bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2654: B cells produce IL-27 in breast cancer to upregulate PD-L1 expression and promote tumor progression
Authors: Yan Hui , Viswanadhapalli Suryavathi , Perez Dariela , Chupp Daniel , Fernandez Maria , Wang Jingwei , Wu Shuai , Rivera Carlos E. , Moroney Justin B. , Taylor Julia , Im John , Luo Yiliao , Liu Junhao , Gangadhara Sareddy , Curiel Tyler , Casali Paolo , Vadlamudi Ratna , Xu Zhenming
Published: September 2020

B cells frequently infiltrate breast cancer (BCa) tumors, but their roles are inadequately understood. As shown by our analyses of the TCGA datasets and human BCa arrays, tumoral expression of B cell hallmark factors (e.g., CD20 and PAX5) coincides with those of the two subunits of pleiotropic cytokine IL-27, EBI3 and IL-27p28, whose expression is associated with poor prognosis in patients. In syngeneic mouse models, B cells promote BCa progression and do so by producing IL-27, but not IL-35 (IL-12/EBI3 heterodimer). Upon treatment by recombinant IL-27 or co-culturing with IL-27-producing B cells, BCa cells as well as targeted B lymphocytesactivate STAT1/STAT3 and upregulate immune checkpoint PD-L1 and chemokine receptor CXCR3, blocking of which synergistically arrest BCa cell proliferation in vitro and tumor growth in vivo, concomitant with reduction in PD-L1+B cells. Finally, B cell-specific deficiency in IL-27 receptor or PD-L1 inhibits BCa tumorigenesis, showing the dual functions of tumor-infiltrating B cells, i.e., IL-27 production for paracrinic PD-L1 upregulation.

Citation Format: Hui Yan, Suryavathi Viswanadhapalli, Dariela Perez, Daniel Chupp, Maria Fernandez, Jingwei Wang, Shuai Wu, Carlos E. Rivera, Justin B. Moroney, Julia Taylor, John Im, Yiliao Luo, Junhao Liu, Sareddy Gangadhara, Tyler Curiel, Paolo Casali, Ratna Vadlamudi, Zhenming Xu. B cells produce IL-27 in breast cancer to upregulate PD-L1 expression and promote tumor progression [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 2654.
Abstract 2667: CaMKK2 knockout in mice challenged by orthotopic GBM leads to upregulation of MHC II on TAMs and an increase in accumulation of CD4+ TILs
Authors: Chakraborty Molly A. , Tomaszewski William H. , Polania Jessica Waibl , Riley Lauren S. , Racioppi Luigi , Sanchez-Perez Luis A. , Sampson John H.
Published: September 2020

Glioblastoma (GBM) is the most lethal primary brain tumor in adults, with the median survival time for GBM patients being only about 15 months. Although immunotherapy treatments have been successful in more immunogenic tumors, it has shown very limited success in GBM. This could be because it is difficult for T cells to infiltrate the tumor, and the tumors have a very immunosuppressive tumor microenvironment (TME) that can often consist of up to about 30 percent tumor associated macrophages (TAMs) by mass. Thus, two ways of possibly improving immunotherapy treatments on GBMs would be to increase the number of tumor infiltrating lymphocytes (TILs) present, and to target tumor-supportive cells such as TAMs. Calmodulin Dependent Protein Kinase Kinase 2 (CaMKK2) is a gene that has been shown to be highly expressed in myeloid cells and is known to alter their phenotype. Recent studies in breast cancer have shown that myeloid cells adopt a more immunostimulatory phenotype in murine knockout CaMKK2 models. However, it is not well understood how CaMKK2 causes this immunosuppressive effect and warrants further exploration. The data from our experiments help to answer this question. High-dimensional flow cytometry with 15 parameters was used in order to determine the identities of immune cells found in the tumor of wildtype and CaMKK2-/- mice. The results of our experiments show that there appear to be more CD4+ TILs present in the TME of knockout mice and that there is an increase in MHC II expression on TAMs found in knockout mice. Since CD4+ TILs recognize peptides presented on MHC II molecules, and there is an increase in the number of CD4+ TILs and expression of MHC II on TAMs in knockout mice, we hypothesize that a stronger immune response could be instigated against the tumor. Therefore, it can be concluded that a possible reason that knocking out CaMKK2 leads to a less immunosupressive TME is because MHC II expression on TAMs is upregulated, and the number of CD4+ TILs in the TME is increased. Whether or not the increase in MHC II expression causes the increase in the number CD4+ TILs requires further exploration. This makes CaMKK2 a promising therapeutic target to treat tumors with immunosuppressive TMEs such as GBM.

