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Background: Previous graduate students and postdoctoral associates from the University of Florida Health Cancer Center, in partnership with the University of Florida Student Science Training Program, implemented a cooperative learning curriculum, providing high school students with a broad overview of cancer topics over six weeks over the summer. To address discussions necessitated by the COVID-19 pandemic on student autonomy, we report lessons learned and outcomes of a cancer biology and therapeutic curriculum modified for a collaborative learning environment., Methods: This pre-post longitudinal observational study conducted in 2023 on a cancer biology and therapeutics course evaluated students' knowledge retention and general awareness and opinions in cancer research. A structured survey was employed for data collection, using learning assessment surveys and the Likert scale ranging from 1 to 10, with 10 being highly likely., Results: Student performance tracked over a 7-year period indicated consistency in performance between years. Post-assessment analysis revealed significant improvements in student benchmark understanding, notably in their ability to define cancer in one sentence (p = 0.0407), identify cancer therapies (p = 0.0040), and recognize cancer hallmarks (p < 0.0001). An increased trend in median response to the likelihood of pursuing cancer research (p = 0.8793) and the possibility of pursuing cancer research (p = 0.4874) were also observed, although not statistically significant. Moreover, feedback from participating students indicated that "the educational activities at the end of class (e.g., escape room, case studies)" and "learning about cancer and getting to work in groups…" the curriculum fostered a positive educational learning environment., Conclusion: Students generally retained the course material presented and upheld a positive perception of the course. Incorporating opportunities for peer-to-peer learning, especially when introducing or discussing complex issues like cancer, may benefit student autotomy., (© 2024. The Author(s).)

Biomimetic tumor microenvironment models bridge the gap between in vitro and in vivo systems and serve as a useful way to address the modeling challenge of how to recreate the cell and system complexity associated with real tissues. Our laboratory has developed an ex vivo rat mesentery culture model, which allows for simultaneous investigation of blood and lymphatic microvascular network remodeling in an intact tissue environment. Given that angiogenesis and lymphangiogenesis are key contributors to the progression of cancer, the objective of this study was to combine tissue and tumor spheroid culture methods to establish a novel ex vivo tumor spheroid-tissue model by verifying its use for evaluating the effects of cancer cell behavior on the local microvascular environment. H1299 or A549 tumor spheroids were formed via hanging drop culture and seeded onto rat mesenteric tissues harvested from adult male Wistar rats. Tissues with transplanted spheroids were cultured in serum-free media for 3 to 5 days. PECAM, NG2, CD11b, and αSMA labeling identified endothelial cells, pericytes, immune cells, and smooth muscle cells, respectively. Time-lapse imaging confirmed cancer cell type specific migration. In addition to increasing PECAM positive capillary sprouting and LYVE-1 positive endothelial cell extensions indicative of lymphangiogenesis, tumor spheroid presence induced the formation of lymphatic/blood vessel connections and the formation of hybrid, mosaic vessels that were characterized by discontinuous LYVE-1 labeling. The results support the application of a novel tumor spheroid microenvironment model for investigating cancer cell-microvascular interactions., (© 2024. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK + samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas., (© 2024. The Author(s).)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 (IP 3 R)-regulated calcium release from endoplasmic reticulum (ER), which is activated by the G q -PLC-IP 3 R 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although full-length fibroblast growth factor 7 (FGF7) blocks cyclophosphamide-induced urothelial apoptosis in mice, limitations include high production costs because of its large size. We previously identified a small peptide derived from FGF2 that mitigated acute radiation syndrome as well as full-length FGF2. Based on the sequence of the FGF2 peptide, we synthesized a corresponding 19 amino acid FGF7 peptide (FGF7p). Our objectives were to determine if systemic FGF7p triggered the downstream targets and protected against cyclophosphamide bladder injury similar to full-length FGF7. We administered FGF7p or vehicle subcutaneously (SQ) to mice subjected to no injury or intraperitoneal (IP) cyclophosphamide and harvested bladders 1 day after injury. We then performed hematoxylin and eosin, TUNEL and immunofluorescence (IF) staining. In uninjured mice, a 20 mg/kg threshold FGF7p dose induced expression of phosphorylated (activated) FRS2α (pFRS2α), and pAKT in urothelium (consistent with cytoprotective effects of FGF7). We then gave FGF7p (20 mg/kg) or vehicle at 72 and 48 h prior to cyclophosphamide. One day after injury, TUNEL staining revealed many more apoptotic urothelial cells with vehicle treatment versus FGF7p treatment. IF for pAKT and readouts of two anti-apoptotic AKT targets (BAD and mTORC1) revealed minimal staining with vehicle treatment, but strong urothelial expression for all markers with FGF7p treatment. In conclusion, FGF7p appears to block bladder urothelial apoptosis via AKT and its targets, similar to FGF7. FGF7p is much more inexpensive to make and has a longer shelf life and higher purity than FGF7., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

An increasing number of studies have demonstrated the significant roles of the interplay between microenvironmental mechanics in tissues and biochemical-genetic activities in resident tumor cells at different stages of tumor progression. Mediated by molecular mechano-sensors or -transducers, biomechanical cues in tissue microenvironments are transmitted into the tumor cells and regulate biochemical responses and gene expression through mechanotransduction processes. However, the molecular interplay between the mechanotransduction processes and intracellular biochemical signaling pathways remains elusive. This paper reviews the recent advances in understanding the crosstalk between biomechanical cues and three critical biochemical effectors during tumor progression: calcium ions (Ca 2+ ), yes-associated protein (YAP), and microRNAs (miRNAs). We address the molecular mechanisms underpinning the interplay between the mechanotransduction pathways and each of the three effectors. Furthermore, we discuss the functional interactions among the three effectors in the context of soft matter and mechanobiology. We conclude by proposing future directions on studying the tumor mechanobiology that can employ Ca 2+ , YAP, and miRNAs as novel strategies for cancer mechanotheraputics. This framework has the potential to bring insights into the development of novel next-generation cancer therapies to suppress and treat tumors.

