bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3847: Dual targeting of EGFL6 and VEGF-A to overcome immune suppression in ovarian cancer
Authors: Umamaheswaran Sujanitha , Mangala Lingegowda S. , Joseph Robiya , Jennings Nicholas B. , Kim Mark S. , Wen Yunfei , Bayraktar Emine , Zhang Ningyan , An Zhiqiang , Sood Anil K.
Published: March 2024

Background: Ovarian carcinomas are known to have a high degree of immune suppression, which may be related, in part, to high levels of vascular endothelial growth factor-A (VEGF-A). Bevacizumab (anti-VEGF antibody) has been shown to increase infiltration of T-cells in the tumor microenvironment (TME). We have also identified EGFL6 as an important angiogenic protein. Here, we examined the biological effects of blocking EGFL6 and VEGF-A in ovarian cancer models.

Methods: We studied the in vitro (endothelial tube formation and migration assays) and in vivo (ID8KO-Trp53;Brca2 and SKOV3-ip1 models) biological effects of EGFL6 and VEGF-A blockade. Tumor-infiltrating immune cells were studied by flow cytometry and immunohistochemistry staining.

Results: Blocking EGFL6 and VEGF-A simultaneously with monoclonal antibodies in RF24 endothelial cells decreased tube formation and migration significantly, in comparison to VEGF-A blockade alone (p<0.001). EGFL6 monotherapy in ID8KO-Trp53;Brca2 tumor bearing syngeneic mice decreased tumor burden (~2.5 fold, p<0.0001) and increased the infiltration of IFNγ+ (~2.75 fold, p<0.0001) and GzmB+ (~1.75 fold, p<0.01) CD8+ T-cells and IFNγ+ (~2 fold, p<0.0001) and GzmB+ (~2 fold, p<0.01) CD4+ T-cells while decreasing PD-L1+ cells (~1.8 fold, p=0.01). Dual blockade of EGFL6 and VEGF-A in SKOV3-ip1 tumor bearing nude mice significantly decreased tumor burden (~3.72 fold, p<0.01) and angiogenesis (~2.36 fold, p<0.05) compared to controls and single-agent blockade. Similar blockade in ID8KO-Trp53;Brca2 tumor bearing mice significantly decreased tumor burden (~4.2 fold, p=0.0004), improved CD8+ (~3.32 fold, p=0.0004) and CD4+ (~2.81 fold, p<0.0001) T-cell infiltration, NK cell infiltration (~2.3 fold, p=0.0005) and decreased myeloid-derived suppressor cells (MDSCs) (~1.92 fold,

p=0.0278) compared to EGFL6 and VEGF-A blockade alone.

Conclusions: Our results demonstrate that simultaneously targeting the angiogenesis drivers EGFL6 and VEGF-A in ovarian cancer significantly decreased tumor burden in vivo and increased the infiltration of T lymphocytes compared to single-agent blockade.

AACR Category: Tumor Microenvironment and Immune Oncology

Key words: ovarian cancer, immune suppression, angiogenesis, VEGF-A, EGFL6

Citation Format: Sujanitha Umamaheswaran, Lingegowda S. Mangala, Robiya Joseph, Nicholas B. Jennings, Mark S. Kim, Yunfei Wen, Emine Bayraktar, Ningyan Zhang, Zhiqiang An, Anil K. Sood. Dual targeting of EGFL6 and VEGF-A to overcome immune suppression in ovarian cancer [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 3847.
Abstract 385: Stress-induced pentraxin 3 enhances anoikis resistant-associated metastasis and post-chemotherapeutic recurrence in head and neck cancer
Authors: Chang Ting-Wei , Chang Wen-Chang , Chen Ben-Kuen
Published: March 2024

Head and neck squamous cell carcinoma (HNSCC) is one of the most aggressive cancer with the properties of local recurrence and lymphatic metastasis. During metastatic process in blood circulation, cancer cells lose the interaction with extracellular matrix and encounter the anoikis stress, resulting in successfully extravasate and form micrometastasis in distal organs. However, most patients develop chemo-drug resistance during the metastatic recurrence which leading to poor prognosis. Thus, it is a pressing matter to elucidate the molecular mechanisms involved in cancer metastasis and the behaviors of the post-chemotherapy treated HNSCC cells. Our previous studies suggest that growth factor signaling stimulated HNSCC cells metastasis by the expression of pentraxin 3 (PTX3). Moreover, recent findings also reveal that chemotherapy mediated oxidative stress induce PTX3 expression and poor progression. Here, we raise the hypothesis that the expression of PTX3 through stress signal may promote cancer cells against from anoikis and promote the post-chemotherapeutic survival. The first, we identified that the secondary messenger-reactive oxygen species (ROS) dominate the gene activation of PTX3 by spheroid culture and platium-based agents’ treatment. The ROS scavenger, NAC, inhibited cisplatin (CDDP)-induced PTX3 expression and rescue the cell viability by decreasing the ROS level. The oxidative stress inducer-H2O2 treatment also verify the PTX3 expression through AKT and NF-κB pathways. In addition, both the spheroid selection and CDDP-induced PTX3 regulated the EMT phenotype and further strengthen the growth factor signaling. In addition, we found that PTX3 also regulate the survival during spheroid selection and drug treatment. The post-selected cells also exhibit the unique glycosylated modification on PTX3. Disrupted the PTM modification of PTX3 could inhibit the spheroid survival and cell invasion. The animal studies revealed that both spheroid selection and CDDP-treated HNSCC cells had stronger extravasation ability than parental cells in NOD-SCID mice. Depletion of PTX3 also repressed the HNSCC cells extravasation ability. The results indicate that PTX3 were essential for oxidative stress-induced tumor metastasis and recurrence. It also speculates that the combination of antioxidants and PTX3 antibody may prevent tumor metastatic recurrence after chemotherapy in HNSCC.

