoma (528ras1, B6ras, and NIH3T3Ras) and carcinoma (DLD-1 and IEC18Ras3) cells were found to release soluble factors capable of suppressing TSP-1 protein, mRNA, and promoter activity in nontumorigenic, immortalized dermal fibroblastic cell lines in culture (e.g., in fibroblasts expressing enhanced green fluorescent protein/TSP-1 reporter). This effect was abrogated in Id1-/- fibroblasts. At least two low molecular weight (<3 kDa), heat-labile, and trypsin-resistant mediators of TSP-1 suppression were found to be released from 528ras1 cells. Their effects on normal fibroblasts were inhibited (albeit to different extents) by pertussis toxin and, in one case, by dimethylsphingosine, none of which affected TSP-1 expression by 528ras1 cells. Collectively, our study suggests that the effect of mutant ras on tumor neovascularization is not limited to changes in angiogenic properties of cancer cells themselves. Rather, mutant ras, through a different signaling mechanism, may modulate the properties of the adjacent normal stroma, thus eliciting a proangiogenic field effect.

Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts.

Vascular endothelial growth factor (VEGF) occurs in at least five different isoforms because of alternative splicing of the gene. To investigate the roles of different VEGF isoforms in tumor blood vessel formation and tumorigenicity, the three major isoforms (VEGF(121), VEGF(165), and VEGF(189)) were overexpressed in an early-stage human melanoma cell line (WM1341B), which is VEGF-negative and nontumorigenic in immunodeficient mice. Although overexpression of VEGF(121) and VEGF(165) resulted in aggressive tumor growth, WM1341B cells transfected with VEGF(189) remained nontumorigenic and dormant on injection. Although tumor growth rate depended on the level and not the isoform of VEGF expressed, striking isoform-specific differences in vascular patterning were associated with VEGF(121)- versus VEGF(165)-dependent tumorigenic conversion of human melanoma. Thus, tumors overexpressing VEGF(165) generated dense, highly heterogeneous vessel networks that were distinctly different from those of tumors expressing VEGF(121) (poorly vascularized and necrotic). Paradoxically, although VEGF(165) expression appears to result in the most effective tumor perfusion, it is the expression of VEGF(121) that is observed during human malignant melanoma progression. Indeed, unbiased selection of spontaneously tumorigenic variants of WM1341B (by coinjection with Matrigel) led to predominant expression of the VEGF(121) isoform. The vascular patterning in these tumors (1341-P3N1, 1341-P3N2) resembled that of the VEGF(121)-transfected WM1341B tumors. These results suggest that, for reasons that remain to be elucidated, a "minimal" program of tumor vascularization may be favored during melanoma progression.

High-frequency Doppler ultrasound monitors the effects of antivascular therapy on tumor blood flow.

The effect of antivascular therapy on blood flow in superficial tumors was monitored using novel high frequency Doppler (HFD) ultrasound techniques. Human melanoma cells (MeWo) were injected orthotopically into the skin of athymic nude mice. Volumetric HFD imaging of established melanomas detected a significant reduction in blood flow 4 h after injection of the tumor vascular targeting agent ZD6126 followed by a recovery of flow by 24 h after injection. Measurements of tumor perfusion in situ by Hoechst 33342 staining correlated with the ultrasound results. This study demonstrates the feasibility of HFD as a noninvasive, quantitative tool for following longitudinally the effects of antivascular therapy on blood flow in superficial tumors.

Host microenvironment in breast cancer development: inflammatory and immune cells in tumour angiogenesis and arteriogenesis.

Breast cancer progression is associated with and dependent upon robust neovascularization. It is becoming clear that tumour-associated 'normal' cells, such as immune/inflammatory cells, endothelial cells and stromal cells, conspire with cancer cells in promoting this process. In particular, infiltrating immune/inflammatory cells secrete a diverse repertoire of growth factors and proteases that enable them to enhance tumour growth by stimulating angiogenesis and, as we suggest here, by promoting 'tumour arteriogenesis' - enlargement of feeding vessels supplying the expanding tumour capillary bed. Macrophages and their chemoattractants (e.g. macrophage chemoattractant protein-1) are critical for the arteriogenic process in ischaemia, and probably also in breast neoplasia. A better understanding of these various cellular and molecular constituents of breast cancer neovascularization may be useful in designing more effective therapies.

