bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6251: Blood brain barrier opening by TTFields: a future CNS drug delivery strategy
Authors: Salvador Ellaine , Kessler Almuth F. , Hörmann Julia , Burek Malgorzata , Brami Catherine T. , Sela Tali V. , Giladi Moshe , Ernestus Ralf-Ingo , Löhr Mario , Förster Carola , Hagemann Carsten
Published: September 2020

Introduction: Although a number of effective drugs are available to treat central nervous system (CNS) disorders, their ability to breach the tight regulation of the blood brain barrier (BBB) still remains a major challenge. Recently, the use of tumor treating fields (TTFields) has become an effective treatment approach for glioblastoma. Furthermore, its combination with chemotherapy significantly improved overall patient survival. Nonetheless, how TTFields could affect the BBB has not yet been studied. Our recent findings exhibit the potential of TTFields administration to open up the BBB in vitro with an optimal frequency of 100 kHz. Consequently, in this study, we therefore aimed to validate our data in vivo.

Experimental procedures: Subsequent to 100 kHz TTFields or heat treatment for 72 h, rats were i.v. injected with Evan´s Blue (EB). Next, they were sacrificed to extract and quantify EB from the brain. In the same manner, rats were injected with TRITC-dextran (TD), after which permeation was visualized in sectioned brains. Cryosections of rat brains were also prepared post-TTFields treatment. These were stained for intercellular junction proteins claudin-5, occludin and PECAM-1 as well as immunoglobulin G (IgG) to assess vessel structure. Finally, serial dynamic contrast-enhanced (DCE) MRI with gadolinium (Gd) contrast agent was performed before and after TTFields administration.

Results: Permeation of both EB and TD was observed in the brain after TTFields application. Moreover, brain cryosections displayed claudin-5 and occludin delocalization but not PECAM-1. Accumulation of IgG in the brain parenchyma was also noted. Confirming these observations, increased Gd in the brain was shown by DCE-MRI post TTFields treatment. A reversion to normal conditions was, however, detected 96 h after end of treatment demonstrated by no difference in contrast enhancement between control and TTFields-treated rats.

Conclusions: Administration of 100 kHz TTFields in rats led to alterations in BBB integrity and permeability, which signal its opening. The subsequent recovery of the BBB at the end of treatment demonstrates transient effects, hence presenting TTFields as a possible novel clinical strategy to open the BBB for enhanced and more effective drug delivery strategy for CNS disorders.

Citation Format: Ellaine Salvador, Almuth F. Kessler, Julia Hörmann, Malgorzata Burek, Catherine T. Brami, Tali V. Sela, Moshe Giladi, Ralf-Ingo Ernestus, Mario Löhr, Carola Förster, Carsten Hagemann. Blood brain barrier opening by TTFields: a future CNS drug delivery strategy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6251.
Abstract 6255: Augmented lung tumor infiltration of chemotherapeutics using synthetic antigen receptor- mesenchymal stem cells (SAR-MSCs)
Authors: Nethi Susheel Kumar , Sehgal Drishti , Panyam Jayanth , Prabha Swayam
Published: September 2020

Targeted delivery can improve drug accumulation in tumor tissue and treatment outcomes. However, currently available options suffer from a lack of selectivity for tumor cells. Mesenchymal Stem cells (MSCs) can be engineered with chemotherapeutic-loaded polymeric nanoparticles (PNPs) for tumor-targeted delivery, owing to their characteristic tumor tropic properties. Although nano-engineered MSCs demonstrated significant anticancer activity in multiple ovarian cancer models, their non-specific accumulation in clearance organs such as lungs and liver remains a concern. Parallel to the concept of chimeric antigen receptor (CAR)-T cell therapy, we advance here a strategy of modifying MSCs with synthetic antigen receptors (SARs) that target specific antigens overexpressed on cancer cells. Our approach consists of stably incorporating recombinant protein G (PG) on the surface of MSCs, followed by binding of a full-length IgG to the PG handle. Because protein G binds to the Fc region of IgG, antigen-binding affinity of the antibody is conserved. Epidermal growth factor receptor (EGFR) has been reported to be overexpressed and implicated in the pathogenesis of several malignancies especially lung cancer. Cetuximab (Cmab) is an anti-EGFR monoclonal antibody used in the treatment of metastatic lung cancer. In the current study, SAR-MSCs were developed by coating the nano-engineered MSCs with palmitated PG, followed by incorporating Cmab on the cell surface. Flow cytometry and confocal microscopy were used to confirm the incorporation of fluorescently labeled PAPG handle on MSC surface. Similarly, the installation of fluorescently labeled antibody on PAPG-functionalized MSCs was confirmed by flow cytometry. Preliminary efficacy studies in a mouse orthotopic A549-luc human lung adenocarcinoma model demonstrated significantly (P<0.05) improved survival of mice treated with Cmab-modified nano-engineered MSCs compared to isotype IgG-modified nano-engineered MSCs control group. These results suggest that the SAR technology could further improve the effectiveness of MSCs as tumor targeted drug delivery vehicles.

