bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3845: Establishment and characterization of patient-derived xenografts of clear cell carcinoma of the ovary
Authors: Watari Hidemichi , Endo Daisuke , Honda Kouhei , Kunitomo Mihoko , Nomura Toshiyuki , Sakuragi Noriaki
Published: July 2017

Background; The patient-derived xenograft (PDX) model is likely to reflect original human cancer biology compared to cultured cell lines, since tumor tissues are directly implanted into animals. PDX models of ovarian cancer have been developed, but were not fully characterized at the molecular level, especially for clear cell carcinoma (CCC), which is more prevalent in Japan than western countries, exhibits chemoresistant phenotype and poor survival. The development of new treatment strategies and the better understanding of biology of CCC both depend on clinically relevant animal models. In particular, molecular annotated PDX models are useful for the preclinical investigation of anticancer drugs and individualized anticancer therapy. Methods; We transplanted 19 parent (primary or metastatic) tumors directly into NOG mice. The histologic characteristics were compared between parental tumors and PDX ones. Among CCC pairs, gene expression analysis, gene mutation analysis were conducted on parental tumors and corresponding PDX tumors. Response to chemotherapeutic agents was analyzed using PDX tumors of CCC, and correlated with clinical outcome. Results; Six of nineteen (31.6%) transplanted tumors could be passaged. Gene expression profiles revealed that engrafted tumors expressed more proliferation-related genes than those of non-englafted ones. Among six PDX models established, two were found to be CCC. Two PDX tumors of CCC maintained the histologic characteristics of parental CCC tumors. Gene expression profile revealed similar results between primary CCC and PDX tumors of CCC except that PDXs lost expression of immune-related genes.. Gene mutational analysis showed similar pattern between primary CCC and PDX of CCC. Drug-sensitivity test showed resistance to carboplatin in two PDX tumors of CCC, which is consistent with their clinical outcomes. Conclusions. PDXs of CCC were established and found to retain histologic and clinical features of original tumors, and can be utilized to screen new target drugs for platinum-resistant CCC.

Citation Format: Hidemichi Watari, Daisuke Endo, Kouhei Honda, Mihoko Kunitomo, Toshiyuki Nomura, Noriaki Sakuragi. Establishment and characterization of patient-derived xenografts of clear cell carcinoma of the ovary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3845. doi:10.1158/1538-7445.AM2017-3845
Abstract 3851: Targeting HER2 in gastric cancer: Hints from a gastric PDX platform
Authors: Corso Simona , Migliore Cristina , Apicella Maria , Menegon Silvia , Pectasides Eirini , Capeloa Tania , Durando Stefania , Cargnelutti Marilisa , Cassoni Paola , Sapino Anna , Giuli Maurizio de , Bass Adam , Giordano Silvia
Published: July 2017

Introduction: Gastric cancer is the 3rd leading cause of cancer mortality worldwide. Surgery is the only curative treatment strategy and conventional chemotherapy has shown limited efficacy. Trastuzumab (a HER2 mAb), is the only therapy targeting molecular alterations approved so far in gastric cancer, for HER2+ patients with advanced disease. However only a fraction (<20%) of HER2 amplified patients benefits from treatment.

Methods: We have recently generated a molecularly annotated colony of gastro-esophageal PDXs (at the moment, > 70 PDXs). The platform also comprises primary cell lines and 3D-cultured organoids derived from gastric cancer PDXs.

Results and Discussion: At present, we have identified several HER2+ PDXs and generated the corresponding in vitro derivatives. Four HER2+ PDXs, showing 3+ HercepTest score and HER2 amplification > 8 copies (thus theoretically sensitive to Trastuzumab), were selected to undergo ‘xenotrials’ with the following anti-HER2 drugs/combos: Trastuzumab (T); Pertuzumab (P, anti-HER2 mAb); Lapatinib (L, a dual HER2-EGFR tyrosine kinase inhibitor). According to RECIST-like criteria, T induced tumor regression in only 1 out of 4 HER2+ PDXs. Interestingly, in all the tumors but one the combos T+P and T+L were able to bypass resistance to T monotherapy and to induce tumor regression. One tumor was resistant to all the tested therapeutic approaches.

Conclusion: We identified PDXs displaying HER2 gene amplification. Only a minor fraction of them benefited from the anti-HER2 mono-therapy, despite the presence of a strong HER2 gene amplification, while the association of two HER2 inhibiting drugs resulted in intense and prolonged response. This suggests that monotherapy with T might not be the most effective therapeutic approach in gastric cancer patients. We are currently investigating the cause of resistance to anti-HER2 drugs to identify mutations/CNV in tumors showing resistance to Trastuzumab.

Note: This abstract was not presented at the meeting.

