Patient-derived Organoid Models Research Articles

Preclinical efficacy of CBR-5884 against epithelial ovarian cancer cells by targeting the serine synthesis pathway

Reprogramming of the serine synthesis pathway (SSP) is intricately linked to the progression of epithelial ovarian cancer (EOC). CBR-5884, a selective small-molecule inhibitor targeting phosphoglycerate dehydrogenase (PHGDH), effectively impedes the de novo synthesis of serine within cancer cells. This study aimed to evaluate the inhibitory effect of CBR-5884 on EOC cells and delineate its specific mechanism, thereby proposing a novel therapeutic approach for treating EOC. The suppression of serine biosynthesis after CBR-5884 treatment was evaluated using RNA sequencing and a serine assay kit, and the results showed that CBR-5884 effectively downregulated serine biosynthesis in EOC cells, particularly those expressing high levels of PHGDH. In vitro studies revealed that CBR-5884 demonstrated significant antitumor effects and suppressed migration and invasion of EOC cells through down-regulation of the integrin subunit beta 4 (ITGB4)/extracellular signal-regulated kinase (ERK)/epithelial–mesenchymal transition signal axis. Additionally, CBR-5884 mitigated the stemness of EOC cells and heightened their sensitivity to chemotherapy. Moreover, in vivo studies revealed that CBR-5884 significantly delayed tumor growth, with histological analysis indicating the safety profile of CBR-5884. Finally, the patient-derived organoid (PDO) models were utilized to explore the preclinical efficacy of CBR-5884 against EOC cells, and the results unveiled that CBR-5884 impeded proliferation and downregulated the expression of ITGB4 in EOC PDO models. Our findings supports the anticancer properties of CBR-5884 in EOC cells exhibiting high PHGDH expression, manifesting through the suppression of proliferation, migration, and invasion, while enhancing chemotherapy sensitivity, suggesting that CBR-5884 holds promise as an efficacious strategy for the treatment of EOC.

Biological impact and therapeutic potential of a novel camptothecin derivative (FLQY2) in pancreatic cancer through inactivation of the PDK1/AKT/mTOR pathway

BackgroundCamptothecin (CPT), a pentacyclic alkaloid with antitumor properties, is derived from the Camptotheca acuminata. Topotecan and irinotecan (CPT derivatives) were first approved by the Food and Drug Administration for cancer treatment over 25 years ago and remain key anticancer drugs today. However, their use is often limited by clinical toxicity. Despite extensive development efforts, many of these derivatives have not succeeded clinically, particularly in their effectiveness against pancreatic cancer which remains modest. Aim of the studyThis study aimed to evaluate the therapeutic activity of FLQY2, a CPT derivative synthesized in our laboratory, against pancreatic cancer, comparing its efficacy and mechanism of action with those of established clinical drugs. MethodsThe cytotoxic effects of FLQY2 on cancer cells were assessed using an MTT assay. Patient-derived organoid (PDO) models were employed to compare the sensitivity of FLQY2 to existing clinical drugs across various cancers. The impact of FLQY2 on apoptosis and cell cycle arrest in Mia Paca-2 pancreatic cancer cells was examined through flow cytometry. Transcriptomic and proteomic analyses were conducted to explore the underlying mechanisms of FLQY2′s antitumor activity. Western blotting was used to determine the levels of proteins regulated by FLQY2. Additionally, the antitumor efficacy of FLQY2 in vivo was evaluated in a pancreatic cancer xenograft model. ResultsFLQY2 demonstrated (1) potent cytotoxicity; (2) superior tumor-suppressive activity in PDO models compared to current clinical drugs such as gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infinitinib, and lenvatinib; (3) significantly greater tumor inhibition than paclitaxel liposomes in a pancreatic cancer xenograft model; (4) robust antitumor effects, closely associated with the inhibition of the TOP I and PDK1/AKT/mTOR signaling pathways. In vitro studies revealed that FLQY2 inhibited cell proliferation, colony formation, induced apoptosis, and caused cell cycle arrest at nanomolar concentrations. Furthermore, the combination of FLQY2 and gemcitabine exhibited significant inhibitory and synergistic effects. ConclusionThe study confirmed the involvement of topoisomerase I and the PDK1/AKT/mTOR pathways in mediating the antitumor activity of FLQY2 in treating Mia Paca-2 pancreatic cancer. Therefore, FLQY2 has potential as a novel therapeutic option for patients with pancreatic cancer.

