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We present a rationale for further clinical development and assessment of metronomic chemotherapy on the basis of unexpected results obtained in translational mouse models of cancer involving treatment of advanced metastatic disease. Historically, mouse cancer therapy models have been dominated by treating established primary tumors or early stage low volume microscopic disease. Treatment of primary tumors is also almost always the case when using genetically engineered mouse models (GEMMs) of cancer or patient-derived xenografts (PDXs). Studies using such models, and others including transplanted cell lines, often yield highly encouraging results which are seldom recapitulated in the clinic, especially when assessed in randomized phase III clinical trials. While there are likely many different reasons for this discrepancy, one is likely the failure to recapitulate treatment of advanced visceral metastatic disease in mice. With this gap in mind, we have developed a number of models of metastatic human tumor xenografts (and more recently, of mouse tumors in syngeneic immunocompetent mice). A pattern of response we have observed with various targeted agents, e.g. VEGF pathway targeting antiangiogenic drugs or trastuzumab, is effective when treating primary tumors in contrast to a complete or severely reduced lack of such efficacy when treating advanced metastatic disease. Interestingly, an exception to this pattern has been observed using various continuous low-dose metronomic chemotherapy regimens, where counterintuitively, superior responses are observed in the metastatic setting, as well as superiority or equivalence of metronomic chemotherapy over standard maximum tolerated dose (MTD) chemotherapy, with lesser toxicity. The basis for these encouraging results may be related to the multiple mechanisms responsible for the anti-tumor effects and longer duration of metronomic chemotherapy regimens made possible by lesser toxicity. These include antiangiogenesis, stimulation of the immune system, stromal cell targeting in tumors, and possibly direct tumor cell targeting, including targeting cancer stem cells (CSCs). In addition, metronomic chemotherapy regimens minimize or even eliminate the problem of chemotherapy-induced host responses that may actually secondarily promote tumor growth and malignancy after causing an initial and beneficial anti-tumor response. We suggest that future preclinical studies of metronomic chemotherapy should be concentrated in the following areas: i) further comparative assessment of anti-tumor efficacy in primary vs metastatic treatment settings; ii) rigorous comparative assessment of conventional MTD chemotherapy vs metronomic chemotherapy using the same agent; iii) assessment of potential predictive biomarkers for metronomic chemotherapy, and methods to determine optimal biologic dose and schedule; and iv) a further detailed assessment of the potential of different chemotherapy drugs administered using MTD or metronomic regimens on stimulating or suppressing components of the innate or adaptive immune systems. [ABSTRACT FROM AUTHOR]

In this issue of Cancer Cell , Seaman et al. demonstrate that antibody drug conjugates (ADCs) against CD276 expressed by tumor cells and tumor vasculature have promising anti-tumor activity while showing little toxicity. Importantly, these agents have the potential to target both angiogenic vessels and non-angiogenic vessels co-opted by tumor cells. [ABSTRACT FROM AUTHOR]

Abstract: Metastatic spread to the central nervous system (CNS) is a common and devastating manifestation of major cancer types. Its incidence is associated with poor prognosis manifested by neurological deterioration leading to diminished quality of life and an extremely short median survival. CNS metastasis is becoming an increasingly important clinical problem. This is especially the case for certain types of cancers for which effective treatments of visceral disease are available. As a result of the present limitations in treating CNS metastases, this manifestation of tumor progression remains an unmet clinical need. Despite its significance, our general understanding of the mechanisms that regulate the brain-metastatic phenotype is currently meager. Both the analysis of mechanistic aspects of brain metastasis and the development of effective treatments necessitate the use of appropriate in vivo models that recapitulate the interaction of the tumor cells with the microenvironment of the brain. Here we review the available preclinical models of CNS metastasis and their use as tools to advance knowledge of the biology of the disease (with the aim of identifying relevant molecular determinants, prognostic biomarkers, and therapeutic targets) as well as examining effective approaches for treatment. [Copyright &y& Elsevier]

The article presents an overview of the use of antiangiogenic therapy for the treatment of cancer. A discussion of the history of antiangiogenic therapy, which began in 1971 with the publication of Folkman's imaginative hypothesis, and of research which was conducted on the effectiveness of the therapy, is presented. The various growth factors and cell types that are associated with cancer are discussed. Drug resistance which has been seen with antiangiogenic drugs, including bevacizumab, is examined.

For more than 50 years, a major goal of research in cancer therapeutics has been to develop universally effective agents that render cancer cells more sensitive to cytotoxic chemotherapy without substantially increasing toxicity to normal cells. The results of recent clinical trials indicate that certain antiangiogenic drugs may produce this long-sought effect. Here, I describe three distinct mechanisms that may help to explain the chemosensitizing activity of these drugs: normalizing tumor vasculature, preventing rapid tumor cell repopulation, and augmenting the antivascular effects of chemotherapy. I then discuss how these potential mechanisms might be exploited to maximize therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Inhibiting angiogenesis is a promising strategy for treatment of cancer and several other disorders, including age-related macular degeneration. Major progress towards a treatment has been achieved over the past few years, and the first antiangiogenic agents have been recently approved for use in several countries. Therapeutic angiogenesis (promoting new vessel growth to treat ischaemic disorders) is an exciting frontier of cardiovascular medicine, but further understanding of the mechanisms of vascular morphogenesis is needed first. [ABSTRACT FROM AUTHOR]

In addition to proliferating cancer cells and various types of normal cells, such as those of the bone marrow, conventional cytotoxic chemotherapeutics affect the endothelium of the growing tumour vasculature. The anti-angiogenic efficacy of chemotherapy seems to be optimized by administering comparatively low doses of drug on a frequent or continuous schedule, with no extended interruptions - sometimes referred to as 'metronomic' chemotherapy. In addition to reduced acute toxicity, the efficacy of metronomic chemotherapy seems to increase when administered in combination with specific anti-angiogenic drugs. Gaining better insight into the mechanisms of these effects could lessen or even eliminate the empiricism used to determine the optimal dose and schedule for metronomic chemotherapy regimens. [ABSTRACT FROM AUTHOR]

