Orthotopic Model Research Articles

Abstract 2679: Myeloid olfactory receptors in melanoma

Abstract Olfactory receptors are commonly found in the nasal cavity and function to detectodorants. However, very recent studies have determined that innate immune cells,including monocytes and macrophages, express olfactory receptors. OLFR2 (OR6A2 inhumans), was recently linked to atherosclerosis development through interaction with itsligand, octanal. Increased levels of octanal are considered as a risk factor foratherosclerosis. Conversely, octanal levels are associated with lower cancer incidence,suggesting that OLFR2 may play a protective role in cancer. Indeed, data from TheCancer Genome Atlas for liver and brain cancers show that OLFR2 expression isassociated with increased patient survival. Here, we utilized an orthotopic melanomamouse model using B16 cancer cells in C57/BL6 mice that had been transplanted witheither wild type or OLFR2 knockout bone marrow to directly test the role of OLFR2 onimmune cells in regulating tumor growth. We found that hematopoietic deletion of OLFR2exacerbated melanoma tumor growth in these mice; again, supporting the notion thatOLFR2 is anti-tumoral. The OLFR2+ cells present in the tumor were myeloid cells.Monocytes, non-classical monocytes specifically, function to detect cancer cells in tumorseeding sites, and recruit NK cells for tumor cell destruction. OLFR2-deficientmacrophages were found to migrate less to tumor cells in an in vitro assay, suggestingthat OLFR2 is involved in the sensing of newly seeding tumor cells, likely through sensingoctanal. Studies are ongoing to determine the exact functions of OLFR2+ monocytes andmacrophages in the tumor microenvironment. OLFR2 may be one of several olfactoryreceptors that is utilized by myeloid cells as a defense mechanism for detection of newlyseeding or metastatic tumor cells. As olfactory receptors are easily druggable targets,understanding their role in cancer prevention could lead to emerging newimmunotherapies for cancer. Citation Format: Elayne M. Benson, Klaus Ley, Marco Orecchioni, Catherine C. Hedrick. Myeloid olfactory receptors in 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 2679.

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal 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 287.

Abstract 3070: CDK12 is a novel therapeutic target in glioblastoma

Abstract Glioblastoma (GBM), the most common primary brain tumor in adults carries a highly unfavorable prognosis of only one to two years despite treatment with the standard of care, involving surgery, temozolomide and radiation. Patient-derived xenograft (PDX) GBM cultures were assessed for their susceptibility to loss of CDK12 function, which was either mediated genetically (shRNA) or by the novel inhibitor, SR-4835. GBM metabolism was measured by utilizing extracellular flux analysis as well as LC/MS based carbon tracing analyses. In vivo efficacy was determined by orthotopic GBM models in mice. Based on Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and shRNA library screens in multiple GBM cell cultures we identified CDK12 as essential for the growth of GBM. Consistently, loss of function of CDK12 (genetically and pharmacologically) led to a reduction of growth of GBM patient-derived xenograft (PDX) lines in vitro. Notably, SR-4835 reduced GBM growth in the low nanomolar range and synergistically enhanced the potency of temozolomide. Extracellular flux analysis and metabolite screening analyses, including carbon tracing (U13C-Glucose, U13C-Glutamine) suggested that loss of function of CDK12 elicited a substantial inhibition of cellular respiration, causing energy deprivation with subsequent cell death with apoptotic features. On the molecular level, loss of function of CDK12 increased ATF4 along with pro-apoptotic Noxa, whereas anti-apoptotic Mcl-1 decreased. In turn, adenoviral mediated ectopic expression of Mcl-1 inhibited cell death mediated by loss of function of CDK12 in GBM cells. In an orthotopic murine PDX model of GBM animals receiving treatment with SR-4835 had a significantly longer overall survival without induction of toxicity as compared to mice treated with vehicle. In summary, these findings suggest that CDK12 is a novel treatment target for GBM and that CDK12 is an important driver of energy metabolism in GBM to partially mediate apoptotic resistance. Moreover, SR-4835 enhanced the potency of temozolomide in GBM models. Therefore, the novel CDK12 inhibitor, SR-4835 should be tested in a clinical trial in patients suffering from GBM. Citation Format: Jeong-Yeon Mun, Chang Shu, Qiuqiang Gao, Mike-Andrew Westhoff, Georg Karpel-Massler, Markus D. Siegelin. CDK12 is a novel therapeutic target in glioblastoma [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 3070.

Abstract 2336: A prototype for a machine-learning assisted image analysis tool for preclinical in vivo studies

Abstract The purpose of this study was to build a software tool to test the feasibility of machine-learning (ML) assisted analysis of in vivo 3D images in oncology research studies. Conventionally, in vivo image segmentations are performed by hand, and are a bottleneck for data analysis, taking time and introducing both inter and intra-user variability. Recently, advances in ML have shown promise in accelerating similar clinical image analysis tasks. Our objective was to work toward making this technology accessible to preclinical cancer researchers. 3D image data across multiple mouse models, organs, modalities, and imaging systems were used. ML segmentation models were trained with user-defined masks on images of mouse bladders (N=134), kidneys (N=131), livers (N=65), and lungs (N=26) from either ultrasound or micro-computed tomography (uCT) scanners. Models were developed using PyTorch/MONAI, hosted in an AWS cloud environment, and served to users via a custom extension for 3D Slicer (www.slicer.org). Performance was assessed as the difference between the human vs. ML organ volumes on a test set unseen by the models during training. Lastly, the functionality of on-the-fly segmentation improvement tools through iterative user feedback was assessed. Results demonstrated that generating segmentations with the prototype ML software took 2-6 seconds per image running on a modern GPU (NVIDIA RTX 3090), compared to 3-7 minutes if performed manually by an expert user (>30x improvement). The Dice scores between the ML generated and human segmentations ranged from 0.81 (liver ultrasound) to 0.84 (bladder ultrasound) but could be improved using ML-based on-the-fly mask improvement tools. Using data from this pilot study, we estimate that the threshold to achieve 90% accuracy on first-pass segmentations is between 200-300 training images, depending on the variance observed in the image data. In summary, the prototype software was able to decrease the manual analysis time across a variety of preclinical in vivo image modalities, which is broadly relevant to preclinical oncology research. The next steps for this project will be to extend the platform to additional orthotopic tumor models (e.g. pancreatic, liver, prostate, etc.) and deploy to the field to enable access to this toolset by preclinical imaging scientists across the world. Citation Format: Adam M. Aji, Thomas M. Kierski, Juan D. Rojas, Hannah Morilak, Olivia J. Kelada, Kathryn H. Gessner, Andrew S. Gdowski, Lucia Kim, William Y. Kim, Ryan C. Gessner, Tomek J. Czernuszewicz. A prototype for a machine-learning assisted image analysis tool for preclinical in vivo studies [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 2336.

