Alterations In Tumor Microenvironment Research Articles

Abstract A30: Oncogenic MCT-1 activation deregulates oxidative metabolism and promotes lung tumor progression and metastasis

Abstract Multiple copies in T-cell malignancy 1 (MCT-1) is involved in transcription regulation and translation initiation. We have identified that MCT-1 plays important roles in cell transformation and survival, catastrophic mitosis and genomic instability. Enhanced MCT-1 activity decreases p53 promoter function, protein stability and activity, thereby overexpressing MCT-1 further promotes tumorigenicity in a p53-null background. Enhanced MCT-1 activation induces SHCs (Src homology 2 domain containing transforming proteins) that transmit EGFR signaling to extracellular-regulated kinase (ERK) and AKT pathway. Here, we identify a novel carcinoma metabolism pathway involving MCT-1-YY1-EGFR-MnSOD axis which confers oxidative resistance, changes tumor microenvironments and promotes tumor development. Inhibiting this oncogenic pathway may be a novel clinical intervention approach to prevent tumor progression and metastasis. Aims: Reactive oxygen species (ROS) promote tumor progression and metastasis, but the underlying mechanism remains unclear. We aim to investigate whether the oncogenic pathway enhances the generation of ROS, alters tumor microenvironment and potentiates metastasis. Results: We found that oncogene MCT-1 (multiple copies in T-cell malignancy 1) stimulated intracellular ROS formation and mitochondrial superoxide production in relationship with the deregulation of antioxidants and redox signaling. Enhanced MCT-1 activation induced the YY1-EGFR-MnSOD signaling cascade which prevented cells against oxidative damage and promoted tumor development. Importantly, MCT-1 overexpressing in lung cancer cells promoted tumor angiogenesis and necrosis along with increase of tumor-promoted M2 macrophages and cancer-associated myofibroblasts in stroma, which potentially provided a malignant microenvironment for tumor progression and metastasis. Conversely, restricting ROS generation and targeting YY1 suppressed the MCT-1-EGFR-MnSOD pathway and invasion ability in lung cancer cells. Clinical results confirm that MCT-1 overexpression is link to poor clinical outcomes and associates with hyper-activation of YY1, EGFR and MnSOD in patients with lung cancer. Importantly, MCT-1 protein enrichment is often identified in late stage and lymph node metastasis of lung cancer. Innovation and Conclusion: Our data reveal a previously unrecognized mechanism of the oxidative metabolism involving MCT-1-YY1-EGFR-MnSOD network which alters tumor microenvironments and promotes tumor progression and metastasis. Citation Format: Hong-Yu Tseng, Yen-An Chen, Jayu Jen, Yi-Ching Wang, Hsin-Ling Hsu. Oncogenic MCT-1 activation deregulates oxidative metabolism and promotes lung tumor progression and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A30.

Abstract B32: GRP55 antagonists alter tumor microenvironment and inhibit tumor growth in a pancreatic tumor xenograft model

Abstract (R,R’)-4’-Methoxy-1-naphthylfenoterol ((R,R’)-MNF) and (R,S’)-MNF are GPR55 inhibitors that reduce proliferation in pancreatic cancer cell lines. The IC50 values for [3H]-thymidine incorporation in PANC-1 cells were 0.11 ± 0.08 µM {(R,S’)-MNF} and 0.65 ± 0.25 µM {(R,R’)-MNF}. (R,R’)-MNF and (R,S’)-MNF attenuate the activities of the β producing decreased expression and nuclear translocation of pyruvate kinase M2, β-catenin and HIF-1α, thereby reducing cyclin D1 expression with concomitant G1/S cell cycle arrest. MNF reduces the expression and function of multidrug exporters in PANC-1 cells and tumor tissues, thereby increasing the sensitivity to other chemotherapeutic agents. The compounds also attenuate expression and function of enzymes and transporters associated with glycolysis including the MCT4 lactate exporter, thereby reducing L-lactate concentration in the tumor microenvironment. We now report the effect of (R,S’)-MNF on the growth of a PANC-1 tumor maintained as a xenograft in mice. Protocol: PANC-1 cells maintained in vitro as monolayer culture were harvested at an exponential growth phase, counted and each mouse inoculated subcutaneously at the right flank region with 5x106 cells in 0.1ml of PBS. On Day 8, mice were weighed and tumor volumes measured (mean tumor size 142mm3), and assigned to groups (n = 10) using randomized block design based on their tumor volumes. The mice received an ip injection (10mL/g) five times per week for 3 weeks of vehicle, 20% hydroxypropyl-beta-cyclodextrin (Control), 20mg/kg (R,S’)-MNF or 40mg/kg (R,S’)-MNF. The animals were checked daily for morbidity and mortality and weight and tumor volume measured twice weekly. On Day 33, the mice were euthanized by cervical extension, body weight, tumor weights and volumes were obtained and the tumors snap frozen. Results: (R,S’)-MNF produced significant dose-dependent inhibition of tumor growth. determined as % change in tumor volume relative to control. In animals treated with 20mg/kg (R,S’)-MNF there was 51.1±5.2% (** p < 0.01) inhibition and a 74.1±2.8% (*** p < 0.005) inhibition in animals receiving 40mg/kg (R,S’)-MNF. No mice were lost to treatment limiting toxicities. There was no significant change in weight between pre-dose and Day 33 for the animals in the control and 20mg/kg (R,S’)-MNF groups while on Day 33 the average weight of the animals receiving 40mg/kg (R,S’)-MNF was significantly lower 9.9±3.7% (*p < 0.05) than their pre-dose weights. The (R,S’)-MNF associated reduction of protein expression in tumor tissues, including pyruvate kinase M2, β-catenin, HIF-1α and MCT4 will be presented as will the changes in tumor/plasma concentrations of L-lactate. Citation Format: Nagendra S. Singh, Irving W. Wainer.{Authors}. GRP55 antagonists alter tumor microenvironment and inhibit tumor growth in a pancreatic tumor xenograft model. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B32.