Potent and specific, or relatively specific, inhibitors of epidermal growth factor receptor (EGFR) signaling, including monoclonal antibodies and small molecular weight compounds, have been successfully developed. Both types of agent have been found to have significant antitumor activity, especially when used in combination with radio- hormone- and chemotherapy in preclinical studies. Because of the potentiation of the conventional drug activity in these combination settings, inhibitors of EGFR signaling have often been referred to as sensitizers for chemotherapy or radiation, as well as drug resistance reversal agents. Phase II clinical trials in head-and-neck as well as lung cancer suggested this concept of chemosensitization might translate into the clinic, but this remains to be definitively proven in randomized, double-blind Phase III trials. Given the extensive preclinical literature on EGFR blocking drugs and the advanced clinical development of such agents, it is surprising that the possibility of development of acquired resistance to the EGFR inhibitors themselves, a common clinical problem with virtually all other currently used anticancer drugs, remains a largely unexplored subject of investigation. Here we summarize some of the possible mechanisms that can result in acquired resistance to EGFR-targeting drugs. Alternative combination therapies to circumvent and delay this problem are suggested. [Copyright &y& Elsevier]

Simple Summary: Advanced prostate cancer (PCa) is a prominent contributor to cancer-related fatalities and is associated with significant morbidity and mortality. Currently, addressing the local recurrence of the disease after radiation therapy (RT) poses a significant clinical challenge. We established the first syngeneic model of radiorecurrent PCa to evaluate the effectiveness of immune checkpoint inhibitors (ICIs) in combination with high-dose ionizing radiation (IR). We observed an enhanced anti-tumor response, which led to a delay in tumor growth and, in some cases, a complete elimination of tumors when combining IR with anti-CTLA4. This improvement was linked to an enhanced activation of total T cells, CD4+ helper T cells, and CD8+ cytotoxic T cells in both the draining lymph node and tumor. These results hold substantial potential for translation into clinical practice, serving as a proof of concept for the application of brachytherapy and immune checkpoint inhibitors (ICIs) in cases of recurrent disease. High-risk prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. Currently, the salvage of local disease recurrence after radiation therapy (RT) is a major clinical problem. Immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with ionizing radiation (IR) in certain advanced cancers. We generated the TRAMP-C2 HF radiorecurrent syngeneic mouse model to evaluate the therapeutic efficacy of ICIs in combination with RT. The administration of anti-PDL1 and/or anti-CTLA4 did not achieve a significant tumor growth delay compared to the control. The combination of IR and anti-PDL1 did not yield additional a growth delay compared to IR and the isotype control. Strikingly, a significant tumor growth delay and complete cure in one-third of the mice were seen with the combination of IR and anti-CTLA4. Immune cells in tumor-draining lymph nodes and tumor-infiltrating lymphocytes from mice treated with IR and anti-CTLA4 demonstrated an upregulation of genes in T-cell functions and enrichment in both CD4+ and CD8+ T-cell populations compared to mice given IR and the isotype control. Taken together, these results indicate enhancement of T-cell response in radiorecurrent PCa by IR and anti-CTLA4. [ABSTRACT FROM AUTHOR]

Summary: There is a large family of known proangiogenic growth factors, many of which can be expressed by a single tumor, especially in advanced stages of disease. Such redundancy, which can be amplified by hypoxia, has long been suspected as a potential cause of acquired resistance when tumors are treated with highly specific targeted antiangiogenic drugs. Definitive preclinical evidence for antiangiogenic drug evasion by alternate pathways of angiogenesis in tumor cells, likely induced by antiangiogenic drug-mediated increases in tumor hypoxia, is reported in this issue of Cancer Cell (); it has major implications for the development of strategies to prolong the effectiveness of antiangiogenic drugs as monotherapies, and for their use as chemosensitizing agents in combination treatment strategies. [Copyright &y& Elsevier]

Angiogenesis is regulated in large part by the balance of various proangiogenic stimulators, such as VEGF, and a diverse group of endogenous inhibitors of angiogenesis, most of which are extrinsic to endothelial cells. With respect to the latter, until recently, none have appeared to be induced as a consequence of a specific, self-regulating, feedback inhibition response. A new inhibitor, called vasohibin, has been uncovered. Vasohibin is selectively induced in endothelial cells by proangiogenic stimulatory growth factors such as VEGF; it appears to operate as an intrinsic and highly specific feed-back inhibitor of activated endothelial cells engaged in the process of angiogenesis (see the related article beginning on page 898). [ABSTRACT FROM AUTHOR]

Editorial. Evaluates cytokine or interleukin 12 as an antiangiogenetic drug. Toxic side effects mediated by anticancer chemotherapeutic drugs; Beneficial effects of normal cell and tissue destruction; Exploitation of the genetic instability of tumor cells.

