bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1044: Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™
Authors: Hwang Kevin , Duro Lauren , Veith Alex , Wedin Nelson , Samusik Nikolay , Lee Je H.
Published: March 2024

Background: Multiplex immunofluorescence (mIF) is an important tool for extracting complex phenotypic information from normal or diseased tissues, and provides accurate and predictive insights on the tissue microenvironment faster. For patient samples in translational and clinical research, high sensitivity, specificity, and reproducibility are critical; however, throughput and cost considerations are also important in designing clinical studies. Enabling an easy-to-use high-performance assay such as InSituPlex® on robust and affordable auto-stainers like the Parhelia Omni-Stainer™ can allow researchers to gain a deeper understanding of the tissue micro-environment through high-quality multiplexed biomarker detection in a fraction of the time and cost.

Methods: We performed Ultivue InSituPlex® assays with OmniVUE™ panels using the Parhelia Omni-Stainer™, a low-cost, high-throughput tissue auto-stainer with reduced requirements for reagents with a minimal dead volume. We compared the whole-slide staining performance across replicates using human tonsil and tumor tissue arrays across multiple independent runs over multiple days. The stained slides were scanned using the Zeiss AxioScan.Z1 high-throughput imaging system, and image batches were processed using Ultivue’s UltiStacker.AI and UltiAnalyzer.AI software.

Results: Tighter temperature control of the slide in the Omni-Stainer supports optimal InSituPlex® signal amplification. Intra-run and inter-day analysis on serial sections of human tonsil and tumor tissue arrays demonstrated a high degree of consistency in InSituPlex® assay performance, specifically with marker-specific signal intensities and cell densities. The Omni-Stainer facilitates the use of low reagent volumes at each step of the assay due to its efficient reagent use, thus lowering the cost of reagents per slide.

Conclusions: This study demonstrated a fast, simple and robust workflow to perform high-performance InSituPlex® mIF assays on the low-cost, high-throughput Parhelia Omni-Stainer™. Ultivue’s configurable OmniVUE™ mIF panels can be run on the Omni-Stainer with reduced associated reagent costs per slide. This workflow can support larger clinical and translational studies in a more cost-effective fashion, leading to in-depth analysis of tissue microenvironment through highly precise multiplexed biomarker detection.

Citation Format: Kevin Hwang, Lauren Duro, Alex Veith, Nelson Wedin, Nikolay Samusik, Je H. Lee. Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1044.
Abstract 1071: Circulation of rare events in the liquid biopsy for early detection of lung mass lesions
Authors: Shah Anya , Resnick Karen , Mason Jeremy , Kuhn Peter , Nieva Jorge , Shishido Stephanie
Published: March 2024

Lung cancer screening with low-dose CT scans (LDCT) has reduced mortality for patients with high-risk smoking histories. However, this method of screening remains imperfect. High rates of false positive scans expose patients to additional morbidity as they pursue subsequent diagnostic evaluation. Real-world application of screening LDCT remains low. Finally, current screening guidelines exclude those without smoking histories, a population which comprises up to a quarter of lung cancer patients. We sought to explore the utility of liquid biopsy (LBx) in lung cancer patients and its use in early cancer screening and diagnosis. Using the third generation High-Definition Single Cell Assay (HDSCA3.0) workflow, we analyzed 99 peripheral blood samples divided into three cohorts: Normal Donors (ND, n=50) who were healthy volunteers with no known pathology, Screening CT patients (CT, n=25) who underwent standard LDCT screening with resultant Lung-RADS 1-2, Biopsy patients (BX, n=24) who had abnormal CT scans requiring tissue biopsy. The ND group was used to characterize baseline epithelial, mesenchymal, endothelial, and immune cells, alongside acellular events (oncosomes). For the CT and BX patients, demographic information including age, gender, and average BMI was roughly equivalent, however average pack-years smoked differed owing to 10 (42%) patients in the BX group who had no prior smoking history. A total of 14 (58%) BX patients were diagnosed with primary lung cancer (BX+), of which 11(75%) were adenocarcinoma, 2 (19%) were squamous cell carcinoma and 1 (6%) was small cell. Across the cohorts we observed intra- and interpatient heterogeneity of rare events including multiple phenotypic rare cell populations and oncosomes. The comparison of the rare event enumerations between the ND, CT, BX+, and BX- cohorts revealed a greater incidence of total events, total rare cells and oncosomes, as well as specific cellular phenotypes in the CT and BX cohorts compared to the ND cohort. LBx analytes were detected at a significant elevation in the BX samples compared to the CT samples, but there was no significant difference between BX+ and BX- samples. The data supports the utility of the LBx in distinguishing patients with an alveolar mass lesion from those without, providing a mechanism for screening prior to LDCT to better inform clinical workup and avoid unnecessary radiation exposure and biopsy.

