bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1083A: An ex vivo 3D bone metastasis model for prostate cancer
Authors: Zilberberg Jenny , Choudhary Saba , Dziopa Eugenia , Mannion Cirian , Kissin Yair , Parasido Erika , Albanese Christopher , Lee Woo
Published: August 2018

Introduction: Bone is the preferred site for prostate cancer (PCa) metastases and there is virtually no cure once the tumor is established within this niche. The development of therapeutic strategies against metastatic PCa has been limited by the lack of an all-human preclinical model to: (1) study PCa-bone interactions under physiologically relevant three dimensional (3D) microenvironments, (2) understand how these interactions result in the development of drug resistance, and (3) explore paradigm-changing precision medicine concepts.

Methods: Conditionally reprogrammed primary PCa cells (CR-PCa, derived from a lymph node metastatic site) were integrated with our ex vivo human 3D bone tissue platform, as a transformative approach for modeling tumor behavior at the bone niche. The 3D bone tissue was engineered using primary osteoblastic (OSB) cells from human bone samples. These cells were biomimetically assembly with biphasic calcium phosphate microbeads (BCPm) in perfusion microfluidic culture devices. BCPm were used to replicate the 3D cellular network of OSTs in human bone. This 3D tissue model was successfully used to support the osteocytic differentiation of primary human OSB cells within the BCPm structure, while maintaining a single cell layer of osteoblasts on the surface of the 3D tissue resembling the bone endosteum.

Results: Integration of the 3D bone tissue with CR-PCa cells resulted in a compromised endosteal layer after only 4 days of coculture, with more apoptotic OSTs in the tissues with CR-PCa vs. no PCa cells controls (P<0.01). For functional analysis, we stained 5 μm sections of the 3D tissues for alkaline phosphatase (ALP, an OSB marker) and sclerostin, an osteocytic marker and major signaling molecule released by embedded OSTs that suppresses osteoblastogenesis. Quantification of immunofluorescence staining revealed that ALP was significantly increased (P<0.05), whereas sclerostin significantly decreased (P<0.01) with the introduction of CR-PCa cells. This OSB phenotype, induced by PCa-bone interactions ex vivo, is entirely consistent with the OSB prostate cancer-bone metastasis niche seen in patients with advanced disease.

Conclusions: Our model recapitulates: (1) an OSB monolayer closely resembling the endosteum at the bone/ bone marrow interface, (2) the complex microphysiological features and functions of 3D-networked OSTs, and (3) the interactions of primary PCa cells with osteoblasts and OSTs.

Citation Format: Jenny Zilberberg, Saba Choudhary, Eugenia Dziopa, Cirian Mannion, Yair Kissin, Erika Parasido, Christopher Albanese, Woo Lee. An ex vivo 3D bone metastasis model for prostate 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 1083A.
Abstract 1086: Phosphoproteomic analysis of signaling pathways driving bone metastasis in prostate cancer
Authors: Abbott Benjamin , Pierce Andrew , Whetton Anthony D. , Townsend Paul A.
Published: August 2018

Osteoblasts play a fundamental role in prostate cancer bone metastasis, whereby dysregulated bone remodelling results in bone pain, fractures and increased mortality. Although it remains unclear how early on in the disease pathway prostate cancer cells populate the endosteal niche, osteoblasts almost certainly play a role in the homing of tumor cells to this microenvironment. In support of this, prostate cancer cells have been shown to home to osteoblast-rich areas in animal models of bone metastasis and human prostate cancer bone metastases are typically osteoblastic on radiography. We have shown that conditioned medium from mature, mineralising human hFOB1.19 osteoblasts (hFOB-CM) is rich in pro-inflammatory cytokines including IL-6, IL-8 and various chemokine ligands. hFOB-CM promotes a strong migratory and invasive phenotype in a panel of prostate cancer cell lines (including androgen-sensitive and -insensitive lines), with invading cells displaying increased actomyosin contractility and amoeboid-like traits, namely a rounded cell morphology, cytoskeletal reorganization and phosphorylation of myosin light chain. We now aim to comprehensively interrogate the intracellular signaling pathways driving endosteal homing in prostate cancer cells by systems-wide ITRAQ mass spectrometry-based phosphoproteomic analysis of prostate cancer cells treated with hFOB-CM. This approach will identify individual signaling pathways or combinations of pathways to target with existing kinase inhibitors to inhibit osteoblast-driven invasion and endosteal homing during prostate cancer metastasis. Ultimately, targeting tumor cell responses to stromal signals may prove critical in the search for better therapies to treat this frequently lethal disease.