Angiogenesis and the role of epigenetics in metastasis.

The major obstacle to devising effective ways to treat cancer is its heterogeneity and genetic instability. It was originally postulated that targeting the process of tumor angiogenesis could circumvent this problem, as it involves genetically stable epigenetically controlled host stroma. Thus, anti-angiogenic approaches should be applicable across various tumor types and organ sites, including metastases. However, early clinical experience with this therapy revealed unexpectedly distinct responses between different tumors and organ sites. Here we propose that the heterogeneity of pre-clinical and clinical results obtained with anti-angiogenic agents stems from the deep functional linkage that may exist between genetic and epigenetic tumor progression. Thus, epigenetic processes regulating tumor associated host blood vessels (such as tumor microenvironment) display unstable, heterogeneous and progressive characteristics to an extent comparable with (and causally linked to) the instability of the cancer cell genome. As well, many known epigenetic factors (such as hypoxia, inflammation, expression of growth factors, etc.) may have genetic causes and consequences (e.g., oncogene expression, loss of tumor suppressor genes). This reciprocal interrelationship and heterogeneity may translate into site and stage specific changes in angiogenesis regulation, and angiogenesis dependence, ultimately to changes in the metastatic ability/efficiency of cancer cells, even in the same patient. A better understanding of the linkage between genetic and epigenetic events in growth and metastasis of various cancers may result in more effective use of anti-angiogenic therapy in future.

High-frequency 3-D color-flow imaging of the microcirculation.

High-frequency (> 20 MHz) ultrasound (US) flow imaging has the potential to be an important tool for assessing microvascular blood flow in superficial tissues noninvasively. In this paper, we describe the development and evaluation of a 3-D US flow imaging system capable of operating at center frequencies in the 20- to 50-MHz range. Flow images are made for tissue volumes of sizes up to 10 mm laterally and 5 mm in depth, permitting a range of scientific and clinical applications. To acquire data sets in a reasonable time, the 2-D sections were derived from data collected with a transducer that was scanning continuously in a direction perpendicular to the beam axis. Due to spectral broadening effects induced by scanning tissue, significant tradeoffs must be made between frame rate, lateral resolution and the minimum detectable blood velocity. 3-D flow images were reconstructed with flow data acquired from a series of adjacent planes. The system was evaluated at a center frequency of 50 MHz, using two PVDF transducers with lateral resolutions of 43 microm and 65 microm and axial resolutions of 66 microm to 72 microm, respectively. Velocity ranges were from below 1 mm/s to 25 mm/s. In vivo validation experiments using the mouse ear demonstrated the ability to follow branching patterns of closely spaced microvessels from 30 microm to 100 microm in diameter. Experiments conducted on mouse tumors successfully imaged microvessel morphology in the tumor microcirculation.

Modulation of the oncogene-dependent tissue factor expression by kinase suppressor of ras 1.

INTRODUCTION: Tissue factor (TF) is the key trigger of the coagulation cascade and the membrane signalling receptor for coagulation protease, factor VIIa. In cancer, TF has been implicated in tumor cell survival, growth, and angiogenesis, and is upregulated as a result of oncogenic transformation. MATERIALS AND METHODS: We assayed TF expression and tumourigenicity in mice in the case of human cancer cell lines expressing oncogenic receptor tyrosine kinases. These cells were also subjected to genetic modulation of the kinase suppressor of ras 1 (KSR1), and treated with oncoprotein inhibitors in vitro and in vivo. RESULTS: Here we show that herceptin, AG1478 and CI-1033, inhibitors of two different members of the ErbB family of oncogenes (HER-2 and EGFR), reduce TF levels in epithelial cancer cells. In EGFR-driven A431 cells, TF upregulation is diminished upon genetic targeting of KSR1, the scaffolding protein involved in EGFR signalling. Conversely, upregulation of KSR1 in A431 cells increases their TF expression and tumourigenicity in mice. The latter property remains dependent on EGFR, as pan-Erb (EGFR) inhibitor, CI-1033, blocks TF promoter activity and inhibits tumour formation by the parental and KSR1 overexpressing A431 cells. CONCLUSIONS: KSR1 emerges, as an important modulator of TF expression in EGFR-driven cancer cells, which also impacts their aggressiveness in vivo.