Inhibition of KPNA4 attenuates prostate cancer metastasis.

Prostate cancer (PCa) is a common cancer in men. Although current treatments effectively palliate symptoms and prolong life, the metastatic PCa remains incurable. It is important to find biomarkers and targets to improve metastatic PCa diagnosis and treatment. Here we report a novel correlation between karyopherin α4 (KPNA4) and PCa pathological stages. KPNA4 mediates the cytoplasm-to-nucleus translocation of transcription factors, including nuclear factor kappa B, although its role in PCa was largely unknown. We find that knockdown of KPNA4 reduces cell migration in multiple PCa cell lines, suggesting a role of KPNA4 in PCa progression. Indeed, stable knockdown of KPNA4 significantly reduces PCa invasion and distant metastasis in mouse models. Functionally, KPNA4 alters tumor microenvironment in terms of macrophage polarization and osteoclastogenesis by modulating tumor necrosis factor (TNF)-α and -β. Further, KPNA4 is proved as a direct target of miR-708, a tumor-suppressive microRNA. We disclose the role of miR-708-KPNA4-TNF axes in PCa metastasis and KPNA4's potential as a novel biomarker for PCa metastasis.

Gold Nanoparticle Reprograms Pancreatic Tumor Microenvironment and Inhibits Tumor Growth.

Altered tumor microenvironment (TME) arising from a bidirectional crosstalk between the pancreatic cancer cells (PCCs) and the pancreatic stellate cells (PSCs) is implicated in the dismal prognosis in pancreatic ductal adenocarcinoma (PDAC), yet effective strategies to disrupt the crosstalk is lacking. Here, we demonstrate that gold nanoparticles (AuNPs) inhibit proliferation and migration of both PCCs and PSCs by disrupting the bidirectional communication via alteration of the cell secretome. Analyzing the key proteins identified from a functional network of AuNP-altered secretome in PCCs and PSCs, we demonstrate that AuNPs impair secretions of major hub node proteins in both cell types and transform activated PSCs toward a lipid-rich quiescent phenotype. By reducing activation of PSCs, AuNPs inhibit matrix deposition, enhance angiogenesis, and inhibit tumor growth in an orthotopic co-implantation model in vivo. Auto- and heteroregulations of secretory growth factors/cytokines are disrupted by AuNPs resulting in reprogramming of the TME. By utilizing a kinase dead mutant of IRE1-α, we demonstrate that AuNPs alter the cellular secretome through the ER-stress-regulated IRE1-dependent decay pathway (RIDD) and identify endostatin and matrix metalloproteinase 9 as putative RIDD targets. Thus, AuNPs could potentially be utilized as a tool to effectively interrogate bidirectional communications in the tumor microenvironment, reprogram it, and inhibit tumor growth by its therapeutic function.

CAPS1 Negatively Regulates Hepatocellular Carcinoma Development through Alteration of Exocytosis-Associated Tumor Microenvironment.

