bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4429: Delivery of miR-143 and miR-506 with novel nano carrier to arrest cell cycle in lung cancer
Authors: Hossian A K M Nawshad , Muthumula Chandra Mohan Reddy , Mattheolabakis George
Published: August 2019

Lung cancer is the leading cause of cancer related deaths worldwide. Although targeting the cell cycle of cancer cells has been a promising approach, no such therapy has translated for the treatment of lung cancer. In our previous work, we identified that the combinatorial treatment of two miRNAs, miR-143 and miR-506, inhibited the cell cycle of lung cancer cells in vitro. These miRs downregulated the CDK1 and CDK4/6 genes, which are involved in the G1/S and G2/M transitions, respectively. Nucleic acid delivery has met formidable challenges, such as short in vivo half-life and rapid degradation in the circulation, as well as limited cell penetration or endosomal escape. We developed a novel nanocarrier, composed of tocopherol (vitamin E), polyethylenenimine, and polyethylene glycol (TPP) in 1:1:1 molar ratio for the delivery of miRNAs. Proper characterization with NMR and FTIR confirmed the synthesis of this molecule. We found it can form nanomicelles, and the positively charged of polyethyleneimine allowed for the complexation of miRNAs, as was detected by a gel retardation assay. Fluorescently tagged TPP micelles were uptaken by cancer cells, and the carrier successfully transfected cancer cells with the combinatorial miRNA treatment inducing gene downregulation. In vivo pharmacokinetics study using a subcutaneous mouse model of lung cancer indicated prolonged accumulation of the TPP-miRNA micelles in the tumor area. In vivo administration of the miR-143/506 in TPP micelles, along with cisplatin co-treatment, inhibited tumor growth in a subcutaneous mouse model of lung cancer. Overall, the promising role of miR-143 and 506 for

the regulation of LC growth may be achieved by this novel TPP-based nanocarrier, which merits further evaluation.

Citation Format: A K M Nawshad Hossian, Chandra Mohan Reddy Muthumula, George Mattheolabakis. Delivery of miR-143 and miR-506 with novel nano carrier to arrest cell cycle in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4429.
Abstract 4430: Simulating delivery of TTFields to the infratentorium in patients with brainstem gliomas
Authors: Ramirez-Fort Marigdalia K. , Cross Brittany , Naveh Ariel , McClelland Shearwood , Mendez Melissa , Santiago Roberto , Mahase Sean S. , Matta Jaime , Bomzon Ze'ev , Lange Christopher S.
Published: August 2019

Purpose/Objective(s): TTFields are FDA-approved for the treatment of recurrent and newly diagnosed Glioblastoma (GBM) in the supratentorial brain. The EF-14 trial showed that combining TTFields with adjuvant chemo-radiation leads to a significant increase in overall survival compared to adjuvant chemo-radiation alone. High-grade gliomas also occur in the infratentorium and brainstem of pediatric and adult patients. Brainstem gliomas have a median survival of 9 to 11 months, necessitating the exploration of novel treatment combinations, such as delivering TTFields to the infratentorial brain.

We recently showed in a simulation-based study that TTFields can be successfully delivered to the infratentorium. Effective delivery was achieved by placement of the arrays on the vertex, bilateral posterolateral occiput, and superior-posterior neck; the array placement results in TTFields at therapeutic intensities throughout the brainstem and cerebellum of realistic computational head models.

The previous simulation-based study was performed using realistic computational models of healthy individuals; it did not provide detailed insight into field distributions within tumor tissue located in the infratentorium. Here, we aim to expand the results of this previous study, by simulating the delivery of TTFields to the infratentorium of adult and pediatric patients with high-grade brainstem gliomas.

Materials/Methods: A realistic computational model was created using MRI data of adult and pediatric patients with high-grade brainstem gliomas. Transducer arrays were placed on the model and the delivery of TTFields to the infratentorial brain was then simulated using a commercial numerical solver. The electric field distribution in the supratentorium, infratentorium, and within the tumor were derived from the simulations, and the dose in the tumor analyzed.

Results: The distribution of calculated isofield lines demonstrates effective delivery of TTFields to infratentorial brain tumors.

Conclusion: Our results provides rationale for clinically investigating the utility of TTFields in treating infratentorial high-grade gliomas.

Citation Format: Marigdalia K. Ramirez-Fort, Brittany Cross, Ariel Naveh, Shearwood McClelland III, Melissa Mendez, Roberto Santiago, Sean S. Mahase, Jaime Matta, Ze'ev Bomzon, Christopher S. Lange. Simulating delivery of TTFields to the infratentorium in patients with brainstem gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4430.
Abstract 4431: Cytotoxic effects of the Jamaican yam tuber in hormone-sensitive cancers and its potential use in green nanotechnology formulation
Authors: Wright Sasha-gay A.
Published: August 2019

