Pancreatic Ductal Adenocarcinoma Organoids Research Articles

OrganoIDNet: a deep learning tool for identification of therapeutic effects in PDAC organoid-PBMC co-cultures from time-resolved imaging data.

Pancreatic Ductal Adenocarcinoma (PDAC) remains a challenging disease due to its complex biology and aggressive behavior with an urgent need for efficient therapeutic strategies. To assess therapy response, pre-clinical PDAC organoid-based models in combination with accurate real-time monitoring are required. We established stable live-imaging organoid/peripheral blood mononuclear cells (PBMCs) co-cultures and introduced OrganoIDNet, a deep-learning-based algorithm, capable of analyzing bright-field images of murine and human patient-derived PDAC organoids acquired with live-cell imaging. We investigated the response to the chemotherapy gemcitabine in PDAC organoids and the PD-L1 inhibitor Atezolizumab, cultured with or without HLA-matched PBMCs over time. Results obtained with OrganoIDNet were validated with the endpoint proliferation assay CellTiter-Glo. Live cell imaging in combination with OrganoIDNet accurately detected size-specific drug responses of organoids to gemcitabine over time, showing that large organoids were more prone to cytotoxic effects. This approach also allowed distinguishing between healthy and unhealthy status and measuring eccentricity as organoids' reaction to therapy. Furthermore, imaging of a new organoids/PBMCs sandwich-based co-culture enabled longitudinal analysis of organoid responses to Atezolizumab, showing an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added. Optimized PDAC organoid imaging analyzed by OrganoIDNet represents a platform capable of accurately detecting organoid responses to standard PDAC chemotherapy over time. Moreover, organoid/immune cell co-cultures allow monitoring of organoid responses to immunotherapy, offering dynamic insights into treatment behavior within a co-culture setting with PBMCs. This setup holds promise for real-time assessment of immunotherapeutic effects in individual patient-derived PDAC organoids.

Screening Splice-Switching Antisense Oligonucleotides in Pancreas-Cancer Organoids.

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC

ObjectiveThe dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of...

Systematic analysis of proteome turnover in an organoid model of pancreatic cancer by dSILO

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

Abstract 6485: Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, commonly characterized by multiple aberrant signaling. Phosphoproteomics provides a direct read-out of these complex signaling networks and the resultant clinically relevant phenotype, as well as a functional scaffold to identify new targets. In the absence of an oncogenic driver, low dose (LD) kinase inhibitor (KI) combinations against multiple (parallel) activated kinases might provide higher efficacy and reduce toxicity as compared to single drug treatment. Aims: 1) To identify targets and test combinations of multiple KIs at LDs for potential synergism in preclinical models. 2) To chart the phosphoproteome of 42 PDAC tumors to reveal signalling pathways that may be involved in PDAC progression. Methods: By using a two step phosphopeptide enrichment with phosphotyrosine immunoprecipitation and immobilized metal affinity chromatography, followed by label free MS analysis, we analyzed phosphoproteome data of 7 immortalized and 2 primary PDAC cell lines, and of 42 PDAC tumors. We used integrative inferred kinase activity (INKA) scoring of the maxquant output to identify hyperactive kinases. For the cell line panel, five KIs were selected based on targeting coverage of the INKA profiles. LD were set as IC20s for 2,3,4 drug combinations. Cell growth inhibition was assessed by SRB assay. Median-effect analysis was used to assess synergy. Functional testing was performed in immortalized 2D, xenoderived 2D and 3D cultures. Effective low dose 3 drug combinations were further validated using patient-derived xenografts. Results: High INKA scoring of multiple activities per cell line without clear outliers of single kinases underscores the need for combination therapies. Multiple LD combinations showed effective growth inhibition (70 to 92%) and synergism, mostly 3 drug combinations, which required targeting of several RTKs and downstream signaling. Different responses were further observed between epithelial and mesenchymal cell lines. These top performing combinations were further validated in PDAC organoids and in vivo. Clinical utility of these kinase targets was then confirmed in 42 tumor phosphoprofiles, which were characterized in different subtypes with distinct therapeutic options. Phosphoproteome signals and kinases activities with potential prognostic value and mutational associations were further described. Conclusion: Our INKA pipeline, which can rank kinase activities in individual tumors, is optimally suited to specifically prioritize actionable kinases with targeting purposes. Tailored LD combination strategies exhibited promising efficacy in preclinical models and may ultimately improve treatment outcomes. Next Steps: Multicellular patient-derived models will be used to further study and target the TME in PDAC tumors. Citation Format: Andrea Vallés Martí, Giulia Mantini, Cynthia Waasdorp, Richard R. de Goeij- de Haas, Alex A. Henneman, Sander R. Piersma, Thang V. Pham, Jaco C. Knol, Joanne Verheij, Frederike Dijk, Hans Halfwerk, Elisa Giovannetti, Connie R. Jiménez, Maarten F. Bijlsma. Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues 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 6485.