Citation Format: Simona Corso, Cristina Migliore, Maria Apicella, Silvia Menegon, Eirini Pectasides, Tania Capeloa, Stefania Durando, Marilisa Cargnelutti, Paola Cassoni, Anna Sapino, Maurizio de Giuli, Adam Bass, Silvia Giordano. Targeting HER2 in gastric cancer: Hints from a gastric PDX platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3851. doi:10.1158/1538-7445.AM2017-3851
Abstract 3882: Identification of targetable EMT markers in cancer stem-like cells derived from therapeutic resistant melanoma
Authors: Crosson William P. , Berlinberg Elyse , Nazarian Ramin
Published: July 2017

Melanoma is the deadliest form of skin cancer, expected to claim approximately 10,000 lives in 2016. An activating mutation in BRAF(V600E) occurs in 50-60% of melanomas and 7% of all cancers. Despite an unprecedented initial response, patients treated with a highly effective BRAF inhibitor, vemurafenib (FDA approved 2011), acquire drug resistance within 6-9 months. Vemurafenib-resistant sub-lines, derived from BRAFV600E isogenic parental melanoma cells, have altered cell morphology and upregulated PDGFRβ expression. Epithelial-to-mesenchymal transition (EMT), a hallmark of cancer-stem-like cells (CSC), is a potential mechanism that melanomas employ to evade targeted therapy. Through immunocytochemical, immunoblot, and flow cytometric analysis, we investigated the expression profile of EMT biomarkers within parental and resistant melanoma cell lines. Our findings indicate CSCs within melanoma populations may serve as progenitor cells for therapy-resistant melanoma sublines through differential expression of key EMT-related biomarkers. Furthermore, resistant cell lines were significantly more invasive in migration assays. Our results implicate notable EMT biomarkers in CSCs that can be used as novel targets for diagnosis and treatment of tumor resistance in melanoma patients.

Citation Format: William P. Crosson, Elyse Berlinberg, Ramin Nazarian. Identification of targetable EMT markers in cancer stem-like cells derived from therapeutic resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3882. doi:10.1158/1538-7445.AM2017-3882
Abstract 3883: Transcriptome analysis reveals mitochondrial gene signature upregulation in gastric cancers expressing the stem cell marker CD133
Authors: Lee Han Hong , Ko Yoon Ho , Kim Tae-Min , Park Won-Sang
Published: July 2017

Gastric cancers expressing the stem cell marker CD133 are more aggressive than those that do not express the marker, and are associated with more unfavorable clinical outcomes. The CD133-associated aggressive behavior of gastric cancer remains poorly understood at the molecular level. In the present study, we performed gene expression profiling of bead-sorted CD133+ and CD133- KATO III cell lines to derive in vitro molecular signatures of CD133 expression in gastric cancer. Three expression signatures associated with CD133 expression ('Mitochondria', 'Cell cycle', and 'Lipid metabolism') were inferred upon study of a module association map of the key molecular signatures associated with CD133 expression in gastric cancer. Using the public gene expression profiles, we show that the patients with overexpression of CD133 show unfavorable clinical outcomes compared to CD133- patients in the context of up-regulation of 'Mitochondria' expression signature. We further showed that CD133+ KATO III cells had a higher mitochondrial function and were more susceptible to the mitochondrial inhibitors rotenone, antimycin A, and NaN3, than were CD133- cells. Together, our findings suggest that transcriptional up-regulation of the 'Mitochondria' gene signature may identify CD133+ gastric cancers; such data are of clinical and pharmacological relevance.

Note: This abstract was not presented at the meeting.

Citation Format: Han Hong Lee, Yoon Ho Ko, Tae-Min Kim, Won-Sang Park. Transcriptome analysis reveals mitochondrial gene signature upregulation in gastric cancers expressing the stem cell marker CD133 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2017-3883
Abstract 3887: GNA13 is a theranostic target that drives drug resistance and cancer stem-like phenotypes in solid tumors
Authors: Rasheed Suhail Ahmed Kabeer , Leong Hui Sun , Lakshmanan Manikandan , Raju Anandhkumar , Dadlani Dhivya , Chong Fui-Teen , Rajarethinam Ravisankar , Skanthakumar Thakshayeni , Tan Ern Yu , Hwang Jacqueline Siok Gek , Lim Kok Hing , Tan Daniel Shao-Weng , Ceppi Paolo , Wang Mei , Tergaonkar Vinay , Casey Patrick J. , Iyer N. Gopalakrishna
Published: July 2017

Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess stem cell-like (CSC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of aggressive phenotypes. Many of the implicated GPCRs signal through the G12 subfamily, comprised of GNA12 and GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in these patients. Consistent with this, we show that GNA13 expression modulates drug resistance through its effect on the CSC sub-population in a panel of patient-derived head and neck (HNSCC) and breast cancer cells. These data were validated in vivo, where GNA13 over-expression in patient-derived xenografts increased tumor initiating capacity, tumorigenicity and drug resistance, with no effect on growth or proliferation. Signaling through NFKB and MAPK pathways appear to be critical to the observed phenotype. Importantly, blockade of GNA13 expression, or select downstream pathways using small-molecule inhibitors, abrogates GNA13-induced CSCs, rendering cells vulnerable to standard-of-care cytotoxic therapy for these cancers. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker, and interfering with GNA13-induced signaling provides a novel strategy to block CSCs and drug resistance in solid tumors.

