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The upregulation of proteolytic enzymes has been demonstrated to promote primary tumor development and metastatic bone cancer. The secreted proteases increase tumor growth and angiogenesis, and potentiate neoplastic cell dissemination. This article reviews the role and mechanisms of cathepsins in normal physiology, cancer, bone remodeling, and the tumor–bone interface, with a specific focus on cathepsins B, D, H, G, L, and K. In this review, we highlight the role of cathepsins in primary bone cancer (i.e., osteosarcoma (OS)), as well as metastatic breast (BCa) and prostate (PCa) cancer. In addition, we discuss the clinical utility and therapeutic potential of cathepsin-targeted treatments in primary and secondary bone cancers.

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

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.

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.

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.

Background: Metastasis is the major cause of therapeutic failure in prostate cancer patients, and hypoxia has been shown to promote metastatic functions. However, whether Src family kinases (SFKs) can be upregulated under hypoxia is unclear. Materials and methods: In the current study, we evaluated the effects of hypoxia on cellular functions and activities of different SFK members (c-Src, Lyn, Fyn) in prostate cancer cells. Prostate cancer cell functions were determined in vitro including migration (wound-healing assay), invasion (Matrigel-based transwell assay) and clonogenic cell survival (colony formation assay). Protein expression was detected by Western blotting and gene knockdown was accomplished by siRNA transfection. Results: SRC, but not LYN and FYN, is associated with overall survival in prostate cancer patients, while all three phosphorylated proteins are highly expressed in tumors compared to normal tissues. Short-term hypoxic exposure significantly enhances cell migration, invasion, clonogenic survival, and consistently, c-Src phosphorylation in both PC-3ML and C4-2B cells. Knockdown of SRC, but not LYN or FYN, abolished hypoxia-induced functions. Finally, small molecule Src inhibitors strongly inhibited cell behaviors and c-Src activation under hypoxic conditions. Conclusion: Our data show that hypoxia is able to enhance metastatic-associated cell functions by activating c-Src in prostate cancer cells. Importantly, SFK inhibition by small molecule inhibitors was able to impair hypoxia-induced metastasis associated cell functions, suggesting a possible role of SFK inhibitors for prostate cancer treatment.

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.

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.

Background: Hemorrhagic cystitis from chemical or radiation bladder injury can cause life-threatening bleeding and/or urothelial cancer. Fibroblast growth factor 7 (FGF7) blocks urothelial apoptosis induced by cyclophosphamide (CPP) or radiation, but there are limitations to its use in patients, including an inability to apply it by direct bladder infusion and the high cost due to the large size of the protein (18.8 kDa). Purpose: We previously identified a small peptide that equaled or exceeded the mitigation effect of full length FGF2 on acute radiation syndrome. Benefits of developing a similar FGF7 biomimetic peptide include lower likelihood of inflammation, longer shelf life, higher purity, and lower cost than the full-length protein. Methods: Based on the 3-dimensional structure of FGF2 peptide, a corresponding FGF7 peptide (FGF7p) consisting of 19 amino acids was identified and synthesized. FGF7p or vehicle was given subcutaneously (SQ) to female mice subjected to sham injury, intraperitoneal (IP) CPP or external beam radiation over the bladder. One day after injury, bladders were harvested. Slides with paraffin embedded tissues underwent H&E staining, TUNEL and immunofluorescence (IF) assays. Results: In uninjured control mice, a 20 mg/kg threshold dose of FGF7p induced expression of phosphorylated (activated) FRS2a (pFRS2a) and pAKT in urothelium (consistent with cytoprotective effects of FGF7, albeit at a 4x higher dose than full length FGF7). Unexpectedly, FGF7p activation of urothelial FRS2a and AKT was delayed 24 hours compared to FGF7. FGF7p (20 mg/kg) or vehicle was given at 72 and 48 hours prior to CPP (150 mg/kg). One day after CPP, TUNEL staining revealed an increase in apoptotic and sloughing urothelial cells in vehicle-treated mice compared to in FGF7p-treated mice. IF for pAKT and its targets, pS6K and pBAD, revealed minimal staining in vehicle-treated mice, but strong urothelial staining for all protective markers in FGF7p-treated mice. Using the same dosing strategy, we subjected anesthetized mice to 10 Gy radiation over the bladder. One day after injury, TUNEL staining revealed many more apoptotic urothelial nuclei in control mice, than in FGF7p-treated mice. Conclusions: FGF7p appears to block bladder urothelial apoptosis via AKT and its targets, in a similar manner to FGF7. FGF7p is 200x less expensive to make vs. FGF7 and is likely to work via direct bladder infusion (avoiding systemic side effects) due to its small size. Our future studies will clarify long term benefits of FGF7p and assess effectiveness of direct bladder infusion. Citation Format: Sridhar Narla, Lori P. Rice, David Ostrov, Steven G. Swarts, Dietmar W. Siemann, Carlton M. Bates. FGF7 peptide (FGF7p) mimetic mitigates urothelial injury from cyclophosphamide or radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5431.

