Patient-derived Organoid Models Research Articles

Abstract 173: Genomic characterization of patient-derived pancreatic cancer organoids

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death with few effective therapies. Patient-derived organoids (PDOs) are a 3-D culture model that allow primary tumour cells to propagate, and have gained considerable traction in many cancer types such as PDAC. However, concerns remain regarding whether these models can predict what occurs in patients. We hypothesize that genomic and transcriptomic drift occurring in PDO models impacts the fidelity of drug response. To investigate, matched WGS and bulk RNAseq was performed on paired PDAC organoids and tumour tissue (n=41). Core biopsies were obtained from in patients with Stage III-IV PDAC enrolled in the COMPASS trial (NCT02750657) and divided for sequencing and organoid generation. Tumour cellularity was enriched by laser capture microdissection. Although alterations in the four major driver genes (KRAS, TP53, SMAD4, CDKN2A) remained consistent, other genomic differences were identified. SNV count was higher in organoids (median 6584 vs 5931, p<0.0001), and enriched in SBS5 mutational signature (p<0.0001). This was not significantly correlated with passage number, and private mutations were identified in both tumours and organoids (median 67% overlap). Two organoids showed significant shifts in ploidy, with both diploid to polyploid shifts and vice versa observed. In the transcriptome, expression of Basal-like genes (KRT5, TP63) was decreased. Bias towards the Classical transcriptomic subtype have previously been observed in PDAC organoid cultures. PDO responses to 5-FU, irinotecan, and oxaliplatin correlated with patient response to FFX (n=22, 72% concordance). However, PDO responses to gemcitabine and paclitaxel were poorly predictive of patient responses to GnP (n=10, 38% concordance), and notably, expression of the biomarker hENT1 was not correlated in matched tumors and PDOs (R=0.17, p=0.40). Despite this, tumour expression of the biomarker hENT1 successfully stratified patient responses to gemcitabine, and organoid hENT1 expression was correlated to gemcitabine response in vitro (R=0.47, p=0.005), indicating that transcriptomic drift may be a major contributor to discrepancies in patient-PDO drug response. In summary, organoids recapitulate major histologic features and driver mutations of patient tumours, but genetic drift and subclonal selection are observed even at lower passages. Further study is required to improve the utility of organoids in translational precision medicine. Citation Format: Irene Y. Xie, Yuanchang Fang, Amy X. Zhang, Karen Ng, Zhen-Mei Liu, Eugenia Flores-Figueroa, Gun Ho Jang, Stephanie Ramotar, Anna Dodd, Julie Wilson, Jennifer J. Knox, Steven Gallinger, Faiyaz Notta. Genomic characterization of patient-derived pancreatic cancer organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 173.

Abstract 192: A novel and rapid patient-derived organoid breast cancer platform for precision medicine

Abstract An increasing number of studies performing correlative drug screens on patient-derived organoids (PDO) are revealing enormous potential for these models in predicting patient response to therapy. Despite this, their future use in a clinical setting is hindered by intrinsic limitations of PDO models, namely low success rates in establishing growing cell cultures from tumor tissue samples and long return times for drug response data that fall outside timescales of clinical actionability. To address these limitations, we leveraged technological advances in emulsion-based microfluidics and droplet generators to develop MicroOrganoSpheres (MOS) - microscale environments containing tumorspheres that retain structural, cellular, and genetic properties of an individual patient’s diseased tissue. Here, we focused on generating MOS from breast cancer tissue biopsies and resections across different subtypes of breast cancer. We established over 30 unique patients’ breast MOS samples at an average of 80% success, with the majority of these samples being hormone receptor positive (ER+/PR+) - the most clinically lethal and challenging subtype to establish. We evaluated MOS dose-responses across several first line standard of care chemotherapies for breast cancer. For this, we created a semi-automated workflow paired with high-content imaging for quantifying dose responses and tracking longitudinal cell dynamics during dosing. We generated dose response data for MOS samples, being able to move from primary tissue samples to MOS to drug dosing data within a clinically relevant timescale of 2-3 weeks. We conclude that our platform demonstrates the feasibility of 1) efficiently establishing MOS from breast cancer patient tumor samples from different subtypes and 2) performing drug dosing studies on these samples with improved turnaround times that enable clinical actionability for the patient. Together, these findings provide a foundation for evaluating this technology as a diagnostic tool in future clinical settings. Citation Format: David M. Graham, Gabrielle Rupprecht, Eric D. Bankaitis, Jeremy M. Force, Wylie Watlington, Steven W. Metzger, Xiling Shen, David Hsu. A novel and rapid patient-derived organoid breast cancer platform for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 192.

Abstract 198: Pan-cancer assay-ready organoid drug screening with robust, reproducible and clinically-relevant output