Background: Triple negative breast cancer (TNBC) is an aggressive malignancy with poor prognosis, in part because of the current lack of any approved molecularly targeted therapy. We evaluated various combinations of three different drugs: nintedanib, an antiangiogenic TKI targeting VEGF receptors, paclitaxel (PTX), or a PD-L1 antibody, using models of orthotopic primary or advanced metastatic TNBC involving a metastatic variant of the MDA-MB-231 human cell line (called LM2–4) in SCID mice and two mouse lines (EMT-6 and a drug-resistant variant, EMT-6/CDDP) in immunocompetent mice. These drugs were selected based on the following: PTX is approved for TNBC; nintedanib combined with docetaxel has shown phase III clinical trial success, albeit in NSCLC; VEGF can act as local immunosuppressive factor; and PD-L1 antibody plus taxane therapy was recently reported to have encouraging phase III trial benefit in TNBC. Methods: Statistical analyses were performed with ANOVA followed by Tukey's Multiple Comparison Test or with Kruskal-Wallis test followed by Dunn's Multiple Comparison Test. Survival curves were analyzed using a Log-rank (Mantel Cox) test. Differences were considered statistically significant when p values were < 0.05. Results: Toxicity analyses showed that nintedanib is well tolerated when administered 5-days ON 2-days OFF; PTX toxicity differed in mice, varied with cell lines used and may have influenced median survival in the metastatic EMT6/CDDP model; while toxicity of PD-L1 therapy depended on the cell lines and treatment settings tested. In the LM2–4 system, combining nintedanib with PTX enhanced overall antitumor efficacy in both primary and metastatic treatment settings. In immunocompetent mice, combining nintedanib or PTX with the PD-L1 antibody improved overall antitumor efficacy. Using the advanced metastatic EMT-6/CDDP model, optimal efficacy results were obtained using the triple combination. Conclusions: These results suggest circumstances where nintedanib plus PTX may be potentially effective in treating TNBC, and nintedanib with PTX may improve PD-L1 therapy of metastatic TNBC. [ABSTRACT FROM AUTHOR]

The article discusses a study which examines the response of doxorubicin on tumour cells. It states that a mouse model was used to determine the effectiveness of the drug on infected mice. Findings reveal that tumour cells respond to the medicine, however, some cells survive and became detectable because only some genes are potential for chemoresistance including the cytokine IL-6 and the Timp-1 protein.

The article presents the authors' insights on cancer host-mediated changes in tumor growth and repopulation caused by reactions to antiangiogenic drugs in advanced cancer therapy. The authors mention two studies related to the topic such as one by P. G. Nikolinakos and colleagues and one by A. Lindauer and colleagues. Furthermore, both studies show that changes in vascular endothelial growth factors (VEGF) and tyrosine kinase inhibitors (TKI) in cytokines were caused by the cancer host.

The article discusses research done on the capability of mouse cancer models to predict phase three clinical trial results. It references a study by M. Singh et al, published within the issue. The failure rate of double-blind, placebo-controlled randomized study is evident in various therapeutic areas but mostly in oncology. Such failure has an impact on the cost of anti-cancer drugs. Due to the desire to upgrade the usefulness of animal models in such study, researchers have started to use genetically engineered mouse models (GEMMs) of spontaneous cancer. X-ray micro-computed tomography and high-resolution micro-ultrasound were used to assess tumor growth and response to treatment.

Robert S. Kerbel responds to a letter to the editor about his article "Tumor Angiogenesis" in the May 8, 2008 issue.

Discusses research which tested an inhibitor of angiogenesis called endostatin on three different mouse tumors. Research by Boehm et al in this issue; Limitations on effectiveness of cancer drugs; Acquired drug resistance; Instability of tumor cells; Basis of using angiogenesis to avoid acquired drug resistance; Development of angiostatin and endostatin; Possibility of new era for cancer treatment. INSET: Tumour growth and blood-vessel supply, by R. S. K..

Backgorund: Prior data suggest pre‐diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple‐negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes. Method: NOD/SCID mice were implanted with HER2+ MDA‐MB‐231/LN/2‐4/H2N, trastuzumab‐resistant HER2+ HCC1954 or a TNBC patient‐derived xenograft (PDX). A daily low‐dose aspirin regimen commenced until primary tumours reached ~250 mm3 and subsequently resected. MDA‐MB‐231/LN/2‐4/H2N mice were monitored for metastasis utilising imaging. To interrogate the survival benefit of pre‐treatment aspirin, 3 weeks post‐resection, HCC1954/TNBC animals received standard‐of‐care (SOC) chemotherapy for 6 weeks. Primary tumour response to aspirin was interrogated using immunohistochemistry. Results: Aspirin delayed time to metastasis in MDA‐MB‐231/LN/2‐4/H2N xenografts and decreased growth of HER2+/TNBC primary tumours. Lymphangiogenic factors and lymph vessels number were decreased in HER2+ tumours. However, no survival benefit was seen in aspirin pre‐treated animals (HCC1954/TNBC) that further received adjuvant SOC, compared with animals treated with SOC alone. In an effort to study mechanisms responsible for the observed reduction in lymphangiogenesis in HER2+ BC we utilised an in vitro co‐culture system of HCC1954 tumour cells and mesenchymal stromal cells (MSC). Aspirin abrogated the secretion of VEGF‐C in MSCs and also decreased the lymph/angiogenic potential of the MSCs and HCC1954 by tubule formation assay. Furthermore, aspirin decreased the secretion of uPA in HCC1954 cells potentially diminishing its metastatic capability. Conclusion: Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo‐preventative agent in the HER2+ or TNBC setting. [ABSTRACT FROM AUTHOR]

Activated EGF receptor (EGFR) plays an oncogenic role in several human malignancies. Although the intracellular effects of EGFR are well studied, its ability to induce and modulate tumor angiogenesis is less understood. We found previously that oncogenic EGFR can be shed from cancer cells as cargo of membrane microvesicles (MVs), which can interact with surfaces of other cells. Here we report that MVs produced by human cancer cells harboring activated EGFR (A431, A549, DLD-1) can be taken up by cultured endothelial cells, in which they elicit EGFR-dependent responses, including activation of MAPK and Akt pathways. These responses can be blocked by annexin V and its homodimer, Diannexin, both of which cloak phosphatidylserine residues on the surfaces of MVs. Interestingly, the intercellular EGFR transfer is also accompanied by the onset of VEGF expression in endothelial cells and by autocrine activation of its key signaling receptor (VEGF receptor-2). In A431 human tumor xenografts in mice, angiogenicendothelial cells stain positively for human EGFR and phospho-EGFR, while treatment with Diannexin leads to a reduction of tumor growth rate and microvascular density. Thus, we propose that oncogene-containing tumor cell-derived MVs could act as a unique form of angiogenesis-modulating stimuli and are capable of switching endothelial cells to act in an autocrine mode. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to an article by Nikos Werner and colleagues about endothelial progenitor cells in the September 8, 2005 issue.