Citation Format: Molly A. Chakraborty, William H. Tomaszewski, Jessica Waibl Polania, Lauren S. Riley, Luigi Racioppi, Luis A. Sanchez-Perez, John H. Sampson. CaMKK2 knockout in mice challenged by orthotopic GBM leads to upregulation of MHC II on TAMs and an increase in accumulation of CD4+ TILs [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 2667.
Abstract 2774: Longitudinal characterization of patient-derived orthotopic xenograft brain tumor models
Authors: Schueler Julia , Heins Mariette , Shatillo Artem , Lehtimäki Kimmo , Peille Anne-Lise , Stenius Taina-Kaisa , Bragge Timo , Rytkönen Jussi , Miszczuk Diana , Huhtala Tuulia
Published: September 2020

Gliomas are the most common primary brain tumors and currently the prognosis is still poor. Due to this, it is one of the main areas in oncological research and drug development programs. Innovative therapies are vital to improve treatment outcomes but must be developed to enable trafficking across the blood brain barrier (BBB). For this advent, animal models provide important information prior to clinical studies. Among the different in vivo models, orthotopic PDX models represent best the tumor microenvironment and natural variability of tumors, hence providing the most reliable results over species.

In the brain tumor field, imaging has a central role in clinical diagnosis and as a prognostic factor to monitor therapy response in patients. Magnetic resonance imaging (MRI) is widely used for clinical diagnosis and disease follow up as it offers unprecedented soft tissue contrast and high spatial resolution non-invasive way. In addition to imaging, molecular profiling, e.g. circulating immune biomarkers and local oncometabolites in the tumor environment may facilitate as important translational biomarkers in development of immunotherapy for gliomas in addition to traditional histopathological readouts.

The purpose of this work was to analyze possible heterogeneity of tumors in vivo, growth rate and volume in stereotactically implanted orthotopic PDX brain tumor models using MRI/MRS imaging. In addition to imaging, neurological scoring was performed to monitor general animal welfare, cytokine profiles from plasma to observe immunological responses over time and determination of oncometabolites in plasma and brain tissues combined with traditional histopathological changes were performed. The data from orthotopic models was also compared to readouts in subcutaneous models.

As a conclusion, translational in vivo imaging techniques were applied to study orthotopic tumor progression combined with circulating immune biomarkers, and general welfare scoring. These readouts provide a powerful and translational research tool together with oncological disease animal models allowing comprehensive evaluation of disease progression and treatment interventions for in vivo studies.

Citation Format: Julia Schueler, Mariette Heins, Artem Shatillo, Kimmo Lehtimäki, Anne-Lise Peille, Taina-Kaisa Stenius, Timo Bragge, Jussi Rytkönen, Diana Miszczuk, Tuulia Huhtala. Longitudinal characterization of patient-derived orthotopic xenograft brain tumor models [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 2774.
Abstract 2793: Application of conditionally reprogrammed cell in in vitro drug sensitivity test and PDX modeling
Authors: Long Yuan , Xi Song , Wen Danyi
Published: September 2020

Tumor cell immortalization through conditional reprogramming, particularly from limited biopsy specimen, is an invaluable tool to generate propagating tumor cells for cell-based drug sensitivity assay and bio-banking in vitro. We have successfully reprogrammed primary tumor cells from a number of tumor types, including lung carcinoma, breast cancer and glioma .etc. The conditionally reprogrammed cells exhibited typical colonized growth, which is well maintained upon cryopreservation. In some cases, the cells can be passaged for multiple times and likely become sable cell lines. Like primary tumor cells, conditional reprogrammed tumor cells are reliable to test drug sensitivity in vitro. We attempt to extend conditional reprogrammed tumor cells to reconstitute and grow PDX models in vivo for testing drug efficacy. The conditional reprogrammed tumor cells could also be used in PDX establishment and the tumor formation rate is much higher than the primary tissue's. In summary, this study explores and expands application of conditional reprogrammed tumor cells in in vitro drug efficacy test and in vivo PDX Modeling.

Citation Format: Yuan Long, Song Xi, Danyi Wen. Application of conditionally reprogrammed cell in in vitro drug sensitivity test and PDX modeling [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 2793.
Abstract 2794: A novel HNSCC organoid-on-a-chip platform for preclinical investigation and drug screening
Authors: Bonilla Silvia , Driehuis Else , Lanz Henriëtte , Clevers Hans , Joore Jos , Queiroz Karla , Vulto Paul
Published: September 2020

Head and neck squamous cell carcinoma (HNSCC) cancer is a broad category of tumor types arising from the inner lining of various anatomic structures including the oral cavity, pharynx, larynx and salivary glands. Treatment often involves an intensive combination of surgery, radiotherapy and chemotherapy. Despite this, tumor recurrence rates remain high and survival rates are relatively poor. Here, we describe a novel high throughput drug screening platform combining the OrganoPlate®, a microfluidic based 3D-culture system, and HN cancer-derived organoids. MIMETAS develops Organ-on-a-Chip-based models for evaluation of new medicines. Our unique microfluidic technology enables testing of compounds on miniaturized 3D organ models in high-throughput. Hereby we show the establishment of HNSCC cancer-derived organoids in 2-lane OrganoPlate®, and its usefulness for phenotypic drug screenings. The aim of the study is to capture differential sensitivity of HN organoid lines to either a common platinum-based chemotherapy or to a targeted drug still not used in the clinic. Organoid lines (T1, T3 and T5) were embedded in the 2-lane OrganoPlate® as single cells in an extracellular matrix gel. At day 2, cultures were treated with cisplatin or PARP-inhibitor niraparib for 120 hours. Additionally, cultures exposed to high doses of cisplatin were evaluated after one week of recovery in order to determine treatment failure and recapitulate potential patient relapse.

