Patient-derived Explants Research Articles

Empowering personalized medicine: unleashing the potential of patient-derived explants in clinical practice.

In recent decades, significant progress has been made in understanding the molecular characteristics of cancer and its microenvironment, leading to the development of life-saving treatments. However, patients often experience side effects from standard therapies, highlighting the need for personalized medicine. Personalized medicine aims to customize drug therapy and preventive care based on individual patients' specific requirements. The heterogeneity within tumors and among patients necessitates personalized medicine approaches. Patient-derived organoids (PDOs), xenografts (PDXs), and explants (PDEs) have emerged as valuable models for studying tumor behaviour and drug response. This paper aims to summarize the latest advancements in patient-derived explants, focusing on their potential utility in the clinic. Different methods for culturing PDEs, including the free-floating approach, the grid method, and sponge scaffolds, are discussed. These approaches provide opportunities for long-term viability, oxygen and nutrient supply, and maintenance of tissue integrity. Additionally, various solid tumor models using PDEs are highlighted, together with assays to study PDE viability, characteristics, and response to drug treatment.

Engineered CAR-Tcells targeting the non-functional P2X purinoceptor 7 (P2X7) receptor as a novel treatment for ovarian cancer.

Recent studies have identified expression of the non-functional P2X7 (nfP2X7) receptor on various malignant cells including ovarian cancer, but not on normal cells, which makes it a promising tumour-associated antigen candidate for chimeric antigen receptor (CAR)-T-cell immunotherapies. In this study, we assessed the cytotoxic effects of nfP2X7-CAR-T cells on ovarian cancer using in vitro and in vivo models. We evaluated the effects of nfP2X7-CAR-T cells on ovarian cancer cell lines (SKOV-3, OVCAR3, OVCAR5), normal peritoneal cells (LP-9) and primary serous ovarian cancer cells derived from patient ascites invitro using monolayer and 3D spheroid assays. We also evaluated the effects of nfP2X7-CAR-T cells on patient-derived tissue explants, which recapitulate an intact tumour microenvironment. In addition, we investigated the effect of nfP2X7-CAR-T cells in vivo using the OVCAR-3 xenograft model in NOD-scid IL2Rγnull (NSG) mice. Our study found that nfP2X7-CAR-T cells were cytotoxic and significantly inhibited survival of OVCAR3, OVCAR5 and primary serous ovarian cancer cells compared with un-transduced CD3+ Tcells in vitro. However, no significant effects of nfP2X7-CAR-T cells were observed for SKOV3 or normal peritoneal cells (LP-9) cells with low P2X7 receptor expression. Treatment with nfP2X7-CAR-T cells increased apoptosis compared with un-transduced Tcells in patient-derived explants and correlated with CD3 positivity.Treatment with nfP2X7-CAR-T cells significantly reduced OVCAR3 tumour burden in mice compared with un-transduced CD3 cells for 7-8 weeks. This study demonstrates that nfP2X7-CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer.

Portable NMR for the investigation of models of mammographic density ex vivo: Androgens antagonise the promotional effect of oestrogen