Tissue factor regulation by epidermal growth factor receptor and epithelial-to-mesenchymal transitions: effect on tumor initiation and angiogenesis.

ErbB oncogenes drive the progression of several human cancers. Our study shows that in human carcinoma (A431) and glioma (U373) cells, the oncogenic forms of epidermal growth factor receptor (EGFR; including EGFRvIII) trigger the up-regulation of tissue factor (TF), the transmembrane protein responsible for initiating blood coagulation and signaling through interaction with coagulation factor VIIa. We show that A431 cancer cells in culture exhibit a uniform TF expression profile; however, these same cells in vivo exhibit a heterogeneous TF expression and show signs of E-cadherin inactivation, which is coupled with multilineage (epithelial and mesenchymal) differentiation. Blockade of E-cadherin in vitro, leads to the acquisition of spindle morphology and de novo expression of vimentin, features consistent with epithelial-to-mesenchymal transition. These changes were associated with an increase in EGFR-dependent TF expression, and with enhanced stimulation of vascular endothelial growth factor production, particularly following cancer cell treatment with coagulation factor VIIa. In vivo, cells undergoing epithelial-to-mesenchymal transition exhibited an increased metastatic potential. Furthermore, injections of the TF-blocking antibody (CNTO 859) delayed the initiation of A431 tumors in immunodeficient mice, and reduced tumor growth, vascularization, and vascular endothelial growth factor expression. Collectively, our data suggest that TF is regulated by both oncogenic and differentiation pathways, and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. Thus, TF could serve as a therapeutic target in EGFR-dependent malignancies.

Atherosclerosis and vascular aging as modifiers of tumor progression, angiogenesis, and responsiveness to therapy.

It is rarely considered that age-related common vascular co-morbidities may affect therapeutic outcomes of antiangiogenic therapy in cancer. Indeed, the accepted model of human disease consists of 4- to 8-week-old (young) tumor-bearing, but otherwise healthy, experimental mice, yet human cancers are diagnosed and treated in later decades of life when atherosclerosis and vascular diseases are highly prevalent. Here we present evidence that tumor growth and angiogenesis are profoundly altered in mice affected by natural aging and with genetically induced atherosclerosis (in ApoE(-/-) mice). Thus, transplantable tumors (Lewis lung carcinoma and B16F1) grew at higher rates in young (4 to 8 weeks old) ApoE(+/+) and ApoE(-/-) nonatherosclerotic syngeneic recipients than in their old (12 to 18 months old) or atherosclerotic (old/ApoE(-/-)) counterparts. These age-related changes were paralleled by reduced tumor vascularity, lower expression of tumor endothelial marker 1, increased acute tumor hypoxia, depletion of circulating CD45(-)/VEGFR(+) cells, and impaired endothelial sprouting ex vivo. Exposure of tumor-bearing mice to metronomic therapy with cyclophosphamide exerted antimitotic effects on tumors in young hosts, but this effect was reduced in atherosclerotic mice. Collectively, our results suggest that vascular aging and disease may affect tumor progression, angiogenesis, and responses to therapy.

Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis.

Tissue factor (TF) is the primary cellular initiator of blood coagulation and a modulator of angiogenesis and metastasis in cancer. Indeed, systemic hypercoagulability in patients with cancer and TF overexpression by cancer cells are both closely associated with tumor progression, but their causes have been elusive. We now report that in human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K). Furthermore, the levels of cell-associated as well as circulating (microvesicle-associated) TF activity are linked to the genetic status of cancer cells. Finally, RNA interference experiments suggest that TF expression is an important effector of the K-ras-dependent tumorigenic and angiogenic phenotype in vivo. Thus, this study establishes a causal link between cancer coagulopathy, angiogenesis, and genetic tumor progression.

Oncogenes and tumor angiogenesis: the question of vascular "supply" and vascular "demand".