Regions of reduced oxygenation (hypoxia) are a characteristic feature of virtually all animal and human solid tumours. Numerous preclinical studies, both in vitro and in vivo, have shown that decreasing oxygen concentration induces resistance to radiation. Importantly, hypoxia in human tumours is a negative indicator of radiotherapy outcome. Hypoxia also contributes to resistance to other cancer therapeutics, including immunotherapy, and increases malignant progression as well as cancer cell dissemination. Consequently, substantial effort has been made to detect hypoxia in human tumours and identify realistic approaches to overcome hypoxia and improve cancer therapy outcomes. Hypoxia-targeting strategies include improving oxygen availability, sensitising hypoxic cells to radiation, preferentially killing these cells, locating the hypoxic regions in tumours and increasing the radiation dose to those areas, or applying high energy transfer radiation, which is less affected by hypoxia. Despite numerous clinical studies with each of these hypoxia-modifying approaches, many of which improved both local tumour control and overall survival, hypoxic modification has not been established in routine clinical practice. Here we review the background and significance of hypoxia, how it can be imaged clinically and focus on the various hypoxia-modifying techniques that have undergone, or are currently in, clinical evaluation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many signaling pathways are dysregulated in cancer cells and the host tumor microenvironment. Aberrant receptor tyrosine kinase (RTK) pathways promote cancer development, progression, and metastasis. Hence, numerous therapeutic interventions targeting RTKs have been actively pursued. Axl is an RTK that belongs to the Tyro3, Axl, MerTK (TAM) subfamily. Axl binds to a high affinity ligand growth arrest specific 6 (Gas6) that belongs to the vitamin K-dependent family of proteins. The Gas6/Axl signaling pathway has been implicated to promote progression, metastasis, immune evasion, and therapeutic resistance in many cancer types. Therapeutic agents targeting Gas6 and Axl have been developed, and promising results have been observed in both preclinical and clinical settings when such agents are used alone or in combination therapy. This review examines the current state of therapeutics targeting the Gas6/Axl pathway in cancer and discusses Gas6- and Axl-targeting agents that have been evaluated preclinically and clinically.

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., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest., (Copyright: © 2021 Wakefield et al.)

Approximately 90% of cancer-related deaths result from cancer metastasis. In prostate and breast cancers, bone is the most common site of cancer cell dissemination. Key steps in the metastatic cascade are promoted through upregulation of critical cell signaling pathways in neoplastic cells. The present study assessed the role of the receptor tyrosine kinase Axl in prostate and breast cancer cell metastasis to bones using (i) Axl knockdown neoplastic cells and osteoclast progenitor cells in vitro, (ii) intracardiac injection of Axl knockdown tumor cells in vivo, and (iii) selective Axl inhibitor BGB324. Axl inhibition in neoplastic cells significantly decreased their metastatic potential, and suppression of Axl signaling in osteoclast precursor cells also reduced the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation and progression of bone metastases. Hence, therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes.

A challenge in cancer research is the lack of physiologically responsive in vitro models that enable tracking of cancer cells in tissue-like environments. A model that enables real-time investigation of cancer cell migration, fate, and function during angiogenesis does not exist. Current models, such as 2D or 3D in vitro culturing, can contain multiple cell types, but they do not incorporate the complexity of intact microvascular networks. The objective of this study was to establish a tumor microvasculature model by demonstrating the feasibility of bioprinting cancer cells onto excised mouse tissue. Inkjet-printed DiI + breast cancer cells on mesometrium tissues from C57Bl/6 mice demonstrated cancer cells' motility and proliferation through time-lapse imaging. Colocalization of DAPI + nuclei confirmed that DiI + cancer cells remained intact postprinting. Printed DiI + 4T1 cells also remained viable after printing on Day 0 and after culture on Day 5. Time-lapse imaging over 5 days enabled tracking of cell migration and proliferation. The number of cells and cell area were significantly increased over time. After culture, cancer cell clusters were colocalized with angiogenic microvessels. The number of vascular islands, defined as disconnected endothelial cell segments, was increased for tissues with bioprinted cancer cells, which suggests that the early stages of angiogenesis were influenced by the presence of cancer cells. Bioprinting cathepsin L knockdown 4T1 cancer cells on wild-type tissues or nontarget 4T1 cells on NG2 knockout tissues served to validate the use of the model for probing tumor cell versus microenvironment changes. These results establish the potential for bioprinting cancer cells onto live mouse tissues to investigate cancer microvascular dynamics within a physiologically relevant microenvironment. Impact statement To keep advancing the cancer biology field, tissue engineering has been focusing on developing in vitro tumor biomimetic models that more closely resemble the native microenvironment. We introduce a novel methodology of bioprinting exogenous cancer cells onto mouse tissue that contains multiple cells and systems within native physiology to investigate cancer cell migration and interactions with nearby microvascular networks. This study corroborates the manipulation of different exogenous cells and host microenvironments that impact cancer cell dynamics in a physiologically relevant tissue. Overall, it is a new approach for delineating the effects of the microenvironment on cancer cells and vice versa.

Background: Osteosarcoma (OS) is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. Liquid biopsies are an alternative to current diagnostic imaging modalities that can be used to monitor treatment efficacy and the development of metastases. This study addresses the use of novel biomarkers to detect circulating osteosarcoma cells., Procedures: Flow cytometry was used to evaluate the relative expression of epithelial cell adhesion molecule (EpCAM), ganglioside 2 and 3 (GD2/3), and cell surface vimentin (CSV) on a panel of OS cell lines. A microfluidic device was used to affirm the efficacy of GD2/3 and CSV to capture CTCs. Once captured, CTCs on the device are enumerated and the capture efficiency for each marker is measured. Patient samples were captured using the LFAM chip., Results: We report the evaluation of GD2, GD3, and CSV as markers for OS cell capture in cell lines and in patient samples. The results of our capture studies correlate with our flow cytometry data and have shown a low capture efficiency of OS cells using EpCAM antibodies, while showing a moderate capture efficiency of OS cells using the GD2, GD3, and CSV antibodies independently. The combination of biomarkers demonstrate a high capture efficiency of approximately 80%. This is further supported by the detection of 1-1.5 CTCs per mL of blood using GD2 + CSV in OS patient samples., Conclusions: The combination of GD2 + CSV significantly increased the capture efficacy of OS cells. The detection of CTCs through routine blood sampling may be used clinically for earlier detection of metastases and monitoring the therapeutic effect of treatments in metastatic osteosarcomas., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier GmbH.)

Receptor tyrosine kinases have been shown to dysregulate a number of pathways associated with tumor development, progression, and metastasis. Axl is a receptor tyrosine kinase expressed in many cancer types and has been associated with therapy resistance and poor clinical prognosis and outcomes. In addition, Axl and its ligand growth arrest specific 6 (Gas6) protein are expressed by a number of host cells. The Gas6/Axl signaling pathway has been implicated in the promotion of tumor cell proliferation, survival, migration, invasion, angiogenesis, and immune evasion. As a result, Axl is an attractive, novel therapeutic target to impair multiple stages of tumor progression from both neoplastic and host cell axes. This review focuses on the role of the Gas6/Axl signaling pathway in promoting the immunosuppressive tumor microenvironment, as immune evasion is considered one of the hallmarks of cancer. The review discusses the structure and activation of the Gas6/Axl signaling pathway, GAS6 and AXL expression patterns in the tumor microenvironment, mechanisms of Axl-mediated tumor immune response, and the role of Gas6/Axl signaling in immune cell recruitment.

