Reduced Tumor Invasion Research Articles

Transforming a toxic drug into an efficacious nanomedicine using a lipoprodrug strategy for the treatment of patient-derived melanoma xenografts

Despite the progress made with the recent clinical use of the anticancer compound cabazitaxel, the efficacy in patients remains unsatisfactory, largely due to the high in vivo toxicity of the agent. Therefore, strategies that achieve favorable outcomes and good safety profiles will greatly expand the repertoire of this potent agent. Here, we propose a combinatorial strategy to reform the cabazitaxel agent and the use of sequential supramolecular nanoassembly with liposomal compositions to assemble a prodrug-formulated liposome, termed lipoprodrug, for safe and effective drug delivery. Reconstructing cabazitaxel with a polyunsaturated fatty acid (i.e., docosahexaenoic acid) via a hydrolyzable ester bond confers the generated prodrug with the ability to be readily integrated into the lipid bilayer of liposomes for systemic administration. The resulting lipoprodrug scaffold showed significantly sustained drug release profiles and improved pharmacokinetics in rats as well as a reduction in systemic toxicity in vivo. Notably, the lipoprodrug outperformed free cabazitaxel in terms of in vivo therapeutic efficacy in multiple separate tumor xenograft-bearing mouse models, one of which was a patient-derived xenograft model. Surprisingly, the lipoprodrug was able to reduce tumor invasiveness and reprogram the tumor immunosuppressive microenvironment by proinflammatory macrophage polarization. Our findings validate this lipoprodrug approach as a simple yet effective strategy for transforming the highly toxic cabazitaxel agent into an efficacious nanomedicine with excellent in vivo tolerability. This approach could also be applied to rescue other drugs or drug candidates that have failed in clinical trials due to poor pharmacokinetic properties or unacceptable toxicity in patients.

Extracellular vesicles originating from glioblastoma cells increase metalloproteinase release by astrocytes: the role of CD147 (EMMPRIN) and ionizing radiation

BackgroundGlioblastoma multiforme is an aggressive primary brain tumor that is characterized by local invasive growth and resistance to therapy. The role of the microenvironment in glioblastoma invasiveness remains unclear. While carcinomas release CD147, a protein that signals for increased matrix metalloproteinase (MMP) release by fibroblasts, glioblastoma does not have a significant fibroblast component. We hypothesized that astrocytes release MMPs in response to CD147 contained in glioblastoma-derived extracellular vesicles (EVs) and that ionizing radiation, part of the standard treatment for glioblastoma, enhances this release.MethodsAstrocytes were incubated with EVs released by irradiated or non-irradiated human glioblastoma cells wild-type, knockdown, or knockout for CD147. Levels of CD147 in glioblastoma EVs and MMPs secreted by astrocytes were quantified. Levels of proteins in the mitogen activated protein kinase (MAPK) pathway, which can be regulated by CD147, were measured in astrocytes incubated with EVs from glioblastoma cells wild-type or knockdown for CD147. Immunofluorescence was performed on the glioblastoma cells to identify changes in CD147 localization in response to irradiation, and to confirm uptake of the EVs by astrocytes.ResultsImmunoblotting and mass spectrometry analyses showed that CD147 levels in EVs were transiently increased when the EVs were from glioblastoma cells that were irradiated with γ rays. Specifically, the highly-glycosylated 45 kDa form of CD147 was preferentially present in the EVs relative to the cells themselves. Immunofluorescence demonstrated that astrocytes incorporate glioblastoma EVs and subsequently increase their secretion of active MMP9. The increase was greater if the EVs were from irradiated glioblastoma cells. Testing MAPK pathway activation, which also regulates MMP expression, showed that JNK signaling, but not ERK1/2 or p38, was increased in astrocytes incubated with EVs from irradiated compared to non-irradiated glioblastoma cells. Knockout of CD147 in glioblastoma cells blocked the increased JNK signaling and the rise in secreted active MMP9 levels.ConclusionsThe results support a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveal a prominent role for ionizing radiation in enhancing the effect. They provide an improved understanding of glioblastoma intercellular signaling in the context of radiotherapy, and identify pathways that can be targeted to reduce tumor invasiveness.1D3uFmTqHFEH8Tq79aQmnJVideo abstractGraphical abstract