The calcium-dependent activator protein for secretion 1 (CAPS1) regulates exocytosis of dense-core vesicles (DCVs) in neurons and neuroendocrine cells. The role of CAPS1 in cancer biology remains unknown. The purpose of this study was to investigate the role of CAPS1 in hepatocellular carcinoma (HCC). We determined the levels of CAPS1 in eight hepatoma cell lines and 141 HCC specimens. We evaluated the prognostic value of CAPS1 expression and its association with clinical parameters. We investigated the biological consequences of CAPS1 overexpression in two hepatoma cell lines in vitro and in vivo. The results showed that loss of CAPS1 expression in HCC tissues was markedly correlated with aggressive tumor phenotypes, such as high-grade tumor node metastasis (TNM) stage (p = 0.003) and absence of tumor encapsulation (p = 0.016), and was associated with poor overall survival (p = 0.008) and high recurrence (p = 0.015). CAPS1 overexpression inhibited cell proliferation and migration by changing the exocytosis-associated tumor microenvironment in hepatoma cells in vitro. The in vivo study showed that CAPS1 overexpression inhibited xenograft tumor growth. Together, these results identified a previously unrecognized tumor suppressor role for CAPS1 in HCC development.

Remodeling of the Tumor Microenvironment Predicts Increased Risk of Cancer in Postmenopausal Women: The Prospective Epidemiologic Risk Factor (PERF I) Study.

An altered tumor microenvironment is one of the earliest signs of cancer and an important driver of the disease. We have seen previously that biomarkers reflecting tumor microenvironment modifications, such as matrix metalloproteinase (MMP)-degraded type 1 collagen (C1M), MMP-degraded type IV collagen (C4M), and citrullinated and MMP-degraded vimentin (VICM), were higher in the serum of cancer patients than in healthy controls. However, it is not known if these biomarkers could predict an increased risk of cancer. The aim of this study was to investigate whether C1M, C4M, and VICM were elevated prior to diagnosis of solid cancers in a large prospective study. Between 1999 and 2001, 5,855 postmenopausal Danish women ages 48 to 89 years enrolled in the Prospective Epidemiologic Risk Factor study. Baseline demographics and serum were collected at the time of registration. Follow up cancer diagnoses were obtained from the Danish Cancer Registry in 2014. Serum C1M, C4M, and VICM levels were measured by competitive ELISAs. A total of 881 women were diagnosed with solid cancers after baseline. C1M, C4M, and VICM levels were significantly elevated in women diagnosed less than 1 year after baseline. C1M and VICM, but not C4M, were independent predictors of increased risk of cancer. C1M, C4M, and VICM are elevated prior to cancer diagnosis. C1M and VICM are both independent predictors of increased cancer risk. C1M and VICM are predictors for increased risk of cancer. Cancer Epidemiol Biomarkers Prev; 25(9); 1348-55. ©2016 AACR.

Functional Flow Patterns and Static Blood Pooling in Tumors Revealed by Combined Contrast-Enhanced Ultrasound and Photoacoustic Imaging.

Alterations in tumor perfusion and microenvironment have been shown to be associated with aggressive cancer phenotypes, raising the need for noninvasive methods of tracking these changes. Dynamic contrast-enhanced ultrasound (DCEUS) and photoacoustic (PA) imaging serve as promising candidates-one has the ability to measure tissue perfusion, whereas the other can be used to monitor tissue oxygenation and hemoglobin concentration. In this study, we investigated the relationship between the different functional parameters measured with DCEUS and PA imaging, using two morphologically different hind-limb tumor models and drug-induced alterations in an orthotopic breast tumor model. Imaging results showed some correlation between perfusion and oxygen saturation maps and the ability to sensitively monitor antivascular treatment. In addition, DCEUS measurements revealed different vascular densities in the core of specific tumors compared with their rims. Noncorrelated perfusion and hemoglobin concentration measurements facilitated discrimination between blood lakes and necrotic areas. Taken together, our results illustrate the utility of a combined contrast-enhanced ultrasound method with photoacoustic imaging to visualize blood flow patterns in tumors. Cancer Res; 76(15); 4320-31. ©2016 AACR.

Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization.