Citation Format: Susheel Kumar Nethi, Drishti Sehgal, Jayanth Panyam, Swayam Prabha. Augmented lung tumor infiltration of chemotherapeutics using synthetic antigen receptor- mesenchymal stem cells (SAR-MSCs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6255.
Abstract 6268: Use of the anti-viral drug ribavirin as a radiosensitizing agent in nasopharyngeal carcinoma
Authors: Huq Sakibul , Casaos Joshua , Serra Riccardo , Peters Michael , Xia Yuanxuan , Ding Andy , Ehresman Jeffrey , Kedda Jayanidhi , Morales Manuel , Gorelick Noah , Zhao Tianna , Ishida Wataru , Perdomo-Pantoja Alexander , Cecia Arba , Ji Chenchen , Suk Ian , Sidransky David , Brait Mariana , Brem Henry , Skuli Nicolas , Tyler Betty
Published: September 2020

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma with a propensity for metastasis, leading many patients to fail available treatments and/or present with advanced-stage disease. There is currently a scarcity of targeted therapies for NPC, despite working knowledge of several proteins with key roles in NPC cancer biology. These include EZH2, Snail, eIF4E, and IMPDH, which are all overexpressed in NPC and correlated with poor patient prognosis. These proteins are known to be targeted by ribavirin, a well-characterized anti-viral drug that has recently been repurposed as an anti-cancer agent in several solid and hematologic malignancies. In the present study, we investigated the potential of ribavirin as a targeted therapy and radiosensitizing agent in five human NPC cell lines. We show in vitro, using cellular growth assays, flow cytometry, BrdU cell proliferation assays, scratch wound assays, and invasion assays, that ribavirin decreases NPC cellular proliferation, migration, and invasion and promotes cell cycle arrest and cell death. Modulation of EZH2, Snail, eIF4E, IMPDH, and mTOR were observed in western blots and enzymatic activity assays in response to ribavirin treatment. In vivo, monotherapy with ribavirin reduced flank tumor growth in multiple NPC xenograft models. Given that radiation therapy is a mainstay of NPC treatment, we next investigated the effects of combining ribavirin with radiation. Using clonogenic assays and flow cytometry, we demonstrate that ribavirin enhanced the cytotoxic effects of radiation on NPC cells in vitro. Most importantly, using a flank tumor xenograft model, we show that pre-treatment with ribavirin potentiated the effects of radiation therapy in vivo. Our work suggests that NPC responds to ribavirin-mediated EZH2, Snail, eIF4E, IMPDH, and mTOR modulation and positions ribavirin for clinical evaluation as a targeted therapy and radiosensitizing agent in this cancer.

Citation Format: Sakibul Huq, Joshua Casaos, Riccardo Serra, Michael Peters, Yuanxuan Xia, Andy Ding, Jeffrey Ehresman, Jayanidhi Kedda, Manuel Morales, Noah Gorelick, Tianna Zhao, Wataru Ishida, Alexander Perdomo-Pantoja, Arba Cecia, Chenchen Ji, Ian Suk, David Sidransky, Mariana Brait, Henry Brem, Nicolas Skuli, Betty Tyler. Use of the anti-viral drug ribavirin as a radiosensitizing agent in nasopharyngeal carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6268.
Abstract 6325: CXCR4 intracellular protein regulates chemotherapy resistance by modulating the expression of death receptor 5
Authors: Nengroo Mushtaq Ahmad , Maheshwari Shrankhla , Singh Akhilesh , Singh Anup Kumar , Chaturvedi Priyank , Verma Ayushi , Arya Rakesh Kumar , Saini Krishan Kumar , Gupta Annapurna , Mishra Anjali , Datta Dipak
Published: September 2020

Chemotherapy resistance is one of the pivotal reasons for cancer-associated huge mortality and morbidity. Cancer cells become therapy resistant by smartly evading the execution of apoptosis. Death Receptors (DR) are critical pro-apoptotic factors that regulate cell death, and paucity of death receptors in cancer cells promotes therapy resistance. High expression of CXCR4 is strongly correlated with poor prognosis and adverse clinical outcomes. Inhibitors of the CXCL12-CXCR4 axis, such as FDA approved drug plerixafor (AMD3100), and Nox-A12 have shown limited clinical success in cancer treatment. Alternately, CXCR7, another CXCL12 binding receptor, could be the reason for the limited success of the CXCR4 blockade in clinics. However, recent preclinical data for blocking both the chemokine receptors failed to explain the pro-tumorigenic functions of these axes. Therefore, CXCR4-CXCL12 signaling may not be the exclusive contributor to its pro-tumorigenic functions. In our continuous effort to understand the chemokine receptor signaling inhibition as cancer therapy, here we unexpectedly discovered that instead of CXCR4-CXCL12 signaling, CXCR4 intracellular protein augments therapy resistance and pro-tumorigenic functions. By utilizing stable gain and loss of function approaches, we observed that CXCR4 selectively resists and sensitizes cancer cells against paclitaxel therapy. Our unbiased apoptosis array followed by immunoblot, FACS, real-time PCR, and ChIP analysis clearly demonstrate that CXCR4 promotes downregulation of pro-apoptotic protein DR5 via modulating differential recruitment of transcription factors p53 and YY1 to the promoter of DR5 gene. Inhibiting CXCR4 mediated signals failed to block the above phenotype. Irrespective of CXCR4 surface expression, three different colon cancer xenograft models evidently exhibited loss of CXCR4 protein results in compromised colon tumor growth in vivo. Finally, performing TCGA data mining, real-time PCR, immunofluorescence microscopy of human cancer patient samples and cell lines, we observed that expression of CXCR4 and DR5 are inversely correlated in human cancer. Together, we showed for the first time that targeting intracellular CXCR4 protein may be critical to dampen the pro-tumorigenic functions of CXCR4.