Among novel promising approaches that have recently entered the scene of anti-cancer therapy angiogenesis inhibition and targeting cancer-causing genes (e.g. oncogenes) are of particular interest as potentially highly synergistic. One reason for this is that transforming genetic lesions driving cancer progression (e.g. mutations of ras and/or p53) are thought to be causative for the onset of tumor angiogenesis and thereby responsible for build up of vascular supply which is essential for cancer cell survival, malignant growth, invasion and metastasis. However, many of the same genetic alterations that emerge during disease progression and repeated rounds of mutagenic and/or apoptosis causing therapy could alter cellular hypoxia-, growth factor- and apoptotic pathways in such a manner, as to also render cancer cells (partially) refractory to the detrimental consequences of poor blood vessel accessibility (density), ischemia, hypoxia and growth factor deprivation. As recent experimental evidence suggests, such cancer cells could therefore display a reduced vascular demand and remain viable even in poorly perfused regions of the tumor as well as possess an overall growth/survival advantage. The latter circumstance may lead to (predict) diminished efficacy of anti-angiogenic agents in certain malignancies. Therefore, we propose that analysis of oncogenic pathways and gene expression profiling of cancer cells may lead to important clues as to potential efficacy of anti-angiogenic therapies, the direct target of which is the host vasculature, but which are ultimately aimed at (indirect) destruction/control of the cancer cells population. We also suggest that oncogene (tumor suppressor)-directed therapies may help reverse diminished vascular demand of highly transformed cancer cells and thereby facilitate (sensitize tumors to) therapies directed against vascular supply of cancers and their metastases.

A paradigm for therapy-induced microenvironmental changes in solid tumors leading to drug resistance.

Intrinsic alterations in the tumor microenvironment are known to contribute to various forms of drug resistance. For example, tumor hypoxia, due to abnormal or sluggish blood flow within areas of solid tumors, can result in both microenvironment-mediated radiation and chemotherapeutic drug resistance. In contrast, acquired resistance to chemotherapy is generally considered to be the result of the gradual selection of mutant subpopulations having genetic mutations and biochemical alterations responsible for the resistant phenotype. Here we present a paradigm for therapyinduced microenvironment-mediated acquired drug resistance. It is based on the results showing that tumor cells appear to be heterogeneous in their relative dependence on adjacent tumor-associated vasculature for survival. Some tumor cells are highly vessel dependent, whereas some are significantly less so, and thus can survive in more hypoxic regions of tumors, distal from such tumor vessels. Hence, it is possible that variant tumor cells that are less vessel dependent may therefore be selected for over time by successful antiangiogenic drug therapies. This results in loss of response or attenuated responses to the therapy. Preliminary evidence is summarized in support of this hypothesis, using paired human colon cancer (HCT116) cell lines that contain two copies of either the wild-type or the disrupted p53 tumor suppressor gene. The mutant cells were found to be less responsive to antiangiogenic therapy, compared to the wild-type cells, and could be progressively selected for in mixed cell populations. Because p53 inactivation can lead to resistance to hypoxia-mediated apoptosis, the results suggest that a protracted and successful antiangiogenic therapy may create more hypoxic tumor microenvironments, thereby creating the necessary conditions to accelerate the selection of mutant tumor cells that are more adept in surviving and growing in such environments. As such, consideration might be given to the combined use of bioreductive hypoxic cell cytotoxic drugs and angiogenesis inhibitors to prolong the efficacy of antiangiogenic therapeutics.

Oncogenes as regulators of tissue factor expression in cancer: implications for tumor angiogenesis and anti-cancer therapy.

Up-regulation of tissue factor (TF) is often observed in cancer. TF is a cell-associated receptor for coagulation factor VII/VIIa, an interaction known to activate the coagulation cascade. At the same time, TF is also known as a mediator of intracellular signaling events that can alter gene expression patterns and cell behavior. Both aspects of TF activity are of possible relevance to tumor growth, metastasis, and angiogenesis, including up-regulation of vascular endothelial growth factor (VEGF). TF up-regulation is often observed on the surfaces of tumor-associated endothelial cells, inflammatory cells, and particularly on cancer cells themselves. In the last case, high TF levels may be associated with poor prognosis and parallel clinical (and genetic) tumor progression. We have proposed elsewhere that TF may be a target of oncogenic events in cancer. Here we discuss our observations suggesting that oncogene-targeting agents may down-regulate TF expression. Such is the effect of treatment with the neutralizing monoclonal antibody (C225) raised against the epidermal growth factor receptor (EGFR) in EGFR-dependent squamous cell carcinoma cells (A431). This two- to threefold TF down-regulation by C225 treatment is paralleled by a decrease in expression of VEGF. It is conceivable that TF participates in signals that regulate VEGF and angiogenesis triggered by activated oncogenic pathways. Therefore, direct targeting of TF in cancer should be considered in combination with other treatment modalities such as oncogene-directed therapies, antiangiogenic agents (e.g., VEGF antagonists), and anti-cancer chemotherapy.