Abstract 1582: Cabozantinib changes the tumor microenvironment to increase the efficacy of immunotherapy in refractory metastatic pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. Tumors evade immune surveillance by expressing PD-L1 that interacts with the T cell protein PD-1, subsequently inhibiting CD8+ cytotoxic T lymphocyte proliferation and effector function. A plausible reason for this observation may be that other, redundant, immune suppressive mechanisms are at play. Our research team has shown that elevated infiltration of polymorphonuclear (PMN)-MDSCs within the PDAC tumor microenvironment (TME) inhibit T cell effector function, regardless of PD-1/PD-L1 inhibition (Holokai et al., 2020). Objective: A Phase II clinical trial was developed to identify the potential therapeutic benefit of combinatorial anit-PD-L1/PD-1 and cabozantinib (cabo) treatment in therapy-resistant (refractory and metastatic) PDAC patients. Methods: NanoString CosMx™ spatial molecular transcriptomic imaging (CosMx™ SMI) was performed using FFPE sections collected from a liver or lung metastatic target lesions at visit 1 (pretreatment) and visit 2 (9 weeks on combinatorial treatment) from individual patients. FFPE sections were prepared from the same target lesion, and fields of view were chosen on the H&E in consultation with GI pathologist. Seurat V 4.0 was used for the analysis and visualization by spatial mapping of each FOV showing identified cell population indicated by color coding in visit 1 and visit 2 within the same patient. Multiplex immunofluorescence (MxIF) of patient FFPE tissues and single cell RNA sequencing (scRNAseq) of PDAC organoids (PDOs) generated from the core biopsies collected at visit 1 and matched visit 2 from each patient were used to validate the CosMx™ SMI data. Results: A uniform manifold approximation and projection showed loss of tumor cell populations within the PDAC TME in response to combinatorial treatment compared to pretreatment visit 1. Comparison between spatial maps of visit 1 to visit 2 demonstrated a shift in cell populations within defined NICHES (local cellular TME) whereby there was 1) a loss of tumor cell populations, 2) increase in CTL infiltration, 3) increase in endothelial cells, 4) decrease immunosuppressive MDSCs, 5) decrease in antigen presenting CAFs, and 6) change in myCAFs to an inflammatory CAF phenotype. Within the iCAF population IL-6, CXCL1, LIFR, ACTA2 and FAK were expressed; correlating with an increase in CD8 perforin and granzyme B positive cells in response to cabo and checkpoint inhibition. MxIF, spectral flow cytometry and scRNAseq analyses of the matched in vitro PDO model showed tumor cell death that correlated with decreased MDSC viability, increased CTL proliferation and a shift in CAF phenotypes. Conclusion: These findings suggest that cabo induces a TME conducive to increased response to immunotherapy in PDAC patients with refractory metastatic disease. Citation Format: Jayati Chakrabarti, Pritha Adhikary, Jiang Wang, Davendra Sohal, Syed A. Ahmad, Junaid Arshad, Aaron J. Scott, Rachna T. Shroff, Yana Zavros. Cabozantinib changes the tumor microenvironment to increase the efficacy of immunotherapy in refractory metastatic 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 1582.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [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 1392.