Citation Format: Anya Shah, Karen Resnick, Jeremy Mason, Peter Kuhn, Jorge Nieva, Stephanie Shishido. Circulation of rare events in the liquid biopsy for early detection of lung mass lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1071.
Abstract 1095: Prox1 transcription factor and its downstream signaling - A new therapeutic target for rhabdomyosarcoma
Authors: Gizaw Nebeyu Y. , Böhling Tom , Sampo Mika , Alitalo Kari , Kivela Riikka M.
Published: March 2024

Rhabdomyosarcoma (RMS) stands as the predominant soft tissue sarcoma in children, characterized by a high-grade neoplasm composed of cells resembling skeletal myoblasts. Despite being prevalent, patients with metastatic or recurrent rhabdomyosarcoma still experience poor outcomes, underscoring the necessity for novel treatments. We have recently shown that PROX1 is a critical factor in RMS tumor growth and myogenic characteristics (Gizaw NY, et al. PNAS 2022). As PROX1 has previously been associated with tumor metabolism in colorectal cancer, we investigated further its role in RMS metabolism. Our findings demonstrate that PROX1 plays a crucial role in regulating RMS cell metabolism. Specifically, silencing of PROX1 resulted in decreased lipid and oxidative metabolism, which was accompanied by an increase in glycolysis and triglyceride accumulation. RNA sequencing and Gene Set Enrichment Analysis revealed downregulation of all genes involved in the cholesterol synthesis pathway in PROX1-silenced RMS cells. Our studies further reveal that inhibiting cholesterol synthesis, akin to PROX1 silencing, can effectively inhibit RMS cell growth. Our results provide novel insights into the role of PROX1 in RMS metabolism and highlight the potential of PROX1-regulated metabolic pathways as promising targets for the development of novel RMS therapies.

Citation Format: Nebeyu Y. Gizaw, Tom Böhling, Mika Sampo, Kari Alitalo, Riikka M. Kivela. Prox1 transcription factor and its downstream signaling - A new therapeutic target for rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1095.
Abstract 1097: CD73 contributes to the pathogenesis of fusion-negative rhabdomyosarcoma through the purinergic signaling pathway
Authors: Hernandez Karla Gabriela Cano , Shah Akansha , Lopez Victor , Tagliabracci Vincent , Chen Kenian , Xu Lin , Bassel-Duby Rhonda , Olson Eric , Liu Ning
Published: March 2024

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-Negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5’ ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while upregulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells, restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.

Citation Format: Karla Gabriela Cano Hernandez, Akansha Shah, Victor Lopez, Vincent Tagliabracci, Kenian Chen, Lin Xu, Rhonda Bassel-Duby, Eric Olson, Ning Liu. CD73 contributes to the pathogenesis of fusion-negative rhabdomyosarcoma through the purinergic signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1097.
Abstract 110: Adoptive transfer of tumor antigen-specific T cells alters the renal tumor microenvironment in the presence and absence of immunotherapy
Authors: Stephens Holly , Elkins Elizabeth , Dempsey Francesca , Swalley Zoey , Li Jing , Zhang Jianqing , Kirkman Richard , Chen Bo-Ruei , Roberts Zachary , Sudarshan Sunil , Sleckman Barry , Tse Hubert , Smith Daniel L. , Norian Lyse A.
Published: March 2024

Immunotherapies are the standard of care for patients with renal cancer, yet their effects on renal tumor antigen-specific CD8 T cell responses and their subsequent ability to modulate the renal tumor microenvironment remain difficult to evaluate. In order to address this gap in the field, we developed a novel murine model to examine tumor-antigen specific CD8 T cell immune responses in orthotopic renal cancer utilizing a new cell line, Renca-tERK-LUC. Parental Renca cells were transduced with a lentivirus expressing the naturally-occurring, mutated tERK peptide found in CMS5 fibrosarcoma cells to create Renca-tERK-LUC tumor cells. The tERK mutation consists of a one base pair change that creates an Sfc1 site, so it can be detected by RT-PCR followed by restriction enzyme digestion. Our data show that Renca-tERK-LUC cells express tERK at high levels and grow progressively in control BALB/c mice, but are rejected in DUC Thy1.1 mice, which contain endogenous TCR transgenic CD8+ T cells specific for tERK peptide/MHC I complexes. We then performed adoptive transfers of 5 × 105 DUC Thy1.1 T cells into mice with established, orthotopic Renca-tERK-LUC tumors to evaluate: 1) the effects of immunotherapy on tumor-antigen-specific T cell responses; and 2) the effects of tumor-antigen-specific T cells on the broader renal tumor microenvironment. Our data indicate that the transfer of relatively low numbers of tumor antigen-specific CD8+ T cells induces transcriptomic and cellular changes in the tumor microenvironment in both the presence and absence of immunotherapy. Defining the parameters surrounding tumor-antigen-specific T cell responses and their ability to modulate the renal tumor microenvironment under the influence of clinically relevant immunotherapeutic intervention has the potential to increase the understanding of immunity to renal tumors, which could ultimately prove beneficial for renal cancer patients.

