bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A10: The hypoxic response in natural killer cells: Linking cytoxicity and tumor immune surveillance to angiogenesis
Authors: Krzywinska Ewelina , Kantari-Mimoun Chahrazade , Castells Magali , Gotthardt Dagmar , Kerdiles Yann , Klose Ralph , Fandrey Joachim , Sexl Veronika , Stockmann Christian
Published: October 2016

Hypoxia-inducible transcription factors (HIFs) are central mediators of cellular adaptation to low oxygen and play a pivotal role in inflammatory responses. Natural Killer (NK) cells, unifying characteristics of innate and adaptive immunity, are cytotoxic innate lymphoid cells with a unique ability to instantly recognize and kill “aberrant” cancer cells while sparing “normal” cells. Owing to these tumoricidal features, NK cells are able to restrict primary tumor growth and limit metastatic spread. By genetic targeting HIFs in NK cells HIFs, we define a crucial role of HIF-1 in NK cell function and cancer immune surveillance. We show that NK cells preferentially infiltrate into hypoxic zones of solid primary tumors and that HIF-1-deficiency in NK cells slows impairs primary tumor growth as well as distant metastasis. This is due to reduced susceptibility of HIF-1-deficient NK cells to tumor cell-derived inhibitory stimuli, resulting in improved recognition and killing of tumor cells.

Furthermore, we define the hypoxic response in NK cells as a critical mediator of tumor angiogenesis. Paradoxically, HIF-1-deficiency in NK cells results in decreased expression of various angiostatic factors within the tumor microenvironment, resulting in unproductive tumor angiogenesis, characterized by immature, non-functional vessel and severe tumor hypoxia. This suggests that the hypoxic response in NK cells slows down overall tumor angiogenesis in order to allow for vessel formation in a more coordinated fashion.

In summary, we define HIF-1 as a critical mediator of NK cell effector function and cancer immune surveillance. Secondly, we show that HIF-1 in NK cells acts as a negative regulator of tumor angiogenesis that ensures the fine-tuning of the angiogenic response. These results indicate that exploiting the hypoxic response in NK cells may represent a novel therapeutic avenue.

Citation Format: Ewelina Krzywinska, Chahrazade Kantari-Mimoun, Magali Castells, Dagmar Gotthardt, Yann Kerdiles, Ralph Klose, Joachim Fandrey, Veronika Sexl, Christian Stockmann. The hypoxic response in natural killer cells: Linking cytoxicity and tumor immune surveillance to angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A10.
Abstract A11: Targeting vascular endothelial growth factor in myeloid cells enhances natural killer cell responses to chemotherapy and ameliorates cachexia
Authors: Castells Magali , Klose Ralph , Gotthardt Dagmar , Putz Eva-Maria , Krzywinska Ewelina , Kantari-Mimoun Chahrazade , Chikdene Naima , Meinecke Anna-Katharina , Schroedter Katrin , Helfrich Iris , Fandrey Joachim , Sexl Veronika , Stockmann Christian
Published: October 2016

Chemotherapy remains a mainstay of cancer treatment but its use is often limited by the development of adverse reactions. Severe involuntary loss of body weight (cachexia) is a frequent cause of death in cancer patients and is exacerbated by chemotherapy. We show that, in contrast to antibody-mediated neutralization of Vascular Endothelial Growth Factor (VEGF)-A, genetic inactivation of VEGF-A in tumor-associated myeloid cells prevents chemotherapy-induced cachexia by inhibiting skeletal muscle loss and the lipolysis of white adipose tissue. It also improves clearance of senescent tumor cells by natural killer cells and inhibits tumor regrowth after chemotherapy. The effects depend on the adipokine and chemoattractant chemerin, which is released by the tumur endothelium in response to chemotherapy. The findings define chemerin as a critical mediator of the immune response elicited by chemotherapy as well as an important inhibitor of cancer cachexia. Efficient targeting of VEGF signaling within the tumor microenvironment should impede weight loss that is frequently associated with chemotherapy, thereby dramatically improving the therapeutic outcome.

Citation Format: Magali Castells, Ralph Klose, Dagmar Gotthardt, Eva-Maria Putz, Ewelina Krzywinska, Chahrazade Kantari-Mimoun, Naima Chikdene, Anna-Katharina Meinecke, Katrin Schroedter, Iris Helfrich, Joachim Fandrey, Veronika Sexl, Christian Stockmann. Targeting vascular endothelial growth factor in myeloid cells enhances natural killer cell responses to chemotherapy and ameliorates cachexia. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A11.
Abstract A15: Mechanisms of CD8+ T cell immunosuppression in triple negative breast cancer
Authors: Gruosso Tina , Gigoux Mathieu , Bertos Nicholas , Saleh Sadiq , Omeroglu Atilla , Zuo Dongmei , Zhao Hong , Souleimanova Margarita , Weaver Valerie , Meterissian Sarkis , Hallett Michael , Park Morag
Published: October 2016