Historically, plant materials have been widely explored for their anti-proliferative effects against a variety of cancer cells, with many of them proving to be very effective. Additionally, their efficacy has been seen in the specific field of targeted cancer therapy. The use of plants for therapeutic purposes in place of or with traditional medicine is collectively referred to as complementary and alternative medicine (C.A.M). Several Jamaican Yam species have shown to be useful as C.A.M in the treatment of certain medical conditions such as diabetes, hypercholesterolemia and cancer. Hormone-sensitive cancers such as breast and prostate cancer are the most frequently diagnosed malignancy and when combined, is the leading cause of cancer related deaths among the Jamaican populace. These statistics unfortunately, are also reflected in the wider Caribbean region. Currently treatment involves: surgery, radiation and androgen deprivation therapy; however, there are some cases in which mutation causes further growth of the cancer cells regardless of treatment. As such, there still remains a dire need of a more effective and lasting therapy. The aim of this study was to assess the antiproliferative effects of yam material and to also deduce a possible mechanism of action in an effort to formulate a means of targeted therapy. The results showed that an alcoholic extract of the Jamaican yam tuber (Dioscorea spp.) demonstrated anti-proliferative effects in androgen insensitive prostate cancer cell lines (IC50 of 18 ppm, 95% C.I. 24-33ppm) and invasive breast cancer cell lines (IC 50 of 28 ppm, 95% C.I 36-54ppm). Western blot assays also indicated that cell death was possibly induced through the cell cycle regulatory protein, cyclin D. The antiproliferative effects and purported mechanism of action of the yam extract against some hormone-sensitive cancer cells makes it an ideal alternative for the formulation of green nanotechnology for targeted therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Sasha-gay A. Wright. Cytotoxic effects of the Jamaican yam tuber in hormone-sensitive cancers and its potential use in green nanotechnology formulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4431.
Abstract 4432: Multi-omics profiling establishes the polypharmacology of FDA Approved CDK4/6 inhibitors and its impact on drug response
Authors: Mills Caitlin E. , Hafner Marc A. , Subramanian Kartik , Chen Chen , Chung Mirra , Boswell Sarah A. , Everley Robert A. , Liu Changchang , Walmsley Charlotte S. , Juric Dejan , Sorger Peter K.
Published: August 2019

FDA approval of multiple drugs with different chemical structures against the same protein target raises the question whether such drugs have sufficiently similar mechanisms of action to be considered functionally equivalent. We compare three recently approved inhibitors of the cyclin-dependent kinases CDK4/6 - palbociclib, ribociclib, and abemaciclib - that have become highly promising therapies for the treatment of breast cancer and potentially other solid tumors. All three drugs have the same nominal targets but differ in selectivity. In humans, abemaciclib uniquely appears to exhibit single-agent activity and has a distinct toxicity profile. We systematically profile targets and activities of these CDK4/6 inhibitors using biochemical assays, mRNA profiling, mass spectrometry-based phospho-proteomics, GR-based dose-response assays, and mouse xenografts. We find that the three drugs differ at a cellular level and that abemaciclib has targets and activities not shared by palbociclib or ribociclib including: induction of cell death (even in pRb-deficient cells), arrest in the G2 phase of the cell cycle, reduced drug adaptation, and unique transcriptional effects in vitro and in vivo. These activities appear to arise from inhibition of CDKs other than CDK4/6 including CDK2/Cyclin A/E and CDK1/Cyclin B. We propose that inhibition of these kinases by abemaciclib target known mechanisms of resistance to CDK4/6 inhibition and thus elicit a response in cell lines that are resistant to palbociclib or ribociclib.

Citation Format: Caitlin E. Mills, Marc A. Hafner, Kartik Subramanian, Chen Chen, Mirra Chung, Sarah A. Boswell, Robert A. Everley, Changchang Liu, Charlotte S. Walmsley, Dejan Juric, Peter K. Sorger. Multi-omics profiling establishes the polypharmacology of FDA Approved CDK4/6 inhibitors and its impact on drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4432.
Abstract 4437: Effects of atypical protein Kinase c inhibitor (DNDA) on lung cancer proliferation and migration by PKC-ι/FAK ubiquination through the Cbl-b pathway
Authors: Bommareddy Raja Reddy , Patel Rekha , Duncan Mildred Acevedo
Published: August 2019

Lung cancer is the second most common cancer and it is the leading cause of cancer death in both men and women. PKC isozymes play an important role in the development and progression of many cancers by regulating the cell cycle, survival, apoptosis, cell motility and malignant transformation. Our focus is to study the role of atypical PKCs (aPKC) in cell proliferation and migration in lung cancer cell lines. We used a novel non-specific inhibitor of aPKC namely DNDA (3,4-amino-2,7napthalenedisulfonic acid). Our hypothesis is that DNDA inhibits cell proliferation and migration of lung cancer cells. Our data from cell viability and flow cytometry showed significant reduction in cell proliferation and induction of apoptosis with DNDA (10µM) in A549 and H1299 lung cancer cells. Additionally, DNDA showed no toxic effect on BEAS-2B normal lung cells. Elevated levels of Focal Adhesion kinase (FAK) are implicated in the progression of cancer and plays a vital role in the invasion and migration of cancer cells. Western blot results showed that the phosphorylation of PKC-ι and phosphorylation of FAK were decreased in A549 lung cancer cells upon DNDA treatment. Moreover, there was no significant reduction in phosphorylation of FAK in H1299 lung cancer cells upon treatment with DNDA. Immunoprecipitation (IP) data revealed an association of PKC-ι with FAK and FAK with Cbl-b. Ubitest results suggests that PKC-ι regulates the cleavage of FAK through its ubiquination by cbl-b and thus inhibits the migration of A549 lung cancer cells which was evident from scratch assay. Our data indicates that DNDA might inhibit the migration of A549 lung cancer cells by PKC-ι/FAK ubiquination via Cbl-b.

