bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1149: Systems pathology by Optra 30 color multiplex immunofluorescence automated whole slide imaging system integrated with image analysis
Authors: Gholap Abhi , Jadhav Anagha P. , Somwanshi Suraj , Kamble Gurunath , Doshi Isha , Huang Jiaoti
Published: August 2019

Background A paradigm shift from single marker immunohistochemistry (IHC) to multiplexing in molecular histopathology has helped better understand the cellular composition and heterogeneity of sub population of cells in tissues. Presently, one needs to manually exchange filters after each multiplex scan for capturing all fluorophores per staining round. This is very cumbersome and inefficient, as this process requires rescanning the same slide multiple times. Moreover, visual assessment and quantification of biomarkers is limited by inter and intra-observer variability. We hereby present an automated multispectral whole-slide imaging system integrated with image analysis for detection of upto 30-color multiplex assay with spectral unmixing and cell segmentation functionality.

Material and Methods

Multiplex IHC whole slide imaging Formalin Fixed Paraffin Embedded rat brain; indirect IHC stained sections with 10 antibodies using photostable fluorophores were obtained. The staining was based on antigenic integrity and antibody binding affinity. Optra's multispectral fluorescent whole slide scanning process was optimized to image sensitive antigens in early cycles and robust antigens in latter cycles. The fully automated fluorescence scanning system imaged weakly expressing biomarkers in the visible range (480-620 nm), highly expressing targets in the violet/blue (405-440 nm), far-red (650-680 nm) and NIR (700-800 nm) range across the entire usable light spectrum (350-800 nm).

ResultsWe could simultaneously detect up to 30 biomarkers within a single tissue section, using 12 staining-imaging-stripping cycles, with 10 antibodies screened per cycle. Software measures enabled separation of spectrally overlapping markers in the multiplex assay. A computer-controlled X-Y-Z stage used autofocusing at 20X/40X magnification. Integrated image analysis solution performed comprehensive evaluation of antigens of interest with accurate quantification of the target proteins based on parameters including but not limited to hue value/width, intensity threshold scoring, morphological characterization, pixel-count thresholding and color saturation.

Conclusion This proposed solution is efficient compared to the current imaging methods to tackle the aforementioned image acquisition and image analysis challenges associated with multiplexing assays.

Note: This abstract was not presented at the meeting.

Citation Format: Abhi Gholap, Anagha P. Jadhav, Suraj Somwanshi, Gurunath Kamble, Isha Doshi, Jiaoti Huang. Systems pathology by Optra 30 color multiplex immunofluorescence automated whole slide imaging system integrated with image analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1149.
Abstract 1150: Targeted immunoPET imaging of prostate cancer using a novel CD133 antibody
Authors: Glumac Paige , LeBeau Aaron
Published: August 2019

Prostate cancer is one of the most frequently diagnosed malignancies and the second leading cause of cancer related death among American men. Aggressive variant prostate cancer (AVPC) accounts for approximately 33% of all prostate cancer-related deaths and is characterized by visceral metastasis, minimal responses to current therapies, and poor overall prognosis. The current disease monitoring strategy for AVPC is positron emission tomography - computed tomography (PET/CT) with [18F]-fluorodeoxyglucose (FDG), which is not specific to prostate cancer cells and thus is inadequate in many cases. As such, novel targeted imaging agents are urgently needed for earlier detection and better monitoring of AVPC. The transmembrane glycoprotein, CD133, is overexpressed in AVPC and we have identified a single chain variable fragment, termed HA10, which preferentially recognizes a glycosylation-independent epitope on CD133. Further characterization of HA10 revealed that it was able to identify CD133 in cells, patient tissue samples, and in vivo xenograft models using fluorescent imaging. The purpose of this study was to investigate the performance of the radioimmunoconjugate, 89Zr-HA10, for µPET/CT imaging in preclinical models of prostate cancer. HA10 was conjugated to p-SCN-Deferoxamine (DFO) and then radiolabeled with [89Zr]Zr-oxalate at room temperature. Parental (CWR-R1) and CD133-transduced (CWR-R1CD133) prostate cancer cell lines were implanted subcutaneously into male athymic nu/nu mice. Mice were administered 180 µCi of 89Zr-HA10 via tail vein injection and µPET/CT imaging was conducted at 24, 48, 72, and 144 hours post-injection. The 89Zr-HA10 construct was successfully labeled with a radiochemical purity >99% as determined by radio-thin layer chromatography. Specificity of 89Zr-HA10 was demonstrated for CD133-positive tumors with strong tumoral uptake detection from 24-72 hours. Furthermore, the margins of the CD133-positive tumors were clearly defined following 3D reconstruction of the µPET/CT images. These studies suggest that 89Zr-HA10 is a promising immunoPET imaging agent for AVPC and potentially other CD133-positive cancer subtypes. Moreover, the use of 89Zr-HA10 as a radiotracer will facilitate a more selective strategy for diagnosis and disease monitoring of these patients compared to the conventional FDG radiotracer. Further studies seek to evaluate the potential of 89Zr-HA10 to detect small metastatic lesions in vivo.