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.

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.

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.

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.

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.

Overexpression of tyrosine kinases c-Src and c-Met has been shown to be highly associated with disease progression and poor clinical outcomes in many types of cancers. Although small molecule Src or Met inhibitors are being individually evaluated in clinical trials, patient data suggest that single treatment with any of these inhibitors only yield limited effects. One possibility is that redundant signaling pathways in tumor cells may allow tumor cell functions less interrupted upon inhibition of only one pathway. We therefore propose that c-Src and c-Met are two compensatory pathways that may cooperate to promote cancer metastasis. In order to delineate this hypothesis, we studied treatment effects of combining inhibitors targeting c-Src (dasatinib) and c-Met (ASLAN002) on multiple functional behaviors associated with tumor metastasis in c-Src and c-Met expressing metastatic cancer cells. Combination of Src- and Met-inhibitors was found to strongly inhibit cell colony formation by clonogenic survival assay. In a wound-healing assay, compared to either treatment alone, the combinatorial treatment exhibited greater suppression on cell motility. In addition, using a Matrigel-based Transwell assay, dasatinib plus ASLAN002 resulted in significant reduction of cell invasion in the presence or absence of HGF. Western blot analysis demonstrated that co-targeting Met and Src kinases lead to not only inhibition on c-Met and c-Src autophosphorylation but also multiple downstream kinases such as focal adhesion kinase (FAK), Akt and extracellular signal-regulated kinase (ERK). These data indicate that combined application of small molecule Src and Met inhibitors cause significant impairment on functional behaviors associated with cancer metastasis in vitro including survival, migration and invasion, suggesting that this combination intervention modality may serve as a potential anti-metastatic strategy that could be further tested in metastatic models in vivo. Citation Format: Yao Dai, Dietmar W. Siemann. Co-targeting c-Met and c-Src pathways to inhibit metastatic cancer cell migration and invasion [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 1477.

Prostate and breast cancers are the most prevalent and the second leading causes of cancer-deaths for men and women in the United States. Cancer survival rates generally worsen significantly with advanced and disseminated disease with nearly 90% of all cancer deaths resulting from metastasis. For breast and prostate cancers, bone is the most common site of metastasis. A key signaling pathway involved in metastatic dissemination of tumor cells is the receptor tyrosine kinase Axl. Axl belongs to the Tyro3, Axl, and MerTK (TAM) subfamily, is expressed in a variety of tumor types, including breast and prostate, and is associated with poor prognosis, metastasis, and outcome. The goal of the present study was to characterize the role of Axl signaling in prostate and breast cancer cell dissemination to bone. This was accomplished using Axl knockdown neoplastic cells, a selective small molecule Axl inhibitor (BGB324), and osteoclast progenitor cells in vitro, as well as intracardiac injection of Axl knockdown tumor cells in vivo. The results showed that Axl inhibition significantly decreases tumor cell migration and invasion in vitro, and suppression of Axl signaling in osteoclast precursor cells reduces the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation of neoplastic bone lesions. Taken together our findings indicate that therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis [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 3154.

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.

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.