Citation Format: Suhail Ahmed Kabeer Rasheed, Hui Sun Leong, Manikandan Lakshmanan, Anandhkumar Raju, Dhivya Dadlani, Fui-Teen Chong, Ravisankar Rajarethinam, Thakshayeni Skanthakumar, Ern Yu Tan, Jacqueline Siok Gek Hwang, Kok Hing Lim, Daniel Shao-Weng Tan, Paolo Ceppi, Mei Wang, Vinay Tergaonkar, Patrick J. Casey, N. Gopalakrishna Iyer. GNA13 is a theranostic target that drives drug resistance and cancer stem-like phenotypes in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3887. doi:10.1158/1538-7445.AM2017-3887
Abstract 3898: Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers
Authors: Tyagi Abhishek , Gupta Shilpi , Kuamr Prabhat , Bharti Alok C. , Das Bhudev C.
Published: July 2017

Background: Activator protein-1 (AP-1) plays a central role in HPV-mediated cervical and tongue cancer (subset of HNSCC). AP-1, a homo/heterodimeric protein composed of Jun and Fos family proteins, has been implicated in chemoradioresistance but the mechanism(s) remained unexplored. In the present study, AP-1 family protein Fra-1 has been shown to play a critical role in promoting radiosensitization in enriched cervical cancer stem-like cells (CaCxSLCs) and can serve as a target for drug intervention in cervical and other HPV associated cancers.

Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating HPV+ve and HPV-ve human cervical cancer cell lines, SiHa, and C33a using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness.

Both CaCxSLCs and Non CaCxSLCs were exposed to different dose of UV irradiation and a herbal compound curcumin, a potent inhibitor of AP-1 was used to examine the role of AP-1 and its family proteins in the radiosensitization of cancer stem cells.

Results: Upon UV irradiation, CaCxSLCs showed a dose-dependent higher proliferation and highly increased AP-1 activity whereas it was completely abolished in non-stem cancer cells. CaCxSLCs also showed differential overexpression of c-Fos and c-Jun at transcript as well as in protein level. The loss of AP-1 activity and expression was accompanied by decrease in cell viability and proliferation in UV irradiated non-stem cancer cells. Interestingly, CaCxSLCs treated with curcumin prior to UV irradiation abolished AP-1 activity and a concomitant reduction in SP cells leading to abrogation of sphere forming ability, loss of proliferation, induction of apoptosis and the cells were poorly tumorigenic. The curcumin pre-treatment abolished the expression of c-Fos and c-Jun but upregulated Fra-1 expression in irradiated CaCxSLCs.

Conclusion: The study suggests a critical role of AP-1 protein in the manifestation of radioresistance but curcumin pretreatment helps in radiosensitizing CaCxSLCs through upregulation of Fra-1 which may serve as a potential target for elimination of radioresistant cancer stem cells in HPV-associated malignancies.

Note: This abstract was not presented at the meeting.

Citation Format: Abhishek Tyagi, Shilpi Gupta, Prabhat Kuamr, Alok C. Bharti, Bhudev C. Das. Emerging role of Fra-1 as a novel target for radiosensitization in HPV-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2017-3898
Abstract 3905: Development of anticancer aptamers targeting cancer stem cells
Authors: Chen Yuh-Ling , Hsieh I-Shan , Hsu Keng-Fu , Hong Tse-Ming
Published: July 2017

Background: Cancer stem cells (CSC), also called tumor initiating cells (TIC), are a small population of cancer cells within a tumor that drive the tumor initiation, metastasis, and resistance to chemotherapeutic drugs. An effective cancer stem cell targeting strategy by oligonucleotide-based approaches such as aptamers in diagnosis and treatment are urgently needed. In this study, we aimed to develop an effective aptamer-based biomarker for CSCs in cancer detection and therapy.

Material and methods: E-cadherin-null lung cancer cells, shEcad-A549, was confirmed with stemness properties and used as targeting cells. The isolation of DNA aptamers binding to CSCs was by using a Cell-SELEX (systematic evolution of ligands by exponential enrichment). By combining the aptamer-pull-down approach with proteomics, we identified the potential binding proteins of CSC-aptamer. Flow cytometry, gene silencing and lung cancer xenograft models were used to explore the functions of CSC-aptamer and its targeted protein in lung cancer formation.

Results: The aptamer AP-9R was identified to bind lung CSCs with high affinity and specificity. AP-9R reduced lung cancer cell stemness and migration but no apparent cytotoxicity. By using proteomic approaches, the target protein of AP-9R in non-small cell lung cancer was further identified and characterized its important role in cancer stemness. For CSCs detection, we developed AP-9R conjugated beads and confirmed their effective captured rate of lung CSCs in vitro. Moreover, administration of AP-9R significantly reduced lung tumor growth in the xenograft model.

Conclusions: Our data are moving closer to understand the significance of cancer stem cells during cancer progression and demonstrate that aptamer-based therapies will be useful as therapeutic and diagnostic agents for lung cancer.