Background: Increased mammographic density (MD) is a strong and independent risk factor for breast cancer. Lifetime oestrogenic exposure is associated with increased MD, however androgenic effects on MD have not been widely investigated. Methods: We studied the effect of 17 β-oestradiol (E2) alone or in combination with an androgen receptor (AR) agonist ( 5 α-dihydrotestosterone [DHT]) or a selective AR modulator (Enobosarm), in modulating MD as measured via single-sided Portable NMR in a patient-derived explant (PDE) model of normal human mammary tissue. Results: We observed an upward trend in explants treated with E2 alone in 3/6 cases, an effect which appeared to be somewhat influenced by menopausal status. Co-treatment of E2 with the AR agonists DHT or Enobosarm however effected a downward trend in regards to MD. E2 significantly upregulated the ER regulated genes (ERGs) CELSR2 and AR, and the AR regulated genes SEC14L2 and GRPC5A, whereas E2 in combination with AR agonist Enobosarm downregulated ERGs SERPINA3, ATP6V1B1, TFF1 and PR regulated gene RANK. Only GREB1 and CLIC6 were upregulated by E2 and downregulated by the combination of E2 with Enobosarm. DHT + E2 exhibited no significant difference in expression of ER, AR or PR genes examined, compared with the effect of E2 alone. A negative trend was observed between ER protein levels and MD increase in PDEs. Since ER protein gets degraded upon hormone activation, this observation supports active ER signalling in the promotion of MD. Conclusions: Collectively, these findings supports the utility of single-sided Portable NMR for the measurement of MD in explanted human mammary tissue to detect MD change in response to exogenous hormone treatments. A mechanism through which activation of AR may reduce MD as a potential mechanism for reducing breast cancer risk associated to high MD is discussed.

Alpelisib decreases nevocytes of congenital melanocytic nevi.

Multiple, large or giant congenital melanocytic nevi (CMN) are uncommon and affected patients can show progressive growth and thickening, associate neurocutaneous melanocytosis or develop melanoma. Current treatment modalities are mostly complex surgeries that frequently do not solve the disease and its risks completely. Thus, investigation on new treatment options for CMN and its complications must continue. MAPK pathway inhibitors are being investigated, also targeting PI3K-AKT. Omipalisib (PI3K inhibitor, with no indications approved yet) has been studied for CMN invitro and in mice with promising results. However, alpelisib, a PI3K inhibitor approved with an adequate safety profile for patients with severe manifestations of PROS (PIK3CA-Related Overgrowth Spectrum), had not yet been tested for CMN. To evaluate the effect of alpelisib in nevocytes of congenital melanocytic nevi. Nevomelanocytic tissue samples of 10 patients were collected prospectively and, following a previously reported preclinical exvivo model, explants were placed in organotypic culture for 5 days, with or without alpelisib. Consecutively, tissue sections were stained and using scanned images with Qupath and ImageJ softwares, representative regions from the dermis were analysed (using Wilcoxon test and Spearman's correlation). When comparing alpelisib-treated explants with respect to control explants, we found a decrease in cell density (p = 0.0273), in density of SOX10+-cells (p = 0.0391) and also in the % of S-100+ area (p = 0.0078), in alpelisib samples. The three markers showed a positive correlation (p < 0.05). This study provides first-time evidence that alpelisib induces nevocyte reduction in CMN from patient-derived explants, probably inducted by autophagy. Alpelisib is an approved drug with an adequate safety profile used in another mosaicism affecting PI3K (PROS). Further studies are needed to evaluate its efficacy in treating CMN and potentially, their complications, either with local or systemic administration, alone or in combination.