Citation Format: Ting-Wei Chang, Wen-Chang Chang, Ben-Kuen Chen. Stress-induced pentraxin 3 enhances anoikis resistant-associated metastasis and post-chemotherapeutic recurrence in head and neck cancer [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 385.
Abstract 386: BACH1 is a key regulator of the tumor hypoxia response
Authors: Nguyen Long Chi , Dann Christopher , Yang Dongbo , Henn Madeline , Shi Emily , Li Thomas , Stock Letícia , Liu Wenchao , Matossian Margarite , Valdespino Andrea , Han Yoo Jane , Zhang Jing , Yerradoddi Geetha Priyanka , Li Yan , Matsumoto Mitsuyo , Olopade Olufunmilayo I. , Igarashi Kazuhiko , Rosner Marsha R.
Published: March 2024

Hypoxia is an important hallmark of aggressive solid tumors and a key driver of metastasis and resistance to therapy. Hypoxic stress induces the activation of various factors, such as hypoxia-inducible factors (HIFs), which facilitate cellular adaptation and promote tumorigenesis. Given the complexity of this mechanism, identifying novel targets that sensitize hypoxic tumors to therapy could have significant clinical impact. Here we show that the pro-metastatic BTB and CNC homology 1 (BACH1) transcription factor is prolyl-hydroxylated by the HIF Prolyl Hydroxylase (PHD), similar to HIFs, in an oxygen-dependent manner. We describe two major prolyl hydroxylation sites in BACH1 and demonstrate that prolyl hydroxylation increases protein turnover and decreases DNA binding in normoxia. We further demonstrate HIF-independent induction of BACH1 within multiple TNBC (Triple-Negative Breast Cancer) cell lines and patient-derived organoids under hypoxic conditions. Utilizing ChIP-seq, bulk, single-cell and spatial transcriptomics across diverse BACH1 knockout tumor models, our study revealed that BACH1 selectively and directly modulates the transcriptional response associated with hypoxia and stress in TNBC. Our findings identify BACH1 as a clinically relevant target for attenuating hypoxia induced, pro-metastatic signaling and therapeutic resistance in cancers.

Citation Format: Long Chi Nguyen, Christopher Dann, Dongbo Yang, Madeline Henn, Emily Shi, Thomas Li, Letícia Stock, Wenchao Liu, Margarite Matossian, Andrea Valdespino, Yoo Jane Han, Jing Zhang, Geetha Priyanka Yerradoddi, Yan Li, Mitsuyo Matsumoto, Olufunmilayo I. Olopade, Kazuhiko Igarashi, Marsha R. Rosner. BACH1 is a key regulator of the tumor hypoxia response [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 386.
Abstract 3873: Understanding spatial organization of cellular plasticity in pancreatic ductal adenocarcinoma
Authors: Zamora Izabella , Parikh Milan , Patel Bidish K. , Bi Lynn , Haviv Doron , Cao Jingyi , Shiau Carina , Hemberg Martin , Hwang William L. , Hacohen Nir , Ting David T. , Mehta Arnav
Published: March 2024

Pancreatic ductal adenocarcinoma (PDAC) is notorious for poor survival rates, underscoring the urgency of understanding factors that drive tumor progression. A key player in this process is epithelial-mesenchymal transition (EMT), known for its role in enhancing cancer cell adaptability, resistance to treatment, and metastasis. Our study focuses on EMT within PDAC, using single-cell spatial transcriptomics to explore how the tumor microenvironment influences changes in cellular states. We introduce a novel method involving Gaussian mixture models (GMM) to categorize EMT stages in single-cell data and utilize deep learning for label transfer of these tumor cell states, including intermediate cell states, onto spatial transcriptomic data with limited gene panels and sparse count profiles. We find strong spatial autocorrelation of classical cells and a dispersion of basal cells alongside other cells at intermediate stages of the EMT spectrum. By leveraging the spatial proximity of clonally related cells, we confirm that cells transition along the EMT spectrum in a gradient and infer transition rates. We next leverage density gradients of tumor cells in different states to identify distinct neighborhoods with varying EMT subtype composition. We observe significant variations in cell density among different EMT states. A focus on cancer-associated fibroblasts (CAFs) and macrophages reveals differences in these cells across density contours. We also examine differential ligand-receptor interactions across cell state gradients to better understand the cellular interactions that drive EMT transitions and alter the tumor environment. By leveraging spatial transcriptomics, this research aims to provide new biological insights into PDAC intratumoral heterogeneity, potentially leading to the identification of novel therapeutic targets to disrupt treatment resistance.