The presence of macrophages within breast tumors correlates with metastatic potential. These tumor-associated macrophages often take on a pro-tumorigenic (M2-like) phenotype resulting in the secretion of growth factors and proteases, including the lysosomal protease cathepsin L. Since cathepsin L also is frequently secreted by breast cancer cells and contributes to tumor invasion, metastasis, and angiogenesis, we hypothesized that secretion of cathepsin L by both tumor-associated macrophages and neoplastic cells would facilitate the metastatic phenotype. Our results showed that the novel cathepsin L/K inhibitors KGP94 and KGP207 could inhibit in vitro M2 macrophage invasion and reduce the macrophage-stimulated invasion of 4T1 murine breast cancer cells. KGP94 and KGP207 treatment also reduced the expression of several M2-associated markers, suggesting that cathepsin L activity may be important for IL-4-driven M0 to M2 differentiation. In addition, cathepsin L shRNA knockdown studies revealed that cathepsin L from both the tumor cell and the macrophage population is important for tumor cell invasion. Thus our data suggest that tumor cells and macrophages may both contribute to the cathepsin L-driven metastatic phenotype of breast cancer. Taken together, these studies highlight the importance of cathepsin L in macrophage functions and suggest that cathepsin inhibition strategies may be therapeutically beneficial by impairing the progression of tumors with high infiltration of M2 macrophages., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest., (Copyright: © 2019 Dykes et al.)

Exercise has long been known to be beneficial to human health. Studies aimed at understanding the effects of exercise specifically focus on predetermined exercise intensities defined by measuring the aerobic capacity of each individual. Many disease models involving animal training often establish aerobic capacity by using the maximal lactate steady state (MLSS), a widely used method in humans that has frequently been used in rodent studies. The MLSS is defined as the highest exercise intensity at which blood lactate concentration remains constant and is roughly equivalent to 70-80% of maximal aerobic capacity. Due to our up-coming experiments investigating the effect of different exercise intensities in specific strains of tumor-bearing mice, the aim of the present study was to determine the MLSS in athymic nude (NCr nu/nu and NMRI), CDF1, and C3H mice by treadmill running at increasing speeds. However, despite thorough exercise acclimation and the use of different exercise protocols and aversive stimuli, less than half of the experiments across strains pointed towards an established MLSS. Moreover, gently prodding the mice during low to moderate intensity running caused a 30-121% (p<0.05) increase in blood lactate concentration compared to running without stimulation, further questioning the use of lactate as a measure of exercise intensity. Overall, MLSS is difficult to determine and large variations of blood lactate levels were observed depending on the exercise protocol, mice handling strategy and strain. This should be considered when planning experiments in mice using forced exercise protocols., Competing Interests: The authors have declared that no competing interests exist.

The growth of primary tumors as well as metastatic neoplastic lesions is strongly dependent on the cancer cells' ability to initiate their own vascular network. This process, angiogenesis, which involves the proliferation, migration, and invasion of endothelial cells, is critically dependent on a variety of signaling molecules that target specific receptors, most notably tyrosine kinases. One receptor tyrosine kinase associated with poor prognosis, metastasis, and outcome in a variety of tumor types, is Axl. Although the role of Axl in tumor cell migration and invasion are well recognized, little is known about the involvement of Axl signaling in the initiation of angiogenesis. Here, we show that Axl inhibition in tumor cells decreases the secretion of pro-angiogenic factors and impairs functional properties of endothelial cells in vitro and in vivo . These data indicate that Axl signaling is an important contributor to tumor angiogenesis., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Physical activity is associated with diminished risk of several cancers, and preclinical studies suggest exercise training may alter tumor cell growth in certain tissue(s) (e.g., adipose). From moderate-intensity exercise-trained rats versus sedentary controls, we hypothesized 1) there will be a decreased prostate cancer cell viability and migration in vitro and, within the prostate, a reduced 5α-reductase 2 (5αR2) and increased caspase-3 expression, and 2) that exercise training in tumor-bearing (TB) animals will demonstrate a reduced tumor cell viability in prostate-conditioned media. Serum and prostate were harvested from sedentary or exercise-trained (treadmill running, 10-11 weeks) immune-competent (Copenhagen; n = 20) and -deficient (Nude; n = 18) rats. AT-1 and PC-3 prostate cancer cells were grown in one or more of the following: serum-supplemented media (SSM), SSM from TB rats (SSM-TB), prostate-conditioned media (PCM) or PCM from TB rats (PCM-TB) for 24-96 h under normoxic (18.6% O 2 ) or hypoxic (5% O 2 ) conditions. Under normoxic condition, there was a decreased AT-1 cell viability in SSM and PCM from the exercise-trained (ET) immune-competent rats, but no difference in PC-3 cell viability in SSM and PCM from ET Nude rats versus the sedentary (SED) group, or in SSM-TB from ET-TB Nude rats versus the SED-TB group. However, there was a decreased PC-3 cell viability in the PCM-TB of the ET-TB group versus SED-TB group. PC-3 cell viability in all conditioned media types was not altered between groups with hypoxia. In the prostate, exercise training did not alter 5αR2 expression levels, but increased caspase-3 expression levels. In conclusion, prior exercise status reduced prostate cancer cell viability in the serum and prostate of trained rats but did not modify several other key prostate tumor cell growth characteristics (e.g., migration, cell cycle except in S phase of PC-3 cells in PCM-TB). Importantly, once the tumor was established, exercise training reduced tumor cell viability in the surrounding prostate, which may help explain the reduced severity of the disease in patients that exercise., Competing Interests: None.

