Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for the biomedical science community to study active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers’ own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. To address this unmet need, we have developed a novel open-source software program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent system that provides proven advantages over existing alternatives to the scientific community. Without extra hardware, AMFIP enables the functional synchronization of the μManager software platform, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to those from Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for myriad hardware and software systems according to user-specific experimental requirements and environments. To validate the intended purposes of developing AMFIP, we applied it to elucidate the question whether single cells, prior to their full spreading, can sense and respond to a soft solid substrate, and if so, how does the interaction depend on the cell spreading time and the stiffness of the substrate. Using a CRISPR/Cas9-engineered human epithelial Beas2B (B2B) cell line that expresses mNeonGreen2-tagged mechanosensitive Yes-associated protein (YAP), we show that single B2B cells develop distinct substrate-stiffness-dependent YAP expressions within 10 hours at most on the substrate, suggesting that cells are able to sense, distinguish, and respond to mechanical cues prior to the establishment of full cell spreading. In summary, AMFIP provides a reliable, open-source, and cost-free solution that has the validated long-term utility to satisfy the need of automatic imaging operations in the scientific community.

Electrically excitable cells such as neurons transmit long-distance calcium or electrical signals to regulate their physiological functions. While the molecular underpinnings and down-stream effects of these intercellular communications in excitable cells have been well appreciated, little is known about whether and how non-excitable cancer cells spontaneously initiate and transmit long-distance intercellular signals. Here we report that non-excitable human colon and prostate cancer cells spontaneously initiate and spread intercellular calcium waves, in vitro and ex vivo. Xenograft model studies suggest that these calcium signals promote the growth rate of tumors in mice. Pharmacological studies elucidated that the inositol-trisphosphate-receptor (IP3R)-regulated calcium release from endoplasmic reticulum (ER), which is activated by the Gq-PLC-IP3R pathway, is a major cause for the initiation of spontaneous calcium transients. Further, the spatial-temporal characteristics of calcium dynamics can be tuned by the culture substrates of different mechanical stiffnesses. Our results provide evidence that calcium dynamics enables long-distance functional communication in non-excitable cancer cells and offer the potential to modulate calcium signaling for new cancer therapies.

Aerobic exercise is receiving increased recognition in oncology for its multiple purported benefits. Exercise is known to induce physiologic adaptations that improve patient quality-of-life parameters as well as all-cause mortality. There also is a growing body of evidence that exercise may directly alter the tumor microenvironment to influence tumor growth, metastasis, and response to anticancer therapies. Furthermore, the physiologic adaptations to exercise in normal tissues may protect against treatment-associated toxicity and allow for greater treatment tolerance. However, the exercise prescription required to induce these beneficial tumor-related outcomes remains unclear. This study characterized the aerobic adaptations to voluntary wheel running in normal tissues and the tumor microenvironment. Female, retired breeder BALB/c mice and syngeneic breast adenocarcinoma cells were utilized in primary tumor and metastasis models. Aerobic exercise was found to induce numerous adaptations across various tissues in these mice, although primary tumor growth and metastasis were largely unaffected. However, intratumoral hypoxia and global metabolism were altered in the tumors of exercising hosts relative to non-wheel running controls. Doxorubicin chemotherapy also was found to be more efficacious at delaying tumor growth with adjuvant aerobic exercise. Additionally, doxorubicin-induced cardiac toxicity was ameliorated in exercising hosts relative to non-wheel running controls. Taken together, these data suggest that the normal tissue and tumor microenvironment adaptations to aerobic exercise can improve doxorubicin efficacy while simultaneously limiting its toxicity.