Drug response was evaluated by assessment of morphology (phase contrast), cell viability (total nuclei count, alamar blue assay), proliferation (EdU incorporation) and DNA damage (phosphorylated histone H2AX). Organoid cultures grow well under medium perfusion in the 2-lane OrganoPlate® and selective sensitivity to cisplatin and niraparib is revealed by the used readouts. The high-throughput, microfluidic 2-lane OrganoPlate® platform offers an attractive method for growing HNSCC cancer-derived organoids, supporting development of individualized tumour models for phenotypic drug screenings.

Citation Format: Silvia Bonilla, Else Driehuis, Henriëtte Lanz, Hans Clevers, Jos Joore, Karla Queiroz, Paul Vulto. A novel HNSCC organoid-on-a-chip platform for preclinical investigation and drug screening [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 2794.
Abstract 2832: Multiplex fluorescent imaging of the metastatic melanoma microenvironment reveals spatially-dependent macrophage interactions modulating tumor-infiltrating T-cells
Authors: Wang Victor G. , Martinek Jan , Gulati Ananya , Boruchov Hannah , Ray Kelly , Palucka Karolina , Chuang Jeffrey H.
Published: September 2020

Tumor-infiltrating lymphocytes (TIL) hold strong prognostic value in determining patient survival as well as response to immune checkpoint blockade across many tumor types, especially metastatic melanoma. Tumors often evolve mechanisms to prevent TIL access and/or function to allow for continued growth. One such mechanism is the recruitment and reprogramming of tumor-associated macrophages (TAM) which suppress TILs through a variety of pathways. The interactions between TAMs and TILs that result in a suppressive tumor microenvironment (TME) have been studied in vitro and with spatially-agnostic sequencing, leaving the location-specific contributions underexplored. Interrogating the TAM-TIL interactions in an intact TME will uncover novel mechanisms responsible for TAM function and may be key to reversing TIL immunosuppression, improving anti-tumor immune responses, survival, and potentially therapy response. We use histocytometry, a multiplex quantitative tissue imaging method capable of capturing whole-slide tumor sections, to explore the human metastatic melanoma TME in situ and resolve the spatial contributions of TAM-TIL interactions. We demonstrate a T-cell preference for physical contact with phagocytic TAMs in the tumor but non-phagocytic TAMs in the stroma. We further model immune synapses from histocytometry to probe the function underlying the TAM-TIL preference based on the directionality of signaling components. The model suggests T-cells in contact with macrophages in the tumor are communicating more frequently with the phagocytic TAMs as compared to their non-phagocytic counterparts, indicating that this interaction extends beyond a simple proximity preference. In addition, proliferating T-cells communicate more frequently with macrophages than non-proliferating T-cells but this effect limited to the intratumor space. Together we demonstrate the functional consequences of physical TAM-TIL interactions with unprecedented spatial resolution. This work unravels important TAM interactions that shape the TME which have not been previously appreciated, providing novel insight into the forces modifying T-cell function in the TME and revealing new TAM biology to explore further.

Citation Format: Victor G. Wang, Jan Martinek, Ananya Gulati, Hannah Boruchov, Kelly Ray, Karolina Palucka, Jeffrey H. Chuang. Multiplex fluorescent imaging of the metastatic melanoma microenvironment reveals spatially-dependent macrophage interactions modulating tumor-infiltrating T-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 2832.
Abstract 2836: Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages
Authors: Shephard Miranda , Pease Nicholas , Vinnedge Lisa M. Privette
Published: September 2020

Breast cancer is the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing murine breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 signaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek−/− mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek−/− tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. Furthermore, we observed increased TSLP expression and angiogenesis, as determined by CD31 staining, in Dek-expressing tumors in vivo. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model.

Citation Format: Miranda Shephard, Nicholas Pease, Lisa M. Privette Vinnedge. Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages [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 2836.
Abstract 2842: Neutrophil migration towards tumor-conditioned media correlates with the malignant potential of breast tumor cells
Authors: Hein Lauren , SenGupta Shuvasree , Xu Yang , Parent Carole
Published: September 2020