Triple negative breast cancer (TNBC), defined as tumors lacking expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), are especially difficult to treat effectively. While ER+ and HER2+ breast cancer subtypes can be treated with Tamoxifen and Herceptin, respectively, there are no targeted therapies for TNBC patients. Furthermore, while only 20-30% of TNBC patients respond to chemotherapy in the neoadjuvant setting, overall outcome remains poor for non-responding patients. However, mounting evidence suggests that immune-checkpoint inhibitor immunotherapies may be especially promising for TNBC patients. We and others have shown that the presence of CD8+ T cells, a crucial component of the cytotoxic arm of the adaptive immune response, is a sign of good clinical outcome in TNBC patients. However, good outcome only correlates with CD8+ T cell invasion of the tumor parenchyma. Here we show that some patients have an accumulation of CD8+ T cells in the surrounding tumor-associated stroma, but not the tumor epithelium, and these patients responded as poorly as patients with no CD8+ T cells at all. Yet how cancer associated fibroblasts (CAFs), a dominant cell type of the tumor-associated stroma, affects CD8+ T cell invasion into the tumor epithelium is still poorly understood.

To identify potential stroma-dependent mechanisms that potentiate or inhibit CD8+ T cells invasion into the tumor epithelium, we performed gene expression profiling of laser-capture microdissected tumor-associated stroma (and matched epithelium) from 56 TNBC cases. Here we identify several key stromal features that may explain the accumulation of CD8+ T cells outside of the tumor epithelium. Preliminary data by immunohistochemistry and immunofluorescence validate some key stromal features and decipher the implication of other immune cell types in CD8+ T cells lack of tumor epithelium infiltration. These key stromal features that impair CD8+ T cell infiltration into the tumor in some patients might explain the relative low efficiency of immunotherapies in TNBC patients (20% of patients respond). One could speculate that targeting these key stromal features would allow a significant CD8+ T cell infiltration into the tumor and thus sensitize patients to immunotherapies.

Citation Format: Tina Gruosso, Mathieu Gigoux, Nicholas Bertos, Sadiq Saleh, Atilla Omeroglu, Dongmei Zuo, Hong Zhao, Margarita Souleimanova, Valerie Weaver, Sarkis Meterissian, Michael Hallett, Morag Park. Mechanisms of CD8+ T cell immunosuppression in triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A15.
Abstract A19: Inhibition of MK2 reduces chronic inflammation and regresses tumors in a mouse model of colorectal cancer
Authors: Ray Anita L. , Castillo Eliseo F. , Nofchissey Robert A. , Beswick Ellen J.
Published: October 2016

Chronic inflammation is a known risk factor for colorectal cancer (CRC). Cancers in patients with increased inflammatory cytokines in tumor tissue are associated with increased drug resistance, metastasis, and mortality. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) as a regulator of pro-inflammatory cytokines and promoter of proliferation in CRC progression. MK2 signaling induces IL-1, IL-6, and TNF-alpha production, which have been associated with CRC development and progression. We investigated this pathway in an inflammation-related model of CRC. This model relies on an initial dose of the carcinogen azoxymethane, followed by administration of dextran sodium sulfate to model chronic inflammation. C57BL/6 mice were found to develop colon tumors by day 54 in this model. Thus, we treated mice with MK2 inhibitors starting at this time point, 3 times a week for 3 weeks. At day 80, neoplasm number and size were measured, as well as pro-inflammatory cytokines and immune cell profiles in the colon using flow cytometry and multiplex bead-based arrays. MK2 inhibition drastically reduced colon pro-inflammatory cytokines, influx of immune cells, and tumor burden compared to vehicle control treated mice. Upon testing two separate MK2 inhibitors, 6/7 and 5/7 mice were completely free of tumors after treatment. We conclude that this pathway is an important contributor to CRC and could be a promising therapeutic target.

Citation Format: Anita L. Ray, Eliseo F. Castillo, Robert A. Nofchissey, Ellen J. Beswick. Inhibition of MK2 reduces chronic inflammation and regresses tumors in a mouse model of colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A19.
Abstract A22: In situ image-based monitoring of extracellular matrix mechanics in 3D pancreatic tumor-stroma co-culture models to inform therapeutic development
Authors: Jones Dustin P. , Hanna William , Cramer Gwendolyn M. , El-Hamidi Hamid , Celli Jonathan P.
Published: October 2016