Auranofin repurposing for lung and pancreatic cancer: low CA12 expression as a marker of sensitivity in patient-derived organoids, with potentiated efficacy by AKT inhibition

BackgroundThis study explores the repurposing of Auranofin (AF), an anti-rheumatic drug, for treating non-small cell lung cancer (NSCLC) adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC). Drug repurposing in oncology offers a cost-effective and time-efficient approach to developing new cancer therapies. Our research focuses on evaluating AF's selective cytotoxicity against cancer cells, identifying RNAseq-based biomarkers to predict AF response, and finding the most effective co-therapeutic agents for combination with AF.MethodsOur investigation employed a comprehensive drug screening of AF in combination with eleven anticancer agents in cancerous PDAC and NSCLC patient-derived organoids (n = 7), and non-cancerous pulmonary organoids (n = 2). Additionally, we conducted RNA sequencing to identify potential biomarkers for AF sensitivity and experimented with various drug combinations to optimize AF's therapeutic efficacy.ResultsThe results revealed that AF demonstrates a preferential cytotoxic effect on NSCLC and PDAC cancer cells at clinically relevant concentrations below 1 µM, sparing normal epithelial cells. We identified Carbonic Anhydrase 12 (CA12) as a significant RNAseq-based biomarker, closely associated with the NF-κB survival signaling pathway, which is crucial in cancer cell response to oxidative stress. Our findings suggest that cancer cells with low CA12 expression are more susceptible to AF treatment. Furthermore, the combination of AF with the AKT inhibitor MK2206 was found to be particularly effective, exhibiting potent and selective cytotoxic synergy, especially in tumor organoid models classified as intermediate responders to AF, without adverse effects on healthy organoids.ConclusionOur research offers valuable insights into the use of AF for treating NSCLC and PDAC. It highlights AF's cancer cell selectivity, establishes CA12 as a predictive biomarker for AF sensitivity, and underscores the enhanced efficacy of AF when combined with MK2206 and other therapeutics. These findings pave the way for further exploration of AF in cancer treatment, particularly in identifying patient populations most likely to benefit from its use and in optimizing combination therapies for improved patient outcomes.

Abstract 305: Bioinspired synthetic hydrogel in pancreatic cancer organoid matrix modeling

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality with a primary hallmark, including dense collagen matrix in the desmoplastic stroma. Organoid technologies often rely on commercially available EHS mouse sarcoma matrix materials, and it remains uncertain how batch-to-batch variability of this matrix impacts the consistency of cancer signaling. Here, we present a novel photo-activated hydrogel tuned to the properties of the desmoplastic stroma in PDAC organoid cultures. Method: We designed multiple synthetic hydrogel materials with tunable mechanical properties, including SP-139 (Stem Pharm, Inc), a polyethylene glycol-based hydrogel functionalized with norbornene and formed with both degradable and non-degradable crosslinking peptides and a cell adhesion peptide (CRGDS). We used rheometry to measure the elastic modulus of the polymerized hydrogel after visible light photoactivation (395 nm) and expanded multiple cancer organoid cultures. RNAseq was performed from patient-derived xenografts to expanded organoids across matrix designs (Cultrex® v. SP-139) and analyzed by GSVA pathway analyses. Result: We optimized the ratio of non-degradable v. degradable crosslinking peptide to support culture expansion and enzymatic digestion with dispase II (125 µg/mL) and collagenase II (1 mg/mL). Using rheometry, SP-139 at 75% v/v had increased elastic modulus versus Cultrex RGF BME matrix Type 2 at 100% v/v (4992±76.3 v. 68.4±5.6 Pa, p<0.0001). Phenotypic growth was confirmed across serial passages (>8) and a diversity of gastrointestinal cancer types (n=5). Baseline growth rates were identical over 48h between SP-139 (75% v/v) and Cultrex (50% v/v) (+13.4% v. +13.0, p=0.75). Growth remained consistent for SP-139 yet increased with Cultrex at interval of 48-96h (+12.9% SP-139 v +22.4% Cultrex, p<0.005) and interval of 96-144h (+13.6% SP-139 v +24.0% Cultrex®, p<0.0001). Therapeutic response was assessed using 3D CellTiter-Glo® using gemcitabine 100 µM (24h) with comparable inhibition of normalized cell viability in SP-139 v. Cultrex® (35.4±1.9% v. 52.3±6.5%, n.s.) and qualitatively with viability staining using calcein AM and Caspase 3/7FITC. RNAseq revealed Cultrex, when compared to SP-139, yielded down regulation of epoxygenase p450 pathways (padj<0.012) and negative regulation of the p38 MAP kinase cascade (padj<0.048). Conclusions: Functionalized PEG-based synthetic hydrogel matrix materials can be tuned for physical properties to mimic cancer tissue microenvironments more accurately. SP-139, designed to mimic the desmoplastic stroma of the pancreatic cancer microenvironment, has favorable properties including controlled growth rates and compatibility with high content imaging by luminescence and fluorescence. RNAseq pathway analysis shows SP-139 maintains pathways of p450 and MAP kinase signaling of importance with therapeutic resistance. Citation Format: Md Shahadat Hossan, Austin Stram, Ethan Samuel Lin, Sean McIlwain, Connie Lebakken, William Richards, Jeremy D. Kratz. Bioinspired synthetic hydrogel in pancreatic cancer organoid matrix modeling [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 305.

