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Anti‐angiogenic drugs targeting the VEGF pathway are most effective in advanced metastatic disease settings of certain types of cancers, whereas they have been unsuccessful as adjuvant therapies of micrometastatic disease in numerous phase III trials involving early‐stage (resectable) cancers. Newer investigational anti‐angiogenic drugs have been designed to inhibit the Angiopoietin (Ang)‐Tie pathway. Acting through Tie2 receptors, the Ang1 ligand is a gatekeeper of endothelial quiescence. Ang2 is a dynamically expressed pro‐angiogenic destabilizer of endothelium, and its upregulation is associated with poor prognosis in cancer. Besides using Ang2 blockers as inhibitors of tumor angiogenesis, little attention has been paid to their use as stabilizers of blood vessels to suppress tumor cell extravasation and metastasis. In clinical trials, Ang2 blockers have shown limited efficacy in advanced metastatic disease settings. This review summarizes preclinical evidence suggesting the potential utility of Ang2 inhibitors or Tie2 activators as neoadjuvant or adjuvant therapies in the prevention or treatment of early‐stage micrometastatic disease. We further discuss the rationale and potential of combining these strategies with immunotherapy, including immune checkpoint targeting antibodies. [ABSTRACT FROM AUTHOR]

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]

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]

Angiopoietin-1 (Ang1) activation of Tie2 receptors on endothelial cells ( ECs) reduces adhesion by tumor cells ( TCs) and limits junctional permeability to TC diapedesis. We hypothesized that systemic therapy with Vasculotide ( VT)-a purported Ang1 mimetic, Tie2 agonist-can reduce the extravasation of potentially metastatic circulating TCs by similarly stabilizing the host vasculature. In vitro, VT and Ang1 treatments impeded endothelial hypermeability and the transendothelial migration of MDA- MB-231∙ LM2-4 (breast), HT29 (colon), or SN12 (renal) cancer cells to varying degrees. In mice, VT treatment inhibited the transit of TCs through the pulmonary endothelium, but not the hepatic or lymphatic endothelium. In the in vivo LM2-4 model, VT monotherapy had no effect on primary tumors, but significantly delayed distant metastatic dissemination to the lungs. In the post-surgical adjuvant treatment setting, VT therapeutically complemented sunitinib therapy, an anti-angiogenic tyrosine kinase inhibitor which limited the local growth of residual disease. Unexpectedly, detailed investigations into the putative mechanism of action of VT revealed no evidence of Tie2 agonism or Tie2 binding; alternative mechanisms have yet to be determined. [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]

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]

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]

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]

Summary: We have been studying the molecular and cellular basis of chronic low-dose, frequently administered, metronomic chemotherapy regimens for the treatment of cancer in a variety of preclinical models, including human breast cancer xenografts. The advantages of metronomic–maintenance-type chemotherapy regimens include significantly reduced host toxicity, potentially reduced costs, increased convenience for patients when oral chemotherapy drugs are used, and the possibility of adopting chronic combination therapies involving conventional chemotherapy drugs and cytostatic molecularly targeted therapies. However, a disadvantage is the empiricism associated with determining the optimal biologic dose (OBD). Recently, we have developed a surrogate biomarker approach involving measurement of circulating endothelial progenitor cells (CEPs) in peripheral blood to help determine the OBD of anti-angiogenic drugs or treatments, including metronomic chemotherapy. Using this approach we determined the OBD for different metronomic chemotherapy regimens and then tested the effect of such drugs for the treatment of established, advanced (high volume) and widespread human breast cancer metastases in immunodeficient mice. This treatment strategy, which was maintained for over 6 months, with no breaks, resulted in marked prolongation of survival and was devoid of overt toxicity. These results suggest the possibility of using metronomic chemotherapy regimens as an adjuvant therapy for early-stage disease, including breast cancer, as was demonstrated recently using long-term daily low-dose UFT for the treatment of early-stage resected non-small cell lung cancer or UFT in combination for early stage breast cancer combined with tamoxifen. [Copyright &y& Elsevier]