The cAMP-responsive element-binding protein (CREB) is involved in the tumorigenicity of HER-2/neu-overexpressing murine and human tumor cells, but a link between the HER-2/neu-mediated CREB activation, its posttranslational modification and localization and changes in the cellular metabolism, due to an altered (tumor) microenvironment remains to be established. The present study demonstrated that shRNA-mediated silencing of CREB in HER-2/neu-transformed cells resulted in decreased tumor formation, which was associated with reduced angiogenesis, but increased necrotic and hypoxic areas in the tumor. Hypoxia induced pCREBSer133, but not pCREBSer121 expression in HER-2/neu-transformed cells. This was accompanied by upregulation of the hypoxia-inducible genes GLUT1 and VEGF, increased cell migration and matrix metalloproteinase-mediated invasion. Treatment of HER-2/neu+ cells with signal transduction inhibitors targeting in particular HER-2/neu was able to revert hypoxia-controlled CREB activation. In addition to changes in the phosphorylation, hypoxic response of HER-2/neu+ cells caused a transient ubiquitination and SUMOylation as well as a co-localization of nuclear CREB to the mitochondrial matrix. A mitochondrial localization of CREB was also demonstrated in hypoxic areas of HER-2/neu+ mammary carcinoma lesions. This was accompanied by an altered gene expression pattern, activity and metabolism of mitochondria leading to an increased respiratory rate, oxidative phosphorylation and mitochondrial membrane potential and consequently to an enhanced apoptosis and reduced cell viability. These data suggest that the HER-2/neu-mediated CREB activation caused by a hypoxic tumor microenvironment contributes to the neoplastic phenotype of HER-2/neu+ cells at various levels.

Constitutively active transforming growth factor β receptor 1 in the mouse ovary promotes tumorigenesis.

Despite the well-established tumor suppressive role of TGFβ proteins, depletion of key TGFβ signaling components in the mouse ovary does not induce a growth advantage. To define the role of TGFβ signaling in ovarian tumorigenesis, we created a mouse model expressing a constitutively active TGFβ receptor 1 (TGFBR1) in ovarian somatic cells using conditional gain-of-function approach. Remarkably, these mice developed ovarian sex cord-stromal tumors with complete penetrance, leading to reproductive failure and mortality. The tumors expressed multiple granulosa cell markers and caused elevated serum inhibin and estradiol levels, reminiscent of granulosa cell tumors. Consistent with the tumorigenic effect, overactivation of TGFBR1 altered tumor microenvironment by promoting angiogenesis and enhanced ovarian cell proliferation, accompanied by impaired cell differentiation and dysregulated expression of critical genes in ovarian function. By further exploiting complementary genetic models, we substantiated our finding that constitutively active TGFBR1 is a potent oncogenic switch in mouse granulosa cells. In summary, overactivation of TGFBR1 drives gonadal tumor development. The TGFBR1 constitutively active mouse model phenocopies a number of morphological, hormonal, and molecular features of human granulosa cell tumors and are potentially valuable for preclinical testing of targeted therapies to treat granulosa cell tumors, a class of poorly defined ovarian malignancies.

405. IL-2 and TNF-a Coding Adenoviruses Enable Adoptive T Cell Therapy in Metastatic, Solid Cancer by Systemically Activating Tumor-Reactive TILs

Adoptive T cell therapy (ACT) using genetically modified T cells has shown exceptional efficacy in the treatment of CD19+ hematological cancers. However, far less impressive results have been achieved in the treatment of solid tumors due to local immunosuppression, rendering tumor-infiltrating T cells (TILs) hypofunctional. We have previously shown that adenovirus (Ad) infection can enhance the efficacy of ACT (Tahtinen et al, CIR 2015) and that intratumoral administration of immunostimulatory cytokines can result in favorable alteration of tumor microenvironment (Tahtinen et al, PLOS One 2015). To combine the benefits of both approaches, we studied if replication-deficient Ad5-vectors coding for interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-a) would affect the activity of adoptively transferred, TCR-transgenic TRP-2(180-188) specific T cells in vivo.To gain clinically relevant mechanism-of-action data, we chose to use ret transgenic mouse model that develops spontaneous malignant skin melanoma which metastasizes into distant organs. Following ACT and intratumoral virus injection, a significant increase in activated PD-1+ CD8+ T cells was seen in both cutaneous lesions and in metastatic lymph nodes. Interestingly, a reverse correlation between tumor weight and the number of tumor-reactive PD-1+ TILs (p=0.0015) was observed, indicating that T-cell hypofunction was overcome and successful tumor lysis was achieved. Local expression of cytokines did not affect the levels of immunosuppressive immune cell subsets such as myeloid-derived suppressor cells (MDSCs) or T regulatory cells (Tregs), latter of which has previously been associated with systemic IL-2 therapy (Ahmadzadeh and Rosenberg, Blood 2006). Instead, Ad5-IL2 treatment induced upregulation of IL-2 receptor α-chain (CD25) in conventional CD4+CD25+Foxp3-cells, suggesting that these helper T cells contributed to CD8+ TIL activation. Finally, beneficial ratios between tumor-reactive PD-1+ CD8+ TILs and Tregs was observed in primary and secondary tumor sites, indicating that IL-2 and TNF-a coding adenoviruses can modify the cellular composition of the tumor microenvironment in favor of adoptively transferred T cells.In conclusion, IL-2 and TNF-a coding adenoviruses can break tumor-associated immunotolerance and significantly increase the levels of active, tumor-reactive T-cells both in injected cutaneous lesions and in non-injected metastatic lymph nodes. Importantly, this triple modality may represent an efficient approach to achieve “CD19-like” clinical responses in the treatment of solid, metastatic cancers currently incurable by standard therapies.