C-Met is a frequently overexpressed or amplified receptor tyrosine kinase involved in metastatic-related functions, including migration, invasion, cell survival, and angiogenesis. Because of its role in cancer progression and metastasis, many inhibitors have been developed to target this pathway. Unfortunately, most c-Met inhibitor clinical trials have failed to show significant improvement in survival of cancer patients. In these trials tumor type, protein overexpression, or gene amplification are the primary selection criteria for patient inclusion. Our data show that none of these criteria are associated with c-Met pathway activation. Hence, it is conceivable that the majority of c-Met inhibitor clinical trial failures are the consequence of a lack of appropriate patient selection. Further complicating matters, c-Met inhibitors are routinely tested in preclinical studies in the presence of high levels of exogenous Hepatocyte Growth Factor (HGF), its activating ligand. In our studies, several tumor cell lines showed sensitivity to a c-Met inhibitor at high HGF concentrations (50 ng/mL). However, when the tumor lines were tested at HGF levels typically detected in human serum (0.4 to 0.8 ng/mL), inhibitor activity was lost. Thus testing c-Met inhibitors at non-physiological concentrations of HGF may lead to incorrect predictions of drug efficacy in vivo ., Competing Interests: CONFLICTS OF INTEREST None.

In 2012, cancer affected 14.1 million people worldwide and was responsible for 8.2 million deaths. The disease predominantly affects aged populations and is one of the leading causes of death in most western countries. In tumors, the aggressive growth of the neoplastic cell population and associated overexpression of pro-angiogenic factors lead to the development of disorganized blood vessel networks that are structurally and functionally different from normal vasculature. A disorganized labyrinth of vessels that are immature, tortuous and hyperpermeable typifies tumor vasculature. Functionally, the ability of the tumor vasculature to deliver nutrients and remove waste products is severely diminished. A critical consequence of the inadequate vascular networks in solid tumors is the development of regions of hypoxia [low oxygen tensions typically defined as oxygen tensions (pO 2 values) < 10 mm Hg]. Tumor cells existing in such hypoxic environments have long been known to be resistant to anticancer therapy, display an aggressive phenotype, and promote tumor progression and dissemination. This review discusses the physiological basis of hypoxia, methods of detection, and strategies to overcome the resulting therapy resistance.

The c-Met/HGF pathway is implicated in cancer progression and dissemination. Many inhibitors have been developed to target this pathway. Unfortunately, most trials have failed to demonstrate efficacy. However, clinical trials have not adequately tested the concept of c-Met pathway inhibition due to the lack of appropriate patient selection criteria., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An imbalance in oxygen delivery to demand in solid tumors results in local areas of hypoxia leading to poor prognosis for the patient. We hypothesize that aerobic exercise increases tumor blood flow, recruits previously nonperfused tumor blood vessels, and thereby augments blood-tumor O2 transport and diminishes tumor hypoxia. When combined with conventional anticancer treatments, aerobic exercise can significantly improve the outcomes for several types of cancers.

Introduction: The src inhibitor Dasatinib has been widely studied as an anti-metastatic agent. The aims of this study were to examine the effect of Src inhibition on the metastatic potential of the 4T1 murine mammary carcinoma., Context: Src is a non-receptor tyrosine kinase well-known to contribute to the metastatic potential of tumour cells. It does so through alteration of signalling pathways important to metastasis. Elevated levels of Src are common in many cancer types, and have been correlated with tumour progression and poor patient prognosis., Aims: This study examined whether disruption of the Src signalling pathway could inhibit metastases formation., Settings and Design: The Src inhibitor Dasatinib was evaluated in vitro and in vivo using the highly metastatic 4T1 murine mammary adenocarcinoma cell line., Methods and Material: In vitro assays included growth curve, western blot, migration, and invasion assays. In vivo assays included intradermal and tail vein injection models., Statistical Analysis Used: In vitro data were analysed using one-way ANOVA with Dunnett's multiple comparisons in GraphPad Prism 6.0. In vivo data were analysed using GraphPad Prism 6.0, using the Wilcoxon matched pairs test., Results: Dasatinib is effective at inhibiting in vitro phosphorylation of Src, migration and invasion in the 4T1 cell line, as well as angiogenesis in vivo . In vitro treatment with Dasatinib impaired the metastatic ability of tumour cells as assessed by a tail vein injection model. However, both the syngeneic BALB/c and the athymic nu/nu mice receiving oral doses of the drug developed significantly higher numbers of 4T1 lung metastases. This effect was not seen in a different breast carcinoma cell line, the MDA-MB-231-4175-LM2, nor was this effect seen in the murine fibrosarcoma KHT cell line., Conclusions: The 4T1 cell line is not an appropriate model to study Src inhibition.

Vascular targeted therapies (VTTs) are agents that target tumor vasculature and can be classified into two categories: those that inhibit angiogenesis and those that directly interfere with established tumor vasculature. Although both the anti-angiogenic agents (AAs) and the vascular disrupting agents (VDAs) target tumor vasculature, they differ in their mechanism of action and therapeutic application. Combining these two agents may realize the full potential of VTT and produce an effective therapeutic regimen. Here, we review AAs and VDAs (monotherapy and in combination with conventional therapies). We also discuss the rationale of combined VTT and its potential to treat cancer.

Carcinoma cells can acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT). However, the significance of EMT in cancer metastasis has been controversial, and the exact fates and functions of EMT cancer cells in vivo remain inadequately understood. Here, we tracked epithelial cancer cells that underwent inducible or spontaneous EMT in various tumor transplantation models. Unlike epithelial cells, the majority of EMT cancer cells were specifically located in the perivascular space and closely associated with blood vessels. EMT markedly activated multiple pericyte markers in carcinoma cells, in particular PDGFR-β and N-cadherin, which enabled EMT cells to be chemoattracted towards and physically interact with endothelium. In tumor xenografts generated from carcinoma cells that were prone to spontaneous EMT, a substantial fraction of the pericytes associated with tumor vasculature were derived from EMT cancer cells. Depletion of such EMT cells in transplanted tumors diminished pericyte coverage, impaired vascular integrity, and attenuated tumor growth. These findings suggest that EMT confers key pericyte attributes on cancer cells. The resulting EMT cells phenotypically and functionally resemble pericytes and are indispensable for vascular stabilization and sustained tumor growth. This study thus proposes a previously unrecognized role for EMT in cancer.