RAPAMYCIN ON THE TREATMENT OF MUCOEPIDERMOID CARCINOMAS AND SQUAMOUS CELL CARCINOMAS

Mucoepidermoid carcinomas (MEC) are the most common malignancy of the salivary glands. Dysfunctional clock signaling is observed in a variety of pathologic conditions. Members of the clock gene family are upregulated in tumor cells. We explored the role of the mTOR signaling pathway, commonly found upregulated in tumors including in MEC, oral squamous cell carcinomas (OSCC), and its population of cancer stem cells (CSC). We also explored the potential implication on the disruption of the mTOR signaling on the expression the clock gene family member BMAL1 in tumor behavior using the specific mTOR inhibitor rapamycin. We showed that MEC and HNSCC are endowed with high expression levels of mTOR and BMAL1. Rapamycin demonstrates potent antiproliferative activity in MEC and HNSCC cells, with the ability to reduce tumor invasion and migration. We also found that rapamycin is an effective modulator of the core clock gene BMAL1. Unexpectedly, inhibition of mTOR signaling resulted in the proliferation of CSC in HNSCC while reduced CSC from MEC. These findings suggest rapamycin have different effects on CSC of different solid tumors. While administration of rapamycin was efficient in treating MEC tumors and depleting its CSC, rapamycin failed in disrupting the population of CSC from OSCC.

Reduced Protumorigenic Tumor-Associated Macrophages With Statin Use in Premalignant Human Lung Adenocarcinoma.

BackgroundStatins have anticancer properties by acting as competitive inhibitors of the mevalonate pathway. They also have anti-inflammatory activity, but their role in suppressing inflammation in a cancer context has not been investigated to date.MethodsWe have analyzed the relationship between statin use and tumor-associated macrophages (TAMs) in a cohort of 262 surgically resected primary human lung adenocarcinomas. TAMs were evaluated by multiplex immunostaining for the CD68 pan-TAM marker and the CD163 protumorigenic TAM marker followed by digital slide scanning and partially automated quantitation. Links between statin use and tumor stage, virulence, and cancer-specific survival were also investigated in a wider cohort of 958 lung adenocarcinoma cases. All statistical tests were two-sided.ResultsWe found a statin dose-dependent reduction in protumorigenic TAMs (CD68+CD163+) in both stromal (P = .021) and parenchymal (P = .003) compartments within regions of in situ tumor growth, but this association was lost in invasive regions. No statistically significant relationship between statin use and tumor stage was observed, but there was a statin dose-dependent shift towards lower histological grade as assessed by growth pattern (P = .028). However, statin use was a predictor of slightly worse cancer-specific survival (P = .032), even after accounting for prognostic variables in a multivariable Cox proportional hazards survival model (hazard ratio = 1.38, 95% confidence interval = 1.04 to 1.84).ConclusionsStatin use is associated with reduced numbers of protumorigenic TAMs within preinvasive lung adenocarcinoma and is related to reduced tumor invasiveness, suggesting a chemo-preventive effect in early tumor development. However, invasive disease is resistant to these effects, and no beneficial relationship between statin use and patient outcome is observed.

Long noncoding RNA ZNF667-AS1 reduces tumor invasion and metastasis in cervical cancer by counteracting microRNA-93-3p-dependent PEG3 downregulation.

Zinc finger protein 667‐antisense RNA 1 (ZNF667‐AS1), located on human chromosome 19q13.43, is a member of the C2H2 zinc finger protein family. Herein, we aimed to analyze the interactions between ZNF667‐AS1, microRNA‐93‐3p (miR‐93‐3p), and paternally expressed gene 3 (PEG3) and to explore their roles in the tumorigenesis of cervical cancer (CC). Differentially expressed long noncoding RNAs and miRNAs related to CC were determined using gene expression datasets sourced from the Gene Expression Omnibus database. Subsequently, the regulatory relationships between ZNF667‐AS1 and miR‐93‐3p and between miR‐93‐3p and PEG3 were identified using the dual‐luciferase reporter gene assay. In addition, the expression of miR‐93‐3p and ZNF667‐AS1 was up‐ or downregulated in CC cells (HeLa), in order to assess their effects on cell cycle distribution and cell invasion in vitro, and tumor growth and metastasis in vivo. MiR‐93‐3p was found to be highly expressed, while ZNF667‐AS1 and PEG3 were poorly expressed in CC. ZNF667‐AS1 could competitively bind to miR‐93‐3p, which targeted PEG3. In addition, miR‐93‐3p downregulation and ZNF667‐AS1 overexpression led to increased expression of PEG3, tissue inhibitor of metalloproteinases, and p16 and decreased expression of cyclin D1, matrix metalloproteinase‐2 and ‐9. MiR‐93‐3p inhibition and ZNF667‐AS1 elevation also inhibited cell cycle entry and cell invasion in vitro, but repressed tumor growth and metastasis in vivo. These key findings demonstrated that upregulation of ZNF667‐AS1 could suppress the progression of CC via the modulation of miR‐93‐3p‐dependent PEG3, suggesting a potential therapeutic target for the treatment of CC.