Anti-tumor effects of DNA vaccine targeting human fibroblast activation protein α by producing specific immune responses and altering tumor microenvironment in the 4T1 murine breast cancer model.

Fibroblast activation protein α (FAPα) is a tumor stromal antigen overexpressed by cancer-associated fibroblasts (CAFs). CAFs are genetically more stable compared with the tumor cells and immunosuppressive components of the tumor microenvironment, rendering them excellent targets for cancer immunotherapy. DNA vaccines are widely applied due to their safety. To specifically destroy CAFs, we constructed and examined the immunogenicity and anti-tumor immune mechanism of a DNA vaccine expressing human FAPα. This vaccine successfully reduced 4T1 tumor growth through producing FAPα-specific cytotoxic T lymphocyte responses which could kill CAFs, and the decrease in FAPα-expressing CAFs resulted in markedly attenuated expression of collagen I and other stromal factors that benefit the tumor progression. Based on these results, a DNA vaccine targeting human FAPα may be an attractive and effective cancer immunotherapy strategy.

Alterations of tumor microenvironment by carbon monoxide impedes lung cancer growth.

We hypothesized that tumor-associated macrophages (TAMs) are controlled by the diffusible gas carbon monoxide (CO). We demonstrate that induction of apoptosis in lung tumors treated with low doses of CO is associated with increased CD86 expression and activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (Erk) 1/2 pathway in tumor microenvironment. Presence of CD86-positive cells was required for the anti-tumoral effects of CO in established A549 xenografts. We show that the effects of CO on tumor stroma and reprogramming of macrophages towards the anti-tumoral phenotype is mediated by reactive oxygen species (ROS)-dependent activation of MAPK/Erk1/2-c-myc pathway as well as Notch 1-dependent negative feedback on the metabolic enzyme heme oxygenase-1 (HO-1). We find a similar negative correlation between HO-1 and active MAPK-Erk1/2 levels in human lung cancer specimens.In summary, we describe novel non-cell autonomous mechanisms by which the diffusible gas CO dictates changes in the tumor microenvironment through the modulation of macrophages.

Abstract P4-04-02: Amplification of chromosome 9p24 targeting PD-L1 and JAK2 correlates with altered tumor microenvironment expression profiles in triple negative breast cancer

Abstract Introduction: In a pilot study, we recently described amplification of chromosome 9p24 encoding PD-L1, PD-L2, and JAK2 (the PDJ amplicon) in 29% of early stage triple negative breast cancers (TNBC). Amplification was associated with poor DFS and OS. The impact of this amplicon on immune function is not known. Methods: Fresh frozen tumor samples from 36 subjects with newly-diagnosed TNBC (stages I-III) were evaluated for copy number aberrations using DNA-based flow cytometry to enrich nuclei based on ploidy status (tetraploidy or aneuploidy), followed by oligonucleotide CGH arrays. Targeted immune expression profiling was performed using the Nanostring PanCancer Immune Profiling Panel (n=770 genes). Results: 23 samples had measurable abnormal ploidy (evidence of sufficient tumor content) for CGH analysis; the remaining samples were omitted from analysis. High level (log2ratio >1) amplification was detected in 6/23 (26.1%) of TNBCs, and low-level copy number gain (CN log2ratio >0 and<1) was detected in 3/23 (13.0%). The remaining samples had no copy number change (n=9) or low level copy number loss (CN log2ratio>-1 and <0, n=5). Increased RNA expression of STAT1, STAT2, STAT3, and STAT6 was associated with PDJ amplification, as well as upregulation of the immune regulatory genes CD47 and IDO1. Conclusion: Amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in a significant proportion of TNBCs. This amplicon is associated with altered gene expression of the tumor microenvironment. Citation Format: Anderson KS, Andreozzi M, Pockaj BA, Hopper M, McCullough AE, Barrett MT. Amplification of chromosome 9p24 targeting PD-L1 and JAK2 correlates with altered tumor microenvironment expression profiles in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-02.

