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Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis and growth in most solid neoplasia.Clonal tumor cell lines expressing varying levels of this pro-angiogenic factor were created via recombinant adeno-associated virus infection of a human (HT29) and rodent (SCCVII) tumor model.The alteration in VEGF expression levels did not significantly impact the in vitro growth rate of the clonal cell lines or the expression levels of other known pro-angiogenic factors. However, the tumors that arose from these clonal cell lines did display significant physiological differences. Upregulation of VEGF expression increased the in vivo growth rate and the intratumoral vessel density of the resulting tumors and decreased the extent of tumor necrosis.Since the tumor vascular network can impact the efficacy of anti-cancer therapies, these results suggest that VEGF expression may be important to consider in the treatment of cancer.

The purpose of this study is to evaluate the effects of low to moderate aerobic exercise on tumor vasculature, perfusion and oxygenation in breast cancer. Poor perfusion and hypoxia are common features of solid tumors that are a result of abnormal tumor angiogenesis. Both chemo- and radiation therapies are negatively affected by tumor hypoxia, with the presence of hypoxia associated with treatment failure and poor outcomes in cancer patients. While strategies to enhance tumor oxygenation and perfusion have been developed, their success in the clinic has been limited. A potential method to increase oxygenation and decrease hypoxia that is safe and well-tolerated is aerobic exercise. The benefits of exercise as a palliative measure are well documented, however the effects of exercise on tumor blood flow and oxygenation have not been extensively characterized. Histological analysis of vessel density (MECA-32), vessel perfusion (Hoechst-33342) and hypoxia (EF5) were quantified in tumor bearing mice subjected to low to moderate intensity treadmill running and compared to sedentary control mice. In a separate model, a murine dorsal skinfold window chamber model and in vivo microscopy of microvessel function was utilized to characterize changes in tumor vascular structure and function in real time. In these studies, the combination of hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence (FPF) imaging of blood transit time were used. Hb saturation imaging reveals the oxygenation of blood within microvessels while FPF imaging gives information regarding blood flow and network connections by recording a fluorescent contrast agent as it is injected into circulation. Spectroscopic analysis was performed in female nude mice with surgically installed titanium window chambers. Mammary tumors were initiated in the windows and once tumors reached a specified volume, animals were subjected to daily treadmill running for up to a week. As a sedentary control, mice were placed on a stationary treadmill for equivalent periods of time. Combination imaging was performed daily to monitor microvessel oxygenation and blood flow. Preliminary results indicate that following one bout of exercise, tumor perfusion is increased, however there was no significant correlation between exercise and tumor microvessel oxygenation. For histological analysis, mammary tumors were initiated in the fat pad of female nude mice. Once tumors nodules were established, animals were subjected to either a single bout or daily bouts of exercise for 1 to 5 weeks. Sedentary controls were exposed to a stationary treadmill for equivalent periods of time. Following exercise protocols, tumors were harvested and sectioned for immunohistochemical analysis. Staining analysis revealed an improvement in tumor oxygenation and an increase in the number of perfused vessels in tumors of exercising animals in comparison to sedentary controls. Our results demonstrate aerobic exercise can increase tumor oxygenation and perfusion in breast cancer, suggesting that exercise can potentially be used as an adjunctive therapy to overcome tumor hypoxia and enhance the efficacy of chemo- and radiation therapy. Citation Format: Jennifer A. Lee, Jennifer Wiggins, Dietmar W. Siemann. Impact of aerobic exercise on tumor oxygenation and perfusion in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B24.

Introduction: Pre-clinical studies have demonstrated that combining angiogenesis inhibitors (AIs) with conventional radiation and chemotherapy treatments can be an effective anti-tumor therapy. However, clinical benefits from such combinations have been more modest. A principal mechanism by which AIs are believed to increase tumor sensitivity to radiation and chemotherapy is vascular normalization, a process which should improve tumor blood perfusion and oxygenation. Here we review the universality of this concept by examining those pre-clinical studies that directly determined the effect of AI treatment on tumor oxygenation. Methods: This review was based on searching the PubMed database using the criteria angiogenesis inhibitors combined with one of each of the following: oxygenation, hypoxia, or normalization coupled with our own knowledge within the fields of tumor hypoxia and vascular targeting. A total of 51 pre-clinical studies were identified. The techniques used to monitor hypoxia/oxygenation included immunohistochemical analysis of hypoxia marker binding (pimonidazole and EF5) or hypoxia gene expression (CA9 and HIF-1alpha), direct oxygenation measurements (Eppendorf electrode, EPR oximetry, Oxylite probe or phosphorous quenching), or radiation response. The AIs included the two principal classes of inhibitors, those targeting VEGF (bevacizumab, DC101) and tyrosine kinase (vandetanib, sunitinib, sorafanib, axitinib, pazopanib, AG-013736, PTK787/ZK222584, SU5416), as well as a variety of other agents (TNP-470, suramin, endostatin, thrombospondin, thalidomide, anginex, arginine deiminase, nucleolin antagonist, nelfinavir). Results: 19 studies demonstrated significant improvements in tumor oxygenation status. These included a decrease in hypoxia marker labelling (bevacizumab, DC101, suramin, endostatin, nucleolin antagonist); an increase in oxygenation determined using the Eppendorf electrode (TNP-470 and an anti-VEGF antibody), EPR oximetry (thalidomide, vandetanib, SU5416), and Oxylite probe (bevacizumab); an increase in hypoxia regulated gene expression (endostatin and SU5416); and enhanced radiation response (thrombospondin). In contrast, 21 studies found a significant decrease in tumor oxygenation using hypoxic markers measured by immunohistochemistry (bevacizumab, DC101, sunitinib, pazopanib, AG-013736, nelfinavir) or PET activity (PTK787/ZK222584), and increased expression of HIF-1alpha (bevacizumab). Moreover, 9 studies reported no change in tumor oxygenation as measured with hypoxic markers (endostatin and SU5416), direct oxygen measurements (anti-VEGF antibody, DC101, arginine deiminase), or radiation response (DC101). Finally, two studies actually reported both increases and decreases when using direct oxygen measurements depending on the time after treatment with bevacizumab or DC101. Conclusions: While there is clear evidence that tumor oxygenation can improve following AI treatment, consistent with vascular normalization occurring, this is clearly not a universal finding. Importantly the effects on tumor hypoxia/oxygenation status show no relationship to either the specific AI or the assay used to monitor these parameters. These findings suggest that for the combination of AIs with conventional anti-cancer therapies to be most effective it will be important to establish the impact the AI has on the tumor vasculature and associated tumor pathophysiology in a given treatment setting. Citation Format: Michael R. Horsman, Dietmar W. Siemann. Is the lack of clinical success with antiangiogenic therapy due to vascular normalization not being a universal phenomenon? [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A04.

The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin and its analogs only exert limited clinical benefits in patients. It has been shown that many tumors either fail to respond to rapamycin initially or are able to acquire resistance after the initial treatment of rapamycin. Such primary and secondary resistances remain as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. We have previously shown that the ATP-competitive dual mTOR kinase inhibitor AZD8055 can significantly inhibit malignant behaviors in parental cells however its functional efficacies in the rapamycin-resistance setting remains unclear. Cancer cells with wildtype PTEN were selected as models of primary rapamycin resistance since the mTOR pathway is usually less abnormal in these cells. To establish cells with acquired rapamycin resistance, PTEN-null prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways were detected by Western blotting. PTEN wildtype and PC-3RR cells essentially failed to respond to rapamycin; IC50 values were greater than 100 nM in PTEN wildtype cells and 1 μM in PC-3RR cells, respectively. However, for both cell types AZD8055 suppressed proliferation at the nanomolar level in a dose dependent manner, with IC50 values less than 30 nM. Similarly, cellular clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E, therefore blocking cap-dependent translation. Further, while the p-S6K (T389) and p-S6 (S235/236) pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 (T37/46) and p-Akt (S473) than did rapamycin in all cells tested. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. However, the PI3K inhibitor wortmannin failed to sensitize cell response to AZD8055, implying such feedback-dependent activation has little impact on the drug efficacy. These findings indicate that dual mTOR kinase inhibitor AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with deactivation of rapamycin-insensitive mTOR downstream components such as Akt and 4E-BP1, but not the upstream feedback pathway. These data provide in vitro evidence in support of future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Li Zhao, Dietmar W. Siemann. Dual mTOR kinase inhibitor reverses rapamycin resistance in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B59.