Citation Format: Mushtaq Ahmad Nengroo, Shrankhla Maheshwari, Akhilesh Singh, Anup Kumar Singh, Priyank Chaturvedi, Ayushi Verma, Rakesh Kumar Arya, Krishan Kumar Saini, Annapurna Gupta, Anjali Mishra, Dipak Datta. CXCR4 intracellular protein regulates chemotherapy resistance by modulating the expression of death receptor 5 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6325.
Abstract 6343: Mitochondrial bioenergetics mediate chemo-resistance of cancer cells
Authors: Zayou Fouzia , Chheda Chintan , Pandol Stephen , Edderkaoui Mouad
Published: September 2020

Background: Resistance to chemotherapy is a major cause of the failure of treatments in many cancers including pancreatic cancer. Accumulated evidence indicates that mitochondrial metabolic alterations are associated with cancer cells growth and proliferation. In this project, we investigated the role of mitochondrial bioenergetics in drug resistance.

Methods: Human pancreatic cancer cell lines MIA PaCa-2 and HPAF-II and mouse pancreatic cancer cell line UN-KPC-961 were treated with increased doses of chemotherapy drug Gemcitabine for 4 months until developing resistance. Mitochondrial proteins, cancer stemness markers and metabolic enzymes were measured by Western and RT-PCR. Cell survival was measured by MTT assay. Mitochondrial bioenergetics were measured by Seahorse Assay.

Results: We found that gemcitabine resistant (GR) cells grow at a much slower rate than wild type (WT) cancer cells; however, they express a significantly higher amount of cancer stemness markers such as Nanog, Oct4 and Sox2. They also expressed higher level of pro-cancer cytokines such as IL-6 and IL-4. Seahorse analysis showed a significantly lower oxygen consumption rate (OCR) in GR cells compared to WT cells. OCR did not change much in GR cells when applying inhibitors of the mitochondrial respiratory chain complex. GR cells produced lower level of ATP and lower spare respiratory capacity. However, GR cells expressed the same amount of mitochondrial proteins such as TOM40, TOM70 and COX4 suggesting the same mass of mitochondria. Pyruvate dehydrogenase-E1 Alpha1 (PDHA1) expression level was significantly lower in GR cells compared to WT cells. PDHA1 inhibition induced WT cells to develop a partial resistance to Gemcitabine; whereas, PDH1 overexpression in GR cells made these cells less resistant to Gemcitabine.

Conclusion: We showed for the first time the difference in mitochondrial bioenergetics between WT and GR cells and showed that GR cells grow independently of the mitochondrial energy production machinery. Furthermore, we identified PDHA1 as a critical mediator of cancer drug resistance.

Citation Format: Fouzia Zayou, Chintan Chheda, Stephen Pandol, Mouad Edderkaoui. Mitochondrial bioenergetics mediate chemo-resistance of cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6343.
Abstract 6374: Novel RAS inhibitors
Authors: Clark Geoffrey Justin
Published: September 2020

The Ras oncoprotein may participate in driving half of human tumors and as such remains the single most significant therapeutic target in cancer. Attempts to develop targeted inhibitors of RAS have proved extremely challenging. We have developed a novel Ras binding molecule that blocks the ability of Ras to interact with its downstream effector components and thus down-regulates Ras signaling. We have also developed the first direct inhibitor of the RAS effector RALGDS. Both compounds suppresses the transforming activity of mutant forms of Ras as well as the wild type protein when it is over-active due to defects in the NF1 protein. The compounds have no detectable toxicity in animals and readily crosses the blood-brain barrier. They are potently active in xenograft models of Ras mediated transformation and can eradicate the ability of Ras addicted tumor cells to form tumors in animals. We propose the compounds may serve as the basis for the development of new treatments for oncogenic RAS driven tumors and for many Rasopathy symptoms.

Citation Format: Geoffrey Justin Clark. Novel RAS inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6374.
Abstract 6452: Exosomal Let 7 and its downstream protein targets may serve as predictive biomarkers in peritoneal carcinomatosis
Authors: Kwong Mei Li M. , Vallejos Paul A. , Andrianarijaona Vola-Masoandro , Choi Audrey , Selleck Matthew J. , Liu Tiantian , Kabagwira Janviere , Wang Charles , Langridge William , Wall Nathan R. , Senthil Maheswari
Published: September 2020

The prognosis for patients with colorectal cancer with peritoneal carcinomatosis (PC) is dismal. Current biomarkers and imaging studies lack sensitivity and specificity to identify PC at early stages. Predictive and diagnostic serum biomarkers that help diagnose PC at early stages is necessary to improve patient outcomes. In the rapidly evolving field of liquid biopsy, exosomes show significant promise. The objective of our study is to identify exosomal biomarkers that are differentially expressed in patients with PC compared to visceral metastases (VM). Proteomic and miRNA analysis were performed using mass spectrometry and next generation sequencing on plasma exosomes from ten patients with colorectal cancer VM and nine patients with PC. Multiple members of the Let-7 family had differential levels of expression between these two patient groups. Specifically, Let-7 isoforms (Let-7a, Let-7d, Let-7e, Let-7f, Let-7g, Let-7i) were upregulated in plasma exosomes of patients with PC compared to those with VM. In addition, 33 exosomal proteins that are downstream targets of Let-7 were also found to be differentially expressed. Correlative investigation was conducted using cell lines representative of PC, VM and control to investigate the possible origins for this exosomal profile. RT-PCR was performed on cell lysates taken from normal colon (CCD-33-CO) and colorectal cancer (SK-CO-1, SNU-C1, and T84) cell lines to further identify Let-7 isoforms in these samples. Let-7 isoforms (Let 7a, Let-7b, Let-7d, Let-7e, Let-7f, Let-7i) were identified at increased levels in the colorectal cancer PC cell line in comparison to cells lines of non-PC colorectal cancer and normal colon. In-vivo and in-vitro studies demonstrate plasma exosomal Let-7 and its downstream protein targets are differentially expressed in colorectal PC compared to VM. Let-7 has been previously implicated in cancer progression and chemoresistance. While this differential expression suggests possible mechanistic differences between colorectal PC and VM, it may also offer a potential liquid biopsy for predicting peritoneal metastasis at a much earlier and treatable time.