Abstract C034: Understanding the role of PARP activity in prostate cancer

Abstract Prostate cancer (PCa) is the second most fatal cancer in men in the United States. African American men have an incidence and death rate two times higher than that of European American men. The androgen receptor (AR) is the primary pro-oncogenic transcription factor responsible for PCa emergence and progression. AR directed therapies including androgen deprivation (ADT), direct receptor antagonism and inhibition of androgen synthesis are the current standard of care for advanced PCa. Adaptive resistance to anti-androgen therapy can occur resulting in castration-resistant prostate cancer (mCRPC). mCRPC remains universally fatal with no curative treatment options, which highlights the need for new treatment regimes. Poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors are approved to treat mCRPC tumors that have homologous recombination (HR) mutations. PARP-1 is an enzyme that recruits DNA damage repair enzymes. PARP-1 enzymatic activity is elevated in mCRPC patient tumors compared to primary tumors. PARP1 enzymatic activity has been implicated in driving pro-survival mechanisms through recruitment of DNA-damage repair enzymes and regulation of oncogenic transcription factors, including AR. Although increased PARP activity has been associated with driving cancer cell survival, the mechanisms and consequences of elevated PARP activity in PCa remains an area to be further explored. The biologically relevant doses of five PARPi were established in regular and steroid depleted growth conditions in CRPC models. PARP enzymatic activity was assessed by immunoblotting for changes in PARlyation, revealing that PARP protein remains stable, while enzymatic activity is attenuated upon challenge with PARPi. Transcriptomic analysis will assess changes in gene expression programs that are associated with inhibition of PARP enzymatic activity as it relates to key oncogenic pathways in PCa. It will be imperative to gain a deeper understanding of how PARP-1 manipulation modulates pro-oncogenic transcriptional networks to better inform clinical response. Patient derived explants (PDEs) from patients with distinct racial backgrounds will be used to examine differential PARPi responses as it relates to race. This will serve to unravel the mechanisms attributed to the increased lethality of PCa observed in African American populations. Collectively, these studies and future studies for this project will further establish the impact of PARP on PCa gene expression programs and provide resources for future studies exploring PARP in PCa. Further, they may help elucidate mechanisms to address PCa racial disparities. Citation Format: Moriah Cunningham, Latese Evans, Salome Tchotorlishvili, Jasibel Vasquez Gonzalez, Candice Bizzaro, Tessa Mulders, Matthew Schiewer. Understanding the role of PARP activity in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C034.

Selective inhibition of CDK9 in triple negative breast cancer

Targeted therapy for triple-negative breast cancers (TNBC) remains a clinical challenge due to tumour heterogeneity. Since TNBC have key features of transcriptionally addicted cancers, targeting transcription via regulators such as cyclin-dependent kinase 9 (CDK9) has potential as a therapeutic strategy. Herein, we preclinically tested a new selective CDK9 inhibitor (CDDD11-8) in TNBC using cell line, patient-derived organoid, and patient-derived explant models. In vitro, CDDD11-8 dose-dependently inhibited proliferation (IC50 range: 281–734 nM), induced cell cycle arrest, and increased apoptosis of cell lines, which encompassed the three major molecular subtypes of TNBC. On target inhibition of CDK9 activity was demonstrated by reduced RNAPII phosphorylation at a CDK9 target peptide and down-regulation of the MYC and MCL1 oncogenes at the mRNA and protein levels in all cell line models. Drug induced RNAPII pausing was evident at gene promoters, with strongest pausing at MYC target genes. Growth of five distinct patient-derived organoid models was dose-dependently inhibited by CDDD11-8 (IC50 range: 272–771 nM), including three derived from MYC amplified, chemo-resistant TNBC metastatic lesions. Orally administered CDDD11-8 also inhibited growth of mammary intraductal TNBC xenograft tumours with no overt toxicity in vivo (mice) or ex vivo (human breast tissues). In conclusion, our studies indicate that CDK9 is a viable therapeutic target in TNBC and that CDDD11-8, a novel selective CDK9 inhibitor, has efficacy in TNBC without apparent toxicity to normal tissues.

Use of patient-derived explants as a preclinical model for precision medicine in colorectal cancer: A scoping review

PurposeWhilst the treatment paradigm for colorectal cancer has evolved significantly over time, there is still a lack of reliable biomarkers of treatment response. Treatment decisions are based on high-risk features such as advanced TNM stage and histology. The role of the tumour microenvironment, which can influence tumour progression and treatment response, has generated considerable interest. Patient-derived explant cultures allow preservation of native tissue architecture and tumour microenvironment. The aim of the scoping review is to evaluate the utility of patient-derived explant cultures as a preclinical model in colorectal cancer.MethodsA search was conducted using Ovid MEDLINE, EMBASE, Web of Science, and Cochrane databases from start of database records to September 1, 2022. We included all peer-reviewed human studies in English language which used patient-derived explants as a preclinical model in primary colorectal cancer. Eligible studies were grouped into the following categories: assessing model feasibility; exploring tumour microenvironment; assessing ex vivo drug responses; discovering and validating biomarkers.ResultsA total of 60 studies were eligible. Fourteen studies demonstrated feasibility of using patient-derived explants as a preclinical model. Ten studies explored the tumour microenvironment. Thirty-eight studies assessed ex vivo drug responses of chemotherapy agents and targeted therapies. Twenty-four studies identified potential biomarkers of treatment response.ConclusionsGiven the preservation of tumour microenvironment and tumour heterogeneity, patient-derived explants has the potential to identify reliable biomarkers, treatment resistance mechanisms, and novel therapeutic agents. Further validation studies are required to characterise, refine and standardise this preclinical model before it can become a part of precision medicine in colorectal cancer.