Citation Format: Raja Reddy Bommareddy, Rekha Patel, Mildred Acevedo Duncan. Effects of atypical protein Kinase c inhibitor (DNDA) on lung cancer proliferation and migration by PKC-ι/FAK ubiquination through the Cbl-b pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4437.
Abstract 4441: Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo
Authors: Ohol Yamini M. , Sun Michael , Leger Paul , Hu Dennis , Biannic Berenger , Rana Payal , Cho Cynthia , Jacobson Scott , Wong Steve , Sanchez Jerick , Han Xinping , Young Kyle , Okano Akinori , Maung Jack , Cutler Gene , Shah Nick , Adusumilli Lavanya , Kaveri Deepika , Talay Oezcan , Pookot Deepa , Abraham Betty , Bradford Delia , Kozon Nathan , Colas Christophe , Kim Andrea , Schwarz Jacob , Wustrow David , Brockstedt Dirk , Kassner Paul
Published: August 2019

USP7 is a deubiquitinase that regulates the levels of multiple downstream targets with roles in cancer progression and immune response. Inhibitors of USP7 may thus decrease oncogene function, increase tumor suppressor function, enhance immune function and sensitize tumor cells to DNA damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple p53-wild type cell lines, including several blood cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of p53-mutant tumor cell lines in vitro. Further, oral administration of our USP7 inhibitors inhibits MM.1S (multiple myeloma; p53-wild type) and H526 (small cell lung cancer; p53-mutant) tumor growth in vivo. Our work confirms that USP7 is a pharmacologically tractable target and future studies will aim to further understand the mechanism of action of USP7 inhibitors in p53-mutant cancers.

Citation Format: Yamini M. Ohol, Michael Sun, Paul Leger, Dennis Hu, Berenger Biannic, Payal Rana, Cynthia Cho, Scott Jacobson, Steve Wong, Jerick Sanchez, Xinping Han, Kyle Young, Akinori Okano, Jack Maung, Gene Cutler, Nick Shah, Lavanya Adusumilli, Deepika Kaveri, Oezcan Talay, Deepa Pookot, Betty Abraham, Delia Bradford, Nathan Kozon, Christophe Colas, Andrea Kim, Jacob Schwarz, David Wustrow, Dirk Brockstedt, Paul Kassner. Discovery of potent and selective inhibitors of USP7 with anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4441.
Abstract 4498: O GlcNAcylation of SOX2 regulates its tumor initiation properties in pancreatic cancer
Authors: Sharma Nikita S. , Gupta Vineet K. , Dauer Patricia , Hadad Roey , kesh Kousik K. , Dudeja Vikas , Saluja Ashok , Banerjee Sulagna
Published: August 2019

Background: Pancreatic Cancer is an extremely aggressive disease with a bad outcome owing to its late detection, high rate of metastasis and self-renewal post-surgical and chemotherapeutic intervention. In cancer the balance between differentiation and self-renewal is tightly regulated by SOX2. Over expression of SOX2, as in pancreatic cancer, can tilt the balance towards self-renewal and thus increase the population of these undifferentiated “stem cells”.

Methods: A CRISPR based OGT knockout kit was purchased from Origene and a knockout cell line was generated with S2VP10 cells. For the tumor initiation studies OGTi (OGT knockout cells) were implanted subcutaneously at a limiting dilution from 500,000 cells to 500 cells. Site directed mutagenesis was performed using theQuick-change Lightning from Agilent.

Results: In the current study we show for the first time that SOX2 undergoes a type of post translational modification known as O GlcNAcylation. We also show for the first time that in humans this modification happens at Serine 246 and mutating this particular site via a side directed mutagenesis leads to a loss of O GlcNAcylation of SOX2.O GlcNAc modification of SOX2 further leads to its activation which then leads to its stabilization in the nucleus. A CRISPR-OGT knockout in pancreatic cancer cell line S2VP10 resulted in a delayed tumor initiation. We further showed that mutation of this site (S246A) prevents the modification of Sox2 and its downstream activity. Our study also demonstrated that targeting OGT in vivo with a small molecule inhibitor OSMI, results in decreased tumor burden, delayed tumor progression and a decreased expression of SOX2 in pancreatic cancer cells.

Conclusion: Our study highlights for the first time that the O-GlcNAc transferase dependent SOX2 glycosylation has a profound effect on the transcriptional activity of SOX2 and is instrumental in determining self-renewal in pancreatic cancer which can be targeted therapeutically.