Abstract 2910: Targeting thioredoxin reductase 1 (TrxR1) suppresses TAZ-driven glioblastoma progression

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults. Poor outcomes for traditional treatments demand targeted therapies based on identified mechanisms that drive tumor development and sustain its malignancy. Molecular pathology studies have classified GBM into subtypes differing in treatment responses and survival rates. Among these subtypes, the mesenchymal (MES) group is associated with the worst prognosis. The Hippo pathway transcriptional co-activator with PDZ-binding motif (TAZ) is one of the three transcriptional regulators that drive the GBM MES gene expression program. Studies from our lab and others have previously shown that TAZ activation is able to promote GBM MES differentiation and progression. Therefore, finding vulnerabilities of TAZ-driven tumors may help to develop targeted therapeutic methods for MES GBM. In an orthotopic GBM mouse model, we found that TAZ-driven tumors show increased expression of thioredoxin reductase 1 (TrxR1), which is responsible for catalyzing the reduction of thioredoxin and maintaining the cellular redox balance. In searching for the cause of increased expression of TrxR1, we found that glucose deprivation can induce TrxR1 expression, suggesting TrxR1 may be an adaptive mechanism for tumor cells to survive in glucose deprivation conditions. Indeed, either depleting TrxR1 genetically or inhibiting TrxR1 pharmacologically can promote tumor cell death under glucose deprivation. Furthermore, knockout TrxR1 in a TAZ-driven mouse GBM model significantly decelerated tumor progression. Collectively, our studies revealed that inhibition of the Trx redox system under glucose deprivation conditions leads to a synergistic cell death induction in GBM cells, therefore making targeting TrxR1 a potential therapeutic strategy for TAZ-driven MES GBM. Citation Format: Miaolu Tang, Kaitlyn Dirks, Soo Yeon Kim, Jessica Thorpe, Wei Li. Targeting thioredoxin reductase 1 (TrxR1) suppresses TAZ-driven glioblastoma 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 2910.

Abstract 4163: Improving the dynamic range of pH low insertion peptides for targeted imaging of pancreatic tumors

Abstract 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 6505: Targeting PINK1 reduces medulloblastoma progression in animal models

Abstract Medulloblastoma is a brain cancer that mainly arises during infancy and childhood. Medulloblastoma is the most common pediatric malignant brain tumor worldwide. It comprises of approximately 20% of all childhood brain tumors and 63% of intracranial embryonic tumors. One of the major effects of proliferating cancer cells is altered mitochondrial metabolism, as well as aberrant metabolism. Medulloblastoma patients suffer severe complications due to intensive and long-term treatments. Thus, we aim to improve applications against medulloblastoma by targeting the PINK1 gene. We hypothesize that PINK1 inactivation slows down medulloblastoma progression. PINK1 is a mitochondrial serine/threonine protein kinase that protects cells by controlling mitochondria quality in response to cellular stress. PINK1 recruits parkin to target damaged mitochondria for degradation through autophagy. In this study, PINK1 has been knocked-out using gene editing technology (CRISPR/Cas9) in two commercial medulloblastoma cell lines (DAOY, and D283 cell lines). We validated PINK1 editing using RT-PCR and Western blot. Then, cell viability was studied in comparison to the control sample. The control sample contained scramble fragments with empty CRISPR/Cas9. We studied apoptosis and performed cell cycle analysis using flow cytometry. Next, orthotopic medulloblastoma mouse model was generated which showed differential survivals comparing the PINK1 knockout group and control. The results showed a low expression of PINK1 within the PINK1 knockout group. In addition, the Western blot showed reductions in PINK1 expression in the knockout group. The knockout group showed more apoptosis (cell death) 40% more than the control. The mouse model showed increased survival of the PINK1 knockout group compared to the control with a p-value of 0.0288 (log-rank). Our analysis shows that survival differences approximately extend human life by one year. We conclude that inhibiting the PINK1 gene in medulloblastoma decreases cell metabolic activities. PINK1 Knockout in medulloblastoma enhances the apoptosis pathways. Engrafted medulloblastoma mouse model treated with PINK1 inhibitor showed superior survival than the control. These results qualify PINK1 to be used as potential therapeutic target for medulloblastoma. The Future work needs additional preclinical studies to investigate the proper dose of PINK1 inhibitor and measurement of toxicity index. Citation Format: Reema N. Alsubaiee, Amani Alghamdi, Bahauddeen M. Alrfaei. Targeting PINK1 reduces medulloblastoma progression in animal 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 6505.