FENDRR reduces tumor invasiveness in prostate cancer PC-3 cells by targeting CSNK1E.

Prostate cancer, one of the most common malignant tumors in urology, now has become a malignant disease that seriously threatens the health of men in China. Although there are a large number of clinical studies on the treatment of patients with prostate cancer, many patients have entered the advanced stage of diagnosis, and little is known about its pathogenesis. We identified a series of ncRNA and TF by differential expression analysis, co-expression analysis, enrichment analysis, connectivity analysis, and hypergeometric test strategies for prostate cancer expression genomes. 53 modules related to prostate cancer PC-3 cells were obtained, involving module focusing of 4448 genes. Based on these modules, we predicted that miR-26a-5p, miR-130a-3p, miR-519d-3p, etc. have important regulatory effects on prostate cancer PC-3 cells. At the same time, a series of transcription factors (relating to RELA, SOX10, TP53, and TWIST2, etc.) were obtained and may play a key regulatory role in prostate cancer PC-3 cell-related modules. These results suggest that FENDRR in prostate cancer may reduce tumor invasion in prostate cancer PC-3 cells by targeting CSNK1E, which may have favourable effort to better understand the underlying pathogenesis of prostate cancer and provide a tough theoretical basis for further studying prostate cancer.

CD73 Promotes Glioblastoma Pathogenesis and Enhances Its Chemoresistance via A2B Adenosine Receptor Signaling.

Glioblastoma (GB) is one of the deadliest brain cancers to afflict humans, and it has a very poor survival rate even with treatment. The extracellular adenosine-generating enzyme CD73 is involved in many cellular functions that can be usurped by tumors, including cell adhesion, proliferation, invasion, and angiogenesis. We set out to determine the role of CD73 in GB pathogenesis. To do this, we established a unique GB mouse model (CD73-FLK) in which we spatially expressed CD73 on endothelial cells in CD73-/- mice. This allowed us to elucidate the mechanism of host CD73 versus GB-expressed CD73 by comparing GB pathogenesis in WT, CD73-/-, and CD73-FLK mice. GB in CD73-/- mice had decreased tumor size, decreased tumor vessel density, and reduced tumor invasiveness compared with GB in WT mice. Interestingly, GBs in CD73-FLK mice were much more invasive and caused complete distortion of the brain morphology. We showed a 20-fold upregulation of A2B AR on GB compared with sham, and its activation induced matrix metalloproteinase-2, which enhanced GB pathogenesis. Inhibition of A2B AR signaling decreased multidrug resistance transporter protein expression, including permeability glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). Further, we showed that blockade of A2B AR signaling potently increased GB cell death induced by the chemotherapeutic drug temozolomide. Together, these findings suggest that CD73 and A2B AR play a multifaceted role in GB pathogenesis and progression and that targeting the CD73-A2B AR axis can benefit GB patients and inform new approaches for therapy to treat GB patients.SIGNIFICANCE STATEMENT Glioblastoma (GB) is the most devastating primary brain tumor. GB patients' median survival is 16 months even with treatment. It is critical that we develop prophylaxes to advance GB treatment and improve patient survival. CD73-generated adenosine has been implicated in cancer pathogenesis, but its role in GB was not ascertained. Here, we demonstrated that host CD73 plays a prominent role in multiple areas of glioblastoma pathogenesis, including promoting GB growth, its angiogenesis, and its invasiveness. We found a 20-fold increase in A2B adenosine receptor (AR) expression on GB compared with sham, and its inhibition increased GB chemosensitivity to temozolomide. These findings strongly indicate that blockade or inhibition of CD73 and the A2B AR are prime targets for future GB therapy.