Citation Format: Nikita S. Sharma, Vineet K. Gupta, Patricia Dauer, Roey Hadad, Kousik K. kesh, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. O GlcNAcylation of SOX2 regulates its tumor initiation properties in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4498.
Abstract 4500: Mechanisms of tumor-associated myeloid cells in modulating host immune microenvironment and metastatic progression
Authors: Ishii Hiroki , Hollander Christine , Greten Tim , Yang Li
Published: August 2019

The immune checkpoint inhibitor blockade takes advantage of the specific receptor-ligand interactions between immune and tumor cells. However, the immunosuppressive microenvironment has been largely ignored, which likely contribute to low efficacy and resistance in immune therapies. Our studies demonstrate that TGF-β signaling in tumor-associated myeloid cells isessential in systemic immune suppression and metastatic progression. Using mouse models of breast cancer metastasis, we further uncover that miR130a and 145 are critical in regulating TGF-β signaling in myeloid cells. miR130a and 145 are down-regulated in these myeloid cells, leading to increased TGF-β RII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decrease tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNgamma-producing cytotoxic CD8 T lymphocytes. Lastly, miR-130a and miR-145 mimics improve systemic anti-tumor immunity and inhibit metastasis in preclinical mouse models. Another important mechanism in myeloid-mediated immune suppression involves a decreased expression of Lamin A/C, nuclear lamina proteins that prominently interact with heterochromatin. Lamin A/C knockout in myeloid cells leads to H3K4me3-mediated upregulation of Gfi1 and C/EBPϵ,critical transcription factors responsible for granulocytic lineage differentiation. In addition, loss of Lamin A/C attenuates antigen-presentation and compromises host anti-tumor immunity. These mechanistic understandings provide options to reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity.

Citation Format: Hiroki Ishii, Christine Hollander, Tim Greten, Li Yang. Mechanisms of tumor-associated myeloid cells in modulating host immune microenvironment and metastatic progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4500.
Abstract 4516: Suppression of metastasis by inhibiting nanoscale physical communication of cancer cell and the endothelium
Authors: Dash Chinmayee , Saha Tanmoy , Khiste Sachin , Sengupta Shiladitya
Published: August 2019

The progression of metastasis proceeds through the physical communication of cancer cells with the endothelium via tunneling nanotubes (TNTs). Restriction on the cancer cell and the endothelium communication by inhibiting TNT formation can lead to breakthrough advancement in the treatment of metastasis. However, the mechanistic interpretation about the formation of TNTs between the cancer cells and the endothelium has been poorly defined in literature. Our study integrates the investigation of mechanism behind the TNT formation and introduction of a pharmacological inhibitor for TNT formation.

The nanobridges are primarily composed of cytoskeletal elements such as actin and tubulin, which originates from the energy dependent remodeling of membrane cytoskeletal elements. Here, we propose that the members of Rho-family of GTPases, Cdc42, Ral and Rac along with exocyst complex, are involved in the actin polymerization based on their localization to the nanobridges between the metastatic cancer cells and the endothelium. A significant decrease in mitochondrial transfer and TNT formation has been observed when the inhibitor for Cdc42/Rac1 GTPase inhibitor (ML141), and a geranylgeranyltransferase 1 inhibitor (L-778,123) has been employed in the co-culture. The amount of mitochondrial transfer was evaluated with flow cytometry and the number of TNT formation was counted from imaging, which in case of inhibitors was highly reduced. Simultaneously the involvement of exocyst complex was confirmed through imaging. The proteins involved in the exocyst complex co-localize with the nanotubes and was further validated by siRNA-based knock down study. The nanotubular pathways are the primary mode of communication between cancer cell and endothelium, which leads to the conversion of healthy endothelium to a pathological one. Hindering the TNT mediated communication via pharmacological inhibitors can reduce the chance of metastasis.

Conclusion: Investigation of molecular mechanism behind the TNT formation and employing innovative ways to have restriction over that can be a potential next generation therapeutic module for controlling metastasis.

Reference: Connor Y, Tekleab S, Nandakumar S, Walls C, Tekleab Y, Reiberger T, Husain A, Gadish O, Sabbisetti V, Kaushik S, Sehrawat S, Kulkarni A, Dvorak H, Zetter B, Jain RK, Edelman E, Sengupta S. Physical nanoscale conduits-mediated communication between tumor cells and endothelium enables a metastatic hijack. Nature Communications (2015) 6:8671.

Citation Format: Chinmayee Dash, Tanmoy Saha, Sachin Khiste, Shiladitya Sengupta. Suppression of metastasis by inhibiting nanoscale physical communication of cancer cell and the endothelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4516.
Abstract 453: A circulating tumor cell assay for dynamic assessment of drug sensitivity in metastatic castration-resistant prostate cancer
Authors: Teng Pai-Chi , Jan Yu Jen , Yoon Junhee , Chen Jie-Fu , Chen Pin-Jung , Yao Nu , Cheng Shirley , Lozano Amber , Freeman Michael R. , You Sungyong , Tseng Hsian-Rong , Posadas Edwin M.
Published: August 2019

Background: Tissue-based gene signatures can predict clinical behavior in prostate cancer (PC). Our objective was to extend their application to circulating tumor cells (CTCs) and to show that changes in the signature were associated with changes in clinical behavior.

Methods: Our approach combined the Thermoresponsive(TR)-NanoVelcro CTC purification system with the Nanostring nCounter system for cellular purification and transcriptomic analysis. The Prostate Cancer Classification System (PCS) panel was modified for use in CTCs. We selected 31 blood samples from 23 PC patients receiving androgen receptor signaling inhibitors (ARSI) and measured the PCS1 Z score (probability). These findings were compared with clinical outcome data (responsiveness/resistance).