During tumor development, neutrophils and other immune cells infiltrate the tumor niche. Neutrophils have been shown to have primarily pro-tumorigenic effects, and high levels of circulating neutrophils are indicative of a poor prognosis. Tumor-associated neutrophils (TANs) migrate to tumors by sensing chemical gradients produced by tumor cells; however, the mechanisms by which tumors and TANs interact and impact each other remain to be determined. It has been shown that malignant breast tumors, such as triple-negative breast tumors, harbor more TANs compared to less aggressive breast tumors. We hypothesize that neutrophil recruitment to the tumor niche depends on tumor-secreted, chemotactic factors that are primarily produced by malignant breast tumors. To test this, we used a panel of breast cancer cells grown in monolayers and more physiologically-relevant tumor spheroids, which were formed over the course of four days using the hanging-drop method. We characterized the size and morphology of the spheroids, which were generated from the poorly metastatic BT474 cells, as well as the highly malignant BT549 cells, MDA-MB-231 cells, and M4 cells of the MCF-10A cell series. We then compared the ability of tumor-conditioned media (TCM) isolated from either monolayers or spheroids to stimulate the migration of primary human neutrophils using transwell assays. We found that, compared to TCM isolated from control or poorly metastatic cell lines, the TCM derived from highly malignant cancer cells robustly stimulated the migration of neutrophils. These data indicate that breast cancer cells secrete factors with unique profiles that have varying potentials to recruit neutrophils. We are in the process of identifying specific tumor-secreted factors in the TCM using mass spectrometry. Additionally, we are assessing how the presence of neutrophils affects cancer cell migration and invasion by utilizing a coculture system in which tumor spheroids and primary neutrophils are imaged in real time. We reason that understanding the role of TANs during breast cancer progression will provide insight into novel therapeutic approaches, particularly in the context of highly metastatic forms of the disease.

Citation Format: Lauren Hein, Shuvasree SenGupta, Yang Xu, Carole Parent. Neutrophil migration towards tumor-conditioned media correlates with the malignant potential of breast tumor 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 2842.
Abstract 2858: Comparative surface proteomic analysis of human breast cancer cell lines
Authors: Benton Betsy , Snovida Sergei , Herting Katherine , Opperman Kay , Kaboord Barbara
Published: September 2020

Enrichment of cell surface proteins is commonly performed by biotinylation using amine-based chemistry; however, this method is often associated with intracellular labeling and difficulty distinguishing the true cell surface proteins from background contaminants. We developed a robust cell surface protein isolation method using amine-based chemistry that is compatible with mass spectrometry (MS). We applied this methodology to evaluate the surface proteomes of three breast cancer cell lines, including hormone responsive MCF-7 cells, and two triple negative breast cancer cell lines (TNBC), BT549 and Hs5789T. Thermo Scientific™ Tandem Mass Tag (TMT) Reagents, combined with high pH reverse phase fractionation of the labeled peptides, were used to multiplex protein identification and quantitative analyses by tandem MS. A heat map of enriched proteins from the three cell lines showed clearly distinct banding patterns of clustered protein groups. Relative quantitative differences in several classes of enriched proteins were observed, including tyrosine kinases, G-Protein-Coupled Receptors (GPCRs), and cell adhesion proteins. The tyrosine kinase EGFR was found to be more highly expressed in TNBC cell lines compared with MCF-7 cells. In contrast, the receptor tyrosine-protein kinase erbB-3 was found to be significantly higher in the MCF-7 cells compared with the TNBC cells. Several GPCRs were identified and found to vary across the three cell lines, including Frizzled 7 which is an emerging target for cancer therapy. Multiple cell adhesion proteins were also identified, including neural cell adhesion molecule L1 which promotes migration of MCF7 cells. Where possible, the data set was found to be consistent with literature reports. In conclusion, we have developed a highly sensitive method for cell surface proteomic analyses that can be effectively used to identify differences in cell types as well as for biomarker discovery and potential drug therapy targets.

Citation Format: Betsy Benton, Sergei Snovida, Katherine Herting, Kay Opperman, Barbara Kaboord. Comparative surface proteomic analysis of human breast cancer cell lines [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 2858.
Abstract 286: Regulation of APOBEC3B gene expression through the Rb/E2F pathway
Authors: Roelofs Pieter A. , Brown William L. , Span Paul N. , Martens John W. , Goh Chai Y. , Chua Boon H. , Kappei Dennis , Harris Reuben S.
Published: September 2020

The propensity of tumors to increase genomic diversity allows for a bottleneck-type adaptation to anti-cancer treatments. The genomic alterations fueling these adaptations in many different tumor types are, in part, brought about by ongoing endogenous activity of the DNA cytosine deaminase APOBEC3B (A3B). The ability of A3B to convert DNA cytosine bases into uracils has been shown to facilitate C-to-T transitions, C-to-G transversions, and DNA breakage. While low in healthy tissues, A3B expression and activity is elevated in tumors and may increase further throughout metastatic disease. However, molecular mechanisms responsible for transcriptional upregulation of A3B in tumor cells are poorly understood. Here, we investigate the possibility that the Rb/E2F pathway, which is often dysregulated in breast cancer, contributes to A3B gene expression. First, we cloned the A3B promoter upstream of a luciferase reporter and used a combination of deletion and site-directed mutagenesis to demonstrate that only one out of five in silico predicted E2F binding sites is functionally relevant, as disrupting this site caused constitutive reporter activation. Moreover, expression of the BK polyomavirus T-antigen, which inactivates Rb, induced the reporter and additional activation was not observed by mutating the functional E2F binding site. Second, SILAC-labelled nuclear protein extracts were subjected to affinity purification coupled to quantitative mass spectrometry to compare transcriptional complexes that bound wild-type promoter sequences, but not sequences with mutant E2F binding site promoter elements. These proteomics experiments showed that proteins belonging to the DREAM and PRC1.6-complexes strongly bind the wild-type but not the mutant E2F binding sites. Third, RNAi-mediated knock-down of candidate E2F family members implicated E2F6, and to a lesser extend E2F4, which are components of the PRC1.6 and DREAM complexes, respectively, in A3B gene repression. Taken together, these studies indicate that A3B expression may be suppressed by the concerted effort of two different E2F binding complexes and that Rb inactivation, by viral T-antigen here and genetic mutation as often seen in breast cancer, is able to upregulate A3B gene expression, promote genomic DNA mutagenesis, and fuel tumor evolution.