Abstract B68: Contributions of STAT3 to pro-tumorigenic alterations within the microenvironment during mammary tumorigenesis

Abstract Inflammation has long been recognized as a possible trigger that contributes to tumor initiation. Numerous studies have shown that cells of both the innate and adaptive immune systems are able to support tumor formation and growth through secretion of growth factors and cytokines and by actively shielding the tumor from wider immune surveillance. As a result, inflammation is generally linked to worse patient outcome. However, depending on factors such as tumor size, extracellular matrix, and the complex composition of immune cell infiltrates, inflammation can promote both pro- and anti-tumor properties. Our studies focus on identifying novel mechanisms through which tumor-derived cytokines act in a paracrine manner to regulate the functions of infiltrating inflammatory cells in the tumor microenvironment. We have previously shown that activation of the fibroblast growth factor receptor-1 (FGFR1) oncogene results in the production of high levels of interleukin 6 (IL-6) family members by mammary epithelial cells. Histological examination of FGFR1-driven tumors revealed high levels of STAT3 activation in both tumor cells and macrophages. Thus, further studies focused on identifying mechanisms through which STAT3 activation in different cellular compartments contributes to mammary tumor growth. Initial studies focused on the consequences of STAT3 activation in epithelial/tumor cells. We found that epithelial cell STAT3 activation resulted in accumulation of the extracellular matrix component hyaluronan, which contributed to epithelial cell proliferation, survival and migration. In macrophages, STAT3 is the transcription factor required for transduction of the anti-inflammatory response initiated by interleukin-10. Ongoing experiments have revealed that in primary STAT3fl/fl cre+ bone marrow-derived macrophages, absence of STAT3 signaling reveals a profoundly enhanced pro-inflammatory phenotype. In vitro, treatment of macrophages lacking STAT3 with recombinant IL-6 results in prolonged activation of the proinflammatory STAT1, as well as upregulation of inflammatory molecules such as TNFα and CCL2. Ongoing experiments are aimed at evaluating the functional consequences of this altered tumor microenvironment in vivo during tumor initiation and growth. As anti-STAT3 therapies have began entering clinical trials for solid tumors (including breast tumors), understanding the mechanisms through which STAT3 promotes tumor growth and progression is critical for developing optimal therapeutic strategies. Citation Format: Polly Chuntova, Laura R. Bohrer, Kaylee L. Schwertfeger. Contributions of STAT3 to pro-tumorigenic alterations within the microenvironment during mammary tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B68.

Autologous Tumor Cell Lysate-Loaded Dendritic Cell Vaccine Inhibited Tumor Progression in an Orthotopic Murine Model for Hepatocellular Carcinoma.

The immune status of the tumor microenvironment influences tumor progression, and hepatocellular carcinoma (HCC) with an immunosuppressive signature often is associated with a poor prognosis. This study examined the impact of a bone marrow-derived dendritic cell (DC) vaccine loaded with autologous tumor cell lysate on tumor progression and the tumor microenvironment using an orthotopic murine HCC model. An orthotopic murine HCC was established by implantation of Hepa1-6 cells in the liver. The impact of DC vaccine loaded with Hepa1-6 cell lysate on tumor progression, survival, and tumor-infiltrating lymphocytes and cytokines was examined. Treating mice with DC vaccine loaded with Hepa1-6 cell lysate inhibited the progression of murine HCC generated through orthotopic implantation of Hepa1-6 cells and resulted in a 90% survival rate by day 60 compared with a survival rate lower than 5% for untreated mice. This anti-tumor response was associated with inhibition of STAT3 phosphorylation within the tumor. The DC vaccine reduced accumulation of Foxp3+CD4+ regulatory T cells within the tumor microenvironment and prevented TGF-β production from the tumor tissue. Tumor cell lysate-loaded DC vaccine prevented HCC progression in a clinically relevant orthotopic murine HCC model. The effect of DC vaccine on the accumulation of Foxp3+CD4+ regulatory T cells within the tumor microenvironment and on the production of TGF-β suggests that tumor regression by DC vaccination may be associated with an altered immunosuppressive tumor microenvironment.