Recognition of the dependence of angiogenesis on proteolytic enzymes has raised significant interest in evaluation of proteases as potential targets to disrupt tumor angiogenesis. While the role of cysteine proteases in tumor metastasis is well understood, their contribution to tumor angiogenesis remains less explored. Cysteine cathepsins, in particular cathepsin L (CTSL) gets are upregulated in a wide range of human cancers. In transformed cells extracellular CTSL levels can increase up to 200-fold and comprise up to 40% of total secreted proteins. The role of CTSL in promoting tumor metastasis is supported by recent studies in our laboratory which have shown that treatment with the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94) significantly impairs the metastatic phenotype of prostate and breast cancer cells. However, the contribution of CTSL to tumor angiogenesis remains less well explored. Thus, the goal of the present studies was to evaluate the role of tumor cell secreted CTSL in tumor cell induced angiogenesis. Upon activation by pro-angiogenic stimuli, endothelial cells migrate and invade through the interstitium to form tubes. The effect of CTSL on the migratory, invasive and tube formation capacity of endothelial cells was therefore investigated. Stimulation of human micro-vascular endothelial cells of the lung (HMVEC-L) with purified human CTSL led to a dose dependent increase in endothelial cell invasion (1.5-fold at 10ng/ml and 2-fold at 100ng/ml). Furthermore, CTSL (100ng/ml) increased the tube forming ability of endothelial cells as demonstrated by an increase in total tube length. Similarly, incubation of HMVEC-L with conditioned media from MDA-MB-231 breast cancer cells, led to a significant increase in their migration, invasion and tube formation capacity (migration 1.3-fold; invasion 2.5-fold). Compared to parental MDA-MB-231 breast cancer cells, CTSL knockdown cells that were inoculated intradermally into ventral skin flaps of female nude mice showed a significant reduction in their ability to induce blood vessel formation (3.5-fold).These proangiogenic endothelial cell functions were accompanied by a significant increase in endothelial cell proliferation upon stimulation with purified CTSL (2-fold). Microarray analyses revealed a significant upregulation of cell cycle related genes by CTSL. Western blot analyses further confirmed upregulation of members of the cyclin family by CTSL. In conclusion, our findings suggest that in addition to its pro-metastatic function, CTSL might further contribute to tumor progression by eliciting various pro-angiogenic endothelial cell functions. Citation Format: Dhivya Sudhan, Belen Rabaglino, Charles Wood, Dietmar Siemann. Role of Cathepsin L in breast cancer angiogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2015-4185

The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin and its analogs only exert limited clinical benefits in patients. It has been shown that many tumors either fail to respond to rapamycin initially or are able to acquire resistance after the initial treatment of rapamycin. Such primary and secondary resistances remain as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. We have previously shown that the ATP-competitive dual mTOR kinase inhibitor AZD8055 can significantly inhibit malignant behaviors however its functional efficacies in the rapamycin-resistance setting remains unclear. Cancer cells with wildtype PTEN were selected as models of primary rapamycin resistance since the mTOR pathway is usually less abnormal in these cells. To establish cells with acquired rapamycin resistance, PTEN-null prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways, including p-S6K (T389)/p-S6 (S235/236), p-4EBP1 (T37/S46) and p-Akt (S473) were detected by Western Blotting. PTEN wildtype and PC-3RR cells essentially failed to respond to rapamycin; IC50 values were greater than 100 nM in PTEN wildtype cells and 1 μM in PC-3RR cells. However, for both cell types AZD8055 suppressed proliferation at the nanomolar level in a dose dependent manner, with IC50 values less than 30 nM. Similarly, cellular clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E, therefore blocking cap-dependent translation. Further, while the p-S6K and p-S6 pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 and p-Akt than did rapamycin in all cells tested. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. However, the PI3K inhibitor wortmannin did not further sensitize cells to AZD8055, implying such feedback-dependent activation is less responsible for weakening the drug efficacy. These findings indicate that AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with the deactivation of rapamycin-insensitive mTOR downstream components such as Akt and 4E-BP1, but not the upstream feedback pathway. These data provide in vitro evidence in support of future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Li Zhao, Dietmar Siemann. Dual mTOR kinase inhibitor overcomes rapamycin resistance in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2015-3578

Tumor cells frequently harbor abnormalities in signaling pathways, leading to increased migration, invasion, survival, angiogenesis, and proliferation. C-Met is one such commonly aberrant pathway. C-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair, and protein levels are often elevated in a large variety of tumors. C-Met is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). HGF is produced by mesenchymal cells and stimulates the c-Met protein, leading to a variety of downstream signaling pathways that result in increased migration, proliferation, survival, and angiogenesis. While many tumor cells express the c-Met receptor, a number of tumor types, including glioblastoma and osteosarcoma, have been observed to co-express both HGF and c-Met, and activate the pathway in an autocrine manner. The purpose of the current study was to determine whether tumor cells utilizing autocrine c-Met signaling would more sensitive to c-Met inhibition than those relying on the paracrine pathway. Experimental procedures include ELISA, western, migration, invasion, and clonogenic cell survival. Cell lines investigated include: two glioblastoma cell lines (U87 and U118), two osteosarcoma cell lines (OS156 and OS521), two fibrosarcoma cell lines (KHT and RIF), a breast (MDA-MB-231) and a prostate cancer cell line (PC-3). The small molecule c-Met inhibitor BMS-777607 and an HGF-neutralizing antibody were used to compare the effects interrupting the c-Met axis in these tumor cell lines. Data obtained using ELISA of conditioned media show that the glioblastoma (U87 and U118) and fibrosarcoma (KHT) cells secrete HGF, while the breast and prostate cells (MDA-MB-231; PC-3) do not. Furthermore western blot analysis showed higher basal levels of phospho-c-Met among the HGF-secreting cell lines in comparison to the non-HGF secreting cell lines, indicating that the KHT, U87, and U118 models activate c-Met in an autocrine manner, while the MDA-MB-231 and PC-3 lines activate c-Met in a paracrine manner. Importantly preliminary assessments indicate the autocrine glioblastoma and fibrosarcoma cell lines to be more sensitive to small molecule c-Met inhibition than the paracrine breast and prostate cancer cell lines. Investigations of additional cancer models are ongoing but our findings to date support the notion that cancer cells that autocrine activate the c-Met pathway may more susceptible to c-Met inhibition than those utilizing primarily paracrine activation of this pathway. Citation Format: Veronica Hughes, Dietmar W. Siemann. C-Met inhibition sensitivity in vitro: Autocrine vs. paracrine activation of the c-Met pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4122. doi:10.1158/1538-7445.AM2015-4122

Background: Src family kinases are often over-expressed and highly active in solid tumors, including prostate cancer. This phenotype is associated with a poor prognosis partly because Src is a key factor in important signaling pathways involved in cell proliferation, angiogenesis, and initiation of metastasis. As a result, several promising small molecule targeting agents have been developed to prevent phosphorylation of key tyrosine residues that produce Src activation. However, these agents are not without side effects. The purpose of this study was to determine if adding a cytostatic soy isoflavone extract (ISF) to the treatment would produce optimal results at lower doses of the Src inhibitor. The ISF used in these studies has been shown to inhibit tumor growth in mice carrying human prostate cancer cell xenografts without causing toxicity to the host. Methods: Aggressive PC-3ML cells (a gift from A. Fatatis, Drexel University), and weakly tumorigenic LNCaP cells were treated for 24 hr with various concentrations of Src inhibitors (dasatinib, saracatinib), ISF (200 ug/ml, NovaSoy), or a combination. To evaluate effects on the metastatic potential of the cells, functional assays of cell growth and motility were performed, including those that assess clonogenic cell survival, cell cycle progression, and transwell migration and invasion activity. Results: Src inhibitors and ISF alone produced very little reduction in cell viability, but significant cytostatic effects, as determined by a reduction in clonogenicity, the number of cells able to form 50-cell colonies. This was likely due, in part, to changes in cell cycle progression. Both dasatinib and saracatinib caused an accumulation of cells in the G1 phase. As expected, ISF treatment resulted in higher numbers of cells in the G2/M phase. When the treatments were combined, cells exposed to dasatinib were observed to accumulate in both phases, with a significant decrease in S-phase cells. The results were not significant for saracatinib-treated cells. Exposure to either a Src inhibitor or ISF significantly reduced the migration of cells in a transwell chamber and their ability to invade through a Matrigel-coated 8 micron-pore membrane towards a chemoattractant (media containing 10% FBS). Dasatinib produced much greater effects, and at lower concentrations, than saracatinib. When combined with ISF, the effects were enhanced, particularly with dasatinib. Conclusions: In vitro studies suggested that combining a Src inhibitor and ISF resulted in greater inhibition of metastatic potential than either alone. This may indicate that including ISF in treatment regimens may allow a lower dose of the targeting agent to be used to achieve optimal response and also decrease toxicity. Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Effects of Src inhibitors and soy isoflavones on human prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2015-777