Hyperactivated Akt/mTOR pathway and Src family kinases (SFKs) pathway commonly occur in prostate cancer that is highly associated with disease progression and poor prognosis. Therefore, small-molecule inhibitors targeting these pathways have been studied extensively for the treatment of advanced prostate cancer. Nevertheless, clinical observation suggests that individual treatment with either inhibitor only yields limited antitumor effects in patients. One possible interpretation is that these signaling pathways are redundant, such that blocking one pathway is not sufficient to yield considerable efficacies. In this regard, we combined mTOR inhibitor rapamycin and Src inhibitor dasatinib to detect functional behaviors in prostate cancer cells. Cell proliferation was detected by MTT, and clonogenic survival was tested by colony formation assay. Cell migration was assessed by “wound-healing” scratch assay. Protein expression was detected by Western blot analysis. We found that rapamycin plus dasatinib showed greater inhibition on cell proliferation and clonogenic survival than either single agent. Such combination effects were additive in PC-3 and synergistic in DU145 cells, respectively, as reflected by combination index calculation. In addition, cell migration was suppressed to a greater extent by combination treatment than either treatment alone. At the molecular level, co-targeting mTOR and SFKs leads to strong inhibition of not only phosphorylation of these two proteins but also their downstream components such as Akt, focal adhesion kinase (FAK), S6K and 4E-BP1. These data indicate that co-targeting mTOR and Src pathways is able to achieve enhanced tumor-suppressing effects in prostate cancer cells with overexpression of both kinases, suggesting that this combinative intervention modality may serve as a potential anticancer strategy that could be further tested in advanced prostate cancer models in vivo. Note: This abstract was not presented at the conference. Citation Format: Yao Dai, Dietmar W Siemann. Combined inhibition of mTOR and Src family kinases enhances treatment effects in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B02.

Although Src inhibitors are able to suppress malignant functions, their clinical outcomes are modest for the treatment of advanced prostate cancer, many of which harbor mutated PTEN. mTOR pathway is commonly hyperactivated in prostate cancer due to frequent PTEN mutation, and it is known that two mTOR complexes are both critical in prostate cancer progression. However, the functional role of mTOR kinase in Src inhibitor-mediated response remains unknown. In the current study, we combined mTOR kinase inhibitor AZD8055 and Src inhibitor dasatinib to evaluate functional behaviors in PTEN-deleted prostate cancer cells. Gene silencing was achieved by siRNA or shRNA transfection. Cell proliferation and clonogenic survival were performed by MTT and colony formation assay, respectively. “Wound-healing” scratch assay was conducted to monitor cell migration, and Matrigel-based transwell assay was applied to assess cell invasion. Protein expression was detected by Western blotting. We found that in PTEN-mt cells, C4-2B showed greater expression of p-Akt, p-S6, p-4EBP1 and p-eIF4E, all of which are mTOR complexes downstream components. Such molecular profile is consistent with functional observation that C4-2B cells are more resistant to dasatinib. Interestingly, single knockdown of either 4EBP1, S6RP or AKT showed little effects of inhibiting cell survival, while double knockdown of both 4EBP1 and AKT, cell survival was significantly inhibited. Consistently, two agents together showed significantly greater inhibition on proliferation and clonogenic survival than either single agent. Such combination effects were either additive or synergistic depending on the cell types, as reflected by combination index calculation. In addition, cell migration and invasion were inhibited in a greater extent by AZD8055 plus dasatinib, compared to either treatment alone. At the molecular level, co-targeting mTOR and Src kinases leads to strong inhibition of mTOR and Src pathways, such as FAK, Akt, S6RP and 4E-BP1. These data indicate that adding AZD8055 to dasatinib may achieve better treatment outcomes in PTEN-mt prostate cancer models. Such combination modality hold potential for future development as a novel strategy for PTEN-mutated prostate cancer patients who are less responsive to Src inhibitors. Citation Format: Yao Dai, Dietmar Siemann. Combination of mTOR kinase inhibitors with Src inhibitors in PTEN-mt prostate cancer cells [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 660.

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.