Abstract Introduction The pharmaceutical industry has mostly relied on 2D cancer cell lines and 3D spheroids for in vitro testing, but poor correlation of pre-clinical and clinical outcomes has driven the development of more predictive models. Patient-derived organoids (PDOs) have emerged as representative in vitro avatars of tumor biology, allowing the generation of biobanks covering a large variety in indications and genetic backgrounds. Here, we assess the robustness of our drug screening platform by testing the reproducibility of our organoid assays within and between organoid batches, read-outs, and different labs. We present an assay-ready organoid platform, allowing short timelines, repeated assays from a single batch of organoids, high throughput assays and large panel screens. Methods Colorectal, breast, lung, pancreatic, ovarian, cervix and melanoma PDO models were selected for subtype and driver mutation variety, banked in large batches, and preserved in assay-ready format. Outgrowth and drug responses of these batches were compared to those of organoids produced in the classic method. Experiments were executed using automated liquid handling equipment to standardize procedures and increase consistency. Performance of the drug screens were assessed by repeated drug sensitivity testing and by calculating intra- and interplate variability, control variability (CV), Z-factor, assay windows and IC50 values. Moreover, drug responses were tested in both celltiterglo-based and high content imaging-based assays. Results Assay performance of the organoid drug testing platform was high, with high Z-factors (>0.6), and low intra- and interplate variability (CV <15%) indicating reproducible assays. Both the classic and assay-ready organoid technologies resulted in highly reproducible IC50outputs, also when assays were executed months apart or in different labs. Timelines were shortened from months to weeks, and the reduced logistical burden allows screening of large organoid panels of >50 models. Drug sensitivity testing in our fast-paced panel screening platform distinguished sensitive from partially and insensitive models, illustrating how organoids allow patient stratification. Conclusion and Discussion The assay-ready organoid technology presented here has further improved our already highly predictive and reproducible organoid drug testing platform. The resulting short timelines and large panel screening capabilities will further unlock the great potential of PDO technology. Using large organoid panels in pre-clinical drug development and in combination with biomarker analysis will allow identification of responsive indications, subtypes, and genotypes, and for early patient stratification. Moreover, as most models presented in our panel screen platform are also available as PDX models, it allows for easy translation into in vivo follow-up studies. Citation Format: Liza Wijler, Jara Garcia Mateos, My Nguyen, Annelot Staes, Linda van Seters, Lama Alhaj Hasan, Victor Tiroille, Bram Herpers, Leo Price, Mariusz Madej, Michiel Fokkelman, Marrit Putker. Pan-cancer assay-ready organoid drug screening with robust, reproducible and clinically-relevant output [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 198.

Abstract 162: Individualizing treatment using patient derived organoids, BH3 profiling and microfluidics: A proof of concept in a patient with low-grade serous ovarian carcinoma

Abstract Background N of 1 treatment paradigms represent the pinnacle of personalized medicine in which a patient’s tumors are profiled to guide treatment. Low-grade serous ovarian cancer (LGSC) is a distinct subtype of ovarian cancer, comprising ~10% of serous carcinomas and typically characterized by a younger age of onset. Molecularly, these tumors are often characterized by alterations within the Ras signaling pathway, including KRAS mutations. Clinically, LGSC is often resistant to standard cytotoxic chemotherapy, but may have sensitivity to hormonal therapy or MEK inhibitors. Here we report on a platform and proof of concept in one LGSC patient to evaluate personalized tumor-directed therapy regimens using patient-derived organoids (PDOs), BH3 profiling and viability evaluation in 3D microfluidic devices. Methods A patient with LGSC presented to the Dana Farber Cancer Institute and was treated with carboplatin and paclitaxel before a total abdominal hysterectomy with a bilateral salpingo-oophorectomy. Tissue was obtained under an IRB approved protocol and PDOs were established. Standard of care and non-standard of care treatments including doxorubicin, abemaciclib, letrozole, alpelisib, tamoxifen, trametinib, venetoclax, and navitoclax were evaluated by two orthogonal assays. First, they were tested for delta priming by BH3 profiling (Bhola et.al., Sci Signal. 2020 ) and second for cell viability using 3D microfluidic devices by TMRM/DRAQ7 dual-color fluorescent staining. Standard of care treatments carboplatin and paclitaxel were evaluated as individual treatments and in combination in 3D microfluidic devices. Results We successfully established a PDO model from the patient’s tumor sample in 14 days. BH3 profiling at 24 hours and viability in 3D microfluidic devices after 6 days in treatment showed that from the eight tested drugs, the model was sensitive to navitoclax and venetoclax. Average percent change in viability was -91.5% and -89.9%, respectively, and the drugs had a dynamic BH3 profiling index of 551.4 AUC (+/- 76.63) and 488.9 AUC (+/- 21.46) with the threshold of response being >175 for BH3 profiling. Trametinib showed a clear response in 3D, with an average percent change of -72.6% compared to the control but no significant response in BH3 profiling. Neither carboplatin and paclitaxel alone, nor in combination, elicited a significant change in viability. This observation was consistent with the patient’s history prior to surgery, where the tumor did not demonstrate significant clinical response to neoadjuvant carboplatin and paclitaxel therapy. Conclusions We describe a proof of concept of a N of 1 response assessment platform for LGSC using PDOs, BH3 profiling and live/dead fluorescent staining in microfluidic devices and demonstrate that BH3 profiling and 3D viability assessment assays show good congruity. Citation Format: Brittany Meisenheimer, Ha V. Vo, Kelley E. McQueeney, Aisha L. Saldanha, Carina Feeney, Courtney H. Qi, Swati Narayan, Jennifer D. Curtis, Marisa R. Nucci, Anthony Letai, Cloud P. Paweletz, Joyce F. Liu, Ursula A. Matulonis, Elena Ivanova. Individualizing treatment using patient derived organoids, BH3 profiling and microfluidics: A proof of concept in a patient with low-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 162.