Citation Format: Izabella Zamora, Milan Parikh, Bidish K. Patel, Lynn Bi, Doron Haviv, Jingyi Cao, Carina Shiau, Martin Hemberg, William L. Hwang, Nir Hacohen, David T. Ting, Arnav Mehta. Understanding spatial organization of cellular plasticity in pancreatic ductal adenocarcinoma [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 3873.
Abstract 3903: Discovery of ULBP6 as a novel immuno-oncology target using pleiotropic signals from 23andMe’s genetic and health survey database
Authors: Tilk Susanne , Fontanillas Pierre , Chung Wei-Jen , Hom Clifford , Shi Shi , Diep Anh , Gerrick Kim , Poggio Mauro , Sood Pranidhi
Published: March 2024

23andMe has assembled one of the largest platforms for human genetics research that combines genotypes from 14 million individuals, 80% of which are consented to participate in research, with billions of phenotypic data points collected from health survey data. Using these large scale genetic and phenotypic datasets, we have developed a unique target discovery platform that has identified hundreds of pleiotropic genetic variants with opposing directionality of effects in cancer and immune diseases. These immune-oncology (I/O) genetic associations point to already clinically validated immune checkpoint receptors (e.g., CTLA4) and those currently in clinical trials (e.g., CD200R1). This suggests that these I/O genetic associations could be linked to genes that are broadly utilized by tumor cells to evade the immune system and could be used to identify novel therapeutic targets involved in regulating anti-tumor immune response. Here, we utilized expression quantitative trait loci (eQTLs) and annotation of coding single nucleotide polymorphisms (SNPs) to link one of these signals to a causal gene, and identified ULBP6, a stress-induced ligand for the activating immunoreceptor NKG2D found on NK and T cells. ULBP6 is known to be shed from the cell surface of tumor cells to block its interaction with NKG2D to evade immune surveillance. Expression profiling of ULBP ligands in various tumor types using MSD, IHC, bulk and single-cell RNA-seq revealed tumor-intrinsic up-regulation of ULBP6 in squamous cancer subtypes. Collectively, these data suggest that ULBP6 is a promising therapeutic target, and that can potentially enhance anti-tumor immunity.

Citation Format: Susanne Tilk, Pierre Fontanillas, Wei-Jen Chung, Clifford Hom, Shi Shi, Anh Diep, Kim Gerrick, Mauro Poggio, Pranidhi Sood. Discovery of ULBP6 as a novel immuno-oncology target using pleiotropic signals from 23andMe’s genetic and health survey database [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 3903.
Abstract 3960: Establishment of humanized mouse models engrafted with luciferase-expressing acute myeloid leukemia cell lines
Authors: Bayon Emilie , Mersni Arije , Wetzel Audrey , Villé Jade , Yin Kiave Yune Ho Wang
Published: March 2024

Introduction: Because acute myeloid leukemia (AML) is a very aggressive and quickly developing cancer, for which actual treatments are either inefficient, poorly tolerated, or leading to resistance, there is a strong need for an effective platform to evaluate novel therapies. Our aim is to provide a relevant humanized mouse model as a precious tool to evaluate innovated treatments to cure AML.

Methods: Mice were humanized with CD34+ hematopoietic stem cells to reconstitute a complete human immune system. Three AML cell lines, namely OCI-AML2, OCI-AML3 and MOLM-14, all expressing mCherry and luciferase, were intravenously engrafted at different doses in non-humanized and CD34-humanized mice. Tumor proliferation was monitored by bioluminescence imaging (BLI) thanks to luciferase expression on tumor cells. Their expression of mCherry was also exploited to determine the presence of tumor cells in blood and tissues using flow cytometry.

Results: All AML cell lines engrafted in mice, but we observed different growth kinetics and homogeneity depending on the humanization of mice and the dose injected. Indeed, tumor grew more slowly and heterogeneously in humanized mice, which underlines the role of human immune cells reconstituted in mice. This was improved by increasing the number of tumor cells injected. Tumor cells were found in femurs, spleen, kidneys, ovary, lungs, and uterus depending on the cell line. Mice survival was also highly affected by the tumor cell line engrafted. We observed that MOLM14 was the most aggressive model, while OCI-AML3 reached a steady state and were tolerated by mice over a long period.

Conclusion: We established 3 different AML cell lines in a model of humanized mice which nicely recapitulates the growth of tumor cells in target organs. These 3 models behave differently in terms of growth kinetic, tolerability by mice, and organs infiltrated, which gives plenty of opportunities to evaluate novel therapies designed to cure AML.