Abstract 1509: Microglial dominance in glioma dissemination: Unraveling TAMs' impact and therapeutic implications through modulating cathepsin activity

Abstract Introduction: It is well known that tumor-associated macrophages/microglia (TAMs) play a vital role in brain tumor progression. Our previous studies suggested that the activated microglia could reduce the cytotoxic effect of chemotherapeutic drugs on glioma cells in a 2D/3D model. This study delves into TAMs within diverse brain TME regions, emphasizing the distinction of resident microglia from infiltrating macrophages in invasion areas and their role in glioma migration and invasion. Material and Method: We employed the murine orthotopic astrocytoma model (ALTS1C1), dissecting brain tumor tissues into normal, invasion, edge, and core regions. Immunofluorescence staining, utilizing TMEM119 and CD68 markers to identify resident microglia and infiltrating macrophages, respectively. The IHC results were analyzed with QuPath software and validated via flow cytometry. Microglia (BV2) and macrophage (RAW264.7) cell lines were directly co-cultured with ALTS1C1 in 2D and 3D migration/invasion models. Bulk RNA sequencing analysis unraveled the differences between BV2 and RAW264.7. Bone marrow-derived macrophages (BMDM) were used for the TAMs activation test, and the cathepsin inhibitor, E64, was applied to elucidate cathepsin's role in microglia within co-culture models. Results: The IHC results reveal more TMEM119+ microglia than CD68+ infiltrating macrophages in the tumor invasion area. On the other hand, there are more CD68+ infiltrating macrophages than TMEM119+ microglia in the tumor core regions, signifying a specific role of microglia in tumor invasion. Flow cytometry further supported that more CD11b+CD45low microglia prevalence in tumor surrounding tissues than in tumor core. ALTS1C1 astrocytoma cells, in the presence of microglia, exhibited more increased invasion and migration ability than the presence of macrophages in 2D/3D models. RNA sequencing highlighted M2 activation and cathepsin gene upregulation in microglia over macrophages. M2-polarized TAMs enhanced 2D migration, while cathepsin inhibition significantly decreased glioma's 3D invasion potential. Conclusion: This study identifies microglia as primary TAMs in brain tumor invasion areas and their roles in glioma cell survival and dissemination. Notable differences in activation states and the critical impact of cathepsin in microglia for their roles on brain tumor invasion and survival offer promising therapeutic avenues. Targeting cathepsin in microglia emerges as a novel glioma prevention and treatment strategy. These findings contribute valuable insights to enhance clinical interventions for glioma patients. Citation Format: Sheng-Yan Wu, Chi-Shiun Chiang. Microglial dominance in glioma dissemination: Unraveling TAMs' impact and therapeutic implications through modulating cathepsin activity [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 1509.

Abstract 4134: The abscopal effect of combination IRE with αPD-1 treatment against PDACs

Abstract Pancreatic ductal adenocarcinoma (PDAC) is associated with a high capacity of metastatic dissemination, which results in a dismal 5-year relative survival rate. Irreversible electroporation (IRE) represents an innovative intervention utilizing short high-voltage electric pulses to induce cell death through either permanent membrane lysis or disruption of cellular homeostasis. Previous studies from our lab have demonstrated that the combination of IRE and immune checkpoint blockade using anti-PD-1 antibody (αPD-1) enhances the infiltration of CD8+ cytotoxic T cells, significantly prolonging survival in an orthotopic murine PDAC model. However, the impact of IRE/αPD-1 on metastatic PDAC tumors, known as the abscopal effect, remains unclear. In this investigation, we bilaterally implanted C57BL/6J mice with subcutaneous KRAS* PDAC tumors, treating the right flank tumors with or without IRE, in conjunction with or without αPD-1 administered intraperitoneally. Tumor growth in both the treated (with or without IRE) and untreated (abscopal) left flank tumors was closely monitored until humane euthanasia or long-term survival. In a separate study, mice serum and tumor tissues were collection on the 5th day post-IRE/αPD-1 treatment. Luminex cytokine assays were conducted on the collected serum, and immunostaining was performed on tumor tissue. Results demonstrated significantly enhanced antitumor activity in both IRE-treated and untreated tumors on mice treated with combined IRE and αPD-1, leading to a marked extension of overall survival. Luminex cytokine assays revealed a substantial increase in crucial cytokines, including IL-2, IFN-gamma, IL-18, IL-19, and IL-3, known for their pivotal roles in fostering cytotoxic T lymphocyte development, promoting T cell differentiation into effector T cells, and facilitating the recruitment of effector CD8+ T cells to the tumor microenvironment. Moreover, immunofluorescence staining of tumor tissue illustrated a notable accumulation of CD169+ macrophages in IRE-treated tumors compared to untreated tumors. Tumors from the combined IRE with αPD-1 group exhibited increased CD8+ T cell accumulation compared to other groups. These data suggest IRE combined with PD-1 blockade promotes robust antitumor immune, leading to systemic antitumor activity and robust abscopal effect. Citation Format: Qizhen Cao, Nicholas A. Egan, Weiyi Peng, Chun Li. The abscopal effect of combination IRE with αPD-1 treatment against PDACs [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 4134.

Abstract 4278: Role of WNK1 in extracellular matrix modification in breast cancer