Citation Format: Paige Glumac, Aaron LeBeau. Targeted immunoPET imaging of prostate cancer using a novel CD133 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1150.
Abstract 1152: Disulfiram targets both proliferating cancer cells and cancer stem-like population in ER-positive breast cancer
Authors: Jang Seojin , Oh Eunhye , Kim Yoon-Jae , Cho Tae-Min , Park Jung Min , Park Soeun , Park Minsu , Seo Jae Hong , Kim Ji Young
Published: August 2019

Drug repositioning can benefit from reduced development risk in terms of safety, toxicity and dosage, as it centers upon drugs that have already been tested in clinical trials. Disulfiram (DSF), a drug for the treatment of alcoholism, has potential anti-cancer activities in a copper (Cu)-dependent manner. In the present study, we investigated the effect of DSF on cell viability, apoptosis, breast cancer stem cells (BCSC)-like properties and p53 activation in ER-positive breast cancer MCF7 and T47D cells. DSF treatment caused a significant suppression of cell viability and marked induction of cell death in a Cu-dependent manner. This response was accompanied by caspase-3/-7 and p53 activation in ER-positive breast cancer cells. The latter phenomenon was associated with G1 phase arrest, coinciding with a significant increase in accumulation of nuclear p21 and downregulation of cyclin D1 and survivin. DSF treatment elicits a significant inhibitory effect on these BCSC-like properties including ALDH1 activity and CD44+/CD24- subpopulations. To examine the effect of DSF on mammosphere-forming ability, MCF7 and T47D cells were cultured in anchorage-independent serum-free culture conditions in the presence or absence of DSF and Cu. DSF considerably suppressed mammosphere formation, as evidenced by significant reduction in the number and volume of mammospheres. Our results demonstrate that DSF induces apoptosis and effectively targets cancer stem-like population, suggesting that disulfiram may be potentially effective for the treatment of ER-positive cancer.

Citation Format: Seojin Jang, Eunhye Oh, Yoon-Jae Kim, Tae-Min Cho, Jung Min Park, Soeun Park, Minsu Park, Jae Hong Seo, Ji Young Kim. Disulfiram targets both proliferating cancer cells and cancer stem-like population in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1152.
Abstract 1153: Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells
Authors: Sharma Nitya V. , Chau Monica , Mukherjee Subhas , Ross James , Miller Brandon , Zhang Changming , Kong Jun , Tucker-Burden Carol , Kaissi Emily , Reitnauer John W. , Dundar Bilge , Olson Cheryl L. , Bix Gregory , Brat Daniel J.
Published: August 2019

Glioblastoma (GBM) is a highly malignant primary brain tumor and has a poor prognosis and is resistant to the most advanced therapies. The development of highly hypoxic centrally located necrotic regions is strongly associated with accelerated GBM progression, yet mechanistic explanations are not complete. Hypoxic regions within GBM are highly enriched for glioma stem cells (GSC), which are critical for tumor initiation, recurrence and therapeutic resistance due to their self-renewing capabilites. While most GBM cells migrate outward, away from hypoxia, GSCs accumulate there, forming a hypercellular border called pseudopalisades that promote their survival. Mechanisms that mediate GSC accumulation within pseudopalisades are unknown. We hypothesize that GSCs are enriched in the pseudopalisades due to the homing of a subpopulation towards the supportive hypoxic microenvironment. This in turn activates critical hypoxia-induced drivers to promote stemness, survival and migratory behavior encouraging the recruitment of more GSCs. Using two genetically distinct patient-derived GBM cell lines, we found that exposure to hypoxia in vitro led to enrichment of GSCs among a heterogeneous population of GSCs and non-GSCs. We found that hypoxia enhanced the expression of stem cell, pro-survival and pro-migratory factors in GSCs, such as the pro-migratory chemokine SDF-1α, and its cognate receptor CXCR4. Importantly, we found hypoxia also induced upregulation of OLIG2, one of four core transcription factors necessary and sufficient to induce stemness, in GSCs and non-GSCs exposed to hypoxia. We determined that OLIG2 was necessary for the expression of stem cell markers, and that its knockdown reduced cellular migration in hypoxia vs normoxia in GSCs. This suggests a critical role for OLIG2 in both hypoxia-induced stemness and homing behavior. We also generated a unique model of localized hypoxia in one brain hemisphere through vaso-cclusion and observed the homing of xenografted GBM cells towards hypoxia, regulated by OLIG2 in vivo. These data shed light on how hypoxic tumor microenvironments facilitate mechanisms vital to GBM progression and therapeutic resistance.