Results: A modified, 16-gene PCS1 signature was established and validated through a rigorous bioinformatics process. We performed analytical validation of our combined CTC-RNA system to ensure reproducibility and specificity. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). The analyzed bloods contained samples from 8 patients who developed resistance to an ARSI allowing for dynamic measurement of gene expression. Our analysis found that the PCS1 Z score increased at the time that ARSI-resistance emerged (Pairwise T-test, P=0.016).

Conclusions:Using this new methodology, contemporary, clinically-relevant gene signatures such as PCS could be measured non-invasively in CTCs. These findings can be used to relate gene expression to clinical drug response. This approach also allowed for measurement of dynamic variations of gene expression in individual patients over time that correlated to ARSI sensitivity.

Citation Format: Pai-Chi Teng, Yu Jen Jan, Junhee Yoon, Jie-Fu Chen, Pin-Jung Chen, Nu Yao, Shirley Cheng, Amber Lozano, Michael R. Freeman, Sungyong You, Hsian-Rong Tseng, Edwin M. Posadas. A circulating tumor cell assay for dynamic assessment of drug sensitivity in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 453.
Abstract 4534: Label free molecular imaging of tumor sections for two and three dimensional tissue classification and pathway mapping
Authors: Jones Emrys A. , Gurung Dipa , Henderson Fiona , Gentry Matthew , McDougall Danielle , Shanneik Yasmin , Langridge James , McMahon Adam , Takats Zoltan
Published: August 2019

Mass spectrometry imaging allows for the presence and abundance of a wide range of bio-molecules to be measured directly from tissue sections without the use of labels or tags. This results in the ability to compare the spatial distribution of hundreds of metabolites, lipids, peptides, and proteins within the same section in a single experiment. Desorption electrospray ionization (DESI) mass spectrometry imaging can differentiate and classify tissue types, and associated disease states, by measuring the unique characteristic molecular fingerprints of the tissues1,2. Furthermore, using histologically annotated databases of molecular profiles, unknown tissues can be classified using the mass spectrometry output coupled to machine learning approaches. A major benefit of DESI is that the analyzed sections are left largely unchanged by the process; the sections can subsequently be stained and the results validated by immunohistochemistry or conventional H&E approaches. During DESI fingerprinting analyses, a full mass spectrum on a cell by cell resolution of 20-50µm pixel size is collected. Chemical changes on very small scales can be distinguished and explored, allowing the chemistry of tumor margins and interface zones to be understood through mapping results onto metabolic pathways. As the data in a DESI experiment is collected and stored, multiple interrogations of the data are possible; as new discoveries are made, previously collected data can be retrospectively mined for additional insights. In this study, the three-dimensional chemical heterogeneity of drug dosed spheroids and tumor xenografts was measured with DESI and compared with MRI and PET imaging modalities and with FT-IR spectroscopic techniques. We demonstrate the power of this multi-analytical approach in glioma models and in colorectal and prostrate tumor samples to uncover metabolic and lipidomic changes during disease progression and to reveal the inherent heterogeneity within a tumor. 1. 1. Calligaris, D.Caragacianu, D.Liu, et al; PNAS, 2014, 111, 15185-151892. Guenther, S., Muirhead, L.J., et al, Cancer Res, 2015, 75, 1828-1837

Citation Format: Emrys A. Jones, Dipa Gurung, Fiona Henderson, Matthew Gentry, Danielle McDougall, Yasmin Shanneik, James Langridge, Adam McMahon, Zoltan Takats. Label free molecular imaging of tumor sections for two and three dimensional tissue classification and pathway mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4534.
Abstract 4545: Quantitative profiling of Cytochrome P450 2S1 in colorectal cancer by PRM assay
Authors: Shaheed Sadr ul , Patterson Laurence H. , Pors Klaus , Sutton Chris W.
Published: August 2019

Introduction: Cytochromes P450s (CYPs) constitute a superfamily of xenobiotic metabolising enzymes responsible for metabolism of many pharmaceuticals in the liver. Elevated mRNA levels of specific isoforms, such as CYP2S1, are associated with poor prognosis in colorectal cancer (CRC), and represent novel therapeutic targets for biotransformation of prodrugs to potent cytotoxics at the cancer site. In order to understand the expression of CYP2S1 protein, we have developed a parallel reaction monitoring mass spectrometry (PRM MS) assay to screen CRC samples.

Method: Peptides (n=3) uniquely associated with CYP2S1 were synthesized and used as standards to optimise analytical performance (fragmentation conditions, LC retention time, LOQ, LOD, dynamic range) in an Orbitrap Fusion-based PRM MS assay. Levels of CYP2S1 were then determined in protein extracts of CRC, relative to the standards.

Results: The PRM MS assay yielded quantitative data over 3 orders of magnitude. CYP2S1 was detected in C106, CaCO2, HCC2998, HT55 and DLD1 cell lines (0.05-1.08pg) and HT55 and DLD1 xenografts (0.29-0.41pg). CYP2S1 was also detected in patient-specific CRC tissues, with elevated levels in Stage III tumours.

Conclusions: The PRM MS assay provides a specific, sensitive, high throughput method for identifying CYP2S1 compared to established enzyme and immunoassays. CYP2S1 levels vary considerably across CRC sources highlighting the importance of screening to identify the correct models for new drug development and the right patients for subsequent treatment.