Citation Format: Mei Li M. Kwong, Paul A. Vallejos, Vola-Masoandro Andrianarijaona, Audrey Choi, Matthew J. Selleck, Tiantian Liu, Janviere Kabagwira, Charles Wang, William Langridge, Nathan R. Wall, Maheswari Senthil. Exosomal Let 7 and its downstream protein targets may serve as predictive biomarkers in peritoneal carcinomatosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6452.
Abstract 6471: Variable expression of ZEB-1, Bcl-2, and E-cadherin in human hepatocholangio and renal cell carcinomas
Authors: Chahine Joeffrey J. , Ko Kyungmin , Kallakury Bhaskar VS , Tuma Pamela L.
Published: September 2020

Although Epithelial-Mesenchymal Transition (EMT) in cancers remains controversial, its relevance in tumor progression is a subject of investigation. The presence of cellular changes associated with EMT in bladder tumor progression have been well investigated, unlike other human carcinomas with very little data available to date. ZEB-1 which targets E-Cadherin repression is a transcriptional regulator that has been implicated in EMT, and its nuclear expression in tumor cells has been associated with aggressive malignancies in some types of cancer. Recently, it was also reported that ZEB-1 inhibits the expression of proapoptotic factor B-cell lymphoma (Bcl)-2 interacting mediator of cell death (BIM) by binding directly to the BIM promoter and repressing its transcription. We tested this hypothesis with a human cohort of 24 Cholangiocarcinoma (CC), 18 Hepatocellular (HCC) and 31 Renal Cell (RCC) human carcinomas. Formalin fixed paraffin embedded tissue sections containing benign and tumor lesions were immunostained using the Dako Autostainers Link 48 along with ZEB-1 monoclonal OT13G6 (ABCAM), Bcl-2 monoclonal 124 (Agilent, Dako), and E-Cadherin monoclonal NCH-38 (Agilent, Dako) antibodies. Nuclear, membraneous and/or cytoplasmic immunoreactivity was scored based on the intensity and percentage of positive cells in both the benign epithelium and adjacent tumor in each case. E-Cadherin was highly expressed in both the adjacent benign and malignant components with no significant variability in all three carcinomas, with ZEB-1 and Bcl-2 evaluated for the any positivity staining in both components for all cases. Results are detailed in the table below.All positive ZEB-1 cases in HCC were in high grade cases unlike the positive ZEB-1 in RCC which were in low grade cases. But more noticeably, 8/10 (80%) positive ZEB-1 were also positive for Bcl-2 in all three carcinoma types. Despite the small number of cases analyzed in this cohort a trend in the expression of ZEB-1 and its co-expression with BCL-2 may have some prognostic implications. We are currently extending this analysis to a larger number of CC, HCC, and RCC to closely examine the significance of ZEB-1, and Bcl-2 expression across differently graded lesions to further test our hypothesis.

Citation Format: Joeffrey J. Chahine, Kyungmin Ko, Bhaskar VS Kallakury, Pamela L. Tuma. Variable expression of ZEB-1, Bcl-2, and E-cadherin in human hepatocholangio and renal cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6471.
Abstract 6539: Bioinformatics algorithm of mRNA-4157 identifies neoantigens with pre-existing TIL reactivities in colorectal tumors
Authors: Zhong Shan , Breton Ben , Zheng Wei , McFadyen Iain , Hopson Kristen , Frederick Joshua , Meehan Robert S. , Zaks Tal
Published: September 2020

Background: Personalized cancer vaccines (PCVs) encode mutation-derived epitopes (neoantigens) predicted from each tumor's unique set of somatic mutations, and can trigger the activation of neoantigen-specific CD4+ and/or CD8+ T cells to target cancer cells. The accurate prediction of immunogenic neoantigens from a tumor's mutanome, which may contain hundreds of somatic mutations, is crucial for designing an effective PCV. In this study, we evaluated the performance of the bioinformatics algorithm for mRNA-4157, an mRNA-based therapeutic PCV, in identifying neoantigens that are recognized by pre-existing tumor infiltrating lymphocytes (TILs) in a cohort of colorectal cancers shared by the National Cancer Institute (NCI) [1].

Methods: Whole exome and transcriptome sequencing data from 29 patients with microsatellite stable, mismatch repair (MMR)-proficient metastatic colorectal cancer were shared by NCI. All patients had received at least one previous systemic therapy, and neoantigen-reactive TIL populations had been identified from these patients by high-throughput immunologic screening [1]. The same bioinformatics algorithm as used for mRNA-4157 was run on the sequencing data, to generate a vaccine design for each patient that included up to 34 neoantigens.

Results: Overall, 41% (26/64) of the neoantigens recognized by CD8+ or CD4+ TILs according to the NCI assay were included in the vaccines designed by the mRNA-4157 bioinformatics algorithm. For 61% (18/29) of the patients, at least one neoantigen with demonstrated CD8+ or CD4+ TIL reactivity was included in the vaccines designed. For 28% (8/29) of the patients, all neoantigens associated with demonstrated CD8+ or CD4+ TIL reactivities were included in the vaccines designed.