Citation Format: Nitya V. Sharma, Monica Chau, Subhas Mukherjee, James Ross, Brandon Miller, Changming Zhang, Jun Kong, Carol Tucker-Burden, Emily Kaissi, John W. Reitnauer, Bilge Dundar, Cheryl L. Olson, Gregory Bix, Daniel J. Brat. Hypoxic microenvironments promote stemness and enhance the directed migration of glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1153.
Abstract 1157: Single-cell transcriptmoics identifies rhamm positive proliferative cells within human colorectal cancer stem cells
Authors: Nakano Michitaka , Yoshikane Kikushige , Miyawaki Kohta , Mizuno Shinichi , Yamaguchi Kyoko , Yamauchi Takuji , Ariyama Hiroshi , Kusaba Hitoshi , Nakamura Masafumi , Maeda Takahiro , Baba Eishi , Akashi Koichi
Published: August 2019

Cancer stem cells (CSCs) can be prospectively isolated based on specific surface marker such as CD44, CD133, LGR5, and CD166, in human colorectal cancer. However, the employment of these CSCs' markers are still insufficient to prospectively isolate the colorectal CSCs. To overcome the cellular heterogeneity of previously reported CSCs fraction, we performed single-cell mRNA sequencing (scRNAseq) of 72 cells within organoid. By conducting scRNAseq, we identified CSCs-like sub-population with high expression of MKI67 and Cyclin families, which was generally expressed in proliferative state of the cells within the organoid. Remarkably, high expression of hyaluronan-mediated motility receptor (HMMR) encoding cell membranous and cytoplasmic molecule, receptor for hyaluronan mediated motility (RHAMM), which was reported to associate with CD44 in hyaluronan binding, was observed in the CSCs-like cells. Prospective isolation of RHAMM+ CD44+ cells, that were sub-population of conventional CSCs fraction, revealed higher organoid- and tumor-forming abilities. Furthermore, comprehensive gene expression analysis also demonstrated the proliferative gene expression within RHAMM+ CD44+ cells. Here, we report RHAMM+ proliferative cells that sub-fraction the conventional CSCs population.

Citation Format: Michitaka Nakano, Kikushige Yoshikane, Kohta Miyawaki, Shinichi Mizuno, Kyoko Yamaguchi, Takuji Yamauchi, Hiroshi Ariyama, Hitoshi Kusaba, Masafumi Nakamura, Takahiro Maeda, Eishi Baba, Koichi Akashi. Single-cell transcriptmoics identifies rhamm positive proliferative cells within human colorectal cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1157.
Abstract 1158: Seven genes were closely associated with ALDH1 in triple negative breast cancer
Authors: Ishikawa Takashi , Narui Kazutaka , Yamada Akimitsu , Kida Kumiko , Shima Hidetaka , Endo Chiho , Adachi Shoko , Sugae Sadatoshi , Miyagi Yohei , Ichikawa Yasushi , Endo Itaru
Published: August 2019

Background: Aldehyde dehydrogenase1 (ALDH1) is widely accepted as a marker of normal and malignant breast stem cells. We reported that ALDH1 expression was associated with aggressive phenotypes and poor clinical outcome of breast cancer patients. However, mechanics of how ALDH1 affects cancer stem cell features is not known.

Aim: To examine genes associated with ALDH1 and find novel druggable targets for the future cancer stem cell therapy.

Methods: ALDH1-positive and ALDH1-negative cells were dissected by a laser capture microdissection from 5 ALDH1 positive triple negative breast cancer samples. A differential analysis of mRNA expression was performed between ALDH1 positive and negative breast cancer cells in each sample by Affymetrix GeneChip microarrays. 1) Microarray analysis suite (MAS5) and robust multi-chip average (RMA) were used as preprocessing algorithms to analyze gene expression data and to extract genes differently expressed between ALDH1 positive and negative cells. 2) The network analysis was performed with extracted genes with MAS5 by the comprehensive information platform “KeyMolnetTM”.

Results: 1) 54,682 genes were extracted and 32,264 genes were selected after eliminating background noise. One hundred and four genes: 63 up-regulated and 41 down-regulated, were extracted in ALDH1-positive cells by MAS5, while only 2 up-regulated genes by RMA. H19 was a gene that was commonly extracted by both algorithms. 2) The network with 104 genes was constructed and BRD4, SMAD4, Pitx3, RARa, MUC1, HASH1 and C/EBPb were identified as genes with the closest distance to ALDH1.

Conclusions: Most of extracted 7 genes have been already known to exert critical roles for maintaining stem cell features, providing a rationale of ALDH1 for the stem cell marker of breast cancer. These genes including H19 could be targets of the cancer stem cell therapy for treating incurable breast cancer.

Citation Format: Takashi Ishikawa, Kazutaka Narui, Akimitsu Yamada, Kumiko Kida, Hidetaka Shima, Chiho Endo, Shoko Adachi, Sadatoshi Sugae, Yohei Miyagi, Yasushi Ichikawa, Itaru Endo. Seven genes were closely associated with ALDH1 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1158.
Abstract 1161: Utility of evidence based mesenchymal gene signatures gene expression panel in predicting outcome in stage matched colorectal cancer patients
Authors: Johnson Benjamin , Ahluwalia Pankaj , Pundkar Chetan , Heneidi Saleh , Jones Kimya , Mondal Ashis , Kolhe Ravindra
Published: August 2019