Citation Format: Sadr ul Shaheed, Laurence H. Patterson, Klaus Pors, Chris W. Sutton. Quantitative profiling of Cytochrome P450 2S1 in colorectal cancer by PRM assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4545.
Abstract 4546: Functional cell surface proteomics of acute myeloid leukemia enables predictive modeling of antibody-drug conjugate cytotoxicity
Authors: Lawrence Robert , Thurman Robert , Biechele Travis , Tognon Cristina , Savage Samantha , Schultz Anna Reister , Tyner Jeffrey , Arthur William
Published: August 2019

Acute myeloid leukemia (AML) remains a significant unmet medical need. The development of targeted therapies for AML is challenging due to the heterogeneity of immunological and genetic disease subtypes and physiological similarities shared by blasts and normal myeloid progenitor cells. The identification of abundant cell surface markers on AML blasts with limited expression on bone marrow progenitors could drive the development of novel antibody-drug conjugates (ADCs) and other antibody-based therapeutics with improved therapeutic windows. Here we used hydrazide chemistry and mass spectrometry-based proteomics to profile the cell surface landscape of 95 primary blast samples from the Beat AML research consortium and normal bone marrow progenitor cells from healthy donors. We measured the abundance of over 5,000 proteins with high reproducibility (R2 = 0.89) and coverage of known cell surface markers (240/371). We also identified more than 2,500 N-glycosylation sites, many of which are predicted by canonical sequence motifs but have not been demonstrated experimentally. These findings thereby expand our knowledge of both the AML and normal hematological cell surface proteome. Additionally, we used pulsed stable isotope labeling coupled with the aforementioned hydrazide method to assess global surface protein abundance and turnover dynamics in a panel of malignant cell lines. We integrated our cell surface characterization with Beat AML’s mRNA expression, mutation, and AML subtype classification data to prioritize proteins for further exploration as candidate targets for ADC therapeutics. We generated a library of ADCs using monoclonal antibodies conjugated with Auristatin T, a highly potent membrane impermeable microtubule inhibitor, and measured ADC cytotoxic activity on a panel of AML cell lines as well as primary bone marrow progenitor cells. We used these integrated datasets to more precisely refine AML subtypes and identify patient populations that could benefit from novel AML ADCs. We report the preclinical validation of CD317 as a proof-of-concept for the utility of cell surface proteomics to identify novel targets for ADC therapeutics.

Citation Format: Robert Lawrence, Robert Thurman, Travis Biechele, Cristina Tognon, Samantha Savage, Anna Reister Schultz, Jeffrey Tyner, William Arthur. Functional cell surface proteomics of acute myeloid leukemia enables predictive modeling of antibody-drug conjugate cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4546.
Abstract 4549: Macrophage-epithelial metabolic crosstalk impairs chemotherapy in pancreatic cancer
Authors: Halbrook Christopher J. , Pontious Corbin , Kovalenko Ilya , Lapienyte Laura , Dreyer Stephan , Zhang Yaqing , Nelson Barbara , Hong Hanna , Chang Daivid , Morton Jennifer P. , Magliano Marina Pasca di , Lyssiotis Costas A.
Published: August 2019

Pancreatic ductal adenocarcinoma (PDA) remains a leading cause of cancer related death, contrasting a relatively low incidence rate. A principle barrier in PDA treatment is the physiology of the tumors, characterized by a densely fibrotic stroma, rich with immune cell infiltration including macrophages. Macrophages are polarized by environmental cues which dictate their function. In PDA, macrophages within the tumor (tumor associated macrophages, or TAMs) are strongly immunosuppressive, inhibiting both infiltration and activation of cytotoxic T-cells. Additionally, TAMs have been shown to drive resistance to chemotherapy, though the mechanism for which remains unclear.

Several pathways have been described by which PDA cells recruit and polarize macrophages into TAMs, and the effects that TAMs have on the tumor microenvironment. These pathways have largely focused on signaling proteins, however, metabolic byproducts also influence the behavior of immune cells within the tumor microenvironment. To explore potential metabolic crosstalk, we have profiled the metabolic factors exchanged between PDA cells and TAMs. Among these, we have found that TAMs release metabolites which can regulate the response of PDA cells to chemotherapy. Importantly, this response is consistent across several murine and patient-derived pancreatic cancer cell lines, and this metabolite release appears to be a general property of anti-inflammatory macrophage metabolism. We further validated these findings in vivo, using a combination of pharmacological and genetic models to modulate myeloid cells within the tumor microenvironment. Taken together, these data suggest that further development of interventions which target either PDA-mediated polarization of TAMs or TAM-mediated inhibition of chemotherapy represent opportunities to improve the efficacy of currently available treatment options.