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 μm and 65 μm and axial resolutions of 66 μm to 72 μm, 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 μm to 100 μm in diameter. Experiments conducted on mouse tumors successfully imaged microvessel morphology in the tumor microcirculation. (E-mail: goetzda@sten.sunnybrook.utoronto.ca) [Copyright &y& Elsevier]

While targeting VEGF has shown success against a number of human cancers, drug resistance has resulted in compromised clinical benefits. In this issue of Cancer Cell, report that tumors resistant to anti-VEGF therapy stimulate tumor-associated fibroblasts to express proangiogenic PDGF-C, implicating it as a potential therapeutic target. [Copyright &y& Elsevier]

Metronomic chemotherapy using the 5-FU prodrug uracil-tegafur (UFT) and cyclophosphamide (CTX) was previously shown to only modestly delay primary tumor growth, but nevertheless markedly suppressed the development of micro-metastasis in an orthotopic breast cancer xenograft model, using the metastatic variant of the MDA-MB-231 cell line, 231/LM2-4. Furthermore, a remarkable prolongation of survival, with no toxicity, was observed in a model of postsurgical advanced metastatic disease. A question that has remained unanswered is the seemingly selective anti-metastatic mechanisms of action responsible for this treatment. We assessed the in vivo effect of metronomic UFT, CTX or their combination, on vascular density, collagen deposition and c-Met (cell mediators or modulators of tumor cell invasion or dissemination) via histochemistry/immunohistochemistry of primary tumor sections. We also assessed the effect of continuous exposure to low and non-toxic doses of active drug metabolites 5-fluorouracil (5-FU), 4-hydroperoxycyclophosphamide (4-HC) or their combination, on 231/LM2-4 cell invasiveness in vitro. In the in vivo studies, a significant reduction in vascular density and p-Met[Y1003] levels was associated with UFT+CTX treatment. All treatments reduced intratumoral collagen deposition. In the in vitro studies, a significant reduction of collagen IV invasion by all treatments was observed. The 3D structures formed by 231/LM2-4 on Matrigel showed a predominantly Mass phenotype under treated conditions and Stellate phenotype in untreated cultures. Taken together, the results suggest the low-dose metronomic chemotherapy regimens tested can suppress several mediators of tumor invasiveness highlighting a new perspective for the anti-metastatic efficacy of metronomic chemotherapy. [ABSTRACT FROM AUTHOR]

Background: There are phase 3 clinical trials underway evaluating anti-PD-L1 antibodies as adjuvant (postoperative) monotherapies for resectable renal cell carcinoma (RCC) and triple-negative breast cancer (TNBC); in combination with antiangiogenic VEGF/VEGFR2 inhibitors (e.g., bevacizumab and sunitinib) for metastatic RCC; and in combination with chemotherapeutics as neoadjuvant (preoperative) therapies for resectable TNBC.Methods: This study investigated these and similar clinically relevant drug combinations in highly translational preclinical models of micro- and macro-metastatic disease that spontaneously develop after surgical resection of primary kidney or breast tumours derived from orthotopic implantation of murine cancer cell lines (RENCAluc or EMT-6/CDDP, respectively).Results: In the RENCAluc model, adjuvant sunitinib plus anti-PD-L1 improved overall survival compared to either drug alone, while the same combination was ineffective as early therapy for unresected primary tumours or late-stage therapy for advanced metastatic disease. In the EMT-6/CDDP model, anti-PD-L1 was highly effective as an adjuvant monotherapy, while its combination with paclitaxel chemotherapy (with or without anti-VEGF) was most effective as a neoadjuvant therapy.Conclusions: Our preclinical data suggest that anti-PD-L1 plus sunitinib may warrant further investigation as an adjuvant therapy for RCC, while anti-PD-L1 may be improved by combining with chemotherapy in the neoadjuvant but not the adjuvant setting of treating breast cancer. [ABSTRACT FROM AUTHOR]

Background: The anti-angiogenic Sorafenib is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance limits its efficacy. An emerging theory to explain intrinsic resistance to other anti-angiogenic drugs is 'vessel co-option,' ie, the ability of tumors to hijack the existing vasculature in organs such as the lungs or liver, thus limiting the need for sprouting angiogenesis. Vessel co-option has not been evaluated as a potential mechanism for acquired resistance to anti-angiogenic agents.Methods: To study sorafenib resistance mechanisms, we used an orthotopic human HCC model (n = 4-11 per group), where tumor cells are tagged with a secreted protein biomarker to monitor disease burden and response to therapy. Histopathology, vessel perfusion assessed by contrast-enhanced ultrasound, and miRNA sequencing and quantitative real-time polymerase chain reaction were used to monitor changes in tumor biology.Results: While sorafenib initially inhibited angiogenesis and stabilized tumor growth, no angiogenic 'rebound' effect was observed during development of resistance unless therapy was stopped. Instead, resistant tumors became more locally infiltrative, which facilitated extensive incorporation of liver parenchyma and the co-option of liver-associated vessels. Up to 75% (±10.9%) of total vessels were provided by vessel co-option in resistant tumors relative to 23.3% (±10.3%) in untreated controls. miRNA sequencing implicated pro-invasive signaling and epithelial-to-mesenchymal-like transition during resistance development while functional imaging further supported a shift from angiogenesis to vessel co-option.Conclusions: This is the first documentation of vessel co-option as a mechanism of acquired resistance to anti-angiogenic therapy and could have important implications including the potential therapeutic benefits of targeting vessel co-option in conjunction with vascular endothelial growth factor receptor signaling. [ABSTRACT FROM AUTHOR]