A number of compelling studies have shown that the mechanical landscape of the tumor microenvironment contributes significantly to regulation of growth behavior, phenotype, and therapeutic response. These findings could be particularly relevant to pancreatic tumors, which are associated with a profound desmolastic reaction. The exceptional abundance of rigid, fibrotic stroma encasing these lethal malignancies has been implicated in obstructing drug delivery and playing multiple tumor-promoting roles. In this context, we use in vitro 3D tumor models with customized stromal composition, combined with in situ quantitative measurements of extracellular matrix (ECM) rigidity via optical microrheology, as a platform for evaluation of therapeutics targeting biomechanical interactions with the microenvironment. Using ECM formulations of calibrated rheological properties (rigidity) in conjunction with optical measurements that provide readouts of dynamic changes provides a window into both sides of a mechanoregulatory dialogue, in which ECM rigidity influences tumor growth and, in turn, cancer cells and stromal fibroblasts remodel their mechanical environment. Here we examine differential therapeutic response to selected chemotherapy agents and photodynamic therapy (PDT) in culture conditions that lead to fibrosis (increasing mechanical strength) as well invasive behavior (local breakdown of ECM). The methodology used here could be more broadly applied to directly quantify impact of stromal depletion therapies and altered ECM remodeling concomitant with therapy.

Citation Format: Dustin P. Jones, William Hanna, Gwendolyn M. Cramer, Hamid El-Hamidi, Jonathan P. Celli. In situ image-based monitoring of extracellular matrix mechanics in 3D pancreatic tumor-stroma co-culture models to inform therapeutic development. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A22.
Abstract A23: Characterization of diverging molecular and phenotypic responses under changing extracellular matrix stiffness in MCF10A cells
Authors: Liu Sonya , Bainer Russell , Yui Yoshihiro , Flinders Colin , Mallick Parag , Weaver Valerie , Mumenthaler Shannon
Published: October 2016

Tissue stiffness has emerged as an important regulator in the maintenance of normal cellular processes. In general, solid tumors are known to be much stiffer than normal tissue, with increased extracellular matrix (ECM) density being indicative of a higher risk for many cancers, including those of the breast. Over the course of breast cancer progression, the ECM becomes increasingly stiffer, a phenomenon which is known to be a driver of malignancy. In a 3D culture system using self-assembling peptide (SAP) gels, we grew MCF10A acini under a dynamic range of stiffness environments (140-5800 Pa). Under normal breast tissue elasticity, mammary epithelial cells form rounded, polarized acini with cleared lumens. However, in tumor-like stiffnesses, the same cells form non-polarized, collapsed structures that invade into the gel. To investigate the specific genes and pathways affected by stiffness, we have conducted tandem mass spectrometry to identify protein expression changes in MCF10A cells grown on SAP gels of increasing stiffness. Interestingly, the resulting analysis showed that cells grown on higher stiffnesses exhibit greater variation in protein expression. These divergent force-regulated molecular pathways are involved in a variety of cellular functions, including cell adhesion and migration, metabolism, and the immune response, among others. This study underscores the significance of mechanical forces exerted by the ECM, and signifies the first large-scale proteomic screen carried out to analyze heterogeneous cellular responses to changing stiffness environments.

Citation Format: Sonya Liu, Russell Bainer, Yoshihiro Yui, Colin Flinders, Parag Mallick, Valerie Weaver, Shannon Mumenthaler. Characterization of diverging molecular and phenotypic responses under changing extracellular matrix stiffness in MCF10A cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A23.
Abstract A25: Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages
Authors: Trinh Bon Q. , Barengo Nicolas , Kim Sang Bae , Lee Ju-Seog , Zweidler-McKay Patrick A. , Naora Honami
Published: October 2016

Thrombocytosis, characterized by high platelet counts, and anemia, caused by low numbers of red blood cells, are associated with poor outcomes in solid tumors and occur in several types of myeloid disorders. The megakaryocyte and erythroid lineages give rise to platelets and red blood cells, respectively, but the mechanisms that cause increased megakaryopoiesis and concurrently decreased erythropoiesis are poorly understood. In this study, we identified that expression of DLX4, a transcription factor encoded by a homeobox gene, increases during megakaryopoiesis and decreases during erythropoiesis. Overexpression and knockdown experiments using normal CD34+ stem/progenitor cells and bipotent K562 cells demonstrated that DLX4 induces lineage markers and morphologic features of megakaryocytes, and concomitantly represses erythroid marker expression and hemoglobin levels. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression induced by DLX4 showed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. Bioinformatic analysis also revealed that DLX4 induces gene signatures that are associated with NF-kappaB signaling. The ability of DLX4 to promote megakaryocyte development and to inhibit erythroid differentiation was diminished by blocking NF-kappaB activity or by repressing IL1B, a transcriptional target of DLX4. These findings indicate that DLX4 promotes megakaryocyte development at the expense of erythroid generation in part by inducing inflammatory signaling. Studies are ongoing to investigate the transcriptional control by homeobox genes of inflammatory signaling in thrombocytosis and anemia that are associated with solid tumors and myeloproliferative diseases.

