Although solid tumors continuously shed cells, only a very small fraction of the neoplastic cells that enter the blood stream are capable of establishing metastases. In order to be successful these cells must attach, extravasate, proliferate, and induce angiogenesis. Preclinical studies have shown that small-molecule ATP-competitive Src kinase inhibitors can effectively impair metastasis associated tumor cell functions in vitro. However, the impact of these agents on the metastatic cascade in vivo is less well understood. The present studies examined the ability of saracatinib, a dual-specific, orally available inhibitor of Src and Abl protein tyrosine kinases, to interfere with the establishment of lung metastases in mice by tumor cells introduced into the blood stream. The results demonstrated that Src inhibition most effectively interfered with the establishment of secondary tumor deposits when treatments were administered while tumor cells were in the initial phases of dissemination.
blood borne tumor cells; metastatic cascade; Src inhibition; saracatinib
Dissemination of cancer cells is strongly associated with reduction in quality of life, worsening of prognosis, and remains the primary cause of therapeutic failure and high mortality in cancer. A crucial factor in the progression of metastases is the ability to establish a functioning blood vessel network. Consequently therapeutic strategies which selectively target tumor vasculature may hold promise for the treatment of metastatic disease. A complicating factor in the assessment of the efficacy of vascular targeting therapies is that the metastatic process can result in multiple neoplastic lesions at various stages of growth and vascularity in a single organ. The goal of this project was to utilize a rodent squamous cell carcinoma (SCCVII) model to characterize the development of metastatic lung lesions and their associated vasculature. Mice were injected with tumor cells via the tail vein to introduce a reproducible number of lung metastases. At various times after cell injection, lungs were removed and serial sections were taken throughout the lobes for morphometric analysis. Tumor volumes were calculated for each nodule using 2 hematoxylin and eosin (H&E) stained sections that were a known distance apart. Sections adjacent to those used for size determination were reserved for immunohistochemical staining with CD31 to identify blood vessels associated with each nodule. The results showed that although the median tumor volume increased from 0.006 to 0.51 mm3 between 7 and 18 days post SCCVII cell injection, a range of tumor sizes existed at all-times. Irrespective of the time of assessment, nodules with volumes ≤ 0.5 mm3 had a constant vessel density while those with volumes >0.5 mm3 showed increasing vessel densities with increasing size. These findings indicate that the methodology outlined in this study can identify metastases in various stages of vascular development and could therefore be applied to evaluate and distinguish therapeutic interventions that seek to prevent the initiation of blood vessel networks and those targeting already established expanding tumor vasculature. Examining the efficacy of such approaches, alone or in combination, in the treatment of metastases in a preclinical model could lead to the development of more effective therapeutic strategies for metastatic disease.
Metastasis; vascular development; carcinoma
The purpose of this study was to investigate two non-invasive methods for determining the treatment efficacy of the vascular disrupting agent (VDA) CA4P: gadolinium enhanced dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for perfusion analysis and ELISA of blood samples. Candidate proteins were identified by Multi-Analyte Profile analysis of plasma from KHT sarcoma-bearing C3H/HeJ mice after CA4P administration. Candidate proteins were further analyzed by ELISA of plasma from treated C3H/HeJ, BALBc, and C57BL6 mice. Changes in selected proteins, tumour perfusion and tumour necrotic fraction after CA4P treatment were then compared in individual animals. The cytokines KC and MCP-1 were observed to increase after CA4P treatment in all tested models. No correlation was found between KC or MCP-1 levels and tumour necrosis. However, tumour perfusion correlated (r=0.89, p<0.00001) with CA4P treatment efficacy as measured by necrotic fraction, suggesting DCE-MRI may have utility in a clinical setting.