Several approaches are being evaluated to improve the historically limited value of studying transplanted primary tumors derived by injection of cells from established cell lines for predicting subsequent cancer therapy outcomes in patients and clinical trials. These approaches include use of genetically engineered mouse models (GEMMs) of spontaneous tumors, or patient tumor tissue derived xenografts (PDXs). Almost all such therapy studies utilizing such models involve treatment of established primary tumors. An alternative approach we have developed involves transplanted human tumor xenografts derived from established cell lines to treat mice with overt visceral metastases after primary tumor resection. The rationale is to mimic the more challenging circumstance of treating patients with late stage metastatic disease. These metastatic models entail prior in vivo selection of heritable, phenotypically stable variants with increased aggressiveness for spontaneous metastasis; they were derived by orthotopic injection of tumor cells followed by primary tumor resection and serial selection of distant spontaneous metastases, from which variant cell lines having a more aggressive heritable metastatic phenotype were established. We attempted to adopt this strategy for breast cancer PDXs. We studied five breast cancer PDXs, with the emphasis on two, called HCI-001 and HCI-002, both derived from triple negative breast cancer patients. However significant technical obstacles were encountered. These include the inherent slow growth rates of PDXs, the rarity of overt spontaneous metastases (detected in only 3 of 144 mice), very high rates of tumor regrowths at the primary tumor resection site, the failure of the few human PDX metastases isolated to manifest a more aggressive metastatic phenotype upon re-transplantation into new hosts, and the formation of metastases which were derived from de novo mouse thymomas arising in aged SCID mice that we used for the experiments. We discuss several possible strategies that may be employed to overcome these limitations. Uncovering the basis of the failure to detect a high rate of overt spontaneous distant metastases having a heritable phenotype in PDX models may reveal new insights into the biology and treatment of advanced metastatic disease. [ABSTRACT FROM AUTHOR]

Acquired evasive resistance is a major limitation of hepatocellular carcinoma (HCC) treatment with the tyrosine kinase inhibitor (TKI) sorafenib. Recent findings suggest that resistance to sorafenib may have a reversible phenotype. In addition, loss of responsiveness has been proposed to be due to a gradual decrease in sorafenib plasma levels in patients. Here, the possible mechanisms underlying reversible sorafenib resistance were investigated using a Hep3B-hCG orthotopic human xenograft model of locally advanced HCC. Tissue and plasma sorafenib and metabolite levels, downstream antitumor targets, and toxicity were assessed during standard and dose-escalated sorafenib treatment. Drug levels were found to decline significantly over time in mice treated with 30 mg/kg sorafenib, coinciding with the onset of resistance but a greater magnitude of change was observed in tissues compared with plasma. Skin rash also correlated with drug levels and tended to decrease in severity over time. Drug level changes appeared to be partially tumor dependent involving induction of tumoral CYP3A4 metabolism, with host pretreatment alone unable to generate resistance. Escalation from 30 to 60 mg/kg sorafenib improved antitumor efficacy but worsened survival due to excessive body weight loss. Microvessel density was inhibited by sorafenib treatment but remained suppressed over time and dose increase. In conclusion, tumor CYP3A4 induction by sorafenib is a novel mechanism to account for variability in systemic drug levels; however, declining systemic sorafenib levels may only be a minor resistance mechanism. Escalating the dose may be an effective treatment strategy, provided toxicity can be controlled. [ABSTRACT FROM AUTHOR]

We compare for the first time, the metastatic aggressiveness of the parental MDA-MB-231 breast cancer cell line and two luciferase-tagged in vivo-derived and selected pro-metastatic variants (LM2-4/luc+ and 164/8-1B/luc+) in SCID, NOD-SCID and NOD-SCID-IL-2Rγnull (NSG) mice following orthotopic implantation and primary tumour resection. The variants are known to be more aggressively metastatic in SCID mice, compared to the parental line which has limited spontaneous metastatic competence in these mice. When 2×106 cells were injected into the mammary fat pad, the growth of the resultant primary tumours was identical for the various cell lines in the three strains of mice. However, metastatic spread of all three cell lines, including the MDA-MB-231 parental cell line, was strikingly more aggressive in the highly immunocompromised NSG mice compared to both NOD-SCID and SCID mice, resulting in extensive multi-organ metastases and a significant reduction in overall survival. While these studies were facilitated by monitoring post-surgical spontaneous metastases using whole body bioluminescence imaging, we observed that the luciferase-tagged parental line showed altered growth and diminished metastatic properties compared to its untagged counterpart. Our results are the first to show that host immunity can have a profound impact on the spread of spontaneous visceral metastases and survival following resection of a primary tumour in circumstances where the growth of primary tumours is not similarly affected; as such they highlight the importance of immunity in the metastatic process, and by extension, suggest certain therapeutic strategies that may have a significant impact on reducing metastasis. [ABSTRACT FROM AUTHOR]

The failure rate of randomized phase III oncology clinical trials is extremely high, even when preceded by encouraging preclinical studies and phase II trial results of the same therapy. Thus, there is considerable effort being made to improve the predictive clinical potential of preclinical models, in addition to improving phase II trial design.With respect to the former, preclinicalmodels have historically relied heavily on treatment of primary spontaneous or transplanted tumors rather than themore common and therapeutically challenging clinical trial circumstance of advancedmetastatic disease.Here,we showthat the oral antiangiogenic tyrosine kinase inhibitor (TKI), sunitinib, which failed to meet primary or secondary survival endpoints in 4 separate phase III metastatic breast cancer (MBC) trials, either alone or with chemotherapy, similarly failed to show monotherapy or combination chemotherapy efficacy in a model of postsurgical advanced MBC using a metastatic variant of the MDA-MB-231 triple-negative human breast cancer. In contrast, the drug was effective when used to treat established orthotopic primary tumors. Similar results were obtained with pazopanib monotherapy, another antiangiogenic oral TKI.However, when an antibody targeting the VEGF pathway (DC101)was tested, it showed a trend inmodestly improving the efficacy of paclitaxel therapy, thus resembling to a degree prior phase III clinical results of bevacizumab plus paclitaxel in MBC. Our results suggest the potential value of treating postsurgical advanced metastatic disease as a possible strategy to improve preclinical models for predicting outcomes in patients with metastatic disease. [ABSTRACT FROM AUTHOR]

