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The treatment of bladder cancers has improved with new immunotherapy such as PD-L1. However, not all bladder cancers respond to PD-L1 immunotherapy which is why new targets are being assessed. Melanoma-associated antigen gene A (MAGE-A) family proteins are expressed in a variety of tumors, with each MAGE-A protein having unique roles in cancer pathogenesis. One advantage of targeting MAGE-A family members is the lack of expression in normal tissues which makes them well-suited for targeted cancer immunotherapy. The challenge with screening the MAGE-A family is to find a specific antibody since the 12-member family has over 60% sequence in homology. In this study, we evaluate multiple MAGE-A3 and MAGE-A4 antibodies using CytoSections, a reproducible alternative to control tissue with an expression of target biomarkers. A series of antibodies specific to each MAGE-A3 and MAGE-A4 protein were discovered and used to screen twenty-five bladder cancer tissues. The results showed that MAGE-A3 was present in 9 of the 25 bladder cancers, while MAGE-A4 was present in 10 of the 25 bladder cancers evaluated; in the meantime, 7 bladder cancers were positive for both targets after double immunofluorescence staining with MAGE-A3 or A4 specific antibodies. The data presented here could be an important development in treatment protocols for pre-screening patients who are at risk. Our findings show that MAGE-A3 and MAGE-A4 can be both present in bladder cancer, suggesting that targeting both genes may be necessary for the success of immunotherapy treatment. Citation Format: Bailey Gilmore, Rachel Gonzalez, Aubrey Su, Dehe Kong, Eden Zewdu, Jina Yom, Tianli Qu, Zhaoying Guo, Xiaomin Hu, Qi Ren, Ranran Zhang, Eric Christenson, Yan Ma, Dezhong Yin, Patrick Yin, Wei Fu, Xuan Liu, Krishnan Allampallam. Colocalization of MAGE-A3 and MAGE-A4 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 999.

Tet-On is a powerful inducible system and a classical tool to regulate gene expression in mammalian cells. It has also been applied to regulate Pol III-driven transcription, such as shRNA or gRNA driven by a U6 or H1 promoter. However, all of the current versions of Tet-On shRNA vectors are based on H1-2O2 or U6-2O2 promoters, which are only compatible with first-generation tetracycline repressor TetR. In addition, these promoters have the problem of driving downstream transcription without the binding of the Tet regulatory protein. Here, we developed a new system that is built upon tetracycline activator protein, Tet-On 3G, combined with a new structure of Tet responsive promoter H1-4O4, which tightly regulates the downstream transcription of gRNA or shRNA. The responsiveness of our system to Doxycycline regulation is dramatically improved compared with the current versions. The new Tet-On system is further optimized into a compact structure to be compatible with the lentivirus package (All-In-One Lenti-Tet-On system), which still keeps the leaky expression at an undetectable level. Combined with all these features, the new generation of the Tet-On system offers broad applications in gene knocking down and genomic editing. Citation Format: Lipeng Wu, Hua Su, Justin Fellows, Mao Fu, Julian Heller, Brian Park, Dezhong Yin. Development of a new All-In-One inducible lentiviral shRNA/gRNA Vector. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3727.

Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide. Hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13), which was newly identified as a liver lipid droplet associated protein, was recently found to be down-regulated in HCC. However, the effects of HSD17B13 on the underlying mechanisms of HCC have not yet been fully explored. For this purpose, HSD17B13 gene was knockdown with CRISPER/Cas9 technique in several HCC cell lines. HSD17B13 expression was detected by Flow cytometry and Western blotting with recombinant rabbit monoclonal antibodies (clone OTIR3G2 and OTIR5C10) against HSD17B13 which were screened for use in immunohistochemistry, western blot, and ELISA application. Overexpressed HSD17B13 induces hepatic cell line G1 capture and increases several G1 arrest-related protein expression, including p21, p27, MMP2 and MMP3. Then the proliferation and cell cycle were suppressed. HSD17B13 gene knockdown will reverse the suppression. These data indicated that HSD17B13 play important roles in the progression of HCC lesions. HSD17B13 might be a potential immune-oncology marker for HCC. Citation Format: Tianli Qu, Lipeng Wu, Jina Yom, Zhaoying Guo, Xiaomin Hu, Qi Ren, Qian Niu, Ruei-Shiuan Lin, Dezhong Yin, Xuan Liu, Fu Wei. Overexpression of HSD17B13 induced G1 arrest in hepatic cell lines detected by anti-HSD17B13 recombinant rabbit monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3695.