Colorectal cancer (CRC) is third deadliest cancer in United States, only behind lung and prostate cancer. Although several therapies are available to treat colorectal cancer but there is need for new prognostic biomarkers to tailor appropriate personalized therapy. Mesenchymal Stem cells (MSCs) are multipotent stem cells which are attracted toward inflammatory sites. In colon cancer, they have been shown to assist in tumor progression. To assess the MSC gene signatures in colon cancer patients, we picked 5 genes from nCounter plexset Mesenchymal Stem cell panel. These genes named: SMAD4, PTK2, GTF3A, CASP3 and HDAC1 were quantified on Nanostring platform. The aim of the study is to quantify these Mesenchymal gene signatures in colorectal cancer patients. Under the IRB approved protocol, a total of 750 colon cancer patients at Medical College of Georgia with 5 years follow-up were initially selected. The patients were stratified on the basis of overall survival in two groups, with higher (> 5 years) and lower survival (<1 year) along with AJCC staging (I to IV), grade, gender and age. A total of 88 patients fitted in our inclusion criteria on the basis of survival duration after diagnosis. The FFPE blocks were acquired from Medical College of Georgia, Augusta. Total RNA was isolated and quantified through Nanodrop method. The statistical analysis of data was performed using student t-test and Pearson correlation.

We found PTK2 levels to be high in stage I patients (p = 0.02*). Also, patients with higher survival in stage I had high expression of PTK2 (p=0.04*). Additionally, PTK2 was expressed in higher amounts in lower surviving patient group (0.00*). GTF3 levels were higher in (p = 0.01) stage I. CASP3 showed higher expression in stage III and IV combined. HDAC1 expression was higher in Grade II patients compared to Grade I. HDAC1 expression was also higher in African American population.

Citation Format: Benjamin Johnson, Pankaj Ahluwalia, Chetan Pundkar, Saleh Heneidi, Kimya Jones, Ashis Mondal, Ravindra Kolhe. Utility of evidence based mesenchymal gene signatures gene expression panel in predicting outcome in stage matched colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1161.
Abstract 1165: Differential expression of CD44 variants drive the progression, invasion, drug-resistance and stemness characteristics in human oral squamous cell carcinoma
Authors: Kashyap Tanushree , Nagini Siddavaram , Rana Ajay , Mishra Rajakishore
Published: August 2019

Purpose: Cluster of differentiation 44 (CD44) is a cell-surface glycoprotein and plays role in the progression and severity of oral squamous cell carcinoma (OSCC). Here we report differential function of CD44 variants in OSCC progression.

Materials and methods: The expression of CD44 standard (CD44s) and variants (CD44v4, CD44v6); the activation of signaling pathways (MAPK, PI3K); the cell viability; and the MMP-9/-2 activity were assessed using RT-PCR, immunohistochemistry, Western blotting, MTT assay and gelatin zymography. These experiments were carried out using fresh human OSCC tissue specimens, including adjacent normal, noninvasive (N0), invasive tumor samples (N1-3) and chemo-radiation resistant tumor samples (RCRT). We also used OSCC cell lines, including parental (SCC9/SCC4) and Cisplatin-resistant (CisR-SCC9/-SCC4) to confirm our observation with human tissues. Knock down of CD44 variants or inactivation of MAPK/PI3K pathways was achieved for in vitro analysis in OSCC cells.

Summary: Differential expressions of CD44 variants (v4and v6) were associated with overall oral cancer aggressiveness. CD44v4 expression was positively correlated with the activation of MAPK pathway causing chemoresistance. Conversely, CD44v6 expression and activation of PI3K-Akt caused invasiveness of OSCC. Finally, an overlapping role of two major signal transduction pathways such as MAPK and PI3K, that impinge on the variants/ isoforms expressions of a single gene i.e. CD44 that leads to the OSCC aggressiveness.

Conclusion: Collectively, these results established that CD44 variants (v4and v6) expression driven by MAPK/PI3K are associated with the overall progression, drug-resistance, invasion, and stemness characteristics leading to the aggressiveness of OSCC. Hence targeting these pathways may be exploited for treating OSCC.

Keywords: OSCC, CD44s, CD44v4/6, MAPK, PI3K, Notch, Chemoresistance, Invasion/migration, stemness, aggressiveness of OSCC.

Note: This abstract was not presented at the meeting.

Citation Format: Tanushree Kashyap, Siddavaram Nagini, Ajay Rana, Rajakishore Mishra. Differential expression of CD44 variants drive the progression, invasion, drug-resistance and stemness characteristics in human oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1165.
Abstract 117: Pharmacological targeting of activated PSC decreases resistance to gemcitabine and increases survival in murine PDA
Authors: Turaga Ravi Chakra , Sharma Malvika , Mishra Falguni , Liu Zhi-Ren
Published: August 2019

Background: Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of cancer owing to its insensitivity to many chemotherapeutic drugs. Gemcitabine remains a cornerstone of PDA treatment despite suboptimal efficacy in clinics. One major mechanism by which PDA becomes resistant to gemcitabine is its conversion to inactive derivative, dFdU by upregulation of the enzyme, cytidine deaminase (Cda) in cancer cells. Activated pancreatic stellate cells (PSC) are the major source of IGF1 in the tumor microenvironment, which may be responsible for an increase in Cda expression in cancer cells. We previously reported a rationally designed protein, ProAgio that targets integrin αVβ3 at a novel site. Here we demonstrate that ProAgio specifically induces apoptosis of integrin αVβ3-expressing activated PSC and therefore circumvents gemcitabine resistance in murine PDA models.

Objective: To test whether targeting activated PSC restores chemotherapeutic sensitivity in previously unresponsive PDA tumors in a murine model.