Objective: Advanced and recurrent diseases are the major causes of death in colon cancer. No standard preclinical model addresses advanced disease and spontaneous metastasis after orthotopic tumour growth. In this study, the authors report the establishment of such standardised orthotopic mouse models of colon cancer and their use in evaluating metronomic topotecan alone or in combination with standard chemotherapy. Design: Human colon cancer cell lines, transfected with human chorionic gonadotropin and luciferase, were injected orthotopically into the caecal wall of severe combined immunodeficient mice, intrasplenically or subcutaneously. For adjuvant therapy, caecal resections were performed 3e5 weeks after tumour cell injection. Chemotherapy drugs tested included uracil/tegafur, folinic acid, oxaliplatin, topotecan, pazopanib and various combinations. Results: Subcutaneous tumours showed exaggerated sensitivity to treatment by delayed tumour growth (p=0.002) and increased survival (p=0.0064), but no metastatic spread. Intrasplenic cell injection resulted in rapid and extensive but artefactual metastasis without treatment effect. Intracaecal cell injection with tumour take rates of 87.5-100% showed spontaneous metastases at clinically relevant rates. Metronomic topotecan significantly polonged survival and reduced metastasis. In the adjuvant setting, metronomic maintenance therapy (after FOLFOX-like induction) prolonged survival compared with vehicle controls (p=0.0003), control followed by topotecan (p=0.0161) or FOLFOX-like therapy (p=0.0003). Conclusion: The refined orthotopic implantation technique proved to be a clinically relevant model for metastasis and therapy studies. Furthermore, metronomic therapy with oral topotecan may be promising to consider for clinical trials of metastatic colon cancer and long-term adjuvant maintenance therapy of colon cancer. [ABSTRACT FROM AUTHOR]

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases. [ABSTRACT FROM AUTHOR]

Tumor-associated macrophages promote tumor growth by stimulating angiogenesis and suppressing antitumor immunity. Thus, therapeutics that inhibit macrophage recruitment to tumors may provide new avenues for cancer therapy. In this study, we showed how chemoattractants stromal cell-derived growth factor 1 alpha (SDF-1α) n di interleukin 1 beta; (IL - 1β) collaborate with myeloid cell integrin-α4β1 to promote tumor inflammation and growth. We found that SDF-1α and IL-1β are highly expressed in the microenvironments of murine lung, pancreatic, and breast tumors; surprisingly, SDF-1α was expressed only by tumor cells, whereas IL-1β was produced only by tumor-derived granulocytes and macrophages. In vivo, both factors directly recruited proangiogenic macrophages to tissues, whereas antagonists of both factors suppressed tumor inflammation, angiogenesis, and growth. Signals induced by IL-1β and SDF-1α promoted the interaction of talin and paxillin with the cytoplasmic tails of integrin-α4β1, thereby stimulating myeloid cell adhesion to endothelium in vitro and in vivo. Inhibition of integrin-α4β1, SDF-1α, or IL-1β was sufficient to block tumor inflammation and growth, and the combined blockade of these molecules greatly accentuated these effects. Furthermore, antagonists of integrin-α4β1 inhibited chemotherapy-induced tumor inflammation and acted synergistically with chemotherapeutic agents to suppress tumor inflammation and growth. These results show that targeting myeloid cell recruitment mechanisms can be an effective approach to suppress tumor progression. [ABSTRACT FROM AUTHOR]

Recombinant granulocyte colony-stimulating factor (G-CSF) is used to accelerate recovery from chemotherapy-induced myelosuppression. G-CSF has been recently shown to stimulate angiogenesis mediated by several types of bone marrow-derived cell populations. To investigate whether G-CSF may alter tumor response to therapy, we studied Lewis lung and EMT/6 breast carcinomas in mice treated with paclitaxel (PTX) chemotherapy in combination with G-CSF. We compared the results obtained to mice treated with PTX and AMD3100, a small-molecule drug antagonist of CXCR4 which, like G-CSF, can be used to mobilize hematopoietic cells. We show that PTX combined with G-CSF treatment facilitates revascularization, leading to an improvement in blood perfusion in LLC tumors, and a decrease in hypoxia in EMT/6 tumors, thus enhancing tumor growth in comparison to PTX or PTX and AMD3100 therapies. We found that hemangiocytes but not Gr-1+ CD11b+ cells colonize EMT/6 tumors after treatment with PTX and G-CSF, but not PTX and AMD3100, and therefore may contribute to angiogenesis. However, increases in hemangiocyte colonization were not observed in LLC PTX and G-CSF-treated tumors, suggesting distinct mechanisms of tumor revascularization after G-CSF. Overall, our observations suggest that despite its known considerable clinical benefits, G-CSF might contribute to tumor revascularization by various mechanisms, and diminish the antitumor activity of chemotherapy, an effect that can be prevented by AMD3100. [ABSTRACT FROM AUTHOR]

Summary: To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression. [Copyright &y& Elsevier]

BACKGROUND: Metronomic chemotherapy involves frequent, regular administration of cytotoxic drugs at non-toxic doses, usually without prolonged breaks. We investigated the therapeutic efficacies of metronomic S-1, an oral 5-fluorouracil prodrug, and vandetanib, an epidermal growth factor receptor and vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, in models of hepatocellular carcinoma (HCC). METHODS: We compared anti-HCC effects and toxicity in the six treatment groups: control (untreated), maximum tolerated dose (MTD) S-1, metronomic S-1, vandetanib, MTD S-1 with vandetanib, and metronomic S-1 with vandetanib. Tumor microvessel density (MVD) and tumor apoptosis were evaluated by immunohistochemistry. The expression of VEGF and thrombospondin-1, an endogenous inhibitor of angiogenesis, was analyzed by Western blot. RESULTS: Metronomic S-1 significantly inhibited tumor growth, which was enhanced by combination with vandetanib. With respect to toxicities, MTD S-1 caused severe body weight loss and myelosuppression, whereas metronomic S-1 did not cause any overt toxicities. Moreover, metronomic S-1 or metronomic S-1 with vandetanib prolonged survival, the latter treatment providing the greatest benefit. Metronomic S-1 and metronomic S-1 with vandetanib decreased MVDs and increased apoptosis in tumor tissues. The expression of VEGF in tumor tissues was upregulated by vandetanib and metronomic S-1 with vandetanib, whereas the expression of thrombospondin-1 was upregulated by metronomic S-1 and metronomic S-1 with vandetanib. CONCLUSION: Metronomic S-1 with an antiangiogenic agent seems to be an effective and safe therapeutic strategy for HCC. [ABSTRACT FROM AUTHOR]