Citation Format: Yuh-Ling Chen, I-Shan Hsieh, Keng-Fu Hsu, Tse-Ming Hong. Development of anticancer aptamers targeting cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3905. doi:10.1158/1538-7445.AM2017-3905
Abstract 3906: Targeting a novel G-protein coupled receptor (GPCR) for elimination of leukemia stem cells (LSC)
Authors: Voli Florida , Yi Hangyu , Gonzales-Aloy Estrella , Wang Jenny Yingzi
Published: July 2017

Acute myeloid leukemia (AML) is a lethal blood cancer. Clinical evidence has highlighted the critical role for leukemic stem cells (LSCs) in the high relapse rate of AML patients. Understanding the signaling pathways critical for LSC function will facilitate the development of new therapies to target LSCs. G protein-coupled receptors (GPCRs) are the most successful drug targets with approximately 36% of currently marketed drugs targeting human GPCRs (Rask-Andersen et al., Nat Rev Drug Discov 2011). Aberrant expression of GPCRs has been observed in various cancers and the importance of GPCRs in cancer stem cells has begun to be appreciated.

In this study, our gene expression profiling analysis identified a novel GPCR (hereafter named NG), which was suppressed in LSCs compared to normal hematopoietic stem cells. Overexpression of NG in pre-LSCs severely impaired leukemia initiation and progression in mice, whereas knockdown of NG significantly accelerated the disease onset. Our data also showed that NG overexpression substantially downregulated several well-known Wnt/β-catenin targets (e.g., Tcf7l2, c-Fos and Ccnd1). These data support a tumor suppressive role for NG in inhibiting leukemogenesis via downregulation of Wnt/β-catenin signaling.

Consistent with these observations, treatment with an agonist that specifically activates the NG signaling pathway induced a marked decrease in cell viability and growth in murine LSCs and in human AML THP-1 cells, but had no effects on human AML MOLM-13 cells. This agrees with our finding that NG specifically targets the Wnt/β-catenin signaling, and the inhibitory effect of the agonist might largely depend on β-catenin expression levels in human AML cells, wherein THP-1 has been reported to express endogenous β-catenin but MOLM-13 exhibits no detectable β-catenin expression (Man CH. et al. Blood 2015).

In summary, our data support a tumour suppressor role for NG in leukemogenesis and the forced expression of NG may provide a means to eradicate LSCs. Thus, restoring NG by the agonist treatment represents a promising therapeutic strategy for AML treatment.

Citation Format: Florida Voli, Hangyu Yi, Estrella Gonzales-Aloy, Jenny Yingzi Wang. Targeting a novel G-protein coupled receptor (GPCR) for elimination of leukemia stem cells (LSC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3906. doi:10.1158/1538-7445.AM2017-3906
Abstract 3907: Targeting cancer stem cells with microRNA therapeutics and particle radiation in challenging cancer models
Authors: Vares Guillaume , Saintigny Yannick , Sai Sei , Sugawara Hirotaka , Nakajima Tetsuo
Published: July 2017

Remarkable progress has been made in the understanding and treatment of human cancer, resulting in greatly improved survival for many patients. However, such achievements remain incomplete or out of reach for some hard-to-treat cancers, such as pancreatic cancer or triple-negative breast cancer. Chondrosarcoma is resistant to conventional radiation therapy and to chemotherapy. Innovative approaches focusing on those challenging cancer models are therefore highly expected. There is now wide acknowledgment that tumors are generally heterogeneous and that cancer treatment failure, relapse or metastasis may be related a small population of stem-like cells that are capable of self-renewing and of causing the different lineage of cancer cells comprising a tumor. These cancer stem cells (CSCs) are more radio-resistant than their non-CSC counterparts and have a distinct molecular signature. CSCs and differentiated cancer cells seem to coexist in a dynamic equilibrium influenced by the micro-environment and by specific micro-RNA expression patterns. The development of new therapeutic strategies (such as micro-RNA modulating strategies) that selectively target CSCs is currently receiving increasing attention. Particle radiation therapy (such as carbon-ion therapy) is effective at treating various radioresistant tumors and has already achieved promising results. Approaches combining high-LET radiation therapy and targeted-therapy have been suggested for tumors with bad prognosis.We have described a new molecular pathway in basal-like breast cells, resulting in the generation of breast CSCs. Maintenance of the CSC phenotype required the downregulation of miR-29 expression and the upregulation of Klf4 expression. Restoring miR-29 with mimic administration might therefore improve breast cancer therapy efficiency by targeting CSCs. For those reasons, we have evaluated the ability of several microRNA-modulating treatments to reverse CSC phenotype in three experimental in vitro models (pancreatic cancer, chondrosarcoma, triple-negative breast cancer), in combination with exposure spread-out Bragg peak (SOBP) carbon-ion beam (at the HIMAC medical accelerator). In triple-negative breast cells, miR-29 mimic administration increased radiation-induced cell death and decreased the proportion of CSCs. We identified several other microRNA mimic treatments capable of improving carbon-ion therapy efficiency by targeting radioresistant CSCs in those experimental models.