TIAM1 promotes chemoresistance and tumor invasiveness in colorectal cancer

Accumulating evidence suggests that cancer cells with stem cell-like features have higher resistance to chemotherapeutic agents. Herein, we identified T-lymphoma invasion and metastasis-inducing protein-1 (TIAM1) as one of the Wnt-signaling associated genes which drives self-renewal and its expression is upregulated by cancer associated fibroblasts (CAFs). TIAM1 expression was assessed in resected colorectal cancer (CRC) tissues from 300 patients who did or did not respond to chemotherapy. siRNA and CRISPR/Cas9 was used to examine whether the inhibition of TIAM1 affects chemosensitivity of CRC. We demonstrate that stemness through Wnt signaling regulates chemosensitivity and this phenomenon occurs exclusively in cancer stem cells. Subsequently, we established patient-derived CAFs and tested whether the drug sensitivity of CRC cell lines is altered with CAF-derived conditioned medium. High-TIAM1 expression correlated significantly with poor prognosis of CRC patients, and was overexpressed in patients who did not respond to chemotherapy. We demonstrated that the inhibition of TIAM1 enhanced sensitivity to chemotherapeutic drugs and reduced tumor invasiveness in a series of experiments in vitro. Moreover, CAF-derived conditioned media increased stemness and chemoresistance in CRC cell lines through TIAM1 overexpression. In addition, we validated TIAM1 associated drug sensitivity using a xenograft model. We have demonstrated that TIAM1 is overexpressed in CRC tumors from patients who did not respond to chemotherapeutic drugs and levels of TIAM1 expression served as an independent prognostic factor. Mechanistically, CAFs enhanced CRC chemoresistance through TIAM1 overexpression. Collectively, these results suggest that TIAM1 regulates chemosensitivity in tumors and stroma and thus may be an attractive therapeutic target.

MSI1 associates glioblastoma radioresistance via homologous recombination repair, tumor invasion and cancer stem-like cell properties

IntroductionGlioblastoma multiforme (GBM) is the most common brain malignancy in adults, and currently available GBM treatments present several unique challenges. It is known that GBM involves cancer stem-like cells (CSCs) and tumor cells that aggressively invade normal brain tissues, and both cell types may cause resistance to radiotherapy (RT) and are thus responsible for therapeutic failure. The radioresistance of GBM cells relies on the efficient activation of the DNA damage response (DDR), but the mechanisms linking this response with stem-cell status and tumor invasion remain unclear. Materials and methodsWe used irradiation to treat patient-derived GBM (Par) cells and then purified radioresistant GBM (R2M2) cells through two rounds of irradiation and an invasion assay. Musashi-1 (MSI1) is a neural stem-cell marker and key oncogenic factor of GBM. We identified MSI1 expression to predict radioresistance through silencing an MSI1-high-expressing R2M2 cell line or inducing overexpression in a Par cell line with low/no MSI1 expression and assessing the subsequent DDR. ResultMSI1 enhances tumor invasion via VCAM1 and modulates GBM radioresistance via the hyperactivation of the DDR through increasing homologous recombination repair and evading apoptosis. MSI1 knockdown induces DNA damage accumulation in irradiated GBM cells and promotes their depletion in vitro; MSI1 knockdown also inhibits the formation of GBMs generated by irradiated xeno-transplanted cells. MSI1 inhibition may radiosensitize tumors, prevent CSC-positive selection induced by RT, and reduce tumor invasion. ConclusionMSI1 may involve in regulating GBM radioresistance, invasion, and recurrence and could be a novel target for GBM treatment.

LGR5, a novel functional glioma stem cell marker, promotes EMT by activating the Wnt/\u03b2-catenin pathway and predicts poor survival of glioma patients

BackgroundTumor recurrence, the chief reason for poor prognosis of glioma, is largely attributed to glioma stem cells (GSCs) and epithelial-mesenchymal transition (EMT). However, the mechanisms among them remain unknown. Here, we determined whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), known as a stem cell marker for colon cancer and gastric cancer, can serve as a novel GSC marker involved in EMT and a therapeutic target in glioma.MethodsStemness properties were examined in FACS-isolated LGR5+/LGR5− cells. Reported stem cell markers, EMT and the Wnt/β-catenin pathway were examined in stable LGR5 knockdown or overexpressed GSCs by Western Blot. The treatment experiment was performed in an intracranial orthotopic xenograft model by knockdown of LGR5 or by using the Wnt/β-catenin pathway inhibitor Wnt-C59. LGR5 expression was determined in 268 glioma specimens by immunohistochemistry.ResultsLGR5+ cells possessed stronger stemness properties compared to LGR5− cells. The expression of SOX2, Nanog, CD133, CD44, CD24 and EpCAM was modulated by LGR5. Both LGR5 knockdown and Wnt-C59 reduced tumor invasion and migration and blocked EMT by inhibiting the Wnt/β-catenin pathway in vitro and suppressed the intracranial orthotopic xenograft growth and prolonged the survival of xenograft mice in vivo. Moreover, LGR5 was positively correlated with Ki67, N-cadherin and WHO grade and negatively correlated with IDH1. Glioma patients with high expression of LGR5 showed significantly poorer prognosis.ConclusionsLGR5 is a new functional GSC marker and prognostic indicator that can promote EMT by activating the Wnt/β-catenin pathway and would thus be a novel therapeutic target for glioma.