An enduring problem in cancer research is the failure to reproduce highly encouraging preclinical therapeutic findings using transplanted or spontaneous primary tumours in mice in clinical trials of patients with advanced metastatic disease. There are several reasons for this, including the failure to model established, visceral metastatic disease. We therefore developed various models of aggressive multi-organ spontaneous metastasis after surgical resection of orthotopically transplanted human tumour xenografts. In this Opinion article we provide a personal perspective summarizing the prospect of their increased clinical relevance. This includes the reduced efficacy of certain targeted anticancer drugs, the late emergence of spontaneous brain metastases and the clinical trial results evaluating a highly effective therapeutic strategy previously tested using such models. [ABSTRACT FROM AUTHOR]

Low-dose metronomic (LDM) chemotherapy is emerging as an alternative or supplemental dosing strategy to conventional maximum tolerated dose (MTD) chemotherapy. It is characterized primarily, but not exclusively, by anti-angiogenic mechanisms of action and the absence of high-grade adverse effects commonly seen with MTD chemotherapy. However, similar to other anticancer therapies, inherent resistance to LDM chemotherapy is common. Moreover, even tumors that initially respond to metronomic regimens eventually develop resistance through mechanisms that are as yet unknown. Thus, we have developed in vivo models of PC-3 human prostate cancer cells resistant to extended LDM cyclophosphamide therapy. Such PC-3 variants show stable resistance to LDM cyclophosphamide in vivo yet retain in vitro sensitivity to 4-hydroperoxy-cyclophosphamide (precursor of the active cyclophosphamide metabolite 4-hydroxy-cyclophosphamide) and other chemotherapeutic agents, namely, docetaxel and doxorubicin. Moreover, LDM cyclophosphamide-resistant PC-3 variants remain sensitive to MTD cyclophosphamide therapy in vivo. Conversely, PC-3 variants made resistant in vivo to MTD cyclophosphamide show varying levels of resistance to metronomic cyclophosphamide when grown in mice. These results and additional studies of variants of the breast cancer cell line MDA-MB-231 suggest that resistance to LDM cyclophosphamide is a distinct phenomenon from resistance to MTD cyclophosphamide and that LDM cyclophosphamide administration does not select for MTD chemotherapy resistance. As such, our findings have various implications for the clinical use of metronomic chemotherapy. [ABSTRACT FROM AUTHOR]

Acquired resistance to antiangiogenic drugs, such as sorafenib, is amajor clinical problem. We studied development of a resistance to sorafenib in new preclinical models of human hepatocellular carcinoma (HCC) along with a strategy to delay such resistance--combination with metronomic chemotherapy. Three different xenograft models were studied using human Hep3B HCC cells, which are highly responsive to sorafenib, namely, orthotopic and subcutaneous transplant in severe combined immunodeficientmice, and an orthotopic transplant in nudemice. The complementary DNA for the β-subunit of human choriogonadotropin was transfected into HCC cells, and urine levels of the protein were monitored as a surrogate of tumor burden. Extended daily treatments, sometimes interrupted by a break period of 3 to 7 days to allow recovery from toxicity at sorafenib doses of 30 to 60 mg/kg, were maintained until and after evidence of tumor relapse. Initially responsive tumors seemed to develop a resistance-like phenotype after long-term daily treatment (e.g., >42 days) at doses of 30 to 60 mg/kg. Transplantation of cell lines established from progressing tumors into new hosts showed that the resistant phenotype was not propagated. Furthermore, a regimen of daily metronomic uracil + tegafur (UFT, an oral 5-fluorouracil prodrug) chemotherapy with a less toxic regimen of sorafenib (15 mg/kg per day) significantly delayed the onset of resistance (>91 days). In conclusion, development of a resistance-like phenotype to sorafenib is reversible, and metronomic UFT plus sorafenib may be a promising and well-tolerated treatment for increasing efficacy by delaying emergence of such resistance. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is an intrinsically chemotherapy refractory malignancy. Development of effective therapeutic regimens would be facilitated by improved preclinical HCC models. Currently, most models consist of subcutaneous human tumor transplants in immunodeficientmice; however, these do not reproduce the extensive liver disease associated with HCC or metastasize. To address this deficiency, we developed an orthotopic model. Human HCC cells were transfectedwith the gene encoding secretable β-subunit human choriogonadotropin (β-hCG),whichwas used as a surrogate marker of tumor burden. The HCC cells were implanted into the left liver lobe of severe combined immunodeficient (SCID) mice, after which the efficacy of different therapies was evaluated on established, but liver-confined human Hep3B cell line HCC. Treatments included sorafenib or metronomic chemotherapy using cyclophosphamide (CTX), UFT, an oral 5-fluorouracil prodrug, or doxorubicin either alone or in various combinations, with or without an antiangiogenic agent, DC101, an anti--vascular endothelial growth factor receptor-2 antibody. Sorafenib inhibited tumor growth in a dose-dependent manner but caused severe weight loss in SCID mice, thus necessitating use of DC101 in subsequent experiments. Although less toxicitywas observed using either single or doubletmetronomic chemotherapy without any added antiangiogenic agent, none, provided survival benefit. In contrast, significantly improved overall survivalwas observed using various combinations ofmetronomic chemotherapy regimens such as UFT+CTXwithDC101. In conclusion, using this model of liver-confined but advanced HCC suggests that the efficacy of a targeted antiangiogenic drug or metronomic chemotherapy can be mutually enhanced by concurrent combination treatment. [ABSTRACT FROM AUTHOR]