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

Aerobic exercise is receiving increased recognition in oncology spheres for its multiple purported benefits. Exercise is known to induce physiologic responses that improve patient quality-of-life parameters as well as all-cause mortality. There is also a growing body of evidence that suggests exercise may directly impact tumor biology and therapy outcomes. Previous studies suggest that exercise can improve tumor perfusion and ameliorate intratumoral hypoxia, increase recruitment of cytotoxic immune cells, and alter metabolic substrate availability. Furthermore, the physiologic adaptations to exercise in normal tissue may protect against treatment-associated toxicity and allow for greater treatment tolerance. With regard to cancer metastasis, reports have been conflicting. The goal of this study was to characterize the effect of exercise on normal tissue adaptations, tumor microenvironment, metastasis, and doxorubicin cardiotoxicity in preclinical breast cancer models. Female, retired breeder BALB/c mice were employed, and aerobic exercise was modeled by singly-housing mice with access to wireless, low-profile voluntary running wheels. Sedentary controls were singly housed with plastic huts. Syngeneic 4T1 or EMT6 breast adenocarcinoma cells were inoculated either intraductally into the 4th mammary duct or intravenously into the tail-vein. In one study, doxorubicin was administered via 3 intraperitoneal (IP) injections over 7 days for a total dose of 11 mg/kg. Exercising mice steadily increased the amount they ran until they reached a plateau of 10-12 km/day from day 10 onward. This exercise regime induced cardiac hypertrophy and oxidative phenotypes in the quadriceps muscle relative to non-wheel running controls. It also prevented the cardiac atrophy and bodyweight loss induced by doxorubicin treatment. In mice bearing intraductal EMT6 tumors, exercise delayed tumor growth and improved survival relative to non-wheel running controls. However, exercise did not alter 4T1 tumor growth or survival. The effect of exercise on metastasis was assessed under three conditions, 1) in mice that ran before and after intravenous 4T1 cell injection, 2) mice that had limited (low exercise) or full (high exercise) access to running wheels after intravenous 4T1 cell injection, and 3) mice than ran after intraductal 4T1 cell injection. Under all three conditions, exercise did not affect the establishment of pulmonary macro-metastases. Taken together, these data suggest that while aerobic exercise may impact tumor growth and metastasis only in some tumor models, the normal tissue adaptations induced by exercise can be sufficient to protect against chemotherapy toxicity, thus warranting further studies of this modality in oncology settings. Citation Format: Zachary Richard Wakefield, Mai Tanaka, Angela Bundy, Sharon Lepler, Christine Pampo, Lori Rice, Dietmar W. Siemann. Investigating the effects of aerobic exercise on preclinical breast cancer outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 905.

Abstract Aerobic exercise enacts physiologic adaptations across multiple tissue types that have positive effects on health and well-being in a number of chronic diseases. In cancer patients, exercise can improve quality of life by improving mood, reducing fatigue, and increasing therapy tolerance. Preclinical evidence suggests that exercise may also have direct effects on the tumor, such as improving perfusion, limiting hypoxia, and increasing immune cell infiltration. Furthermore, physiologic adaptations to exercise may protect against treatment toxicities. The goal of this project was to analyze the effect of aerobic exercise on breast cancer growth and doxorubicin-induced toxicity in a preclinical setting. These studies utilized retired breeder, female BALB/c mice. Aerobic exercise was modeled by singly-housing mice in cages with access to wireless, low-profile voluntary wheel running. Sedentary controls were singly-housed with plastic huts. To assess the impact of exercise on tumor growth, mice were intraductally injected with murine EMT6 breast cancer cells (103 cells in 5 µL) and then randomized into sedentary or exercising groups. Tumor volume was measured, and endpoint was defined as tumor volume of 1200 mm3. To examine the effect of exercise on doxorubicin-induced toxicity, non-tumor bearing mice were randomized into sedentary or exercising groups and given 3 intraperitoneal injections of doxorubicin (total dose 11 mg/kg) on days 10, 13, and 16 after randomization. The experiment was terminated on day 21. In both studies, mice were euthanized at endpoint and tissues were harvested, weighed, and either snap-frozen, or OCT- or paraffin-embedded. The results showed that aerobic exercise delayed tumor growth and improved survival in the intraductal EMT6 model. Proteomic profiling with a cytokine array revealed differential expression of Reg3G, MMP-9, and Tissue Factor between sedentary and exercising tumors. Moreover, chemotherapy treatment trended to decrease skeletal and cardiac muscle masses relative to untreated controls, and exercise prevented these effects. Doxorubicin also induced differential cardiac gene expression of α-MHC, β-MHC, and SERCA 2a relative to untreated controls, and exercise prevented these effects in 2 of 3 markers. In conclusion, aerobic exercise delayed tumor growth and improved survival in an intraductal model of breast cancer. Exercise also suppressed doxorubicin-induced skeletal and cardiac muscle toxicities. These findings suggest that aerobic exercise may have the potential to improve therapy outcomes by affecting both tumor growth and chemotherapy toxicities when implemented during treatment. Citation Format: Zachary Wakefield, Angela Bundy, Sharon Lepler, Christine Pampo, Lori Rice, Dietmar Siemann. Aerobic exercise delays intraductal breast cancer growth and suppresses doxorubicin-induced skeletal and cardiac muscle toxicities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2965.