Citation Format: Emilie Bayon, Arije Mersni, Audrey Wetzel, Jade Villé, Kiave Yune Ho Wang Yin. Establishment of humanized mouse models engrafted with luciferase-expressing acute myeloid leukemia cell lines [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 3960.
Abstract 3971: Progesterone impacts regulatory T cell activity to promote immunosuppression in the mammary gland
Authors: Chowanec Eilidh I. , Werner Lauryn R. , Hagan Christy R.
Published: March 2024

Estrogen and progesterone signaling together drive hormone-receptor positive breast cancer growth and progression. However, there have been limited studies on the independent role that progesterone and its receptor, the progesterone receptor (PR), play in the development of breast cancer. Studies in the Hagan lab have shown that progesterone signaling promotes mammary tumor growth and changes the immune landscape of the mammary gland towards immunosuppression. Regulatory T-cells (Tregs) are immune cells known to promote immunosuppression in the tumor microenvironment. The data from our lab shows that among hormone receptor positive breast cancers, higher levels of PR expression correlates to higher levels of Tregs in the tumor microenvironment. To determine if progesterone signaling increases the number of Tregs in the tumor-bearing mammary gland, we used a syngeneic orthotopic murine tumor model, E0771 cells, engineered to express mouse PR or control empty vector. Mice were treated with progesterone or placebo for 7 days before implantation of the modified E0771 cells. The results of this study show that progesterone promoted tumor growth, and flow cytometry analysis at tumor endpoint showed that PR-positive tumors treated with progesterone harbored more Tregs than mice treated with placebo. Additionally, initial studies using flow cytometry and RNA-seq indicate increased Treg activity following progesterone treatment in PR+ tumor-bearing mice. As progesterone treatment promoted tumor growth and Treg recruitment in progesterone receptor-positive tumors, these data imply that progesterone treatment may promote the release of secreted factors impacting the immune cells in the mammary microenvironment. To determine if progesterone treatment results in cytokine or chemokine changes that may affect Treg recruitment, cytokine arrays and enzyme-linked immunosorbent assays (ELISAs) were performed using cell culture supernatant from murine mammary tumor cells expressing progesterone receptor (E0771-PR) treated with progesterone (0-72 hours). E0771-PR cells treated with progesterone showed higher levels of cytokines and chemokines that have been linked to Treg recruitment and differentiation, such as MCSF. Our findings demonstrate that progesterone signaling promotes an immune suppressed microenvironment in the mammary gland through an increase in Tregs. This evidence provides rationale to investigate the use of anti-progestins as a mechanism to overcome the immune-cold tumor microenvironment seen in progesterone receptor positive breast tumors.

Citation Format: Eilidh I. Chowanec, Lauryn R. Werner, Christy R. Hagan. Progesterone impacts regulatory T cell activity to promote immunosuppression in the mammary gland [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 3971.
Abstract 3976: Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach
Authors: Chenchik Alex , Liu Tianbing , Makhanov Mikhail , Hu Dongfang , Kobzik Lester , Ghias Khadija , Diehl Paul
Published: March 2024

Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods.

Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) ɑ/β or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these ɑ/β and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases.

Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Lester Kobzik, Khadija Ghias, Paul Diehl. Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach [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 3976.
Abstract 4035: Lipid-based nanoparticle combination immunotherapy with sitravatinib for the systemic treatment of high-risk neuroblastoma
Authors: Diaz-Infante Miranda , Kane Griffin , Lusi Christina , Brassil Meghan , Wigglesworth Kim , Alarcon Christian , Shohet Jason , Atukorale Prabhani
Published: March 2024

We seek to develop a systemic, innate immunomodulatory nanoparticle therapy that combines synergistically with sitravatinib for the treatment of high-risk neuroblastoma, where metastasis is the largest hurdle for current treatment modalities. Here, we utilize a unique lipid-based nanoparticle (LNP) system comprised of co-encapsulated agonists of the Stimulator of Interferon Genes (STING) and Toll-like Receptor 4 (TLR4) pathways (immuno-NPs) that can be safely administered in the systemic blood circulation for delivery to the tumor microenvironment (TME) and uptake by perivascular innate antigen-presenting cells (APCs). STING/TLR4 agonists together promote the synergistic production of Type I interferon β (IFNβ), a key activator of anti-tumor immunity from the TME itself. Here, we also focus on sitravatinib, an investigational drug shown to inhibit receptor tyrosine kinases such as discoidin domain receptor 2 (DDR2), which can inhibit the sustained remodeling of collagen around primary tumors. The inhibition of such is vital to treatment as sustained collagen remodeling can facilitate the metastasis of cancer cells via epithelial-mesenchymal transition (EMT). We hypothesize that the downstream effects of sitravatinib treatment will augment tumor clearance and increase the therapeutic efficacy of immuno-NPs with respect to recruiting APCs. Our early pilot studies in humanized mice bearing orthotopic SH-5YSY tumors show that immuno-NPs are effective at tumor clearance. We found that mice inoculated subcutaneously with syngeneic 9464D-GD2 tumors showed decreased tumor growth when treated with our novel combination therapy in comparison to monotherapy controls. In both humanized and syngeneic tumor models, our ongoing mechanistic studies involve quantification of EMT in primary tumors, systemic nanoparticle deposition at metastatic sites, and the mechanistic impact of combination therapy on IFNβ-promoted elimination of metastasis. Here, we have proposed and tested an immunotherapy with great promise for treating neuroblastoma. However, the impact of nanoparticle-based immunotherapy reaches far beyond neuroblastoma: it has the potential to be a platform for the development of immunotherapies specific to other highly metastatic cancer types.