MP20-16 NOVEL FUNCTION OF PTUPB IN AKR1C3/AR/AR-V7 AXIS INHIBITION AND ENZALUTAMIDE SENSITIZATION IN ADVANCED PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023MP20-16 NOVEL FUNCTION OF PTUPB IN AKR1C3/AR/AR-V7 AXIS INHIBITION AND ENZALUTAMIDE SENSITIZATION IN ADVANCED PROSTATE CANCER Joy Yang, Pengfei Xu, Bo Chen, Shu Ning, Hans Adomat, Sung-Hee Hwang, Allen Gao, Martin Gleave, Bruce Hammock, Christopher Evans, and Chengfei Liu Joy YangJoy Yang More articles by this author , Pengfei XuPengfei Xu More articles by this author , Bo ChenBo Chen More articles by this author , Shu NingShu Ning More articles by this author , Hans AdomatHans Adomat More articles by this author , Sung-Hee HwangSung-Hee Hwang More articles by this author , Allen GaoAllen Gao More articles by this author , Martin GleaveMartin Gleave More articles by this author , Bruce HammockBruce Hammock More articles by this author , Christopher EvansChristopher Evans More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The steroidogenic enzyme Aldo-keto Reductase 1C3 (AKR1C3) is a major factor that contributes to the progression of castration-resistant prostate cancer (CRPC) and treatment failure. However, none of the available AKR1C3 inhibitors has shown promising results in clinical settings. Thus, there is an unmet need to develop superior AKR1C3 inhibitors for clinical testing. METHODS: PTUPB was synthesized by linking two pharmacophores, celecoxib and TPPU. In vitro and in vivo pharmacological activities of PTUPB in combination with enzalutamide were evaluated in several enzalutamide-resistant cell lines, conditional reprogrammed cell cultures, and patient-derived organoid models. The mechanism of action of PTUPB was examined via RNA sequence analysis, immunoblot analysis, co-immunoprecipitation, steroid measurement, and enzyme activity assays. Animal studies using PTUPB in combination with enzalutamide were performed in the castration-relapse VCaP tumor model. RESULTS: AKR1C3 levels are reciprocally regulated by the full-length androgen receptor (AR-FL) by binding to the distal enhancer region of AKR1C3. A novel function of PTUPB in AKR1C3 inhibition was discovered: at the same dose (0.5 µM) PTUPB suppressed the AKR1C3 activity more than indomethacin (60% vs. 40%) and celeboxib (60% vs. 0, p<0.05). And at the same dose (20 µM), PTUPB was more effective than indomethacin (98% vs. 22% of inhibition, p<0.05) and celecoxib (98% vs. 60% inhibition, p<0.05) in inhibiting CRPC cell growth. PTUPB synergizes with enzalutamide treatment for tumor suppression and gene signature regulation. Combination treatment with PTUPB and enzalutamide provides benefits by blocking AR and its variant (AR-V7) signaling, which inhibits the growth of VCaP castration-relapse tumors with 2.8-fold more inhibition (p<0.05) in tumor weights between the single (PTUPB) and combination treatments (Enza+PTUPB). There was also more inhibition in the growth of the patient-derived xenograft organoids by PTUPB alone or in combination with Enza. CONCLUSIONS: Targeting the ARK1C3/AR/AR-V7 axis with PTUPB and enzalutamide may overcome drug resistance to AR signaling inhibitors in advanced prostate cancer. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C. Liu) and R01CA251253 (C. Liu) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e281 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joy Yang More articles by this author Pengfei Xu More articles by this author Bo Chen More articles by this author Shu Ning More articles by this author Hans Adomat More articles by this author Sung-Hee Hwang More articles by this author Allen Gao More articles by this author Martin Gleave More articles by this author Bruce Hammock More articles by this author Christopher Evans More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

PD04-02 USING GENOMIC AND TRANSCRIPTOMIC PROPERTIES TO DETERMINE ANDROGEN RESPONSE IN DUCTAL PROSTATE CANCERS AND DETERMINE EFFICACY OF POLY(ADP-RIBOSE) POLYMERASE INHIBITORS WITH ANDROGEN SIGNALLING INHIBITORS THERAPY IN VITRO