Melanoma-associated antigen gene A (MAGEA) family proteins are expressed in a variety of tumors with each MAGEA protein having unique roles in cancer pathogenesis. The MAGEA members lack of expression in normal tissue and their role in cancer make them well suited for targeted cancer immunotherapy. Thus, knowing which MAGEA protein expression exists in a tumor is important. However, the MAGEA family shares sequence similarity that makes it difficult to find antibodies that are specific to just one family member. It is also challenging to source tissue generally with HIPAA regulation and even more so to get tissue representation of all 12 MAGEA family members. Here we present CytoSections as an alternative to patient control tissues. CytoSections are formalin fixed, paraffin embedded (FFPE) section of over-expression cell pellets. These cells were transfected with expression plasmids coding for genes of individual MAGES family. In this study, CytoSections are used to screen antibodies to all 12-member MAGEA family of proteins. The MAGEA family members were initially shown positive by targeted protein expression in western and immunohistochemistry using DDK tag antibody. Then multiple MAGEA3, MAGEA4, MAGEA9 antibodies were assessed by IHC to determine their specificity to their intended target. Using MAGEA family as a pilot project, we show that CytoSections is a verified, reproducible, and renewable alternative to human control tissues and serves as an ideal tool for antibody specificity assessment. Citation Format: Eden B. Zewdu, Rachel M. Gonzalez, Yan Ma, Derek Ling, Xiaomin Hu, Qi Ren, Ranran Zhang, Li-Hui Lei, Krishnan Allampallam, Dezhong Yin, Xuan Liu, Wei Fu. A renewable IHC control tool CytoSections࣪ for defining MAGEA3, MAGEA4, and MAGEA9 antibody specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2803.

Three-dimensional “organoid” growth of tumors may represent a more physiologically relevant in vitro model system than traditional two-dimensional monolayer cultures of cancer cell lines. With the increased availability of cryopreserved human cancer organoids generated by academic laboratories, large-scale biobanking initiatives, and commercial sources, there is an unmet need for simplified, standardized, and cost-effective methods for preparation of the complex growth media required by these models. Human organoid culture media contains a variety of recombinant proteins, small molecules, and other growth factors that are costly to purchase in small-scale, time consuming to reconstitute and aliquot, and have varying stability and shelf life once prepared. Organoid culture media often also utilizes undefined conditioned media (CM) from one or more engineered cell lines that must be cultured separately, requiring additional time and resources to maintain. These lines secrete critical growth factors and the CM generated must be carefully prepared, collected, and stored. CM is subject to variability in activity levels due to batch-to-batch and protocol-to-protocol differences that can affect subsequent organoid culture performance. To address these challenges and to facilitate the wide-spread adoption of human organoid culture we are developing reagent kits containing essential growth medium components in an individually lyophilized format for long-term storage and easy single-use preparation of organoid growth media for a variety of tissue types including human colon, mammary, and lung. We compared growth medium formulated with our components with small-scale “homebrew” preparations and found equivalent or better culture performance (as calculated by doubling times) and similar morphology. Both approaches were able to generate media capable of supporting growth for at least three passages (~30 days) in 15 different organoid models. Additionally, we compared protocols and cell lines for CM generation to support organoid culture and found that an optimized, scaled-up production method could generate Wnt-3a and R-spondin1 CM with higher activity levels (up to 52%) and reduced lot-lot variability in comparison with commonly used small-scale protocols. We found that the optimized protocols produced CM that was stable for at least 2 months at 4°C and at least 6 months at -20°C, and was resilient against freezing and thawing. Previously frozen CM was also able to support organoid growth in culture. These results show that our tissue-specific kits and ready-to-use CM can provide a simplified, cost effective method to support the long-term propagation of human cancer organoids from multiple tissues and cancer types. Citation Format: James Clinton, Allison Ruchinskas, Himanshi Desai, Nanda Mahashetty, Dezhong Yin. Propagation of human colon, mammary, and lung cancer organoids in growth medium utilizing tissue-specific reagent kits and ready-to-use Wnt-3a and R-Spondin1 conditioned media [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 51.

Exosomes are subcellular particles 50-200 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases, including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor-derived exosomes have been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for the isolation of large quantities of highly purified, ready to use exosomes from cell culture supernatant. Exosomes from a variety of cancer cell lines representing the most prevalent cancer types, including prostate (LNCaP), colorectal (HCT116), breast (MDA-MB-231, MCF-7), lung (A549), cervical (HeLa), and glioblastoma (U87-MG) were isolated and characterized in this study. We employed a stringent quality control approach in order to define and characterize these exosomes. Our method generated a high yield (>1×1010 exosomes/mL) and average protein equivalent of 2 mg/mL. Our data demonstrated expression of a number of different characteristic exosomal proteins including tetraspanin, which was confirmed through western blot and PCR. Isolated exosomes had a median size of 102 nm through nanoparticle tracking analysis. Western blot data revealed cell line specific protein marker expression of CD63, CD81, flotillin 1, Alix and TSG101 protein. We assessed the functionality of these purified exosomes through cellular uptake, migration assay, and anchorage independent growth assay in this study. We also evaluated exosomal RNA from these purified exosomes for the presence of common mutations associated with cancer through PCR. Here we report that exosomes isolated by our method not only had high exosome yield and quality, but also retained relevant functionality and thereby make ideal reference standards/controls in the field of cancer research and diagnostics development. Citation Format: Siddhartha Paul, Nanda Mahashetty, Joy Wells, Dezhong Yin, Fang Tian. Development and characterization of cancer cell line exosomes as reference standards in cancer research [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 LB-038.