MRI radiomics captures early treatment response in patient-derived organoid endometrial cancer mouse models

BackgroundRadiomics can capture microscale information in medical images beyond what is visible to the naked human eye. Using a clinically relevant mouse model for endometrial cancer, the objective of this study was to develop and validate a radiomic signature (RS) predicting response to standard chemotherapy.MethodsMice orthotopically implanted with a patient-derived grade 3 endometrioid endometrial cancer organoid model (O-PDX) were allocated to chemotherapy (combined paclitaxel/carboplatin, n=11) or saline/control (n=13). During tumor progression, the mice underwent weekly T2-weighted (T2w) magnetic resonance imaging (MRI). Segmentation of primary tumor volume (vMRI) allowed extraction of radiomic features from whole-volume tumor masks. A radiomic model for predicting treatment response was derived employing least absolute shrinkage and selection operator (LASSO) statistics at endpoint images in the orthotopic O-PDX (RS_O), and subsequently applied on the earlier study timepoints (RS_O at baseline, and week 1-3). For external validation, the radiomic model was tested in a separate T2w-MRI dataset on segmented whole-volume subcutaneous tumors (RS_S) from the same O-PDX model, imaged at three timepoints (baseline, day 3 and day 10/endpoint) after start of chemotherapy (n=8 tumors) or saline/control (n=8 tumors).ResultsThe RS_O yielded rapidly increasing area under the receiver operating characteristic (ROC) curves (AUCs) for predicting treatment response from baseline until endpoint; AUC=0.38 (baseline); 0.80 (week 1), 0.85 (week 2), 0.96 (week 3) and 1.0 (endpoint). In comparison, vMRI yielded AUCs of 0.37 (baseline); 0.69 (w1); 0.83 (week 2); 0.92 (week 3) and 0.97 (endpoint). When tested in the external validation dataset, RS_S yielded high accuracy for predicting treatment response at day10/endpoint (AUC=0.85) and tended to yield higher AUC than vMRI (AUC=0.78, p=0.18). Neither RS_S nor vMRI predicted response at day 3 in the external validation set (AUC=0.56 for both).ConclusionsWe have developed and validated a radiomic signature that was able to capture chemotherapeutic treatment response both in an O-PDX and in a subcutaneous endometrial cancer mouse model. This study supports the promising role of preclinical imaging including radiomic tumor profiling to assess early treatment response in endometrial cancer models.

Enhancing the Efficacy of HIPEC Through Bromelain: A Preclinical Investigation in Appendiceal Cancer.

Appendiceal cancer (AC) excessive mucin production is a barrier to heated intraperitoneal chemotherapy (HIPEC) drug delivery. Bromelain is a pineapple stem extract with mucolytic properties. We explored bromelain treatment effects against mucinous AC in a patient-derived tumor organoid (PTO) model and an AC cell line. PTOs were fabricated from tumor specimens obtained from patients with AC undergoing cytoreductive surgery with HIPEC. PTOs underwent HIPEC treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37°C and 42°C with and without bromelain pretreatment. From October 2020 to May 2023, 16 specimens were collected from 13 patients with low-grade (12/16, 75%) and high-grade AC (4/16, 25%). The mucin-depleting effects of bromelain were most significant in combination with N-acetylcysteine (NAC) compared with bromelain (47% versus 10%, p = 0.0009) or NAC alone (47% versus 12.8%, p = 0.0027). Bromelain demonstrated > 31% organoid viability reduction at 60 min (p < 0.001) and > 66% in 48 h (p < 0.0001). Pretreatment with bromelain increased cytotoxicity of both cisplatin and MMC HIPEC conditions by 31.6% (p = 0.0001) and 35.5% (p = 0.0001), respectively. Ki67, CK20, and MUC2 expression decreased after bromelain treatment; while increased caspase 3/7 activity and decreased Bcl-2 (p = 0.009) and Bcl-xL (p = 0.01) suggest induction of apoptosis pathways. Furthermore, autophagy proteins LC3A/B I (p < 0.03) and II (p < 0.031) were increased; while ATG7 (p < 0.01), ATG 12 (p < 0.04), and Becline 1(p < 0.03), expression decreased in bromelain-treated PTOs. Bromelain demonstrates cytotoxicity and mucolytic activity against appendiceal cancer organoids. As a pretreatment agent, it potentiates the cytotoxicity of multiple HIPEC regimens, potentially mediated through programmed cell death and autophagy.

Abstract PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-28-12.