Abstract Aberrant blood vessel networks in solid tumors lead to impaired tissue perfusion and areas of hypoxia (pO2 < 10 mmHg). Tumor hypoxia is associated with aggressive progression, dissemination, and therapeutic resistance. It is found in 40% of breast cancers, and can constitute a major obstacle to anticancer therapy. Exercise is associated with improvements in cardiovascular and respiratory function, aerobic capacity and overall health. In cancer patients undergoing therapy, exercise has been shown to decrease treatment related side effects and general fatigue. The goal of the present investigation was to determine whether aerobic exercise could be applied to improve tumor perfusion and oxygenation in a breast cancer model. Such modulation of the tumor physiology and host environment would be expected to lead to enhanced antitumor efficacy when combined with radiotherapy or chemotherapy. The effects of a single and daily bouts of moderate intensity treadmill running were studied in mice bearing the syngeneic murine mammary carcinoma 4T1. The exercise intensity was determined by measuring the anaerobic threshold, which was assessed by measuring the steady rise in blood lactate during exercise. Initial studies examined the effect of a single moderate bout of exercise (18 m/min) in mice bearing ~500 mm3 orthotopic tumors. In subsequent experiments mice were exercised at 18 m/min, 5 days a week, for 8 weeks prior to orthotopic injection of tumor cells and continued exercise (5 days a week) for 2 weeks during tumor growth. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. At the end of each of the exercise regimen, tumors were harvested, sectioned, stained, and tile mapped to assess physiological changes by immunofluorescence. The detection of open blood vessels (Hoechst-33342) was used as an indirect indicator of perfusion. Tumor hypoxia was determined using the 2-nitroimidazole (EF5). Blood vessels were stained using the endothelial cell marker CD-31. All markers were quantified using Photoshop and ImageJ NIH software. Our results indicate that in the 4T1 breast cancer model moderate intensity exercise did not significantly alter tumor growth, oxygenation or blood vessel number. However, daily bouts of exercise did significantly increase the number of open tumor vessels indicating improved tumor perfusion. These results suggest that a daily exercise regimen may have the potential to improve drug delivery to mammary tumors Citation Format: Jennifer M. Wiggins, Sharon Lepler, Christine Pampo, Lori Rice, Jennifer A. Lee, Dietmar Siemann. The impact of daily exercise on tumor perfusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5913. doi:10.1158/1538-7445.AM2017-5913

Abstract Intratumoral hypoxia (pO2 < 10mmHg) is associated with aggressive tumor progression, radiation resistance, suppression of the immune anti-tumor response and poor patient outcome. It is often found in solid tumors, and can constitute a major obstacle to anticancer therapy. The primary cause of abnormal oxygen tensions in tumors is the aberrant tumor vasculature, characterized by tortuous, leaky and immature vessels. A number of intervention strategies designed to overcome tumor hypoxia and improve patient outcome have been investigated including high oxygen content breathing and the use of bioreductive prodrugs, but these have had only moderate success in the clinic. The goal of the present investigations was to determine whether aerobic exercise could be applied to improve tumor perfusion, oxygenation and the immune anti-tumor response in breast cancer and fibrosarcoma models. Such modulation of the tumor physiology and host environment would be expected to lead to enhanced antitumor efficacy when combined with radiotherapy or chemotherapy. The effects of mild and moderate intensity treadmill running were studied in mice bearing syngeneic murine mammary carcinomas (4T1 and EMT6) or fibrosarcomas (KHT). The exercise intensities were determined by measuring the anaerobic threshold, which was assessed by measuring the steady rise in blood lactate during an exercise bout. Mice were orthotopically injected with tumor cells and exercise commenced when tumors reached a size of ∼500 mm3 (single) or ∼200 mm3 (repeated bouts). Once size was attained, mice were exposed to either mild (12 m/min) or moderate (18 m/min) intensity exercise. Controls for each treatment consisted of sedentary mice exposed to a stationary treadmill for the equivalent amount of time. At the end of the exercise period tumors were analyzed by histology to assess for physiological changes. Specifically, blood was collected and tumors were harvested, sectioned and evaluated by immunofluorescence. The detection of open blood vessels (Hoechst-33342) was used as an indirect indicator of perfusion and while the hypoxia marker (EF5) was used to determine the level of tumor hypoxia. Both markers were quantified using a Chalkley counter and ImageJ NIH software. In addition, the evaluation of tumor infiltrating immune cells after exercise compared to rest is under active investigation by histological analysis. Furthermore, plasma samples from exercised mice are being tested for immune related cytokines, chemokines and growth factors and compared to those from sedentary controls. Results to date indicate no difference in tumor growth rate between sedentary and exercising mice. Mild daily bouts of aerobic exercise do not affect the total number of tumor blood vessels but do increase the number of blood vessels that are actively perfused. These findings suggest that exercise may have potential utility in overcoming the aberrant microenvironmental conditions associated in solid tumors with therapeutic resistance. Citation Format: Jennifer M. Wiggins, Jennifer Lee, Lori Rice, Sharon Lepler, Christine Pampo, Dietmar Siemann. The impact of aerobic exercise on the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 732.