Effect of p53 status on tumor response to antiangiogenic therapy.

The p53 tumor suppressor gene is inactivated in the majority of human cancers. Tumor cells deficient in p53 display a diminished rate of apoptosis under hypoxic conditions, a circumstance that might reduce their reliance on vascular supply, and hence their responsiveness to antiangiogenic therapy. Here, we report that mice bearing tumors derived from p53(-/-) HCT116 human colorectal cancer cells were less responsive to antiangiogenic combination therapy than mice bearing isogenic p53(+/+) tumors. Thus, although antiangiogenic therapy targets genetically stable endothelial cells in the tumor vasculature, genetic alterations that decrease the vascular dependence of tumor cells can influence the therapeutic response of tumors to this therapy.

A role for survivin in chemoresistance of endothelial cells mediated by VEGF.

Although standard anticancer chemotherapeutic drugs have been designed to inhibit the survival or growth of rapidly dividing tumor cells, it is possible to enhance the efficacy of such drugs by targeting the proliferating host endothelial cells (ECs) of the tumor vasculature. A theoretical advantage of this strategy lies in the possibility of circumventing, or significantly delaying, acquired drug resistance driven by the genetic instability of tumor cells. Here, we show that both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor significantly reduce the pro-apoptotic potency of chemotherapy on both micro- and macrovascular ECs. This cytoprotection to drug toxicity was found to be phosphatidylinositol 3-kinase-dependent and could be recapitulated in the absence of VEGF by overexpressing the dominant-active form of the serine/threonine kinase protein kinase B/Akt. Downstream of phosphatidylinositol 3-kinase, we also show that survivin plays a pivotal role in VEGF-mediated EC protection by preserving the microtubule network. In this respect, its induction effectively protects ECs against chemotherapeutic damage, whereas overexpression of its dominant-interfering mutant (C84A) abrogates the protective effects of VEGF. Accordingly, the potency of VEGF as a chemoprotectant was more pronounced with drugs that interfere with microtubule dynamics than those that damage DNA. These studies implicate a role for survivin up-regulation as a novel mechanism of EC drug "resistance" and support the notion that angiogenic factors that induce the expression of survivin may act to shield tumor ECs from the apoptotic effects of chemotherapy. Thus, exploiting chemotherapeutic drugs as antiangiogenics is likely to be compromised by the high concentrations of proangiogenic survival/growth factors present in the tumor microenvironment; targeting EC survival pathways should improve the antiangiogenic efficacy of antineoplastic agents, particularly microtubule-inhibitor drugs.

Restoration of thrombospondin 1 expression in tumor cells harbouring mutant ras oncogene by treatment with low doses of doxycycline.

Oncogenes act as inducers of tumor neovascularization, at least in part through suppression of endogenous angiogenesis inhibitors, e.g., thrombospondin 1 (TSP-1/THSP1). Therefore, restoration of TSP-1 levels can be viewed as a possible means to inhibit tumor angiogenesis. We observed that low concentrations (0.1-10 microg/ml) of doxycycline (but not those of related tetracycline) restore TSP-1 expression in H-ras oncogene-expressing tumor cell lines (528ras1, MT-Ras). Interestingly, this effect was relatively ras-specific, as doxycycline did not alter TSP-1 expression in several cell lines (e.g., 528neu2 fibrosarcoma, B16F1 melanoma, and Lewis lung carcinoma) harbouring other types of transforming alterations. Doxycycline-induced reversal of TSP down-regulation was abrogated under hypoxic conditions. Therefore, we conclude that, in vivo, TSP-1 is likely under dual and/or synergistic control of oncogenes and hypoxia-related pathways. Disruption of both components may be necessary for the 'rescue' of TSP-1 expression in ras-driven cancers.