Breast cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related death for women in the United States. Approximately 90% of cancer-related death results from metastasis, and the bone is the most common site of metastasis for breast cancer. Upregulation of signaling pathways can promote the metastatic phenotypes of neoplastic cells, including cell migration, invasion, proliferation and angiogenesis. In addition, when breast cancer cells disseminate to the bone, neoplastic cells upregulate signaling pathways to activate osteoclast-induced bone resorption. A key pathway involved in metastatic spread of cancer cells is the receptor tyrosine kinase Axl. Genetic and pharmacologic Axl inhibition in both human and rodent breast cancer cell lines (MDA-MB-231 and 4T1) impaired tumor cell migration and invasion. Intracardiac inoculation of Axl knockdown (KD) MDA-MB-231 cells significantly decreased the number of bone metastases compared to the scrambled control MDA-MB-231 cells. Another mechanism by which Axl inhibition impaired bone metastasis is through the modulation of osteoclast maturation and activity. Co-culture of Axl KD breast cancer cells with murine osteoclast precursor cells (Raw264.7) significantly reduced the number of tumor cell-induced osteoclast resorption pits on bone slices. In addition, genetic and pharmacologic inhibition of Axl in Raw264.7 cells as well as bone marrow derived osteoclast precursor cells, significantly reduced osteoclast formation and activity. Scrambled control and Axl KD Raw264.7 cells were cultured in serum free medium for 24 hours, and their conditioned media were collected. The conditioned media of Axl KD Raw264.7 cells revealed significantly decreased levels of monocyte chemoattractant protein 1 (MCP-1), which known to promote osteoclastogenesis, compared to the scrambled control Raw264.7 cells. In summary, we have demonstrated that inhibition of Axl decreases breast cancer metastasis to the bone through modulation of the host and neoplastic cell functions. Collectively, these data indicate that Axl signaling may be a promising therapeutic target to impair tumor progression and metastasis. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cells utilize Axl for breast cancer bone metastasis [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 6114.

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.

A challenge in cancer research is the lack of a physiologically responsive in vitro model that allows for the investigation of cancer cells in a tissue-like environment. A model that enables real-time investigation of cancer cell migration, fate, and function during microvascular network growth does not currently exist. While current models such as 2D in vitro models or microfluidic systems incorporate real-time cell tracking and multiple cell types, they do not mimic the complexity of intact networks and tissue environments. The objective of this study was to establish a novel tumor-microvasculature model by demonstrating the feasibility of bioprinting cancer cells onto excised mouse mesometrium tissues. Prelabeled DiI mouse breast cancer (4T1) cells were inkjet-printed onto mouse mesometrium tissues. The cell ink for printing comprised 2% Na-alginate mixed in minimum essential media with 1% PenStrep (MEM) containing 15 million 4T1 cells. A single cancer cell spot per tissue was created by printing 10 drops of cell ink in the same location. MEM was added on top of tissue 30 seconds after printing and then incubated for 5 minutes before being plated into 6-well plates containing MEM supplemented with 20% serum. Tissues were cultured for 5 days, with media being changed every day. The spot of DiI+ cells was imaged every 24 hours to then quantify cell number and area for Day 0, 1, and 2. At Day 2 or 5, tissues were fixed in methanol and labeled with platelet endothelial cell adhesion molecule (PECAM), and E-cadherin, to identify endothelial cells and cancer cells, respectively. Co-localization of DAPI+ nuclei confirmed that DiI+ cells remained intact post-printing. Printed DiI+ 4T1 cells also remained viable after printing on Day 0 and after culture on Day 5. Time-lapse imaging over 5 days in culture enabled tracking of cell motility and proliferation. The number of cells (Day 0: 159 +/- 40, Day 1: 370 +/- 78, Day 2: 889 +/- 184, Day 5: 18,031 +/- 1,695) and cell area (Day 0: 0.72 +/- 0.19, Day 1: 1.89 +/- 0.33, Day 2: 2.92 +/- 0.44, Day 5: 5.93 +/- 0.75 mm2) were significantly increased over time. Moreover, a proliferation assay of anti-BrdU on Day 2 also highlighted that a subset of E-cadherin+ cells are in the S-phase of the cell cycle, contributing to the increase in cell number and cell area. Also, microvessels in the tissue were angiogenic evident by PECAM+ sprouts. These results corroborate that cancer cells are mobile and proliferative in this novel ex vivo model. Further, they demonstrate the potential for bioprinting cancer cells onto live, intact tissues to investigate cancer dynamics within a physiologically relevant microenvironment. Citation Format: Ariana D. Suarez-Martinez, Marc Sole-Gras, Samantha S. Dykes, Zachary R. Wakefield, Kevin Bauer, Arinola Lampejo, Dietmar W. Siemann, Yong Huang, Walter L. Murfee. A novel tumor microenvironment model that combines bioprinting and tissue culture to investigate cancer cell and microvascular interactions [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A23.