Citation Format: Pieter A. Roelofs, William L. Brown, Paul N. Span, John W. Martens, Chai Y. Goh, Boon H. Chua, Dennis Kappei, Reuben S. Harris. Regulation of APOBEC3B gene expression through the Rb/E2F pathway [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 286.
Abstract 2931: VGF as a theranostic marker in neuroendocrine lung cancer
Authors: Yang Li-Hao , Yu Chia-Cherng , Chou Yu-Ting
Published: September 2020

Activating EGFR mutations are predictive markers for response in lung cancer patients under the treatment of EGFR-tyrosine kinase inhibitors (TKIs), whereas acquired resistance to EGFR-TKIs inevitably occurs in patients. Neuroendocrine transformation is one of the mechanisms behind the development of acquired resistance to EGFR-TKIs. In this study, we report that VGF, a secreted factor highly expressed in neurons and neuroendocrine tissues, is a potential theranostic marker in neuroendocrine lung tumors. Gene expression profiling analysis revealed that VGF levels were elevated during the differentiation of neuronal stem cells to neurons with surged H3K27ac modification at the VGF locus. We observed that VGF was highly expressed in lung neuroendocrine cancer cells. Moreover, VGF expression was low in lung adenocarcinoma but was enriched during EGFR-TKI selection. Correlation analysis showed that VGF was positively associated with chromogranin A, chromogranin B, and synaptophysin expression in lung tumors. ChIP-qPCR assays showed that the signal of H3K27ac modification at the VGF locus was increased in EGFR-TKI resistant cells compared to their sensitive counterparts. Clonogenic analysis and annexin-V staining assays revealed that VGF silencing attenuated cellular grown with increased apoptosis in neuroendocrine lung cancer cells as well as in EGFR-TKI resistant cells, indicating the potential of VGF as a therapeutic target. Kaplan-Meier analysis showed that high VGF expression predicted poor survival outcome in patients with lung tumors. Our findings provide an insight into the oncogenic role of VGF and suggest the potential of VGF as a theranostic for lung cancer.

Citation Format: Li-Hao Yang, Chia-Cherng Yu, Yu-Ting Chou. VGF as a theranostic marker in neuroendocrine 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 2931.
Abstract 2939: Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes
Authors: Figel Sheila , Birkemeier Meaghan , Dharma Sanam , Steinmetz Emma , Ciesielski Michael , Fenstermaker Robert
Published: September 2020

Background: Previously we found that survivin is expressed on the surface of cancer cells in vitro, and is targetable using certain survivin-specific antibodies. The survivin gene is alternatively spliced into two alternative isoforms, ΔEX3 and 2B, which diverge structurally from the canonical isoform in their C-termini but retain a conserved BIR (Baculovirus IAP Repeat) domain.

Methods: In order to characterize plasma membrane survivin (PM-SVN), tagged proteins representing canonical survivin (isoform 1), Δex3 and 2B were transiently expressed in multiple cancer cell line backgrounds. Relative surface expression was measured by flow cytometry staining of intact cells. Mutant constructs were used to determine the structural requirements for translocation. Imaging flow cytometry (Imagestream) was used to visualize survivin isoform localization, and to detect co-localization on the cell surface. Secreted exosomes were examined for expression of survivin isoforms.

Results: Isoforms 1, Δex3 and 2B could be detected on the surface of HEK293T embryonic kidney, HeLa cervical adenocarcinoma, U87 glioblastoma, and A1207 glioblastoma cells. PM-SVN expression was variable, however Δex3 showed robust membrane expression across all cell lines. Deletion of a predicted transmembrane sequence in Δex3's unique C-terminus did not impair its translocation, and deletion of the first 10 amino acids, thought to be required for dimerization, disrupted surface expression only in HEK293T cells. Imaging flow cytometry analysis of cells revealed polar localization of PM-SVN, with specific punctate areas seen in 13-20% of cells. Staining with antibodies targeting both isoform 1 and Δex3 showed co-immunofluorescence in cell membrane puncta, indicating the formation of heterodimers. Stressing cells by heat shock increased the proportion of PM-SVN in 293T and A1207 cells; in 293T, this was accompanied by enrichment of survivin protein in secreted exosomes.

Conclusions: In addition to canonical survivin, isoforms Δex3 and 2B are displayed on the surface of cancer cells, and can be secreted as exosomal cargo. Membrane translocation may be stress-induced and appears to require the conserved BIR domain. PM-SVN shows a polar distribution on the cell membrane, and isoforms 1 and Δex3 heterodimerize within these puncta.