You have accessJournal of UrologyCME1 Apr 2023PD04-02 USING GENOMIC AND TRANSCRIPTOMIC PROPERTIES TO DETERMINE ANDROGEN RESPONSE IN DUCTAL PROSTATE CANCERS AND DETERMINE EFFICACY OF POLY(ADP-RIBOSE) POLYMERASE INHIBITORS WITH ANDROGEN SIGNALLING INHIBITORS THERAPY IN VITRO Weranja Ranasinghe, Melbourne, Australia Patricia Troncoso, Mitchell Lawrence, Nicholas Choo, Birunthi Niranjan, Melissa Papargiris, Hong Wang, Andrew Ryan, Peter Shepherd, Bradley Broom, Ganiraju Manyam, Renea Taylor, Timothy Thompson, Nora Navone, Gail Risbridger, and Brian Chapin Weranja RanasingheWeranja Ranasinghe More articles by this author , Melbourne, Australia Patricia TroncosoMelbourne, Australia Patricia Troncoso More articles by this author , Mitchell LawrenceMitchell Lawrence More articles by this author , Nicholas ChooNicholas Choo More articles by this author , Birunthi NiranjanBirunthi Niranjan More articles by this author , Melissa PapargirisMelissa Papargiris More articles by this author , Hong WangHong Wang More articles by this author , Andrew RyanAndrew Ryan More articles by this author , Peter ShepherdPeter Shepherd More articles by this author , Bradley BroomBradley Broom More articles by this author , Ganiraju ManyamGaniraju Manyam More articles by this author , Renea TaylorRenea Taylor More articles by this author , Timothy ThompsonTimothy Thompson More articles by this author , Nora NavoneNora Navone More articles by this author , Gail RisbridgerGail Risbridger More articles by this author , and Brian ChapinBrian Chapin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The histologic variant prostatic ductal adenocarcinoma (DAC) has an aggressive biology with predisposition to metastasize early to visceral organs at low PSA levels. In contrast to acinar adenocarcinoma of the prostate (PAC), DACs have no effective systemic therapies, responding poorly to androgen deprivation therapy and chemotherapy. Poly(ADP-ribose) polymerase inhibitors (PARPi) may synergize with androgen signaling inhibitors (ARSI) inducing a “BRCAness” phenotype in castrate resistant prostate cancer (CRPC). A recent clinical trial demonstrated the efficacy of this combination therapy in CRPC, regardless of HR status. We aimed to characterize the genomic and transcriptomic properties of DAC to determine the androgen response and assess efficacy of PARPi+ARSI therapy in vivo. METHODS: 20 DAC and 10 PAC tumors, matched for stage, grade and volume, were micro-dissected from radical prostatectomy samples. Whole exome sequencing, RNA sequencing and Hallmark pathway analyses were performed. Validated BRCA2 heterozygous mutant (201.1A-Cx) and HR-proficient (287R) patient-derived DAC organoid models were treated with PARPi, enzalutamide and the combination of PARPi+enzalutamide. RESULTS: DACs had higher rates of mutations compared to matched PAC tumors (Figure 1). Unsupervised hierarchical clustering of RNA expression identified a distinct DAC cluster, with downregulation of AR pathways and enrichment of HR pathways, compared to PAC. Niraparib+Enzalutamide significantly reduced organoid viability of BRCA2 heterozygous mutant (201.1A-Cx) and HR proficient (287R) cells compared to control, Niraparib or Enzalutamide alone. Moreover, PARPi and ARSI were synergistic in these DAC models. Similarly, Talazoparib + Enzalutamide reduced the viability of the HR proficient 287R organoid compared to control, Talazoparib or Enzalutamide (65% vs 100% vs 91% vs 78%, p<0.01). CONCLUSIONS: In contrast to PACs, primary DACs have a unique genomic and transcriptomic landscape with high rates of mutations associated with intrinsic androgen resistance. PARPi+ARSI were more effective at decreasing DAC organoid viability than either treatment alone, regardless of the HR status. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e142 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Weranja Ranasinghe More articles by this author Melbourne, Australia Patricia Troncoso More articles by this author Mitchell Lawrence More articles by this author Nicholas Choo More articles by this author Birunthi Niranjan More articles by this author Melissa Papargiris More articles by this author Hong Wang More articles by this author Andrew Ryan More articles by this author Peter Shepherd More articles by this author Bradley Broom More articles by this author Ganiraju Manyam More articles by this author Renea Taylor More articles by this author Timothy Thompson More articles by this author Nora Navone More articles by this author Gail Risbridger More articles by this author Brian Chapin More articles by this author Expand All Advertisement PDF downloadLoading ...

N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer

A genome-wide PiggyBac transposon-mediated screen and a resistance screen in a PIK3CAH1047R-mutated murine tumor model reveal NF1 loss in mammary tumors resistant to the phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor alpelisib. Depletion of NF1 in PIK3CAH1047R breast cancer cell lines and a patient-derived organoid model shows that NF1 loss reduces sensitivity to PI3Kα inhibition and correlates with enhanced glycolysis and lower levels of reactive oxygen species (ROS). Unexpectedly, the antioxidant N-acetylcysteine (NAC) sensitizes NF1 knockout cells to PI3Kα inhibition and reverts their glycolytic phenotype. Global phospho-proteomics indicates that combination with NAC enhances the inhibitory effect of alpelisib on mTOR signaling. In public datasets of human breast cancer, we find that NF1 is frequently mutated and that such mutations are enriched in metastases, an indication for which use of PI3Kα inhibitors has been approved. Our results raise the attractive possibility of combining PI3Kα inhibition with NAC supplementation, especially in patients with drug-resistant metastases associated with NF1 loss.

MP04-18 LEVERAGING PATIENT-DERIVED RENAL CELL CARCINOMA ORGANOIDS TO ENRICH TUMOR-REACTIVE T CELLS AND IDENTIFY NOVEL IMMUNE CHECKPOINTS