Abstract Introduction: Breast cancer (BCa) is the most common cancer and the second leading cause of cancer related deaths in women in the United States. Metastatic dissemination of cancer cells remains the leading cause of mortality in BCa. Therefore, determination of the mechanisms that promote metastasis is important for development of new therapeutic strategies. The extracellular matrix (ECM), which is the main non-cellular component of the tumor microenvironment (TME), plays an important role in BCa progression and metastasis. The cancer-associated fibroblasts (CAFs) are the principal cell types that together with BCa cells drive the modification of ECM during cancer development. Lysine deficient receptor protein kinase 1 (WNK1), a widely expressed serine threonine kinase that regulates blood pressure and electrolyte balance in the body, and is highly overexpressed in BCa microenvironment. The aim of the present study was to investigate the role of WNK1 in modification of ECM in BCa. Methods: The clinical relevance of WNK1 overexpression in BCa was determined by immunohistochemically staining human BCa sections, analyzing data from publicly available databases and creating orthotopic mouse models of BCa using 4T1 mouse BCa cells followed by treatment of the tumor bearing mice with WNK1 inhibitors, WNK-IN-11 and WNK463 (10 mg/kg , oral for 10 days). In vitro assays were conducted using 4T1 cells and mouse embryonic fibroblasts (MEFs) to determine the regulatory role of WNK1 on cancer cells and fibroblasts. Results and Conclusion: Our results indicated that WNK1 is strongly expressed in BCa cells and fibroblasts, the two most abundant cell types in the BCa microenvironment. Furthermore, WNK1 expression strongly correlated with expressions of matrix regulatory and cross-linking genes. Our results demonstrated that WNK1 inhibitors significantly reduced orthotopic 4T1 growth and metastasis. WNK1 inhibition also significantly inhibited collagen deposition in tumor tissues, which is a negative prognostic indicator in BCa. In vitro proliferation and migration assays using 4T1 cells and MEFs identified that WNK1 inhibitors (1-100nM) can significantly inhibit the proliferation and migration of 4T1 cells and MEFs. Results from co-culture studies with 4T1 cells and MEFs further indicated that WNK1 inhibition in MEFs could significantly reduce the invasive properties of 4T1 cells. Taken together, our results suggest that blocking the actions of WNK1 in BCa may be a promising therapeutic approach. Citation Format: Prabhat Suman, Sooraj Kakkat, Suhas S. Patil, Veronica Ramirez, Elba A. Turbat-Herrera, Seema Singh, Chandrani Sarkar, Debanjan Chakroborty. Role of WNK1 in extracellular matrix modification in breast 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 4278.

Abstract 2367: STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic adenocarcinoma

Abstract Background: Immunotherapy with CLDN18.2/CD3 BiTEs (bispecific T-cell engagers) has shown clinical activity in pancreatic adenocarcinoma. However, only few patients benefit from the treatment, and most responders develop acquired resistance. STING agonists may promote the development of stem-like central memory CD8+ T cells and enhance antitumor responses. This study aimed to investigate the synergistic effect and related mechanism of STING agonist plus CLDN18.2/CD3 BiTEs regimen. Methods: Single-cell RNA sequencing and flow cytometry were used to examine the tumor immune landscape in humanized PDX mouse models. By qPCR, western blot and flow cytometry, an orthotopic transplantation mouse model of pancreatic cancer and an in vitro tumor-PBMC coculture system were established to reveal the potential molecular mechanism of STING agonist ADU-S100 in optimizing the effects of CLDN18.2/CD3 BiTEs (IBI389). Results: In PDAC organoids, IBI389 induced potent, specific cytotoxicity, the CLDN18.2 BiTEs significantly engaged human CD3+ T cells with tumor cells in an organoid-PBMC coculture system in vitro, resulting in the formation of the immunological synapse (IS). IBI389 monotherapy significantly suppressed tumor growth and improved survival in vivo in hCD3e C57BL/6 and humanized hCD34+ PDX mouse models. Further, single-cell RNA sequencing and flow cytometry analysis revealed that the intertumoral TCF1+PD-1+ stem-like T cells and STING signaling activity of T cells were enriched in the early stage of tumor development and sharply decreased in the late stage during CLDN18.2 BiTEs administration. Furthermore, we discovered that the STING agonist ADU-S100 could significantly induce the enrichment of TCF1+PD-1+ stem-like CD8+ T cells in vitro and in vivo, increasing IBI389’s therapeutic effects. Furthermore, humanized PDX mice that were given the combination of IBI389 and ADU-S100 and survived after complete tumor rejection were rechallenged with PDX tumors. Humanized PDX tumors and hCD3e KPC orthotopic tumors from mice treated with the combination contained increased numbers of TCF1+PD-1+ stem-like CD8+ T cells, CD4+CD127+ memory T cells, CD8+CD127+ memory T cells, and fewer numbers of CD4+PD-1+Tim-3+ exhausted T cells, CD8+PD-1+Tim-3+ exhausted T cells and CD4+CD25+ Treg cells. Conclusions: Pancreatic tumors appear to evade immune response by inducing immune-suppressive tumor microenvironment development. In mice, the combination of STING agonist ADU-S100 and CLDN18.2 BiTEs increased the proportion of memory and stem-like CD4+ and CD8+ T cells while decreasing the proportion of regulatory and exhausted T cells in pancreatic tumors, eradicating all detectable tumors. This data may be utilized to formulate immune-based combination therapies for PDAC. Citation Format: Tianxing Zhou, Jingrui Yan, Bin Wang, Yongjie Xie, Chao Yang, Jun Yu, Jihui Hao. STING agonist ADU-S100 improves the antitumor response of CLDN18.2/CD3 BiTEs by enhancing stem-like tumor-reactive CD8+ T cells in pancreatic 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 2367.