Citation Format: Bon Q. Trinh, Nicolas Barengo, Sang Bae Kim, Ju-Seog Lee, Patrick A. Zweidler-McKay, Honami Naora. Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A25.
Abstract A45: Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways in triple-negative breast cancer
Authors: Wu Tony J. , Nabet Barzin Y. , Xu Bihui , Boelens Mirjam C. , Jonkers Jos , Minn Andy J.
Published: October 2016

Breast cancer cells can achieve protection from DNA damage both through cell-autonomous mechanisms and intercellular communication with the tumor microenvironment. Previously, we described an Interferon-Related DNA Damage Resistance Signature comprising of a network of interferon-stimulated genes (ISGs) that promotes and clinically predicts chemotherapy and radiation resistance in breast cancer. Here, we examine the heterotypic tumor-stroma interactions that regulate ISGs. We show that STAT1 and other ISGs are induced in breast cancer cells following interaction with stroma. STAT1 induces NOTCH3 expression, explaining a requirement for juxtacrine signaling. Moreover, we report that stroma upregulates ISG expression in breast cancer cells through exosomes. Exosomes transferred from the stroma and ISG induction are both dependent on RAB27B. In mice, targeting these pathways abrogate stroma-mediated resistance and results in long-term tumor-free survival. Analysis of primary human tumors supports the role of anti-viral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance. To determine whether these observations can further predict clinical efficacy, cell lines derived from a genetically engineered mouse model for p53-induced breast cancer, K14cre;Brca1F/F;p53F/F, were tested. We show that stroma induces breast cancer ISGs through exosomes, which in turn can induce signaling to activate NOTCH3 and regulate DNA damage resistance.

Citation Format: Tony J. Wu, Barzin Y. Nabet, Bihui Xu, Mirjam C. Boelens, Jos Jonkers, Andy J. Minn. Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A45.
Abstract A47: miR-30c: a TGF beta-regulated miRNA that coordinates vascular-directed fibrinolysis and tumor angiogenesis
Authors: McCann James V. , Xiao Lin , Khan Omar , Kowalski Piotr , Azam Salma , Pecot Chad V. , Anderson Daniel G. , Dudley Andrew C.
Published: October 2016

Tumor-associated endothelial cells (TECs) are dysfunctional and leak fibrin which is resolved (fibrinolysis) and replaced with collagen in a process that closely resembles wound healing. Because degraded perivascular fibrin also promotes angiogenesis and creates scaffolds for invasive cancer cells, vascular-directed fibrinolysis in solid tumors is a fundamental spark during tumor progression. Using a miRNA screen of freshly isolated TECs, we identified a TGF beta-regulated miRNA that controls vascular-mediated fibrinolysis in tumors. miR-30c is increased in TECs relative to normal endothelial cells (NECs) whereas its target PAI-1 (a fibrinolysis inhibitor) is strikingly decreased. TECs secrete less PAI-1 and they rapidly degrade and migrate through fibrin scaffolds resulting in uncoordinated vessel sprouting. However, TGF beta and miR-30c antagomiRs reduce miR-30c expression which re-instates PAI-1 secretion, diminishes vascular-specific fibrinolysis, and “normalizes” TEC sprouting. miR-30c antagomiRs coupled to vascular-tropic nanoparticles reduce TEC migration in fibrin and enforce vascular expression of PAI-1 in vitro and in vivo. Thus, vascular-specific expression of miR-30c in tumors may be manipulated in vivo using this nanoparticle-based strategy. We also show that TGF beta promotes the export of miR-30c in extracellular vesicles (EVs) which are received by recipient stromal cells. TGF beta-mediated extravascular mobilization of miR-30c may down regulate PAI-1 expression in nearby stromal cells or cancer cells thereby programing fibrinolysis in these non-vascular recipients. Taken together, we propose an axis between TGF beta, miR-30c, and PAI-1 in TECs controls the rate of perivascular fibrinolysis and is therefore an important effector of tumor emergence.

Citation Format: James V. McCann, Lin Xiao, Omar Khan, Piotr Kowalski, Salma Azam, Chad V. Pecot, Daniel G. Anderson, Andrew C. Dudley. miR-30c: a TGF beta-regulated miRNA that coordinates vascular-directed fibrinolysis and tumor angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A47.
Abstract B06: Characterizing the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets in prostate cancer cell lines and donor immune cells in culture
Authors: Adams Bruce A. , Lim James , Chow Tianna , Bernstein Susan , Ryan Charles J.
Published: October 2016

Introduction: Cancer immunotherapy has changed the paradigm of cancer treatment by harnessing the immune system to identify and eradicate tumors leading to significant improvements in the objective response and long term survival of patients. However, more than 50% of patients do not respond to the most advanced immunotherapeutics in clinical trials, suggesting that further research is needed to understand the mechanism of action and resistance to immune-modulating agents in patients. The current hypothesis is that the tumor microenvironment can modulate the reprogramming of stromal cells caused by chronic inflammation, leading to a suppressed immune system that is highlighted by T-cell exhaustion and anergy. Hypoxia and high interstitial fluid pressure within tumors are elements that may contribute to gene and protein expression changes in stromal, immune and tumor cells in the microenvironment to inhibit immune function and promote cancer proliferation and metastasis.