Citation Format: Holly Stephens, Elizabeth Elkins, Francesca Dempsey, Zoey Swalley, Jing Li, Jianqing Zhang, Richard Kirkman, Bo-Ruei Chen, Zachary Roberts, Sunil Sudarshan, Barry Sleckman, Hubert Tse, Daniel L. Smith, Lyse A. Norian. Adoptive transfer of tumor antigen-specific T cells alters the renal tumor microenvironment in the presence and absence of immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 110.
Abstract 1102: Amentoflavone enhances regorafenib-induced apoptosis and -inhibited invasion via inactivating STAT3/NF-ΚB signaling pathway in Hepatocellular carcinoma
Authors: Tsai Yan ting , Chen Ying-Tzu , Hsu Fei-Ting , Tsai Jai-Jen , Liu Yu-Chiang
Published: March 2024

Hepatocellular carcinoma (HCC) is an aggressive tumor and ranks as the third leading cause of cancer-related deaths worldwide. Amentoflavone, a biflavonoid compound, possesses antiviral, angiogenesis-inhibitory, and anticancer properties. Prior studies have illustrated that amentoflavone effectively restrains tumor growth and promotes apoptosis in various cancers, including brain cancer and osteosarcoma. Regorafenib, a small molecule inhibitor of multi-kinases, can inhibit angiogenesis, tumor growth, and anti-tumor metastasis. While regorafenib is a targeted drug approved for the treatment of metastatic colorectal cancer and has also been approved for use in HCC, its effectiveness in HCC is limited. Consequently, our goal is to investigate whether amentoflavone may enhance the sensitivity of regorafenib in treating HCC and explore the underlying mechanisms. In our study, through the combined treatment and combination index analysis, we proved that the combination of regorafenib and amentoflavone may induce cytotoxicity more effectively compared to monotherapy, as assessed by the MTT assay in Huh7 and HepG2 cells. The invasion and migration assays also demonstrated a superior inhibitory effect in the combination group. Additionally, apoptosis markers such as "cleaved caspase-3, -8, -9, Fas and FasL" assayed by flow cytometry, confirm that the combination treatment may induce the extrinsic/intrinsic apoptosis pathway in HCC. In our Western blotting analysis, we observed that the phosphorylation of STAT3 and NF-κB proteins was effectively suppressed by the combination of regorafenib and amentoflavone in Huh7 and HepG2 cells, respectively. Furthermore, our in vivo study confirmed a superior tumor inhibition rate and control rate with the combination of regorafenib and amentoflavone. Importantly, no signs of general toxicity were observed in any of the treatment groups, as evidenced by body weight monitoring and Hematoxylin and Eosin stain (H&E) pathology evaluation. We believe that the combined treatment of these two drugs effectively triggers cell death and suppresses metastatic patterns through the inactivation of the STAT3/NF-κB signaling pathway. These results suggest that amentoflavone has the potential to serve as an adjuvant that sensitizes regorafenib for the treatment of HCC without causing any side effects.

Citation Format: Yan ting Tsai, Ying-Tzu Chen, Fei-Ting Hsu, Jai-Jen Tsai, Yu-Chiang Liu. Amentoflavone enhances regorafenib-induced apoptosis and -inhibited invasion via inactivating STAT3/NF-ΚB signaling pathway in Hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1102.
Abstract 1147: Sub-cellular spatial resolution of immune and stromal interactions underlying prostate cancer progression
Authors: Mo Chia-Kuei , Jayasinghe Reyka G. , Abedin do Ateih , Li Xiang , Weimholt Cody , Kim Eric H. , Pachynski Russell , Chen Feng , Ding Li
Published: March 2024

Prostate cancer boasts a complex morphological structure and diverse cellular composition, yet current technology falls short in resolving the detailed spatial interactions among cells within the environment. In this study, we employed the Xenium platform, coupled with the 3D reconstruction tool Mushroom, to examine high-resolution spatial cellular interactions within the prostate cancer microenvironment. Our investigation focused on the transition of molecular characteristics from normal to high-grade prostatic intraepithelial neoplasia (HGPIN, pre-cancer), spanning the spectrum of Gleason Grades 3 to 4 and advancing to metastatic prostate cancer. We identified genes with unique spatial and grade-dependent expression profiles, including MYC, CP, and UHCL1. Evaluating sub-cellular imaging facilitated a comprehensive characterization and quantification of immune and stromal interactions, unveiling distinct spatial niches across transition stages. These findings underscore the nuanced immune interactions within the tumor microenvironment and offer crucial insights into the development of immunotherapeutic approaches tailored to the specific spatial and immune dynamics observed in advanced prostate cancer. This research contributes to the evolving understanding of prostate cancer progression, providing a foundation for the development of targeted therapies that disrupt specific cellular interactions.