The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithehal cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGFβ, plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGFβ-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithehium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA 1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis. [ABSTRACT FROM AUTHOR]

Abstract: Circulating endothelial cells (CECs) and circulating endothelial progenitors (CEPs) are currently being investigated in a variety of diseases as markers of vascular turnover or damage and, also in the case of CEPs, vasculogenesis. CEPs appear to have a “catalytic” role in different steps of cancer progression and recurrence after therapy, and there are preclinical and clinical data suggesting that CEC enumeration might be useful to select and stratify patients who are candidates for anti-angiogenic treatments. In some types of cancer, CECs and CEPs might be one of the possible hidden identities of cancer stem cells. The definition of CEC and CEP phenotype and the standardization of CEC and CEP enumeration strategies are highly desirable goals in order to exploit these cells as reliable biomarkers in oncology clinical trials. [Copyright &y& Elsevier]

Summary: Herein we report that the VEGFR/PDGFR kinase inhibitor sunitinib/SU11248 can accelerate metastatic tumor growth and decrease overall survival in mice receiving short-term therapy in various metastasis assays, including after intravenous injection of tumor cells or after removal of primary orthotopically grown tumors. Acceleration of metastasis was also observed in mice receiving sunitinib prior to intravenous implantation of tumor cells, suggesting possible “metastatic conditioning” in multiple organs. Similar findings with additional VEGF receptor tyrosine kinase inhibitors implicate a class-specific effect for such agents. Importantly, these observations of metastatic acceleration were in contrast to the demonstrable antitumor benefits obtained when the same human breast cancer cells, as well as mouse or human melanoma cells, were grown orthotopically as primary tumors and subjected to identical sunitinib treatments. [Copyright &y& Elsevier]

Summary: Several hypotheses have been proposed to explain how antiangiogenic drugs enhance the treatment efficacy of cytotoxic chemotherapy, including impairing the ability of chemotherapy-responsive tumors to regrow after therapy. With respect to the latter, we show that certain chemotherapy drugs, e.g., paclitaxel, can rapidly induce proangiogenic bone marrow-derived circulating endothelial progenitor (CEP) mobilization and subsequent tumor homing, whereas others, e.g., gemcitabine, do not. Acute CEP mobilization was mediated, at least in part, by systemic induction of SDF-1α and could be prevented by various procedures such as treatment with anti-VEGFR2 blocking antibodies or paclitaxel treatment in CEP-deficient Id mutant mice, both of which resulted in enhanced antitumor effects mediated by paclitaxel, but not by gemcitabine. [Copyright &y& Elsevier]

We determined whether phosphorylated epidermal growth factor receptor (EGFR) expressed on tumor-associated endothelial cells is a primary target for therapy with EGFR tyrosine kinase inhibitors (TKIs). Human colon cancer cells SW620CE2 (parental) that do not express EGFR or human epidermal growth factor receptor 2 (HER2) but express transforming growth factor α (TGF-α) were transduced with a lentivirus carrying nontargeting small hairpin RNA (shRNA) or TGF-α shRNA. The cell lines were implanted into the cecum of nude mice. Two weeks later, treatment began with saline, 4-[R]-phenethylamino-6-[hydroxyl] phenyl-7H-pyrrolo [2,3-D] -pyrimidine (PKI166), or irinotecan. Endothelial cells in parental and nontargeting shRNA tumors expressed phosphorylated EGFR. Therapy with PKI166 alone or with irinotecan produced apoptosis of these endothelial cells and necrosis of the EGFR-negative tumors. Endothelial cells in tumors that did not express TGF-α did not express EGFR, and these tumors were resistant to treatment with PKI166. The response of neoplasms to EGFR antagonists has been correlated with EGFR mutations, HER2 expression, Akt activation, and EGFR gene copy number. Our present data using colon cancer cells that do not express EGFR or HER2 suggest that the expression of TGF-α by tumor cells leading to the activation of EGFR in tumor-associated endothelial cells is a major determinant for the susceptibility of neoplasms to therapy by specific EGFR-TKI. [ABSTRACT FROM AUTHOR]

Cancer patients treated with antiangiogenic multitargeted receptor tyrosine kinase (RTK) inhibitors show increased levels of plasma VEGF and placental growth factor and decreased levels of soluble VEGF receptor-2, thus implicating these overall changes as a possible class effect of such drugs and raising the possibility of their exploitation as surrogate biomarkers for pharmacodynamic drug activity/exposure and patient benefit. A postulated mechanism for these changes is that they are tumor-dependent, resulting from drug-induced decreases in vascular function, increases in tumor hypoxia, and changes in hypoxia-regulated genes. However, here we report that an identical pattern of change is observed in normal nontumor-bearing mice treated with SU11248/sunitinib, a small-molecule inhibitor of VEGF and PDGF RTKs. The changes were dose-dependent, plateaued after 4 days of consecutive treatment, reversed after discontinuation of therapy, and correlated with antitumor activity. Altered protein expression was found in a broad variety of tissues, and dose-depen- dent elevations were observed of several plasma proteins previously unassociated with this class of inhibitor, including G-CSF, SDF-1α, SCF, and osteopontin. Our results suggest that observed sunitinib-induced molecular plasma changes, including those both directly and indirectly targeted by drug, represent a systemic tumor-independent response to therapy and may correlate with the most efficacious antitumor doses, potentially having utility for defining the optimal biologic dose range for this drug class but not as predictive markers of tumor response or clinical benefit. They may also be relevant to drug-associated toxicities, drug resistance, and observed rapid tumor (re)growth seen after cessation of therapy. [ABSTRACT FROM AUTHOR]