Citation Format: Benjamin Abbott, Andrew Pierce, Anthony D. Whetton, Paul A. Townsend. Phosphoproteomic analysis of signaling pathways driving bone metastasis in prostate 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 1086.
Abstract 1099: Molecular drivers of neutrophil recruitment to primary non-small cell lung cancer
Authors: Wang Claire , Rayes Roni , Mouhanna Jack , Giannias Betty , Chandrasekaran Arvind , Mot Rachel , Moraes Christopher , Huang Sidong , Cools-Lartigue Jonathan , Bertos Nicholas , Ferri Lorenzo , Spicer Jonathan
Published: August 2018

Neutrophils are associated with developing cancer lesions and are the main immune component of primary non-small cell lung cancer (NSCLC). Multiple studies support the notion that tumor associated neutrophils (TANs) can promote tumor progression. We hypothesize that there is a hierarchy of molecular cues produced by developing lung cancers that guide circulating neutrophils to infiltrate the tumor microenvironment and become TANs. Identifying these cues may permit modulation of neutrophil infiltration within developing lung cancers and may thereby act as an immunotherapeutic tool to suppress cancer progression and improve response to existing therapeutics. To this end, we profiled 5 established NSCLC cell lines representing the common NSCLC subtypes using a qRT-PCR 84 gene panel (A549; KRAS mutant, PC9; EGFR mutant, H1993; MET amplification, H3122; EML4-ALK translocation, HCC78; ROS1 translocation). We focused on 4 of the most commonly upregulated genes in all cell lines, which were osteopontin (Spp1), vascular endothelial growth factor A (VEGF-A), macrophage inhibitory factor (MIF), and C-X-C motif ligand 1 (CXCL1). After confirming protein expression of these targets by western blot, we performed shRNA knock down (KD) of these genes and tested the migration of neutrophils towards treated and control cell lines in a novel microfluidic device that allows increased throughput studies of neutrophil attractants. Findings from KD experiments were confirmed via antibody-mediated inhibition. We observed a 3-fold increase of neutrophil migration to the A549 cancer cell line compared to the serum free control (p=0.0265). Furthermore, this increase was inhibited in Spp1 (64% decrease), MIF (84%), VEGF (82%) KDs and their corresponding neutralizing antibodies. We have therefore identified 4 proteins that play a key role in neutrophil recruitment to NSCLC cell lines in vitro and have demonstrated the application of a simple microfluidic device to test neutrophil migration patterns. This data provides the basis for in vivo investigations to elucidate the key molecular cues for neutrophil infiltration within developing lung cancers.

Citation Format: Claire Wang, Roni Rayes, Jack Mouhanna, Betty Giannias, Arvind Chandrasekaran, Rachel Mot, Christopher Moraes, Sidong Huang, Jonathan Cools-Lartigue, Nicholas Bertos, Lorenzo Ferri, Jonathan Spicer. Molecular drivers of neutrophil recruitment to primary non-small cell lung 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 1099.
Abstract 1100: Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer metastasis
Authors: Korkaya Hasan , Lee Eunmi , Piranioglu Raziye , Albers Thomas , Ouzounova Maria , Weeks Charlie , Rodier Riley , Korkaya Ahmet K. , Hassan Khaled A.
Published: August 2018

We recently demonstrated that infiltration of granulocytic MDSCs in distant organs is a critical step in metastatic process. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice are regulated a distinct set of genes. Two genes, S100A8 and S100A9 were highly upregulated in gMDSCs isolated from mice with metastatic 4T1 tumors compared to mice with non-invasive tumors. Using the recombinant single S100A8, S100A9 proteins or the S100A8/S100A9 heterotetramer (calprotectin), we demonstrated that calprotectin is a critical player in gMDSC induction and infiltration in distant organs particularly in lungs. Furthermore, injection of recombinant calprotectin but not single S100A8 or S100A9 proteins via tail vein enhanced the metastatic ability of nan-invasive EMT6 tumors in mice. We therefore performed computational screen of NCI compounds against the crystal structure of calprotectin and identified over 100 lead compounds. In vitro gMDSC differentiation assay identified 3 compounds with strong gMDSC inhibitory activity. We therefore designed a pilot experiment to test these compounds in preclinical mouse models. We also compared the activity of these compounds against tasquinimod, a small molecule targeting S100A9 only. However, tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. In line with these data, one of the compounds (#10) showed a potent activity against gMDSCs in 4T1 tumor-bearing mice and significantly inhibited pulmonary metastasis. Moreover, this compound eliminated the cytotoxicity caused by cyclophosphamide and animals were free of cancer. In summary, we have identified a molecular target which regulates gMDSC induction and play critical role in metastatic process and our findings from in vitro screens and preclinical testing provide a strong rationale for targeting MDSCs.

Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Thomas Albers, Maria Ouzounova, Charlie Weeks, Riley Rodier, Ahmet K. Korkaya, Khaled A. Hassan. Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer 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 1100.
Abstract 1115: A CRISPR/Cas9 kinome screen identifies GRK5 as a novel regulator of rhabdomyosarcoma (RMS) self-renewal
Authors: Pham Thao Q. , Vleeshouwer-Neumann Terra , Chen Eleanor
Published: August 2018

Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue sarcoma. Clinical outcome for patients with relapsed or metastatic disease remains poor. However, current treatment options remain relatively unchanged over the last two decades, so there remains an urgent need for more effective treatment options for disease relapse. Cancer stem cells (CSCs), also known as tumor-propagating cells (TPCs), have the capacity for self-renewal and have been proposed to be responsible for tumor relapse and metastasis. RMS has been shown to follow the CSC model of tumor progression. We have previously performed an siRNA library screen against the human kinome (714) and a secondary CRISPR/Cas9 screen of top candidates to narrow down to 11 novel kinase candidates that are potentially essential for self-renewal of human RMS cells. GRK5, a G-protein coupled receptor kinase, was among the top candidate genes. Preliminary data from in vitro cell-based assays and in vivo limiting-dilution experiments in xenografts showed that CRISPR/Cas9-mediated targeted disruption of GRK5 resulted in significant reduction in the self-renewal capacity of RMS cells. GRK5-deficient RMS cells also displayed significant reduction in growth secondary to cell cycle arrest at the G2/M checkpoint. The functional effects on tumor growth and self-renewal appeared unique to GRK5 among the GRK kinase subfamily, as targeted disruption of closely-related GRK4 and GRK6 did not significantly affect RMS tumor cell growth and self-renewal. We propose that targeted disruption of GRK5 by CRISPR/Cas9 results in elevated p53 protein stability and activity, leading to inhibition of proteins required for cell cycle progression. We are also characterizing the loss-of-function effects of GRK5 in a RMS TPC reporter cell line created using a CRISPR/Cas9 knockin approach that will allow for direct assessment of GRK5 and other candidate gene function within the TPC population. By characterizing the biologic role of TPC as well as the function of GRK5 in RMS, we hope to provide new biologic insights into cancer stem cell biology and GRK5 as a novel therapeutic target for the treatment of advanced RMS.

Citation Format: Thao Q. Pham, Terra Vleeshouwer-Neumann, Eleanor Chen. A CRISPR/Cas9 kinome screen identifies GRK5 as a novel regulator of rhabdomyosarcoma (RMS) self-renewal [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 1115.
Abstract 1119: Quaking (Qki) loss enhances stemness of glioma stem cells through regulating intracellular vesicle trafficking system
Authors: Hu Jian , Shingu Takashi
Published: August 2018

Glioma stem cells (GSCs) have higher self-renewal capacity than neural stem cells (NSCs), yet the underlying molecular mechanism is less clear. Here we report the generation of a novel conditional KO allele of Quaking (Qki), a tumor suppressor in glioblastoma with RNA binding activity. When Qki was deleted in Nestin-CreERT2;QkiL/L mice, the NSC population, which is characterized by long-term BrdU retention and GFAP+/Nestin+ double positive staining, was greatly increased, indicating that Qki deletion enhances NSC self-renewal. To determine whether Qki deletion promotes gliomagenesis, we generated a Nestin-CreERT2;QkiL/L;PtenL/L;p53L/L cohort, in which 92% of the mice developed glioblastoma with a median survival of 105 days. However, the Nestin-CreERT2;PtenL/L;p53L/L cohort did not develop any glioma up to a year; therefore Qki deletion greatly promotes gliomagenesis. Transcriptomic and proteomic profiling coupled with PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) analyses revealed that genes involved in intracellular vesicle trafficking were greatly enriched by Qki deletion. Specifically, 34% of the genes up-regulated by Qki deletion are involved in receptor delivery, and 39% of the genes down-regulated by Qki deletion are subunits of receptor degradation machineries such as endosomes and lysosomes. High level of receptor delivery and low level of receptor degradation concomitantly enrich receptors on the membrane and enhance the activity of the receptors that are involved in maintaining stemness, including RTK, Notch1 and Frizzled. Lower lysosomal activity induced by Qki deletion also leads to defective mitophagy, which consequently leads to accumulation of damaged mitochondria, high level of ROS, and genomic instability in Qki-deficient NSCs. We identified that genomic instability induced by Qki deletion led to accumulation of classical glioblastoma-associated mutations such as copy number gains of PDGFRa, Cyclin D1 and Cyclin D3. Taken together, our data suggest that Qki loss-induced endolysosomal defects promotes gliomagenesis through both reducing receptor degradation and inducing genomic instability.

Citation Format: Jian Hu, Takashi Shingu. Quaking (Qki) loss enhances stemness of glioma stem cells through regulating intracellular vesicle trafficking system [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 1119.
Abstract 1124: Wnt-producing niche cells support cancer stem-like cells in pancreatic ductal adenocarcinoma
Authors: Wu Katherine , Grbovic-Huezo Olivera , Hartmann Griffin , Jacks Tyler , Tammela Tuomas
Published: August 2018

Despite modern advances in cancer therapy, the prognosis for pancreatic ductal adenocarcinoma (PDAC) remains dismal, highlighting the need for better understanding of the biologic underpinnings of the disease. Well established as a pathway for growth and regeneration, Wnt signaling maintains subpopulations of cancer cells in a stem-like state, contributing to the heterogeneous composition of tumors. Our previous work demonstrated that Porcupine, an essential enzyme for secretion of paracrine Wnt signals, can be used to mark Wnt-producing cells in normal and tumor tissues. We detected the presence of nuclear beta-catenin, a biomarker of active Wnt-signaling, in a subpopulation of cancer cells adjacent to Porcupine+ niche cells in murine PDAC tissue. Furthermore, genetic inactivation of Porcupine translated to a 25% increase in survival in PDAC-bearing mice. In vitro, we see a marked increase in spheroid formation and proliferation of the stem-like subpopulation over the rest of the primary tumor cells. Ongoing lineage-tracing experiments in an autochthonous murine model of PDAC will determine whether these cancer stem cells are able to produce their Wnt-secreting niche in vivo. Our current efforts are focused on the preclinical evaluation of Wnt inhibitors in the treatment of mouse and human PDAC as single agents or in combination with chemotherapy. Such findings open the door for interrogation of the Wnt pathway as a possible target for improving the clinical outcome of pancreatic cancer.