Background: Intratumoral hypoxia (pO2 Purpose: The goal of the present investigations was to determine whether aerobic exercise could be applied as a means to improve tumor physiology and decrease tumor hypoxia in breast cancer models. Methods: The effects of single and repeated bouts of moderate intensity exercise (treadmill running) were studied in mice bearing syngeneic murine mammary carcinoma models (EMT6 and 4T1). The exercise intensities were determined by measuring the anaerobic threshold, which is reflected by the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and once the tumors reached a size of 250 mm3, the mice were exposed to either a single bout of exercise or daily exercise bouts performed for 5 consecutive days. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. Following cessation of each exercise regime tumors were harvested, sectioned and evaluated by immunofluorescence. Vessel density (MECA-32), blood perfusion (Hoechst-33342) and hypoxia (EF5) were quantified and significance was established at p Results: Preliminary data indicate that even a single exercise bout may begin to demonstrate improvements in blood vessel perfusion and tumor oxygenation. Importantly, after 5 daily bouts of exercise, there was a significant increase in the number of patent vessels and improvement in oxygenation in the tumors of exercising mice compared to those of sedentary controls. Conclusion: The results indicate that moderate aerobic exercise can improve the oxygenation status of solid tumors. These findings suggest that such a non-toxic intervention may have potential utility in overcoming hypoxia-associated treatment resistance in cancer patients and improve patient outcome. Citation Format: Jennifer M. Wiggins, Jennifer A. Lee, Lori Rice, Dietmar Siemann. The impact of aerobic exercise on oxygenation and vascularity in breast cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3198. doi:10.1158/1538-7445.AM2015-3198

4-1BB (CD137) is a member of the tumor necrosis factor receptor superfamily. It interacts with 4-1BB ligand (4-1BBL) and delivers a costimulatory signal for T cell activation. The immune response induced by 4-1BB monoclonal antibodies (mAbs) has been shown to have a marked augmentation of tumor-selective cytolytic T cell activity.The antitumor efficacy of agonistic 4-1BB mAbs (BMS-469492, Bristol-Meyer Squibb), used alone or in combination with radiation therapy, was evaluated in murine lung (M109) and breast (EMT6) carcinoma models.Treatment with BMS-469492 led to only a modest growth retardation in M109 tumors (3 days, p0.05), but a significant growth delay in EMT6 tumors (12.5 days, p0.05). When BMS-469492 was administered in conjunction with single dose radiation therapy (5, 10 or 15 Gy), enhanced tumor responses were noted only at the highest evaluated radiation dose (15 Gy). In contrast, in the EMT6 model, BMS-469492 treatment resulted in enhanced antitumor effects at all radiation doses. In addition, the response of EMT6 tumors to fractionated radiotherapy also was significantly increased when BMS-469492 was included in the treatment protocol.These results suggest that monoclonal antibodies against 4-1BB may not only be efficacious in cancer immunotherapy, but may also have utility when applied in combination with conventional anticancer treatments, such as radiation therapy.

Purpose: As first-generation small-molecule vascular disrupting agents (VDA) have begun to enter clinical trials, second-generation agents are under active development. One such agent is the combretastatin A4 disodium phosphate (CA4P) analogue OXi4503 (CA1P). Experimental Design: C3H/HeJ mice bearing KHT sarcomas were treated with CA4P and OXi4503 and the effect on tumor vasculature was determined by evaluating the extent of vascular shutdown (Hoechst-33342 vessel staining) and tumor perfusion inhibition (dynamic contrast-enhanced magnetic resonance imaging). Dynamic contrast-enhanced magnetic resonance imaging and tumor necrosis end points also were used to examine the pathophysiologic tumor effects following repeated exposures to these agents. Results: Single doses of either agent (CA4P, 100 mg/kg; OXi4503, 25 mg/kg) resulted in an 80% to 90% reduction in tumor perfusion 4 hours after treatment. Whereas recovery in tumor perfusion was observed 48 hours posttreatment, this recovery was significantly slower in mice treated with OXi4503. Tumors re-treated with either VDA 72 hours after the first drug exposure showed a similar reduction and recovery in tumor perfusion. Histologic evidence showed the presence of a smaller viable rim after exposure to OXi4503 than that observed after CA4P treatment. Furthermore, the extent of recovery of tumor necrosis 72 hours after drug treatment was less for OXi4053. Conclusions: The present studies show that the second-generation VDA OXi4503 possesses significant antivascular effects in solid tumors. Importantly, the vasculature of tumors of mice that had received an initial dose this agent was as responsive to a subsequent treatment.

There is increasing interest in small-molecule drugs that can selectively disrupt the abnormal vasculature associated with disease processes such as cancer and macular degeneration. These agents are distinct from anti-angiogenic strategies, which do not target existing vessels but prevent additional vessel growth, althouglh they may potentially be complimentary with these antiangiogenic strategies. Several vascular disrupting agents (VDAs) are now undergoing clinical evaluation. The main focus of research has been on two drug classes: the first is comprised of agents that bind reversibly with tubulin and prevent microtubule assembly; the second are the flavanoids, which can induce intratumor cytokine release. Data from phase I studies have established that these agents can selectively reduce tumor blood flow at well-tolerated doses. Preclinical data indicate that VDAs can improve the tumor response to cytotoxic chemotherapy, radiation and antiangiogenic treatments. This activity has been attributed to the ability of these agents to selectively destroy the central regions of tumors, areas widely believed to contain cell populations resistant to cytotoxic therapies. The VDA compounds combretastatin A4 phosphate (CA4P) and 5,6-dimethylxantlenone-4-acetic acid (DMXAA) are being evaluated in phase II clinical trials in combination with conventional cytotoxic therapies for the potential treatment of cancer. This review discusses the small-molecule VDAs in clinical development. In addition, the potential for combination therapy with VDAs and traditional anticancer therapies, such as radiation, chemotherapy and anti-angiogenics is described.

Historically, approaches to improve cancer therapy have focused primarily on achieving increased tumor cell kill. Recently however another treatment approach has garnered considerable attention. Rather than targeting the neoplastic cell population directly, this strategy endeavors to impair the tumor’s nutritional support system by target the tumor blood vessel network. Vascular targeting approaches are based on the recognition that a continuously expanding vasculature is an essential requirement for tumor initiation, progression and metastasis. Indeed it is generally well accepted that most tumors remain dormant and fail to develop beyond a few millimeters in size in the absence of angiogenic growth. The therapeutic potential of targeting the tumor vasculature is therefore abundantly clear. As a consequence the fi eld of vascular targeting has expanded rapidly and led to a large number of investigational drugs, many of which have now begun to undergo clinical evaluation. These agents are quite distinct from conventional anticancer treatments such as radiation therapy and cytotoxic drugs. Indeed the application of vascular targeting strategies as adjuvants to standard therapeutic modalities may offers unique opportunities to develop even more effective cancer therapies.

Recombinant adeno-associated viral vectors serotype 2 (rAAV2) can transduce several tissues with high efficiency.The transduction efficiency of rAAV2 in pancreatic and colon cancer was compared to that of recombinant adenovirus (rAd) in vitro and in vivo using green fluorescent protein (GFP).With the exception of SU.86.86, the percentage of GFP-positive cells was below 10% at a multiplicity of infection (MOI) of 100 for rAAV2. At the same MOI, almost 100% of cells expressed GFP when rAd was used. However, the transduction efficiency for rAA V2 was comparable to that of rAd when coinfected with wt adenovirus, leading to a dramatic increase in the amount of double-stranded rAAV2 DNA. Similar results were obtained in vivo. While widespread GFP expression was readily detected in all xenografts injected with rAd, only one section of all tumors injected with rAAV2 contained GFP-positive cells.The results of this study indicate that rAAV2 might be useful for an ex vivo approach in cancer gene therapy, but it does not seem to be feasible for the in vivo treatment of malignant tumors.

Purpose In this study, the efficacy of combining ZD6474 (Zactima), a vascular endothelial growth factor (VEGF) receptor 2–associated tyrosine kinase inhibitor currently undergoing Phase II clinical trial evaluation, with single and fractionated dose radiation exposures was examined in a human colorectal carcinoma model (HT29). Methods and Materials HT29 xenograft–bearing mice were treated with either single-dose (10 Gy) or multifraction (2 Gy/day for 2 weeks) radiotherapy alone or in conjunction with a 2-week course of ZD6474 (25 mg/kg). In the single-dose investigation, ZD6474 treatment followed radiotherapy, whereas in the fractionated dose studies the antiangiogenic therapy was given before, after, or concurrent with the radiation. Tumor response was determined by tumor growth delay. Results ZD6474 increased the response of HT29 xenografts to both single and fractionated dose radiotherapy. In the fractionation studies sequencing of therapies had little impact on treatment outcomes; the time for the median tumors in each of the treatment groups to grow to five times the starting size was 53, 53.5, and 49 days, respectively. Conclusions These studies indicate that ZD6474, when used in conjunction with radiation therapy, has a clear therapeutic advantage, providing a rationale for considering the combination of this agent with radiotherapy in the clinic.