Conclusion: The bioinformatics algorithm for mRNA-4157 is able to predict and select neoantigens with pre-existing TIL reactivities based on human ex vivo assays for vaccine designs with high accuracy.

[1] Parkhurst MR, et al. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers. Cancer Discov. 2019 Aug;9(8):1022-1035.

Citation Format: Shan Zhong, Ben Breton, Wei Zheng, Iain McFadyen, Kristen Hopson, Joshua Frederick, Robert S. Meehan, Tal Zaks. Bioinformatics algorithm of mRNA-4157 identifies neoantigens with pre-existing TIL reactivities in colorectal tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6539.
Abstract 6557: Aescin, a triterpene saponin induces apoptosis & oxidative damage in human oral & laryngeal cancer cells via notch signaling
Authors: Kathiresan Suresh , Ramadoss Hemavardhini , Gupta Sanjay
Published: September 2020

The incidence of Head and Neck cancer in increasing worldwide. The major risk factor for these cancers includes viral infection, smoking, alcohol and tobacco consumption. Natural agents have often exhibited medicinal properties with usefulness in treating various human ailments including cancer. Aescin is one of the most prominent constituent of Aesculus hippocastanum L. (Hippocastanaceae) seeds has been conventionally used as medicinal herb. We determined the anticancer effect of aescin on human oral carcinoma (KB) cells and human laryngeal carcinoma (HEp-2) cells. Treatment of KB cells with 25µM and HEp-2 cells with 22µM aescin for 24 hours suppressed proliferation in these cells and induced morphological changes consistent with apoptosis. Exposure of both cell lines to aescin resulted in marked increase in reactive oxygen species, loss of cell viability, and reduction of cellular glutathione levels resulting in depolarization of mitochondrial membrane potential thereby increasing oxidative DNA damage in KB and HEp-2 cells. Furthermore, aescin-mediated apoptosis in these cells progress via notch signaling as shown by downregulation of Notch1, Notch2, Jagged-1, HES1, and HEY1 expression. Taken together, our results demonstrate that aescin initiate apoptosis in KB and HEp-2 cells by increasing oxidative damage implying that aescin might be developed as a potential chemotherapeutic agent for the treatment of human oral and laryngeal cancer.

Key word: Oral carcinoma, Aescin, apoptosis, anti-proliferative, Notch signaling

Citation Format: Suresh Kathiresan, Hemavardhini Ramadoss, Sanjay Gupta. Aescin, a triterpene saponin induces apoptosis & oxidative damage in human oral & laryngeal cancer cells via notch signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6557.
Abstract 6666: Evaluation of the complexity of the immune response to CMP-001, a TLR9 agonist
Authors: Sabree Shakoora , Voigt Andrew , Rogers Laura , Blackwell Sue , Lemke-Miltner Caitlin , Weiner George J.
Published: September 2020

CMP-001 is a virus-like particle containing a TLR9 agonist (class A CpG ODN) encapsulated by the Qβ capsid. Preclinical studies and early phase clinical trials of intralesional injection with CMP-001, combined with anti-PD1 antibody, demonstrate promising results. Prior work by our group has demonstrated that the immunostimulatory effects of CMP-001 are dependent on generation of an anti-Qβ antibody response which results in opsonization of CMP-001 by plasmacytoid dendritic cells (pDCs) and production of IFNα. While a variety of cell types including monocytes and B cells can take up CMP-001, the immune response to CMP-001 is based in large part on rapid induction of IFNα production by pDCs. The current studies were designed to assess the downstream effects of CMP-001 and anti-Qβ antibody on various arms of the immune response using normal donor human peripheral blood mononuclear cells (PBMCs) as a model.

Stimulation of PBMCs with CMP-001 and anti-Qβ antibody induced production of IFNα from pDCs as previously observed. This IFNα production occurred very rapidly (within 2 hours) and was short lived, but had long term secondary effects, including induction of CXCL10 production by monocytes. Single cell RNA sequencing done 24 hours after stimulation demonstrated additional downstream changes including the following:

Upregulation of CXCL10, MIP1-α, IL1-RA and IDO by monocytes.

Downregulation of CD14 by monocytes.

Upregulation of a broad variety of interferon response genes in various cell types including NK cells and T cells.

We conclude the primary effect of in situ immunization with CMP-001 is to induce rapid production of IFNα production by pDCs. This results in significant downstream effects on various cell populations that contribute to the anti-tumor immune response. Understanding these pathways could help identify the most promising combination immunotherapies involving CMP-001. Ongoing studies are exploring the kinetics of these responses and whether similar changes are observed in the tumor microenvironment.

Citation Format: Shakoora Sabree, Andrew Voigt, Laura Rogers, Sue Blackwell, Caitlin Lemke-Miltner, George J. Weiner. Evaluation of the complexity of the immune response to CMP-001, a TLR9 agonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6666.
Abstract 6673: B7-H3-targeted 4-1BB activation potentiates CD8 T cell-dependent antitumor immunity without systemic toxicity
Authors: You Gihoon , Lee Yangsoon , Kang Yeon-Woo , Park Han Wook , Park Kyeongsu , Jung Jaeho , Lee Seung-Woo
Published: September 2020