Citation Format: Katherine Wu, Olivera Grbovic-Huezo, Griffin Hartmann, Tyler Jacks, Tuomas Tammela. Wnt-producing niche cells support cancer stem-like cells in pancreatic ductal adenocarcinoma [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 1124.
Abstract 113: Gemcitabine treatment induces immunosuppressive microenvironment in pancreatic cancer by promoting the infiltration, growth, and polarization of macrophages
Authors: Deshmukh Sachin Kumar , Bhardwaj Arun , Tyagi Nikhil , Khan Mohammad Aslam , Srivastava Sanjeev K. , AL-Ghadhban Ahmed , Dugger Kari , Carter James E. , Singh Ajay P. , Singh Seema
Published: August 2018

Resistance to chemotherapeutic drugs remains a major cause of therapeutic failure in pancreatic cancer (PC) patients. Efficacy of chemotherapy is further limited by its debilitating effects on immune system. Apart from genetic abnormalities in tumor cells, the tumor immune- microenvironment plays a crucial role in PC chemoresistance. However, it is unclear how chemotherapy affects the cancer immunity or how it affects the tumor microenvironment (TME). In the present study, we demonstrate gemcitabine induces an increased infiltration of CD68+ M2 macrophages in pancreatic tumors isolated from an orthotopic xenograft murine model. Moreover, the conditioned media from PC cell lines cultured with gemcitabine (Gem-CM) promotes migration, invasion, and growth of RAW264.7 macrophage. Interestingly, Gem-CM induces an upregulation of tumor-associated or M2-polarized macrophage markers, arginase-1 and TGF-β1. Additionally, gemcitabine treatment of PC cell lines induces the expression of M2-polarization associated cytokines, growth factors and chemokines, including IL-8. IL-8 exhibits the greatest upregulation after culture with Gemcitabine. Further, IL-8 neutralization in Gem-CM diminished its ability to induce growth, migration, and invasion of RAW264.7 macrophages. Together, these findings suggest an indirect effect of gemcitabine on increasing macrophage trafficking and M2-polarization that potentially supports a pro-tumor microenvironment. These actions may contribute to the chemoresistance of PC. Prevention of gemcitabine-induced macrophage infiltration, growth and M2-polarization will offer a better strategy to counter chemotherapy-induced immunosuppression in PC.

Citation Format: Sachin Kumar Deshmukh, Arun Bhardwaj, Nikhil Tyagi, Mohammad Aslam Khan, Sanjeev K. Srivastava, Ahmed AL-Ghadhban, Kari Dugger, James E. Carter, Ajay P. Singh, Seema Singh. Gemcitabine treatment induces immunosuppressive microenvironment in pancreatic cancer by promoting the infiltration, growth, and polarization of macrophages [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 113.
Abstract 1132: PAX6 drives cancer cells toward a stem like state via GLI-SOX2 signaling axis in lung adenocarcinoma
Authors: Oki Akira , Hoque Mohammad O.
Published: August 2018

PAX6 is an essential transcription factor for embryonic stem cell maintenance, but its contribution to lung cancer stem cells (CSCs) remains unexplored. We demonstrate that PAX6 acts as an oncogene responsible for induction of cancer stemness properties and plays a biological role in tumor initiation and CSC expansion in lung adenocarcinoma (LUAD). Mechanistically, PAX6 activates Hedgehog-GLI signaling in a SMO-independent noncanonical manner, resulting in SOX2 upregulation directly by the binding of GLI to the proximal promoter region of the SOX2 gene. The overexpressed SOX2 enhances the expression of key pluripotent factors (OCT4 and NANOG) and suppresses differentiation lineage factors (HOPX and NKX2-1). Thus, PAX6 drives cancer cells toward a stem-like state via the GLI-SOX2 signaling axis in LUAD. In contrast, in the differentiated non-CSCs that represent most of the tumor cell population, PAX6 is transcriptionally silenced by its promoter methylation. In human lung cancer tissues, the positive linear correlations of PAX6 expression with GLI and SOX2 expression and its negative correlations with HOPX and NKX2-1 expression were observed. Therapeutically, the blockade of the PAX6-GLI-SOX2 signaling axis elicits a long-lasting therapeutic efficacy by limiting CSC expansion following chemotherapy. Our findings provide a rationale for targeting the PAX6-GLI-SOX2 signaling axis with chemotherapy as an effective therapeutic strategy.

Citation Format: Akira Oki, Mohammad O. Hoque. PAX6 drives cancer cells toward a stem like state via GLI-SOX2 signaling axis in lung adenocarcinoma [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 1132.
Abstract 1135: SOX9 regulates cancer stem-like cells and chemotherapy response in non-small cell lung cancer
Authors: Voronkova Maria , Luanpitpong Sudjit , Rojanasakul Yon
Published: August 2018