Vascular disrupting agents (VDAs) are designed to cause a rapid and selective shutdown of the established tumor vasculature, which leads to secondary ischemic tumor cell death.We examined the efficacy of a novel VDA, MN-029, in the rodent KHT sarcoma model.A significant reduction in the functional vessel number was observed after intraperitoneal injection of MN-029 at a dose of 100 mg/kg. Histological evaluation showed extensive necrosis (approximately 90%) by 24 h. MN-029 treatment to the tumor-bearing mice also resulted in a dose-dependent tumor cell killing. When used in combination with radiation or cisplatin chemotherapy, a 100 mg/kg dose of MN-029 significantly enhanced tumor killing compared to that seen with radiation or cisplatin alone.The results demonstrated that MN-029 could cause rapid vascular shutdown in solid tumors, dose-dependent secondary tumor cell killing, and effective enhancement of the antitumor effects of radiation and cisplatin chemotherapy.

The antitumor efficacy of combination therapy of the vascular disrupting agent ZD6126 and antiangiogenic agent ZD6474 was evaluated in the models of human colorectal (HT29) and ovarian carcinoma (OW1).HT29 and OW1 xenograft-bearing mice were treated with ZD6126 and ZD6474 either alone or in combination. ZD6126 therapy consisted of three doses of 100 mg/kg administered 1, 3 and 5 days after the tumor reached the starting size. ZD6474 was administered daily at a dose of 25 mg/kg on days 1-5.ZD6126 and ZD6474 treatment alone only resulted in modest tumor growth delay. However, significantly enhanced antitumor effects were observed in the combination treatment.The combination of the vascular disrupting with the antiangiogenic agent led to significant enhancement of antitumor efficacy in HT29 and OW1 human tumor models. Such combination therapy may have significant therapeutic benefit even in tumors insensitive to either treatment alone.

The therapeutic potential of targeting the tumor vascular supply is now widely recognized. Intense research and development activity has resulted in a variety of investigational agents, a number of which are currently in clinical development. As these novel agents are quite distinct from the cytotoxic drugs conventionally used in the treatment of solid tumors, it will be particularly important to ensure early differentiation of these vascular-targeted therapies in order to encourage widespread understanding of their potential benefits and application in the clinic. Two distinct groups of vascular-targeted therapies have evolved: antiangiogenic agents and vascular-disrupting approaches. These differ in three key respects: their physiologic target, the type or extent of disease that is likely to be susceptible, and the treatment scheduling. Inhibitors of angiogenesis interfere with new vessel formation and therefore have a preventative action, require chronic administration, and are likely to be of particular benefit in early-stage or asymptomatic metastatic disease. Vascular-disrupting agents target the established tumor blood vessels, resulting in tumor ischemia and necrosis. These agents are therefore given acutely, show more immediate effects, and may have particular efficacy against advanced disease. It is essential that these agents can be readily distinguished from conventional therapies and that an understanding of key differences between the two types of vascular-targeted therapies is fostered. Here, a simple taxonomy and nomenclature is proposed in anticipation that the therapeutic potential of this novel class can be realized as these approaches advance in clinical settings and a new anticancer strategy becomes available in the clinic.

BACKGROUND Tumor endothelium represents a valuable target for cancer therapy. The vasculature plays a critical role in the survival and continued growth of solid tumor masses; in addition, the inherent differences between tumor blood vessels and blood vessels associated with normal tissue make the tumor vasculature a unique target on which to base the design of novel therapeutics, which may allow highly selective treatment of malignant disease. Therapeutic strategies that target and disrupt the already formed vessel networks of growing tumors are actively being pursued. The goal of these approaches is to induce a rapid and catastrophic shutdown of the vascular function of the tumor so that blood flow is arrested and tumor cell death due to the resulting oxygen and nutrient deprivation and buildup of waste products occurs. METHODS Biologic approaches and small-molecule drugs that can be used to damage tumor vasculature have been identified. Physiologic, histologic/morphologic, and immunohistochemical assessments have demonstrated that profound disruption of the tumor vessel network can be observed minutes to hours after treatment. The small-molecule agents that have made the greatest advances in the clinical setting (5,6-dimethylxanthenone-4-acetic acid [DMXAA], combretastatin A4 disodium phosphate [CA4DP], and ZD6126) are the focus of the current review. RESULTS Loss of patent blood vessels, decreased tumor blood flow, extensive necrosis, and secondary ischemia-induced tumor cell death have been well documented in a variety of preclinical tumor models treated with agents such as DMXAA, CA4DP, and ZD6126. The use of such agents in conjunction with irradiation and other chemotherapeutic agents has led to improved treatment outcomes. CONCLUSIONS The targeting of tumors' supportive blood vessel networks could lead to improvements in cancer cure rates. It is likely that this approach will prove to be most efficacious when used in concert with conventional treatment strategies. Cancer 2004. © 2004 American Cancer Society.

Purpose To evaluate the antitumor efficacy of a vascular targeting strategy that combines an agent that disrupts established tumor blood vessels (ZD6126) with one that interferes with new vessel formation (ZD6474) in models of human renal cell carcinoma (Caki-1) and Kaposi's sarcoma (KSY-1). Methods and materials Caki-1 and KSY-1 xenograft-bearing nude mice were treated with ZD6126 and ZD6474 either as single agents or in combination when the tumors reached a size of ∼200 mm 3 . ZD6126 therapy consisted of three doses of 100 mg/kg administered 1, 3, and 5 days after the tumor reached the starting size. ZD6474 was administered daily (25 mg/kg) on Days 1–5. In the combination studies, ZD6474 treatment began immediately after the first dose of ZD6126. The tumor response to treatment was evaluated using a regrowth delay endpoint. Results Significant tumor growth delays were observed in both tumor models with either agent with the treatment regimen used. In the Caki-1 and KSY-1 models, respectively, ZD6126 treatment resulted in a tumor growth delay of 23 and 26 days and ZD6474 produced a tumor growth delay of 24.5 and 14.5 days. When ZD6126 and ZD6474 were combined, the tumor growth delays increased to 55 (Caki-1) and 86 (KSY-1) days. In the KSY-1 model, the combination therapy also resulted in 3 of 8 long-term tumor-free survivors. Conclusion These results indicate that statistically significant antitumor efficacy can be achieved using a treatment strategy that combines a therapy that targets the established tumor blood vessels with one that interferes with the process of angiogenesis.

Vascular endothelial growth factor (VEGF) is one of the most important factors involved in tumor angiogenesis.Antisense phosphorothiolate oligodeoxynucleotides (PS-ODNs) were used to reduce VEGF production while the small molecule PD0203359-0002 (PD203359) was used to inhibit VEGF/bFGF receptor tyrosine kinase activity.PD203359 exposure was found to profoundly impair the growth of human endothelial cells (HMVEC-L) at doses 20-fold less than those affecting human renal cell carcinoma (Caki-1) cell growth. In vivo, treatment with PD203359 inhibited tumor cell-induced angiogenesis and resulted in a significant tumor growth delay. Treatment with VEGF antisense PS-ODNs also significantly increased the time for tumors to grow to five times the starting size. Most importantly, when the PD203359 and VEGF antisense treatments were combined, a greater antitumor response than could be achieved with either therapy alone was observed.Simultaneously targeting VEGF production and VEGF receptor signaling enhances the anticancer efficacy of either therapy alone.

Current and potential shortfalls in the number of radiation scientists stand in sharp contrast to the emerging scientific opportunities and the need for new knowledge to address issues of cancer survivorship and radiological and nuclear terrorism. In response to these challenges, workshops organized by the Radiation Research Program (RRP), National Cancer Institute (NCI) (Radiat. Res. 157, 204-223, 2002; Radiat. Res. 159, 812-834, 2003), and National Institute of Allergy and Infectious Diseases (NIAID) (Nature, 421, 787, 2003) have engaged experts from a range of federal agencies, academia and industry. This workshop, Education and Training for Radiation Scientists, addressed the need to establish a sustainable pool of expertise and talent for a wide range of activities and careers related to radiation biology, oncology and epidemiology. Although fundamental radiation chemistry and physics are also critical to radiation sciences, this workshop did not address workforce needs in these areas. The recommendations include: (1) Establish a National Council of Radiation Sciences to develop a strategy for increasing the number of radiation scientists. The strategy includes NIH training grants, interagency cooperation, interinstitutional collaboration among universities, and active involvement of all stakeholders. (2) Create new and expanded training programs with sustained funding. These may take the form of regional Centers of Excellence for Radiation Sciences. (3) Continue and broaden educational efforts of the American Society for Therapeutic Radiology and Oncology (ASTRO), the American Association for Cancer Research (AACR), the Radiological Society of North America (RSNA), and the Radiation Research Society (RRS). (4) Foster education and training in the radiation sciences for the range of career opportunities including radiation oncology, radiation biology, radiation epidemiology, radiation safety, health/government policy, and industrial research. (5) Educate other scientists and the general public on the quantitative, basic, molecular, translational and applied aspects of radiation sciences.

Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines.We compared the efficacy of the CMV promoter to the promoter of the elongation factor-1 alpha (EF-1 alpha) for the generation of stable colon carcinoma cell lines (HT-29). Green fluorescent protein (GFP) expression cassettes were delivered by recombinant adeno-associated virus (AAV) which is known for its ability to stably transduce cells. Stable cell lines were characterized in vitro by FACS and in vivo after HT-29 clones were grown as xenografts in nude mice.Stable HT-29 clones with97% of all cells homogeneously expressing GFP were generated with the EF-1 alpha promoter. In contrast in clones carrying the CMV promoter, only up to 60% of the cells were GFP-positive with expression levels varying widely between cells. Superinfection with wild-type adenovirus induced GFP expression in more than 90% of the cells indicating that the CMV promoter was silenced. In vivo the tumors carrying the EF-1 alpha promoter were homogeneously GFP-positive, whereas the CMV promoter gave rise to a scattered pattern of GFP expression.This study underlines the importance of the promoter for the generation of stable cell lines. In addition it demonstrates that recombinant AAV can effectively be used as a gene delivery system for this purpose.

It has been well established that the vascularization of solid tumors is a prerequisite if a clinically relevant size is to be reached. For progressive tumor growth, the vessel network must continuously expand to satisfy the neoplastic cells' nutritional needs and waste product removal requirements. This utter reliance of the tumor on its vasculature provides a logical target for new approaches to cancer therapy. Indeed, there currently exists a great deal of enthusiasm for the development of interventions that compromise the growth and/or function of the tumor neovasculature. Two primary directions are being pursued. Inhibitors of angiogenesis seek to interrupt the angiogenic process to prevent new vessel formation. Antivascular approaches aim to cause direct damage to the tumor endothelium and thus lead to extensive secondary neoplastic cell death. The application of such strategies as adjuvants to conventional radiation treatments offers unique opportunities to develop more effective cancer therapies.

The tumor vessel support network offers a tantalizing target for cancer therapy, given the complete dependence of a solid neoplasia on the vasculature for both the delivery of oxygen and other nutrients, as well as the effective removal of wasteproducts. Attacking a tumor's supportive blood vessel network offers ameans of improving cancer cure rates on the basis of two principles. The first reflects evidence indicating that physiological conditions in tumors, arising primarily as a consequence of inadequate and non-uniform vascular networks, are significant contributors to resistance to non-surgical anticancer treatments. The second involves the recognition that the inherent differences between blood vessels in tumors and those associated with normal tissues provide a variety of unique targets for the design of novel therapeutics, highly selective for neoplastic growth. Therapeutic approaches that aim to destroy the tumor endothelium are being actively pursued. The application of such vascular targeting strategies as adjuvants to conventional therapeutics such as radiotherapy, offers unique opportunities to develop even more effective cancer therapies.

Purpose: The present report reviews the preclinical data on combined chemotherapy/vascular targeting agent treatments. Basic principles are illustrated in studies evaluating the antitumor efficacy of the vascular targeting agent ZD6126 ( N -acetylcochinol- O -phosphate) when combined with the anticancer drug cisplatin in experimental rodent (KHT sarcoma) and human renal (Caki-1) tumor models. Methods and Materials: C3H/HeJ and NCR/nu-nu mice bearing i.m. tumors were injected i.p. with ZD6126 (0–150 mg/kg) or cisplatin (0–20 mg/kg) either alone or in combination. Tumor response to treatment was assessed by clonogenic cell survival. Results: Treatment with ZD6126 was found to damage existing neovasculature, leading to a rapid vascular shutdown. Histologic evaluation showed dose-dependent morphologic damage of tumor cells within a few hours after drug exposure, followed by extensive central tumor necrosis and neoplastic cell death as a result of prolonged ischemia. ZD6126 doses that led to pathophysiologic effects also enhanced the tumor cell killing of cisplatin when administered either 24 h before or 1–24 h after chemotherapy. In both tumor models, the administration of a 150 mg/kg dose of ZD6126 1 h after a range of doses of cisplatin resulted in an increase in tumor cell kill 10–500-fold greater than that seen with chemotherapy alone. In contrast, the inclusion of the antivascular agent did not increase bone marrow stem cell toxicity associated with this anticancer drug. Conclusion: The results obtained in the KHT and Caki-1 tumor models indicate that ZD6126 effectively enhanced the antitumor effects of cisplatin therapy. These findings are representative of the marked enhancements generally observed when vascular targeting agents are combined with chemotherapy in solid tumor therapy.

Purpose: The aim of this study was to evaluate the antitumor efficacy of the novel vascular targeting agent ZD6126 ( N -acetylcochinol- O -phosphate) in the rodent KHT sarcoma model, either alone or in combination with single- or fractionated-dose radiation therapy. Methods: C3H/HeJ mice bearing i.m. KHT tumors were injected i.p. with ZD6126 doses ranging from 10 to 150 mg/kg. Tumors were irradiated locally in unanesthetized mice using a linear accelerator. Tumor response to ZD6126 administered alone or in combination with radiation was assessed by clonogenic cell survival assay or tumor growth delay. Results: Treatment with ZD6126 led to a rapid tumor vascular shutdown as determined by Hoechst 33342 diffusion. Histologic evaluation showed morphologic damage of tumor cells within a few hours after drug exposure, followed by extensive central tumor necrosis and neoplastic cell death as a result of prolonged ischemia. When combined with radiation, a 150 mg/kg dose of ZD6126 reduced tumor cell survival 10–500-fold compared with radiation alone. These enhancements in tumor cell killing could be achieved for ZD6126 given both before and after radiation exposure. Further, the shape of the cell survival curve observed after the combination therapy suggested that including ZD6126 in the treatment had a major effect on the radiation-resistant hypoxic cell subpopulation associated with this tumor. Finally, when given on a once-weekly basis in conjunction with fractionated radiotherapy, ZD6126 treatment was found to significantly increase the tumor response to daily 2.5 Gy fractions. Conclusion: The present results demonstrated that in the KHT sarcoma, ZD6126 caused rapid tumor vascular shutdown, induction of central tumor necrosis, tumor cell death secondary to ischemia, and enhancement of the antitumor effects of radiation therapy.

Combretastatin A4 disodium phosphate (CA4DP) was evaluated in a xenograft model of AIDS-KS. KS xenografts were highly vascular, showing brisk mitotic activity, focal areas of necrosis, and intervening fibrovascular septae. Neoplastic cells were large or spindle-shaped, with vesicular nuclei and modest pleomorphism. Multiple junctions, microvillous-like projections, abortive lumina and rare Weibel Palade bodies were revealed by electron microscopy. Treatment with CA4DP (100 mg/kg) resulted in rapid onset of vascular effects that within 4 h resulted in an almost complete vascular shutdown in these tumors. Histological evaluation showed morphological damage within a few hours after treatment, followed by extensive necrosis which increased to approximately 90% by 24 h. At this time, viable tumor cells were evident only at the periphery of the tumor. These findings demonstrate not only the marked susceptibility of the KS model to CA4DP but also its potential application in studies related to the pathogenesis and therapy of AIDS-KS.

The utility of combining the vascular targeting agents 5,6-dimethyl-xanthenone-4 acetic acid (DMXAA) and combretastatin A-4 disodium phosphate (CA4DP) with the anticancer drugs cisplatin and cyclophosphamide (CP) was evaluated in experimental rodent (KHT sarcoma), human breast (SKBR3) and ovarian (OW-1) tumor models. Doses of the vascular targeting agents that led to rapid vascular shutdown and subsequent extensive central tumor necrosis were identified. Histologic evaluation showed morphologic damage of tumor cells within a few hours after treatment, followed by extensive hemorrhagic necrosis and dose-dependent neoplastic cell death as a result of prolonged ischemia. Whereas these effects were induced by a range of CA4DP doses (10–150 mg/kg), the dose response to DMXAA was extremely steep; doses ≤ 15 mg/kg were ineffective and doses ≥ 20 mg/kg were toxic. DMXAA also enhanced the tumor cell killing of cisplatin, but doses > 15 mg/kg were required. In contrast, CA4DP increased cisplatin-induced tumor cell killing at all doses studied. This enhancement of cisplatin efficacy was dependent on the sequence and interval between the agents. The greatest effects were achieved when the vascular targeting agents were administered 1–3 hr after cisplatin. When CA4DP (100 mg/kg) or DMXAA (17.5 mg/kg) were administered 1 hr after a range of doses of cisplatin or CP, the tumor cell kill was 10–500-fold greater than that seen with chemotherapy alone. In addition, the inclusion of the antivascular agents did not increase bone marrow stem cell toxicity associated with these anticancer drugs, thus giving rise to a therapeutic gain. © 2002 Wiley-Liss, Inc.