vascular targeting; CA4P KHT sarcoma; DCE-MRI; cytokines
Prostate cancer that has progressed to metastatic disease remains largely untreatable. Nearly 90% of patients with advanced prostate cancer develop skeletal metastases, resulting in a substantial reduction in the quality of life and a drastic worsening of patient prognosis. The mechanisms involved in prostate cancer cell dissemination, however, remain poorly understood. We previously reported the identification of a highly tumorigenic E-cadherin positive prostate tumor stem cell subpopulation that expressed the embryonic stem cell markers SOX2 and OCT3/4. We herein demonstrate that this subpopulation is also highly invasive and, importantly, is capable of altering its E-cadherin expression during the process of invasion. The non-tumorigenic E-cadherin negative subpopulation which minimally expresses SOX2 or OCT3/4 was found to be poorly invasive. In addition, targeted knockdown of SOX2 or OCT3/4 markedly suppressed the invasion of prostate cancer cells. Taken together, these findings indicate that the expression of SOX2 or OCT3/4 is required for invasive cell capacity, but the ability to modulate E-cadherin is the key permissive factor enabling cancer stem cell invasion in vitro. We therefore propose a model in which the post-epithelial to mesenchymal transition phenotype progresses to a frank, aggressive, and invasive phenotype by a process requiring the acquisition of E-cadherin plasticity. Considering the clinical significance of the metastatic complications of prostate adenocarcinoma, the identification of factors that promote the dissemination of the malignant prostate phenotype is essential to establish effective therapies to combat this disease in future.
Prostate cancer invasion; cancer stem cell; E-cadherin; SOX2; OCT3/4
This study evaluated the therapeutic efficacy of combining vascular disrupting agents with antiangiogenic agents.
Materials and Methods
Human clear cell renal carcinoma (Caki-1) tumors were established in nude mice. Treatments consisted of Avastin (2 mg/kg) administered twice a week; CA4P (100 mg/kg) or OXi4503 (25 mg/kg) administered 3 times a week for a period of 2 weeks; or a combination of Avastin and CA4P or OXi4503. Tumor response was assessed by growth delay.
The tumor growth delays were 8, 6, and 18 days for Avastin, CA4P, and OXi4503, respectively. When the two therapies were combined, there was a significantly greater tumor response than what was achieved with single-agent treatments. For example, Avastin plus CA4P led to a growth delay of 13 days, and 27 days for Avastin plus OX14503.
Vascular-directed therapies that include both antiangiogenic and vascular disrupting therapeutics can result in significantly enhanced antitumor effects.
Antiangiogenic therapy; vascular disrupting agents; combination therapy; renal cell carcinoma
The goal of this study was to examine the therapeutic potential of the vascular endothelial growth factor (VEGF) signaling inhibitor cediranib in a human model of renal cell carcinoma (Caki-1).
Methods and Materials
The effects of cediranib treatment on in vitro endothelial cell function (proliferation, migration, and tube formation), as well as in vivo angiogenesis and tumor growth, were determined.
In vitro, cediranib significantly impaired the proliferation and migration of endothelial cells and their ability to form tubes, but had no effect on the proliferation of Caki-1 tumor cells. In vivo, cediranib significantly reduced Caki-1 tumor cell–induced angiogenesis, reduced tumor perfusion, and inhibited the growth of Caki-1 tumor xenografts.
The present results are consistent with the notion that inhibition of VEGF signaling leads to an indirect (i.e., antiangiogenic) antitumor effect, rather than a direct effect on tumor cells. These results further suggest that inhibition of VEGF signaling with cediranib may impair the growth of renal cell carcinoma.
Antiangiogenic therapy; Caki-1 renal cell carcinoma; Cediranib (Recentin, AZD2171)
VEGF is the key player in tumor angiogenesis. In the current study, the impact of VEGF expression on the response of tumors to the VEGFR2 associated tyrosine kinase inhibitor vandetanib was evaluated.
Materials and Methods
Human colon carcinoma (HT29) and murine squamous carcinoma (SCCVII) clonal cell lines expressing varying levels of VEGF were established and their response to vandetanib was assessed in tissue culture and as solid tumors.