Citation Format: Chia-Kuei Mo, Reyka G. Jayasinghe, Ateih Abedin do, Xiang Li, Cody Weimholt, Eric H. Kim, Russell Pachynski, Feng Chen, Li Ding. Sub-cellular spatial resolution of immune and stromal interactions underlying prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1147.
Abstract 1217: Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma
Authors: Carvalho Larissa A. , Phadke Manali S. , Smalley Keiran S. M.
Published: March 2024

Introduction: Mutations in the small GTPase NRAS are the second most common oncogenic driver in cutaneous melanoma, being found in ~25% of cases. Targeted therapy strategies are currently lacking for NRAS-mutant melanoma. Here, we characterized the anti-tumor activity and immune effects of RMC-7977, a RASMULTI(ON) preclinical tool compound, representative of the investigational agent RMC-6236, in preclinical mouse and human models of NRAS-mutant melanoma.

Methods: The in vitro potency of RMC-7977 was determined using mouse and human NRAS-mutant melanoma cell lines. Recovery of signaling was quantified by Western Blot and receptor tyrosine kinase (RTK) arrays. In vivo anti-tumor activity of RMC-7977 was determined in two syngeneic mouse melanoma models (SW1 NRASG13D and NRAS#5 NRASQ61R), and tumor immune infiltrate quantified using multiplexed IHC and flow cytometry. Mechanisms of resistance were defined using RNA-seq.

Results: RMC-7977 had good activity in vitro against human cancer cell lines and in mouse models of NRAS-mutant melanoma. Time-course studies demonstrated RMC-7977 rapidly inhibited phospho-ERK signaling, which then began to recover after 8 hr of treatment. There was also evidence of adaptive receptor tyrosine kinase signaling by 48 hr. In syngeneic mouse models of NRAS-mutant melanoma, RMC-7977 led to a rapid inhibition of tumor growth, increased expression of tumor MHC class I and PD-L1 and an increase in CD4+ and CD8+ T cell infiltrate into tumors. Therapeutic escape was associated with decreased CD4+ and CD8+ T cell infiltration and increased numbers of immunosuppressive MDSCs. Cells collected from tumors resistant to RMC-7977 in vivo did not show any shift in sensitivity to the inhibitor when re-challenged in vitro, suggesting that the resistance observed in vivo resulted from mechanisms specific to the tumor microenvironment, for example adaptive RTK signaling or immune escape. RNA-seq and IHC analysis suggested that resistance was associated with decreased MHC expression and antigen presentation, as well as reduced co-stimulation of T cells. Combination studies of RMC-7977 with anti-PD-1 are ongoing to identify potential synergistic interactions in these preclinical models.

Conclusion: RMC-7977 has anti-tumor activity in syngeneic mouse models of NRAS-mutant melanoma, associated with a robust immune response.

Citation Format: Larissa A. Carvalho, Manali S. Phadke, Keiran S. M. Smalley. Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1217.
Abstract 1271: PMEPA1 acts as a switch to modulate cooperative cellular invasion and drive NSCLC tumor progression
Authors: Khatib Tala O. , Pedro Brian A. , Knippler Christina M. , Matsuk Veronika Y. , Webster Sarah F. , Bombin Sergei , Dwivedi Bhakti , Kowalski Jeanne , Johnston Henry R. , Mouw Janna K. , Marcus Adam I.
Published: March 2024

Metastatic disease drives cancer patient mortality. One primary mode of metastasis is collective invasion, whereby cohesive groups of cells invade into the adjacent stroma while maintaining cell-cell contacts. Importantly, these cellular packs harbor genetically and phenotypically heterogeneous subpopulations that cooperate to drive invasion. To deconstruct how phenotypic cellular heterogeneity facilitates distinct molecular profiles within a single tumor, we established the technique Spatiotemporal Cellular and Genomic Analysis (SaGA). SaGA is an image-guided approach that optically highlights phenotypically defined cell(s) for live fluorescence-activated cell sorting and analysis. To elucidate the transcriptional heterogeneity across invasive packs within a 3D physiologically-relevant microenvironment, we combined SaGA with single cell RNA sequencing for isolation and analysis of actively invading cells. This novel single cell transcriptomics approach reveals that - during cellular invasion into the microenvironment - the follower subpopulation supplies TGFβ to heterogeneously stimulate both autocrine canonical and paracrine noncanonical signaling pathways. This cooperative exchange facilitates a symbiotic relationship between tumor subpopulations in non-small cell lung carcinoma both in vitro and in vivo. In vitro, we determine that PMEPA1 protein levels mitigate TGFβ downstream effector signaling within heterogeneous tumor cells to modulate JAG1 expression and activity. In vivo, we show that a reduction in JAG1 levels decreases primary tumor cell invasion and progression. We mechanistically define a TGFβ-PMEPA1-JAG1 signaling axis across leader and follower subpopulations critical for tumor cell invasion and cancer progression. Together, these data highlight the importance of cellular heterogeneity and subpopulation communication required for tumor progression.