4-1BB is a costimulatory receptor on activated T and NK cells, and the stimulation of 4-1BB by the natural ligand or agonistic mAb enhances cellular proliferation and effector functions. Immunotherapy targeting 4-1BB has been tested for cancer patients; however, dose-limiting toxicities of 4-1BB agonists restrict further clinical development. B7-H3 (CD276) is overexpressed on the cell surface of multiple cancers and tumor-associated endothelial cells, yet barely on healthy adult tissues. To restrict 4-1BB stimulation activity in tumors, we have developed an FcγR-binding null bispecific IgG1 antibody consisting of B7-H3-targeting IgG and two anti-4-1BB single-chain variable fragments. The B7-H3x4-1BB bispecific antibody (B5x1D8) shows a potent in vitro T cell costimulatory activity in the presence of B7-H3 on the tumor cells. B5x1D8 rapidly accumulates in B7-H3-overexpressing tumors compared to 4-1BB agonistic mAb. B5x1D8 elicits a 4-1BB-dependent anti-tumor response in three different B7-H3-overexpressing murine tumor models. More importantly, in contrast to 4-1BB mAb, B5x1D8 does not induce any observable systemic abnormalities. Treatment of B5x1D8 increases the density, cytokine production, and proliferation of CD8 T cells in the tumor. Characterization of TILs indicates that B5x1D8 increases the number of PD-1+TIM-3+ “terminal effector” CD8 T cells for eliminating tumor cells. Furthermore, a combination of B5x1D8 and immune checkpoint blockade (ICB), anti-PD1, synergistically inhibits tumor growth. The human 4-1BB-targeting bispecific antibody also induces B7-H3-dependent 4-1BB costimulation and inhibits tumor growth in human 4-1BB transgenic mice. In sum, our data suggest that the B7-H3x4-1BB bispecific antibody represents an alternative form of IgG-based 4-1BB agonistic mAb for effective and safe cancer immunotherapy against B7-H3 positive cancers as monotherapy and combination therapy with other immunotherapy, like ICB.

Citation Format: Gihoon You, Yangsoon Lee, Yeon-Woo Kang, Han Wook Park, Kyeongsu Park, Jaeho Jung, Seung-Woo Lee. B7-H3-targeted 4-1BB activation potentiates CD8 T cell-dependent antitumor immunity without systemic toxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6673.
Abstract 6693: Metronomic treatment of orally available oxaliplatin and pemetrexed to potentiate the efficacy of anti-PD-1 by modulating tumor immunity
Authors: Maharjan Ruby
Published: September 2020

The rational combination of metronomic chemotherapy (MCT) with immunotherapy has the potential of immune modulation inducing enhancement of host immunity, thereby overcome tumor induced immunosuppressive defense. In this study, oral formulation of oxaliplatin (OXA) and pemetrexed (PMX) were developed by complexing ionically with Nα-deoxycholyl-l-lysyl-methylester (DCK) as intestinal permeability enhancer to form OXA/DCK-NE and PMX/DCK-OP respectively. OXA/DCK-NE and PMX/DCK-OP were combined for oral MCT and its anti-tumor effect was examined against maximum tolerated dose (MTD) of same drug combination by intravenous administration in colon cancer (CT26) syngeneic mouse model. MCT was able to modulate immunity in CT26 mouse model compared to MTD, such as enhancement of effector T-cell population in tumor, elevation of tumor antigen uptake, and activation of dendritic cells as analyzed by flow cytometry of tumor tissue and draining lymph nodes (TDLN). Moreover, cytotoxic T-cell and helper T-cell populations in lymphoid organs such as blood, spleen, and TDLN were found to be upregulated in MCT group but not in MTD group. Furthermore, on combining MCT with αPD-1, the tumor suppression was more effective showing 97.8% decrease in tumor volume compared to that of control leading to tumor free mice (95% complete response). Mice that achieved complete antitumor response upon re-challenge, demonstrated that combination therapy of MCT and αPD-1 induced memory response which was further confirmed by 1.5-fold enhanced antigen specific memory response compared to αPD-1 only treated group as detected by secretion of INF-γ.

In this study, we observed that combination of OXA/DCK-NE and PMX/DCK-OP with αPD-1 therapy synergized immunomodulatory effect by enhancing the populations of effector T-cells thereby inducing complete response and antigen specific memory response. Thus, this suggests the rationality of combination treatment with oral metronomic OXA and PMX with αPD-1 to elicit effective immune modulation.

Citation Format: Ruby Maharjan. Metronomic treatment of orally available oxaliplatin and pemetrexed to potentiate the efficacy of anti-PD-1 by modulating tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6693.
Abstract 683: Identification of a macrocyclic peptide that blocks the CD47-SIRPα interaction and its antitumor effect
Authors: Hazama Daisuke , Murata Yoji , Okamoto Takeshi , Tanaka Daisuke , Sakamoto Mariko , Kakuchi Yuka , Saito Yasuyuki , Kotani Takenori , Nishimura Yoshihiro , Matozaki Takashi
Published: September 2020

Much recent attention has focused on the modification of immune responses as a basis for new cancer treatments. Immune checkpoint inhibitors, such as monoclonal antibodies (mAbs) to CTLA-4, PD-1, and PD-L1, have provided clinical benefit in patients with a variety of types of cancer, and a growing number of agents targeting immune checkpoints are in preclinical and clinical development. SIRPα is a transmembrane protein that is abundant in myeloid cells, such as macrophages and dendritic cells. SIRPα interacts with another transmembrane protein CD47 through their ectodomains, and they constitute the cell-cell communication system (the CD47-SIRPα system). We previously demonstrated that the interaction of SIRPα on macrophages with CD47 on tumor cells prevented the phagocytosis of tumor cells by macrophages. In contrast, blockage by anti-SIRPα antibodies of the CD47-SIRPα interaction enhanced the phagocytic activity of macrophages against antibody-opsonized tumor cells, suggesting that the CD47-SIRPα axis acts as an immune checkpoint pathway involved in tumor escape from immunosurveillance. Here, by the use of random nonstandard peptide integrated discovery (RaPID) system, we have identified novel macrocyclic peptides that bind to the extracellular domain of SIRPα with high affinity. These SIRPα-binding macrocyclic peptides inhibited the CD47-SIRPα interaction. Moreover, one of the SIRPα-binding macrocycles promoted the phagocytosis of antibody-opsonized tumor cells by macrophages in vitro and enhanced the antitumor effect of mAbs against tumor antigens in xenograft or syngeneic mouse models of cancer. Our results thus suggest that macrocyclic peptides against SIRPα hold promise as a novel tool for cancer immunotherapy.