Methods: To study the effect of ProAgio and gemcitabine combination in PDA, we used two murine PDA models: a) KP orthotopic mouse model where KPC cells were orthotopically injected into the pancreas and, b) LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model. Mice were treated with 20 doses of ProAgio (10mg/kg; i.p.) and 10 doses of gemcitabine twice weekly (50mg/kg; i.p.). IGF1 and Cda levels were analyzed by ELISA and immunohistochemistry. The intratumoral levels of the gemcitabine prodrug 2′,2′-difluorodeoxcytidine (dFdC) and the inactivated and activated metabolites (2′,2′-difluorodeoxyuridine (dFdU) and gemcitabine triphosphate (dFdCTP) respectively were determined by LCMS.

Results: Our data in murine PDA models clearly demonstrate that pharmacological targeting of activated PSC by ProAgio can stabilize PDA, and when combined with gemcitabine significantly increased the survival benefit by 3-fold (p<0.001). Pharmacological targeting of activated PSC by ProAgio consequently decreases IGF1R signaling in cancer cells. As a result, there is a decrease in Cda expression leading to an increase in active metabolite, dfdCTP in the tumor, which contributes to the enhanced efficacy of gemcitabine. In conclusion, our findings for the first time demonstrate the mechanistic approach to target activated PSC, resulting in decreased Cda expression and enhanced gemcitabine delivery, thereby restoring chemotherapeutic sensitivity in murine PDA models.

Citation Format: Ravi Chakra Turaga, Malvika Sharma, Falguni Mishra, Zhi-Ren Liu. Pharmacological targeting of activated PSC decreases resistance to gemcitabine and increases survival in murine PDA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 117.
Abstract 1182: New bioluminescent strategies to measure kinetics of immune cell-mediated cytotoxicity
Authors: Bach Maggie L. , Karassina Natasha , Hofsteen Peter , Leippe Donna , Cali James , Vidugiriene Jolanta
Published: August 2019

The number of immunotherapeutic strategies for treating cancer have expanded in recent years, increasing the need for assays that can be used to assess their effectiveness. To facilitate studies of these immunotherapies, we developed bioluminescent methods that could rapidly and sensitively measure target cell cytotoxicity. Two approaches were developed. The first method utilizes a bioluminescent enzymatic activity assay for a well-accepted cytotoxicity marker, lactate dehydrogenase (LDH), which is rapidly released from dead cells. With the improved sensitivity of the bioluminescent assay, lower levels of LDH can be measured, resulting in the detection of fewer dead cells (LOD< 10 cells). Cytotoxicity in both 2D and 3D cell cultures can be monitored over time by removing samples of medium at different time points. Here we present examples in which this assay was used to test the effectiveness of immunotherapeutic treatments including antibody-drug conjugates (ADC) and antibody-dependent cell-mediated cytotoxicity (ADCC). In ADCC models, cell killing was detectable at early time points (by 2 hours). Increases in the cytotoxicity of individual samples could be followed over time, from early time points to 24 hours. In the second approach we developed a novel bioluminescent assay based on the specific labeling of target cells. With this method, intracellular proteins in live target cells are covalently labeled. Upon target cell death, the labeled proteins are released from the cell and the label is quantitated using bioluminescence detection reagents. Using this assay, we were able to quantitate the killing of target cells in both ADCC and CAR-T applications. By adding the detection reagents directly to the medium at the start of the ADCC reaction, cell death was monitored in real-time for up to 6 hours. Neither assay requires genetic engineering and both involve no or minimal target cell preparation. They are applicable to multiple cell types and the non-lytic nature of these approaches provides kinetic capabilities and opportunities for multiplexing. The ability to rapidly quantitate cytotoxicity will facilitate the functional analysis of immunotherapies.

Citation Format: Maggie L. Bach, Natasha Karassina, Peter Hofsteen, Donna Leippe, James Cali, Jolanta Vidugiriene. New bioluminescent strategies to measure kinetics of immune cell-mediated cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1182.
Abstract 1187: Toward high-throughput, high-multiplex FFPE tumor tissue assays for translational research: 7-color, whole slide imaging
Authors: Bares Amanda J. , Wheeler Eloise M. , Manesse Maël
Published: August 2019

Background: Immuno-oncology research and predictive biomarkers discovery requires the ability to rapidly identify, quantify, and spatially map many cell sub-types of the tumor environment in FFPE tissue sections. Multiplexed immunofluorescence (IF) enables identification of multiple targets within separate spectral, or color, channels, which in turn enables more in-depth analysis of rare tissue material relative to traditional methods. Although many methods for multiplexed IF exist, they are often costly and require time-consuming assay development and long imaging times. The Ultivue® InSituPlex® technology enables rapid, optimized staining of multiple targets utilizing widely-used immunohistochemistry (IHC) staining and fluorescence imaging instrumentation. Here, we demonstrate the application of InSituPlex technology to image 6 targets and a nuclear counterstain in a 7-color single whole-slide scan, without linear unmixing-based image reconstruction. This technique expands the subtyping depth that can be achieved in a single imaging round and on a single section, and also increases sample-to-answer throughput with a streamlined, single-day workflow.