The secreted glycolytic enzyme GPI/AMF stimulates glioblastoma cell migration and invasion in an autocrine fashion but can have anti-proliferative effects.

Aerobic glycolysis confers several advantages to tumor cells, including shunting of metabolites into anabolic pathways. In glioblastoma cells, hypoxia induces a flux shift from the pentose phosphate pathway toward glycolysis and a switch from proliferation to migration. The mechanistic link between glycolysis and migration is poorly understood. Since glucose-6-phosphate isomerase (GPI) is identical to the secreted cytokine autocrine motility factor (AMF), we investigated whether GPI/AMF regulates glioblastoma cell invasion. The expression and hypoxic regulation of GPI/AMF and its receptor AMFR were analyzed in glioblastoma tissue and cell lines. Functional effects were studied in vitro and in xenograft models. High GPI/AMF expression in glioblastomas was found to be associated with a worse patient prognosis, and levels were highest in hypoxic pseudopalisades. Hypoxia upregulated both GPI/AMF and AMFR expression as well as GPI/AMF secretion in vitro. GPI/AMF stimulated cell migration in an autocrine fashion, and GPI/AMF expression was upregulated in migratory cells but reduced in rapidly proliferating cells. Knockdown or inhibition of GPI/AMF reduced glioblastoma cell migration but in part stimulated proliferation. In a highly invasive orthotopic glioblastoma model, GPI/AMF knockdown reduced tumor cell invasion but did not prolong survival. In a highly proliferative model, knockdown tumors were even larger and more proliferative than controls; however, perivascular invasion, provoked by simultaneous bevacizumab treatment, was reduced. GPI/AMF is a potent motogen for glioblastoma cells, explaining in part the association between glycolysis and migration. Targeting GPI/AMF is, however, problematic, since beneficial anti-invasive effects may be outweighed by unintended mitogenic effects. 1.Increased glycolysis is linked with increased cell migration and invasion in glioblastoma cells. 2.The glycolysis enzyme GPI/AMF may serve as a target for antimetabolic and anti-invasive therapy. 3.Despite reducing tumor invasion, GPI/AMF targeting may have unwanted growth stimulatory effects.

Abstract 5226: Tiam1 is a key modulator of chemoresistance in colorectal cancer

Abstract Purpose: Emerging evidence indicates that a small subpopulation of cancer cells with stem cell-like properties are responsible for acquisition of drug resistance in colorectal cancer (CRC). Intriguingly, the acquisition of such a stem-like state and subsequent drug resistance has been shown to be induced by the tumor microenvironment. In particular, cancer associated fibroblasts (CAFs) have been identified as a key component of tumor microenvironment which promote cancer stemness. Wnt signaling is a well-recognized pathway that drives self-renewal, and recently one of the Wnt-responsive genes, Tiam1, a guanine nucleotide exchange factor specific for Rac1, was shown to be overexpressed in CRC and associated with an aggressive cancer phenotype. Herein, we have investigated Tiam1 as a key modulator of chemoresistance in CRC. Methods: Tiam1 expression was assessed in resected CRC tissues from 300 patients who did or did not respond to chemotherapy. We used siRNA and CRISPR/CAS9 approach in three cell lines and animal models to examine whether inhibition of Tiam1 affects chemosensitivity in CRC. In addition, we established CRC patient derived-CAFs and examined whether the drug sensitivity of CRC cells was altered by supplementation of CAF-derived conditioned medium. Results: High Tiam1 expression significantly correlated with poor prognosis in CRC patients (P=0.032), and emerged as an independent prognostic factor (HR 5.06, 95% CI 1.10-23.2, P=0.038). In addition, Tiam1 overexpression is associated with patients who did not respond to chemotherapy (P=0.0005). We demonstrated that siRNA-mediated inhibition of Tiam1 enhanced sensitivity to three chemotherapeutic drugs (5-fluorouracil, oxaliplatin and irinotecan), and reduced tumor invasiveness in HCT116, SW480 and SW620 cell lines. Silencing of Tiam1 resulted in downregulation of stemness-related genes and spheroid formation ability. Furthermore, we validated that Tiam1-knockdown enhanced drug sensitivity and decreased the expression of stemness-related genes in xenograft tumors. Finally, we illustrated that the conditioned medium (CM) derived from CAFs led to increased stemness and chemoresistance in CRC cells through Tiam1 overexpression, and silencing of Tiam1 in CAFs resulted in reversal of stemness and chemoresistance properties. From a mechanistic standpoint, the target genes of Wnt signaling were also upregulated during co-culture with CM in CRC cells. Conclusion: We have firstly demonstrated that Tiam1 is overexpressed in CRC patients who did not respond to chemotherapeutic drugs; and furthermore, high Tiam1 expression emerged as an independent prognostic factor in CRC. Mechanistically, CAFs enhanced CRC chemoresistance through Tiam1 overexpression. Collectively, these results suggest that Tiam1 suppression in stroma may sensitize CRC cells to chemotherapeutic agents, suggesting that Tiam1 may be an attractive therapeutic target in colorectal cancer. Citation Format: Daisuke Izumi. Tiam1 is a key modulator of chemoresistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5226.