Low tumor accumulation following systemic delivery remains a key challenge for advancing many cancer nanomedicines. One obstacle in engineering nanoparticles for high tumor accumulation is a lack of techniques to monitor their stability and mobility in situ. One way to monitor the stability and mobility of magnetic nanoparticles biological fluids in situ is through dynamic magnetic susceptibility measurements (DMS), which under certain conditions provide a measure of the particle's rotational diffusivity. For magnetic nanoparticles modified to have commonly used biomedical surface coatings, we describe a systematic comparison of DMS measurements in whole blood and tumor tissue explants. DMS measurements clearly demonstrated that stability and mobility changed over time and from one medium to another for each different coating. It was found that nanoparticles coated with covalently grafted, dense layers of PEG were the only ones to show good stability and mobility in all settings tested. These studies illustrate the utility of DMS measurements to estimate the stability and mobility of nanoparticles in situ, and which can provide insights that lead to engineering better nanoparticles for in vivo use.

Breast cancer in the United States is the second most commonly diagnosed cancer in women. About 1 in 8 women will develop invasive breast cancer over the course of her lifetime and breast cancer remains the second leading cause of cancer-related death. In pursuit of novel therapeutic strategies, researchers have examined the tumor microenvironment as a potential anti-cancer target. In addition to neoplastic cells, the tumor microenvironment is composed of several critical normal cell types, including fibroblasts, vascular and lymph endothelial cells, osteoclasts, adipocytes, and immune cells. These cells have important roles in healthy tissue stasis, which frequently are altered in tumors. Indeed, tumor-associated stromal cells often contribute to tumorigenesis, tumor progression, and metastasis. Consequently, these host cells may serve as a possible target in anti-tumor and anti-metastatic therapeutic strategies. Targeting the tumor associated host cells offers the benefit that such cells do not mutate and develop resistance in response to treatment, a major cause of failure in cancer therapeutics targeting neoplastic cells. This review discusses the role of host cells in the tumor microenvironment during tumorigenesis, progression, and metastasis, and provides an overview of recent developments in targeting these cell populations to enhance cancer therapy efficacy.

OBJECTIVES/SPECIFIC AIMS: The objective of our collaboration is to develop a strong trans-disciplinary team consisting of microfluidics engineers, cancer biologists, and clinicians, to identify a universal marker to detect circulating osteosarcoma cells (COC) using microfluidic devices. Our goals are 3 fold: 1) Identify cell surface markers unique to osteosarcoma (OS) for COC isolation, 2) Develop a Geometrically Enhanced Mixing (GEM) device to isolate COCs, and 3) Evaluate the efficacy of GEM device to detect COCs in patients with OS. The long term goal of this collaboration is to utilize this cell detection approach to evaluate treatment efficacy and correlate the presence of circulating osteosarcoma cells with metastatic incidence. METHODS/STUDY POPULATION: In this phase of our study, we have identified an abundant and conserved cell surface marker across a panel of OS cell lines. Flow cytometry was used to evaluate the relative expression of Epithelial Cell Adhesion Molecule (EpCAM), and Ganglioside 2 or/and 3 (GD2/3) on a panel of OS cell lines. An antibody coated GEM microfluidic device is used to affirm the efficacy of GD2/3 to capture COCs. Further capture studies will be conducted using OS cell spiked blood samples. Analysis of variance (ANOVA) will be used to determine any significant difference in capture efficiency between EpCAM, GD2/3 cell surface markers. RESULTS/ANTICIPATED RESULTS: Our results demonstrate that EpCAM is not a suitable marker for COC detection. Results from our flow cytometry studies demonstrate that GD2/3 expression is significantly higher than EpCAM expression, across all OS cell lines within our panel. The cell capture efficiency strongly correlates with the cell surface expression data obtained from flow cytometry analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: OS is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. At diagnosis, 80% of patients will present with metastasis, however only 20% of these cases are clinically detectable. Innovative strategies to identify patients at risk of metastasis would allow for stratification of intervention therapies. Liquid biopsies are a novel alternative to current diagnostic imaging systems to monitor metastatic incidence and treatment efficacy. The detection of circulating tumor cells (CTCs) through routine blood sampling has the potential to be used clinically for earlier detection, monitoring the treatment of metastatic cancers and surveying the effect of therapeutic interventions on metastasis. To date, the majority of the studies on CTCs have evaluated their presence in carcinomas. Although sarcomas are rare, they generally have a poorer prognosis. This study will address one of the unmet medical needs in the field of CTC detection; the identification of cell surface OS makers to improve binding specificity, increase purity, and maintain a high capture efficiency.