Pharmacological inhibition of KDM1A/LSD1 enhances estrogen receptor beta-mediated tumor suppression in ovarian cancer

Ovarian cancer (OCa) is the most lethal gynecologic cancer. Emerging data indicates that estrogen receptor beta (ERβ) functions as a tumor suppressor in OCa. Lysine-specific histone demethylase 1A (KDM1A) is an epigenetic modifier that acts as a coregulator for steroid hormone receptors. However, it remain unknown if KDM1A interacts with ERβ and regulates its expression/functions in OCa. Analysis of TCGA data sets indicated KDM1A and ERβ expression showed an inverse relationship in OCa. Knockout (KO), knockdown (KD), or inhibition of KDM1A increased ERβ isoform 1 expression in established and patient-derived OCa cells. Further, KDM1A interacts with and functions as a corepressor of ERβ, and its inhibition enhances ERβ target gene expression via alterations of histone methylation marks at their promoters. Importantly, KDM1A-KO or -KD enhanced the efficacy of ERβ agonist LY500307, and the combination of KDM1A inhibitor (KDM1Ai) NCD38 with ERβ agonist synergistically reduced the cell viability, colony formation, and invasion of OCa cells. RNA-seq and DIA mass spectrometry analyses showed that KDM1A-KO resulted in enhanced ERβ signaling and that genes altered by KDM1A-KO and ERβ agonist were related to apoptosis, cell cycle, and EMT. Moreover, combination treatment significantly reduced the tumor growth in OCa orthotopic, syngeneic, and patient-derived xenograft models and proliferation in patient-derived explant models. Our results demonstrate that KDM1A regulates ERβ expression/functions, and its inhibition improves ERβ mediated tumor suppression. Overall, our findings suggest that KDM1Ai and ERβ agonist combination therapy is a promising strategy for OCa.

Targeting hyaluronan-mediated motility receptor (HMMR) enhances response to androgen receptor signalling inhibitors in prostate cancer.

Resistance to androgen receptor signalling inhibitors (ARSIs) represents a major clinical challenge in prostate cancer. We previously demonstrated that the ARSI enzalutamide inhibits only a subset of all AR-regulated genes, and hypothesise that the unaffected gene networks represent potential targets for therapeutic intervention. This study identified the hyaluronan-mediated motility receptor (HMMR) as a survival factor in prostate cancer and investigated its potential as a co-target for overcoming resistance to ARSIs. RNA-seq, RT-qPCR and Western Blot were used to evaluate the regulation of HMMR by AR and ARSIs. HMMR inhibition was achieved via siRNA knockdown or pharmacological inhibition using 4-methylumbelliferone (4-MU) in prostate cancer cell lines, a mouse xenograft model and patient-derived explants (PDEs). HMMR was an AR-regulated factor that was unaffected by ARSIs. Genetic (siRNA) or pharmacological (4-MU) inhibition of HMMR significantly suppressed growth and induced apoptosis in hormone-sensitive and enzalutamide-resistant models of prostate cancer. Mechanistically, 4-MU inhibited AR nuclear translocation, AR protein expression and subsequent downstream AR signalling. 4-MU enhanced the growth-suppressive effects of 3 different ARSIs in vitro and, in combination with enzalutamide, restricted proliferation of prostate cancer cells in vivo and in PDEs. Co-targeting HMMR and AR represents an effective strategy for improving response to ARSIs.