Abstract 3283: Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib resistance

Abstract Introduction: Despite numerous available treatments, metastatic renal cell carcinoma (mRCC) remains highly lethal, with over 90% of patients succumbing within 5 years. Sorafenib (SF), a second-line treatment for mRCC, exhibits modest efficacy, and the underlying molecular basis for its failure is elusive. Hyaluronic acid (HA), implicated in promoting cell proliferation, invasion, and motility through CD44 and RHAMM receptors, represents a potential player in SF resistance. Hymecromone (HC), a non-toxic dietary supplement, is known to inhibit HA synthesis and downregulate CD44 and RHAMM mRNA levels. This study investigates the elusive mechanisms of SF resistance and evaluates the effectiveness of the HC+SF combination as a targeted therapy for advanced RCC, providing insights into a novel and potentially impactful treatment approach. Methods: HA levels were measured in normal and tumor kidney specimens using ELISA-like assay and IHC. Similarly, HAS3 and UGT1A9 (A9) levels and SF glucuronidation were measured by qPCR and a glucuronidation assay using tumor microsomes. Response to SF+HC combination was examined in A9 and HAS3 knockdown and overexpression cell lines (transfectants) and primary RCC spheroids by proliferation, apoptosis, motility, and invasion assays. Target analysis was performed by immunoblot. Treatment efficacy was evaluated in subcutaneous and orthotopic xenograft mouse models, with bioluminescence imaging. Results: SF failure in mRCC was linked due to its glucuronidation and inactivation by A9. Tumor microsomes from patients who developed mRCC glucuronidated SF 5-fold higher than from non-mRCC patients. A9 levels were elevated (8-10 folds) in mRCC patients’ tumor along with HA and HAS3 (5-10 folds) as compared to non-mRCC patients and normal kidney tissues. HC treatment downregulated A9 promoter activity and inhibited SF-glucuronidation in RCC cells. SF (5uM) and HC (20, 40ug/ml) combination inhibited RCC cell and primary tumor spheroid proliferation (>90%), motility/invasion (>80%), and induced apoptosis (5-fold) and downregulated HAS3 expression (>80%), and HA synthesis in RCC cells. While A9 and HAS3 transfectants both were resistant to SF+HC treatment, A9 and HAS3 - KO transfectants were sensitive to SF. Endothelial cells co-cultured with either A9 or HAS3 transfectants were resistant to SF+HC treatment. SF+HC upregulated apoptosis effectors, but downregulated CD44, RHAMM, and phospho-Met levels in EV but not in A9 or HAS3 transfectants. HC (100 or 200mg/kg) and SF (30mg/kg) oral combination abrogated Caki-1 tumor growth (>80%), without serum/tissue toxicity. HC+SF treatment decreased A9, HA and HAS3 levels, Ki67 index and microvessel density. Both A9 and HAS3 overexpressing tumors were resistant to treatment. Conclusion: This study demonstrates the mechanism of SF failure in mRCC and proposes a safe, oral adjunct combination for effective treatment. Citation Format: Anuj Kumar Sharma, Karina Aguilar, Chandramukhi S. Panda, Vinata B. Lokeshwar. Role of HA, HAS3, UGT1A9 in metastatic renal cell carcinoma and sorafenib resistance [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 3283.

Abstract 2824: Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line

Abstract Prostate cancer is the second most common cancer in men with new cases diagnosed in 1.4 million men each year. Luckily the 5-year survival rates in developed countries are high reaching over 90%. However, approximately one third will develop a metastatic disease. In metastatic patients, prostate cancer almost always progresses despite the androgen deprivation therapy and is then referred to as “castration-resistant (mCRPC)”. Bone is involved in 80% of patients with metastasis, and the bone lesions are typically osteoblastic. The 22Rv1 human prostate carcinoma cell line is androgen sensitive and shown to express androgen receptor (AR). They also express low levels of prostate-specific membrane antigen (PSMA). The aim of this study was to establish orthotopic and intratibial preclinical in vivo prostate cancer models that can be used in drug development when targeting cancer cells and their local environment. Male athymic nude mice (5-6 weeks old) and NOG mice (over 20 weeks old) were used in the study. 2.5 × 105 22Rv1 cells (ATCC) were inoculated orthotopically into the prostate of NOG mice, and 5 × 105 cells were inoculated intratibially into the bone marrow cavity of athymic nude mice. Tumor growth was followed by PSA measurements every second week using Human PSA ELISA assay. In addition, in the intratibial model, tumor-induced bone changes were monitored by X-ray imaging of the hind limbs. Prostates and hind limbs were collected at sacrifice, fixed in 10% NBF, and processed to paraffin blocks for further histological analysis. Sections of prostate were stained with hematoxylin-eosin (HE) and AR. Tumor-bearing and contralateral (healthy) tibias were stained with HE - OrangeG, MGT, TRAP, and AR. The observed tumor take rate was 92% in the orthotopic and 100% in the intratibial models. Confirmed by IHC, tumors formed in both the orthotopic and intratibial models expressed AR. 22Rv1 cells formed osteoblastic - lytic mixed bone lesions in the intratibial model. Based on lesion areas, randomization of the tumor bearing mice in the intratibial model can be done after 2 weeks from cancer cell inoculation. Taken together, the 22Rv1 prostate cancer models are relatively fast growing, express AR and present good take rates. Furthermore, the intratibial model represents robust tumor-induced changes in bone with strong osteoblastic component. Thus, these models have several advantages in efficacy studies of novel drug candidates for prostate cancer. Citation Format: Justyna Zdrojewska, Katja Fagerlund, Jenni H. Mäki-Jouppila, Mari I. Suominen. Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line [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 2824.

Abstract 4015: Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Overall survival rates are low for metastatic RMS, emphasizing the urgent need of improved therapies. Chimeric Antigen Receptor (CAR) Ts have shown remarkable results against hematological malignancies, but CAR T cell therapy remains challenging in solid tumors, mainly due to a lack of ideal tumor specific targets, and of the immunosuppressive tumor microenvironment (TME). To maximize antitumoral activity against RMS and to address optimal CAR T persistence in vivo, novel CAR constructs were investigated against upregulated RMS targets. By surfaceome profiling, we recently identified L1CAM as promising candidate for RMS CAR T cell therapy, and confirmed CD276 and FGFR4 expression. CD276- (CD276.CD28HD.28TM.28CSD.3z) and Dual-CAR Ts targeting both CD276 and FGFR4 (F8-FR4.CD28HD.28TM.28CSD.3z) showed tumor eradication in an orthotopic RMS model, whereas FGFR4-CAR Ts exhibited only a partial response in vivo. Moreover, CD276-CAR Ts showed a limited activity against RMS tumors expressing lower amounts of CD276, eradicating the tumor in 1/5 mice, underlying the need of improved CAR T cell potency against low-density target-expressing tumors. First, to improve FGFR4-CAR Ts activity, we optimized the recognition of FGFR4 epitope by modifying the CAR moiety “hinge”. We replaced the CD28-derived hinge with various hinges of different length: no hinge, IgG4 (12aa), IgG4CH3 (119aa), and IgG4CH2CH3 (229aa). Luciferase assays showed effective in vitro killing capacity of FGFR4-CAR Ts expressing an IgG4-derived hinge, with a tumor cell survival less than 10% at Effector to Target (E:T) ratio of 5:1. They also released the double amount of IFN-γ (50pg/ml) compared to the other constructs, when co-incubated with Rh4 cells. Next, to verify the potential of L1CAM as CAR T target for RMS, we generated a panel of L1CAM-CARs expressing either a long (IgG4CH2CH3, 229aa) or a short (CD28, 35aa) Hinge, and a CD28- or 41BB-derived costimulatory domain (CSD). L1CAM.IgG4CH2CH3Hinge.28TM.28CSD.3z CAR Ts outperformed the other constructs, killing ~60% of Rh30 at the E:T ratio of 1:1 and more than 90% at the E:T ratio of 5:1. They also exhibited a 3-fold higher IFN-γ release (120 pg/ml) compared to the other constructs. However, although our examination of patient-derived TMA revealed a strong immunoreactivity for L1CAM, more expressed in aggressive RMS subtypes, the selected L1CAM-CAR Ts against Rh30 cells, exhibiting ~10’000 L1CAM copies, showed limited activity in an in vivo pilot study. Based on these results and on the RMS TME investigation, which revealed consistent collagen (COL4A1, COL4A2 and COL18A1) deposition in RMS-derived mice tumor xenografts, we hypothesize that co-expressing a collagenase, able to partially degrade the dense extracellular matrix surrounding tumors, will lead to an improved CAR T cell infiltration and activity in vivo. Citation Format: Caroline Piccand, Andrea Timpanaro, Stenija Anton-Joseph, Michele Bernasconi, Jochen Karl Rössler. Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in 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 4015.