Citation Format: Tala O. Khatib, Brian A. Pedro, Christina M. Knippler, Veronika Y. Matsuk, Sarah F. Webster, Sergei Bombin, Bhakti Dwivedi, Jeanne Kowalski, Henry R. Johnston, Janna K. Mouw, Adam I. Marcus. PMEPA1 acts as a switch to modulate cooperative cellular invasion and drive NSCLC tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1271.
Abstract 1278: Differential urinary microbiome and its metabolic footprint in bladder cancer patients following BCG treatment
Authors: Piao Xuan-Mei , Min Kyungchan , Byun Young Joon , Zheng Chuang-Ming , Kim Seon-Kyu , Park Seong-Hwan , Moon Sungmin , Kim Kyeong , Kang Ho Won , Kim Won Tae , Yun Seok Joong , Park Hansoo
Published: March 2024

Introduction & Objectives: Recent insights reveal a urinary microbiome, challenging prior beliefs about urine's sterility. Using 16S rRNA gene sequencing, a pivotal tool in mapping this microbiome, our study delves into the interplay between bladder cancer (BCa), BCG therapy, and the urinary microbiome. We analyzed bacterial profiles in benign and malignant samples, considering various clinical parameters.

Materials & Methods: A total of 88 patients participated in the study, comprising 30 individuals with benign diseases and 58 BCa patients. Urine samples were collected from patients with benign conditions and from BCa patients before and after BCG treatment. Malignant samples obtained before the completion of the intravesical BCG induction cycle were labeled 'Pre-BCG', while those collected after this cycle were termed 'Post-BCG'. Following collection, DNA was extracted and underwent 16S rRNA gene sequencing. The sequencing data was then utilized for bioinformatic analyses, which included metagenomic profiling and metabolic pathway inference.

Results: Significant differences in bacterial compositions were observed between benign and malignant samples, with a metabolic signature suggesting toluene degradation pathway may mitigate bladder cancer development. In the post-BCG samples, we inferred elevations of 2-oxoglutarate, a BCG derivative, and L-methionine, which is associated with therapeutic benefits, compared to their pre-BCG counterparts. Furthermore, post-BCG samples showed microbial differences in responders and increased quinolone synthesis compared to non-responders. This is of significance due to quinolone's role in modulating DNA topoisomerase and mitochondrial functions, both pivotal in bladder cancer progression.

Conclusions: Utilizing 16S rRNA gene sequencing, we drew comparisons between the urinary microbiomes of benign and malignant samples and suggested a potential pathway for bladder cancer occurrence. Following intravesical BCG treatment, certain microbial metabolic pathways seemed to offer therapeutic benefits to responders. Our study provides insights into the microbial influences on bladder cancer development and the varying responses to BCG treatment.

Citation Format: Xuan-Mei Piao, Kyungchan Min, Young Joon Byun, Chuang-Ming Zheng, Seon-Kyu Kim, Seong-Hwan Park, Sungmin Moon, Kyeong Kim, Ho Won Kang, Won Tae Kim, Seok Joong Yun, Hansoo Park. Differential urinary microbiome and its metabolic footprint in bladder cancer patients following BCG treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1278.
Abstract 1324: Superior anti-tumor activity of GL205, an allogeneic anti-CD5 CAR-NK for treating T-cell malignancies
Authors: Jung Miyoung , Kim Seung Min , Lee Eunsol , Sun Hyunseung , Yoo Jaeyoung , Choi Hwi Wan , An Subin , Cho Sunglim , Min Bokyung
Published: March 2024

T cell malignancies are rare and intractable diseases with fewer treatment options and higher unmet needs compared to B-cell lymphomas. Chemotherapy such as CHOP has been used as the standard therapy for treatment of T cell malignancies, but clinical outcome is not a promising and alternative treatments are needed. Currently, Brentuximab Vedotin, an antibody-drug conjugate that targets CD30 is widely used for Hodgkin lymphoma, ALCL and CTCL. However, the application is limited to specific subtypes because CD30 expression is restricted to some subsets of T-cell lymphoma. CD5 is an attractive target for cancer immunotherapy because it is commonly expressed in most of T cell malignancies. Although CD5 CAR-T cell products have demonstrated the anti-cancer efficacy, there are several disadvantages including 1) challenges due to difficulties in manufacturing process caused by fratricide, 2) product contamination by CAR-introduced patient’s malignant T cells, 3) severe side effects such as CRS and neurotoxicity. Therefore, CD5 CAR-NK as allogeneic anti-tumor therapy can be an alternative for treatment of T cell malignancies. GL205, cord blood-derived CD5 CAR-NK, is genetically modified to express CD5-directed CAR and simultaneously produce IL-15 which supports the NK survival and proliferation. GL205 is ex-vivo expanded, cryopreserved cell therapy product comprising of pure NK cells (> 99%) with high CD5 CAR expression (> 90%). GL205 showed significantly higher in vitro direct-killing efficacy against CD5+ tumor cells compared to donor matched CBNK. IFN-γ and IL-15 release from GL205 was significantly increased after co-incubation with CD5+ tumor cells. In xenograft mouse model, GL205 administration exhibited a greater survival rate and low tumor burden compared to vehicle group. In addition, GL205 was detected in blood for 3 months, demonstrating in vivo long-term persistence. In conclusion, GL205 could be a valuable and off-the-shelf allogeneic therapeutic option for treating T cell malignancies.