Lung cancer is the leading cause of cancer-related death worldwide in both men and women. A major limitation of the lung cancer chemotherapy effectiveness is drug resistance and tumor relapse, which both have been attributed to the acquisition of cancer stem-like cells (CSCs). Thus, it is crucial to identify specific markers and drug targets for this cell population. Our work is focused on the embryonic transcription factor SOX9, which has been implicated in CSC regulation in a number of cancer types, but little is known about its role in non-small cell lung cancer (NSCLC), the predominant type of lung cancer. In addition, the regulatory mechanisms and downstream targets of SOX9 as well as its role in chemoresistance are largely unknown. We hypothesized that SOX9 plays a key role in lung cancer progression and chemoresistance by stimulating cancer stem-like properties. To test this hypothesis, we genetically manipulated SOX9 expression in various NSCLC cell lines and evaluated its effects on CSC formation, biomarkers expression, and drug resistance. Our results showed that SOX9 depletion decreases the number of tumor spheres formed by NSCLC cells, thus supporting its role in CSC regulation in lung cancer. We also showed that SOX9 depletion dramatically downregulates the expression of a functional CSC regulator ALDH1A1. Consistent with this finding, SOX9 overexpression in non-cancer lung epithelial cells promotes tumor sphere formation. Moreover, we noticed that SOX9 expression goes up in response to chemotherapy exposure. Notably, SOX9 knockdown increases cell sensitivity to the chemotherapeutic cisplatin, whereas SOX9 overexpression decreases it. Depletion of SOX9 also increases CSC sensitivity to cisplatin, as demonstrated by the tumor sphere formation assay. Analysis of the Cancer Genome Atlas (TCGA) data showed that high SOX9 expression correlates with poor survival in NSCLC patients. Taken together, our results indicate the essential role of SOX9 in the regulation of CSCs in NSCLC. Since CSCs are a key driver of chemoresistance and relapse, our findings may have important implications in the development of novel therapeutic strategies for chemoresistant and recurrent lung cancer.

Citation Format: Maria Voronkova, Sudjit Luanpitpong, Yon Rojanasakul. SOX9 regulates cancer stem-like cells and chemotherapy response in non-small cell lung 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 1135.
Abstract 1137: ANO1 confers the maintenance of stemness and radioresistance in glioma stem cells by stabilizing EGFRvIII
Authors: Lee Dae-Hee , Kim Hee-Jin , Kim Jeong-Yub , Park Jea-Yong , Park Myung-Jin
Published: August 2018

Anoctamin-1 (ANO1), also known as TMEM16A, is a Ca2+-activated chloride channel known to mediate a number of physiologic functions such as mediating acute and chronic pain and regulating blood vessels and airway smooth muscle. In particular, ANO1 expression is amplified in a variety of cancers, and overexpression of ANO1 is not favorable for clinical prognosis. Previous studies have reported that ANO1 is associated with tumorigenesis, proliferation, migration, and invasion of cancer cells, but the mechanism is not clear yet. In this study, we examined role of ANO1 in the maintenance of stemness and radio-resistance in glioma stem cells (GSCs). Knockdown of ANO1 with shRNA and treatment of ANO1 specific inhibitors (CaCCAo1 and TAO1) suppressed the expression of Notch intracellular domain, Nestin and EGFRvIII and decreased self-renewal activity and invasion in GSCs. Suppression of ANO1 increased radiation sensitivity by inducing ROS-mediated GSCs death. However, inhibition of chloride channel activity, the main function of ANO1, was found to be unaffected in this event. Interestingly, we found that ANO1 retained stemness by direct binding to EGFRvIII, which is specifically expressed in GSCs, and that the chaperone property of ANO1 function may affect the stability of EGFRvIII. In intracranial mouse model of GSCs, knockdown of ANO1 increased the survival rate of mice by suppressing tumor formation. Taken together, we suggest that ANO1 is a crucial molecule involved in the maintenance of stemness and radiation resistance in GSCs and may be a good therapeutic target for treating glioblastoma.

Citation Format: Dae-Hee Lee, Hee-Jin Kim, Jeong-Yub Kim, Jea-Yong Park, Myung-Jin Park. ANO1 confers the maintenance of stemness and radioresistance in glioma stem cells by stabilizing EGFRvIII [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 1137.
Abstract 1141: A novel regulatory gene subnetwork of glioma stem-like cells
Authors: Deng Changwang , Le Son B. , Tran David D.
Published: August 2018

Glioblastoma (GBM) is the most common and deadliest malignant brain tumor in adults. GBM is enriched in glioma stem-like cells (GSCs) that contribute to tumor initiation and treatment resistance and thus a natural focus for therapeutic development. However, targeting GSCs has been a challenge because of a lack of master regulators specific to GSCs and not to normal brain cells. Therefore, systematic identification of the GSC-specific common regulatory program will improve our understanding of GSCs and provide an opportunity to develop GSC-specific therapeutics that are efficacious and safe. To accomplish these objectives, we have recently developed two innovative tandem computational platforms, GeneRep and nSCORE, which were subsequently experimentally validated. The tandem platforms complement each other and allow us to 1) interrogate large-scale gene expression profiles of GSCs, GBM-differentiated cells, normal neuronal precursor cells (NPCs) and astrocytes (NAs) to extract candidate GSC-specific gene regulatory networks; and 2) functionally rank GSC-specific core master regulators, respectively. A cluster of 8 interrelated master subnetworks emerged, which were functionally grouped into the stemness and cancer pathways, some of which belonged to the Sox, Olig and Myc families. By enforcing the expression of this cluster in NAs, we were able to reprogram these normal cells into GSCs as measured by the neurosphere formation and in vivo tumorigenesis assays, confirming the critical requirement of this cluster for the GSC identity. Furthermore, this network cluster was present and functioned cooperatively to maintain GSCs in several patient-derived GSC lines examined to date. Survival and neurosphere formation rates of GSCs were profoundly suppressed when at least two nodes in this cluster were depleted by siRNA compared to depletion of the individual nodes. Our data establish a novel gene regulatory cluster controlling GSCs and provide a compelling rationale for its therapeutic targeting to reduce tumor recurrence.