A variety of approaches has demonstrated that interfering with tumor-induced angiogenesis may be an effective strategy in cancer therapy. However, it is likely that to be most effective such strategies will require extended suppression of the angiogenic process. Gene therapy offers a possible approach to achieve sustained release of a therapeutically potent transferred gene product. In the present study the angiogenesis inhibitor endostatin was expressed through a recombinant adeno-associated viral (rAAV) vector and shown to be biologically active in vitro and in vivo. Intramuscular injection of rAAV-HuEndo (1 x 10(9) i.u.) led to a sustained serum endostatin level of approximately 35-40 ng/mL. This endostatin level was sufficient to inhibit tumor cell-induced angiogenesis and to suppress both the initiation and subsequent growth of a human colorectal cancer model.

The failure of standard of care treatment for patients diagnosed with glioblastoma (GBM) coupled with the highly vascularized nature of this solid tumor has led to the consideration of agents that target vascular endothelial growth factor (VEGF) or its receptors, as alternative therapeutic strategies for this disease. While early clinical studies determined that recurrent GBMs respond favorably to bevacizumab (BVZ), progression of tumors after anti-VEGF targeted therapy is inevitable and in many instances the disease presents with a more aggressive phenotype. Recently completed phase III trials evaluating BVZ in combination with chemoradiation in newly diagnosed GBM also failed to establish an overall survival benefit. Hence there is a critical need to understand why anti-angiogenic therapies are ineffective in GBM patients. In particular, knowledge of the mechanisms responsible for adaptive resistance to VEGF/VEGFR targeting agents, that characterize recurring tumors, will be needed if significant advancement in prolonging survival rates of GBM patients is to be achieved. The results reported herein, suggest a potential mechanism by which anti-VEGF/VEGFR therapies regulate the enhanced invasive phenotype through a pathway that involves transforming growth factor beta (TGFβ) receptor (TGFβR) and chemokine receptor CXCR4. The VEGFR signaling inhibitors (Cediranib and Vandetanib) elevated the expression of CXCR4 in VEGFR-expressing primary patient-derived GBM cell lines and tumors, and enhanced the in vitro migration of these lines toward CXCL12. The combination of Cediranib and the CXCR4 antagonist AMD3100/Plerixafor provided a greater survival benefit to tumor-bearing animals, compared to monotherapies with these agents. The upregulation of CXCR4 by VEGFR inhibitors was dependent on TGFβ/TGFβR, but not HGF/MET, signaling activity, suggesting a mechanism of crosstalk among VEGF/VEGFR, CXCL12/CXCR4, and TGFβ/TGFβR pathways in the malignant phenotype of recurrent tumors after anti-VEGF/VEGFR therapies. Thus, the combination of VEGFR, CXCR4, and TGFβR inhibitors could provide an alternative strategy to halt GBM progression.

Background: Efforts to impede early steps in the metastatic cascade have led to the development of promising small molecule targeting agents. The purpose of this study was to evaluate the use of Src tyrosine kinase inhibitors known to interfere with cell migration and invasion, in combination with a cytostatic soy isoflavone extract (ISFs) on metastatic activity in prostate cancer cells. Methods: PC-3ML cells (a gift from A. Fatatis, Drexel University), and LNCaP cells treated with a Src inhibitor, ISFs, or a combination, were evaluated for clonogenic cell survival, cell cycle progression, and transwell migration and invasion activity. Results: Src inhibitors and ISFs induced an accumulation of cells in the G1 and G2/M phases of the cell cycle, respectively, with little change in cell viability or survival. Invasion studies with PC-3 ML and LNCaP cells suggest that ISFs can reduce metastatic activity by 39% and 35%, respectively. In addition, ISFs enhance the effectiveness of the Src inhibitors to reduce invasiveness. This may indicate that including ISFs in treatment regimens may allow a lower dose of the targeting agent to be used to achieve optimal response and decrease toxicity. Conclusions: In vitro studies suggested that combining a Src inhibitor and ISFs resulted in greater inhibition of metastatic activity than either alone. Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Combining tyrosine kinase inhibitors and isoflavones to target metastatic activity in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4037. doi:10.1158/1538-7445.AM2014-4037

It is estimated that ∼85% of patients with advanced prostate cancer develop skeletal metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of the prognosis. The development of novel therapeutic strategies that impair the metastatic process and associated skeletal adversities is therefore critical to improving prostate cancer patient survival. Tumor cells rely on proteolytic enzymes for successful establishment of metastases. Cathepsin L (CTSL) is a lysosomal cysteine protease that is up regulated in a wide range of human cancers including prostate cancer. The elevated secretion of CTSL by aggressively metastasizing tumor cells plays a key role in metastatic spread of tumor cells through proteolytic degradation of extracellular matrix and basement membrane components. In addition, tumor secreted cytokines have been shown to increase osteoclastic secretion of CTSL to promote bone resorption. Intervention strategies targeting CTSL may therefore serve a dual purpose by inhibiting both metastatic dissemination as well as bone resorption. The goal of present studies was to evaluate the anti-metastatic and anti-bone resorptive efficacy of CTSL inhibition using the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94). KGP94 treatment significantly impaired the metastatic phenotype of highly metastatic prostate cancer PC-3ML cells. Treatment with 25µM KGP94 decreased the migratory and invasive capacities of PC-3ML cells by 78 and 53% respectively. To validate the anti-metastatic efficacy of KGP94 in-vivo, luciferase labelled PC-3ML cells were injected into the left ventricle of male nu/nu mice followed by daily treatment with 20mg/kg KGP94. Metastatic progression was monitored by weekly bioluminescence imaging using firefly luciferase reporter system. KGP94 treatment led to a significant reduction in metastatic tumor burden (65% reduction) and a reduction in metastatic incidence. Histological assessment of bones further confirmed the reduction in metastatic burden upon KGP94 treatment. CTSL inhibition also led to a significant increase in overall survival of tumor bearing mice (p=0.01). In vitro assessment of anti-resorptive function of KGP94 was performed using osteoclastogenesis and pit formation assays. KGP94 treatment of RAW264.7 osteoclast precursor cells led to a significant decrease (72%) in osteoclast formation as demonstrated by a decrease in the number of TRAP+ multinucleate cells. KGP94 also inhibited the bone resorptive activity of osteoclasts as indicated by a significant reduction in the number of resorptive pits formed by osteoclasts on bone slices. In conclusion, our findings suggest that CTSL inhibition using small molecule KGP94 can significantly impair metastatic dissemination of prostate cancer cells and alleviate bone resorption. Citation Format: Dhivya R. Sudhan, Dietmar W. Siemann. In-vivo evaluation of the anti-metastatic efficacy of small molecule Cathepsin L inhibitor KGP94 in a prostate cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4030. doi:10.1158/1538-7445.AM2014-4030

Purpose: The cysteine cathepsin proteases are known to play an important role in tumor progression, invasion and angiogenesis. The present study evaluated the anti-tumor activity and pharmacokinetics of the small-molecule cathepsin L inhibitor KGP94. Materials and Methods: Male CDF1 or C3H/HeNHsd mice were inoculated on the right rear foot with a C3H mammary carcinoma or a SCCVII carcinoma, respectively. A solution of KGP94 was prepared each day by dissolving in a mixture of 10% Tween 80 and 90% HEPES-buffer. It was intraperitoneally injected at 0.01 ml/g mouse bodyweight. Various doses (1-20 mg/kg) were administered between 1-20 days, starting from the day of tumor inoculation. Anti-tumor activity was assessed by determining the tumor growth time, which was the time in days to reach a volume of 500 mm3 (TGT500). Pharmacokinetics of KGP94 were investigated by drawing blood samples before, and up to 24 hours after KGP94 injection. Results are listed as Mean (± Standard Error). One-way ANOVA comparison of group means was performed, and a P Results: The TGT500 for control animals was 18.0 days (± 0.3) for the C3H mammary carcinoma and 13.6 days (± 0.7) for the SCCVII carcinoma. Treating tumors with KGP94 significantly increased the TGT500 for both tumor models to around 21 and 17 days respectively, when doses were at 10.0 mg/kg or higher. At a dose of 5.0 mg/kg the TGT500 of the C3H mammary carcinoma was significantly increased to 21.4 days (± 1.1), whereas the TGT500 of the SCCVII carcinoma was unchanged at 13.8 (± 0.2). At lower KGP94 doses neither tumor model showed significant growth delay. Additional experiments with the C3H mammary carcinoma model showed that treatment was most effective when started immediately after tumor inoculation, and that treatment for 5 days was just as effective as 20 days. Pharmacokinetic analysis of KGP94 levels in mouse plasma is ongoing. Conclusion: In the C3H mammary carcinoma KGP94 significantly delayed tumor growth, but the effect was primarily on the early phase following tumor cell inoculation. This suggests that KGP94 may have a more significant role in inhibiting the establishment of metastasis rather than influencing the growth of established tumors. Significant growth delay was also observed in the SCCVII tumor model. However, it occurred with higher doses of KGP94 compared to the C3H mammary carcinoma. The higher dose could be explained by the more aggressive nature of the SCCVII tumor model. Supported by The Danish Cancer Society, The Danish Council for Independent Research: Medical Sciences, and OXiGENE Inc. Citation Format: Thomas R. Wittenborn, Michael Stratford, Mary Lynn Trawick, Kevin G. Pinney, David J. Chaplin, Dietmar W. Siemann, Michael R. Horsman. Assessment of anti-tumor activity of the cathepsin L inhibitor, KGP94. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1816. doi:10.1158/1538-7445.AM2014-1816