Citation Format: Christopher J. Halbrook, Corbin Pontious, Ilya Kovalenko, Laura Lapienyte, Stephan Dreyer, Yaqing Zhang, Barbara Nelson, Hanna Hong, Daivid Chang, Jennifer P. Morton, Marina Pasca di Magliano, Costas A. Lyssiotis. Macrophage-epithelial metabolic crosstalk impairs chemotherapy in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4549.
Abstract 4562: Intra-tumor immune activity is linked to genetic diversity of tumor infiltrating lymphocyte and impact clinical outcomes in hepatocellular cancer
Authors: Kawaguchi Tstutomu , Yan Li , Takahashi Hideo , Qi Qianya , Peng Xuan , Takabe Kazuaki , Otsuji Eigo
Published: August 2019

Hepatocellular carcinoma (HCC) remains the second leading cause of the cancer-related death worldwide. Ongoing clinical trials with immune checkpoint inhibitors (CPIs) for HCC have revealed considerable amount of patients do not respond. Hence, to identify the responder is imminent need for immunotherapy in HCC. We aimed to examine the immune landscape of HCC, using the publically available data set The Cancer Genome Atlas (TCGA). Clinicopathological and genomic expression data in 371 patients with HCC were obtained from TCGA. CYT was defined by GZMA and PRF1 expression, and CIBERSORT and TIMER were used to evaluate intra-tumoral immune cell composition. Kaplan-Meier curve for overall survival (OS), disease free interval (DFI), progression free interval (PFI), and disease-specific survival (DSS) were obtained and Cox Progression Hazards model was used for multivariable analysis. Gene Set Enrichment Analysis (GSEA) was also conducted to analyze the gene sets enriched in CYT-High HCC patients. High CYT was associated with high levels of activated CD8+ T cells, gamma-delta T cells, M1 macrophages, and memory CD4+T cells on CYBERSORT. A similar result was also obtained on TIMER. CYT-high HCC patients had significantly improved DFI, PFI, DSS, and OS, compared to CYT-low HCC patients. The levels of immune checkpoint molecules (ICMs), including programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), T cell immunoglobulin and mucin domain 3 (TIM3), indoleamine 2,3-dioxygenase 1 (IDO1), and IDO2, correlated significantly with CYT. High-CYT tumors showed increased somatic copy number alterations (SCNA). Gene expression of APOBEC3 family was significantly higher in high-CYT tumors compared to low-CYT tumors. T-cell and B-cell receptors was more diverse in High-CYT tumors compared to low-CYT tumors. Multivariate survival analysis demonstrated high CYT was an independent protective factor for prognosis in patients with HCC. High CYT is associated with significantly improved survival in HCC, secondary to enhanced immunity and increased cytolytic activity by T cell and M1 macrophage. Additionally, high CYT is associated with ICMs, and CYT can be a potential biologic marker for CPIs.

Note: This abstract was not presented at the meeting.

Citation Format: Tstutomu Kawaguchi, Li Yan, Hideo Takahashi, Qianya Qi, Xuan Peng, Kazuaki Takabe, Eigo Otsuji. Intra-tumor immune activity is linked to genetic diversity of tumor infiltrating lymphocyte and impact clinical outcomes in hepatocellular cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4562.
Abstract 4564: Tumor infiltrating lymphocytes are associated with p16 protein expression in acral lentiginous melanoma
Authors: Nino Marco A. Galvez , Castillo Miluska , Bernabe Luis A. , Casavilca sandro , Villegas Valeria , Sanchez Joselyn , Dunstan Jorge , Abugatas Julio , Castaneda Carlos , Gomez Henry
Published: August 2019

Background: The loss of function of gene p16(INK4a)/CDKN2 is associated with the development of several neoplasm and tumor progression, likewise, the level of tumor infiltrating lymphocytes is a predictive and prognostic biomarker of survival. This study aims to evaluate the association between composition of tumor infiltrating lymphocytes (TILs) and expression of p16 in acral lentiginous melanoma (ALM), and their impact on prognosis.

Materials and methods: A cohort of 148 surgical pathology specimens of ALM was studied. TILs were evaluated by immunohistochemical detection of CD3 and CD8, along with CD20, CD4, CD68, and CD163 in a subset of 43 cases, likewise, we classify the level of TILs in grades by combining density and distribution. p16 protein expression by immunohistochemistry was also investigated in all cases.

Results: The median age was 66 years, median Breslow thickness was 6.0 mm, Grade III of TILs was found in 28.4% and lymph nodes were involved in 44.6%. Breslow thickness (p<0.001), stage I-II (p<0.001), negative lymph nodes (p<0.001) and <10% p16 (p<0.001) were associated with longer survival. Grade III of TILs was associated with thinner Breslow thickness (p=0.008) and lower mitosis (p=0.047). A higher density of CD3 TILs was associated with male gender (p=0.008), thinner Breslow thickness (p=0.047), negative lymph node (p=0.031), early stage (p=0.046), and p16 nuclear expression of >10% (p=0.045). Higher CD8 TIL was associated with >p16 (p=0.03). Survival analysis found that longer survival doesn’t associated with high grade of TILs (p=0.094). Levels of CD3+ and CD8+ cells were correlated with those of CD4+, CD20+, CD68+ and CD163+ immune cells.

Conclusions: Loss of expression of p16 is associated with lower levels of CD3+ and CD8+ TIL, indicating a probable relationship between p16 and TILs immune response in ALM.