Citation Format: Sheila Figel, Meaghan Birkemeier, Sanam Dharma, Emma Steinmetz, Michael Ciesielski, Robert Fenstermaker. Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes [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 2939.
Abstract 2964: Saffron has enhanced anti-proliferative effect against colorectal cancer cells with deficient DNA mismatch repair
Authors: Amin Amr , Soleimani Akbar , Murali Chandraprabha , Beresova Lucie , Shoraka Hamid , Brim Hassan , Ashktorab Hassan
Published: September 2020

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. A subtype of CRC is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy, however, these roles in the context of of dMMR have not been explored. In this study, we aimed to study the effects of saffron in CRC cell lines with dMMR.

Methods: Saffron crude extracts and specific compounds (crocin, crocetin and safranal) were used in colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MH3) and HCT116+3+5 (Inserted MLH1 and MSH3). CDC25b, p-H.H2AX, TPDP1, and GAPDH were analyzed by Western blot. Cell cycle was analyzed by FACS. Proliferation and cytotoxicity were analyzed by MTT. Wound and migration assays were also performed.

Results: Saffron crudes restricted the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. Wound healing assay indicates that deficient MMR cells are doing better than proficient MMR cells. The expression of CDC25b and TDP1 were upregulated in proficient MMR cell compared to deficient MMR cell, while H2AX was significantly upregulated in both cells type particularly at >10 mg/ml saffron in a concentration-dependent manner (after normalization with GADPH as control).The reduction in cellular proliferation and motility might be due to cell cycle arrest at G2-phase rather than to apoptosis. The major active saffron compounds, safranal and crocin reproduced total saffron crudes effects but with lesser outcome.

Conclusion: Saffron's anti-proliferative and healing effects is significant in cells with deficient MMR MLH1 and MSH3 genes. This effect may have a therapeutic implications and benefits for the CRC MSI tumors that are generally not recommended for 5FU-based treatment.

Citation Format: Amr Amin, Akbar Soleimani, Chandraprabha Murali, Lucie Beresova, Hamid Shoraka, Hassan Brim, Hassan Ashktorab. Saffron has enhanced anti-proliferative effect against colorectal cancer cells with deficient DNA mismatch repair [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 2964.
Abstract 2995: Dissecting renal cell carcinoma vulnerabilities: Using CRISPR/Cas9 to study resistance to glutaminase inhibition
Authors: Raczka Aleksandra M. , Reynolds Paul A.
Published: September 2020

The aim of this study was to identify genes, which when knock out confer resistance to a novel glutaminase inhibitor in Renal Cell Carcinoma (RCC). RCC represents around 3% of all malignancies worldwide, with its most common type, clear cell RCC (ccRCC), making up around 70-80% of all RCC. The most frequent genomic alteration in ccRCC involves loss of VHL gene in >90% of ccRCC cases. Large-scale cancer genomics sequencing studies have identified several driver genes beyond VHL, particularly PBRM1 (40%), SETD2 (15%), and BAP1 (10%). RCC is characterised by significant tumour heterogeneity and inherent (in 25-30% cases) or acquired resistance to available chemotherapeutics. Glutamine addiction is a potential new therapeutic target for RCC as it has been recently shown not only to rely on glutamine for energy generation and maintenance of redox homeostasis, but also de novo pyrimidine synthesis. CB-839 is a small, orally administered reversible inhibitor of human kidney-type glutaminase (GLS). CB-839 is currently in early phase clinical trials and shows promising results. Given the significant incidence of resistance to previously approved therapies, we have applied a genome-scale CRISPR/Cas9 approach in a cell culture model of ccRCC (786-0 cell line) to identify candidate genes, which when knocked down confer resistance to CB-839. Next generation sequencing data analysis of drug-selected sgRNA library representation from two timepoints was performed using the MAGeCKFlute bioinformatics workflow. We conducted the screen in two biological replicates and verified successful genome-scale sgRNA library coverage at baseline in both cases. As a result, we identified candidate genes and pathways that are implicated in tumour metabolism, autophagy and metastasis. Additionally, this analysis identified KD genes, which in combination with CB-839 are synthetic lethal. We have prioritized the top 12 candidate genes, which we are validating in four cell lines (786-0, A498, A704 and Caki-1). After establishing CRISPR/Cas9 single gene knockdown (KD) cell lines, KD is confirmed using RT-qPCR and Western Blot, followed by functional analysis. KD cells were treated with increasing concentrations of the drug in parallel with a cell line containing a scrambled sgRNA to detect growth changes between the two. After in vitro validation, we performed ex vivo validation of the candidate hits. Our data show that the genome-scale CRISPR/Cas9 approach is effective in identifying candidates modulating drug resistance to CB-839.