You have accessJournal of UrologyCME1 Apr 2023MP04-18 LEVERAGING PATIENT-DERIVED RENAL CELL CARCINOMA ORGANOIDS TO ENRICH TUMOR-REACTIVE T CELLS AND IDENTIFY NOVEL IMMUNE CHECKPOINTS Elliot Merritt, Julie-Ann Cavallo, John Sfakianos, Amir Horowitz, Laura Zuluaga, Jordan Rich, Indu Saini, Kennedy E. Okhawere, Burak Upcinar, Ketan Badani, Benjamin Hopkins, and Anna Tocheva Elliot MerrittElliot Merritt More articles by this author , Julie-Ann CavalloJulie-Ann Cavallo More articles by this author , John SfakianosJohn Sfakianos More articles by this author , Amir HorowitzAmir Horowitz More articles by this author , Laura ZuluagaLaura Zuluaga More articles by this author , Jordan RichJordan Rich More articles by this author , Indu SainiIndu Saini More articles by this author , Kennedy E. OkhawereKennedy E. Okhawere More articles by this author , Burak UpcinarBurak Upcinar More articles by this author , Ketan BadaniKetan Badani More articles by this author , Benjamin HopkinsBenjamin Hopkins More articles by this author , and Anna TochevaAnna Tocheva More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite that renal cell carcinomas (RCC) are heavily infiltrated by intratumoral T cells, >70% of RCC patients fail to respond to immune checkpoint inhibitors (ICIs) immunotherapy. Our knowledge of the cellular and molecular mechanism underlying ICI efficacy in RCC is limited. Tumor cell-intrinsic IFNg responsivity has vital yet opposing functions in ICI efficacy by inducing the expression of antigen presenting molecules and T cell recruiting chemokine ligands from tumor cells, while in parallel increasing the expression of therapeutically relevant immune checkpoint ligands (IC-L) such as PD-L1. Major obstacles to improving ICI efficacy in RCC are the lack of (1) knowledge as it pertains to tumor-intrinsic IFNg responsivity; and (2) human models that recapitulate tumor cell heterogeneity and ICI responses. METHODS: We leverage patient-derived organoids (PDO) to determine IFNg-responsivity by analyzing the expression of antigen presenting molecule and IC-Ls. For each PDO, we generated blood and tumor derived PDO-reactive T cell lines that were phenotyped to determine T cell subset composition and inhibitory receptor expression. We performed PDO/T cell co-culture assays (PDOTs) and measured intracellular cytokine production to determine the increase in anti-tumor T cell reactivity in response to PD1 ICI and in combination with PDO relevant IC-Ls. RESULTS: We identified patient-specific intratumoral heterogeneity in IFNg responsivity to reveal IFNg-dependent and independent IC-L expression and soluble mediator secretion. Paired PDO IC-L expression and TIL checkpoint receptor expression data were used to identify ICI strategies in patient-specific manner. PDOTs were used to identify patient-specific therapeutically relevant IC-Ls, which in combination with PD-1 ICI improved anti-tumor T cell responses in autologous PDOT co-cultures. Functional immunophenotyping showed that in addition to CD8 T cells, intratumoral double negative T cells play a key role in ICI responses in RCC. CONCLUSIONS: PDO model intra-tumoral and inter-patient heterogeneity in the tumor-immune phenome and IFNg responsivity. Our paired autologous PDOT biobank enables patient-specific identification of targetable immune checkpoints and PDOT co-cultures enable characterization of patient-specific anti-tumor response to rationally design ICI combination therapies that augment anti-tumor T cell responses in RCC. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e40 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Elliot Merritt More articles by this author Julie-Ann Cavallo More articles by this author John Sfakianos More articles by this author Amir Horowitz More articles by this author Laura Zuluaga More articles by this author Jordan Rich More articles by this author Indu Saini More articles by this author Kennedy E. Okhawere More articles by this author Burak Upcinar More articles by this author Ketan Badani More articles by this author Benjamin Hopkins More articles by this author Anna Tocheva More articles by this author Expand All Advertisement PDF downloadLoading ...

Novel Human Meningioma Organoids Recapitulate the Aggressiveness of the Initiating Cell Subpopulations Identified by ScRNA-Seq.

High-grade meningioma has an unsatisfactory outcome despite surgery and postoperative radiotherapy; however, the factors driving its malignancy and recurrence remain largely unknown, which limits the development of systemic treatments. Single-cell RNA sequencing (scRNA-Seq) technology is a powerful tool for studying intratumoral cellular heterogeneity and revealing the roles of various cell types in oncogenesis. In this study, scRNA-Seq is used to identify a unique initiating cell subpopulation (SULT1E1+ ) in high-grade meningiomas. This subpopulation modulates the polarization of M2-type macrophages and promotes meningioma progression and recurrence. A novel patient-derived meningioma organoid (MO) model is established to characterize this unique subpopulation. The resulting MOs fully retain the aggressiveness of SULT1E1+ and exhibit invasiveness in the brain after orthotopic transplantation. By targeting SULT1E1+ in MOs, the synthetic compound SRT1720 is identified as a potential agent for systemic treatment and radiation sensitization. These findings shed light on the mechanism underlying the malignancy of high-grade meningiomas and provide a novel therapeutic target for refractory high-grade meningioma.

Discovery of YH677 as a cancer stemness inhibitor that suppresses triple-negative breast cancer growth and metastasis by regulating the TGFβ signaling pathway

Triple-negative breast cancer (TNBC) has a poor prognosis due to the lack of specific and highly effective therapeutic agents. Cancer stem cells (CSCs) are one of the main factors contributing to TNBC relapse and metastasis. Therefore, targeting CSCs selectively with small molecules is a novel strategy for drug development. In this study, the natural product harmine (HM) was identified as a hit compound from 2632 natural product monomers based on phenotypic screening of a 2D assay and patient-derived organoid (PDO) model that was established from a patient who had multiple drug resistance and various visceral and contralateral breast metastases. Next, harmine was further modified and optimized to obtain a lead compound (YH677) with a tetrahydro-β-carboline scaffold. YH677 showed potent antiproliferative and antimigratory activities against several TNBC cell lines in vitro. In addition, YH677 inhibited epithelial mesenchymal transition (EMT) and stem cell marker expression in a dose-dependent manner. More importantly, YH677 suppressed breast cancer growth and metastasis in orthotopic, metastatic xenograft and patient-derived xenograft (PDX) models in vivo. Mechanistic studies showed that YH677 inhibits the expansion of CSCs by regulating the TGFβ/Smad signaling pathway. These preclinical data provide a basis for the development of YH677 as a lead compound for TNBC treatment.