Citation Format: Miranda Diaz-Infante, Griffin Kane, Christina Lusi, Meghan Brassil, Kim Wigglesworth, Christian Alarcon, Jason Shohet, Prabhani Atukorale. Lipid-based nanoparticle combination immunotherapy with sitravatinib for the systemic treatment of high-risk neuroblastoma [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 4035.
Abstract 4037: Optimizing the efficacy of anti-VEGF/cytotoxics/anti-PD-1 scheduling in a lung cancer mice model through B and T cells monitoring
Authors: Sicard Guillaume , Protzenko Dorian , Giacometti Sarah , Fanciullino Raphaelle , Barlesi Fabrice , Ciccolini Joseph
Published: March 2024

The exact modalities of combinatorial regimens associating Immune Checkpoint Inhibitors (ICI) with other treatments are yet to be defined. Understanding how and when cytotoxics best reshape tumor immunity could help optimizing the scheduling of ICI’s. In vivo studies were performed on C57BL/6 mice grafted with LL/2lung cancer cells. A longitudinal immunomonitoring study on tumor, spleen and blood after multiple treatments including Cisplatin, Pemetrexed and anti-VEGF as single agents and in combination was performed for up to 21 days to determine the best time-window during which immune checkpoint inhibitors should be added. Finally, an efficacy study was conducted comparing the antiproliferative performance of various schedules of anti-VEGF, Pemetrexed-Cisplatin doublet, plus anti-PD-1 (i.e., immunomonitoring-guided scheduling, concurrent dosing or a random sequence). Immunomonitoring showed marked differences between treatments, organs and time-points. However, harnessing tumor immunity (i.e., promoting CD8 lymphocytes or increasing the CD8/Treg ratio) started on D7 and peaked on D14 with the anti-VEGF followed by cytotoxics combination. Therefore, a two-week delay between anti-VEGF/cytotoxic and anti-PD1 administration was considered the best sequence to test. Efficacy studies then confirmed that this sequence achieved higher anti-proliferative efficacy compared to other treatment modalities (i.e. -71% in tumor volume compared to control at the end of the study). Anti-VEGF and cytotoxic agents show time-dependent immunomodulatory effects, suggesting that sequencing is a critical feature when combining these agents with immune checkpoint inhibitors. Our efficacy study confirmed that sequencing treatments further enhances anti-proliferative effects in lung cancer models compared to concurrent dosing.

Citation Format: Guillaume Sicard, Dorian Protzenko, Sarah Giacometti, Raphaelle Fanciullino, Fabrice Barlesi, Joseph Ciccolini. Optimizing the efficacy of anti-VEGF/cytotoxics/anti-PD-1 scheduling in a lung cancer mice model through B and T cells monitoring [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 4037.
Abstract 4047: Unraveling synergistic therapeutic strategies involving FAK inhibition and immunotherapy using anti-PD1 antibody in KRAS and LKB1-driven lung cancer
Authors: Koo Junghui , Tucker-Burden Carol , Roy Melissa , Huang Chunzi , Zhou Wei , Gilbert-Ross Melissa , Fu Haian , Ramalingam Suresh S. , Marcus Adam I.
Published: March 2024

Lung cancer presents a global health challenge demanding innovative preclinical models forresearch and therapeutic advancement. Genetically engineered mouse models (GEMMs) have seenremarkable growth in complexity and application over the past two decades. Our work leveragesthese models and is specifically designed to address the pressing need for effectiveimmunochemotherapies in the context of KRAS and LKB1 (KL) mutant lung adenocarcinoma,which shows resistance to most immunotherapies. Our study centers around a preclinicalsyngeneic orthotopic mouse model we developed using mouse lung and metastatic tumor cell linesderived from KL-GEMM. This model, optimized to mimic human lung cancer closely, providesan ideal platform for exploring the intricate interplay between the immune system and tumorprogression. Furthermore, we have previously shown the LKB1 mutant cell lines and tumormodels are uniquely sensitive to FAK inhibitors, and therefore we wanted to test whether thecombination of a FAK inhibitor with anti-PD1 therapy can improve sensitivity. The combinatoryadministration of FAK inhibitor (VS-6063) with anti-PD1 mAb synergistically increased anti-tumor response and survival outcome compared with single-agent therapy in the KL-mediatedsyngeneic orthotopic lung cancer model. By using GEMM-derived syngeneic cell lines, we haveevaluated the potential for creating innovative immunochemotherapies, customized for KL co-mutant lung adenocarcinomas with promising clinical potential. Furthermore, using this modelenables a deeper understanding of the KL mutant disease's pathogenesis, the impact of sex-biasedcancer development, and the assessment of novel immunochemotherapy treatment strategies in theKL genetic sub-type of lung adenocarcinoma.