Delivery of a BET protein degrader via a CEACAM6-targeted antibody–drug conjugate inhibits tumour growth in pancreatic cancer models

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody–drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

Whole-genome bisulfite sequencing identifies stage- and subtype-specific DNA methylation signatures in pancreatic cancer

In pancreatic ductal adenocarcinoma (PDAC), no recurrent metastasis-specific mutation has been found, suggesting that epigenetic mechanisms, such as DNA methylation, are the major contributors of late-stage disease progression. Here, we performed the first whole-genome bisulfite sequencing (WGBS) on mouse and human PDAC organoid models to identify stage-specific and molecular subtype-specific DNA methylation signatures. With this approach, we identified thousands of differentially methylated regions (DMRs) that can distinguish between the stages and molecular subtypes of PDAC. Stage-specific DMRs are associated with genes related to nervous system development and cell-cell adhesions, and are enriched in promoters and bivalent enhancers. Subtype-specific DMRs showed hypermethylation of GATA6 foregut endoderm transcriptional networks in the squamous subtype and hypermethylation of EMT transcriptional networks in the progenitor subtype. These results indicate that aberrant DNA methylation contributes to both PDAC progression and subtype differentiation, resulting in significant and reoccurring DNA methylation patterns with diagnostic and prognostic potential.

An alternative splicing signature defines the basal-like phenotype and predicts worse clinical outcome in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extremely poor prognosis. PDAC presents with molecularly distinct subtypes, with the basal-like one being associated with enhanced chemoresistance. Splicing dysregulation contributes to PDAC; however, its involvement in subtype specification remains elusive. Herein, we uncover a subtype-specific splicing signature associated with prognosis in PDAC and the splicing factor Quaking (QKI) as a determinant of the basal-like signature. Single-cell sequencing analyses highlight QKI as a marker of the basal-like phenotype. QKI represses splicing events associated with the classical subtype while promoting basal-like events associated with shorter survival. QKI favors a plastic, quasi-mesenchymal phenotype that supports migration and chemoresistance in PDAC organoids and cell lines, and its expression is elevated in high-grade primary tumors and metastatic lesions. These studies identify a splicing signature that defines PDAC subtypes and indicate that QKI promotes an undifferentiated, plastic phenotype, which renders PDAC cells chemoresistant and adaptable to environmental changes.

Abstract B099: Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations

Abstract Introduction: Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. Moreover, as glycans constitute the outermost layer of cells, diverse alterations during carcinogenesis affect the multiple processes of invasion and metastasis, making glycosylation a keystone mechanism in cancer progression. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells for comprehensive cell surface glycan expression profile using lectin microarray and transcriptomic analyses and then analyzed the relationship between altered glycans and glycan-related genes, with a particular focus on the status of KRAS mutations. Materials and methods: Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system containing extracellular matrix and specific niche factors. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. Results: We successfully established 16 3D-organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS mutations (7 G12V, 5 G12D, 1 Q61L) were found in 13 organoids, while wild-type KRAS without mutations was detected in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Pairings of a PDAC organoid with a non-PDAC counterpart were available from 2 patients; one was a PDAC-normal MPD pair and the other was PDAC-IPMN pair, eliminating the influence of individual variations in glycan expression status for those pairs. Lectin reactivity of AAL and AOL with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. And also, in the two cases with PDAC and non-PDAC organoids pairs, the reactivity of AAL were significantly elevated in the KRAS mutants, with FUT6 and FUT3 elevated. Conclusion: PDAC 3D-cultured organoids with KRAS mutations were covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers. Citation Format: Hiromitsu Nakahashi, Tatsuya Oda, Osamu Shimomura, Yoshimasa Akashi, Kazuhiro Takahashi, Yoshihiro Miyazaki, Tomoaki Furuta, Yukihito Kuroda, Pakavarin Louphrasitthiphol, Bryan J. Mathis, Hiroaki Tateno. Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B099.