Citation Format: Daisuke Hazama, Yoji Murata, Takeshi Okamoto, Daisuke Tanaka, Mariko Sakamoto, Yuka Kakuchi, Yasuyuki Saito, Takenori Kotani, Yoshihiro Nishimura, Takashi Matozaki. Identification of a macrocyclic peptide that blocks the CD47-SIRPα interaction and its antitumor effect [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 683.
Abstract 707: Combinatorial assessment of ctDNA release and mutational burden predicts clinical outcome from anti-PD-(L)1 therapies in non-small-cell lung cancer
Authors: Fang Wenfeng , Ma Yuxiang , Yin Jiani C. , Zhou Huaqiang , Wang Fufeng , Bao Hua , Wang Ao , Wu Xue , Hong Shaodong , Yang Yunpeng , Huang Yan , Zhao Hongyun , Shao Yang W. , Zhang Li
Published: September 2020

Background: Blood-based detection of circulating tumor DNA (ctDNA) is a non-invasive and clinical accessible source of biomarkers that enables selection of non-small-cell lung cancer (NSCLC) patients for treatment with anti-PD-(L)1 therapies. However, new approaches are necessary to improve enrichment of responders.

Patients and Methods: Pretreatment plasma ctDNA from 97 advanced-stage NSCLC patients who underwent anti-PD-(L)1 therapy from December 2015 to August 2017, with matched tissue samples from 66 patients, were profiled using a targeted next-generation sequencing (NGS) panel (Geneseeq) encompassing 422 cancer-relevant genes. External validation was performed using two large randomized trials, POPLAR and OAK. Results: In the 72 patients with adequate ctDNA release (maximum somatic allele frequency, MSAF ≥2%), high blood tumor mutational burden (bTMB) was associated with significantly prolonged median PFS (110 vs 60 days, HR=0.54 [95%CI, 0.31-0.92]; log-rank p = 0.02) and a trend towards improved DCB (34.8% vs 22.5%, p=0.39). While a high concordance was observed between bTMB and tTMB (Spearman's ρ= 0.71), the greatest progression-free survival (PFS) improvement was achieved with concomitantly high bTMB and tTMB (mPFS, 156 vs 59 days, HR=0.47 [95%CI, 0.24-0.91], log-rank p=0.02). Remarkably, the subset of patients with low ctDNA release (MSAF<2%), and consequently non-evaluable bTMB, derived similar PFS benefit as those with high bTMB. The additive predictive value of MSAF and bTMB was externally validated by results from the two large randomized trials.

Conclusions: Our findings validated panel-assessed bTMB as a promising biomarker that demonstrated added value over tissue testing alone for benefit with anti-PD-(L)-1 therapies. Our findings also provided the first clinical evidence suggesting that matched tissue and blood testing might be necessary to refine the predictive value of TMB. Moreover, we identified low ctDNA MSAF as an important and robust predictor of improved immunotherapy outcome.

Citation Format: Wenfeng Fang, Yuxiang Ma, Jiani C. Yin, Huaqiang Zhou, Fufeng Wang, Hua Bao, Ao Wang, Xue Wu, Shaodong Hong, Yunpeng Yang, Yan Huang, Hongyun Zhao, Yang W. Shao, Li Zhang. Combinatorial assessment of ctDNA release and mutational burden predicts clinical outcome from anti-PD-(L)1 therapies in non-small-cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 707.
Abstract 739: A disintegrin and metalloproteinase 8 as a potential blood biomarker for early diagnosis of gastric cancer
Authors: Choi Jae-Il , Park Joon Seong , Chung Hye Won , Lim Jong-Baeck
Published: September 2020

Purpose: We evaluate the clinical significance of A Disintegrin and Metalloproteinase 8 (ADAM 8) as a potential blood biomarker for gastric cancer (GC).

Materials & Methods: Blood ADAM 8 was measured by ELISA and CEA, Cytokines/chemokines (IL-23, SDF-1α/CXCL12, IL-8, and sCD40L) were measured by chemiluminescent immunoassay. They were compared among 5 groups; normal/gastritis, high-risk, early GC (EGC), advanced GC (AGC) without distant metastasis and AGC with distant metastasis by one-way analysis of variance in both training (n=80) and validation dataset (n=241). Clinicopathological features of GC and GC-associated cytokines were evaluated for their correlations with blood ADAM 8. To evaluate the diagnostic accuracy to predict GC, receiver operating characteristic (ROC) curve and logistic regression were used.