Citation Format: Junghui Koo, Carol Tucker-Burden, Melissa Roy, Chunzi Huang, Wei Zhou, Melissa Gilbert-Ross, Haian Fu, Suresh S. Ramalingam, Adam I. Marcus. Unraveling synergistic therapeutic strategies involving FAK inhibition and immunotherapy using anti-PD1 antibody in KRAS and LKB1-driven lung cancer [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 4047.
Abstract 4097: LNP-mRNA neoantigen vaccines to intercept progression of pre-invasive to invasive lung adenocarcinoma
Authors: He Yongfeng , Zhong Sijin Luo , Bhinder Bhavneet , Markowitz Geoffrey , Singh Arshdeep , Gardner Eric , Ko Myung Soo , Altorki Nasser K. , Jiang Shaoyi , Mittal Vivek
Published: March 2024

The widespread use of chest computed tomography in lung cancer screening has led to the detection of ground-glass opacity (GGO) nodules, which typically harbor pre-invasive or minimally invasive adenocarcinoma and 20-40% of GGO nodules progress to invasive lung adenocarcinoma within 4 years. Lack of approved treatments to intercept disease progression has imposed challenges in the clinical management of patients with GGOs. Previous studies showed that GGO nodules have substantially lower rates of HLA deletions than invasive/metastatic lung cancer, suggesting that GGO nodules constitute a critical window of vulnerability for immune interception. We therefore sought to identify the most immunogenic and cytotoxic neoantigens in patients with GGO nodules and to develop LNP-mRNA cancer vaccines to intercept the disease progression from pre-invasive to invasive adenocarcinoma. Whole exome and RNA sequencing of a large cohort (>300) patients identified potential neoantigen candidates, with KRAS-G12C, KRAS-G12D and EGFR-L858R as the most frequently predicted shared neoantigens consistent with >40% GGO patient population harboring either mutant KRAS or EGFR driver oncogenes. LNP-mRNA vaccines targeting KRAS-G12C and EGFR-L858R showed marked immunogenicity as determined by ELISPOT assays. Moreover, KRAS-G12C vaccination induced CD4+ T cell responses whereas EGFR-L858R vaccination triggered CD8+ T cell responses. Both these vaccines are being tested in GGO immunoprevention studies. Our data suggest the potential of KRAS and EGFR vaccines in the treatment of early disease, particularly in EGFR mutant patients who fail to respond to immunotherapy.

Citation Format: Yongfeng He, Sijin Luo Zhong, Bhavneet Bhinder, Geoffrey Markowitz, Arshdeep Singh, Eric Gardner, Myung Soo Ko, Nasser K. Altorki, Shaoyi Jiang, Vivek Mittal. LNP-mRNA neoantigen vaccines to intercept progression of pre-invasive to invasive lung adenocarcinoma [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 4097.
Abstract 4130: Targeting epigenetically-driven inflammation for PDAC metastasis prevention in high-risk individuals
Authors: Handler Jesse , Kalhor Reza
Published: March 2024

Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dire prognosis, in large part due to the late stage at which it is diagnosed in most patients. High-risk individuals (HRIs) identified through family history or germline genetic testing are candidates for imaging-based screening, yet even with intensive surveillance, metastasis often occurs between scans. Our study aims to address this critical clinical challenge by investigating the epigenetic determinants of PDAC metastasis and developing novel strategies for metastasis prevention in HRIs.

Methods: Innovative transplantation models of PDAC liver and peritoneal metastasis incorporating DNA barcodes for subclonal tracking were developed to facilitate the identification of subclones with high or low metastatic potential. An in-depth epigenomic characterization and comparison of these subclones was performed utilizing assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq.

Results: Metastasis-high subclones demonstrate widespread chromatin accessibility differences relative to their metastasis-low peers. These differences are linked to expression changes for genes involved in motility, differentiation, survival, and particularly inflammation. Top epigenetically activated genes in metastasis-high subclones include the pro-inflammatory cytokines IL-1α and IL-23α and the pro-inflammatory cytokine receptor IL-18R1.

Discussion: These findings highlight the role of epigenetic changes in PDAC metastasis, especially activation of inflammatory pathways resulting from altered chromatin accessibility. These insights provide a foundation for exploring anti-inflammatory agents for PDAC metastasis prevention. Targeting inflammation, alongside imaging-based screening, could significantly improve outcomes for HRIs.