Abstract 4189: Cationic polymer inhibits pancreatic cancer invasion and metastasis

Abstract Background and Hypothesis: Pancreatic cancer (PC) has the poorest prognosis of all major cancers, with a 5-year survival of less than 5%. Cell-free DNA (cfDNA) has been well validated as a biomarker that correlates with tumor burden. More recently, a role for cfDNA in tumor progression has also been identified. The latter occurs through activation of Toll-like receptors (TLRs) in both tumor cells and the host environment, which can upregulate prometastatic signaling pathways. In addition, tumor-derived extracellular vesicles (EVs) such as microparticles and exosomes have also been implicated in promoting metastasis by activating proinvasive pathways in tumor cells and preconditioning secondary sites for metastatic establishment. Our laboratory has previously shown that cationic polymers can neutralize cfDNA and abrogate inflammation in disease models of autoimmunity and infection. We investigated the ability of the cationic polymer, PAMAM-G3, to bind and inhibit cfDNA and EVs and thereby inhibit tumor invasion in vitro and metastasis in vivo. Methods and Results: Transwell-Matrigel invasion assays were performed using multiple PC cell lines. Cells were plated in the upper chamber with no serum +/- cfDNA, EVs, or PAMAM-G3. The bottom chamber was plated with complete media as a chemo-attractant. Cells treated with cfDNA, EVs, or PAMAM-G3 were also analyzed for activation of pro-inflammatory pathways (NFkB, MAPK, etc.). The in vivo model was performed in C57B6 mice that were injected with bioluminescent murine PC cells into their spleens and followed for liver metastases with bioluminescent imaging. Experimental mice were treated biweekly with intraperitoneal PAMAM-G3 (20 mg/kg) or saline starting 48 hours after pancreatic tumor cell implantation. PAMAM-G3 significantly inhibited in vitro invasion of pancreatic cancer cell lines in response to cancer patient serum-derived cfDNA and extracellular vesicles. Moreover, PAMAM-G3 (20 mg/kg) treatment led to a significant reduction in liver metastasis without affecting primary tumor growth in vivo. Serum derived from saline-treated mice induced higher levels of invasion than serum from polymer-treated mice and exogenous polymer blocked this effect (**** = p<0.0001, *** = p<0.001, ** = p<0.01, NS = not significant by t-test). Discussion: Cationic polymers such as PAMAM-G3 may represent a novel class of therapeutics to combat pancreatic cancer metastasis. These polymers can bind and neutralize cfDNA and tumor-derived EVs, thereby blocking activation of pro-inflammatory pathways within tumor cells, and reducing tumor invasion and metastasis. Cationic polymers will be tested in other preclinical models of cancer and in dosing studies for potential progression to clinical translation. Citation Format: Ibtehaj A. Naqvi, Ruwan Gunaratne, Jessica McDade, Douglas Rouse, Jen Jen Yeh, David Pisetsky, Jaewoo Lee, Rebekah White, Bruce Sullenger. Cationic polymer inhibits pancreatic cancer invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4189.

Development of a Function-Blocking Antibody Against Fibulin-3 as a Targeted Reagent for Glioblastoma.

Purpose: We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFκB signaling.Experimental Design: We used deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy.Results: We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFκB signaling in GBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3-expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3.Conclusions: To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using "anti-ECM" strategies toward more efficient targeted therapies for malignant glioma. Clin Cancer Res; 24(4); 821-33. ©2017 AACR.