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.

Epigenetic alteration is a hallmark of all cancers. Such alterations lead to modulation of fundamental cancer-related functions, such as proliferation, migration, and invasion. In particular, methylation of Histone H3 Lysine 4 (H3K4), a histone mark generally associated with transcriptional activation, is altered during progression of several human cancers. While the depletion of H3K4 demethylases promotes breast cancer metastasis, the effect of H3K4 methyltransferases on metastasis is not clear. Nevertheless, gene duplications in the human SETD1A (hSETD1A) H3K4 methyltransferase are present in almost half of breast cancers. Herein, expression analysis determined that hSETD1A is upregulated in multiple metastatic human breast cancer cell lines and clinical tumor specimens. Ablation of hSETD1A in breast cancer cells led to a decrease in migration and invasion in vitro and to a decrease in metastasis in nude mice. Furthermore, a group of matrix metalloproteinases (including MMP2, MMP9, MMP12, MMP13, and MMP17) were identified which were downregulated upon depletion of hSETD1A and demonstrated a decrease in H3K4me3 at their proximal promoters based on chromatin immunoprecipitation analysis. These results provide evidence for a functional and mechanistic link among hSETD1A, MMPs, and metastasis in breast cancer, thereby supporting an oncogenic role for hSETD1A in cancer. Implications: This study reveals that hSETD1A controls tumor metastasis by activating MMP expression and provides an epigenetic link among hSETD1A, MMPs, and metastasis of breast cancer. Mol Cancer Res; 13(3); 461–9. ©2014 AACR.

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. 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.

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

Cancer is the second leading cause of death in the United States and approximately 90% of cancer-related deaths result from metastasis, the spread of cancer cells to secondary sites. In tumors, the upregulation of certain signaling pathways promotes the metastatic phenotypes of cancer cells characterized by enhanced invasion, migration, survival, proliferation and induction of angiogenesis. A key pathway involved in metastasis is the receptor tyrosine kinase Axl. Axl is expressed in a variety of tumor types and is associated with poor prognosis, metastasis, and outcome. The purpose of the current study was to evaluate the efficacy of genetic and pharmacologic inhibitions of Axl on the metastatic phenotypes, specifically cell migration, invasion and angiogenesis. Human breast and prostate cancer cell lines (MDA-MB-231 and PC3-ML, respectively), and human lung microvascular endothelial cell (HMVEC-L) were used in this study. Stable Axl short hairpin RNA (shRNA) knockdown MDA-MB-231 and PC3-ML cell lines were generated by Mission Lentiviral transduction particles (Sigma). The efficacy of genetic and pharmacologic inhibition of Axl on tumor cell migration and invasion was evaluated by transwell migration and invasion assays. The effect of Axl knockdown on prostate cancer cell metastasis in vivo was evaluated by an intracardiac bone metastasis model. Conditioned media of Axl knockdown or control MDA-MB-231 cells were analyzed for angiogenic factors using the human angiogenesis array. The effect of Axl knockdown conditioned medium on angiogenic properties in vitro were assessed by endothelial cell tube formation and sprouting. Induction of angiogenesis in vivo was measured by performing intradermal assay. Genetic and pharmacologic inhibition of Axl decreases tumor cell migration and invasion. Axl knockdown inhibits metastatic disease burden of prostate cancer. Axl knockdown conditioned medium secretes less pro-angiogenic factors compared to its scramble control cells, suggesting that tumor cells utilize Axl pathway to promote the induction of angiogenesis. Axl knockdown conditioned medium impairs angiogenic properties, including endothelial cell tube formation and sprouting. Axl knockdown of tumor cells suppress tumor cell-induced angiogenesis in vivo. Collectively, these data indicate that Axl is involved in multiple steps of the metastatic pathway and that Axl is a promising anti-metastatic agent to inhibit tumor cell dissemination and initiation of angiogenesis. Further studies are ongoing to understand the downstream molecular signaling of Axl. Citation Format: Mai Tanaka, Dietmar W. Siemann. Axl: A promising therapeutic target that leads to multimodal inhibition of the metastatic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2849.