Anti-angiogenic therapies target the tumor vasculature, impairing its development and growth. It was hypothesized over 40 years ago by the late Judah Folkman and Julie Denekamp that depriving a tumor of oxygen and nutrients, by targeting the tumor vasculature, could have therapeutic benefits. Identification of growth factors and signaling pathways important in angiogenesis subsequently led to the development of a series of anti-angiogenic agents that over the past decade have become part of the standard of care in several disease settings. Unfortunately not all patients respond to the currently available anti-angiogenic therapies while others become resistant to these agents following prolonged exposure. Identification of new pathways that may drive angiogenesis led to the development of second-generation anti-angiogenic agents such as those targeting the Ang-2/Tie2 axis. Recently, it has become clear that combination of first and second generation agents targeting the blood vessel network can lead to outcomes superior to those using either agent alone. The present review focuses on the current status of VEGF and Ang-2 targeted agents and the potential utility of using them in combination to impair tumor angiogenesis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

It is estimated that approximately 90% of patients with advanced prostate cancer develop bone metastases; an occurrence that results in a substantial reduction in the quality of life and a drastic worsening of 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. Recognition of the importance of Cathepsin L (CTSL) to metastatic dissemination of cancer cells has led to the development of several CTSL inhibition strategies. The present investigation employed intra-cardiac injection of human PC-3ML prostate cancer cells into nude mice to examine tumor cell dissemination in a preclinical bone metastasis model. CTSL knockdown confirmed the validity of targeting this protease and subsequent intervention studies with the small molecule CTSL inhibitor KGP94 resulted in a significant reduction in metastatic tumor burden in the bone and an improvement in overall survival. CTSL inhibition by KGP94 also led to a significant impairment of tumor initiated angiogenesis. Furthermore, KGP94 treatment decreased osteoclast formation and bone resorptive function, thus, perturbing the reciprocal interactions between tumor cells and osteoclasts within the bone microenvironment which typically result in bone loss and aggressive growth of metastases. These functional effects were accompanied by a significant downregulation of NFκB signaling activity and expression of osteoclastogenesis related NFκB target genes. Collectively, these data indicate that the CTSL inhibitor KGP94 has the potential to alleviate metastatic disease progression and associated skeletal morbidities and hence may have utility in the treatment of advanced prostate cancer patients., (© 2016 UICC.)

A significant proportion of breast cancer patients harbor clinically undetectable micrometastases at the time of diagnosis. If left untreated, these micro-metastases may lead to disease relapse and possibly death. Hence, there is significant interest in the development of novel anti-metastatic agents that could also curb the growth of pre-established micrometastases. Like primary tumor, the growth of metastases also is driven by angiogenesis. Although the role of cysteine protease Cathepsin L (CTSL) in metastasis associated tumor cell functions such as migration and invasion is well recognized, its role in tumor angiogenesis remains less explored. The present study examines the contribution of CTSL to breast cancer angiogenesis and evaluates the anti-angiogenic efficacy of CTSL inhibitor KGP94. CTSL semi-quantitative RT-PCR analysis on breast tissue panels revealed significant upregulation of CTSL in breast cancer patients which strongly correlated with increased relapse and metastatic incidence and poor overall survival. Preclinically, CTSL ablation using shRNA or KGP94 treatment led to a significant reduction in MDA-MB-231 tumor cell induced angiogenesis in vivo. In-vitro assessments demonstrated a significant decrease in various angiogenic properties such as endothelial cell sprouting, migration, invasion, tube formation and proliferation in the presence of KGP94. 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. Collectively, these data indicate that CTSL is an important contributor to tumor angiogenesis and that the CTSL inhibition may have therapeutic utility in the treatment of breast cancer patients.

SOX2 is an embryonic stem cell marker that in prostate cancer has been associated not only with tumorigenesis but also metastasis. Furthermore hypoxia in primary tumors has been linked to poor prognosis and outcomes in this disease. The goal of the present study was to investigate the impact of hypoxia on SOX2 expression and metastasis-associated functions in prostate cancer cells. A tissue microarray of 80 samples from prostate cancer patients or healthy controls was employed to examine the expression of HIF-1α and its correlation with SOX2. The role of SOX2 and HIF-1/2α in the regulation of cell invasion and sphere formation capacity under hypoxic conditions was investigated in vitro using short hairpin RNA (shRNA)-mediated knockdown in three human prostate cancer cell lines. HIF-1α expression was significantly elevated in malignant prostate tissue compared to benign or normal tissue, and in tumor samples its expression was highly correlated with SOX2. In prostate cancer cells, acute and chronic exposures to hypoxia that resulted in elevated expression levels of HIF-1α and HIF-2α, respectively, also induced SOX2. Genetic depletion of SOX2 attenuated hypoxia-induced cell functions. Knockdown of HIF-1α, but not HIF-2α, decreased acute hypoxia-mediated cell invasion and SOX2 up-regulation, whereas only HIF-2α gene silencing reduced sphere formation capacity and chronic hypoxia-mediated SOX2 up-regulation. Enhanced SOX2 expression and HIF-1α or HIF-2α associated phenotypes are dependent on the time duration of exposure to hypoxia. The present results indicate that SOX2 may be a key mediator of hypoxia-induced metastasis-associated functions and hence may serve as a potential target for therapeutic interventions for metastatic prostate cancer.