Oxypalmatine regulates proliferation and apoptosis of breast cancer cells by inhibiting PI3K/AKT signaling and its efficacy against breast cancer organoids

BackgroundBreast cancer (BC) is known as the most common cancer in women. Discovering novel and effective drugs is a priority for the treatment of BC. Oxypalmatine (OPT) is a natural protoberberine-type alkaloid isolated from Phellodendron amurense Rupr. (Rutaceae) with potential anti-cancer activity. PurposeThis investigation aimed to elucidate the biological role and potential mechanisms of OPT in BC cells, and intended to assess the therapeutic potential of OPT in BC patient-derived organoid models. MethodsCCK-8 and EdU assays, and flow cytometry were used to test the activity of OPT against BC cells. In addition, patient-derived organoid models were constructed to assess the therapeutic efficiency of OPT in BC. Besides, network pharmacological analysis and RNA sequencing analysis were performed to predict the underlying anti-BC mechanism of OPT. Moreover, Western blot analysis was applied to test the expression of genes modulated by OPT. ResultsOPT attenuated the proliferation and DNA replication, and induced apoptosis in multiple BC cells. Interestingly, OPT also exerted a cytotoxic effect on BC organoids characterized as luminal A, HER2-overexpressing, and triple-negative subtypes, indicating that OPT was a potential broad-spectrum anticancer drug. Network pharmacological analysis suggested that OPT might affect signals contributing to BC progression, including PI3K/AKT, MAPK, and VEGFA-VEGFR2 signaling pathways. Moreover, bioinformatics analysis of data from our RNA sequencing suggested that PI3K/AKT was a downstream pathway of OPT in BC. Finally, OPT was shown to inactivate PI3K/AKT signaling pathway in BC cells by Western blot analysis. ConclusionsCollectively, our study demonstrated that OPT suppressed proliferation and induced apoptosis through mitigating the PI3K/AKT signaling pathway in BC cells. Moreover, our work first adopted BC organoid models to confirm OPT as an effective and promising drug, laying a foundation for the potential use of OPT in BC treatment.

M5C-dependent cross-regulation between nuclear reader ALYREF and writer NSUN2 promotes urothelial bladder cancer malignancy through facilitating RABL6/TK1 mRNAs splicing and stabilization

The significance of 5-methylcytosine (m5C) methylation in human malignancies has become an increasing focus of investigation. Here, we show that m5C regulators including writers, readers and erasers, are predominantly upregulated in urothelial carcinoma of the bladder (UCB) derived from Sun Yat-sen University Cancer Center and The Cancer Genome Atlas cohort. In addition, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) as a methyltransferase and Aly/REF export factor (ALYREF) as a nuclear m5C reader, are frequently coexpressed in UCB. By applying patient-derived organoids model and orthotopic xenograft mice model, we demonstrate that ALYREF enhances proliferation and invasion of UCB cells in an m5C-dependent manner. Integration of tanscriptome-wide RNA bisulphite sequencing (BisSeq), RNA-sequencing (RNA-seq) and RNA Immunoprecipitation (RIP)-seq analysis revealed that ALYREF specifically binds to hypermethylated m5C site in RAB, member RAS oncogene family like 6 (RABL6) and thymidine kinase 1 (TK1) mRNA via its K171 domain. ALYREF controls UCB malignancies through promoting hypermethylated RABL6 and TK1 mRNA for splicing and stabilization. Moreover, ALYREF recognizes hypermethylated m5C site of NSUN2, resulting in NSUN2 upregulation in UCB. Clinically, the patients with high coexpression of ALYREF/RABL6/TK1 axis had the poorest overall survival. Our study unveils an m5C dependent cross-regulation between nuclear reader ALYREF and m5C writer NSUN2 in activation of hypermethylated m5C oncogenic RNA through promoting splicing and maintaining stabilization, consequently leading to tumor progression, which provides profound insights into therapeutic strategy for UCB.

Response and acquired resistance to MET inhibitors in de novo MET fusion-positive advanced non-small cell lung cancer

ObjectivesDe novo mesenchymal-to-epithelial transition (MET) gene fusions in non-small cell lung cancer (NSCLC) are a promising target for MET tyrosine kinase inhibitors (TKIs). We aimed to examine the response to targeted therapy with MET TKIs and resistance mechanisms in de novo MET fusion-positive NSCLC as these have not been comprehensively explored. Materials and MethodsWe examined the MET fusions in 4,429 patients with advanced-stage NSCLC using targeted next-generation sequencing and validated the results using RT-PCR. We analyzed cellular models harboring MET fusions and established a patient-derived organoid (PDO) model. ResultsWe identified 13 (0.29 %, 13/4429) patients with de novo MET fusions and found EPHB4, THAP5, TNPO3, and DST as novel MET fusion partners. The most common concomitant gene with MET fusions was TP53 mutations. Among 12 patients receiving MET TKI treatment, two achieved stable disease, six achieved partial response, and four underwent progressive disease. An in vitro study showed that EPHB4-MET is a functional driver gene. MET inhibitors significantly inhibited the proliferation and phosphorylation of downstream STAT3, AKT, and ERK1/2 in EPHB4-MET overexpressing cells. Acquired MET D1228H/N or D1246N mutations were found in patients harboring MET fusions after acquiring resistance to MET TKIs. Tivantinib showed optimal suppression efficacy in a PDO model with an acquired MET D1228N mutation. ConclusionMET fusions occur in a rare subset of patients with NSCLC and represent a promising therapeutic target. MET secondary mutations D1228H/N or D1246N present the potential resistance mechanisms of MET inhibitors in patients with de novo MET fusions.