Citation Format: Aleksandra M. Raczka, Paul A. Reynolds. Dissecting renal cell carcinoma vulnerabilities: Using CRISPR/Cas9 to study resistance to glutaminase inhibition [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 2995.
Abstract 3102: Analysis of plasma and serum as a source of circulating tumor DNA for hotspot mutations detection in liquid biopsy
Authors: Pittella-Silva Fabio , Chin Yoon Ming , Chan Hui Ting , Nagayama Satoshi , Miyauchi Eisaku , Low Siew-Kee , Nakamura Yusuke
Published: September 2020

Blood-based analysis of ctDNA is a promising tool for cancer screening, drug selection, detection of minimum residual disease, monitoring release/recurrence and response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess highly-valuable serum samples collection and comparative studies on the use of serum vs plasma as ctDNA source are still essential. Here we analyzed cell free DNA (cfDNA) from matched plasma and serum samples from healthy donors, colorectal and lung cancer patients to evaluate the differences of serum and plasma on mutation detection efficacy and reproducibility by targeted NGS. We initially addressed the difference of both analytes on the quality of cfDNA recovery and on sequencing performance. We then compared mutation detection using matched samples from healthy individuals spiked-in with standard oligonucleotides including common mutations. The amount of cfDNA was more abundant in serum than plasma and the presence of longer DNA fragments was significantly higher in serum samples. Spiked-in mutated nucleotides were detected in both plasma and serum samples, but allele frequencies (AF) were approximately half in serum samples compared with plasma samples, suggesting ctDNA from serum is more diluted in DNAs of noncancerous origins. We further confirmed this difference in detection efficiency using matched plasma and serum samples from cancer patients and found that mutations with low AF are more frequently missed in serum samples. In addition, concordance with tissue-biopsy mutations found at diagnosis was better in plasma samples. Finally, we examined the use of fragment size selection as an alternative tool to improve mutation detection in serum samples. Our findings clearly demonstrated that the use of serum in retrospective studies should consider the limitations for detecting low AF mutations and indicate the advantages of plasma samples for prospective clinical applications of liquid biopsy.

Citation Format: Fabio Pittella-Silva, Yoon Ming Chin, Hui Ting Chan, Satoshi Nagayama, Eisaku Miyauchi, Siew-Kee Low, Yusuke Nakamura. Analysis of plasma and serum as a source of circulating tumor DNA for hotspot mutations detection in liquid biopsy [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 3102.
Abstract 3270: Dual blockade of PD-L1 and LAG-3 using a bispecific antibody improves anti-tumor immunity
Authors: Ni Haiqing , Qiu Yajing , Jing Hua , Zhang Pan , Chen Bingliang , Wu Zhihai , Zhou Shuaixiang , Yu Michael , Liu Junjian
Published: September 2020

Background: Anti-PD-1/PD-L1 treatments are effective in a fraction of patients with advanced malignancies. However, the majority of patients still do not respond. Resistance to cancer immunotherapy can be mediated by additional immune checkpoints. Lymphocyte-activation gene 3 (LAG-3) is a member of the Ig superfamily and participates in immune suppression. Combination of anti-PD-1 and anti-LAG-3 showed synergistic anti-tumor effect in preclinical mouse models and early clinical trials. Here, we develop IBI323, an anti-PD-L1/LAG-3 bispecific antibody, which preserves the dual pathway blockade function while processes new biological functions.

Material & Methods: The binding affinity of IBI323 to antigens were measured by Fortebio and confirmed at cellular level using PD-L1 overexpressing cells and primary T cell. IBI323 was also assessed by ELISA for PD-1 and LAG-3 pathway blocking function. IBI323 mediated PD-L1+ and LAG-3+ cells bridging was evaluated by flow cytometry. Anti-tumor activity of IBI323 was evaluated in human PD-L1 knock-in mice bearing PD-L1 overexpressing MC38 tumors. Besides tumor growth, the frequencies and quality of the tumor-specific T cells were assessed by flow cytometry. In addition, florescence labeled antibodies was used for detecting the biodistribution of IBI323 in vivo.

Results: IBI323 bound to PD-L1 and LAG-3 with high affinity (KD <1 nm). At cellular level, IBI323 bound to PD-L1 overexpressing cells and LAG-3+ T cells in a similar manner to parental antibodies. IBI323 was able to block PD-1/PD-L1 and LAG3/MHCII interactions simultaneously. Moreover, bridging between PD-L1+ cells and LAG-3+ cells was observed in the presence of IBI323 in vitro. Correspondingly, IBI323 was mainly distributed at the location of PD-L1+ tumor in vivo. In mice harboring established PD-L1+ MC38 tumor, IBI323 treatment was superior to combination of individual anti-PD-L1 and LAG-3 antibodies. The better activity of IBI323 treatment was reflected by an enhanced number of tumor-specific CD8+ cytotoxic T cells.

Conclusions: We developed a novel bispecific antibody against PD-L1 and LAG-3, which resulted in dramatic tumor growth inhibition. The anti-tumor efficacy of IBI323 was better than either monotherapy or anti-PD-L1 and LAG-3 combination treatment. Based on the encouraging preclinical results, IBI323 could soon be evaluated in clinical trials.