Results: In both training and validation dataset, blood ADAM 8 increased along the GC carcinogenesis (ANOVA, all of p<0.01). Blood ADAM 8 were significantly higher in EGC and AGC compared with high-risk and normal (post-hoc Bonferroni, all of p<0.01). However, there was no significant difference among cancer groups. Blood ADAM 8 was correlated with N-stage (Spearman's correlation, γs,=0.320, p=0.011), but not correlated with T-stage or M-stage. Pearson's correlations showed blood ADAM 8 was closely correlated with pre-inflammatory cytokines (IL-23, SDF-1α/CXCL12, all of p<0.05), but not correlated with pro-angiogenic cytokines (IL-8, sCD40L). ROC curve and logistic regression demonstrated serum ADAM 8 showed higher diagnostic accuracy (sensitivity, 73.7%; specificity, 86.2%) than CEA (sensitivity, 23.1%; specificity, 91.4%). Combination of ADAM 8 and CEA more increased the diagnostic accuracy to predict GC (sensitivity, 81.8%; specificity, 84.0%).

Conclusion: Blood ADAM 8 is a promising biomarker for early detection of GC.

Citation Format: Jae-Il Choi, Joon Seong Park, Hye Won Chung, Jong-Baeck Lim. A disintegrin and metalloproteinase 8 as a potential blood biomarker for early diagnosis of gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 739.
Abstract 809: nCounter for detection of clinically relevant alterations in exosomes of non-small cell lung cancer cells and patients
Authors: Capitán Ana Giménez , Bracht Jill , Huang Chung-Ying , Teixidó Cristina , Reguart Noemí , Boykin Rich , Warren Sarah , Beechem Joseph M , Ramirez Santiago Viteri , García Juan José , Aguilar Andrés , Costa Rafael Rosell , Gerlach Jay , Molina-Vila Miguel Angel
Published: September 2020

Background: ALK, ROS1 and RET fusions and MET exon 14 skipping variant (METex14) are present in 10-15% of advanced non-small-cell lung cancer (NSCLC) patients and their accurate identification is critical to guide targeted therapies. In a significant number of cancer patients, the tumor tissue available is insufficient for genetic analysis and repeated tissue biopsies for monitoring the course of the disease and the emergence of resistance are not feasible. Liquid biopsies constitute the only alternative available in these cases, but NGS techniques have shown insufficient sensitivity for fusion detection in blood samples. The nCounter technology has been adapted to detect fusions and skipping variants in FFPE tumor biopsies and we aimed to validate it for exosomes.

Methods: Exosomes were purified using a miRCURY kit (Qiagen) and RNA was extracted using the TRI reagent (MRC Inc). A customized nCounter panel (Nanostring) for detection ALK, ROS1 and RET fusion transcripts and MET ex14 mRNA was used with a 10-cycles preamp step. First, proof-of-concept experiments were run by testing exosomes isolated from the culture medium of cell lines. Next, we tested exosomes isolated from the blood of NSCLC patients with know genotypes.

Results: nCounter fusion probes successfully detected ALK, RET and ROS1 fusion transcripts in exosomes isolated from the culture medium of the cell lines H3122 (EML4-ALKv1), H2228 (EML4-ALKv3), HCC78 (SLC34A2-ROS1) and LC/2-Ad (CCDC6-RET). Exosomes from a cell line established from a patient progressing to alectinib were also positive for EML4-ALKv1and showed high MET expression levels, while exosomes from the fusion-negative cell lines A549 and H23 tested negative. Finally, fusion transcripts were detected in exosomes purified from the blood of fusion positive NSCLC patients but not in fusion negative cases.

Conclusions: nCounter can detect ALK, RET and ROS1 fusion transcripts in exosomes purified from the blood of advanced NSCLC patients

Citation Format: Ana Giménez Capitán, Jill Bracht, Chung-Ying Huang, Cristina Teixidó, Noemí Reguart, Rich Boykin, Sarah Warren, Joseph M Beechem, Santiago Viteri Ramirez, Juan José García, Andrés Aguilar, Rafael Rosell Costa, Jay Gerlach, Miguel Angel Molina-Vila. nCounter for detection of clinically relevant alterations in exosomes of non-small cell lung cancer cells and patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 809.
Abstract 835: Novel framework for quantifying synergy in high-throughput drug combination cytotoxicity experiments
Authors: Thurman Robert , Arthur Bill
Published: September 2020

Combination therapies are a cornerstone of cancer treatment, enabling a strategy of engaging multiple mechanisms of action. The in vitro testing of drug combinations is therefore a key step in drug discovery, and plate-based assays and robotics now allow for high-throughput cytotoxicity experiments that test combinations at doses diluted at high resolution. While additivity models such as Bliss, Loewe and Highest Single Agent (HSA) are well-established for gauging the synergy or antagonism of combinations, new frameworks are required for incorporating these models in the context of hundreds of experiments testing dozens of dose combinations per experiment. We have developed a framework that compares observed cytotoxicity results against the predictions from additivity models on a dose-by-dose basis across an entire matrix of dose combinations. Visualizations readily highlight any synergistic or antagonistic deviations from additivity. Statistical tests are then performed using 3x3 blocks of adjacent combinations. Such an approach mutes the effects of outliers and rewards consistency of activity in nearby doses. To capture the overall trend in an experiment and to facilitate comparisons across multiple experiments, we propose a summary metric, TSDA (total significant deviation from additivity) that takes into account both the absolute deviations from additivity and their statistical significance. Generally, positive values of TSDA indicate overall synergy, while negative values indicate overall antagonism. Null distributions for TSDA have been generated using simulations of sham experiments (same drug for both axes in a combination). We have applied this framework to over 1500 individual combination experiments spanning a range of cell lines and therapeutic mechanisms. The framework has been used to successfully guide follow-up in vivo experiments and to inform clinical plans.

Citation Format: Robert Thurman, Bill Arthur. Novel framework for quantifying synergy in high-throughput drug combination cytotoxicity experiments [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 835.