Vandetanib treatment had no effect on tumor cell clonogenic cell survival in vitro but doses ≥10 nM significantly reduced endothelial cell migration. In vivo, tumors derived from cell clones expressing high levels of VEGF displayed significantly enhanced angiogenesis and more aggressive growth. An intradermal angiogenesis assay was used to demonstrate that a 4-day treatment with vandetanib (50 mg/kg/day) was able to significantly inhibit blood vessel growth induced by both parental and high VEGF-expressing tumor cell clones. In the HT29 tumor model, treatment response to vandetanib (50 mg/kg/day, Monday-Friday for 2 weeks) was greatest in xenografts derived from the highest VEGF-expressing cell clones. A similar trend was noted in the SCCVII tumor model. The present findings indicate that vandetanib therapy effectively counteracted the aggressive feature of tumor growth resulting from VEGF over-expressing tumor cells and suggest that such tumors may be particularly well suited for anti-VEGF interventions.
VEGF; tumor vascularity; angiogenesis; anti-angiogenic therapy; vandetanib; ZACTIMA™; ZD6474
The purpose of this study was to examine the pathophysiologic impact of CA4P treatment in regions of tumors that ultimately either necrose or survive treatment with such agents.
Methods and Materials
Proliferation, perfusion, vessel density, and VEGF expression were analyzed in the KHT tumor model following CA4P treatment. Analyses were conducted in the whole tumor and the tumor periphery.
Perfusion in the tumor periphery decreased 4 hours after treatment but returned to baseline 20 hours later. Whole tumor perfusion also decreased 4 hours after treatment, but did not return to baseline. Vessel density decreased in the tumor as a whole, but not in the tumor periphery. No significant effect on VEGF expression was observed, but a decrease in proliferation in the whole tumor and the periphery was noted.
The present studies have shown that those areas of the tumor that survive CA4P treatment are affected by CA4P exposure, though only transiently. The decrease in perfusion could negatively impact therapies utilizing the combination of CA4P and conventional anticancer agents by decreasing drug delivery and tissue oxygenation.
These findings suggest that the timing of CA4P treatments when used in conjunction with conventional anticancer therapies should be considered carefully.
vascular targeting; KHT sarcoma; tumor perfusion; CA4P
A tumor’s dependence on angiogenesis for survival and growth has led to the advancement of a variety of blood vessel directed anticancer treatment strategies. Over expression of angiopoietin-2 (Ang-2) in tumor vasculature and its crucial role in angiogenesis, i.e. the destabilization of endothelial/peri-endothelial cell interactions, now raises the possibility of additional novel anti-angiogenic therapeutics. The present study utilized a co-culture sphere model to (i) demonstrate the destabilizing effect of Ang-2 on endothelial/smooth muscle cell contact and (ii) evaluate the impact of the investigational Ang-2 antibody MEDI3617 on endothelial/smooth muscle cell dissociation. Real time imaging of spheres showed both exogenous Ang-2 and PMA induced endogenous Ang-2 secretion resulting in sphere destabilization (loss of endothelial cells from smooth muscle cell core). The presence of MEDI3617 inhibited this process. To assess the anti-angiogenic potential of MEDI3617 in vivo, nude mice were injected intradermally with human renal cell carcinoma cells (Caki-1, Caki-2) and the number of blood vessels induced over a 3 day period was scored. MEDI3617 (2, 10, 20 mg/kg) significantly reduced the initiation of blood vessels for both tumor models at all doses investigated. These data indicate that MEDI3617 treatment significantly impairs the initiation of angiogenesis by inhibiting the Ang-2 mediated disruption of endothelial/muscle cell interaction associated with blood vessel destabilizing and thereby reduces tumor cell induced angiogenesis. The results support the notion that targeting the angiopoietin/Tie2 axis may offer novel anti-angiogenic strategies for cancer treatment.
Angiopoietin-2; Angiogenesis; Anti-angiogenic therapy; Antibody therapy; Spheroids
Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5′-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model.