Citation Format: Changwang Deng, Son B. Le, David D. Tran. A novel regulatory gene subnetwork of glioma stem-like cells [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 1141.
Abstract 1142: STK31 contributes to lung carcinogenesis through induction of stemness and resistance to EGFR-TKI therapy
Authors: Lau Eunice Yuen-Ting , Chin Isabella Kit-Nam , Fong Alvin Hong-Wai , Ma Victor Wan-San , Cho William Chi-Shing
Published: August 2018

Lung adenocarcinoma (LUAD), the most common histological subtype of lung cancer, remains one of the leading causes of cancer-related deaths worldwide. With improving knowledge in the molecular aberrations underlying lung carcinogenesis, a large number of targeted therapies are being developed to better patient's survival. EGFR-activating mutations are one of the most frequent genetic alterations in advanced lung adenocarcinoma, and patients harbouring certain types of EGFR activating mutation confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Despite promising initial responses, majority of patients develop resistance to treatment and the mechanism of resistance remains unknown in up to 30% of patients. Recent studies revealed that cancer stem cells (CSCs) endowed with stem cell-like properties confer resistance to EGFR-TKI treatment. Identification of pathways maintaining stemness properties potentially provide novel drug targets to overcome treatment resistance. Protein phosphorylation is the most common form of reversible post-translational modification and is frequently involved in the deregulation of signal transductions associated with cancer development and progression. In this study, we have identified Serine/Threonine Kinase 31 (STK31) to be upregulated in enriched lung CSC populations, as well as in the erlotinib resistant derivatives from two LUAD cell lines. Through analysing The Cancer Genome Atlas (TCGA) data, mRNA expression of STK31 is observed to be higher in LUAD tumor when compared with paired normal tissues (p<0.001). High mRNA expression of STK31 was also found to be associated with poorer disease-free survival (p=0.0324) and overall survival (p=0.0012) in LUAD patients. Functional studies using shRNA based knockdown of STK31 in lung cancer cell lines revealed its regulatory role on cancer and stem cell-like properties in vitro and in vivo including tumorigenicity, self-renewal, drug resistance and metastasis. Repressing the expression of STK31 in erlotinib resistant cells re-sensitized the cells to drug treatment. Further studies will be done to delineate the downstream mechanism by which STK31 regulates lung CSCs and erlotinib resistance.

Citation Format: Eunice Yuen-Ting Lau, Isabella Kit-Nam Chin, Alvin Hong-Wai Fong, Victor Wan-San Ma, William Chi-Shing Cho. STK31 contributes to lung carcinogenesis through induction of stemness and resistance to EGFR-TKI therapy [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 1142.
Abstract 1151: A unique layered 3D stromal tissue model for in vitro evaluation of immunologic agents and immune cell infiltration into cancer microenvironment
Authors: Morimura Rii , Tsukamoto Kei , Kitano Shiro , Shinozaki Eiji
Published: August 2018

Background; The modulation of the immune system is a proven key target for cancer therapy, however, the complex interplay between immune cells and cancer cells in cancer microenvironment remains only partially understood. In addition, the currently available in vitro cancer microenvironment models are not designed to study the immunologic agents and a role of immune cells. Here, we developed a unique layered 3D stromal tissue model comprising an allogeneic immune, stromal and cancer cells.

Method; Human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts (NHDF) were coated with collagen and heparin to construct Extracellular matrix (ECM) nanofilm and cultured to form multi-layers. Lung cancer cells were placed between stromal cells. Furthermore, we added Peripheral Blood Mononuclear Cell (PBMC) and Programmed cell death 1 (PD-1) blockade with Nivolumab and evaluated cytotoxic effects of immune cells on cancer cells and immune cell infiltration by immunohistochemistry.

Result; The co-cultured stromal tissues were multi-layered (approximately 20 layers), and the presence of lung cancer cells between stromal cells was observed. Immunohistochemistry revealed CD8 positive immune cell infiltration into the fibroblast layers. PBMC with Nivolumab treatment significantly decreased the viability of lung cancer cells in this 3D co-culture compared with the results in 2D mono-culture. High level of programmed cell death ligand 1 (PD-L1) expression on lung cancer cell lines correlated with a higher response to PD-1 blockade in vitro as well as clinical trials.

Conclusion; In summary, we present a unique 3D cell culture system based on an allogeneic stromal/cancer/immune cell setting to study immunologic agents and immune cell infiltration in cancer microenvironment. This 3D model has the ability to reflect in vivo conditions closer than 2D model and provide a tool for in vitro immunologic agent testing.