Abstract B100: Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression

Abstract Background: Nerves are a major component of the tumor microenvironment, with active crosstalk between cancer cells and nerves that may contribute to cancer progression. Netrin1 (Ntn1) is an axon guidance molecule that is important during early neural development. Recent studies suggest that Ntn1 may also play a role in tumorigenesis but its role in pancreatic cancer is not well understood. We hypothesized that Ntn1 plays a pro-tumorigenic role in pancreatic ductal adenocarcinoma (PDAC) progression and metastasis in part through modulation of nerves in the microenvironment. Methods: Expression of Ntn1 and its receptor Neogenin1(Neo1) were investigated in mouse models of spontaneous PDAC (LSL-Kras+/G12D/Pdx1-Cre; KC, LSL-Kras+/G12D/LSL-Trp53+/R172H/Pdx1-Cre; KPC). The influence of Ntn1 on pancreatic cancer progression and innervation was evaluated by KC mice with or without conditional knockout of Ntn1 (LSL-Kras+/G12D/Pdx1-Cre/Netrin1fl/fl; KCN). Murine PDAC cell lines and organoids were used to examine the functional role of Ntn1 and Neo1 through genetic and pharmacological modulations. The capacity of these cell lines to generate metastasis and the effect on neurite outgrowth was studied in a co-culture system with Dorsal Root Ganglia (DRG). Results: Expression of Ntn1 was not observed in normal acinar cells or ductal cells, but was found in both PanIN lesions and PDAC. Neo1 expression was similar to Ntn1 and there was a positive correlation between the expression of Ntn1 and Neo1. A lower rate of PanIN progression was observed in KCN mice compared to KC mice. In the metastatic model, overexpression of Ntn1 increased tumor burden and decreased survival while injections of a neutralizing Ntn1 antibody or genetic knockdown of Neo1 improved both endpoints. Ntn1 and Neo1 were upregulated in pancreatic organoids upon KRAS activation. Recombinant Ntn1 (rNTN1) promoted organoid forming capacity and upregulate Sox2 and Sox9 gene expression, which was inhibited by genetic knockdown of Neo1 or an anti-Neo1 antibody, supporting a direct autocrine role in modulating stemness. Nerve fibers were significantly increased at the site of PDAC liver metastasis, with Ntn1 showing the highest expression level among neurotrophins, as it was significantly increased in metastatic PDAC cells. Consistent with this observation, we observed less innervation in KCN mice, which correlated with reduced PanIN progression. Co-culture of DRGs with PDAC cells that overexpressed Ntn1 resulted in much greater axonal elongation consistent with a role of Ntn1 in promoting innervation. Conclusion: The Netrin1/Neogenin1 interaction is an important regulator of PDAC progression, acting both directly on PDAC cells to promote stemness, and indirectly via modulating tumor innervation. Blockade of Ntn1/Neo1 interaction reduces the capacity of PDAC to metastasize to the liver and improves survival. Overall, targeting the Ntn1/Neo1 interaction may represent a potential new therapeutic approach for the treatment of pancreatic cancer. Citation Format: Yosuke Ochiai, Sunagawa Masaki, Ermanno Malagola, Hiroki Kobayashi, Feijing Wu, Ruth A. White, Leah B. Zamechek, Timothy C. Wang. Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B100.

Abstract B080: Mutant-selective KRASG12D(ON) inhibitor suppresses proliferation in vitro and tumor growth in vivo of KrasG12D GEMM-derived PDAC organoids