Vascular disrupting strategies impair a tumor’s blood vessel network which is essential for tumor progression and metastasis. Vascular disrupting agents cause a rapid and selective vascular shutdown in tumors to produce extensive secondary neoplastic cell death due to ischemia. A lead agent in this therapeutic strategy is the tubulin depolymerizing agent combretastatin-A4 phosphate (CA4P). Used alone CA4P induces extensive necrosis in a wide variety of preclinical cancer models and significant blood flow reductions in the patient tumors. Preclinical and clinical data further indicate that CA4P can effectively be combined with chemotherapy or radiotherapy. Finally, the potential of combining VDAs with antiangiogenic therapies has shown considerable promise in preclinical models and such combinations are now beginning to be evaluated in patients.
Vascular disrupting agents; CA4P (Zybrestat™; fosbretabulin); antiangiogenic therapy; tubulin depolymerization
ASA404, a flavonoid tumor-vascular disrupting agent (Tumor-VDA), is in clinical trial for the treatment of non-small cell lung cancer. Its action differs from both that of the tubulin binding class of Tumor-VDAs and antiangiogenic agents. In mice, ASA404 induces a sequence of changes in tumor tissue, starting within one hour with increased vascular permeability, increased endothelial apoptosis and decreased blood flow. Later effects include the release of serotonin, induction of tumor necrosis factor and other cytokines and chemokines, as well as induction of nitric oxide. This cascade of events induces sustained effects on blood flow, tumor hypoxia, vascular failure, inflammatory responses and, in some tumors, complete regression. One feature of the action of ASA404 against murine tumors is its ability to potentiate the effects of radiation and a variety of chemotherapeutic agents. The flavonoid class appears to be unique because of its dual action on vascular endothelial function and innate immunity. The translation of preclinical to clinical results demands an understanding of both the mechanisms underlying the dual effects and the species differences in ASA404 activity. Clinical trials indicate that the future of ASA404 as an effective agent relies on a deep appreciation of its cellular action.
DMXAA; ASA404; vadimezan; antivascular; tumor necrosis factor; toll-like receptors; serotonin; ceramide
This study investigated the anti-tumour effects of the novel vascular disrupting agent plinabulin (NPI-2358) when given alone or combined with radiation.
Materials and Methods
Foot implanted C3H mammary carcinomas or leg implanted KHT sarcomas were used, with plinabulin (Nereus Pharmaceuticals, San Diego, USA) injected intraperitoneally. Dynamic contrast-enhanced magnetic resonance imaging measurements were made with gadolinium-DTPA on a 7-tesla magnet. Treatment response was assessed using either a regrowth delay (C3H mammary carcinoma) or clonogenic survival (KHT sarcoma) assay, or histological estimates of necrosis for both models.
Plinabulin (7.5 mg/kg) significantly reduced both IAUC and Ktrans within 1-hour after drug injection, reaching a nadir at 3-hours, but returning to normal within 24-hours. A dose-dependent decrease in IAUC and Ktrans, was seen at 3-hours. No significant anti-tumour effects were seen in the C3H mammary carcinoma until doses of 12.5 mg/kg and above were achieved, but started at 1.5 mg/kg in the KHT sarcoma. Irradiating tumours 1-hour after injecting plinabulin enhanced response in both models.
Plinabulin induced a time and dose dependent decrease in tumour perfusion. The KHT sarcoma was more sensitive than the C3H mammary carcinoma to the anti-tumour effects of plinabulin, while radiation response was enhanced in both models.
Plinabulin (NPI-2358); Vascular targeting; Radiation; C3H mammary carcinoma; KHT sarcoma; Magnetic Resonance Imaging
Intratumoral hypoxia is known to be associated with radioresistance and metastasis. The present study examined the effect of acute and chronic hypoxia on the metastatic potential of prostate cancer PC-3, DU145 and LNCaP cells.