Citation Format: Rii Morimura, Kei Tsukamoto, Shiro Kitano, Eiji Shinozaki. A unique layered 3D stromal tissue model for in vitro evaluation of immunologic agents and immune cell infiltration into cancer microenvironment [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 1151.
Abstract 1152: Therapeutic response to PD1/PDL1 axis in a disseminated C1498 syngeneic tumor model
Authors: Mahajan Vivek , Bui Thi , Belle Ian , Wood Edgar , Usher Lindsay , Rainbolt Elizabeth , Kidder Shasta , Synnott Aidan , Harris David
Published: August 2018

The majority of the leukemia cases are diagnosed in adults, of which, acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) are the most common subtypes. Treatment options include chemotherapy, stem cell transplantation and more recently the use of checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4 to enhance antitumor responses. In this study we developed a disseminated AML disease model using PD-L1+, C1498 tumor cells to evaluate the therapeutic response to immune checkpoint inhibitors, PD-1, PD-L1 and CTLA-4 and to examine the effect of treatment on infiltration of immune cell populations in selected tissues. C1498 cells were transduced with a lentivirus vector construct containing luciferase and GFP reporter genes. Upon establishment of a clonal population with stable reporter expression, C1498-GFP-Luc expressing cells were implanted intravenously into syngeneic albino C57BL/6 mice and growth of the leukemic tumor cells was monitored via in vivo bioluminescence imaging (BLI) using IVIS Spectrum CT®. In vehicle-treated animals tumor growth was progressive and most animals reached endpoint within 30 days. Luciferase expression was widespread with metastatic development in peripheral organs and tissues. An efficacious response to therapy directed against the PD-1/PD-L1 axis was observed, with anti-PD-L1 showing greater overall efficacy than anti-PD-1. Combination of either PD-1/PD-L1 with anti-CTLA-4 further improved the therapeutic efficacy as measured by prolonged survival and reduced tumor growth. To gain mechanistic insights into how these immunotherapies function in suppressing tumor growth we performed additional studies utilizing flow cytometry and immunohistochemistry to assess the composition and phenotype of immune cell infiltrates in hematological and non-hematological tissues. In summary, comprehensive immunological characterization of the C1498 model revealed the importance of PD1 / PD-L1 pathway in immune evasion and provides a rationale for therapeutic intervention with checkpoint blockade.

Citation Format: Vivek Mahajan, Thi Bui, Ian Belle, Edgar Wood, Lindsay Usher, Elizabeth Rainbolt, Shasta Kidder, Aidan Synnott, David Harris. Therapeutic response to PD1/PDL1 axis in a disseminated C1498 syngeneic tumor model [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 1152.
Abstract 1157: The B-NDG mouse is a perfect tool for immune system humanization and patient derived xenograft transplantation
Authors: Zhang Meiling , Guo Chaoshe , Shen Yuelei , Guo Yanan
Published: August 2018

The B-NDG mice (NOD-Prkdcscid Il2rgtm1/Bcgen), independently developed by Biocytogen, display a severe immunodeficiency phenotype, with no mature T, B or functional NK cells, and deficient in cytokine signaling. The B-NDG mouse is one of the best models for reconstitution of human immune system using peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (CD34+). Human PBMC reconstituted well in B-NDG mice, with the percentage of hCD45+ cells was over 50% in five weeks. To further analyze the subsets of immune cell composition, we found the majority of human cells were CD8+ T lymphocytes. Other than PBMC, human CD34+ hematopoietic stem cells also demonstrated a high-engraftment efficacy in B-NDG mice. Successful engraftment of CD34+ cells develops all major cell types via myeloid and lymphoid lineage to reconstitute functional human immune system. At 10 weeks post CD34+ cell implantation, the percentage of hCD45+ cells is over 30% with detectable human T, B and NK cells. Therefore, the immuno-humanized B-NDG mice are useful tools for studying hematopoiesis, immunology, infectious disease, and ES/iPS cell research. With barely any rejection to human derived cells, the B-NDG mouse is an ideal host for transplantation of PDX (Patient-Derived Xenograft), and retains the high heterogeneity characteristics of tumor tissues. Each PDX model can be considered as a single tumor patient and fully embodies the patient group characteristics. We have a collection of various PDX models including leukemia, breast cancer, gastric, lung, pancreatic, and colon et al. The humanized PDX model by implantation of PDX on the immuno-humanized B-NDG mice will be a promising tool to evaluate the in vivo efficacy of immune checkpoint antibodies in monotherapy or in combination. Therefore, therapeutic efficacy studies using B-NDG based PDX models are more meaningful and predictive in preclinical drug development and discovery.

Citation Format: Meiling Zhang, Chaoshe Guo, Yuelei Shen, Yanan Guo. The B-NDG mouse is a perfect tool for immune system humanization and patient derived xenograft transplantation [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 1157.
Abstract 1164: Pdx model of pleural effusion of lung cancer patient for testing on targeted drug sensitivity and resistance
Authors: Zhang Feifei , Zhou Zhen , Long Yuan , Xu Wenhua , Zhao Shizhu , Yang Yang , Liu Hui , Cheng Jijun , Lu Shun , Wen Danyi
Published: August 2018

Advanced lung cancer of late stage patients is selectively sensitive to available targeted therapeutics and often develops resistance to early sensitive drug treatment. Surgery tumor tissues are usually not available to establish patient-derived xenograft (PDX) models which is critical to evaluate drug sensitivity and resistance. We applied CD45 magnet beads to enrich the tumor cell population from pleural effusion specimens of advanced lung cancer. Valuable drug-resistant PDX models were successfully established both from pleural effusion and from routine surgery tissues. Genetic profiling of the PDX models revealed TKI drug-associated EGFR mutations (L858R, T790M, Exon19del, C797S). In particular we successfully established a clinically AZD9291-resistant EGFR triple mutant (Ex19del, T790M, C797S) PDX model. In vivo efficacy results with gefitinib, erlotinib and AZD9291 well matched corresponding gene mutations among the models. Interestingly, we successfully induced AZD9291-resistant clones in vivo using an EGFR double mutant (L858R, T790M) PDX model, and NGS revealed mutations in kinases involved in other signaling pathways, indicating potential therapeutic options against resistance emergence. In conclusion, these precious PDX models provide insight into resistance mechanisms as well as serve as models to test potency of many drugs including current investigational TKIs.