Citation Format: Jesse Handler, Reza Kalhor. Targeting epigenetically-driven inflammation for PDAC metastasis prevention in high-risk individuals [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 4130.
Abstract 4133: Hyaluronidase inhibitor delphinidin inhibits cancer metastasis
Authors: McGuire Jeremy Jonathan , Taguchi Taketo , Tombline Greg , Paige Victoria , Janelsins Michelle , Gilmore Nikesha , Seluanov Andrei , Gorbunova Vera
Published: March 2024

Cancer remains a formidable global health challenge, with metastasis being a key contributor to its lethality. Abundant high molecular weight hyaluronic acid, a major non-protein component of extracellular matrix, protects naked mole rats from cancer and reduces cancer incidence in mice. Hyaluronidase plays a critical role in degrading hyaluronic acid and is frequently overexpressed in metastatic cancer. We investigated the potential of targeting hyaluronidases to reduce metastasis. High throughput screening identified delphinidin, a natural plant compound found in fruits and vegetables, as a potent hyaluronidase inhibitor. Delphinidin-mediated inhibition of hyaluronidase activity led to an increase in high molecular weight hyaluronic acid in cell culture and in mouse tissues, and significantly reduced proliferation, migration and invasion of breast (EO771), prostate (RM1), and melanoma (B16-F10) metastatic cell lines. Addition of exogenous hyaluronidase partially reversed all of these effects. Moreover, delphinidin treatment suppressed melanoma (B16-F10) metastasis in a spontaneous mouse model of metastasis. These results provide a proof of principle that inhibition of hyaluronidase activity suppresses cancer cell migration, invasion and metastasis. These results combined with the lack of adverse effect make delphinidin an attractive potential therapeutic option for inhibition of metastasis. We believe further investigation is warranted to fully elucidate the mechanisms and optimize the use of delphinidin in cancer therapy.

Citation Format: Jeremy Jonathan McGuire, Taketo Taguchi, Greg Tombline, Victoria Paige, Michelle Janelsins, Nikesha Gilmore, Andrei Seluanov, Vera Gorbunova. Hyaluronidase inhibitor delphinidin inhibits cancer 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 4133.
Abstract 4159: Noninvasive measurement of murine glioblastoma in vivo with a benchtop ultrasound instrument
Authors: Kierski Thomas M. , Rojas Juan D. , Thang Morrent , Hingtgen Shawn D. , Durham Phillip G. , Dayton Paul A. , Czernuszewicz Tomek J. , Gessner Ryan C.
Published: March 2024

The purpose of this study was to evaluate a new in vivo approach for rapidly measuring tumor volumes in rodent models for glioblastoma (GBM). GBM is the most common primary brain tumor with <5% of patients surviving >5 years post diagnosis. These poor outcomes underscore the importance of researching new treatment options, and murine models play a vital role in this. Noninvasive measurements of murine GBM tumors are commonly performed using CT and MRI but these tools are costly and time-consuming to use. BLI can quickly detect the presence of GBM cells in vivo, but image data can be challenging to correlate to tumor volume. In this study we demonstrate the feasibility of imaging GBM in vivo through the intact mouse skull using a benchtop robotic ultrasound scanner. This approach could offer researchers a means to quickly size tumors in the murine brain noninvasively. To test feasibility of this approach, a female athymic nude mouse aged 16 weeks was implanted with 2e5 U87 GBM cells in the brain. The tumor was given 25 days to develop prior to ultrasound imaging. After imaging, the brain was harvested for ex vivo validation of tumor presence and volume. Imaging was performed using a Vega ultrasound system (Revvity, Waltham, MA, USA) with the mouse in supine position and fur removed from head with depilatory cream. In vivo imaging consisted of a contrast-enhanced acoustic angiography (AA) scan to assess tumor perfusion. An ex vivo B-mode scan of the brain was collected after sacrificing the animal to validate the location and size of the tumor without interference from the skull. The tumor volumes were measured in each image and compared. 3D segmentations in the ex vivo and in vivo images yielded volumes of 43.1 mm3 and 44.6 mm3, respectively, a 3.5% variance. In the in vivo AA image, the borders of the tumor were adequately well-defined for volume estimation, despite some shadowing from the sagittal suture of the skull. In the B-mode image of the excised brain, the tumor appeared hyperechoic compared to the contralateral hemisphere. Images were acquired in < 2 min. This pilot study demonstrates a potential alternative to CT and MRI for monitoring of GBM volumes in vivo.