Refractory disease is the greatest challenge in treating patients with acute myeloid leukemia (AML). Blood vessels may serve as sanctuary sites for AML. When AML cells were co-cultured with bone marrow endothelial cells (BMECs), a greater proportion of leukemia cells were in G0/G1. This led us to a strategy of targeting BMECs with tubulin-binding combretastatins, causing BMECs to lose their flat phenotype, degrade their cytoskeleton, cease growth, and impair migration despite unchanged BMEC viability and metabolism. Combretastatins also caused downregulation of BMEC adhesion molecules known to tether AML cells, including vascular cell adhesion molecule (VCAM)-1 and vascular endothelial (VE)-cadherin. When AML-BMEC co-cultures were treated with combretastatins, a significantly greater proportion of AML cells dislodged from BMECs and entered the G2/M cell cycle, suggesting enhanced susceptibility to cell cycle agents. Indeed, the combination of combretastatins and cytotoxic chemotherapy enhanced additive AML cell death. In vivo mice xenograft studies confirmed this finding by revealing complete AML regression after treatment with combretastatins and cytotoxic chemotherapy. Beyond highlighting the pathologic role of BMECs in the leukemia microenvironment as a protective reservoir of disease, these results support a new strategy for using vascular-targeting combretastatins in combination with cytotoxic chemotherapy to treat AML., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Proteolytic enzymes may serve as promising targets for novel therapeutic treatment strategies seeking to impede cancer progression and metastasis. One such enzyme is cathepsin L (CTSL), a lysosomal cysteine protease. CTSL upregulation, a common occurrence in a variety of human cancers, has been widely correlated with metastatic aggressiveness and poor patient prognosis. In addition, CTSL has been implicated to contribute to cancer-associated osteolysis, a debilitating morbidity affecting both life expectancy and the quality of life. In this review, we highlight the mechanisms by which CTSL contributes to tumor progression and dissemination and discuss the therapeutic utility of CTSL intervention strategies aimed at impeding metastatic progression and bone resorption., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 μM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre-clinical drug candidates., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The tumor microenvironment is increasingly recognized as a major factor influencing the success of therapeutic treatments and has become a key focus for cancer research. The progressive growth of a tumor results in an inability of normal tissue blood vessels to oxygenate and provide sufficient nutritional support to tumor cells. As a consequence the expanding neoplastic cell population initiates its own vascular network which is both structurally and functionally abnormal. This aberrant vasculature impacts all aspects of the tumor microenvironment including the cells, extracellular matrix, and extracellular molecules which together are essential for the initiation, progression and spread of tumor cells. The physical conditions that arise are imposing and manifold, and include elevated interstitial pressure, localized extracellular acidity, and regions of oxygen and nutrient deprivation. No less important are the functional consequences experienced by the tumor cells residing in such environments: adaptation to hypoxia, cell quiescence, modulation of transporters and critical signaling molecules, immune escape, and enhanced metastatic potential. Together these factors lead to therapeutic barriers that create a significant hindrance to the control of cancers by conventional anticancer therapies. However, the aberrant nature of the tumor microenvironments also offers unique therapeutic opportunities. Particularly interventions that seek to improve tumor physiology and alleviate tumor hypoxia will selectively impair the neoplastic cell populations residing in these environments. Ultimately, by combining such therapeutic strategies with conventional anticancer treatments it may be possible to bring cancer growth, invasion, and metastasis to a halt., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The failure of standard treatment for patients diagnosed with glioblastoma (GBM) coupled with the highly vascularized nature of this solid tumor has led to the consideration of agents targeting VEGF or VEGFRs, as alternative therapeutic strategies for this disease. Despite modest achievements in survival obtained with such treatments, failure to maintain an enduring survival benefit and more invasive relapsing tumors are evident. Our study suggests a potential mechanism by which anti-VEGF/VEGFR therapies regulate the enhanced invasive phenotype through a pathway that involves TGFβR and CXCR4. VEGFR signaling inhibitors (Cediranib and Vandetanib) elevated the expression of CXCR4 in VEGFR-expressing GBM cell lines and tumors, and enhanced the in vitro migration of these lines toward CXCL12. The combination of VEGFR inhibitor and CXCR4 antagonist provided a greater survival benefit to tumor-bearing animals. 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, TGFβ/TGFβR, and CXCL12/CXCR4 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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

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.

Vascular targeting agents on their own have been shown to be insufficient for complete treatment of solid tumors, emphasizing the importance of studying the vascular effects of these drugs for their use with conventional therapies in the clinic. First-pass fluorescence imaging combined with hyperspectral imaging of hemoglobin saturation of microvessels in the murine dorsal window chamber model provides an easily implementable, low cost method to analyze tumor vascular response to these agents in real-time. In this study, the authors utilized these methods to spectroscopically demonstrate distinct vessel structure, blood flow and oxygenation changes in human Caki-2 renal cell carcinoma following treatment with OXi4503 alone, Sunitinib alone and both drugs together. We showed that treatment with OXi4503 plus Sunitinib destroyed existing tumor microvessels, inhibited blood vessel recovery and impaired Caki-2 tumor growth significantly more than either treatment alone.

Background: Previous studies have hypothesized that tumor blood flow may be elevated or reduced during exercise, which could impact the tumor microenvironment. However, to date technical limitations have precluded the measurement of tumor blood flow during exercise. Using an orthotopic preclinical model of prostate cancer, we tested the hypotheses that during exercise tumors would experience 1) diminished vascular resistance, 2) augmented blood flow, 3) increased numbers of perfused vessels, and 4) decreased tissue hypoxia and, furthermore, that the increased perfusion would be associated with diminished vasoconstriction in prostate tumor arterioles., Methods: Dunning R-3327 MatLyLu tumor cells were injected into the ventral prostate of male Copenhagen rats aged 4 to 6 months randomly assigned to tumor-bearing (n = 42) or vehicle control (n = 14) groups. Prostate tumor blood flow, vascular resistance, patent vessel number, and hypoxia were measured in vivo in conscious rats at rest and during treadmill exercise, and vasoconstrictor responsiveness of resistance arterioles was investigated in vitro., Results: During exercise there was a statistically significant increase in tumor blood flow (approximately 200%) and number of patent vessels (rest mean ± standard deviation [SD] = 12.7±1.3; exercise mean ± SD = 14.3±0.6 vessels/field; Student t test two-sided P = .02) and decreased hypoxia compared with measurements made at rest. In tumor arterioles, the maximal constriction elicited by norepinephrine was blunted by approximately 95% vs control prostate vessels., Conclusions: During exercise there is enhanced tumor perfusion and diminished tumor hypoxia due, in part, to a diminished vasoconstriction. The clinical relevance of these findings are that exercise may enhance the delivery of tumor-targeting drugs as well as attenuate the hypoxic microenvironment within a tumor and lead to a less aggressive phenotype.

Systemic drug delivery to solid tumors involving macromolecular therapeutic agents is challenging for many reasons. Amongst them is their chaotic microvasculature which often leads to inadequate and uneven uptake of the drug. Localized drug delivery can circumvent such obstacles and convection-enhanced delivery (CED)--controlled infusion of the drug directly into the tissue--has emerged as a promising delivery method for distributing macromolecules over larger tissue volumes. In this study, a three-dimensional MR image-based computational porous media transport model accounting for realistic anatomical geometry and tumor leakiness was developed for predicting the interstitial flow field and distribution of albumin tracer following CED into the hind-limb tumor (KHT sarcoma) in a mouse. Sensitivity of the model to changes in infusion flow rate, catheter placement and tissue hydraulic conductivity were investigated. The model predictions suggest that 1) tracer distribution is asymmetric due to heterogeneous porosity; 2) tracer distribution volume varies linearly with infusion volume within the whole leg, and exponentially within the tumor reaching a maximum steady-state value; 3) infusion at the center of the tumor with high flow rates leads to maximum tracer coverage in the tumor with minimal leakage outside; and 4) increasing the tissue hydraulic conductivity lowers the tumor interstitial fluid pressure and decreases the tracer distribution volume within the whole leg and tumor. The model thus predicts that the interstitial fluid flow and drug transport is sensitive to porosity and changes in extracellular space. This image-based model thus serves as a potential tool for exploring the effects of transport heterogeneity in tumors.