Feasibility of establishing and drug screening patient-derived rectal organoid models from pretreatment rectal cancer biopsies.

176 Background: Response to neoadjuvant chemotherapy and radiation therapy in the treatment of locally advanced rectal cancer is heterogenous and prognostic of clinical outcomes, necessitating the need for predictive biomarkers to guide personalized treatment recommendations. Sensitivity to a given chemotherapy in patient-derived organoids predicts patient response to that chemotherapy, establishing it as a promising model for efforts to ascertain predictive biomarkers and personalize treatment decisions. This study assessed the feasibility of obtaining patient-derived rectal organoids from standard of care pre-treatment proctoscopy biopsies. Methods: In this clinical trial (NCT04371198), biopsies were obtained from patients with stage II rectal adenocarcinoma prior to receipt of neoadjuvant therapy. Tissue samples were mechanically and enzymatically dissociated to obtain a single cell suspension. Cells were then mixed with matrigel at a ratio of 2,000 cells:5 µL Matrigel in a 50ul dome and plated on a 24 well tissue culture plate with colorectal cancer organoid media at 37oC/5% CO2. Established patient-derived organoids were then used to perform drug screens with clinically-applicable chemotherapeutics including oxaliplatin, irinotecan and 5-FU, followed by high throughput drug screen using our recently published MicroOrganoSpheres platform using the NCI Approved Oncology Drugs Set VI* library. Results: Of the 20 patients enrolled, 17 (85%) patient-derived organoids were created from pre-treatment specimens. 15 (88%) of these samples were successfully established as defined by the ability to passage organoids for at least two passages. All established samples were used to perform standard of care drug screens and high throughout drug screens, which demonstrated differences in drug sensitivities among the samples. Moreover, within two weeks of receiving the sample, four established quickly enough to complete drug screening with oxaliplatin, SN38, and 5-Fluorouracil. Conclusions: These results demonstrate the feasibility of establishing patient-derived rectal organoids from biopsy specimens obtained by proctoscopy, and reinforce the utility of patient-derived organoids as a tractable ex vivo platform to personalize rectal cancer treatment. Planned future directions include in vitro determination of radiation therapy sensitivity as well as systematic assessment of the correlation between individual patients and their organoid model. Clinical trial information: NCT04371198 .

DOX-loaded hydroxyapatite nanoclusters for colorectal cancer (CRC) chemotherapy: Evaluation based on the cancer cells and organoids.

It is meaningful to find suitable in vitro models for preclinical toxicology and efficacy evaluation of nanodrugs and nanocarriers or drug screening and promoting clinical transformation of nanocarriers. The emergence and development of organoids technology provide a great possibility to achieve this goal. Herein, we constructed an in vitro 3D organoid model to study the inhibitory effect of nanocarriers on colorectal cancer. And designed hydroxyapatite nanoclusters (c-HAP) mediated by polydopamine (PDA) formed under alkaline conditions (pH 9.0), then used c-HAP to load DOX (c-HAP/DOX) as nanocarrier for improved chemotherapy. In vitro, drug release experiments show that c-HAP/DOX has suitable responsive to pH, can be triggered to the facile release of DOX in a slightly acidic environment (pH 6.0), and maintain specific stability in a neutral pH value (7.4) environment. c-HAP/DOX showed an excellent antitumor effect in the two-dimensional (2D) cell model and three-dimensional (3D) patient-derived colon cancer organoids (PDCCOs) model. In addition, c-HAP/DOX can release a sufficient amount of DOX to produce cytotoxicity in a slightly acidic environment, entering efficiently into the colorectal cancer cells caused endocytosis and induced apoptosis. Therefore, organoids can serve as an effective in vitro model to present the structure and function of colorectal cancer tissues and be used to evaluate the efficacy of nanocarriers for tumors.

P074 Interferon-γ promotes antigen processing and presentation pathway in the intestinal epithelium of patients with Crohn’s disease

Abstract Background Interferon-γ (IFN-γ) is a major driver of excessive immune response, leading to intestinal mucosal damage and contributing the pathogenesis of Crohn’s diseases (CD). In contrast to the inflammatory cells and immune system, IFN-γ-induced cellular responses has not been well documented in the intestinal epithelium. The intestinal epithelium consists of multiple cell types and patient-derived intestinal organoid model may be appropriate to evaluate the IFN-γ-induced epithelial cellular responses in patients with CD. Methods To evaluate the IFN-γ-mediated epithelial cellular responses, human small intestinal organoids (enteroids) derived from the controls (n=6) and patients with Crohn’s disease (CD, n=10) were constructed. We focused on the IFN-γ-induced cell death mechanism upregulated in CD enteroids and validated them in surgically resected small intestine in patients with active CD. Results As the concentration of IFN-γ increased in the culture medium, the organoid-forming efficiency decreased steadily, which did not differ between control and CD enteroids. The enteroid viability measured using MTT was estimated to be 83.10 pg/ml for EC50. In control and CD enteroids treated with 100 pg/ml IFN-γ, PANoptosis was the main inflammatory cell death mechanism. Bulk and single-cell RNA sequencing revealed that IFN-γ induced the expression of MHC class II-associated genes, co-inhibitory molecule CD274 (PD-L1), and IDO1 in control and CD enteroids, which may be associated with peripheral tolerance in the absence of exogenous antigens. Gene set enrichment analysis identified an up-regulated antigen processing and presentation pathway with increased expression of MHC class I molecules in IFN-γ-treated CD enteroids compared to IFN-γ-treated control enteroids (normalized enrichment score=2.13, p=0.002, q=0.005). IFN-γ-induced MHC class I pathway could result in T cell-mediated cytotoxicity in CD enteroids. The selective JAK1 inhibitor, upadacitinib, inhibited the IFN-γ-induced PANoptosis and T cell-mediated cytotoxicity in CD enteroids. The expression of HLA-A and B was higher in the inflamed small intestine in patients with CD than those in patients with ischemic enteritis. Conclusion IFN-γ promotes antigen processing and presentation pathway in the intestinal epithelium. In patients with CD, increased expression of MHC class I molecules may induce T cell-mediated cytotoxicity, which could contribute to the development of CD.