Abstract 4731: Targeting Lon protease as novel strategy to treat aggressive IDH mutant diffuse malignant astrocytoma

Abstract While isocitrate dehydrogenase (IDH) wild-type and mutant malignant astrocytoma (IDHwt, IDHmt) are diffuse CNS tumors that have a high degree of parallelism with one another, including microvascular proliferation and necrosis, the IDHwt tumors clinically presents with a higher tumor grade and thus poorer prognosis and survival rate. Isocitrate dehydrogenase mutation (IDH) hinders the hypoxia response in gliomas and promotes an advantageous reduction in both tumor growth and treatment resistance. Since recurrence is high, adequate characterization and identification of potential novel molecular targets in IDHmt mutant diffuse glioma is integral for developing new therapeutic strategies for better control of tumor burden and, thus, improved outcomes. Our previous published work has shown that the hypoxia responsive LONP1 mitochondrial protease is involved in glioma growth and treatment resistance and that LONP1 inhibitors can augment the effect of chemotherapy in IDHwt models. Here, we highlight the downstream activity of LONP1 via the NRF2 pathway and how synergistically, along with the IDH mutation, LONP1 overexpression can promote the subsequent proneural to mesenchymal transition (PMT) induced by stress and other tumor microenvironment signals. By characterizing the interplay between LONP1 and IDH mutation in in-vitro models, we demonstrate that oxidative stress modulates both PMT and TMZ drug resistance. When translated to an in-vivo patient-derived, intracranial orthotopic xenograft model, LONP1 overexpression is associated with decreased survival.Based on our findings, future clinical translation includes the possibility of using LONP1 as a novel target to optimize the standard of care treatment in IDH mutant diffuse astrocytoma. Citation Format: Christopher Douglas, Shashi Jain, Naomi Lomeli, Thao Vu, James Pham, Daniela A. Bota. Targeting Lon protease as novel strategy to treat aggressive IDH mutant diffuse malignant astrocytoma [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 4731.

Abstract 6342: Anti-tumor in vivo efficacy of different classes of anti-CLEC-1 myeloid checkpoint antibodies in monotherapy and in combination with chemotherapeutic agents

Abstract The c-type lectin receptor CLEC-1 is a pattern recognition receptor [1] expressed by endothelial and myeloid cells in mice, non-human primates, and humans. While genetic deletion of CLEC-1 in mice does not lead to any developmental defect, CLEC-1 deletion or CLEC-1 targeting promotes anti-tumor response by increasing cross-presentation of necrotic or damaged cells by cDC1 dendritic cells and by enhancing T-cell activation [2]. To date, the identification of CLEC-1 endogenous ligand(s) and their biological activity into myeloid or endothelial cells remained to be fully investigated. We aimed to further characterize ligands that are involved into CLEC-1 activation and develop monoclonal antagonist anti CLEC-1 antibody anti-tumor therapy that are able fully counteract inhibitory signaling induced into myeloid cells. Using LC/MS campaign, we confirmed CLEC-1 specific binding to the E3 ubiquitin ligase TRIM21and to the secreted histidine rich glycoprotein (HRG) as previously described [2] and also identify several novel intra-cellular and cell surface proteins that bind to CLEC-1 receptor in protein specific-manner. To block interaction of CLEC-1 ligands, we developed different class of antagonist monoclonal antibodies and evaluated their anti-tumor efficacy in humanized CLEC-1 KI orthotopic and ectopic tumor mouse models fully resistant or partially resistant to ICI therapy. While blocking of CLEC-1 binding to its secreted ligand HRG moderately increases anti-tumor responses, inhibition of CLEC-1 binding to its cytoplasmic and membrane ligands significantly impairs MC38 tumor growth (n=12, p=0.04). Importantly, high anti-tumor efficacy was observed in combination therapy with cyclophosphamide in MC38 mouse model. Additionally, antagonist anti-CLEC-1 antibodies significantly increase overall survival of Hepa1.6-bearing mice (n=32 for each treatment, p<0.002), as compared to corresponding isotype control treatment in 3 independent experiments. Altogether, our results further dissect the mechanism of action of the myeloid checkpoint CLEC-1 in its ability to impair anti-tumor immunity and support its use as a novel and highly promising anti-CLEC antagonist antibody for cancer immunotherapy. Citation Format: Aurore Morello, Mylène Deramé, Marion Drouin, Emmanuelle Wilhelm, Stéphanie Neyton, Irène Baccelli, Caroline Mary, Vanessa Gauttier, Isabelle Girault, Kévin Biteau, Cécile Batty, Géraldine Teppaz, Ariane Desselle, Marion Colonello, Marine Malloci, Elise Chiffoleau, Nicolas Poirier. Anti-tumor in vivo efficacy of different classes of anti-CLEC-1 myeloid checkpoint antibodies in monotherapy and in combination with chemotherapeutic agents [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 6342.