Citation Format: Thomas M. Kierski, Juan D. Rojas, Morrent Thang, Shawn D. Hingtgen, Phillip G. Durham, Paul A. Dayton, Tomek J. Czernuszewicz, Ryan C. Gessner. Noninvasive measurement of murine glioblastoma in vivo with a benchtop ultrasound instrument [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 4159.
Abstract 4163: Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors
Authors: Bynum Ryan , Sanderson Emma , Edil Barish H. , Jain Ajay , McNally Lacey R.
Published: March 2024

Objective: Current intraoperative imaging modalities such as near-infrared fluorescent imaging (NIR) or ultrasound are limited due to operator experience and depth, and targeted imaging of pancreatic tumors would be beneficial. However, specific pancreatic tumor biomarkers available for targeting in a novel imaging protocol are lacking. As such, we focus on developing a novel targeted imaging paradigm utilizing multispectral optoacoustic imaging (MSOT) to target the acidic microenvironment of pancreatic tumors. Peptides know as pH low insertion peptides (pHLIPs) when bound to dye allow for contrasted imaging selectively based on pH using MSOT. We have identified the need for a pHLIP with improved dynamic range to improve imaging accuracy. Here, we synthesize the novel pHLIP V7FS for comparison to V3 with aims of improving the dynamic range of imaging with MSOT in pancreatic cancer.

Methods: Using microwave chemistry, V7FS and V3 were synthesized with >90% purity. Peptides were conjugated to HiLyte 750 dye, and peptide-dye conjugation confirmed via spectroscopy. Intraoperative measurements of human pancreatic tumor and surrounding uninvolved tissue were obtained using a microsensor. Near-infrared fluorescent imaging was used to measure probe uptake in S2VP10 and S2013 pancreatic cancer cells lines grown in pH specific media (7.4, 6.8, 6.6) in replicates. MSOT was then used to image probes in phantoms mimicking tissue and then in mice with pancreatic tumors from orthotopically implanted S2VP10. Statistical analysis was performed using ANOVA.

Results: Compared to surrounding uninvolved pancreatic tissue, human pancreatic tumor was more acidic with pH 6.2-6.7 vs 7.2-7.3 (p<0.01). Signal at pH 7.4, 6.8 and 6.6 was higher for V7FS-750 compared to V3-750 (p=0.0105). Signal for V7FS-750 was centered around pH 6.6 and signal for V3-750 centered around pH 6.8. Interaction between probe and pH did not reach significance (p=0.745). Peak uptake in orthotopic murine models was observed at 3 hours following intravenous injection.

Conclusions: V7FS-750 was shown to have higher signal accumulation compared to V3-750 favoring a more acidic environment (pH 6.6 vs pH 6.8). This novel pHLIP was demonstrated to be viable for use in an imaging paradigm utilizing MSOT in an orthotopic murine model. Continued improvement in the dynamic range of these probes is promising for the use of pHLIPS in imaging of pancreatic cancer with MSOT.

Citation Format: Ryan Bynum, Emma Sanderson, Barish H. Edil, Ajay Jain, Lacey R. McNally. Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors [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 4163.
Abstract 4167: Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes
Authors: Michie Megan S. , Du Junwei , Ye Jiayu (Jennifer) , Duncan Kathleen , Berezin Mikhail Y. , Achilefu Samuel
Published: March 2024

Tumors consist of a complex mixture of resident and infiltrating cells that can support their survival and evasion of treatment. Selective detection of genetically stable cells in the tumor microenvironment (TME) offers insight into tumor biology beyond the malignant cells, which are the focus of most molecular imaging approaches to cancer detection. Motivated by the goal of non-invasively understanding the primary breast TME, we have developed fluorescent molecular probes that target cancer-associated fibroblasts (CAFs), a major component of the stromal compartment of many tumors, and emit light in the near-infrared (NIR) spectral window, providing access to deeper tissues than visible probes. We synthesized and characterized a series of CAF-targeted antibody- and small molecule-based fluorescent imaging agents utilizing a highly hydrophilic NIR dye and ligands that target Fibroblast Activation Protein (FAP). FAP is a key cell-surface marker of breast cancer CAFs with an S1 phenotype, which are modulators of immunosuppression, metastatic spread, and are associated with worse outcomes, especially in HER2+ and triple-negative breast cancer. Modifications to the chemical structure of the linker and targeting ligands allowed us to alter the specificity and brightness of the CAF-probes in cellulo. Imaging of a co-implanted tumor model of SKBR3 human breast cancer cells and FAP expressing fibroblasts after administering our lead small-molecule CAF imaging agent revealed selective tumor targeting of CAF-rich tumors compared to cancer cell predominant tumors with favorable pharmacokinetics and tumor-to-background ratio (TBR) greater than 2. Similar imaging studies with MMTV-PyMT transgenic mice, an established murine model of breast cancer, showed analogous enhanced uptake in mammary tumors (TBR > 2) and clearance over 24 hours. NIR fluorescence also correlated with CAFs isolated from PyMT tumor tissue via flow cytometry, consistent with our hypothesis that this agent can specifically detect endogenous CAFs. This study identified a novel, bright, and water-soluble FAP-specific NIR agent for in vivo imaging, setting the stage for simultaneous optical imaging of malignant cells and activated fibroblasts in the TME to elucidate cancer-host interactions.

Citation Format: Megan S. Michie, Junwei Du, Jiayu (Jennifer) Ye, Kathleen Duncan, Mikhail Y. Berezin, Samuel Achilefu. Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes [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 4167.