Methods: We have developed a novel primary cell culture bioreactor to modulate both oxygen level (0.1% to 19%) and hydrostatic pressure levels (26 to 260 mmHg / 0.5 psig to 5 psig) to study the effects on gene and protein expression profiles of prostate cancer cell lines and peripheral blood mononuclear cells (PBMCs) derived from donors. We performed high-resolution immunofluorescence imaging and western blot protein expression analysis of primary immuno-therapy targets CTLA-4, PD-1, and PD-L1. In addition, whole transcriptome sequencing was performed on cells across a range of physiologically-relevant culturing conditions to mimic various tumor microenvironments found within the body.

Conclusions: We report that prostate cancer cell lines and PBMCs differentially express immunotherapeutic targets when cultured at physiologically relevant tumor microenvironment conditions, resulting in reduced expression of key targets. Analysis of mRNA-seq data revealed alterations in gene expression profiles of immunotherapeutic and drug-target pathways involving CTLA-4 and AR signaling. In contrast, we observed increased CD47 and CD44 expression at low oxygen and high pressure culturing conditions in cancer cell lines and PBMCs. These data highlight the importance of using physiologically relevant microenvironment conditions that include hypoxia and pressure to identify biomarkers and drug target candidates.

Citation Format: Bruce A. Adams, James Lim, Tianna Chow, Susan Bernstein, Charles J. Ryan. Characterizing the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets in prostate cancer cell lines and donor immune cells in culture. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B06.
Abstract B19: Vascular normalization suppresses tumor progression and potentiates docetaxel chemotherapy in prostate cancer
Authors: Kang You Hyun , Jin Hyun A , Heo Jun Hyeok , Kim Ki Hong , Han Kyung Seok , Hong Sung Joon
Published: October 2016

Background: Tumor requires excessive oxygen and nutrients due to their rapid growth. Tumor is generally vascular but the vasculature is not physiologic but leaky, dilated, and tortuous. The inefficiency of tumor vessels induces hypoxic stress that makes the tumor more aggressive. Here, we investigated the role of normalizing tumor vasculature on tumor progression and treatment efficacy of conventional chemotherapy in prostate cancer.

Methods: Human vascular endothelial cell (HUVEC) was used to evaluate the effects of SAC-1004 on endothelial cells. Immunofluorescent confocal imaging with immunocytochemistry was performed for E-cadherin expression in HUVECs. MTS assay was performed to assess cell proliferation of HUVECs and PC-3 cells after SAC-1004 treatment. Immunohistochemical staining with anti-CD31 and anti-SMA antibodies was performed to assess vasculature in vivo. PC-3 subcutaneous xenografts were developed to evaluate the effects of SAC-1004 on tumor vasculature and tumor growth in prostate cancer. Magnetic resonance images were taken to assess vascular perfusion in xenografts. SAC-1004 was administrated via tail vein once a day for 7 days. Docetaxel was administrated intravenously once a week.

Results: SAC-1004 restored junction protein E-cadherin in vitro in endothelial cells and significantly reduced VEGF-induced retinal vascular leakage. SAC-1004 promoted proliferation of HUVECs in vitro in a dose-dependent manner. However, proliferation of cancer cells was not significantly affected by SAC-1004 treatment. Reduced vascular leakiness and improved hypoxia were found in PC-3 xenografts treated with SAC-1004 treatment and magnetic resonance imaging after SAC-1004 treatment for 7 days confirmed improved perfusion in the PC-3 xenografts treated with SAC-1004 compared with control. Interestingly, SAC-1004 treatment suppressed tumor growth in PC-3 xenografts but SAC-1004 followed by intravenous docetaxel treatment showed the most potent tumor suppression compared with control in PC-3 xenografts.

Conclusions: SAC-1004 improves hypoxic tumor microenvironment and restores abnormal leak vasculature of tumor by restoring endothelial cell junction in prostate cancer. Vascular normalization induced tumor regression and docetaxel combination following vascular normalization sensitized prostate cancer to docetaxel treatment.