Citation Format: Feifei Zhang, Zhen Zhou, Yuan Long, Wenhua Xu, Shizhu Zhao, Yang Yang, Hui Liu, Jijun Cheng, Shun Lu, Danyi Wen. Pdx model of pleural effusion of lung cancer patient for testing on targeted drug sensitivity and resistance [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 1164.
Abstract 1171: Quantitative evaluation of hepatobiliary transporter density with microscopic fluorescence imaging using indocyanine green
Authors: Meng Hui , Gao Yuan , Yin Lin , Ma Xibo , Wang Kun , Tian Jie
Published: August 2018

The sodium-dependent taurocholate cotransporter polypeptide (NTCP) has been identified as a predictive marker for post-surgery survival rate of hepatocellular carcinoma (HCC) patients. Previous studies indicated that the expression of NTCP was associated with tumor differentiation. The ability to quantify protein expression is imperative to ensure successful personalized medicine including treatment stratification and therapeutic monitoring. Current standards for transporter analysis are based on immunohistochemistry and immunofluorescence. These measurements are complicated and too time consuming. Here, we endeavored to identify the expression of NTCP by quantitative microscopic fluorescence imaging (MFI) features, that could be used for investigation of tumor heterogeneity. Quantitative image features including the intensity, texture and wavelet texture were extracted from microscopic fluorescence images. Using multiple xenograft tumor models with varying NTCP expression, we determined the NTCP density in each model with MFI using indocyanine green. The MFI-calculated transporter density was correlated with ex vivo immunohistochemistry. Overall, our results argue that MFI provides a promising measure of transporter expression equivalent to immune-staining, with implications for use in investigation of tumor heterogeneity.

Citation Format: Hui Meng, Yuan Gao, Lin Yin, Xibo Ma, Kun Wang, Jie Tian. Quantitative evaluation of hepatobiliary transporter density with microscopic fluorescence imaging using indocyanine green [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 1171.
Abstract 1173: The identification of circulating tumor cells in the cerebrospinal fluid of gastric cancer patients with brain metastasis and the evaluation of intra-patient tumor heterogeneity
Authors: Cho Jang Ho , Kim Sun Young , Kim Kyung , Kim Jusun , Lee Su Jin , Lee Jeeyun , Park Se Hoon , Park Joon Oh , Park Young Suk , Lim Ho Yeong , Kang Won Ki , Kim Seungtae
Published: August 2018

PURPOSE : The purpose of this study was to identify circulating tumor cells (CTC) and evaluate tumor heterogeneity in cerebrospinal fluid (CSF) of advanced gastric cancer (GC) patients with leptomeningeal seeding. METHOD : We used the DEPArray system to identify and align single enabled CTCs in CSF based on multi-parallel fluorescence analysis. CSF were stained with an antibody cocktail recognizing cytokeratin, EpCAM, and HER2. CTC subpopulations of CSF were quantified as both absolute cell count and relative frequency. Based on the stained morphology of each single CSFTCs, five groups (Group A~E) were categorized.

RESULTS : CSF samples were obtained from a HER2 positive GC patient with leptomeningeal seeding. Each single CTC was collected according to the characteristics of morphology and the difference of marker expression. Group A CTC revealed HER2 positive, CK positive and EpCAM positive cells. The ratio of group A cells to total cells was 56.7%. Although the tumor of patient was HER2 positive cancer, HER2 negative CTCs in CSF (Group D + E) accounted for 21.8% of the total cells. Among CTCs we detected in CSF, there was no CTC revealing HER2 positive, CK negative and EpCAM negative.

CONCLUSIONS : DEPArray analysis might be a novel tool to capture CTC in CSF samples and evaluate the intra-patient's tumor heterogeneity. These findings suggested that DEPArray analysis could be used as a useful tool for the precision medicine.

Characterization of HER2 + CSFTCs on gastric cancer patientHER2CytokeratinEpCamCell#%/total cellGroupPositivePositivePositive13556.7APositivePositiveNegative20.8BPositiveNegativePositive4920.6CPositiveNegativeNegative00.0NegativePositivePositive52.1DNegativePositiveNegative00.0NegativeNegativePositive4719.7E

Citation Format: Jang Ho Cho, Sun Young Kim, Kyung Kim, Jusun Kim, Su Jin Lee, Jeeyun Lee, Se Hoon Park, Joon Oh Park, Young Suk Park, Ho Yeong Lim, Won Ki Kang, Seungtae Kim. The identification of circulating tumor cells in the cerebrospinal fluid of gastric cancer patients with brain metastasis and the evaluation of intra-patient tumor heterogeneity [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 1173.