Background Angiogenesis is an essential process during tumor development and growth. The murine dorsal skinfold window chamber model has been used for the study of both tumor microvasculature and other vascular diseases, including the study of anti-angiogenic agents in cancer therapy. Hyperspectral imaging of oxygen status of the microvasculature has not been widely used to evaluate response to inhibition of angiogenesis in early tumor cell induced vascular development. This study demonstrates the use of two different classes of anti-angiogenic agents, one targeting the Vascular Endothelial Growth Factor (VEGF) pathway involved with vessel sprouting and the other targeting the Angiopoietin/Tie2 pathway involved in vascular destabilization. Studies evaluated the tumor microvascular response to anti-angiogenic inhibitors in the highly angiogenic renal cell carcinoma induced angiogenesis model. Methods Human renal cell carcinoma, Caki-2 cells, were implanted in the murine skinfold window chamber. Mice were treated with either VEGF pathway targeted small molecule inhibitor Sunitinib (100 mg/kg) or with an anti-Ang-2 monoclonal antibody (10 mg/kg) beginning the day of window chamber surgery and tumor cell implantation. Hyperspectral imaging of hemoglobin saturation was used to evaluate both the development and oxygenation of the tumor microvasculature. Tumor volume over time was also assessed over an 11-day period post surgery. Results The window chamber model was useful to demonstrate the inhibition of angiogenesis using the VEGF pathway targeted agent Sunitinib. Results show impairment of tumor microvascular development, reduced oxygenation of tumor-associated vasculature and impairment of tumor volume growth compared to control. On the other hand, this model failed to demonstrate the anti-angiogenic effect of the Ang-2 targeted agent. Follow up experiments suggest that the initial surgery of the window chamber model may interfere with such an agent thus skewing the actual effects on angiogenesis. Conclusions Results show that this model has great potential to evaluate anti-VEGF, or comparable, targeted agents; however the mere protocol of the window chamber model interferes with proper evaluation of Ang-2 targeted agents. The limitations of this in vivo model in evaluating the response of tumor vasculature to anti-angiogenic agents are discussed.

The interaction between 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and radiation was investigated in two different mouse tumor models and a normal mouse tissue. C3H mouse mammary carcinomas transplanted in the feet of CDF1 mice and KHT mouse sarcomas growing in the leg muscles of C3H/HeJ mice were used. DMXAA was dissolved in saline and injected intraperitoneally. Tumors were irradiated locally in nonanesthetized mice, and response was assessed using tumor growth for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. DMXAA alone had an antitumor effect in both tumor types, but only at doses above 15 mg/kg. DMXAA also enhanced radiation damage, and again there was a threshold dose. No enhancement was seen in the C3H mammary carcinoma at 10 mg/kg and below, while in the KHT sarcoma, doses above 15 mg/kg were necessary. This enhancement of radiation damage was also dependent on the sequence of and interval between the treatments with DMXAA and radiation. Combining radiation with DMXAA at the maximum tolerated dose (i.e., the highest dose that could be injected without causing any lethality) of either 20 mg/kg (CDF1 mice) or 17.5 mg/kg (C3H/HeJ mice) gave an additive response when the two agents were administered simultaneously. Even greater antitumor effects were achieved when DMXAA was administered 1-3 h after irradiation. However, when administration of DMXAA preceded irradiation, the effect was similar to that seen for radiation alone, suggesting that appropriate timing is essential to maximize the utility of this agent. When such conditions were met, DMXAA was found to increase the tumor response significantly in the absence of an enhancement of radiation damage in normal skin, thus giving rise to therapeutic gain.

Background and purpose : The ability of combretastatin A-4 disodium phosphate (CA4DP) to induce vascular damage and enhance the radiation response of murine tumors was investigated. Materials and methods : A C3H mouse mammary carcinoma transplanted in the foot of CDF1 mice and the KHT mouse sarcoma growing in the leg muscle of C3H/HeJ mice were used. CA4DP was dissolved in saline and injected intraperitoneally. Tumor blood perfusion was estimated using 86 RbCl extraction and Hoechst 33342 fluorescent labelling. Necrotic fraction was determined from histological sections. Tumors were locally irradiated in non-anaesthetised mice and response assessed by local tumor control for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. Results : CA4DP decreased tumor blood perfusion and increased necrosis in a dose-dependent fashion in the C3H mammary carcinoma, which was maximal at 250 mg/kg. The decrease in perfusion and induction of necrosis by CA4DP was more extensive in the KHT sarcoma. CA4DP enhanced radiation damage in both tumor types. In the KHT sarcoma this enhancement was independent of whether the drug was given before or after irradiating, whereas for C3H mammary carcinoma the enhancement was only significant when administered at the same time or after the radiation, with no enhancement seen if CA4DP was given before. These effects were drug-dose dependent. CA4DP did not enhance radiation damage in normal skin. Conclusions : CA4DP enhanced radiation damage in the two tumor models without enhancing normal tissue damage. These radiation effects were clearly consistent with the anti-vascular action of CA4DP.

Background and purpose : The neovascularization of tumor cells is a prerequisite if a clinically relevant tumor size is to be reached. A continuously expanding vessel network supplying nutritional requirements and removing waste products is essential for continued tumor development, growth and survival. Results : In many tumors, the growing endothelium is unable to fully support the demands of the neoplastic cell population. As a consequence of the inadequacies of the resulting aberrant vasculature, microenvironmental conditions develop in tumors which are not only detrimental to the response of tumors to conventional anticancer treatments, but may lead to or predispose cells to genetic modifications resulting in more aggressive phenotypes and higher metastatic potential. Yet the utter dependence of the tumor on its induced vessel formation for growth, survival and spread has also created a great deal of enthusiasm for developing therapeutic approaches to specifically targeting the tumor microcirculation. Conclusions : The application of such strategies as adjuvants to conventional radiation treatments offers unique opportunities to develop more effective cancer therapies.

The effect of etoposide (VP-16) on a human epidermoid carcinoma cell line (HEp3) has been evaluated under different growth conditions. A significant increase in resistance to this agent from exponential to unfed plateau cells has been observed. Variations in the cell cycle distribution appear not to be sufficient to explain the difference in cell killing. Rather, our studies suggest the presence of an important proportion of quiescent (Q) cells in the plateau phase cultures which may determine the treatment outcome.

Our laboratory has concentrated on the possible regulation the benzamides and nicotinamides may have on the processes of DNA repair and apoptosis. Recent reports [14-16] have suggested that both apoptosis and inflammation are regulated by the transcription factor NF-kB. We have initiated studies regarding the hypothesis that the benzamides and nicotinamides could inhibit the production of tumor necrosis factor alpha (TNFalpha) and the inflammatory response as well as induce apoptosis via inhibition of NF-kB. Our data have shown that nicotinamide and two N-substituted benzamides, metoclopramide (MCA) and 3-chloroprocainamide (3-CPA), gave dose dependent inhibition of lipopolysacharide induced TNFalpha in the mouse within the dose range of 10-500 mg/kg. Moreover, lung edema was prevented in the rat by 3 i 50 mg/kg doses of 3-CPA or MCA, and 100-200 μM doses of MCA could also inhibit NF-kB in Hela cells. Taken together these data strongly support the notion that benzamides and nicotinamides have potent anti-inflammatory and antitumor properties, because their primary mechanism of action is regulated by inhibition at the gene transcription level of NF-kB, which in turn inhibits TNFalpha and induces apoptosis.