Abstract KRAS is the most frequently mutated oncogene in pancreatic ductal adenocarcinoma (PDAC), with KRASG12D driving initiation and progression of ~40% of tumors. Mutant KRAS proteins exist predominantly in the GTP-bound (RAS(ON)) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. RMC-9805 is an orally bioavailable, mutant-selective covalent inhibitor of the active state of KRASG12D. RM-044, used here, is the corresponding representative preclinical tool compound. RM-044, KRASG12D and cyclophilin A form a stable, high affinity tri-complex resulting in the disruption of interactions with downstream KRAS effectors and suppression of KRASG12D(ON) signaling. RM-044 decreased RAS pathway activity and inhibited proliferation in multiple human KRASG12D PDAC cell lines in vitro. While these data are encouraging, we sought to study models that more faithfully represent the heterogeneity and complex tumor microenvironment found in the tumors of patients with KRASG12D PDAC. To address this, we generated and characterized pancreatic organoids from the lox-STOP-lox-KrasG12D genetically engineered mouse model (GEMM) and introduced expression of the KrasG12D mutation and p53 loss (KP) through a vector containing a p53 sgRNA and Cre recombinase. GEMM-derived KP pancreatic organoid lines treated with RM-044 showed concentration-dependent proliferation inhibition in vitro. KP organoids were implanted orthotopically into mouse pancreas leading to the development of PDAC tumors in immunocompetent mice. Histopathological examination of KP organoid derived tumors revealed molecular and phenotypic features translatable to the tumors of patients with PDAC. In this orthotopic model, oral administration of RM-044 inhibited MAPK pathway activation and led to deep and durable tumor growth suppression at a well-tolerated dose. In summary, mutant-selective, covalent inhibition of KRASG12D(ON) inhibits cell proliferation and drives tumor growth suppression in both human and murine preclinical models of KRASG12D PDAC. The generation of preclinical models that allow for in-depth translational analyses of KRASG12D(ON) inhibition in vitro and in vivo can support identification of optimal therapeutic regimens. Collectively, these preclinical results indicate that direct targeting of KRASG12D(ON) in PDAC has the potential to translate into clinical benefit for patients with pancreatic cancer. Citation Format: Mark P. Labrecque, Lingyan Jiang, Nicole Nasholm, Biswadeep Nayak, Xinxing Yu, Avian Song, Caroline Weller, James W. Evans, Maria Paz Zafra, Marie Parsons, Marie Menard, Lukas E. Dow, Jingjing Jiang, Ida Aronchik, Mallika Singh. Mutant-selective KRASG12D(ON) inhibitor suppresses proliferation in vitro and tumor growth in vivo of KrasG12D GEMM-derived PDAC organoids [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B080.

A pancreatic cancer organoid platform identifies an inhibitor specific to mutant KRAS

KRAS mutations, mainly G12D and G12V, are found in more than 90% of pancreatic ductal adenocarcinoma (PDAC) cases. The success of drugs targeting KRASG12C suggests the potential for drugs specifically targeting these alternative PDAC-associated KRAS mutations. Here, we report a high-throughput drug-screening platform using a series of isogenic murine pancreatic organoids that are wild type (WT) or contain common PDAC driver mutations, representing both classical and basal PDAC phenotypes. We screened over 6,000 compounds and identified perhexiline maleate, which can inhibit the growth and induce cell death of pancreatic organoids carrying the KrasG12D mutation both invitro and invivo and primary human PDAC organoids. scRNA-seq analysis suggests that the cholesterol synthesis pathway is upregulated specifically in the KRAS mutant organoids, including the key cholesterol synthesis regulator SREBP2. Perhexiline maleate decreases SREBP2 expression levels and reverses the KRAS mutant-induced upregulation of the cholesterol synthesis pathway.

Single-organoid analysis reveals clinically relevant treatment-resistant and invasive subclones in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases, characterized by a treatment-resistant and invasive nature. In line with these inherent aggressive characteristics, only a subset of patients shows a clinical response to the standard of care therapies, thereby highlighting the need for a more personalized treatment approach. In this study, we comprehensively unraveled the intra-patient response heterogeneity and intrinsic aggressive nature of PDAC on bulk and single-organoid resolution. We leveraged a fully characterized PDAC organoid panel (N = 8) and matched our artificial intelligence-driven, live-cell organoid image analysis with retrospective clinical patient response. In line with the clinical outcomes, we identified patient-specific sensitivities to the standard of care therapies (gemcitabine-paclitaxel and FOLFIRINOX) using a growth rate-based and normalized drug response metric. Moreover, the single-organoid analysis was able to detect resistant as well as invasive PDAC organoid clones, which was orchestrates on a patient, therapy, drug, concentration and time-specific level. Furthermore, our in vitro organoid analysis indicated a correlation with the matched patient progression-free survival (PFS) compared to the current, conventional drug response readouts. This work not only provides valuable insights on the response complexity in PDAC, but it also highlights the potential applications (extendable to other tumor types) and clinical translatability of our approach in drug discovery and the emerging era of personalized medicine.