Ultrastructural analysis of prostate cancer tissue provides insights into androgen-dependent adaptations to membrane contact site establishment.

Membrane trafficking and organelle contact sites are important for regulating cell metabolism and survival; processes often deregulated in cancer. Prostate cancer is the second leading cause of cancer-related death in men in the developed world. While early-stage disease is curable by surgery or radiotherapy there is an unmet need to identify prognostic biomarkers, markers to treatment response and new therapeutic targets in intermediate-late stage disease. This study explored the morphology of organelles and membrane contact sites in tumor tissue from normal, low and intermediate histological grade groups. The morphology of organelles in secretory prostate epithelial cells; including Golgi apparatus, ER, lysosomes; was similar in prostate tissue samples across a range of Gleason scores. Mitochondrial morphology was not dramatically altered, but the number of membrane contacts with the ER notably increased with disease progression. A three-fold increase of tight mitochondria-ER membrane contact sites was observed in the intermediate Gleason score group compared to normal tissue. To investigate whether these changes were concurrent with an increased androgen signaling in the tissue, we investigated whether an anti-androgen used in the clinic to treat advanced prostate cancer (enzalutamide) could reverse the phenotype. Patient-derived explant tissues with an intermediate Gleason score were cultured ex vivo in the presence or absence of enzalutamide and the number of ER-mitochondria contacts were quantified for each matched pair of tissues. Enzalutamide treated tissue showed a significant reduction in the number and length of mitochondria-ER contact sites, suggesting a novel androgen-dependent regulation of these membrane contact sites. This study provides evidence for the first time that prostate epithelial cells undergo adaptations in membrane contact sites between mitochondria and the ER during prostate cancer progression. These adaptations are androgen-dependent and provide evidence for a novel hormone-regulated mechanism that support establishment and extension of MAMs. Future studies will determine whether these changes are required to maintain pro-proliferative signaling and metabolic changes that support prostate cancer cell viability.

Critical role of antioxidant programs in enzalutamide-resistant prostate cancer.

Therapy resistance to second-generation androgen receptor (AR) antagonists, such as enzalutamide, is common in patients with advanced prostate cancer (PCa). To understand the metabolic alterations involved in enzalutamide resistance, we performed metabolomic, transcriptomic, and cistromic analyses of enzalutamide-sensitive and -resistant PCa cells, xenografts, patient-derived organoids, patient-derived explants, and tumors. We noted dramatically higher basal and inducible levels of reactive oxygen species (ROS) in enzalutamide-resistant PCa and castration-resistant PCa (CRPC), in comparison to enzalutamide-sensitive PCa cells or primary therapy-naive tumors respectively. Unbiased metabolomic evaluation identified that glutamine metabolism was consistently upregulated in enzalutamide-resistant PCa cells and CRPC tumors. Stable isotope tracing studies suggest that this enhanced glutamine metabolism drives an antioxidant program that allows these cells to tolerate higher basal levels of ROS. Inhibition of glutamine metabolism with either a small-molecule glutaminase inhibitor or genetic knockout of glutaminase enhanced ROS levels, and blocked the growth of enzalutamide-resistant PCa. The critical role of compensatory antioxidant pathways in maintaining enzalutamide-resistant PCa cells was validated by targeting another antioxidant program driver, ferredoxin 1. Taken together, our data identify a metabolic need to maintain antioxidant programs and a potentially targetable metabolic vulnerability in enzalutamide-resistant PCa.