Citation Format: You Hyun Kang, Hyun A Jin, Jun Hyeok Heo, Ki Hong Kim, Kyung Seok Han, Sung Joon Hong. Vascular normalization suppresses tumor progression and potentiates docetaxel chemotherapy in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B19.
Abstract B20: Obesity-induced Nlrc4 inflammasome promotes angiogenesis in breast cancer
Authors: Kolb Ryan , Phan Liem , Borcherding Nicholas , Liu Yinghong , Yuan Fang , Janowski Ann M. , Xie Qing , Markan Kathleen R. , Li Wei , Potthoff Matthew J. , Fuentes-Mattei Enrique , Ellies Leslie G. , Knudson Michael , Lee Mong-Hong , Yeung Sai-Ching Jim , Cassel Suzanne L. , Sutterwala Fayyaz S. , Zhang Weizhou
Published: October 2016

Obesity, which impacts 36% of adults in the U.S., is associated with an increased risk of ER-positive breast cancer in postmenopausal women, increased incidence of the more aggressive triple negative breast cancer, increased risk of developing invasive ductal carcinoma (IDC) versus ductal carcinoma in situ (DCIS; non-invasive) and a worse clinical outcome. There have been many hypotheses to explain this link between obesity and breast cancer, but a causal mechanism is still largely missing. Here, using two syngeneic orthotopic transplant models in C57Bl/6 mice, we examined the role of obesity-associated inflammasome activation/IL-1 signaling in obesity-driven breast cancer progression. Our results show that the tumor microenvironment under the conditions of obesity promotes Nlrc4 inflammasome activation in tumor-infiltrating myeloid cells, which in turn leads to IL-1β activation which promotes tumor progression. Further studies show that obesity-induced Nlrc4 inflammasome activation/IL-1β promotes increased angiogenesis and upregulation of Vegfa, primarily in adipocytes. Finally, treatment of mice with Metformin inhibited obesity-associated angiogenesis and breast cancer progression. This data provides evidence for obesity-induced Nlrc4 inflammasome mediated activation of IL-1β as a causal mechanism for obesity-driven breast cancer progression and a rationale for blockade of IL-1 signaling, the use of anti-angiogenesis therapies, or treatment with metformin in obese breast cancer patients.

Citation Format: Ryan Kolb, Liem Phan, Nicholas Borcherding, Yinghong Liu, Fang Yuan, Ann M. Janowski, Qing Xie, Kathleen R. Markan, Wei Li, Matthew J. Potthoff, Enrique Fuentes-Mattei, Leslie G. Ellies, Michael Knudson, Mong-Hong Lee, Sai-Ching Jim Yeung, Suzanne L. Cassel, Fayyaz S. Sutterwala, Weizhou Zhang. Obesity-induced Nlrc4 inflammasome promotes angiogenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B20.
Abstract B22: Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system
Authors: MacDonald Amanda , Priess Michelle , Curran Jennifer , Krause Silva
Published: October 2016

Pancreatic cancer is one of the most aggressive types of cancer, with only about 5% of patients surviving 5 years past the initial diagnosis. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal due to the cancer's complex pathology. Novel multi-targeted agents or therapeutic combinations able to disrupt the aggressive fibrotic microenvironment and restrict tumor proliferation are a current focus in the field. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, and adhesion molecules in cancer progression and metastasis and as such has been shown to modulate multiple pathways in tumors. In order to further explore how necuparanib affects the tumor and its microenvironment, a 3-dimensional (3D) culture system mimicking pancreatic cancer was developed containing a co-culture of human pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. Morphological and histological assessment of the 3D tumor spheroids demonstrated that necuparanib inhibits a) pancreatic tumor cell growth, b) pancreatic tumor cell invasion, and c) pancreatic stellate cell growth. Necuparanib's activity in these assays was shown to be dose-dependent. Moreover, using a negative control for necuparanib engineered to lack the ability to bind growth factors, we established that necuparanib's activity is growth factor binding dependent. The ability to inhibit both tumor and stromal cells demonstrates that necuparanib is a very promising multi-targeting agent for difficult-to-treat cancers such as desmoplastic pancreatic tumors. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support the current Phase 2 safety and efficacy study being performed in patients with metastatic pancreatic cancer.

Citation Format: Amanda MacDonald, Michelle Priess, Jennifer Curran, Silva Krause. Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B22.
Abstract B24: Targeting hyaluronan sensitizes breast cancer-associated fibroblasts to lapatinib and overcomes stromal resistance
Authors: Marusyk Andriy , Tabassum Doris , Guerriero Jennifer , Place Andrew , Rozhok Andrii , Letai Antony , Polyak Kornelia
Published: October 2016

Tumors represent abnormal organs comprised not only of neoplastic cells, but also of normal cells and extracellular matrix (ECM). Therefore, clinical progression as well as response to therapies depends not only on properties of neoplastic cells, but also on the interactions between different types of cells within tumors. Cancer-associated fibroblasts (CAFs) are the major non-nenoplastic cell component of tumors and major the producers of ECM. Despite extensive research, mechanisms and consequences of interactions between breast carcinoma cells and CAFs remain insufficiently understood.