Abstract PO4-14-12: Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids

Abstract Background: Activating mutations in ERBB2 (HER2) are enriched &amp;gt;4-fold in invasive lobular carcinoma (ILC) with a rate of up to 19% in metastatic ILC. ILC is a histologic subtype of breast cancer characterized by loss of E-cadherin (CDH1), suggesting a potential interaction between loss of CDH1 and mutations in ERBB2. Recent trials have demonstrated promising single agent efficacy using the irreversible pan-HER tyrosine kinase inhibitor (TKI), neratinib, in patients with metastatic ERBB2 mutant ILC. However, further studies on combination therapies with other anticancer agents are needed to increase response rate and progression free survival for these patients. HER2-targeted antibody drug conjugates (ADC), particularly trastuzumab deruxtecan (T-DXd), have shown great promise in HER2-low metastatic breast cancer. Yet, their efficacy as a single agent or with HER2 TKIs in HER2-low and -mutant ILC is unknown and warrants investigation. Methods: Past studies analyzing ERBB2 mutations used overexpression of ERBB2 mutant cDNA, but this approach does not faithfully recapitulate the human disease. To model ERBB2 missense mutations as found in human breast cancers, we used CRISPR-based prime editing to generate a panel of isogenic ILC cell lines and patient-derived organoids (PDO) harboring ERBB2 wild-type (WT) or ERBB2 mutations (S310F or V777L). Both of these ERBB2 mutations have been previously characterized and are known to be activating mutations. We then used them to test neratinib and other TKIs with ADCs, including T-DXd and trastuzumab emtansine (T-DM1). Results: We successfully introduced single copy, heterozygous activating ERBB2 mutations (S310F or V777L) into two ERBB2-nonamplified metastatic ILC cell lines (MDA-MB-134 and SUM44PE) and one ERBB2-nonamplified metastatic ILC PDO (IPM-BO-053). Positive clones carrying the mutations were verified by Sanger sequencing and droplet digital PCR and subsequently pooled together. We further demonstrated that these mutations hyperactivated HER2 and downstream signaling pathways. ILC cell lines harboring these mutations showed enhanced sensitivity to HER2 TKIs but not ADCs. In contrary, ERBB2 mutations did not alter responses of IPM-BO-053 PDOs to HER2 TKIs but significantly increased responses to ADCs. Interestingly, we also observed accelerated HER2 protein degradation upon heregulin stimulation in ERBB2 mutant ILC PDOs, suggesting activating ERBB2 mutations may enhance ADC/HER2 complex internalization, degradation, and release of payloads. Lastly, we explored drug synergy between HER2 TKIs and ADCs in isogenic IPM-BO-053 PDOs and found that combination of T-DXd and neratinib or afatinib showed synergy (Combination Index &amp;lt; 1). Conclusions: Although the reason for the discrepancies in drug response between ILC cell lines and PDOs is not clear, we hypothesize that response in 3D PDOs might be more faithfully representing response seen in patients. We will generate additional ILC PDOs with knock-in ERBB2 mutations to validate our findings. Irreversible HER2 TKIs, such as neratinib and afatinib, showed synergy with T-DXd in ERBB2 mutant ILC PDOs. This holds important therapeutic implications in light of current treatment options for ILC. In future experiments, we will test if neratinib or afatinib increases endocytic uptake of T-DXd using a fluorescently labeled endocytosis tracker. Further, an in-depth molecular characterization of our isogenic cell lines and PDOs models is ongoing to gain mechanistic insights into how activating ERBB2 mutations increase HER2 internalization and degradation. Our in vitro studies provide a strong foundation for in vivo testing of neratinib or afatinib with T-DXd for ERBB2 mutant ILC. Citation Format: Jie Bin Liu, Danielle Tseng, Jagmohan Hooda, Daniel Brown, Adrian Lee, Steffi Oesterreich. Predicting Response to HER2 Tyrosine Kinase Inhibitors and Antibody Drug Conjugates in HER2 Mutant Invasive Lobular Carcinoma Using CRISPR/Cas9 Knock-in Cell lines and Patient-derived Organoids [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-14-12.

Abstract PS09-09: Multiomics profiling and molecular classification refine precision treatment strategies for HER2-positive breast cancer

Abstract Background: Anti-HER2 targeted therapy has achieved a series of breakthroughs. However, the current treatment strategy regarding HER2-positive breast cancer remains indiscriminate and lacks specificity, which limits the further improvement of overall treatment response and may lead to overtreatment and extra cost for some patients. Our study aims to reveal the molecular heterogeneity of HER2-positive breast cancer to guide a more precise treatment. Patients and methods: We selected HER2-positive breast cancer patients treated at Fudan University Shanghai Cancer Center between 2013 and 2014 and conducted genomic, transcriptomic, proteomic and metabolomic profiling. We then applied a non-negative matrix factorization algorithm on transcriptomic data to obtain an unsupervised classification. And we further studied the correlation between subtypes and corresponding treatment strategies in multiple cohorts of adjuvant and neoadjuvant therapy. For clinical accessibility, we developed convolutional neural network models through deep learning algorithm based on digital pathology to identify different subtypes. Additionally, we explored novel treatment strategies using the patient-derived organoids (PDOs) models. Results: We established a novel multiomics cohort of 180 HER2- breast cancer patients and classified them into four clinically significant molecular subtypes: (1) A classical HER2-enriched (HER2-CLA, N=51) subtype characterized by strong ERBB2 signaling and remarkable sensitivity to anti-HER2-targeted therapy (pathologic complete response with dual-targeted therapy: 93%). (2) an immunomodulatory (HER2-IM, N=36) subtype characterized by an immune-activated microenvironment and excellent prognosis with current treatment (no relapse in 97% of patients with a median follow-up of 86 months). Tumors of this subtype were therefore candidates for de-escalatory treatment. (3) A luminal-like (HER2-LUM, N=55) subtype distinguished by activated estrogen receptor signaling and (4) a basal/mesenchymal-like (HER2-BM, N=38) subtype enriched in activated receptor tyrosine kinase pathways. HER2-LUM and HER2- BM showed limited benefit from anti-HER2 therapy, and thus, add-on therapies might be needed. The overall area under the curve (AUC) of the convolutional neural network model based on digital pathology for identifying different subtypes is 0.77. In the exploration of novel treatment strategies, we found in the PDO model that the HER2-LUM subtype is more sensitive to a treatment regimen combining standard (chemotherapy and targeted therapy) with subsequent endocrine therapy and CDK4/6 inhibitors compared to other subtypes. Additionally, the HER2-BM subtype demonstrated greater sensitivity to treatment with a combination of EGFR inhibitors, PDGFR inhibitors or VEGFR inhibitors. Conclusion: We uncovered a high degree of molecular heterogeneity in HER2-positive breast cancer and illustrated its impact on treatment response. More precise treatment can be given according to the characteristics of different subtypes, which may achieve good efficacy and simultaneously reduce overtreatment and extra cost. The comprehensive profiling of HER2-positive breast cancers could also serve as an important resource for further exploration. Key Words: HER2-positive breast cancer cohort; molecular classification; targeted therapy; precision treatment; de-escalatory treatment. Citation Format: Yu-Wei Li, Ding Ma, Xiang-Rong Wu, Lei-Jie Dai, Shen Zhao, Yu-Zheng Xu, Xi Jin, Xiao Yi, Ying Wang, Cai-Jin Lin, Yi-Fan Zhou, Tong Fu, Wen-Tao Yang, Ming Li, Hong Lv, Yi-Zhou Jiang, Zhi-Ming Shao. Multiomics profiling and molecular classification refine precision treatment strategies for HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS09-09.