Osteosarcoma is a highly fatal cancer, with most patients ultimately succumbing to metastatic disease. The purpose of this study was to evaluate the effects of the antirheumatoid drug aurothiomalate on canine and human osteosarcoma cells and on canine osteosarcoma growth and metastasis in a mouse xenograft model. We hypothesized that aurothiomalate would decrease osteosarcoma cell survival, tumor cellular proliferation, tumor growth, and metastasis. After performing clonogenic assays, aurothiomalate or a placebo was administered to 54 mice inoculated with canine osteosarcoma. Survival, tumor growth, embolization, metastasis, histopathology, cell proliferation marker Ki67, and apoptosis marker caspase-3 were compared between groups. Statistical analysis was carried out using the Kaplan-Meier method with the log-rank test and one-way analysis of variance with the Tukey's test or Dunn's method. Aurothiomalate caused dose-dependent inhibition of osteosarcoma cell survival (P<0.001) and decreased tumor growth (P<0.001). Pulmonary macrometastasis and Ki67 labeling were reduced with low-dose aurothiomalate (P=0.033 and 0.005, respectively), and tumor emboli and pulmonary micrometastases were decreased with high-dose aurothiomalate (P=0.010 and 0.011, respectively). There was no difference in survival, tumor development, ulceration, mitotic indices, tumor necrosis, nonpulmonary metastases, and caspase-3 labeling. Aurothiomalate treatment inhibited osteosarcoma cell survival and reduced tumor cell proliferation, growth, embolization, and pulmonary metastasis. Given aurothiomalate's established utility in canine and human medicine, our results suggest that this compound may hold promise as an adjunctive therapy for osteosarcoma. Further translational research is warranted to better characterize the dose response of canine and human osteosarcoma to aurothiomalate.

Regular physical exercise is considered to be an integral component of cancer care strategies. However, the effect of exercise training on tumor microvascular oxygenation, hypoxia, and vascular function, all of which can affect the tumor microenvironment, remains unknown. Using an orthotopic preclinical model of prostate cancer, we tested the hypotheses that, after exercise training, in the tumor, there would be an enhanced microvascular Po2, increased number of patent vessels, and reduced hypoxia. We also investigated tumor resistance artery contractile properties. Dunning R-3327 AT-1 tumor cells (10(4)) were injected into the ventral prostate of 4-5-mo-old male Copenhagen or Nude rats, which were randomly assigned to tumor-bearing exercise trained (TB-Ex trained; n = 15; treadmill exercise for 5-7 wk) or sedentary groups (TB-Sedentary; n = 12). Phosphorescence quenching was used to measure tumor microvascular Po2, and Hoechst-33342 and EF-5 were used to measure patent vessels and tumor hypoxia, respectively. Tumor resistance artery function was assessed in vitro using the isolated microvessel technique. Compared with sedentary counterparts, tumor microvascular Po2 increased ∼100% after exercise training (TB-Sedentary, 6.0 ± 0.3 vs. TB-Ex Trained, 12.2 ± 1.0 mmHg, P < 0.05). Exercise training did not affect the number of patent vessels but did significantly reduce tumor hypoxia in the conscious, resting condition from 39 ± 12% of the tumor area in TB-Sedentary to 4 ± 1% in TB-Ex Trained. Exercise training did not affect vessel contractile function. These results demonstrate that after exercise training, there is a large increase in the driving force of O2 from the tumor microcirculation, which likely contributes to the considerable reduction in tumor hypoxia. These results suggest that exercise training can modulate the microenvironment of the tumor, such that a sustained reduction in tumor hypoxia occurs, which may lead to a less aggressive phenotype and improve patient prognosis.

Metastasis remains the major cause of therapeutic failure, poor prognosis and high mortality in breast and prostate cancer patients. Aberrant microenvironments including hypoxia and acidic pH are common features of most solid tumors that have been long associated with enhanced metastasis and poor patient outcomes. Novel approaches to reduce metastatic incidences and improve overall survival of cancer patients clearly are needed. The crucial role of Cathepsin L (CTSL) in the dissemination of tumor cells has led to the development of novel cathepsin L inhibition strategies. The present study evaluated the ability of KGP94, a small molecule inhibitor of CTSL, to impair the metastatic phenotype of prostate (PC-3ML) and breast (MDA-MB-231) cancer cells both under normal and aberrant microenvironmental conditions. To assess the role of CTSL in hypoxia and acidosis triggered metastasis associated cell functions, secreted CTSL levels were determined under conditions pertinent to the tumor microenvironment. Acute exposures to hypoxic or acidic conditions significantly elevated secreted CTSL levels either through an increase in intracellular CTSL levels or through activation of lysosomal exocytosis or both, depending on the tumor type. Increases in CTSL secretion closely paralleled enhanced tumor cell migration and invasion suggesting that CTSL could be an essential factor in tumor microenvironment triggered metastasis. Importantly, KGP94 treatment led to marked attenuation of tumor cell invasion and migration under both normal and aberrant microenvironmental conditions suggesting that it may have significant utility as an anti-metastatic agent.

Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a subpopulation of malignant cells within a tumor mass and possess the ability to self-renew giving rise to heterogeneous tumor cell populations with a complex set of differentiated tumor cells. CSC may be the cause of metastasis and therapeutic refractory disease. Because few markers exist to identify and isolate pure CSC, we used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to create DNA aptamers that can identify novel molecular targets on the surfaces of live CSC. Out of 22 putative DNA sequences, 3 bound to ~90% and 5 bound to ~15% of DU145 prostate cancer cells. The 15% of cells that were positive for the second panel of aptamers expressed high levels of E-cadherin and CD44, had high aldehyde dehydrogenase 1 activity, grew as spheroids under nonadherent culture conditions, and initiated tumors in immune-compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers., (Copyright © 2012 UICC.)