Citation Format: Guillaume Vares, Yannick Saintigny, Sei Sai, Hirotaka Sugawara, Tetsuo Nakajima. Targeting cancer stem cells with microRNA therapeutics and particle radiation in challenging cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3907. doi:10.1158/1538-7445.AM2017-3907
Abstract 3913: CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer
Authors: Wang Dingzhi , Sun Haiyan , DuBois Raymond N.
Published: July 2017

Emerging evidence suggests that the primary tumor induces the formation of pre-metastatic niches in certain distant organs. However, the mechanisms underlying the contributions of the primary tumor to pre-metastatic niche formation are not fully understood. Here we demonstrate that colorectal carcinoma cells secrete VEGF-A that stimulates tumor-associated macrophages to produce CXCL1 in the primary tumor. Elevation of CXCL1 in pre-metastatic liver tissue recruits CXCR2-positive myeloid-derived suppressor cells to form a pre-metastatic niche that ultimately promotes liver metastases. Importantly, we found that pre-metastatic liver-infiltrating MDSCs induce tumor cell survival without involvement of innate or adaptive immune responses. Our work not only reveals a novel mechanism by which the interactions of malignant cells with their stromal counterparts in the primary tumor contribute to the formation of a pre-metastatic niche that favors tumor cell survival, but also provides a rational for development of CXCR2 antagonists to inhibit or prevent metastatic spread of disease. Overall, our study provides the first evidence we know of showing that primary malignant cell-secreted VEGF-A stimulates tumor-associated macrophages to produce CXCL1 that recruits CXCR2-positive MDSCs to form a pre-metastatic niche which promotes liver metastases. Our findings not only shed light on how the tumor microenvironment contributes to pre-metastatic niche formation at distant sites, but also provide comprehensive insights into how MDSCs are recruited to other organs where they contribute to metastatic spread of disease.

Citation Format: Dingzhi Wang, Haiyan Sun, Raymond N. DuBois. CXCL1 is critical for pre-metastatic niche formation and metastasis in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3913. doi:10.1158/1538-7445.AM2017-3913
Abstract 3925: Integrative platform for the reconstruction and modeling of mechanically arrested single cells in the brain vasculature under spatial constraint and shear stress
Authors: Barrios Wendy V. Alvarez , Lu Huijie , Cowdrick Kyle R. , Galarneau Michelle , Jiang Lan , Lake Melinda , Yang Lin , Chen Danny , Hoelzle David , Peng Zhangli , Zhang Siyuan
Published: July 2017

During metastasis of breast cancer, tumor cells dissociate from the primary tumor, disseminate through the vasculature, and colonize secondary organs such as the brain, lungs, liver and bones. The mechanical arrest of circulating tumor cells (CTCs) in capillary beds is an essential step in the metastatic cascade for the successful early colonization of secondary organs. During mechanical arrest, tumor cells are subjected to extreme physical stresses such as shear stress and spatial constraint exerted by blood flow, as well as from compression by the constricting capillary on the restricted cell. These mechanical stresses may critically impact metastatic success. However, the precise physical and mechanical response of the disseminated tumor cells at the time of arrest in the vasculature to the forces of spatial constraint and shear stress remain largely unknown. Here we present an integrative platform that combines the use of a microfluidic device and computational model as a tunable system to reliably reproduce the mechanical arrest of single cells and quantitatively determine their physical response to precisely controlled mechanical forces of shear stress and spatial constraint. Using confocal microscopy and computational modeling, we found that during mechanical arrest, single tumor cells undergo extreme morphological deformations. Furthermore, the cell morphology is dependent on the magnitude of shear stress applied; with magnitudes above 60Pa inducing elongation, blebbing, and rupture of the cell membrane. Our results demonstrate that our integrative platform is a viable model system for the qualitative and quantitative study of physical cell behavior precisely at the metastatic stage of mechanical arrest. Particularly, our results highlight the direct influence of physical forces on the restricted tumor cell, and suggest the importance of these external cues as dynamic determinants of cellular processes and ultimately of metastatic success. We anticipate our platform to be a starting point for future studies seeking to understand the reciprocal molecular and biophysical interplay imposed by mechanical forces at the greatly understudied stage of mechanical arrest in metastasis.

Citation Format: Wendy V. Alvarez Barrios, Huijie Lu, Kyle R. Cowdrick, Michelle Galarneau, Lan Jiang, Melinda Lake, Lin Yang, Danny Chen, David Hoelzle, Zhangli Peng, Siyuan Zhang. Integrative platform for the reconstruction and modeling of mechanically arrested single cells in the brain vasculature under spatial constraint and shear stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3925. doi:10.1158/1538-7445.AM2017-3925
Abstract 3928: Association of calcium sensing receptor polymorphisms at rs1801725 with circulating calcium in breast cancer patients
Authors: Whalen Diva , Wang Li , Widatalla Sarrah , Ochieng Josiah , Richmond Ann , Sakwe Amos
Published: July 2017