Methods: InSituPlex technology was used to perform 6-plex immune profiling of different FFPE tumor sections. Slides were stained with a cocktail of primary antibodies using a Leica® Bond® RX autostainer and imaged on commercially available fluorescent slide scanners. Subsequent image analysis of the whole slide scans was carried out using Indica Labs HALO® software.

Results: In this poster, we present an assay for pathology research that enables fast immuno-profiling in tumor tissues, using a streamlined staining workflow and 7-color imaging. Newly selected fluorophores were used with corresponding optical filter pairs, which increased the number of spectral channels available on commercially available slide scanners while limiting channel cross-talk. Analysis led to the identification of key immune cell phenotypes through marker colocalization without significant ambiguity between targets tagged with spectrally adjacent fluorescent labels.

Conclusion: The newly-expanded fluorophore configuration of the InSituPlex technology enables high throughput identification of 6 targets and a nuclear counterstain on whole tumor sections, empowering translational and immuno-oncology research.

Citation Format: Amanda J. Bares, Eloise M. Wheeler, Maël Manesse. Toward high-throughput, high-multiplex FFPE tumor tissue assays for translational research: 7-color, whole slide imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1187.
Abstract 1189: Revisiting immune exhaustion in Hodgkin’s lymphoma
Authors: Colombo Anthony R. , Hav Monirath , Gerdtsson Erik , Bisnesto-Villasboas Jose , Ansell Stephen , Hicks James , Kuhn Peter , Merchant Akil
Published: August 2019

Clinical successes with immune check-point blockers have demonstrated the potency of the immune system in controlling cancers, most strikingly in Hodgkin lymphoma (HL), where overall response rates to PD1/L1 inhibitors approach 90%. Complete or durable responses, however, are uncommon, therefore targeting the PD1/L1 axis alone is not sufficient. Recent work analyzing the spatial arrangement of PD1 and PDL1 expressing cells has given us new insight into the mechanism of action of PD1/L1 inhibitors, however this work limited itself to studying a single check point marker on a subset of cells. We hypothesize that comprehensive profiling of the frequency and spatial arrangement of immune cells in the Hodgkin lymphoma tumor immune microenvironment (TME) will provide new insights into the mechanism of checkpoint blockers and identify novel targets for immune therapy. Until now, multiparameter spatial analysis of the immune microenvironment was limited by technical challenges. Flow and mass cytometry are able to identify immune subsets of interest but spatial information is lost. Multiplex tissue imaging methods are limited to 6-8 simultaneous markers and cannot capture the full complexity of the immune phenotypes. The Fluidigm Hyperion imaging mass cytometry (IMC) system combines a CyTOF mass cytometer with a laser ablation system allowing for 40+ parameter simultaneous immunophenotyping on a single slide of FFPE tissue, with sub-cellular resolution. We have developed a panel of 34 antibodies that allow for comprehensive characterization of CD4, CD8 and myeloid cells components in the TME of Hodgkin lymphoma using IMC. Here we report on our spatial analysis of TIM3 and LAG3 expressing CD4+ lymphocytes. Our data suggests LAG3+CD4+ and TIM3+CD4+ lymphocytes had shorter mean nearest distance to PDL1+Hodgkin Reed-Sternberg (HRS) cells upon comparison to PDL1- HRS cells (t-test, p=1.703e-08,p=1.126e-14). Future studies should explore multiple exhausted marker models that seeks to further understand the presence of simultaneous exhaustion signals in the niche environment. These data suggest that therapies that target TIM3 and/or LAG3 should be tested in Hodgkin Lymphoma and that spatial analysis of immune subsets by IMC should be explored as selective and pharmacodynamic biomarkers.

Citation Format: Anthony R. Colombo, Monirath Hav, Erik Gerdtsson, Jose Bisnesto-Villasboas, Stephen Ansell, James Hicks, Peter Kuhn, Akil Merchant. Revisiting immune exhaustion in Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1189.
Abstract 1191: PD-L1 expression and the tumor immune microenvironment in NUT carcinoma
Authors: Rooper Lisa M. , London Nyall R. , Taube Janis M. , Westra William H. , Bishop Justin A. , Kang Hyunseok
Published: August 2019