Methods and Materials
Cell proliferation and clonogenicity were tested by MTT assay and colony formation assay, respectively. “Wound-healing” and Matrigel-based chamber assays were used to monitor cell motility and invasion. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was tested by Western blot and HIF-1-target gene expression was detected by real time PCR. Secretion of matrix metalloproteinases (MMPs) was determined by gelatin zymography.
When PC-3 cells were exposed to 1% oxygen (hypoxia) for various periods of time, chronic hypoxia (≥24 h) decreased cell proliferation and induced cell death. In contrast, prostate cancer cells exposed to acute hypoxia (≤6 h) displayed increased motility, clonogenic survival and invasive capacity. At the molecular level, both hypoxia and anoxia transiently stabilized HIF-1α. Exposure to hypoxia also induced the early expression of MMP-2, an invasiveness-related gene. Treatment with the HIF-1 inhibitor YC-1 attenuated the acute hypoxia-induced migration, invasion, and MMP-2 activity.
The length of oxygen deprivation strongly impacted the functional behavior of all 3 prostate cancer cell lines. Acute hypoxia in particular was found to promote a more aggressive metastatic phenotype.
acute hypoxia; prostate cancer; metastasis; HIF-1α; MMP-2
The c-Met receptor tyrosine kinase is aberrantly activated in many solid tumors. In a prior study we showed that prostate cancer PC-3 cells exhibit constitutively activated c-Met without exogenous hepatocyte growth factor (HGF); however whether this characteristic is due to an endogenous HGF/c-Met autocrine loop remains controversial. In the current study we examined the response of PC-3 cells to an anti-HGF neutralizing antibody or a small molecule Met kinase inhibitor (BMS-777607).
Cell scattering was tested by monitoring cell morphology after HGF stimulation. Cell migration was examined by both “wound-healing” and transwell assasy and invasion was detected by Matrigel-coated transwell assay. Proliferation, survival and anoikis were determined by MTT, colony formation and trypan blue exclusion assay, respectively. Gene and protein expression were assessed by real-time PCR and Western blot, respectively.
Although HGF mRNA could be detected in PC-3 cells, the molecular weight of secreted “HGF” protein was inconsistent with the functional recombinant HGF. Furthermore, conditioned medium from PC-3 cell cultures was ineffective at triggering either motogenic behavior or c-Met signaling in DU145, another prostate cancer cell line expressing c-Met but lacking basal c-Met activation. PC-3 cells also were not responsive to the anti-HGF neutralizing antibody in experiments assessing proliferation, migration, or c-Met signaling. BMS-777607 treatment with micromolar doses nonetheless led to significant inhibition of multiple PC-3 cell functions including proliferation, clonogenicity, migration and invasion. At the molecular level, BMS-777607 suppressed autophosphorylated c-Met and downstream c-Src and Akt pathways.
These results suggest that the constitutive c-Met activation in PC-3 is independent of autocrine stimulation. Because PC-3 cells were responsive to BMS-777607 but not the anti-HGF antibody, the findings also indicate that under circumstances where c-Met is constitutively hyperactive in the absence of functional HGF, targeting the c-Met receptor remains a viable therapeutic option to impede cancer progression.
BMS-777607; c-Met; HGF; Neutralizing antibody; Prostate cancer
The vasculature of solid tumors is fundamentally different from that of normal vasculature and offers a unique target for anti-cancer therapy. Direct vascular-targeting with Tumor-Vascular Disrupting Agents (Tumor-VDAs) is distinctly different from anti-angiogenic strategies, and offers a complementary approach to standard therapies. Tumor-VDAs therefore have significant potential when combined with chemotherapy, radiotherapy, and angiogenesis-inhibiting agents. Preclinical studies with the different Tumor-VDA classes have demonstrated key tumor-selective anti-vascular and anti-tumor effects.