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Abstract Treatment of triple-negative breast cancer (TNBC) remains challenging. Cancer stem cells (CSCs) are the most intractable subpopulation of TNBC cells, which has associated with a high risk of relapse and poor prognosis. However, eradication of CSCs continues to be difficult. Here, we integrated the multiomics data of our large TNBC cohort (n=360) to identify vital markers of CSCs. We discovered that EMSY, inducing a BRCAness phenotype, was preferentially expressed in breast CSCs, promoted the enrichment of ALDH+ cells and was positively correlated with poor relapse-free survival. Mechanistically, EMSY competitively bound to the Jmjc domain, which was critical for the enzyme activity of the KDM5B-specific H3K4 demethylase to reshape methionine metabolism and promote CSC self-renewal and tumorigenesis in an H3K4 methylation-dependent manner. Moreover, EMSY accumulation in TNBC cells sensitized them to PARP inhibitors against bulk cells and methionine deprivation against CSCs. These findings indicate that clinically relevant eradication of CSCs could be achieved with a strategy that targets CSC-specific vulnerabilities in amino acid metabolism. EMSY was highly expressed in BCSCs and positively correlated with TNBC malignancy a. Heatmap with mRNA expression characteristics of non-CSC and CSC subtypes. The top 2,000 most differentially expressed genes used for clustering are plotted. Samples are also annotated on top by the FUSCC subtypes5. b. Volcano plot showing genes targeted by a siRNA inducing a significant change (Fold change (FC) represented the proportion of ALDH+ cells compared to the control)34. c. Overlap analysis of the upregulated genes in CSC subtype (Gene cluster in A) and the genes via siRNA (Gene cluster in B) inducing a significant reduction of the ALDH+ cell ratio. d. The mRNA expression of EMSY in non-CSC and CSC TNBC transcriptomic subtypes (n = 360, Mann-Whitney’s test). Each boxplot showed the median and 95% confidence intervals (95% CI). e. The CNV frequency of EMSY in non-CSC and CSC subtypes. f, g. EMSY mRNA expression of samples with different EMSY copy number in FUSCC (n=302) and TCGA (n=71) TNBC cohorts. h, i. Immunohistochemistry analysis of EMSY expression in specimens of patients between non-CSC (n=45) and CSC (n=20) subtypes. Representative images (h) and the graph of H Scores (i, mean ± SEM, Mann-Whitney’s test) were shown. Scale bar, 4μm (left), 20μm (right). j, k. Kaplan–Meier RFS plots of EMSY in FUSCC (n=360) and GSE234669 (n=442) TNBC (https://kmplot.com/analysis/). Accumulation of EMSY enhanced CSC self-renewal and tumorigenesis a. The mRNA (left) and protein (right) expression of EMSY in FACS sorted ALDH+ and ALDH− cells. b-d. ALDH activity detected by ALDEFLUOR assay and shown as mean ± SEM (n=3/group, one-way ANOVA’s test) in EMSY-knockdown cells (b, c) and EMSY-overexpressing cells (d). e, f. Self-renewal ability determined by primary mammosphere formation (e, n=6 biological independent experiments, one-way ANOVA’s test) and secondary mammosphere formation (f, n=6 biological independent experiments, one-way ANOVA’s test) in EMSY-knockdown SUM149 and MDA-MB-453 cells. Scale bar, 200 μm. g. Self-renewal ability determined by primary mammosphere formation and secondary mammosphere formation in EMSY-overexpressing SUM159 (n=6 biological independent experiments, one-way ANOVA’s test). Scale bar, 200 μm. h-l. EMSY-knockdown SUM149 were injected into mammary fat pads with extreme limiting dilutions (5 x 104, 5 x 103 cells/fat pad). Tumor growth curves (n=5/group, two-way ANOVA’s test) (h, i) were shown. Tumor cell ALDH activity was determined by ALDEFLUOR assay and the bar plot was shown as mean ± SEM (n=5/group, one-way ANOVA test) (j). Frequency of breast CSC was calculated based on the positive tumor sites per group by ELDA (ELDA: Limiting Dilution Analysis for stem cell research (wehi.edu.au)) (k, l). Dairy methionine deprivation, alone or in combination with PARPi, as a therapeutic strategy for EMSY-amplification tumors. a. Immunohistochemistry analyses of EMSY expression in specimens of patients and representative images were shown. b. Patient-derived organoid (PDO) models were performed with fresh isolated primary tissues derived from four independent TNBC patients according to the mRNA level of EMSY. PDO was cultured with fresh medium with (Complete) or without methionine (methionine deprivation, MD) and tumor sphere numbers were determined (n=6/group, two-way ANOVA test). c-e. Effect of MD and olaparib on CSCs and tumor progression. Mice bearing patient-derived xenograft model (PDX, FD-009) were treated with PARPi (50mg/kg, i.p., once every day), dairy MD, and their combination. Tumor volume (c), tumor cell ALDH activity (d) and absolute ALDH+ cancer cells in tumor tissue (e) were assessed (n=5/group, one/two-way ANOVA test). f. Single breast cancer cells from the primary tumors were implanted for a second tumor formation in a limited dilution assay (1000 and 10,000 cells/fat pad); frequency of breast CSC was calculated based on the positive tumor sites per group by ELDA (ELDA: Limiting Dilution Analysis for stem cell research (wehi.edu.au)). Citation Format: Cui-Cui Liu, Ke-da Yu. EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-07.