Activation of the HGF/c-Met axis in the tumor microenvironment: A multispecies model

The tumor microenvironment is an integral component in promoting tumor development. Cancer-associated fibroblasts (CAFs), which reside in the tumor stroma, produce Hepatocyte Growth Factor (HGF), an important trigger for invasive and metastatic tumor behavior. HGF contributes to a pro-tumorigenic environment by activating its cognate receptor, c-Met, on tumor cells. Tumor cells, in turn, secrete growth factors that upregulate HGF production in CAFs, thereby establishing a dynamic tumor-host signaling program. Using a spatiotemporal multispecies model of tumor growth, we investigate how the development and spread of a tumor is impacted by the initiation of a dynamic interaction between tumor-derived growth factors and CAF-derived HGF. We show that establishment of such an interaction results in increased tumor growth and morphological instability, the latter due in part to increased cell species heterogeneity at the tumor-host boundary. Invasive behavior is further increased if the tumor lowers responsiveness to paracrine pro-differentiation signals, which is a hallmark of neoplastic development. By modeling anti-HGF and anti-c-Met therapy, we show how disruption of the HGF/c-Met axis can reduce tumor invasiveness and growth, thereby providing theoretical evidence that targeting tumor-microenvironment interactions is a promising avenue for therapeutic development.

3D Mathematical Modeling of Glioblastoma Suggests That Transdifferentiated Vascular Endothelial Cells Mediate Resistance to Current Standard-of-Care Therapy.

Glioblastoma (GBM), the most aggressive brain tumor in human patients, is decidedly heterogeneous and highly vascularized. Glioma stem/initiating cells (GSC) are found to play a crucial role by increasing cancer aggressiveness and promoting resistance to therapy. Recently, cross-talk between GSC and vascular endothelial cells has been shown to significantly promote GSC self-renewal and tumor progression. Furthermore, GSC also transdifferentiate into bona fide vascular endothelial cells (GEC), which inherit mutations present in GSC and are resistant to traditional antiangiogenic therapies. Here we use three-dimensional mathematical modeling to investigate GBM progression and response to therapy. The model predicted that GSCs drive invasive fingering and that GEC spontaneously form a network within the hypoxic core, consistent with published experimental findings. Standard-of-care treatments using DNA-targeted therapy (radiation/chemo) together with antiangiogenic therapies reduced GBM tumor size but increased invasiveness. Anti-GEC treatments blocked the GEC support of GSCs and reduced tumor size but led to increased invasiveness. Anti-GSC therapies that promote differentiation or disturb the stem cell niche effectively reduced tumor invasiveness and size, but were ultimately limited in reducing tumor size because GECs maintain GSCs. Our study suggests that a combinatorial regimen targeting the vasculature, GSCs, and GECs, using drugs already approved by the FDA, can reduce both tumor size and invasiveness and could lead to tumor eradication. Cancer Res; 77(15); 4171-84. ©2017 AACR.

3D Mathematical modeling of glioblastoma suggests that transdifferentiated vascular endothelial cells promote resistance to current standard-of-care therapy.

e13535 Background: Glioblastoma (GBM), the most aggressive brain tumor in human patients, is highly heterogeneous and intensively vascularized. Glioma stem/initiating cells (GSC) are found to play a crucial role by increasing cancer aggressiveness and by promoting resistance to therapy. Recently, crosstalk between GSCs and vascular endothelial cells that line capillaries has been shown to considerably promote GSC self-renewal and tumor progression. GSCs have been shown to also transdifferentiate into bona-fide vascular endothelial cells (GEC). GECs inherit mutations present in GSCs and are resistant to traditional anti-angiogenic therapies. Methods: We develop a multispecies mathematical model to investigate the 3D spatiotemporal dynamics of vascularized GBM progression and response to cancer therapies. Results: The model predicts GSCs drive invasive fingering and that GECs spontaneously form a network within the hypoxic core, consistent with published experimental findings. We demonstrate that standard-of-care treatments using DNA-targeted therapy (radiation/chemo) together with anti-angiogenic therapies reduce GBM tumor sizes but increase invasiveness. Anti-GEC treatments block the GEC support of GSCs and reduce tumor sizes but can lead to increased invasiveness. Anti-GSC therapies that promote differentiation or disturb the stem cell niche effectively reduce tumor invasiveness and sizes, but are ultimately limited in reducing tumor sizes because GECs can maintain GSCs. Anti-GEC therapies are required to remove the tumor completely. Conclusions: Our results suggest that a combinatorial regimen targeting the vasculature, GSCs and GECs, using drugs already approved by the FDA, can reduce both tumor sizes and invasiveness and could lead to tumor eradication without recurrence when the treatment is stopped.

CBX7 negatively regulates migration and invasion in glioma via Wnt/β-catenin pathway inactivation.