Citation Format: Marco A. Galvez Nino, Miluska Castillo, Luis A. Bernabe, sandro Casavilca, Valeria Villegas, Joselyn Sanchez, Jorge Dunstan, Julio Abugatas, Carlos Castaneda, Henry Gomez. Tumor infiltrating lymphocytes are associated with p16 protein expression in acral lentiginous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4564.
Abstract 4571: Pro-inflammatory and pro-angiogenic properties of tumor associated natural killer cells in prostate cancer
Authors: Baci Denisa , Gallazzi Matteo , Mortara Lorenzo , Noonan Douglas M. , Bruno Antonino
Published: August 2019

Prostate cancer (PCa) represents the second cause of male cancer death worldwide. Immune cells can acquire pro-tumor phenotype and functions as consequence of their plasticity. Natural killer (NK) cells are cellular mediators of the innate immunity, primarily involved in tumor recognition and elimination. Altered NK phenotype and functions have been observed in different tumors including PCa. Here, we provide new insights on phenotype and functional characterization of peripheral blood tumor-associated NK cells (TANKs) from benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (ADK) patients. We also investigated NK interaction with other components of the tumor microenvironment. NK cell phenotype and functional characterization was performed by multicolour flow cytometry for surface antigens on BPH and PCa TANKs. Interactions of TANKs with other TME components (endothelial cells, macrophages) were investigated using released products from BPH and PCa TANKs for functional studies of angiogenesis, macrophage recruitments and polarization. The effects of TANKS on macrophage polarization state were evaluated by gene expression analysis RT-PCR (qPCR).We found that NK cells from peripheral blood of BPH and PCa patients acquire a pro-angiogenic/ decidual-like phenotype, identified as CD56+CD9+CD49a+CD69+ NKs. These results were confirmed also exposing heathy-donor derived NKs to conditioned media of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP). In addition, polarization of NKs with the PCa cell line conditioned media resulted in downregulation of IFNγ, TNFα and Granzyme A and increased production of pro-angiogenic factors, such CXCL8, Angiopoietin1 (Angiop1) and Angiogenin (ANG). Secretome analysis revealed the ability of NKs from BPH and PCa patients to release higher level of pro-angiogenic molecules (CXCL8, MMP-1, MMP-9 and uPAR) and cytokines/chemokines involved in macrophage recruitment, CCL1, CCL2, CCL5, CCL13, CXCL1, CXCL11 and GM-CSF. Conditioned media from BPH and PCa NKs were able to promote angiogenesis in vitro, by inducing the formation of capillary-like structures on human umbilical-vein endothelial cells (HUVEC), recruit the THP-1 monocyte cell line and polarize THP-1 differentiated macrophage towards M2-like tumor associated macrophages (TAM).Our data demonstrate that NK cells from PCa patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. Therefore, we propose tumor associated NKs as a new hallmark and potential target in prostate cancer.

Citation Format: Denisa Baci, Matteo Gallazzi, Lorenzo Mortara, Douglas M. Noonan, Antonino Bruno. Pro-inflammatory and pro-angiogenic properties of tumor associated natural killer cells in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4571.
Abstract 4573: Immune infiltrate profiling for progressive malignant stages of cutaneous squamous cell carcinoma
Authors: Alam Md Maksudul , Khandelwal Alok , Moore-Medlin Tara , Savage Hillary , Ma Xiaohui , Nathan Cherie-Ann
Published: August 2019

Cutaneous squamous cell carcinoma (cSCC) is the most common human cancer, affecting more than 300,000 individuals in the United States annually. Although, most cSCC can be treated successfully by surgery and/or radiation, a significant percentage of cSCC are aggressive and often unresectable and/or can metastasize to the lymph nodes. Organ transplant patients are at a significantly higher risk for cSCC due to a compromised immune system. Recently, the PD-1 inhibitor cemiplimab was approved for the treatment of patients with metastatic cSCC or patients with locally advanced cSCC who are not candidates for curative surgery or curative radiation. This suggests a critical role of immune cells in modulation of disease progression in cSCC. Hence, immune profiling of cSCC is important in identifying novel agents that could benefit patients. Our objective was to employ immunohistochemistry (IHC) to characterize the immune cell infiltrate in normal skin and compare it to actinic keratosis (AK), cSCC and metastatic cSCC (cSCC-M), in order to investigate the immune phenotype associated with progressive stages of malignancy. Our results suggest that AK, cSCC and cSCC-M exhibit significantly higher levels of CD3+, CD4+ and CD8+ T-cells compared to normal skin. Although, the number of CD3+ T-cells are comparable in AK, cSCC and cSCC-M, the CD8+ T-cells show higher infiltration in cSCC-M compared with cSCC and AK. The FOXP3+ Regulatory T-cells (T-reg) display higher abundance with progressive stages of carcinogenesis. However, surprisingly no significant alterations were observed in the abundance of CD4+ T-cells among AK, cSCC and cSCC-M. We also found that the CD68+ Tumor-associated macrophages (TAMs) exhibit a trend of increasing abundance with progressive disease stage. Our study suggests that the CD8+ T-cell population in cSCC-M could be potentially exhausted in the immune hostile tumor micro-environment (TME) constituted by T-reg and TAMs. Alternatively, CD8+ T-cells themselves might express FOXP3 and other immune suppressive factors, thereby reaching a state of anergy. Immunotherapy continues to be a burgeoning field and holds exciting therapeutic prospects in the treatment of cSCC. Moreover, transplant patients with aggressive cSCC are a unique patient population that could benefit from immunotherapy.

Citation Format: Md Maksudul Alam, Alok Khandelwal, Tara Moore-Medlin, Hillary Savage, Xiaohui Ma, Cherie-Ann Nathan. Immune infiltrate profiling for progressive malignant stages of cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4573.