Objective-To investigate the effects of bevacizumab, a human monoclonal antibody against vascular endothelial growth factor, on the angiogenesis and growth of canine osteosarcoma cells xenografted in mice. Animals-27 athymic nude mice. Procedures-To each mouse, highly metastasizing parent osteosarcoma cells of canine origin were injected into the left gastrocnemius muscle. Each mouse was then randomly allocated to 1 of 3 treatment groups: high-dose bevacizumab (4 mg/kg, IP), low-dose bevacizumab (2 mg/kg, IP), or control (no treatment). Tumor growth (the number of days required for the tumor to grow from 8 to 13 mm), vasculature, histomorphology, necrosis, and pulmonary metastasis were evaluated. Results-Mice in the high-dose bevacizumab group had significantly delayed tumor growth (mean ± SD, 13.4 ± 3.8 days; range, 9 to 21 days), compared with that for mice in the low-dose bevacizumab group (mean ± SD, 9.4 ± 1.5 days; range, 7 to 11 days) or control group (mean ± SD, 7. 2 ± 1.5 days; range, 4 to 9 days). Mice in the low-dose bevacizumab group also had significantly delayed tumor growth, compared with that for mice in the control group. Conclusions and Clinical Relevance-Results indicated that bevacizumab inhibited growth of canine osteosarcoma cells xenografted in mice, which suggested that vascular endothelial growth factor inhibitors may be clinically useful for the treatment of osteosarcoma in dogs. Impact for Human Medicine-Canine osteosarcoma is used as a research model for human osteosarcoma; therefore, bevacizumab may be clinically beneficial for the treatment of osteosarcoma in humans.

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5'-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model.

VEGF is a key regulator of normal and pathologic angiogenesis. Although many trans-activating factors of VEGF have been described, the transcriptional repression of VEGF remains much less understood. We have previously reported the identification of a SCAN domain-containing C2H2 zinc finger protein, ZNF24, that represses the transcription of VEGF. In the present study, we identify the mechanism by which ZNF24 represses VEGF transcription. Using reporter gene and electrophoretic mobility shift assays, we identify an 11-bp fragment of the proximal VEGF promoter as the ZNF24-binding site that is essential for ZNF24-mediated repression. We demonstrate in 2 in vivo models the potent inhibitory effect of ZNF24 on the vasculature. Expression of human ZNF24 induced in vivo vascular defects consistent with those induced by VEGF knockdown using a transgenic zebrafish model. These defects could be rescued by VEGF overexpression. Overexpression of ZNF24 in human breast cancer cells also inhibited tumor angiogenesis in an in vivo tumor model. Analyses of human breast cancer tissues showed that ZNF24 and VEGF levels were inversely correlated in malignant compared with normal tissues. These data demonstrate that ZNF24 represses VEGF transcription through direct binding to an 11-bp fragment of the VEGF proximal promoter and that it functions as a negative regulator of tumor growth by inhibiting angiogenesis.

Although solid tumors continuously shed cells, only a small fraction of the neoplastic cells that enter the blood stream are capable of establishing metastases. In order to be successful, these cells must attach, extravasate, proliferate and induce angiogenesis. Preclinical studies have shown that small-molecule ATP-competitive Src kinase inhibitors can effectively impair metastasis-associated tumor cell functions in vitro. However, the impact of these agents on the metastatic cascade in vivo is less well understood. In the present studies, we have examined the ability of saracatinib, a dual-specific, orally available inhibitor of Src and Abl protein tyrosine kinases, to interfere with the establishment of lung metastases in mice by tumor cells introduced into the blood stream. The results demonstrate that Src inhibition most effectively interferes with the establishment of secondary tumor deposits when treatments are administered while tumor cells are in the initial phases of dissemination.

Background: The c-Met receptor tyrosine kinase is aberrantly activated in many solid tumors. In a prior study we showed that prostate cancer PC-3 cells exhibit constitutively activated c-Met without exogenous hepatocyte growth factor (HGF); however whether this characteristic is due to an endogenous HGF/c-Met autocrine loop remains controversial. In the current study we examined the response of PC-3 cells to an anti-HGF neutralizing antibody or a small molecule Met kinase inhibitor (BMS-777607)., Methods: Cell scattering was tested by monitoring cell morphology after HGF stimulation. Cell migration was examined by both "wound-healing" and transwell assasy and invasion was detected by Matrigel-coated transwell assay. Proliferation, survival and anoikis were determined by MTT, colony formation and trypan blue exclusion assay, respectively. Gene and protein expression were assessed by real-time PCR and Western blot, respectively., Results: Although HGF mRNA could be detected in PC-3 cells, the molecular weight of secreted "HGF" protein was inconsistent with the functional recombinant HGF. Furthermore, conditioned medium from PC-3 cell cultures was ineffective at triggering either motogenic behavior or c-Met signaling in DU145, another prostate cancer cell line expressing c-Met but lacking basal c-Met activation. PC-3 cells also were not responsive to the anti-HGF neutralizing antibody in experiments assessing proliferation, migration, or c-Met signaling. BMS-777607 treatment with micromolar doses nonetheless led to significant inhibition of multiple PC-3 cell functions including proliferation, clonogenicity, migration and invasion. At the molecular level, BMS-777607 suppressed autophosphorylated c-Met and downstream c-Src and Akt pathways., Conclusions: These results suggest that the constitutive c-Met activation in PC-3 is independent of autocrine stimulation. Because PC-3 cells were responsive to BMS-777607 but not the anti-HGF antibody, the findings also indicate that under circumstances where c-Met is constitutively hyperactive in the absence of functional HGF, targeting the c-Met receptor remains a viable therapeutic option to impede cancer progression.

A tumor's dependence on angiogenesis for survival and growth has led to the advancement of a variety of blood vessel directed anticancer treatment strategies. Overexpression of angiopoietin-2 (Ang-2) in tumor vasculature and its crucial role in angiogenesis, i.e. the destabilization of endothelial/peri-endothelial cell interactions, now raises the possibility of additional novel anti-angiogenic therapeutics. The present study utilized a co-culture sphere model to (i) demonstrate the destabilizing effect of Ang-2 on endothelial/smooth muscle cell contact and (ii) evaluate the impact of the investigational Ang-2 antibody MEDI3617 on endothelial/smooth muscle cell dissociation. Real time imaging of spheres showed both exogenous Ang-2 and PMA induced endogenous Ang-2 secretion resulted in sphere destabilization (loss of endothelial cells from smooth muscle cell core). The presence of MEDI3617 inhibited this process. To assess the anti-angiogenic potential of MEDI3617 in vivo, nude mice were injected intradermally with human renal cell carcinoma cells (Caki-1, Caki-2) and the number of blood vessels induced over a 3 day period was scored. MEDI3617 (2, 10, 20 mg/kg) significantly reduced the initiation of blood vessels for both tumor models at all doses investigated. These data indicate that MEDI3617 treatment significantly impairs the initiation of angiogenesis by inhibiting the Ang-2 mediated disruption of endothelial/muscle cell interaction associated with blood vessel destabilization and thereby reduces tumor cell induced angiogenesis. The results support the notion that targeting the angiopoietin/Tie2 axis may offer novel anti-angiogenic strategies for cancer treatment., (Copyright © 2012 Elsevier Inc. All rights reserved.)