Abstract 3339: TTFields for the management of spinal metastases in in vitro and in vivo models

Abstract Spine metastases often affect cancer patients, leading to significant morbidity and mortality. Their prevalence has increased over time due to the progressive improvements in cancer management which led to a longer survival of these individuals. Radiation therapy is the mainstay treatment for local control of these lesions, with surgery reserved for selected cases (e.g., spinal instability, neurological deficits secondary to neural compression requiring urgent decompression, separation surgery). Chemotherapy addresses the diffusely metastatic spread. However, when spinal metastases progress despite using all the appropriate available treatment, there follows a progressive decline in function and further treatment options are limited, highlighting the need for development of novel therapies. Tumor treating fields (TTFields) has demonstrated to impair tumor cell replication and are influenced by surrounding tissue, such as bone (presents non-conductive properties). Our goal was to investigate the effects of TTFields in in vitro and in vivo spinal metastasis models. We hypothesize that the deposition of TTFields is increased in bone, and this would ultimately result in vertebral metastasis growth suppression. A computational model using a simulated spine resection cavity with metallic hardware was used to investigate the TTFields intensity in various tissues. For in vitro studies, luciferase tagged KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized human bone graft. These were treated with TTFields and cell viability was quantified. In vivo assays were performed using a previous validated murine orthotopic tumor model. The effects of TTFields in these animals were monitored with imaging (bioluminescence and MRI) and clinical findings (progressive neurological deficits milestones that corresponds to the degree of spinal cord compression). At the end of the study, tissue was collected for histologic analysis. Sham stimulation was used for control groups. Computational simulation demonstrated an elevated TTFields intensity in the tumor-bone interface of the resected vertebral body. In all experimental models, TTFields significantly suppressed tumor cell growth. In vivo models, this effect on cell proliferation prevented tumor growth into the spinal canal, delaying, or even preventing, neurological deficits. TTFields inhibited tumor growth in three-dimensional cultures and orthotopic murine models of spinal metastases, preventing functional loss. The bony environment surrounding the tumor demonstrated to enhance the intensity of electric fields on a computational simulation, which stands as an advantage with the use of TTFields for vertebral tumors. These results provide solid evidence supporting future clinical trials investigating the effects of this novel and promising technology for the treatment of spine metastases. Citation Format: Romulo Augusto Andrade de Almeida, Daniel Ledbetter, Xizi Wu, Ariel Naveh, Chirag Patel, Queena Gonzalez, Thomas H. Beckham, Robert Y. North, Laurence Rhines, Christopher A. Alvarez-Breckenridge, Claudio E. Tatsui. TTFields for the management of spinal metastases in in vitro and in vivo 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 3339.

Abstract 715: Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment

Abstract The stroma of solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell-derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Increasing evidence demonstrates their ability to hamper cancer patients' response to most treatments currently applied in the clinic, including immunotherapy. Therefore, strategies to counteract negative effects of TAMs are nowadays gaining momentum at preclinical, translational, and clinical levels. Here, we present the development and evaluation of nanoparticles (NPs) loaded with TLR agonists and/or inhibitors of immunosuppressive pathways to reprogram TAMs and the tumor microenvironment (TME) to unleash an effective immune response to fight against the tumor. Firstly, we have evaluated in vitro the ability of poly(I:C) and/or R848, agonists of TLR3 and TLR7/8 respectively, to reprogram TAMs into antitumor effector cells, and also the efficacy of Stattic and/or Galunisertib, inhibitors of STAT3 and TGF-β pathways respectively, to inhibit immunosuppression by cancer cells and/or TAMs. The best combinations of these drugs were encapsulated in nanoemulsions or polymeric nanocapsules for improved TAM-targeting and pharmacokinetics in vivo. These NPs were characterized for their physicochemical properties and also tested in vitro using primary human macrophages. For in vivo evaluation, subcutaneous and orthotopic murine models of lung cancer (CMT167) were used, showing antitumoral efficacy and TME reprogramming as evaluated by FACS, RNA analysis and multiplex immunofluorescence. Protamine-NCs-loaded with poly(I:C)+R848 and coated with an additional layer of hyaluronic acid functionalized with mannose were used to target the CD206 receptors, showing antitumoral efficacy mediated by TAM reprogramming, evaluated as higher CD86 while lower CD206 and Arg1 expression. Nanoemulsions with a PEGylated surfactant, encapsulating Stattic+Galunisertib+R848 showed faster antitumoral activity versus the free drugs. Experiments performed in IFN-γ KO mice and immune deficient mice (NSG and Balb/c nude) revealed that a fully functional immune system is crucial for the response to the treatment. In conclusion, our work demonstrates the antitumoral efficacy and reprogramming of the tumor microenvironment by combinations of TLR agonists and/or inhibitors of immunosuppression, which can be improved by nanotechnological approaches. Further investigations are ongoing to assess their antitumoral efficacy in other tumor models (i.e. breast and pancreatic cancer), with the final aim of clinical translation. Citation Format: Fernando Torres Andon, Alba Pensado-López, Clement Anfray, Aldo Ummarino, Iago Fernández-Mariño, Lucia Sanjurjo, Jose Crecente-Campo, Eduardo Fernández-Megía, Flavia Castro, Maria Jose Oliveira, Alfonso Calvo, Rosario García Campelo, Alberto Mantovani, Maria Jose Alonso, Paola Allavena. Nanomedicines loaded with TLR agonists and inhibitors of immunosuppression to reprogram the tumor microenvironment [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 715.