Using organotypic 3d co-cultures between breast carcinoma cell lines representing different sub-types of the disease and CAFs, we interrogated changes in gene expression and metabolism that result from the interaction. We found that while contact with fibroblats induces robust changes in expression and metabolism, the majority of changes are subtype and cell line specific. Furthermore, we found that fibroblasts can provide a dramatic protection against dual EGFR/HER2 inhibitor lapatinib. Protective effect requires close contact between the two cell types in context of 3d culture, and is mediated by both reduced accumulation of the drug within carcinoma cells and elevated apoptotic threshold. Using synthetic lethality screens, we have identified several signaling pathways whose inhibition enhances cytotoxic effects of lapatinib. Interrogation of functional relevance of ECM has revealed that the stromal protection can be reversed by targeting hyaluronan, a major ECM component produced by fibroblasts. Rather than simply enhancing cytotoxic activity of lapatinib against carcinoma cells, hyaluronidase treatment sensitized CAFs to lapatinib, thereby removing the protective niche. Our studies shed light on molecular changes induced by the interaction with CAFs and offer novel approaches for overcoming microenvironmental protection by therapeutically targeting CAFs.

Citation Format: Andriy Marusyk, Doris Tabassum, Jennifer Guerriero, Andrew Place, Andrii Rozhok, Antony Letai, Kornelia Polyak. Targeting hyaluronan sensitizes breast cancer-associated fibroblasts to lapatinib and overcomes stromal resistance. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B24.
Abstract B25: Characterization, targeting, and modulation of carbonic anhydrase IX activity for the development of small-molecule inhibitors to treat triple-negative breast cancer
Authors: Mboge Mam Y. , Chen Zhijuan , Mahon Brian P. , Lamas Nicole , Tu Shingkuang , Carta Fabrizio , Superan Claudiu T. , McKenna Robert , Frost Susan C.
Published: October 2016

The microenvironment within a solid tumor is usually heterogeneous with certain regions being acidic and hypoxic. These acidic and hypoxic regions arise from rapidly proliferating cells combined with poor tumor perfusion. Cancer cells cope with these hostile changes in their microenvironment by expressing genes that are essential for survival. One of the coping mechanisms is an upregulation of pH regulatory factors, including carbonic anhydrase IX (CAIX). The action of CAIX helps to maintain physiological pH inside the cell (pHi) while regulating extracellular acidification (pHe). Extracellular acidification of the tumor microenvironment promotes local invasion, metastasis and decreases the effectiveness of adjuvant therapies, thus contributing to poor clinical outcome. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in a triple negative breast cancer cell line. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors using X-ray crystallography methods. These structures show the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity measured as 18O exchange between H2O16 and H13C18O3 . We have also investigated the effect CAIX inhibition on cancer cell metabolism and extracellular acidification using Seahorse technology. This reveals that CAIX may contribute to the “non-glycolytic” acidification process. In total, these observations indicate that CAIX is a viable small molecular drug target and contribute to our understanding of the function of CAIX in modulating pH in cancer cells.

Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Nicole Lamas, Shingkuang Tu, Fabrizio Carta, Claudiu T. Superan, Robert McKenna, Susan C. Frost. Characterization, targeting, and modulation of carbonic anhydrase IX activity for the development of small-molecule inhibitors to treat triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B25.
Abstract B27: Blockade of protein kinase A (PKA) localization augments the trafficking and anti-tumor efficacy of adoptively transferred chimeric antigen receptor-expressing T cells
Authors: Newick Kheng , O'Brien Shaun , Sun Jing , Kapoor Veena , Maceyko Steve , Lo Albert , Pure Ellen , Moon Ed K. , Albelda Steven M.
Published: October 2016

Infusion of T cells expressing chimeric antigen receptors (CAR T cells) is still relatively ineffective in solid tumors due to the presence of immunosuppressive mediators (such as prostaglandin E2 (PGE2) and adenosine), and poor T cell trafficking. Since PGE2 and adenosine activate protein kinase A (PKA), which then inhibits T cell receptor (TCR) activation, we generated CAR T cells that expressed a small peptide called the “regulatory subunit I anchoring disruptor” (RIAD) that inhibits the association of protein kinase A (PKA) with ezrin; this interaction is required for the negative effects of PKA on TCR activation. After exposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showed increased TCR signaling, more cytokine release, and enhanced killing of tumor cells compared to CAR T cells. When injected into tumor-bearing mice, murine and human CAR-RIAD T cells demonstrated enhanced anti-tumor efficacy compared to CAR T cells due to increased T cell infiltration of established tumors, and attenuated tumor-induced hypofunction. Subsequent in vitro assays showed that both mouse and human CAR-RIAD cells migrated more efficiently than CAR cells in response to the chemokine CXCL10 and also adhered better to various matrices. Our data therefore show that the addition of the RIAD peptide to adoptively transferred CAR T cells augments their efficacy by increasing their effector function and by improving trafficking into tumor sites. This treatment strategy should therefore be considered for clinical application in treating solid tumors.