Abstract PL07-01: Altered metabolism in leukemic microenvironment

Abstract Interactions of leukemia cells and their bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells. It has been postulated that specific niches provide a sanctuary where subpopulations of leukemic cells evade chemotherapy-induced death and acquire a drug-resistant phenotype. Understanding the cellular and molecular biology of the leukemia stem cell (LSC) niche and of microenvironment/leukemia interactions may provide new targets that allow destruction of LSCs without adversely affecting normal stem cell self-renewal. Key emerging therapeutic targets include chemokine receptors such as CXCR4 and hypoxia-related proteins, as well as the metabolic abnormalities of the leukemia-associated stroma. We have recently reported that CXCR4 inhibition causes leukemia cell dislodgement from CXCL12-producing marrow niches, reduced proliferation and induction of differentiation of AML cells in an in vivo model of AML, translating into pronounced anti-leukemia effects. Studies in murine leukemia models using the hypoxia probe pimonidazole demonstrated extensive areas of hypoxia within leukemic, but not healthy normal, bone marrow. Time-course analysis of bone marrow spaces within calvaria and femurs by multiphoton intravital microscopy (MP-IVM) demonstrated lodging of p190-Bcr/Abl tdTomato cells in close proximity to blood vessels, followed by accumulation of leukemia cells localized within the sinusoidal and marrow spaces resulting in the demise of the animals within 3 weeks. In this model, pimonidazole detected hypoxic areas despite abundant vascular supply in the marrow cavities. In vivo magnetic resonance imaging with hyperpolarized pyruvate showed higher pyruvate-lactate conversion (high glycolytic flux) in leukemic marrows. These findings were supported by significant pimonidazole uptake by the diseased bone marrow in patients with acute leukemia, causing stabilization of HIF-1α in 55% (76/138) of primary AML patients and of its target CA9. Paradoxically, AML cells become highly dependent on mitochondrial oxidative phosphorylation (OXPHOS) for their survival, and inhibition of OXPHOS with the novel small molecule complex I inhibitor IACS-10759 inhibits oxygen consumption, eliminates hypoxia in vivo and inhibits AML growth. These findings suggest that altered tumor metabolism underlies the hypoxia observed in leukemias. We postulate that the altered tumor microenvironment within the hypoxic niche cells will influence leukemia development and response to therapy. Hence, targeting key metabolic alterations should be considered in the armamentarium of anti-AML therapies. IACS-10759 is presently completing IND enabling safety/toxicity studies with first in human studies targeting relapsed/refractory AML planned for 2016. Citation Format: Marina Y. Konopleva, Tomasz Zal, Niki M. Zacharias Millward, Byoung-Sik Cho, Karine Harutyunyan, Anna Zal, Hong Mu, Sergej Konoplev, Juliana Benito, Juliana Velez, Carlos Bueso-Ramso, Jennifer Molina, Pratip K. Bhattacharya, Maria Emilia Di Francesco, Joseph Marszalek, Michael Andreeff. Altered metabolism in leukemic microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-01.

Exosomes: Potential in Cancer Diagnosis and Therapy.

Exosomes are membrane-bound, intercellular communication shuttles that are defined by their endocytic origin and size range of 30–140 nm. Secreted by nearly all mammalian cell types and present in myriad bodily fluids, exosomes confer messages between cells, proximal and distal, by transporting biofunctional cargo in the form of proteins, nucleic acids, and lipids. They play a vital role in cellular signaling in both normal physiology and disease states, particularly cancer. Exosomes are powerful progenitors in altering target cell phenotypes, particularly in tumorigenesis and cancer progression, with the ability to alter tumor microenvironments and to assist in establishing the pre-metastatic niche. Many aspects of exosomes present them as novel means to identify cancer biomarkers for early detection and therapeutic targets, and using intrinsic and engineered characteristics of exosomes as therapeutic devices to ameliorate the progression of the disease. This review outlines some of the recent and major findings with regard to exosomes in cancer, and their utilization as therapeutic tools.

New Antiangiogenetic Therapy Beyond Bevacizumab in the Treatment of Advanced Non Small Cell Lung Cancer.

Lung cancer is the leading cause of death from cancer worldwide with limited available treatment options in absence of specific molecular alteration. New therapeutic approaches for addressing non small cell lung cancer (NSCLC) are urgently needed. Angiogenesis plays a central role in the tumor growth and metastatic dissemination which stimulates multiple cells to build new abnormal microvessels and leads to tumor microenvironment alterations. This process involves many factors, such as, the vascular endothelial growth factor (VEGF) that has a dominant role, the fibroblast growth factors (FGFs) and the plateled-derived growth factor (PDGF) that together contribute to resistance to VEGF/VEGFR- directed therapy. To date, bevacizumab is currently the only angiogenesis inhibitor that has been approved for the treatment of patients with advanced NSCLC in first-line setting. Moreover, in the last year, two new antiangiogenic agents have been approved for the treatment of patients with advanced NSCLC in second line setting. This review describes the new antiangiogenic agents in the treatment of advanced NSCLC.