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]

Abstract: Historically, preclinical tumor therapy models in mice have frequently been deficient in predicting subsequent clinical activity; over-prediction of clinical anti-tumor efficacy is common. Several approaches are being made in an attempt to improve the clinical relevance of preclinical models, and include the use of genetically engineered mouse models (GEMMs) of cancer or patient derived xenografts (PDXs). Here we summarize, in the context of breast cancer, another approach, namely, the development of postsurgical models of either macroscopic or microscopic metastatic disease to mimic metastatic or adjuvant therapy. To do so we used in vivo selected metastatic variants of established human breast cancer cell lines such as MDA-MB-231. Testing antiangiogenic drugs such as the oral tyrosine kinase inhibitor (TKI) sunitinib alone or combined with chemotherapy in models involving treatment of established primary tumors invariably resulted in demonstrable anti-tumor activity. In contrast, identical treatments of postsurgical mice with advanced metastatic disease did not: survival times were not prolonged. This reflects multiple failed phase III trials of sunitinib based therapies in metastatic breast cancer patients. However, using a VEGF pathway targeting antibody drug instead of a TKI, with (paclitaxel) chemotherapy, resulted in anti-tumor activity in the metastatic setting, partially reflecting prior clinical results of the E2100 phase III trial of weekly paclitaxel plus bevacizumab. Other experiments involving postsurgical adjuvant treatment of early stage disease foreshadowed the phase III clinical trial failures of adjuvant bevacizumab in colorectal or breast cancer. In contrast, some investigational metronomic oral chemotherapy protocols alone or in combination with an antiangiogenic drug demonstrated potent activity in the advanced metastatic setting; these encouraging results have yet to be validated in randomized phase III clinical trials, which are underway based on some encouraging phase II clinical trial results. We have also observed circumstances where mice with advanced systemic disease, when successfully treated so as to prolong survival, sometimes relapse with brain metastases, reflecting a similar clinical phenomenon. Given our overall findings, we suggest that using preclinical mouse tumor models which mimic postsurgical adjuvant or metastatic therapy may be a promising strategy to help improve the ability to predict subsequent clinical outcomes. [Copyright &y& Elsevier]

Summary: Phase III trials of antiangiogenic drugs for metastatic breast cancer have either had only limited success, e.g. the monoclonal anti-VEGF antibody bevacizumab when used with various conventional chemotherapy regimens, or have failed altogether, e.g. the small molecule oral tyrosine kinase inhibitor (TKI) sunitinib. No phase III trial has yet demonstrated an overall survival benefit and the progression free survival (PFS) benefits, when attained with bevacizumab are short, with perhaps one exception. Together, these results call for a reappraisal of using antiangiogenic drugs for breast cancer and possible strategies to improve their efficacy. Among the reasons to help explain the limited benefits observed thus far include the possibility that angiogenesis may not be a major driver of breast cancer growth, compared to some other types of cancer; that acquired resistance may develop rapidly to VEGF-pathway targeting antiangiogenic drugs, in part due to angiogenic growth factor redundancy; that optimal chemotherapy regimens have not been used in conjunction with an antiangiogenic drug; and that antiangiogenic drugs may secondarily aggravate biologic aggressiveness of the tumors, thereby reducing their overall efficacy after inducing an initial benefit. Several possible strategies are discussed for improving the efficacy of antiangiogenic drugs, including combination with different chemotherapy regimens, e.g. long term and less toxic metronomic chemotherapy protocols; validation of predictive biomarkers to individualize patient therapy; development of improved preclinical therapy models, e.g. involving advanced metastatic breast cancer, and combination with other types of anti-cancer agents especially biologies such as trastuzumab for Her2-positive breast cancer. Reasons for the current concern regarding use of antiangiogenic drug treatments for early stage cancers, including breast cancer, are also discussed. [Copyright &y& Elsevier]

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]

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]

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]