Patients with metastatic or end-stage breast cancer (BC) inevitably develop hypercalcemia, while up to 30% of BC patients develop cancer-induced hypercalcemia (CIH) in the absence of metastases or bone diseases. The commonly diagnosed mild increase in circulating calcium activates the calcium sensing receptor (CaSR) and has been shown to be associated with larger and more aggressive breast tumors in postmenopausal and premenopausal patients respectively. Whether differences in circulating calcium and/or specific inactivating CaSR variants play any role in disparities in BC outcomes remains unclear. DESIGN METHODS: We identified 199 BC cases and 384 age and genetic ancestry-matched controls with calcium assay and genotyping data from the Vanderbilt University DNA biorepository (BioVU) linked to de-identified electronic medical records. The linear mixed effects and codominant models were used to assess the relationship between inactivating CaSR mutations at rs1801725 (codon 986) and rs1801726 (codon 1011) and either circulating calcium levels or risk of high calcium-driven aggressive BC outcomes. RESULTS: We observed that circulating calcium levels were significantly higher in BC cases compared to control subjects (p=0.001) and interestingly, in subjects of African ancestry compared to Caucasians (p=0.001). The A986S mutant CaSR is common among Caucasians while the Q1011E mutant receptor is common among African Americans. However, only inactivating mutations at rs1801725 locus were significantly associated with higher calcium levels (p=0.006) and a higher (69%) risk of high calcium-driven aggressive BC outcomes compared to the wild type receptor. We also demonstrate that invasive BC cells are tolerant to sustained high calcium and that their adaptation to high calcium occurs via up-regulation of calcium-activated early response and malignancy-associated genes. CONCLUSION: These data suggest that inactivating CaSR polymorphisms at rs1801725 predispose BC patients to hypercalcemia and that high circulating calcium-driven aggressive disease outcomes occur via calcium modulated malignancy-associated genes such as MAGEC2/CT10.

Citation Format: Diva Whalen, Li Wang, Sarrah Widatalla, Josiah Ochieng, Ann Richmond, Amos Sakwe. Association of calcium sensing receptor polymorphisms at rs1801725 with circulating calcium in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2017-3928
Abstract 3946: Chromosome instability drives phenotypic switching to metastasis
Authors: Gao Chong-Feng , Su Yanli , Koeman Julie , Essenberg Curt , Dykema Karl , Hudson Eric , Khoo Sok Kean , Woude George Vande
Published: July 2017

Chromosome instability (CIN) is the most striking feature of human cancers. However, how CIN drives tumor progression to metastasis remains elusive. Here we studied the role of chromosome content changes in generating the phenotypic dynamics that is required for metastasis. We isolated epithelial (E) and mesenchymal (M) clones from human carcinoma cell lines and showed that the E clones were able to generate mesenchymal variants, which had the potential to further produce epithelial revertants autonomously. The successive acquisition of invasive mesenchymal and then epithelial phenotypes recapitulated the steps in tumor progression to metastasis. Importantly, the generation of mesenchymal variants from clonal epithelial populations was associated with subtle changes in chromosome content, which altered the chromosome transcriptome and influenced the expression of genes encoding intercellular junction (IJ) proteins, while the loss of chromosome 10p, which harbors the ZEB1 gene, was frequently detected in epithelial variants generated from M clones. Knocking down these IJ genes in E cells induced a mesenchymal phenotype, while knocking down the ZEB1 gene in M cells induced an epithelial phenotype, demonstrating a causal role of chromosome content changes in phenotypic determination. Thus, our studies suggest a novel paradigm of tumor metastasis: primary epithelial carcinoma cells that lose chromosomes harboring IJ genes acquire an invasive mesenchymal phenotype, and subsequent chromosome content changes such as loss of 10p in disseminated mesenchymal cells generate epithelial variants, which can be selected for to generate epithelial tumors during metastatic colonization.

Citation Format: Chong-Feng Gao, Yanli Su, Julie Koeman, Curt Essenberg, Karl Dykema, Eric Hudson, Sok Kean Khoo, George Vande Woude. Chromosome instability drives phenotypic switching to metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3946. doi:10.1158/1538-7445.AM2017-3946
Abstract 3959: CCL18-recruited naïve CD4+ T cells are converted to tumor-infiltrating regulatory T cells in breast cancer and suppress antitumor immunity
Authors: Su Shicheng , Liao Jianyou , Liu Jiang , Liu Qiang , Song Erwei
Published: July 2017

Tumor-infiltrating regulatory T cells (Tregs) play a central role in tumor immunosuppression. However, it remains unclear whether they are directly recruited from peripheral blood or converted from infiltrating naïve T cells. Here, full-length TCR α/β repertoire analysis of different T cell subsets from peripheral blood, primary tumors and draining lymph nodes in patents suggests that Tregs in human breast cancer are mainly converted from tumor-infiltrating naïve CD4+ T cells. Infiltration of naïve CD4+ T cells and Tregs are closely correlated, both indicating poor prognosis for breast cancer patients. Naïve CD4+ T cells in the tumors are recruited by tumor-associated macrophages (TAMs) via CCL18. In addition, naïve T cells and memory T cells exhibit distinctive chemotactic response due to different expression of regulator of G-protein signaling 1(RGS1). Specific silencing CCL18 receptor-PITPNM3 in naïve CD4+ T cells using CD4 aptamer-siRNA blocks their chemotaxis, and thus reduces infiltrating Tregs and inhibits tumor progression in humanized mice. By comparison, silencing RGS1 in memory CD8+ T cells using CD8 aptamer-siRNA enhance their recruitment to tumors and anti-tumor immune response in vivo. These findings provide mechanistic insights for Treg enrichment in breast cancer and suggest that modification of the CCL18-PITPNM3-RGS1 signaling pathway may be an attractive strategy for anticancer immunotherapy.