Purpose: One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background. Methods: The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol. Results: The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to ∼200 nmol/min/mg, and the NQO1 activity in KHT/iv increased ∼10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis. Conclusions: Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.

Purpose: The aim of this study was to evaluate the antitumor efficacy of combretastatin A-4 disodium phosphate (combretastatin prodrug) in the rodent KHT sarcoma model either alone or in combination with radiation therapy. Methods: KHT tumors were grown in C3H/HeJ mice. Combretastatin A-4 prodrug was injected intraperitoneally at doses ranging from 10 to 100 mg/kg. Tumors were irradiated in unanesthetized mice using a 137 Cs source. Tumor response to combretastatin A-4 prodrug was assessed by histological evaluations as well as an in vivo to in vitro cell survival assay. Results: Histological evaluation showed morphological damage of tumor cells within a few hours after drug treatment, followed by extensive central necrosis. Administering increasing doses of combretastatin A-4 prodrug to tumor-bearing mice resulted in a dose-dependent increase in cell killing irrespective of whether the tumors were irradiated or not. When combined with radiation, a 100 mg/kg dose of combretastatin A-4 prodrug reduced tumor cell survival 10–500-fold lower than that seen with radiation alone. Further, the shape of the cell survival curve observed following the combination therapy suggested that including combretastatin in the treatment had a major effect on the radiation-resistant hypoxic cell subpopulation associated with this tumor. Conclusion: The present results demonstrated that in the KHT sarcoma, combretastatin A-4 prodrug caused rapid vascular shutdown, a concentration-dependent direct cell killing, and effective enhancement of the antitumor effects of radiation therapy.

Purpose: Cathepsins are a class of proteolytic enzymes recognized to play an important role in tumour progression, invasion and angiogenesis. The present study evaluated the anti-tumour activity of the small-molecule cathepsin L inhibitor KGP94 in a murine breast tumour model. Materials and Methods: Male CDF1 mice were inoculated on the right rear foot with a C3H mammary carcinoma. A solution of KGP94 was prepared each day by dissolving in a mixture of 10% Tween 80 and 90% HEPES-buffer. It was intraperitoneally injected at 0.01ml/g mouse bodyweight. Various doses (5-20mg/kg) were administered between 5-20 days, starting either from the day of tumour implant or when tumours had reached a volume of 200mm3. Tumour response was assessed by determining the tumour growth time, which was either the time in days to reach a volume of 500mm3 (TGT500) for animals treated from the day of tumour implant, or 1000mm3 (TGT1000) for animals treated when tumours were at a volume of 200mm3. Results are listed as Mean (± Standard Error). One-way ANOVA with a posthoc Student-Newman-Keuls comparison of group means was performed, and a P Results: The TGT500 for control animals was 18.3 days (± 0.4). Treating tumours with 10mg/kg for 5 days significantly increased the TGT500 to 21.9 days (± 0.7). When the time of treatment with this dose was increased to 10 or 20 days, no additional growth inhibition was observed. Varying the drug dose for this 5 days treatment period showed that the TGT500 with 5mg/kg and 20mg/kg were 23.5 days (± 1.9) and 23.4 days (± 1.1), respectively. For experiments in which treatments were started when tumours had already reached 200mm3 an effect on TGT1000 was only observed with the highest drug dose at 20mg/kg; the respective TGT1000 being 5.9 days (± 0.2) for controls and 8.3 days (± 0.7) for KGP94. Conclusion: This synthetic, small-molecule cathepsin L inhibitor significantly delayed tumour growth, but the effect was primarily on the early phase following tumour inoculation. Such an effect suggests that KGP94 may have a role to play in inhibiting metastatic development rather than influencing the growth of established tumours. Supported by The Danish Cancer Society, The Danish Council for Independent Research: Medical Sciences, and OXiGENE Inc. Citation Format: Thomas Wittenborn, Mary Lynn Trawick, Kevin G. Pinney, David J. Chaplin, Dietmar W. Siemann, Michael R. Horsman. KGP94, a small-molecule cathepsin L inhibitor with antitumor activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2013-5071

Overexpression of tyrosine kinases c-Src and c-Met has been shown to be highly associated with prostate cancer progression and poor clinical outcomes, therefore small molecule Src or Met inhibitors are being extensively evaluated in clinical trials for advanced prostate cancer. However clinical data suggest that single treatment with any of these inhibitors only yield limited effects in patients. One possibility is that redundant signaling pathways in tumor cells exist that allows tumor cell uninterrupted upon blockade of purely one pathway. We hypothesize that c-Src and c-Met are two compensatory pathways that may cooperate to promote prostate cancer progression, hence here we studied treatment effects of combining inhibitors targeting c-Src (dasatinib, saracatinib) and c-Met (ASLAN002, also named BMS-777607) on multiple functional behaviors associated with tumor growth and metastasis in c-Src and c-Met expressing prostate cancer cells (PC-3, DU145). Combination of Src inhibitors and ASLAN002 was found to inhibit cell proliferation and colony formation more strongly than either single agent, as assessed by MTT and clonogenicity, respectively. In a wound-healing assay, compared to either treatment alone, the combinatorial treatment exhibited greater suppression on cell motility induced by hepatocyte growth factor, the only known ligand of c-Met. Similarly, using a Matrigel-based Transwell assay, Src inhibitors plus ASLAN002 resulted in significant reduction of cell invasion in the presence or absence of HGF. Western blot analysis demonstrated that co-targeting Met and Src kinases lead to not only inhibition on c-Met and c-Src autophosphorylation but also greater blockade of multiple downstream kinases such as focal adhesion kinase (FAK), Akt and extracellular signal-regulated kinase (ERK). These data indicate that combined application of small molecule Src and Met inhibitors cause significant impairment on various phenotypes associated with prostate cancer progression in vitro including proliferation, survival, migration and invasion, suggesting that this combination intervention modality may serve as a potential anti-metastatic strategy that could be further tested in metastatic prostate cancer models in vivo. Citation Format: Yao Dai, Kyung-Mi Bae, Dietmar W. Siemann. Combined targeting c-Src and c-Met pathways to impair prostate cancer cell growth and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 927. doi:10.1158/1538-7445.AM2013-927

Angiogenesis, the process of new blood vessel formation from pre-existing ones, is crucial to tumor growth and progression. Understanding the interplay between tumor cells and surrounding vasculature is of significant interest towards the identification of novel pro-angiogenic factors and the development of anti-angiogenic therapies. Cathepsin L (CTSL) is a cysteine protease that is up regulated in a wide range of human cancers. In transformed cells extracellular CTSL levels can increase up to 200-fold and comprise up to 40% of total secreted proteins. The role of CTSL in promoting tumor metastasis is supported by recent studies in our laboratory which have shown that treatment with the small molecule CTSL inhibitor 3-bromophenyl-3-hydroxyphenyl-ketone thiosemicarbazone (KGP94) significantly impairs the metastatic phenotype of prostate and breast cancer cells. However, the contribution of CTSL to tumor angiogenesis remains less well explored. Thus, the goal of the present studies was to evaluate the role of tumor cell secreted CTSL in tumor cell induced angiogenesis and to determine whether KGP94 treatment could inhibit this process. Upon activation by pro-angiogenic stimuli, endothelial cells migrate and invade through the interstitium to form tubes. The effect of CTSL on the migratory, invasive and tube formation capacity of endothelial cells was therefore investigated. Stimulation of human micro-vascular endothelial cells of the lung (HMVEC-L) with purified human CTSL led to a dose dependent increase in endothelial cell invasion (1.5 fold at 10ng/ml and 2 fold at 100ng/ml). Furthermore, CTSL (100ng/ml) increased the tube forming ability of endothelial cells as demonstrated by an increase in total tube length, average length of individual tube and number of tubes formed. Tumor cells secrete high levels of CTSL; therefore the effect of tumor cell conditioned media on endothelial cell migratory and invasive potential was evaluated. Incubation of HMVEC-L with conditioned media from MDA-MB-231 breast cancer cells, led to a significant increase in their migration, invasion and tube formation capacity (migration 1.3 fold; invasion 2.5 fold). These pro-angiogenic endothelial cell functions were significantly impaired in HMVEC-L treated with 10 or 25μM KGP94. For example, there was a 55% reduction in conditioned media stimulated invasiveness at 10μM and a 68% reduction at 25μM. Similarly, KGP94 treatment also significantly impaired purified CTSL and conditioned media stimulated tube formation. Interestingly, KGP94 treatment alone, did not affect the migratory or invasive capacity of unstimulated endothelial cells, suggesting that this agent might selectively impede tumor induced angiogenesis. Citation Format: Dhivya R. Sudhan, Dietmar W. Siemann. Tumor angiogenesis: The role of Cathepsin L and its therapeutic intervention by the small molecule inhibitor KGP94. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2013-5093