KRT17High/CXCL8+ tumor cells display both classical and basal features and regulate myeloid infiltration in the pancreatic cancer microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is generally divided in two subtypes, classical and basal. Recently, single cell RNA sequencing has uncovered the co-existence of basal and classical cancer cells, as well as intermediary cancer cells, in individual tumors. The latter remains poorly understood; here, we sought to characterize them using a multimodal approach. We performed subtyping on a single cell RNA sequencing dataset containing 18 human PDAC samples to identify multiple intermediary subtypes. We generated patient-derived PDAC organoids for functional studies. We compared single cell profiling of matched blood and tumor samples to measure changes in the local and systemic immune microenvironment. We then leveraged longitudinally patient-matched blood to follow individual patients over the course of chemotherapy. We identified a cluster of KRT17-high intermediary cancer cells that uniquely express high levels of CXCL8 and other cytokines. The proportion of KRT17High/CXCL8+ cells in patient tumors correlated with intra-tumoral myeloid abundance, and, interestingly, high pro-tumor peripheral blood granulocytes, implicating local and systemic roles. Patient-derived organoids maintained KRT17High/CXCL8+cells and induced myeloid cell migration in an CXCL8-dependent manner. In our longitudinal studies, plasma CXCL8 decreased following chemotherapy in responsive patients, while CXCL8 persistence portended worse prognosis. Through single cell analysis of PDAC samples we identified KRT17High/CXCL8+ cancer cells as an intermediary subtype, marked by a unique cytokine profile and capable of influencing myeloid cells in the tumor microenvironment and systemically. The abundance of this cell population should be considered for patient stratification in precision immunotherapy.

Incorporation of human iPSC-derived stromal cells creates a pancreatic cancer organoid with heterogeneous cancer-associated fibroblasts

The aggressiveness of pancreatic ductal adenocarcinoma (PDAC) is affected by the tumor microenvironment (TME). In this study, to recapitulate the PDAC TME exvivo, we cocultured patient-derived PDAC cells with mesenchymal and vascular endothelial cells derived from human induced pluripotent stem cells (hiPSCs) to create a fused pancreatic cancer organoid (FPCO) in an air-liquid interface. FPCOs were further induced to resemble two distinct aspects of PDAC tissue. Quiescent FPCOs were drug resistant, likely because the TME consisted of abundant extracellular matrix proteins that were secreted from the various types of cancer-associated fibroblasts (CAFs) derived from hiPSCs. Proliferative FPCOs could re-proliferate after anticancer drug treatment, suggesting that this type of FPCO would be useful for studying PDAC recurrence. Thus, we generated PDAC organoids that recapitulate the heterogeneity of PDAC tissue and are a potential platform for screening anticancer drugs.

Establishment of organoid models for pancreatic ductal adenocarcinoma and screening of individualized therapy strategy.

Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and receive effective chemotherapy have the best chance for long-term survival. Unfortunately, because of the heterogeneity of pancreatic cancer, it is difficult to find a personalized treatment strategy for patients. Organoids are ideal preclinical models for personalized medicine. Therefore, we explore the cultivation conditions and construction methods of PDAC organoid models to screen the individualized therapy strategy. Fresh PDAC tissues from surgical resection were collected and digested with digestive enzymes; then the tumor cells were embedded in Matrigel with a suitable medium to establish the PDAC organoid models. The genetic stability of the organoids was analyzed using whole exon sequencing; hematoxylin and eosin staining and immunohistochemistry of organoids were performed to analyze their consistency with the pathological morphology of the patient's tumor tissue; After 2 days of organoid culture, we selected four commonly used clinical chemotherapy drugs for single or combined treatment to analyze drug sensitivity. Two cases of PDAC organoid models were successfully established, and the results of their pathological characteristics and exome sequencing were consistent with those of the patient's tumor tissue. Both PDAC organoids showed more sensitivity to gemcitabine and cisplatin, and the combined treatment was more effective than monotherapy. Both organoids better retained the pathological characteristics, genomic stability, and heterogeneity with the original tumor. Individual PDAC organoids exhibited different sensitivities to the same drugs. Thus, this study provided ideal experimental models for screening individualized therapy strategy for patients with PDAC.