Note: This abstract was not presented at the meeting.

Citation Format: Shicheng Su, Jianyou Liao, Jiang Liu, Qiang Liu, Erwei Song. CCL18-recruited naïve CD4+ T cells are converted to tumor-infiltrating regulatory T cells in breast cancer and suppress antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3959. doi:10.1158/1538-7445.AM2017-3959
Abstract 3966: Targeting integrin αvβ3-expressing cancer stem cells to manipulate tumor-associated macrophages
Authors: Wettersten Hiromi I. , Minami Toshiyuki , Kaneda Megan M. , Seguin Laetitia , Varner Judith A. , Weis Sara M. , Cheresh David A.
Published: July 2017

Tumor associated macrophages are involved in regulation of cancer growth and aggressiveness. Whereas M1 macrophages trigger an inflammatory response and inhibit tumor growth, M2 macrophages secrete pro-tumor cytokines into the microenvironment to support tumor progression. A macrophage switch from M1 to M2 has been associated with lung cancer progression, and cancer stem cells have been implicated as a driver of this reprogramming.

We recently reported that integrin αvβ3 expression is induced on lung adenocarcinoma cells during drug resistance and is both necessary and sufficient to reprogram these tumors to a stem-like state. Given the role that cancer stem cells play in switching M1 to M2 macrophages, we asked whether αvβ3 expression on lung adenocarcinoma cells account for this macrophage conversion. The M1/M2 macrophage ratio in αvβ3-positive tumors was markedly decreased relative to tumors lacking αvβ3. We next treated mice bearing αvβ3-positive tumors with a monoclonal antibody (LM609) targeting this receptor to assess its ability to alter the macrophage phenotype within these tumors. LM609 was able to selectively eliminate the αvβ3-positive cancer stem cells via antibody-dependent cell-mediated cytotoxicity (ADCC), and this not only increased the M1 macrophage population, but also markedly enhanced the sensitivity of these tumors to the effects of therapy.

These findings reveal that αvβ3-expressing cancer stem cells favor the pro-tumor M2 macrophage phenotype. Eliminating αvβ3-positive cancer stem cells via ADCC serves to both increase pro-inflammatory macrophages within the tumor microenvironment and prolong tumor sensitivity to therapy.

Citation Format: Hiromi I. Wettersten, Toshiyuki Minami, Megan M. Kaneda, Laetitia Seguin, Judith A. Varner, Sara M. Weis, David A. Cheresh. Targeting integrin αvβ3-expressing cancer stem cells to manipulate tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2017-3966
Abstract 3968: Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth
Authors: Achyut Bhagelu R. , Bradford Jennifer , Angara Kartik , Rashid Mohammad , Jain Meenu , Borin Thaiz , Iskander ASM , Ara Roxan , Arbab Ali
Published: July 2017

Glioblastoma (GBM) development and therapeutic resistance has been accompanying with the tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). TAMs are heterogeneous cell populations of immune regulatory myeloid-derived suppressor cells (MDSCs) and polarization of anti-tumor macrophages (M1) into pro-tumor macrophages (M2). We investigated the role of myeloid cell NF-κB signaling in orthotopic GBM model using immune deficient and immune competent hosts. Interestingly, conditional deletion of canonical NF-κB signaling (p65) with Lysm-Cre (p65 KO) in myeloid cells, significantly inhibited syngeneic GL261 tumor growth in immune-competent mice compared to control mice. We studied the TAMs recruitment to the tumor and their polarization under the influence of TME. P65 KO mice displayed decreasing trend of immune cell infiltration (CD45), which phenotyped as decreased F4/80+, CD68+, CD206+ (M2) and Gr1+CD11b+ (MDSCs) macrophages, compared to control mice. This was associated with the increased CD80+ (M1) macrophages, increasing trend of CD4+ and CD8+ cytotoxic T cells, and decreased CD44+ mesenchymal cancer stem cells (CSCs) populations in the TME. Cytokine array data indicated that loss of canonical NF-κB signaling within the TAMs was implicated in increased production of IFNγ, IGF1, MCP1, MIP1α, and TNFα cytokines. Co-culture of T cells with p65 KO or control MDSCs identified increased proliferation of T cells with p65 KO MDSCs compared to control MDSCs. Conversely, GBM patient-derived xenografts and U251 GBM cell line-derived tumors showed increasing trend of growth in immune-deficient mice, following the transplantation of p65 KO bone marrow (BM) compared to control BM. Pro-tumor macrophages and CSCs were increased and T cell populations were decreased in human tumors grown in immune deficient mice transplanted with p65 KO BM, compared with control BM. In addition, analysis of human data set revealed higher expression of p65 subunit of NF-κB complex in brain tumor stroma compared to the tumor cells. The study suggests that canonical NF-κB signaling in TAMs is required for the tumor-promoting macrophage polarization and GBM growth in immunocompetent host compared to immune deficient host. Therefore, targeting myeloid-specific NFκB signaling in GBM could inhibit the immune suppressive TAMs and improve the anti-tumor immunity.