Citation Format: Miyoung Jung, Seung Min Kim, Eunsol Lee, Hyunseung Sun, Jaeyoung Yoo, Hwi Wan Choi, Subin An, Sunglim Cho, Bokyung Min. Superior anti-tumor activity of GL205, an allogeneic anti-CD5 CAR-NK for treating T-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1324.
Abstract 1327: Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia
Authors: Kizerwetter Monika , Yang Huilin , Rahnama Ruyan , Bonifant Challice , Spangler Jamie
Published: March 2024

Acute myeloid leukemia (AML) is a highly prevalent blood and bone marrow cancer characterized by the uncontrolled growth of abnormal myeloblasts. Current treatments for AML often result in systemic toxicities for patients, many of whom will still experience relapse. There is thus an unmet need to develop safer and more effective therapies targeting AML. Chimeric antigen receptor (CAR) T cells, which are engineered to express a cancer-targeting extracellular antibody single-chain variable fragment (scFv) linked to an intracellular T cell-activating domain, have shown promise in treating B cell leukemias, but their success has been limited in myeloid leukemias. This is due in part to the lack of AML-specific target antigens. Moreover, impaired T cell function, a hallmark of AML, is also problematic when relying on autologous T cell activation upon reinfusion. We overcame the limitations of CAR T therapy in AML by developing CAR-expressing natural killer (NK) cells targeting CD123. While present on both healthy and AML cells, CD123 is expressed at >10-fold higher levels on malignant cells. Furthermore, use of NK allows use of fully functional allogeneic cells from healthy donors, since NK cells do not induce graft-versus-host-disease. To optimize the tumor cell selectivity of CAR NK cells, we employed the yeast display directed evolution platform to isolate anti-CD123 scFvs with varying affinities and are identifying and characterizing clones that maximize the interaction with CD123high AML cells over healthy CD123low cells, as determined by binding, cytotoxicity, cytokine secretion, and intracellular signaling studies. Collectively, our efforts introduce a new paradigm for engineered cell therapeutics with significant promise for AML treatment.

Citation Format: Monika Kizerwetter, Huilin Yang, Ruyan Rahnama, Challice Bonifant, Jamie Spangler. Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1327.
Abstract 1398: The role of ST8SIA2 & ST8SIA4 in melanoma progression and metastasis
Authors: Amirfallah Amirali , Gallant Kayla , Cavallo Maria , Huang Zongguan , Hartsough Edward
Published: March 2024

Melanoma is the deadliest form of skin cancer; while early detection affords patients a 5-year survival rate greater than 90 percent, survival for metastatic patients is poor. Metastatic dissemination is reliant on melanoma’s ability to escape immune surveillance and migrate to distant organs. These processes are heavily influenced by cell surface proteins, which themselves can be regulated by post-translational modifications (PTM) like protein glycosylation. An understudied glycosylation that may affect immune evasion and cancer progression is polysialylation (polySia). Here, a long-chained polymer of sialic acid moieties is formed by the polysialyltransferases ST8SIA2 and ST8SIA4. In silico analysis suggests that compared to normal skin, primary cutaneous melanoma has elevated expression of both ST8SIA2 and ST8SIA4. Interestingly, although ST8SIA4 is upregulated in metastatic samples, its expression is associated with increased survival. Further analysis revealed a positive correlation between CD8+ T cell infiltrates and ST8SIA4 expression. Furthermore, analysis of ST8SIA2 expression shows an inverse correlation with survival and T Cell CD8+ cells infiltration, suggesting ST8SIA2 and -4 differentially regulate the tumor immune microenvironment. One potential polySia modified cell surface protein, mediating these effects is Cell Adhesion Molecule 1 (CADM1). CADM1 is a target of ST8SIA2 and may also be modified by ST8SIA4. Loss of CADM1 increases the invasive properties of melanoma cells and our recent point mutational analysis reveals that the adhesive function of CADM1 might be regulated by polySia at Asp 113. In addition to its role in cancer migration and invasiveness, CADM1 expression may also affect the tumor immune environment. Further investigations are needed to untangle the complex relationship between PolySia modification and melanoma immune evasion and metastasis.