Abstract PO2-23-08: High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative breast cancer

Abstract Background: Hormone receptor-positive and HER2-negative breast cancer, characterized by its significant intratumor heterogeneity (ITH), poses increased recurrence, metastasis risk, and endocrine resistance. A comprehensive understanding of the genomic and biological characteristics underlying ITH is crucial for the development of effective therapeutic strategies. Methods: We leveraged the multiomics data from our large cohort of 381 hormone receptor-positive and HER2-negative breast tumors, supplemented with single-cell sequencing (scRNA-seq) data from 9 individual samples, to investigate ITH and its associated molecular features. Mechanistic investigations were conducted using Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) proteomics, co-immunoprecipitation (Co-IP), Chromatin Immunoprecipitation followed by quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assays. We specifically focused on the role of Proteasome activator subunit beta (PSME2), a potential regulator of drug resistance and metastasis. Results: We quantified the degree of ITH at sample level using multi-omics and scRNA-seq data and observed a correlation between higher ITH levels, increased genomic instability, upregulation of the STING type-I-interferon-dependent pathway, and poor efficacy of endocrine therapy. Both bulk and single-cell data revealed an enrichment of PSME2, associated with type-I interferon, in groups exhibiting high ITH. Patients with elevated PSME2 expression manifested a poorer prognosis and diminished responsiveness to endocrine therapy. Within the context of ITH, we discovered that IFN-I transcriptionally activated PSME2 via STAT1. Further investigation revealed a complex interplay among PSME2, PARP1, and EWS, wherein PSME2-mediated degradation of EWS relieved the transcriptional repression of PARP1, resulting in its overexpression. This series of events, orchestrated by PSME2, led to ERα PARylation and promoted tamoxifen resistance. In subsequent clinical translation studies employing mouse xenograft, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models, we demonstrated that PARP1 inhibitors could effectively mitigate tamoxifen resistance induced by high PSME2 expression, providing new insights into therapeutic strategies. Conclusions: Our study highlights the importance of understanding ITH in hormone receptor-positive and HER2-negative breast cancer and identifies PSME2 as a key player in endocrine resistance and patient prognosis. The application of PARP1 inhibitors shows promise in improving treatment outcomes in tumors with elevated ITH and PSME2 expression. Working Modle Citation Format: Li-Ping Ge, Zi-Yu Wang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-23-08.

Nanodrug modified with engineered cell membrane targets CDKs to activate aPD-L1 immunotherapy against liver metastasis of immune-desert colon cancer

Immunotherapy based on the PD-1/PD-L1 axis blockade has no benefit for patients diagnosed with colon cancer liver metastasis (CCLM) for the microsatellite stable/proficient mismatch repair (MSS/pMMR)) subtype, which is known as an immune-desert cancer featuring poor immunogenicity and insufficient CD8+ T cell infiltration in the tumor microenvironment. Here, a multifunctional nanodrug carrying a cyclin-dependent kinase (CDK)1/2/5/9 inhibitor and PD-L1 antibody is prepared to boost the immune checkpoint blockade (ICB)-based immunotherapy against MSS/pMMR CCLM via reversing the immunosuppressive tumor microenvironment. To enhance the MSS/pMMR CCLM-targeting efficacy, we modify the nanodrug with PD-L1 knockout cell membrane of this colon cancer subtype. First, CDKs inhibitor delivered by nanodrug down-regulates phosphorylated retinoblastoma and phosphorylated RNA polymerase II and meanwhile arrests the G2/M cell cycle in CCLM to promote immunogenic signal release, stimulate dendritic cell maturation, and enhance CD8+ T cell infiltration. Moreover, CDKi suppresses the secretion of immunosuppressive cytokines in tumor-associated myeloid cells sensitizing ICB therapy in CCLM. Notably, the great efficacy to activate immune responses is demonstrated in the patient-derived xenograft model and the patient-derived organoid model as well, revealing a clinical application potential. Overall, our study represents a promising therapeutic approach for targeting liver metastasis, remolding the tumor immune microenvironment (TIME), and enhancing the response of MSS/pMMR CCLM to boost ICB immunotherapy.