To meet the need for improved approaches to study cancer in vitro, there has been a surge in the development of novel research models utilizing advanced culture methods. These methods permit in vitro growth of cancer types previously not possible, and/or models with enhanced in vivo relevancy compared to traditional continuous cell lines. However, availability of these early-stage research models is currently limited and there is a lack of data on the ability to scale up production of these models to support the needs of the global research community. We sought to investigate the protocols, expansion capacity, cryopreservation ability, genetic stability, and feasibility of larger-scale bioproduction of a subset of the models generated by the Human Cancer Models Initiative (HCMI), an international collaborative effort between the National Cancer Institute, the foundation Hubrecht Organoid Technology, Cancer Research UK, and the Wellcome Trust Sanger Institute. The HCMI's initial goal is the development of 1,000 novel human cancer models, paired with bioinformatics and patient clinical data, particularly from rare or underrepresented cancer types. One advanced culture method being utilized, three-dimensional organoid “microtissue” culture, potentially poses challenges for traditional large-scale bioproduction processes. It requires growth embedded within an undefined extracellular matrix and complex media formulations containing multiple small molecules and recombinant proteins with unknown stability and shelf-life. Additionally, organoid growth media typically includes multiple sources of undefined conditioned media containing critical growth factors. We cultured organoid models derived from human colon, pancreas, esophagus, and mammary tissues developed by laboratories contributing to the HCMI. Multiple unique donors were available for all tissues and both cancer and non-cancer models were available for two tissue types. Most models were maintained in culture continuously for at least 60 days (7-27 population doublings, > 10 passages). Tissue and donor variability was evident in model characteristics, including morphology (assessed by microscopy and immunocytochemistry), growth rate, and genetic stability (measured by short tandem repeats analysis). All models were amenable to scale up beyond multiwell plates, and all models could recover from cryopreservation. While organoid culture represents a significant divergence from typical two-dimensional monolayer culture of continuous cell lines, our results show that these next-generation in vitro models are suitable for larger-scale bioproduction. This is vital to ensure the widespread availability of these models within the research community to facilitate applications like pre-clinical drug discovery and basic cancer research. Citation Format: James Clinton, Penney McWilliams-Koeppen, Siddhartha Paul, Allison Ruchinskas, Dezhong Yin, Robert Newman. Feasibility of manufacturing-scale bioproduction of novel next-generation 3D organoid cancer models in support of the Human Cancer Models Initiative [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5012.

How to balance high efficiency of transgene expression with low cytotoxicity is a central concern for cell biology. Developing lipid-based transfection reagents that have those attributes and can be conveniently used for multiple cell types is critical for studies in cancer biology and cell biology. To address these concerns, ATCC has developed two lipid-based transfection reagents, GeneXPlus and TransfeX™ that provide a powerful tool for efficiently transfecting a broad spectrum of cell types including continuous cancer cell lines, stem cells, immortalized cells, and primary cells with little or no cytotoxicity. For recognized hard-to-transfect cancer cell lines, like THP-1, SH-SY5Y, and Raw 264.7 cells, we have achieved over 30% GFP expression without obvious cytotoxicity by using the GeneXPlus and an EF1α-GFP vector. TransfeX™ has been found to be highly suitable for the transfection of stem cells, immortalized cell lines, and many types of primary cells such as mammary, renal and bronchial epithelial cells, dermal microvascular endothelial cells, and human dermal fibroblasts. We have developed optimized transfection protocols for the TransfeX™ transfection reagent for different cell types and achieved over 50% transfection efficiency and low cytotoxicity. ATCC will provide data showing the broad spectrum of cell types successfully transfected with either GeneXPlus or TransfeX™. Citation Format: Dezhong Yin, Joy Wells, James Clinton, Lysa-Anne Volpe, Kevin Grady, Jeanmarie Curley. Development of lipid-based transfection reagents for efficient expression of transgenes in hard-to-transfect cell types. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-248. doi:10.1158/1538-7445.AM2014-LB-248