CBX7, a member of the Polycomb-group proteins, plays a significant role in normal and cancerous tissues and has been defined as a tumor suppressor in thyroid, breast and pancreatic cancers. However, its function in glioma remains undefined. CBX7 expression is decreased in glioma, especially in higher grade cases, according to data in the CGGA, GSE16001 and TCGA databases. Further experimental evidence has shown that exogenous CBX7 overexpression induced apoptosis and inhibited cell proliferation, colony formation and migration of glioma cells. In this study, we show that the invasive ability of glioma cells was decreased following CBX7 overexpression and CBX7 overexpression was associated with Wnt/β-catenin pathway inhibition, which also decreased downstream expression of ZEB1, a core epithelial-to-mesenchymal transition factor. This reduction in Wnt signaling is controlled by DKK1, a specific Wnt/β-catenin inhibitor. CBX7 enhances DKK1 expression by binding the DKK1 promoter, as shown in Luciferase reporter assays. Our data confirm that CBX7 inhibits EMT and invasion in glioma, which is manifested by influencing the expression of MMP2, MMP9, E-cadherin, N-cadherin and Vimentin in LN229, T98G cells and primary glioma cells (PGC). Furthermore, as a tumor suppressor, CBX7 expression is pivotal to reduce tumor invasion and evaluate prognosis.

Abstract IA11: Targeting the mechanosensing machinery to limit glioblastoma initiation and invasion

Abstract Glioblastoma (GBM) is the most common and deadly primary brain cancer, with a median survival time of less than two years from diagnosis, even with aggressive multimodal care. A pathophysiological hallmark of GBM is the intimate infiltration of tumor cells within the brain, which complicates surgical resection and contributes to the adaptive-evasive response to anti-angiogenic agents. In this sense, GBM may be regarded as a disease of cell migration, raising the possibility that one could target the machinery of cell migration to limit tumor progression. Over the past decade, we have shown that GBM migration is strongly sensitive to biophysical inputs from the environment including tissue stiffness1 and physical confinement.2 We have also shown that microenvironmental stiffness-based cues can act synergistically with canonical mitogenic signaling pathways (e.g. EGFR) to drive GBM progression.3 These efforts, together with seminal studies from other laboratories, have helped stimulate interest in mining the physical microenvironment and associated cellular mechanosensing systems for novel prognostic and therapeutic targets. In this presentation, I will start by discussing our recent efforts to investigate these ideas specifically in the context of patient-derived GBM tumor initiating cells (TICs), a small subset of tumor cells with stem-like properties that are thought to drive tumor recurrence and drug resistance. Surprisingly, we find that TICs are comparatively insensitive to microenvironmental mechanical inputs, escaping the block on motility normally present in highly compliant matrix environments. We show that this stiffness-sensing can be restored through overexpression of contractile agonists, including Rho GTPase, Rho-associated kinase, and myosin light chain kinase. This intervention not only restores suppression of motility on compliant matrices but also significantly retards 3D invasion in vitro and reduces diffuse infiltration in a mouse orthotopic xenograft model. The latter effect is accompanied by a 30% extension in survival. This provides direct evidence that manipulation of the cellular mechanosensing machinery can be used to successfully reduce GBM progression in a preclinical model.4 These and other results have motivated us to develop next-generation culture platforms for tumor mechanobiology that enable discovery and screening in a more scalable and high-throughput platform than traditional approaches. For example, I will discuss a platform we recently introduced that allows orthogonal patterning of extracellular matrix stiffness and adhesive ligand density. We have used this platform to both identify microRNAs whose expression is sensitive to matrix biophysical parameters and explore the role played by macrophage-secreted factors in driving GBM adhesion and mechanosensing.5

Current knowledge on the active form of Vitamin D synthesized in the skin and its effects on malignant melanoma.

The link between sunlight and skin cancer is a frequently discussed topic. However, ultraviolet radiation also induces the production of Vitamin D in the body. Keratinocytes and their ability to synthesize the active form of Vitamin D, which is consumed at the place of its origin in the skin, have a unique place in this discussion. We observe a remarkable sunshine-related paradox when we monitor the relationship between the dose of solar radiation and one type of skin cancer - malignant melanoma. Recent knowledge of the non-calcemic effects of Vitamin D, which include growth regulation, DNA repair, differentiation, apoptosis, membrane transport, metabolism, cell adhesion and oxidative stress, could help to further clarify this relationship. In this context, adjuvant Vitamin D therapy is currently being considered in patients with malignant melanoma, and this is expected to reduce tumor invasiveness and micrometastases and thus improve patient prognosis and reduce the risk of relapse.