Citation Format: Kheng Newick, Shaun O'Brien, Jing Sun, Veena Kapoor, Steve Maceyko, Albert Lo, Ellen Pure, Ed K. Moon, Steven M. Albelda. Blockade of protein kinase A (PKA) localization augments the trafficking and anti-tumor efficacy of adoptively transferred chimeric antigen receptor-expressing T cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B27.
Abstract B34: Targeting M2-tumor associated macrophages (M2-TAMs) in prostate cancer
Authors: Zarif Jelani C. , Hernandez James R. , Pienta Kenneth J.
Published: October 2016

Prostate cancer is a leading cause of cancer-related deaths of men in the U.S., and in the past year, over 30,000 men died from this disease. While localized prostate cancer is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Alternatively activated macrophages, also known as M2-macrophages, primarily scavenge debris and in the process, promote angiogenesis and wound repair. M2-macrophages are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors such as prostate cancer to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. The purpose of this project is to develop novel therapeutics that will directly target M2-TAMs for destruction and subsequently attenuate prostate tumor growth, progression and metastasis. The central hypothesis of this study is to determine if targeting of M2-TAMs using specific surface antigens will be an effective therapy for lethal prostate cancer and potentially, other cancers. Our study elucidated M2-Macrophage biology including creating homogenous populations of both M1 and M2 macrophages using a new strategy as to allow for identification of novel surface antigen markers. We then assessed novel markers of macrophage populations and assessed whether these surface antigens that are expressed on M2-macrophages are also expressed in human prostate cancers indicative of myeloid infiltration. Validation studies using metastatic prostate cancer tissues from rapid autopsies and surgical specimens supplied by the Department of Urology at Johns Hopkins University School of Medicine were also be performed. Complementary studies will be executed to target these markers for anti-tumor cell efficacy using these relevant model systems. By identifying and targeting specific markers on M2-TAMs, we predict that this targeting will provide a better prognosis for patients who have been diagnosed with lethal prostate cancer.

Citation Format: Jelani C. Zarif, James R. Hernandez, Kenneth J. Pienta. Targeting M2-tumor associated macrophages (M2-TAMs) in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B34.
Abstract B39: Identifying molecular signatures of tumor dormancy as a basis for the rational design of precision nanomedicines
Authors: Tiram Galia , Ferber Shiran , Satchi-Fainaro Ronit
Published: October 2016

Tumor progression is dependent on a number of sequential steps, including initial tumor-vascular interactions and recruitment of blood vessels (i.e., the “angiogenic switch”), as well as an established interaction of tumor cells with their surrounding microenvironment and its different immune, endothelial and connective cellular and extra-cellular components. Failure of a microscopic tumor, either primary, recurrent or metastatic, to complete one or more of these early stages may lead to delayed clinical manifestation of the cancer (i.e., tumor dormancy). Micrometastasis, dormant tumors, and residual tumor cells - referred to as minimal residual disease, contribute to the occurrence of relapse, and constitute fundamental clinical manifestations of tumor dormancy that together are responsible for the vast majority of cancer deaths. However, although the tumor dormancy phenomenon has critical implications for early detection and treatment of cancer, it is one of the most neglected areas in cancer research and the associated biological mechanisms are still mostly unknown.

We have created several models of patient-derived xenografts mimicking pairs of dormant vs fast-growing, primary vs metastatic and drug-sensitive vs resistant cancers. We investigated the molecular and cellular changes in tumor-host interactions that govern tumor progression. Those led to the discovery of novel targets and provided important tools for cancer theranostics (therapy and diagnostics). Based on the acquired knowledge, we designed a new strategy to improve treatment outcomes of patients with bone neoplasms, glioblastoma, brain metastases, melanoma, breast and prostate cancers. We have identified molecular signatures which following their selective delivery into target cells, can potentially induce a dormant-like phenotype. This goal was achieved by utilizing polymeric nanomedicines and guidance by high resolution, intravital non-invasive imaging techniques.

A better understanding of tumor dormancy and the availability of relevant markers will most likely change the way we diagnose and treat the disease using novel combined theranostic nanomedicines.

Citation Format: Galia Tiram, Shiran Ferber, Ronit Satchi-Fainaro. Identifying molecular signatures of tumor dormancy as a basis for the rational design of precision nanomedicines. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B39.