Citation Format: Haiqing Ni, Yajing Qiu, Hua Jing, Pan Zhang, Bingliang Chen, Zhihai Wu, Shuaixiang Zhou, Michael Yu, Junjian Liu. Dual blockade of PD-L1 and LAG-3 using a bispecific antibody improves anti-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 3270.
Abstract 3361: Discovery and preclinical characterizations of a humanized anti-claudin 18.2 antibody SPX-101
Authors: Zhu Guidong , Ma Jichun , Ye Jingdong , Qin Jingdong , Cai Yi
Published: September 2020

Claudin 18.2 (CLDN 18.2) is a major component of the tight junctions and is highly expressed in several cancers including gastric (80%), esophageal (80%), pancreatic (60%) and lung cancers, but only expressed at very low levels in healthy tissues. The chimeric IgG1 CLDN 18.2 antibody zolbetuximab significantly improved PFS and OS in gastric cancer patients in a CLDN 18.2-dependent manner on the background of EOX. This research aimed to produce humanized anti-claudin18.2 antibodies with enhanced activities and improved pharmaceutical characteristics compared to known CLDN 18.2 antibodies. We used several immunization methods to produce mouse anti-CLDN18.2 antibodies with high affinity and excellent selectivity against CLDN 18.1. Humanization of a selected clone led to a humanized anti-CLDN18.2 antibody (h533o) with significantly improved binding affinity and cellular activity in ADCC and CDC assays compared to zolbetuximab. The binding affinity of h533o was relatively insensitive to elevated temperatures or acidity, suggesting the robustness of target engaging ability in tumor microenvironment. Maturation and selection by the off-rate approach, using phage libraries that systematically modified the CDR regions, identified multiple antibodies with comparable affinity and selectivity. Mutational scan of the Fc region in h533o identified SPX-101 with reduced ADA effect in mice while retaining other favorable properties. SPX-101 suppressed tumor growth in several xenograft mouse models including NUGC4 and HEK293 cells that stably expressed CLDN18.2. In summary, SPX-101 represents a humanized anti-CLDN18.2 antibody with high affinity and specificity, high effector cell-mediated cytotoxicity, low immunogenicity, and in vivo tumor control efficacy in xenograft rodent models.

Citation Format: Guidong Zhu, Jichun Ma, Jingdong Ye, Jingdong Qin, Yi Cai. Discovery and preclinical characterizations of a humanized anti-claudin 18.2 antibody SPX-101 [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 3361.
Abstract 3366: In vivo efficacy of a PD-L1 targeted, antigen seeding engineered toxin body
Authors: Ramos Hilario J. , Brieschke Brigitte , LeMar Sara , Dekker Joseph D. , Iberg Aimee , Robinson Garrett L. , Sarkar Asis , Anand Banmeet , Singh Melissa M. , Zhao Jay , Higgins Jack P. , Willert Erin K.
Published: September 2020

Engineered toxin bodies (ETBs) comprised of a proprietarily engineered Shiga-like Toxin A subunit (SLTA) genetically fused to antibody-like binding domains work through novel mechanisms of action and can force internalization, self-route through intracellular compartments to the cytosol, and induce potent cell-kill via the enzymatic and permanent inactivation of ribosomes. Our PD-L1 targeted ETB, MT-6402, includes antigen seeding technology (AST), and additionally delivers a viral antigen for presentation in complex with MHC-I to resident viral-specific cytotoxic T lymphocytes (CTLs). The fusion protein provides a powerful, dual mechanism of action to specifically target and destroy PD-L1 positive tumor and inhibitory immune cells. In vitro testing of human tumor cell lines has demonstrated that cell surface PD-L1 expression is required for activity of the PD-L1 targeted ETBs. MT-6402 demonstrated effective depletion of PD-L1 expressing cells across a panel of immortalized cancer cell lines from multiple origins, including lung, skin, breast, and ovary. In a co-culture assay of PD-L1 target cells and antigen specific T-cells, PD-L1 targeted MT-6402 delivers a viral antigen to the target cells and can activate cytokine secretion and T-cell mediated killing. Additionally, MT-6402 treatment of human peripheral blood mononuclear cells (PBMCs) leads to selective depletion of PD-L1 positive cells (IFN-γ stimulated monocytes) in the absence of depletion of PD-L1 low/negative lymphocytes. Murine models, including patient derived xenografts, have demonstrated that the PD-L1 targeted ETBs are efficacious in vivo. Additional in vivo tumor models to further describe the effect of MT-6402 are being explored. Exploratory studies in a primate model have shown that MT-6402 can be tolerated at levels shown to induce T cell activation (type 1 cytokines). MT-6402 delivers a powerful and unique dual mechanism of action. The combination of a PD-L1 specific direct cell kill and redirection of a robust effector T cell response to the tumor has potential benefit in solid tumor indications, including in the relapsed setting, when disease has progressed after checkpoint and/or other therapies.

Citation Format: Hilario J. Ramos, Brigitte Brieschke, Sara LeMar, Joseph D. Dekker, Aimee Iberg, Garrett L. Robinson, Asis Sarkar, Banmeet Anand, Melissa M. Singh, Jay Zhao, Jack P. Higgins, Erin K. Willert. In vivo efficacy of a PD-L1 targeted, antigen seeding engineered toxin body [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 3366.