The combination of breast cancer PDO and mini-PDX platform for drug screening and individualized treatment.

The majority of advanced breast cancers exhibit strong aggressiveness, heterogeneity, and drug resistance, and currently, the lack of effective treatment strategies is one of the main challenges that cancer research must face. Therefore, developing a feasible preclinical model to explore tailored treatments for refractory breast cancer is urgently needed. We established organoid biobanks from 17 patients with breast cancer and characterized them by immunohistochemistry (IHC) and next generation sequencing (NGS). In addition, we in the first combination of patient-derived organoids (PDOs) with mini-patient-derived xenografts (Mini-PDXs) for the rapid and precise screening of drug sensitivity. We confirmed that breast cancer organoids are a high-fidelity three-dimension (3D) model invitro that recapitulates the original tumour's histological and genetic features. In addition, for a heavily pretreated patient with advanced drug-resistant breast cancer, we combined PDO and Mini-PDX models to identify potentially effective combinations of therapeutic agents for this patient who were alpelisib + fulvestrant. In the drug sensitivity experiment of organoids, we observed changes in the PI3K/AKT/mTOR signalling axis and oestrogen receptor (ER) protein expression levels, which further verified the reliability of the screening results. Our study demonstrates that the PDO combined with mini-PDX model offers a rapid and precise drug screening platform that holds promise for personalized medicine, improving patient outcomes and addressing the urgent need for effective therapies in advanced breast cancer.

Inhibition of the transmembrane transporter ABCB1 overcomes resistance to doxorubicin in patient-derived organoid models of HCC.

Transarterial chemoembolization is the first-line treatment for intermediate-stage HCC. However, the response rate to transarterial chemoembolization varies, and the molecular mechanisms underlying variable responses are poorly understood. Patient-derived hepatocellular carcinoma organoids (HCCOs) offer a novel platform to investigate the molecular mechanisms underlying doxorubicin resistance. We evaluated the effects of hypoxia and doxorubicin on cell viability and cell cycle distribution in 20 patient-derived HCCO lines. The determinants of doxorubicin response were identified by comparing the transcriptomes of sensitive to resistant HCCOs. Candidate genes were validated by pharmacological inhibition. Hypoxia reduced the proliferation of HCCOs and increased the number of cells in the G0/G1 phase of the cell cycle, while decreasing the number in the S phase. The IC50s of the doxorubicin response varied widely, from 29nM to >1µM. Doxorubicin and hypoxia did not exhibit synergistic effects but were additive in some HCCOs. Doxorubicin reduced the number of cells in the G0/G1 and S phases and increased the number in the G2 phase under both normoxia and hypoxia. Genes related to drug metabolism and export, most notably ABCB1, were differentially expressed between doxorubicin-resistant and doxorubicin-sensitive HCCOs. Small molecule inhibition of ABCB1 increased intracellular doxorubicin levels and decreased drug tolerance in resistant HCCOs. The inhibitory effects of doxorubicin treatment and hypoxia on HCCO proliferation are variable, suggesting an important role of tumor-cell intrinsic properties in doxorubicin resistance. ABCB1 is a determinant of doxorubicin response in HCCOs. Combination treatment of doxorubicin and ABCB1 inhibition may increase the response rate to transarterial chemoembolization.

Comparison of Surgical and Colonoscopy Tissue to Establish Colorectal Patient-derived Organoids.

Patient-derived organoids (PDOs) are ex vivo models that retain the functions and characteristics of individualized source tissues, including a simulated tumor microenvironment. However, the potential impact of undiscovered differences between tissue sources on PDO growth and progression remains unclear. This study aimed to compare the growth and condition of PDO models originating from surgical resection and colonoscopy and to provide practical insights for PDO studies. Tissue samples and relevant patient clinical information were collected to establish organoid models. PDOs were derived from both surgical and colonoscopy tissues. The growth of the organoids, including their state, size, and success rate of establishment, was recorded and analyzed. The activity of the organoids at the end stage of growth was detected using calcein-AM fluorescence staining. The results showed that the early growth phase of 2/3 colonoscopy-derived organoids was faster compared to surgical PDOs, with a growth difference observed within 11-13 days of establishment. However, colonoscopy-derived organoids exhibited a diminished growth trend after this time. There were no significant differences observed in the terminal area and quantity between the two types of tissue-derived organoids. Immunofluorescence assays of the PDOs revealed that the surgical PDOs possessed a denser cell mass with relatively higher viability than colonoscopy-derived PDOs. In the establishment of colorectal patient-derived organoids, surgically derived organoids require a slightly longer establishment period, while colonoscopy-derived organoids should be passaged prior to growth inhibition to preserve organoid viability.