Citation Format: Amirali Amirfallah, Kayla Gallant, Maria Cavallo, Zongguan Huang, Edward Hartsough. The role of ST8SIA2 & ST8SIA4 in melanoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1398.
Abstract 1421: The HDAC6-specific inhibitor AVS100 (SS208) blocks M2 polarization of tumor associated macrophages and potentiates immunotherapy in preclinical tumor models
Authors: Kovalovsky Damian , Noonepalle Satish , Suresh Manasa , Kumar Dileep , Berrigan Michael , Horvath Anelia , Kim Allen , Quiceno-Torres David , Musunuri Karthik , Villagra Alejandro
Published: March 2024

Inhibition of HDAC6 was associated with an increased proinflammatory tumor microenvironment and an antitumoral response. Here, we show that a highly specific HDAC6 inhibitor AVS100 (SS208), blocks M2 polarization in murine and human macrophages while partially affecting M1 polarization. AVS100 effects were observed as blocked upregulation of M2-related gene signature under M2 polarizing conditions and blocked generation of CD206+ and Arg1+ macrophages. Oral administration of AVS100 had an antitumoral effect in SM1 melanoma and CT26 colon cancer models and increased the efficacy of anti-PD1 treatment, leading to complete remission in melanoma and increased response in colon cancer. Flow cytometry and scRNAseq analysis of tumor-infiltrating immune cells revealed an increase of proinflammatory/anti-inflammatory ratio in tumor-associated macrophages as well as an increase of intratumoral CD8 effector T-cells after AVS100 treatment. Interestingly, cured mice didn’t relapse and became resistant to a subsequent tumor challenge, suggesting acquired antitumoral T-cell immunity. T-cell repertoire analysis of effector/memory T-cells in cured mice revealed a higher number of immunodominant T-cell clones after AVS100 treatment, indicating increased T-cell expansion. Finally, AVS100 has demonstrated no mutagenicity and a strong safety profile in rats and dogs, leading to its recent U.S. FDA clearance of an Investigational New Drug (IND) application and planned initiation of Phase Ia/b clinical trials targeting locally advanced or metastatic solid tumors in the first half of 2024. Altogether, we have performed the preclinical characterization of a novel small molecule inhibitor targeting HDAC6 for solid cancers. AVS100 had an antitumoral effect as single agent and improved the efficacy of immune checkpoint inhibition by blocking the immunoregulatory tumor microenvironment and increasing T-cell immunity.

Citation Format: Damian Kovalovsky, Satish Noonepalle, Manasa Suresh, Dileep Kumar, Michael Berrigan, Anelia Horvath, Allen Kim, David Quiceno-Torres, Karthik Musunuri, Alejandro Villagra. The HDAC6-specific inhibitor AVS100 (SS208) blocks M2 polarization of tumor associated macrophages and potentiates immunotherapy in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1421.
Abstract 1422: A novel gut microbiota therapeutic approach for enhancing immune checkpoint inhibitor therapy
Authors: Valle Priscilla Del , Nguyen Gabriella , Choi Yongbin , Sabaeifard Parastoo , li Wenng , Lichterman Jake , Coughlin Laura , Poulides Nicole , Sanjuan Angelica , Koh Andrew Y.
Published: March 2024

Immune checkpoint inhibitor therapy (ICT) has revolutionized cancer care, but up to 50% of patients do not respond to ICT. A host factor implicated in variable ICT response is the gut microbiome: a) germfree/antibiotic-treated mice do not respond to ICT; b) human ICT responders have distinct gut microbiome signatures compared to non-responders; and c) fecal microbiota transplants are safe and potentially effective in cancer patients previously unresponsive to ICT. We have previously established that ICT induces translocation of gut microbiota into secondary lymphoid tissues (particularly the mesenteric lymph nodes) and tumor which is required to enhance extraintestinal anti-tumor immunity (PMID: 36867675). Here, we have developed a novel gut microbiota therapy (GMT) which circumvents the challenges and limitations of conventional microbiome therapies that use live microbes. GMT utilizes specific components derived from commensal gut microbiota identified in our published clinical study (PMID:28923537) that can be administered parenterally (subcutaneous/intravenous) to safely and reproducibly enhance ICT efficacy in multiple preclinical cancer models (B16-F10 with anti-PD-1 and/or CTLA-4, MC38 with or without anti-PD-1, humanized mouse with human melanoma and human anti-PD-1). GMT acts by agonizing the pattern recognition receptors hTLR1/2/6, hNOD2 and hSTING. GMT induces anti-tumor cytokines (IL-12) but does not significantly increase sepsis associated cytokines (IL-6, TNFa) in both human immune cells and in non-human primates. Mechanistically, GMT acts via dendritic cells, and potentially through increased expression of delta-like ligand protein 4 (DLL4), a Notch signaling ligand that activates Notch 1/4 in T cells and promotes Th1 and Th17 differentiation. Current efforts to identify the active components of GMT are underway in which we have used serial liquid-liquid extractions (ethyl acetate followed by water:methanol:chloroform) and subsequent reverse phase chromatography to identify the most active fractions and identification of the bacterial components/constituents within these fractions. Together, these results demonstrate that GMT has the potential to serve as an innate immune adjuvant (akin to vaccine adjuvants) that enhances the effect of immunotherapy for possibly many cancer types.