Abstract B059: Understanding the role of poly (ADP-ribose) polymerase 1 (PARP-1) activity in prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed cancer in men in the United States. African American men have two times higher incidence rate and death rate from PCa than European American men. In PCa, the androgen receptor (AR) acts as a pro-oncogenic transcription factor. Androgen deprivation therapy (ADT) and AR antagonists are the current standard of care for advanced PCa. Late stage of disease is recognized as metastatic castrate resistant prostate cancer (mCRPC) due to adaptive resistance to ADT. mCRPC remains universally fatal with no durable therapeutic options. Poly (ADP-ribose) polymerase 1 (PARP-1) has been identified as a driver of cancer cell survival through the recruitment of DNA-damage-repair enzymes and regulation of oncogenic transcription factors, such as AR, in PCa. PARP-1 enzymatic activity is elevated in mCRPC patient tumors as compared to primary tumors. Although increased PARP activity in these tumors has been shown to be a driver of cancer cell survival, the mechanisms and consequences of elevated PARP activity in PCa remains an active research program in our lab, especially regarding PARP-responsive transcriptional networks. PARP inhibitors (PARPi) have been approved for use in mCRPC with alterations in homologous recombination (HR) genes. The experiments below are designed to better understand the mechanisms by which PARP enzymes may transcriptionally regulate and promote pro-tumorigenic functions. Five different PARP inhibitors were analyzed in a CRPC cell line model to assess their relative potencies and develop IC50 values. A cell growth assay using trypan blue exclusion was used to compare the effectiveness of this IC50 value in regular growth conditions as it compares to steroid depleted growth conditions used to mimic ADT. Immunoblot analyses show that there is differential impact on PARP activity (PARylation) in response to the PARPi tested at the respective IC50 concentrations. It will be imperative to better understand how PARP-1 manipulation regulates gene expression. Tissue microarray (TMA) data was analyzed to assess the level of PARP-1, PAR &amp; gH2AX levels in tissue derived from African American versus European American patients. Although not statistically significant, higher PARylation levels were present in African American TMAs compared to European American prostate tumors. It is essential to obtain TMAs representing a more diverse patient demographic to further assess this difference in PARylation levels As well as TMA data, Patient-Derived Explants (PDEs) from patients with different racial backgrounds were assessed for differential impact of PARPi based on race. Collectively, these studies and future studies for this project will further establish the impact of PARP on PCa gene expression programs and provide resources for future studies exploring PARP in PCa. Further, they may help elucidate mechanisms to address PCa racial disparities. Citation Format: Moriah Cunningham, Latese Evans, Salome Tchotorlishvili, Jasibel Vasquez Gonzalez, Candice Bizzaro, Matthew Schiewer. Understanding the role of poly (ADP-ribose) polymerase 1 (PARP-1) activity in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B059.

Making In Vitro Tumor Models Whole Again (Adv. Healthcare Mater. 14/2023)

In Vitro Tumor Models The success of personalized cancer therapy hinges on a timely clinical decision and a preclinical model that can recapitulate tumor and stromal heterogeneity for clinically relevant drug profiling. In article 2202279, Eliza Li Shan Fong and co-workers review how by transcending the existential reductionist approaches, bioengineered patient-derived explant (PDE) models can truly replicate the tumor features in vivo and allow clinicians to rapidly offer and strategize specific care and treatment for each cancer patient.

Comparative assessment of primary cancer cell culture techniques and cellular composition analysis in non-small cell lung cancer

Preclinical models are required to study individual therapy responses to improve all cancer treatments, particularly non-small cell lung cancer (NSCLC). Patient-derived explants (PDEs) culture model is of great importance in terms of the possibility of tumor cell culture with the microenvironment, and the development of molecular mechanisms and personalized treatment methods. In our study, primary tumor culture with microenvironment was performed using different methods from tumor tissues obtained from 51 patients with NSCLC. To identify the most efficient method, mechanical, enzymatic, and tumor fluid techniques were applied. While the malignant cell rate was > 95% in 3 of these cases, the cancer-associated fibroblasts (CAF) microenvironment was high in 46 (80–94%) and low in 2 (1–79%). Subtyping of cells obtained from culture was performed using the light microscope and, if necessary, additional immunohistochemical markers. Consequently, using different techniques, here we successfully performed primary cell culture from patients with NSCLC with microenvironment. Depending on the cell type and culture conditions, proliferation rate was shown to be altered.