The presence of macrophages within breast tumors is associated with metastatic potential. Tumor-associated macrophages are often stimulated by Interleukin-4, resulting in a pro-tumorigenic (M2-like) phenotype and the secretion of growth factors and proteases, including the lysosomal protease cathepsin L (CTSL). CTSL is often secreted by breast cancer cells and stromal cells and contributes to tumor invasion, metastasis, and angiogenesis. We hypothesized that secretion of the proteolytic enzyme cathepsin L by both tumor-associated macrophages and neoplastic cells facilitate tumor invasion and dissemination. Initial studies found that inhibiting CTSL using the novel cathepsin L/K inhibitors KGP94 and KGP207 prevented in vitro M2 macrophage invasion and reduced macrophage-stimulated invasion of 4T1 murine breast cancer cells. Interestingly, KGP94 and KGP207 also reduced the expression of several M2-associated markers, suggesting that CTSL activity may be important for Interleukin-4-driven M0 to M2 differentiation. Furthermore, CTSL shRNA knockdowns revealed that CTSL supplied from both the tumor cell and the macrophage population is important for tumor cell invasion. These data suggest that tumor cells and macrophages may both contribute to the CTSL-driven metastatic phenotype of breast cancer. Taken together, these studies highlight the importance of CTSL 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. Citation Format: Samantha S. Dykes, Henrietta O. Fasanya, Dietmar W. Siemann. Host and neoplastic cell secretions of proteolytic enzymes potentiate metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3759.

Src inhibitors have been actively evaluated in the clinic for the treatment of advanced prostate cancer however the effects are modest. One interpretation is that oncogenic signaling pathways are redundant and blocking one pathway is not sufficient to yield considerable efficacies. mTOR pathway is commonly hyperactivated in prostate cancer. However, the functional role of mTOR in Src inhibitor-mediated response remains largely unknown. In the current study, we combined mTOR inhibitor rapamycin and Src inhibitor dasatinib to detect functional behaviors in prostate cancer cells. Cell proliferation and clonogenic survival were detected by MTT and colony formation assay, respectively. Wound-healing” scratch assay was conducted to monitor cell migration, and Matrigel-based transwell assay was applied to assess cell invasion. Protein expression was detected by Western blotting. Gene silencing was detected by siRNA transfection. We found that two agents combined together showed greater inhibition on proliferation and clonogenic survival than either agent alone. Such combination effects were additive or synergistic depending on the cell types, as reflected by combination index calculation. In addition, cell migration and invasion were inhibited in a greater extent by rapamycin plus dasatinib, compared to either treatment alone. At the molecular level, co-targeting mTOR and Src lead to clear inhibition of Src and its downstream pathway focal adhesion kinase (FAK), Akt and mTOR downstream components S6RP and 4E-BP1. Interestingly, knockdown 4EBP1, but not S6RP, attenuated dasatinib-mediated functional inhibition. These data indicate that adding mTOR inhibitors to Src-based intervention may achieve better treatment outcomes. Such combination could be evaluated as a novel treatment strategy for prostate cancer patients who are less responsive to Src inhibitors. In addition, 4EBP1 may be used as a candidate predictive biomarker for monitoring clinical effectiveness of Src inhibitors in prostate cancer. Citation Format: Yao Dai, Dietmar W. Siemann. mTOR pathway components dictate cell response to Src inhibitors in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3914.

Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a sub-population 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 non-adherent culture conditions, and initiated tumors in immune-compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers.

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.

Angiogenesis, the growth of new vessels from pre-existing ones, is an important feature of tumor growth that has been exploited as a therapeutic target in oncology. Given its key role in facilitating blood vessel sprouting, VEGF has been a major focus of anti-angiogenic strategies, but the observation of resistance in some clinical trials utilizing such agents has led to a search for new or complementary targets in angiogenesis process. The Angiopoietin/Tie2 pathway and in particular the Angiopoietin-2 (Ang-2) ligand which is critically involved in the destabilization of normal vasculature, has been identified as one such target. The current study investigated the potential benefits of combining an Ang-2 targeted therapy with small molecule VEGF targeted agents (Sunitinib, Cediranib) in a human renal cell carcinoma model. The results showed that while both Ang-2 and VEGF interference on their own impaired tumor growth and new blood vessel formation, the combination of agents that targeted both pathways resulted in significantly superior anti-tumor and anti-angiogenic effects.

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.