Citation Format: Priscilla Del Valle, Gabriella Nguyen, Yongbin Choi, Parastoo Sabaeifard, Wenng li, Jake Lichterman, Laura Coughlin, Nicole Poulides, Angelica Sanjuan, Andrew Y. Koh. A novel gut microbiota therapeutic approach for enhancing immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1422.
Abstract 1430: Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity
Authors: Rajpurohit Surendra K. , Gossman Taitum , Nasanally Sai , Bhatt Keshu , Van Derhei Aiden , Hotwani Simran , Manhiani Devan-Anmol , Pasi Rohan , Mi Jiaqi , Arora Vishal , Lokeshwar Balakrishna
Published: March 2024

Cardiotoxicity is the second leading cause of death among cancer survivors and can manifest as arrhythmia, acute coronary syndrome, acute heart failure, or pericarditis/myocarditis. Chronic myocyte damage and apoptosis can transition from an asymptomatic phase to adaptive myocardial hypertrophy, followed by decreased left ventricular ejection fraction, and eventual heart failure. Cardio-oncology is an emerging field that studies cancer therapy-associated cardiotoxicities. The development of reliable and effective preclinical models for cardiotoxicity prediction and prevention is essential to improving the long-term cardiac health of cancer patients. Zebrafish are a promising animal model for the field of cardio-oncology and have emerged as an invaluable tool for investigating genetic and molecular aspects of cardiovascular research. Zebrafish heart cells are like human heart cells at the molecular level, making them an ideal system for studying the genetic underpinning of cardiac function. Zebrafish myl7 gene, a myosin light chain-7 gene, is a regulatory gene of heart orthologs to human MYL7. In-vivo imaging is limited due to the opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating the blocking of pigmentation. The Zebrafish Casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. In this study, we have developed a novel transparent transgenic zebrafish model and established time-lapse in-vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes. The newly developed homozygous transparent transgenic Casper/myl7:RFP; annexin-5:YFP/NF-kB: GFP/mpeg1:mcherryFP, allows us to study cell death process (Annexin5) and inflammatory activity (NF-kB) in microglia and cardiomyocytes. We have successfully established in vivo fluorescence and confocal microscopy in this newly developed strain, enabling the study of cellular morphology and function changes under various conditions. This will allow us to elucidate cell death patterns, and cardio-inflammatory and cardiotoxicity pathways.

Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simran Hotwani, Devan-Anmol Manhiani, Rohan Pasi, Jiaqi Mi, Vishal Arora, Balakrishna Lokeshwar. Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1430.
Abstract 1434: Modeling the 3q26 OncCassette using genetically engineered mice
Authors: Meneses Kayleah M. , Liu Yi , Weems Capella R. , Jamieson Lee , Nguyen Duy T. , Justilien Verline , Murray Nicole R. , Fields Alan P.
Published: March 2024

Lung squamous cell carcinoma (LUSC) is a devastating disease, accounting for ~30% of all lung cancer diagnoses and ~40,000 deaths annually in the United States alone. LUSC patients suffer from high prevalence of relapse and a dismal 5-year survival of only ~24%. The lack of animal models that reflect the salient features of human disease to assess the safety and efficacy of drugs, and to explore the underlying molecular mechanisms of LUSC tumor biology, is a major impediment for LUSC patients. We have identified concomitant 3q26 copy number gain (CNG) and TP53 loss as a defining genetic alteration present in ~90% of LUSC tumors. Furthermore, we have identified and characterized three 3q26 oncogenes (PRKCI, ECT2 and SOX2) that genetically and biochemically collaborate to drive LUSC tumor formation. Using syngeneic mouse modeling, we have demonstrated that coordinate overexpression of Prkci, Ect2 and Sox2, and loss of Trp53 in ex vivo cultures of lung basal stem cells (LBSCs; a cell of origin for LUSC) drives transformation of these cells into tumors of exclusively LUSC histopathology. These findings lead to the hypothesis that PRKCI, ECT2 and SOX2 represent a multigenic driver of 3q26 CNG-driven LUSC, which we term the 3q26 OncCassette. Based in these findings, we have developed and begun characterizing the first autochthonous genetically engineered mouse model (GEMM) to study 3q26 OncCassette-driven tumorigenesis. Our novel immune competent GEMM will provide critical insight into tumor initiation and progression of preneoplastic lesions to malignant LUSC and allow characterization of the immune landscape in these tumors.

Citation Format: Kayleah M. Meneses, Yi Liu, Capella R. Weems, Lee Jamieson, Duy T. Nguyen, Verline Justilien, Nicole R. Murray, Alan P. Fields. Modeling the 3q26 OncCassette using genetically engineered mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1434.