Abstract 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

Abstract 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

Abstract Background: In advanced prostate cancer (PCa), cells escape from the primary tumor and enter the bloodstream. These circulating cells preferentially target the axial skeleton and can form bone metastases. This results in weakened bones, spinal compressions, and fractures. Patients experience pain and other bone-related events that increase mortality and decrease quality of life. Therefore, identifying factors that lead to cancer progression, such as areas of hypoxia in the tumor microenvironment, will be crucial to developing strategies to inhibit the spread of malignant cells to distant sites. Efforts to interfere with early steps in the metastatic cascade have led to the development of promising small molecule targeting agents. One such agent is the tyrosine kinase inhibitor (TKI), saracatinib, shown to inhibit activation of Src and other kinases thought to be involved in cancer cell dissemination. The aim of these studies was to use aggressive, human PCa cells (PC-3ML), selected for their ability to form bone tumors in mice, to investigate the effects of various agents shown to interfere with metastatic activity. Methods: PC-3ML cells (a gift from A. Fatatis, Drexel University), expressing Green Fluorescent Protein (GFP) and luciferase (Luc), were injected into the left ventricle of male nude mice, using a closed-chest technique, to ensure that they bypass the lungs and circulate throughout the body. Prior to injection, cells were subjected to either hypoxia for 6 or pretreated for 24 hr with saracatinib. Immediately after injection, mice were placed in an in vivo imaging system (Xenogen IVIS, Caliper Life Sciences) to verify that the cells had disseminated throughout the body. This ensured that only successfully injected mice were randomization to treatment groups. Additional images were collected weekly thereafter to monitor the location and development of tumor nodules. Three weeks post-injection, mice were euthanized and tissues expressing Luc were collected, including bones of the legs, mandible and spine. The bones were fixed and decalcified, followed by flash freezing in OCT freezing medium. Serial frozen sections were then evaluated for GFP expressing cells using a fluorescent stereoscope. Results: Tumor cell deposits were almost always found in bone, frequently in the tibial-femoral joint, scapula-humerus joint, spine, and mandible. Mice injected with PC-3ML cells pretreated with saracatinib had fewer metastases and cells exposed to hypoxia for 6 hr prior to injection had more metastases, on average, than control cells, as predicted by in vitro assays. Conclusions: Intracardiac injection of GFP- and Luc- labeled PC-3ML cells resulted in skeletal metastases that could be monitored in mice over time using an in vivo imaging system and in tissue sections by fluorescent microscopy. The studies verified in vitro data showing that hypoxia can enhance, and saracatinib inhibit, metastatic activity. Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Saracatinib inhibits human prostate cancer cell bone metastases in a xenograft model. [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 3946. doi:10.1158/1538-7445.AM2013-3946