Modelling esophageal adenocarcinoma and Barrett's esophagus with patient-derived organoids.

Currently, esophageal adenocarcinoma (EAC) research is hindered by a dearth of adequate models to study this disease. Traditional cell line and genetically engineered mouse models are lacking in biological and physiological significance, whilst the inefficiency of patient-derived xenografts limit their potential applications. This review describes the landscape of EAC research using patient-derived organoids (PDOs). Here, we detail the methods of establishment and optimization of EAC PDO cultures, as well as current and prospective applications of these models. We further highlight a crucial knowledge gap in the mechanisms of EAC transformation from its precursor lesion, Barrett's esophagus (BE). As such, we also describe the culture requirements of BE PDOs and attempts to model tumorigenesis using PDO models.

Targeting the MCP-GPX4/HMGB1 Axis for Effectively Triggering Immunogenic Ferroptosis in Pancreatic Ductal Adenocarcinoma.

Induction of ferroptosis can inhibit cancer cells in vitro, however, the role of ferroptosis in treatment in vivo is controversial. The immunosuppressive cells activated by the ferroptotic tumor cells can promote the growth of residual tumor cells, hindering the application of ferroptosis stimulation in tumor treatment. In this study, a new strategy is aimed to be identified for effectively triggering immunogenic ferroptosis in pancreatic ductal adenocarcinoma (PDAC) and simultaneously stimulating antitumor immune responses. Toward this, several molecular and biochemical experiments are performed using patient-derived organoid models and a KPC mouse model (LSL-KrasG12D /+, LSL-Trp53R172H/+,Pdx-1-Cre). It is observed that the inhibition of macrophage-capping protein (MCP) suppressed the ubiquitin fold modifier (UFM)ylation of pirin (PIR), a newly identified substrate of UFM1, thereby decreasing the transcription of GPX4, a marker of ferroptosis, and promoting the cytoplasmic transportation of HMGB1, a damage-associated molecular pattern. GPX4 deficiency triggered ferroptosis, and the pre-accumulated cytosolic HMGB1 is released rapidly. This altered release pattern of HMGB1 facilitated the pro-inflammatory M1-like polarization of macrophages. Thus, therapeutic inhibition of MCP yielded dual antitumor effects by stimulating ferroptosis and activating antitumor pro-inflammatory M1-like macrophages. The nanosystem developed for specifically silencing MCP is a promising tool for treating PDAC.

Abstract LB450: Mechanisms of acquired resistance to AKT inhibitor capivasertib in AKT1 mutant patient derived breast cancer models

Abstract Capivasertib, a pan-AKT inhibitor, has recently been approved in combination with fulvestrant to treat ER+ HER2- breast cancer patients with one or more of PI3KCA/AKT1/PTEN alterations. However, as with many targeted therapies acquired drug resistance remains a challenge. In this study, we investigated the mechanisms of acquired capivasertib resistance in AKT1 mutant breast cancer models. We established two estrogen receptor positive (ER+) AKT1 E17K mutant breast cancer patient derived organoids (PDOs), that were sensitive to capivasertib in vitro. PDOs were chronically exposed to 1µM capivasertib. PDOs were sequenced (DNA &amp; RNA) at multiple timepoints of treatment (3 days, 4 and 9 weeks after development of resistance). Using CRISPR-Cas9 editing we also engineered CAMA1 (ER+) cells to express AKT1 E17K, and generated derived resistant AKT1 mutant and parental models in response to continuous capivasertib exposure. CAMA1 AKT1 mutant and parental resistant were screened with an siRNA Kinome library supplemented with genes from the PDO RNA-sequencing. Phosphoproteomics was performed on capivasertib resistant E17K models in the presence and absence of capivasertib. PDO models persisting after either 4 or 9 weeks of capivasertib treatment showed no acquisition of genetic driver events, or changes in AKT copy number. Resistant organoids at 9 weeks treatment showed increased expression of ERBB3 (HER3), EGFR3 and LPAR5 as well as increased expression of MAPK signalling components. In siRNA screens on CAMA1 derived resistant models, siRNA targeting ERBB2, CDK4, PLK4 and CHEK1, ESR1 and LPAR5 modulated sensitivity to capivasertib. Analysis of phosphoproteins in capivasertib resistant models revealed reactivation of mTORC1 signalling despite sustained AKT inhibition, with dramatic upregulation of PDK1 and mTORC2. Consistent with the siRNA screens, drug combinations identified fulvestrant as a synergistic agent with capivasertib in the resistant E17K models. Sensitivity to mTOR inhibition was maintained in capivasertib resistant models, with mTOR inhibition re-sensitizing cells to capivasertib. In these models, acquired resistance to capivasertib occurs through upregulation of ER signalling and rewiring of pathway signalling to bypass AKT inhibition and reactivate mTORC1. Targeting these mechanisms could prolong the use of capivasertib, with future work focusing on validating these mechanisms and re-sensitizing drug combinations in other AKT1 mutant models &amp; PDOs. Citation Format: Sarah Mearns, Alex Pearson, Li-Xuan Sim, Rosalind Cutts, Prithika Sritharan, Heena Shah, Aditi Gulati, Stuart Williamson, Simon Barry, Elza De-Bruin, Nicholas C. Turner. Mechanisms of acquired resistance to AKT inhibitor capivasertib in AKT1 mutant patient derived breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB450.