Abstract 3237: KDM1A/LSD1 inhibition enhances chemotherapy response in ovarian cancer

Abstract Background: Ovarian cancer (OCa) is the deadliest of all gynecological cancers. The standard treatment for OCa is platinum-based chemotherapy, such as carboplatin or cisplatin in combination with paclitaxel. Most patients are initially responsive to these treatments; however, nearly 90% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. Recent studies have revealed that the epigenetic modifier lysine-specific histone demethylase (KDM1A/LSD1) is highly overexpressed in OCa. However, whether KDM1A plays a role in chemoresistance and whether its inhibition enhances chemotherapy response in OCa is unknown. In this study, we explored the hypothesis that KDM1A inhibition will sensitize OCa cells to standard-of-care chemotherapy. Methods: Transduction of KDM1A-specific shRNA was used to generate KDM1A knockdown (KD) cells. Cell viability, colony formation, and apoptosis assays were used to determine the impact of KDM1A KD on sensitization of SKOV3, OVCAR8, and OVCAR5 OCa cells to cisplatin, carboplatin, and paclitaxel. The combination effect of KDM1A inhibitors (NCD38 and SP2509) and chemotherapy drugs was further determined. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, and Western blotting. In vivo efficacy ofKDM1A knockdown with or without chemotherapy was determined using OCa orthotopic xenograft models. Results: Analysis of TCGA datasets revealed that KDM1A expression is negatively correlated with OCa survival, and its expression is high in patients who do not respond to chemotherapy. Further, Western blotting results show that treatment with chemotherapy drugs cisplatin, carboplatin, and paclitaxel increased KDM1A expression in OCa cells. KDM1A-KD or KDM1A inhibition sensitized OCa cells to chemotherapy drugs in reducing cell viability, survival, and enhancing apoptosis. Moreover, knockdown of KDM1A sensitized carboplatin-resistantA2780-CP70 cells to carboplatin treatment. RNA-seq studies revealed that KDM1A-KD and cisplatin-regulated genes showed negative correction with epithelial-mesenchymal transition (EMT) and positive correlation with apoptosis-related pathways. Importantly, KDM1A KD in combination with cisplatin significantly reduced the tumor growth in the orthotopic OCa xenograft model. Conclusion: These results suggest that the combination of KDM1A inhibitors with chemotherapy is a viable therapeutic approach for the treatment of OCa Citation Format: Jessica D. Johnson, Yihong Chen, Sridharan Jayamohan, Yi He, Diksha Jamwal, Neetesh Pandey, Edward Kost, Ratna K. Vadlamudi, Gangadhara R. Sareddy. KDM1A/LSD1 inhibition enhances chemotherapy response 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 3237.

Abstract 6657: Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial Ovarian Cancer

Abstract Objective: Epithelial Ovarian cancer (EOC) has the highest mortality rate among gynecologic malignancies, necessitating novel therapeutic approaches. Viral oncolytic immunotherapy with recombinant oncolytic vaccinia virus (OVV) offers promise for advanced-stage cancer treatment. OPCML is a GPI-anchored 3 Ig-domain tumor suppressor inactivated by somatic methylation in 87% of EOC. OVV with OPCML (OVV-OPCML) gene insertion may have potential as a therapeutic agent. This study explores the oncolytic, signaling-inhibitory, anti-angiogenic and synergistic immunotherapeutic properties of OVV-OPCML in EOC treatment, evaluating its therapeutic potential. Methods: To create OVV-OPCML, the virulent vaccinia virus Copenhagen strain F3L site was modified with OPCML cDNA, confirmed by qPCR and western blot. Oncolytic potential was assessed through in vitro and in vivo EOC cell infection with OVV-OPCML. Real-time cell analysis (RTCA) assessed cell proliferation. Xenograft tumor models in nude mice were used to evaluate in vivo oncolytic activity, while OVV-OPCML combined with anti-PD-1 treatment was tested using a syngeneic orthotopic EOC mouse model (ID8) in C57BL/6 immunocompetent mice. Serum IFN-γ and TNF-α were quantified via ELISA. Receptor Tyrosine Kinases, angiogenesis and phosphorylation together with immune markers were examined by western blotting and by multiplex immunohistochemistry (mIHC) analysis of tumor sections. Results: OVV-OPCML selectively infected OC cells, leading to viral replication and tumor cell death, while sparing normal ovarian cells in-vitro. OPCML protein expression increased post-infection. OVV-OPCML reduced ERBB2, EGFR, and FGFR1 expression in SKVO3 OV cells. Intratumoral or intraperitoneal (IP) OVV-OPCML administration in human ovarian cancer nude mouse models reduced tumor growth and improved survival. IP in-vivo OVV-OPCML in combination with anti-PD-1 treatment in vivo, in the IP ID8 syngeneic model demonstrated synergy with survival improvement. H&E staining showed tumor cell damage without significant harm to normal tissues. Protein expression of ERBB2, EGFR, FGFR1, and their phosphorylated forms decreased. CD31, VEGFR2, p-VEGFR2 (Tyr1175), and p-VEGFR2 (Tyr1214) downregulation suggested tumor angiogenesis disruption. OVV-OPCML treatment elevated serum TNF-α and INF-γ levels and increased CD8+ T cells and PD-L1 expression in OVV-OPCML treated syngeneic tumors. Conclusions: OPCML gene enhances OVV selectivity against OC cells through direct oncolysis and OPCML protein production, inhibiting specific RTKs, suppressing angiogenesis, and activating antitumor immune responses synergistically with anti-PD-1. OVV-OPCML therapy shows promise as a targeted strategy for ovarian cancer treatment. Citation Format: Yong Tang, Yingzhao Liu, Mingjing Deng, Fei Huang, Jiahu Wang, Paul Blake, Hani Gabra, Qi Wang. Oncolytic Vaccinia Virus Carrying OPCML Tumor Suppressor is active in Epithelial 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 6657.