SRC, a non-receptor tyrosine kinase, is frequently over-expressed and highly activated in blood as well as solid tumors in various organs, including prostate, and has been associated with aggressive disease and a poor patient prognosis. Prostate cancer patients with a high risk of developing metastases have few treatment options, none of which can result in a durable cure. Therefore, the aim of the present study was to examine the impact of a SRC inhibitor, dasatinib, on the ability of human prostate cancer cell to complete key steps in the metastatic process, including invasion and angiogenesis. Dasatinib treatment impaired the metastatic phenotypes of the human prostate cancer cell lines, PC-3, DU-145, and LNCaP, by significantly reducing migration and invasion in modified Boyden chambers. Inhibition of phosphorylation, and therefore enhanced activation, of SRC and key downstream signaling pathway elements, including FAK, STAT3, Paxillin, and Akt, as determined by Western blotting, also was observed. This suggests that dasatinib interferes with critical cell functions associated with the metastatic cascade. Dasatinib also had direct effects on the ability of microvascular endothelial cells to form tubes in vitro and impaired the ability of PC-3 cells to induce angiogenesis in vivo. In conclusion, the present findings suggest that SRC inhibition by dasatinib may have utility in reducing the metastatic spread of prostate cancer cells.

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 24h 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.

BACKGROUND:Src, a non-receptor tyrosine kinase frequently overexpressed and highly activated in malignancies, has been associated with a poor patient prognosis. The aim of the present studies was to examine the impact of an Src inhibitor (saracatinib) on a highly metastatic murine sarcoma cell line (KHT).MATERIALS AND METHODS:Phosphorylation of Src and downstream effectors was determined using Western immunoblotting. Cell cycle was analyzed by flow cytometry using propidium iodide DNA staining, migration and invasion in modified Boyden chambers, activated MMP-9 by gel zymography, and visualization of pSrc and pFAK by confocal immunofluorescence. The number of KHT lung nodules in saracatinib-treated mice was compared to controls.RESULTS:Saracatinib inhibited major pathways in the metastatic cascade in vitro, including Src and FAK activation. Functions required for metastasis, such as migration and invasion, were reduced when cells were exposed to 0.5 μM and 1.0 μM saracatinib, respectively (p<0.0001). Pretreatment of KHT cells with either 1 μM or 5 μM saracatinib prior to tail vein injection decreased lung colonies in mice from 13.0 to 5.0 (p<0.05) and less than 1.0 (p<0.01), respectively.CONCLUSION:These findings suggest that Src inhibition by saracatinib may reduce the metastatic activity of tumor cells.

2119 Vascular endothelial growth factor (VEGF) acts as a survival factor for newly-formed vasculature, enhances the permeability of blood vessels and has been implicated as the key factor in the angiogenic process of solid tumors. Thus, interfering with VEGF signaling has become a major strategy to inhibit tumor growth and spread. One approach is to inhibit VEGF receptor-2 (VEGFR-2)-associated tyrosine kinase signaling. The present studies report on the efficacy of AZD2171, an oral, highly potent and selective VEGF signaling inhibitor of VEGFR-1, -2, and -3 tyrosine kinases, in human renal cell carcinoma (Caki-1) cells. In vitro treatment with AZD2171 was found to have a significant impact on endothelial cell (EC) but not renal cell carcinoma proliferation. AZD2171 50-100 nM inhibited the growth of human microvascular endothelial cells (lung) but doses ≤5 μM, had little impact on Caki-1 cell growth. AZD2171 10 nM significantly impaired (by a factor of 2) the number of ECs that migrated into a denuded area over a 48-hour period. An intradermal assay was used to study the effect of AZD2171 treatment on Caki-1 tumor cell-induced angiogenesis in vivo. Nude mice received AZD2171 (3, 6, or 9 mg/kg/day) or vehicle by oral gavage commencing on the day prior to tumor cell inoculation. Caki-1 cells (1 x 105) were injected intradermally at four sites on the ventral surface of each of four mice. Three days post-inoculation, the mice were humanely culled and the skin flap containing the inoculation site was excised and the number of blood vessels induced was counted. Compared with controls, skin flaps from AZD2171-treated mice showed a significant and dose-dependent reduction in the number of blood vessels induced by the tumor cells. The antitumor efficacy of AZD2171 was assessed in mice bearing Caki-1 xenografts. When tumor volume reached ~200 mm3, mice were randomly assigned to receive either no treatment or AZD2171 6 mg/kg/day po for 2 weeks (Mon-Fri). In animals receiving AZD2171, tumors took twice as long to reach five times the starting size compared with untreated controls. These results suggest that inhibition of VEGF signaling with AZD2171 may impair the growth of renal cell carcinoma.