Nuclear protein in testis (NUT) carcinoma is a rare, highly aggressive malignancy that is defined by translocations of the NUTM1 gene and has a median survival of less than one year. Although immunotherapy is an attractive novel therapeutic option, the immune microenvironment has not yet been characterized in this tumor type. We performed immunohistochemistry for PD-L1, IDO1, CD8, CD68, FOXP3 and myeloperoxidase on whole-slide sections from 9 cases of NUT carcinoma, quantified immune cell subsets using Halo image analysis software, manually scored percentage of PD-L1 and IDO1 expression in tumor cells, and classified immune cell PD-L1 expression as focal (≤5%) or non-focal (>5%). The 9 NUT carcinomas included 6 sinonasal, 2 mediastinal, and 1 soft tissue primaries. Median survival was 11 months (range 2-19); 7 patients (78%) died of disease while 2 (22%) had no evidence of disease at last follow up (12 and 19 months). All tumors demonstrated a dominant population of myeloperoxidase+ neutrophils, with a mean density of 954/mm2 (range 14-2311). There were also a mean 59/mm2 CD8+ cytotoxic T-cells (range 15-107), 75/mm2 FOXP3+ T-regulatory cells (range 9-349), and 54/mm2 CD68+ macrophages (range 0-417). Immune cell density did not correlate with survival. Four tumors (44%) expressed PD-L1, with mean 17% positivity (range 0-70%). PD-L1 expression was associated with improved survival (p=0.02), with higher mean PD-L1 levels in surviving patients (65% vs. 4%, p<0.001). Two tumors (22%) expressed non-focal PD-L1 on tumor-infiltrating lymphocytes, which was also associated with survival (100% vs. 0%, p=0.03); whereas all tumors showed non-focal PD-L1 on neutrophils and macrophages. PD-L1+ tumors also had more CD8+ T-cells per mm2 (81 vs 41, p=0.07). Three tumors (33%) expressed IDO1, which was associated with improved survival (p=0.01). Overall, NUT carcinoma demonstrates an immunosuppressive microenvironment with a dominant population of tumor-infiltrating neutrophils that also show PD-L1 expression. However, evidence of PD-L1 and IDO1 expression on tumor cells with associated cytotoxic T-cell response and improved prognosis suggests that adaptive immunity plays an important role in NUT carcinoma outcomes, raising the possibility that immunotherapy might be harnessed to augment this response.

Note: This abstract was not presented at the meeting.

Citation Format: Lisa M. Rooper, Nyall R. London, Janis M. Taube, William H. Westra, Justin A. Bishop, Hyunseok Kang. PD-L1 expression and the tumor immune microenvironment in NUT carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1191.
Abstract 1200: AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells
Authors: Chouaib Sallem , Terry Stephane , Buart Stephanie , Engelsen Agnete , Gausdal Gro , Lorens James , Thiery Jean Paul , Mami-Chouaib Fathia
Published: August 2019

As immunotherapies are now used to treat a large proportion of NSCLC patients, defining mechanisms of immune resistance is critical. Immune resistance may arise from both genetic instability and tumor heterogeneity driven by microenvironmental stresses such as hypoxia that promotes carcinoma cell plasticity as well as extrinsic or intrinsic mechanisms of immune resistance. AXL, a member of the TAM receptor tyrosine kinase family is widely expressed human cancers and increasingly recognized for its role in cell plasticity and drug resistance. In this study, we used a model of hypoxia-induced tumor plasticity to generate multiple lung cancer clones with mesenchymal and epithelial features to address mechanisms of immune resistance. We demonstrate that AXL expression is dramatically increased in mesenchymal lung cancer clones. Moreover, expression of AXL in the cells was correlated with an increased cancer cell intrinsic resistance to both NK and CTL-mediated killing, Notably, small molecule AXL targeting potently sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance to immune cells involved a novel molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Congruently, higher ICAM1 and ULBP1 tumor expression, correlated with improved patient survival in two NSCLC cohorts. These results reveal a novel AXL-mediated immune escape regulatory pathway, suggest AXL as a novel candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

Citation Format: Sallem Chouaib, Stephane Terry, Stephanie Buart, Agnete Engelsen, Gro Gausdal, James Lorens, Jean Paul Thiery, Fathia Mami-Chouaib. AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1200.
Abstract 121: S100A9 inhibitor Tasquinimod: A novel strategy to inhibit small cell lung cancer progression and metastasis
Authors: Sassi Salha E. , Ahirwar Dinesh , Ganju Ramesh K.
Published: August 2019

Small cell lung cancer (SCLC) treatment is a major clinical challenge at present as it is highly refractory to available drugs. The MDSCs/macrophages are known to help SCLC develop resistant to available therapies. S100A9 (Migration inhibitory factor-related protein 14 (MRP14) is an EF-hand calcium-binding protein that has been involved in cell migration, invasion, proliferation, and tumor metastasis in various type of cancers, however not much is known about its role in SCLC. In this study, we found that S100A9 protein is highly up-regulated in various types of pulmonary neuroendocrine carcinomas (NEC) patient tissues compared to normal using tissue microarrays. We also observed that SCLC patients with higher S100A9 expression have significantly increased numbers of macrophage in the stroma. We have also shown that pre-treatment of the cells with S100A9 inhibitor (Tasquinimod) suppressed in-vitro cell migration, invasion, and colony formation. In addition, we analyzed the efficacy of S100A9 inhibitor against SCLC using in vivo mouse models. S100A9 inhibitor significantly reduces tumor growth and metastasis in SCLC in xenograft mouse models. We further observed that S100A9 inhibitor suppressed myeloid-derived suppressor cells (MDSC) populations and TAMs of the M2-polarized phenotype in SCLC. Moreover, we found myeloid cells sequestered from tumors of treated mice expressed were MI type as they showed higher levels of inducible nitric oxide synthase (iNos), and lower levels of arginase-1. Molecular analysis revealed that Tasquinimod decreases expression of IL6, IL10, and TGF-β1 in the cancer cells which helps inhibit macrophage activation to TAMs. Reduced proliferation and vascularization were observed in the tumors obtained from animals treated with S100A9 inhibitor. We also observed S100A9

inhibitor suppressed osteolytic bone formation in ex-vivo resorption assay. Overall, our studies, for the first time, show that Tasquinimod that targets S100A9 signaling could be used as a novel therapeutic strategy against SCLC.