Antibody-Exatecan Conjugates with a Novel Self-immolative Moiety Overcome Resistance in Colon and Lung Cancer.

ADCs combining a novel self-immolative moiety and topoisomerase I inhibitor exatecan as payload show deep and durable response in low-target-expressing and MDR+ tumors resistant to DXd/SN-38 ADCs without increasing toxicity. This new class of ADCs has the potential to benefit an additional patient population beyond current options. See related commentary by Gupta et al., p. 817. This article is highlighted in the In This Issue feature, p. 799.

Blockade of NMT1 enzymatic activity inhibits N-myristoylation of VILIP3 protein and suppresses liver cancer progression

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. Identification of the underlying mechanism of HCC progression and exploration of new therapeutic drugs are urgently needed. Here, a compound library consisting of 419 FDA-approved drugs was taken to screen potential anticancer drugs. A series of functional assays showed that desloratadine, an antiallergic drug, can repress proliferation in HCC cell lines, cell-derived xenograft (CDX), patient-derived organoid (PDO) and patient-derived xenograft (PDX) models. N-myristoyl transferase 1 (NMT1) was identified as a target protein of desloratadine by drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) assays. Upregulation of NMT1 expression enhanced but NMT1 knockdown suppressed tumor growth in vitro and in vivo. Metabolic labeling and mass spectrometry analyses revealed that Visinin-like protein 3 (VILIP3) was a new substrate of NMT1 in protein N-myristoylation modification, and high NMT1 or VILIP3 expression was associated with advanced stages and poor survival in HCC. Mechanistically, desloratadine binds to Asn-246 in NMT1 and inhibits its enzymatic activity, disrupting the NMT1-mediated myristoylation of the VILIP3 protein and subsequent NFκB/Bcl-2 signaling. Conclusively, this study demonstrates that desloratadine may be a novel anticancer drug and that NMT1-mediated myristoylation contributes to HCC progression and is a potential biomarker and therapeutic target in HCC.

Papillary thyroid cancer organoids harboring BRAFV600E mutation reveal potentially beneficial effects of BRAF inhibitor-based combination therapies

BackgroundsPapillary thyroid cancer (PTC), which is often driven by acquired somatic mutations in BRAF genes, is the most common pathologic type of thyroid cancer. PTC has an excellent prognosis after treatment with conventional therapies such as surgical resection, thyroid hormone therapy and adjuvant radioactive iodine therapy. Unfortunately, about 20% of patients develop regional recurrence or distant metastasis, making targeted therapeutics an important treatment option. Current in vitro PTC models are limited in representing the cellular and mutational characteristics of parental tumors. A clinically relevant tool that predicts the efficacy of therapy for individuals is urgently needed.MethodsSurgically removed PTC tissue samples were dissociated, plated into Matrigel, and cultured to generate organoids. PTC organoids were subsequently subjected to histological analysis, DNA sequencing, and drug sensitivity assays, respectively.ResultsWe established 9 patient-derived PTC organoid models, 5 of which harbor BRAFV600E mutation. These organoids have been cultured stably for more than 3 months and closely recapitulated the histological architectures as well as mutational landscapes of the respective primary tumors. Drug sensitivity assays of PTC organoid cultures demonstrated the intra- and inter-patient specific drug responses. BRAFV600E inhibitors, vemurafenib and dabrafenib monotherapy was mildly effective in treating BRAFV600E-mutant PTC organoids. Nevertheless, BRAF inhibitors in combination with MEK inhibitors, RTK inhibitors, or chemotherapeutic agents demonstrated improved efficacy compared to BRAF inhibition alone.ConclusionsThese data indicate that patient-derived PTC organoids may be a powerful research tool to investigate tumor biology and drug responsiveness, thus being useful to validate or discover targeted drug combinations.

Intergenic CircRNA Circ_0007379 Inhibits Colorectal Cancer Progression by Modulating miR-320a Biogenesis in a KSRP-Dependent Manner.

Circular RNAs (circRNAs) are covalently closed RNA structures that play multiple roles in tumorigenesis and progression. Compared with exon‒intron circRNAs, the biological functions and implications of intergenic circRNAs in human cancer are still poorly understood. Here, we performed circRNA microarray analysis and identified an intergenic circRNA, circ_0007379, that was significantly downregulated in patients with colorectal cancer (CRC). The biogenesis of circ_0007379 was mediated by reverse complementary matches (RCMs) and was negatively regulated by the RNA helicase DHX9. Functionally, circ_0007379 suppressed CRC cell growth and metastasis in cell culture as well as in patient-derived organoid and xenograft models. Mechanistically, circ_0007379 acted as a scaffold to facilitate the processing of both pri-miR-320a and pre-miR-320a in a KSRP-dependent manner, leading to miR-320a maturation and subsequent repression of transcription factor RUNX1 expression. Thus, our findings establish a previously unrecognized function of circRNA in inhibiting CRC progression.