Citation Format: Bhagelu R. Achyut, Jennifer Bradford, Kartik Angara, Mohammad Rashid, Meenu Jain, Thaiz Borin, ASM Iskander, Roxan Ara, Ali Arbab. Canonical NFkB signaling in myeloid cells is required for the glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3968. doi:10.1158/1538-7445.AM2017-3968
Abstract 397: Whole genome copy number variation analysis using a SNP-focused targeted sequencing panel for tumor analysis
Authors: Wang Jiashi , Giorda Kristina , Lai Zhongwu , Stetson Daniel , Jarosz Mirna
Published: July 2017

Accurate genome-wide copy number variation (CNV) analysis is critical for disease and cancer research. Current approaches for CNV analysis include fluorescence in situ hybridization (FISH), array comparative genomic hybridization (array CGH), and SNP arrays. Unfortunately, these methods are not sensitive enough for real world cancer samples because of tumor ploidy, purity and heterogeneity. NGS-based targeted sequencing is increasingly being used for CNV analysis due to throughput, coverage, cost, and sample input requirements. For CNV analysis, detection power is improved by combining both read depth and SNP allele frequency analysis, particularly for copy-neutral events such as loss of heterozygosity. A custom xGen Lockdown CNV backbone panel was developed for broad, uniform genome coverage and to enrich for population-based SNPs. We demonstrate use of the panel as an addition to the xGen Exome Research Panel and a custom cancer focused panel. Downstream analysis incorporates both read depth and observed minor allele frequencies to determine CNVs with enhanced sensitivity. To increase the resolution for large-scale alterations of chromosome 7, a hot-spot for disease-associated CNVs, probe density was increased 6 fold. A known standard, NA12878, was used to validate the panel’s ability to detect heterozygous SNPs with high confidence. In addition, mixtures of cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) were tested with varying levels of background copy-neutral genomic DNA. The sensitivity and specificity of the panel to detect CNV and LOH events with was assessed using deep exome and Affymetrix SNP array data. The ability to detect copy number alterations with high resolution and accuracy would be a valuable resource for disease and cancer research.

Citation Format: Jiashi Wang, Kristina Giorda, Zhongwu Lai, Daniel Stetson, Mirna Jarosz. Whole genome copy number variation analysis using a SNP-focused targeted sequencing panel for tumor analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2017-397
Abstract 3978: B7-H1/PD-L1 soluble expression in multiple cancer subtypes: High sensitivity measurement by immunoassay
Authors: Haworth Justin , Grant Rainer , Thayer Samuel , Floren Melissa , Steere Nathan , O'Brien Isabel , Younge Paul , Anderson Michael , Wegner Greta , Brumbaugh Kathryn
Published: July 2017

Aberrant expression of the B7-H1/PD-L1 immunomodulatory molecule is evident in many forms of cancer. The evidence for increased protein levels of B7-H1/PD-L1 comes largely from immunohistochemistry analysis of surgical and biopsy specimens. While several studies attempt to correlate B7-H1/PD-L1 expression with poor prognosis, there are studies that conclude no correlation based on technical difficulties associated with detecting B7-H1/PD-L1 protein expression on the cell surface. We were able to quantifiably measure soluble B7-H1/PD-L1 in conditioned medium from tumor cell cultures using two new immunoassays, a Quantikine® ELISA and a Simple Plex™ Assay. We show that breast, prostate, glioma, and non-Hodgkin’s lymphoma cells express various levels of B7-H1/PD-L1 which correlate to their PTEN or proliferation status. This is in agreement with previous findings showing that increases in B7-H1/PD-L1 expression levels are frequently coincident with loss of the PTEN tumor suppressor or increased activation of the phosphoinositide 3-kinase (PI 3-K) pathway. The androgen-responsive LNCaP prostate cancer cell line does not exhibit measurable B7-H1/PD-L1 in any instance, while the androgen-independent prostate cancer cell line PC-3 expresses measurable B7-H1/PD-L1 that is further inducible with PMA treatment. In all cell lines included in our study, incubation with PMA increases the level of B7-H1/PD-L1 released into the culture media, while pretreatment with the PI 3-K inhibitor, LY294002, abrogates this increase. Importantly, the Simple Plex platform is able to quantify very low levels of B7-H1/PD-L1 that are below detection by ELISA. Our data suggest that combining the Quantikine ELISA and Simple Plex platforms is a powerful strategy for reliably and quantitatively assessing soluble B7-H1/PD-L1 levels.

Citation Format: Justin Haworth, Rainer Grant, Samuel Thayer, Melissa Floren, Nathan Steere, Isabel O'Brien, Paul Younge, Michael Anderson, Greta Wegner, Kathryn Brumbaugh. B7-H1/PD-L1 soluble expression in multiple cancer subtypes: High sensitivity measurement by immunoassay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2017-3978