Citation Format: Salha E. Sassi, Dinesh Ahirwar, Ramesh K. Ganju. S100A9 inhibitor Tasquinimod: A novel strategy to inhibit small cell lung cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 121.
Abstract 1212: Anti-SIRPα antibodies as a potential weapon for cancer immunotherapy via accelerated screening in humanized mouse models
Authors: Jin James , Yang Benny(Yi) , Guo Chaoshe , Shen Yuelei , Ni Jian
Published: August 2019

Signal regulatory protein α (SIRPα) is a transmembrane protein with an extracellular region comprising three Ig-like domains and a cytoplasmic region containing immunoreceptor tyrosine-based inhibition motifs which mediate binding of the protein tyrosine phosphatases SHP1 and SHP2. SIRPα is especially abundant in myeloid cells such as macrophages and DCs, whereas it is expressed at very low levels in T, B, NK, and NK T cells. SIRPα inhibits phagocytosis in macrophages upon interacting with its ligand CD47, which is commonly upregulated on the surface of malignant cells. Thus, antibodies that block the CD47-SIRPα interaction should enhance macrophage phagocytosis in the tumor microenvironment and inhibit tumor growth, making anti-SIRPα antibodies promising as potential new tools for cancer immunotherapy. We are interested in subverting the negative feedback of the CD47-SIRPa axis, in order to treat cancer patients. Using Biocytogen’s antibody research platform, we obtained several high-affinity anti-human SIRPα mAb by high-throughput flow cytometry screening. Next, these murine antibodies were screened directly in single or double humanized mice models (B-hSIRPα or B-hSIRPα/hCD47) bearing a syngeneic humanized cancer cell line (MC38-hCD47), where we discovered candidates that have potent anti-tumor activity in vivo. Chimeric antibodies and humanized antibodies based on these candidates were generated and further examined in vivo. In addition, we uncovered synergistic anti-tumor activity of anti-human SIRPα mAbs in combination with anti-mouse PD-1 mAb in B-hSIRPα/hCD47 mice. In brief, our existing studies demonstrate that SIRPα is an ideal target for preclinical cancer models. Clinical evaluation of the translational merits for these novel anti-SIRPα antibodies is pending.

Citation Format: James Jin, Benny(Yi) Yang, Chaoshe Guo, Yuelei Shen, Jian Ni. Anti-SIRPα antibodies as a potential weapon for cancer immunotherapy via accelerated screening in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1212.
Abstract 1217: Exploiting host pre-existing immunity for melanoma cancer immunotherapy
Authors: Briquez Priscilla S. , Hauert Sylvie , Repond Grégoire , Swartz Melody A. , Hubbell Jeffrey A.
Published: August 2019

Vaccines are considered one of greatest successes of modern medicine; indeed, prophylactic vaccinations allow tight control of disease spreading and even eradication of some diseases. Vaccines immunize patients against known antigens derived from infectious agents, so that the immune system can efficiently block pathogen entry and mount a faster and better immune reaction in case of infection. In the context of cancer, finding good antigens for immunization against tumor cells can be challenging since tumor cells are derived from the self, and thus are not always efficiently recognized as malignant by the immune system. Here, we seek to develop a cancer immunotherapy that delivers a pre-encountered antigen to tumor cells to redirect the host pre-existing immunity against cancer. As a vaccine model, we used ovalbumin (OVA) antigen adjuvanted with CpG-B oligodeoxynucleotides to pre-immunize C57BL/6 mice, developing both T cell and B cell immunity against OVA. At least one month later, mice were challenged with B16-OVA melanoma cells. We observed that tumor growth in pre-immunized mice was slightly delayed compared to growth in naïve mice, but this effect was modest, leading to an overall 1-day increased survival. We reasoned that this effect could be improved by modifying the cellular localization of OVA antigen; indeed, OVA is expressed intracellularly in B16-OVA cells, thus excluding potential antibody-mediated anti-tumoral immune response. Therefore, we created a B16 melanoma cell line that overexpresses a membrane-bound OVA (B16memOVA), using similar design as published elsewhere (DiLillo et al., J. Immunol. 2010). In vitro, B16memOVA cells behaved similarly to the parental B16 cell line, and the growth of B16memOVA tumors in OVA-expressing mice (which are tolerant to OVA) was similar to the growth of B16 tumors, thus validating our B16memOVA tumor model. When implanted in naïve mice, B16memOVA tumors were able to grow, despite the onset of a neo-immune reaction against OVA, which slows tumor growth. In contrast, mice pre-immunized against OVA totally rejected B16memOVA tumors. Our efforts are now focusing on the development of an antigen-delivery method to trigger the expression of membrane-bound OVA in B16 tumors after implantation, to apply this approach in a therapeutic setup.

Citation Format: Priscilla S. Briquez, Sylvie Hauert, Grégoire Repond, Melody A. Swartz, Jeffrey A. Hubbell. Exploiting host pre-existing immunity for melanoma cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1217.