Abstract 414: Prospective profiling of systemic and loco-regional alterations during intraperitoneal chemotherapy for ovarian cancer, results from GOG 271

Abstract Objectives: The concept of personalized approaches for recurrence in ovarian cancer has progressed at a modest pace, compared to other cancers. This is partly due to our limited understanding of the local tumor microenvironment during and immediately following chemotherapy-induced tumor involution, which requires sampling of the peritoneal cavity. Most studies to date have focused on chemo-naive tumors and on the more easily accessible peripheral blood samples, yet these are often not reflective of local changes. We hypothesize that sequential sampling and analysis of blood and intraperitoneal (IP) fluid during IP chemotherapy is feasible and will provide molecular insight into chemotherapy-induced systemic and local changes in the tumor environment. Methods: Peritoneal fluid aspiration or wash and blood samples were obtained from patients (n = 13) at three time points (baseline and following the first and second rounds of chemotherapy, respectively) Nanostring was performed to profile miRNA from peritoneal samples and plasma. RNA extraction from peripheral blood mononuclear cells (PBMC) followed by Nanostring was performed to evaluate changes in 511 immune genes during chemotherapy. Flow cytometry for surface and intracellular immune markers was performed on PBMCs. Results: From 13 patients, 30 blood, 12 intraperitoneal fluid (PF), and 20 intraperitoneal wash (PW) samples were obtained. Despite small volumes, cell counts were measurable in 7 of 12 (58%) PF and 11 of 20 (55%) PW samples. Nanostring results revealed 12 immune genes and 29 miRNAs differentially expressed during chemotherapy (p<0.05). Additionally, changes amongst miRNA were noted to occur earlier than immune genes in which the majority was differentially expressed only after the second round of chemotherapy. The miRNA tended to be significantly changed, in the same direction, throughout chemotherapy. Bioinformatics analyses revealed the top immune pathways most affected by chemotherapy and included cell-to-cell signaling, immune cell trafficking, and inflammatory response among the top functions. Conclusions: Sampling of the peritoneal cavity through IP catheters during chemotherapy is feasible and can be implemented through standardized protocols. Despite a small sample size, we were able to identify chemotherapy-induced molecular changes in immune genes and miRNAs systemically and loco-regionally. The findings suggest that miRNA alterations may be more suggestive of early tumor microenvironment alterations compared to immune genes in which changes are seen after subsequent cycles of chemotherapy. Larger studies are needed for validation to assess these changes as therapeutic targets. Citation Format: Shannon Grabosch, Anda M. Vlad, Tianzhou Ma, Jyothi T. Mony, Mary Strange, Joan Brozick, Julia Thaller, George Tseng, Xin Huang, Kathleen Moore, Kunle Odunsi, Robert P. Edwards. Prospective profiling of systemic and loco-regional alterations during intraperitoneal chemotherapy for ovarian cancer, results from GOG 271. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 414. doi:10.1158/1538-7445.AM2015-414

Inhibition of tumor progression by oral piceatannol in mouse 4T1 mammary cancer is associated with decreased angiogenesis and macrophage infiltration

Piceatannol, a polyphenol which exhibits anticancer activities, is found in grapes, red wine and berries. It has been shown to inhibit several transcription factor pathways. The present study was conducted to determine whether oral administration of piceatannol inhibits mammary tumor progression. 4T1 mammary carcinoma cells were injected into the mammary fat pad of syngeneic female BALB/c mice. Starting 1 day later, piceatannol (10- or 20-mg/kg body weight/day) was administered by oral gavage for 30 days. Piceatannol treatment reduced tumor growth. In tumor tissues, piceatannol treatment reduced the expression of transcription factors P-NFκB p65, P-STAT3 and HIF-1α and multiple proteins involved in regulation of cell cycle progression (Ki67, cyclin D1, cyclin A, CDK2, CDK4), angiogenesis (VEGF-A, VEGFR-2, VE-cadherin, CD31) and lymphangiogenesis (VEGF-C, LYVE-1), as well as macrophage infiltration. Piceatannol significantly increased apoptotic cells and expression of both Bax and cleaved caspase-3 but reduced Bcl-2 expression in tumor tissues. In addition, piceatannol reduced the number and volume of pulmonary tumor nodules and expression of MMP-9 in both lung and tumor. It also reduced tissue levels of cytokines/chemokines, including M-CSF and MCP-1. In vitro results revealed that piceatannol inhibited migration of 4T1 cells and monocytes, as well as secretion of MCP-1 and M-CSF by 4T1 cells. 4T1 cell-conditioned medium stimulated monocyte migration, which was suppressed by a CCR2 antibody. These results indicate that alteration in tumor microenvironment (macrophages, transcription factors, etc.) is an important mechanism by which piceatannol inhibits tumor proliferation, angiogenesis and lymphangiogenesis, leading to suppression of mammary tumor growth and metastasis.