Tumor-vascular disrupting agents; Tumor-VDAs; ASA404; Tubulin-binding agents; CA4P; Tumor vasculature; Anti-vascular therapy
Unlike normal blood vessels, the unique characteristics of an expanding, disorganized and leaky tumor vascular network can be targeted for therapeutic gain by vascular disrupting agents (VDAs), which promote rapid and selective collapse of tumor vessels, causing extensive secondary cancer cell death. A hallmark observation following VDA treatment is the survival of neoplastic cells at the tumor periphery. However, comparative studies with the second generation tubulin-binding VDA OXi4503 indicate that the viable rim of tumor tissue remaining following treatment with this agent is significantly smaller than that seen for the lead VDA, combretastatin. OXi4503 is the cis-isomer of CA1P and it has been speculated that this agent's increased antitumor efficacy may be due to its reported metabolism to orthoquinone intermediates leading to the formation of cytotoxic free radicals. To examine this possibility in situ, KHT sarcoma-bearing mice were treated with either the cis- or trans-isomer of CA1P. Since both isomers can form quinone intermediates but only the cis-isomer binds tubulin, such a comparison allows the effects of vascular collapse to be evaluated independently from those caused by the reactive hydroxyl groups. The results showed that the cis-isomer (OXi4503) significantly impaired tumor blood flow leading to secondary tumor cell death and >95% tumor necrosis 24 h post drug exposure. Treatment with the trans-isomer had no effect on these parameters. However, the combination of the trans-isomer with combretastatin increased the antitumor efficacy of the latter agent to near that of OXi4503. These findings indicate that while the predominant in vivo effect of OXi4503 is clearly due to microtubule collapse and vascular shut-down, the formation of toxic free radicals likely contributes to its enhanced potency.
OXi4503; combretastatin; vascular disrupting agents; endothelial cells; magnetic resonance imaging; viable rim; tubulin-binding agents
4T1 mouse mammary adenocarcinomas and Caki-1 human renal cell carcinomas grown in mouse dorsal window chambers were serially treated with the vascular disrupting agent (VDA) OXi4503 and their responses compared. The real-time in vivo response was assessed using spectral imaging of microvascular hemoglobin saturation. To our knowledge this is the first use of spectral imaging technology for investigation of vascular disrupting agents. Previous research showing tumor size dependence in the treatment response to VDAs suggested that for the size of tumors used in this study only a moderate response would be observed; however, the tumors unexpectedly had very different responses to treatment. Caki-1 tumors showed little permanent vessel damage and experienced transient vessel collapse with time-dependent oxygenation changes followed by recovery starting at 6 h after treatment. Caki-1 tumors did not manifest necrotic avascular regions even after repeated treatments. These results are consistent with those obtained using other imaging modalities and histology. In contrast, similarly sized 4T1 tumors showed extensive vessel disintegration, minor vascular collapse, and a drop in tumor oxygenation up to 6 h post-treatment, after which reperfusion of collapsed vessels and extensive vascular remodeling and neovascularization of the tumor rim occurred from 8–48 h. The completely disintegrated vessels did not recover and left behind avascular and apparently necrotic regions in the tumor core. Spectral imaging appears to be a useful technique for in vivo investigation of vascular disrupting agents. The differential responses of these two tumor-types suggest that further investigation of the mechanisms of action of VDAs and individual characterization of tumor VDA-responses may be needed for optimal clinical use of these agents.
angiogenesis; oxygen; spectral imaging; vascular targeting; window chamber
Neovascularization is intimately involved in tumor survival, progression, and spread, factors known to contribute significantly to treatment failures. Thus, strategies targeting the tumor blood vessel support network may offer not only unique therapeutic opportunities in their own right, but also novel means of enhancing the efficacies of conventional anticancer treatments. This article reviews one such therapeutic approach directed at the tumor blood vessel support network. Vascular disrupting therapies seek the destruction of the established neovasculature of actively growing tumors. The goal of these therapies is to cause a rapid and catastrophic shutdown in the vascular function of the tumor in order to arrest the blood flow and produce tumor cell death as a result of oxygen and nutrient deprivation and the build up of waste products.
Tumor vasculature; Vascular disrupting agents; Small-molecule vascular disrupting agents; Conventional anticancer therapies; Combined modality; Treatments