Human patient derived organoids: an emerging precision medicine model for gastrointestinal cancer research.

Gastrointestinal cancers account for approximately one-third of the total global cancer incidence and mortality with a poor prognosis. It is one of the leading causes of cancer-related deaths worldwide. Most of these diseases lack effective treatment, occurring as a result of inappropriate models to develop safe and potent therapies. As a novel preclinical model, tumor patient-derived organoids (PDOs), can be established from patients' tumor tissue and cultured in the laboratory in 3D architectures. This 3D model can not only highly simulate and preserve key biological characteristics of the source tumor tissue in vitro but also reproduce the in vivo tumor microenvironment through co-culture. Our review provided an overview of the different in vitro models in current tumor research, the derivation of cells in PDO models, and the application of PDO model technology in gastrointestinal cancers, particularly the applications in combination with CRISPR/Cas9 gene editing technology, tumor microenvironment simulation, drug screening, drug development, and personalized medicine. It also elucidates the ethical status quo of organoid research and the current challenges encountered in clinical research, and offers a forward-looking assessment of the potential paths for clinical organoid research advancement.

Sialyl-Tn serves as a potential therapeutic target for ovarian cancer

BackgroundOvarian cancer remains the deadliest of the gynecologic cancers in the United States. There have been limited advances in treatment strategies that have seen marked increases in overall survival. Thus, it is essential to continue developing and validating new treatment strategies and markers to identify patients who would benefit from the new strategy. In this report, we sought to further validate applications for a novel humanized anti-Sialyl Tn antibody-drug conjugate (anti-STn-ADC) in ovarian cancer.MethodsWe aimed to further test a humanized anti-STn-ADC in sialyl-Tn (STn) positive and negative ovarian cancer cell line, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. Furthermore, we sought to determine whether serum STn levels would reflect STn positivity in the tumor samples enabling us to identify patients that an anti-STn-ADC strategy would best serve. We developed a custom ELISA with high specificity and sensitivity, that was used to assess whether circulating STn levels would correlate with stage, progression-free survival, overall survival, and its value in augmenting CA-125 as a diagnostic. Lastly, we assessed whether the serum levels reflected what was observed via immunohistochemical analysis in a subset of tumor samples.ResultsOur in vitro experiments further define the specificity of the anti-STn-ADC. The ovarian cancer PDO, and PDX models provide additional support for an anti-STn-ADC-based strategy for targeting ovarian cancer. The custom serum ELISA was informative in potential triaging of patients with elevated levels of STn. However, it was not sensitive enough to add value to existing CA-125 levels for a diagnostic. While the ELISA identified non-serous ovarian tumors with low CA-125 levels, the sample numbers were too small to provide any confidence the STn ELISA would meaningfully add to CA-125 for diagnosis.ConclusionsOur preclinical data support the concept that an anti-STn-ADC may be a viable option for treating patients with elevated STn levels. Moreover, our STn-based ELISA could complement IHC in identifying patients with whom an anti-STn-based strategy might be more effective.

Stem cell dynamics and cellular heterogeneity across lineage subtypes of castrate resistant prostate cancer.

To resist lineage-dependent therapies such as androgen receptor inhibition, prostate luminal epithelial adenocarcinoma cells often adopt a stem-like state resulting in lineage-plasticity and phenotypic heterogeneity. Castrate resistant prostate adenocarcinoma can transition to neuroendocrine and occasionally to amphicrine, co-expressed luminal and neuroendocrine, phenotypes. We developed CRPC patient-derived organoid models that preserve heterogeneity of the originating tumor, including an amphicrine model displaying a range of luminal and neuroendocrine phenotypes. To gain biological insight and to identify potential treatment targets within heterogeneous tumor cell populations, we assessed the lineage hierarchy and molecular characteristics of various CRPC tumor subpopulations. Transcriptionally similar stem/progenitor cells were identified for all lineage populations. Lineage tracing in amphicrine CRPC showed that heterogeneity originated from distinct subclones of infrequent stem/progenitor cells that produced mainly quiescent differentiated amphicrine progeny. By contrast, adenocarcinoma CRPC progeny originated from stem/progenitor cells and self-renewing differentiated luminal cells. NEPC was composed almost exclusively of self-renewing stem/progenitor cells. Amphicrine subpopulations were enriched for secretory luminal, mesenchymal, and enzalutamide treatment persistent signatures that characterize clinical progression. Finally, the amphicrine stem/progenitor subpopulation was specifically depleted with an AURKA inhibitor, which blocked tumor growth. These data illuminate distinct stem cell characteristics for subtype-specific CRPC in addition to demonstrating a context for targeting differentiation-competent prostate stem cells.

A novel dihydroacridine derivative targets epidermal growth factor receptor-expressing cancer cells in vitro and in vivo

A novel dihydroacridine derivative targets epidermal growth factor receptor-expressing cancer cells in vitro and in vivo