Recent and advanced therapy for oral cancer.

Oral cancer is a common and deadly kind of tissue invasion, has a high death rate, and may induce metastasis that mostly affects adults over the age of 40. Most in vitro traditional methods for studying cancer have included the use of monolayer cell cultures and several animal models. There is a worldwide effort underway to reduce the excessive use of laboratory animals since, although being physiologically adequate, animal models rarely succeed in exactly mimicking human models. 3D culture models have gained great attention in the area of biomedicine because of their capacity to replicate parent tissue. There are many benefits to using a drug delivery approach based on nanoparticles in cancer treatment. Because of this, in vitro test methodologies are crucial for evaluating the efficacy of prospective novel nanoparticle drug delivery systems. This review discusses current advances in the utility of 3D cell culture models including multicellular spheroids, patient-derived explant cultures, organoids, xenografts, 3D bioprinting, and organoid-on-a-chip models. Aspects of nanoparticle-based drug discovery that have utilized 2D and 3D cultures for a better understanding of genes implicated in oral cancers are also included in this review.

Abstract 3440: Targeting the mutant estrogen receptor in metastatic breast cancer

Abstract Introduction: Estrogen receptor gene (ESR1) drives approximately 70-80% of breast cancer cases, which are primarily treated with endocrine therapies. Mutations in the ESR1 exclusively occur in the ligand binding domain (LBD) and are significantly enriched following endocrine therapy. These mutations retain the estrogen receptor (ER) expression thus conferring ligand-independent growth, which is a paramount contributor to endocrine therapy resistant disease. Worse progression-free survival is consistently associated with ESR1 mutation status; therefore, there is an unmet need for therapies against these mutations. To this end, we recently developed TX-245, a bis-benzamide molecule that hampers the function of the two most common ESR1 mutations - Y537S and D538G. Methods: TX-245 was evaluated in vitro using MTT cell proliferation assay and colony formation assay carried out in multiple ER+ breast cancer cells harboring wild type (WT) ESR1 or mutant (MT) ESR1. Approaches such as tumor spheroid cultures were also implemented to study the effect of TX-245 compared to selected SERDs/SERMs via CellTiter-Glo 3D viability assay. Mechanistic insights were obtained performing TR-FRET assay, fluorescence polarization assay, RNA-seq, ChIP-seq, western blot, and qRT-PCR. The therapeutic efficacy in vivo was evaluated using cell xenografts, patient derived xenografts (PDX), patient derived explants (PDE), and metastatic mouse models of MT-ER breast cancer. Results: TX-245 demonstrated greater anti-proliferative dose-dependent activity in models harboring WT and MT ESR1, including those resistant to fulvestrant and tamoxifen, when compared to other SERDs/SERMs. RNAseq data suggests that TX-245 dramatically alters the transcription of ERα-regulated genes, with repression of canonical estradiol-upregulated and induction of estradiol-repressed genes. TX-245 was able to antagonize ER-chromatin interaction and ER-transcriptional signaling in breast cancer cells driven by ESR1 mutation. TX-245 treatment downregulated ER protein expression and decreased expression of ER targeted genes (i.e., GREB and PGR) in multiple ER+ cell lines. TX-245 treatment as a single agent or in combination with palbociclib resulted in tumor regression in xenografts expressing WT, Y537S or D538G ESR1 mutation. PDX and PDE models consistently exhibited substantial tumor growth inhibition as well as decreased proliferation (Ki67 staining). Metastases studies using intracardiac and intratibial injection demonstrated that TX-245 inhibited the progression of established metastatic niches in mice injected with Y537S or D538G ER+ breast cancer cells. Conclusion: Our results indicate that TX-245 inhibits cell proliferation in cell lines that express WT or mutant ESR1, induce ER degradation, suppress ER-mediated signaling and reduce tumor burden in mouse models. These studies highlight the utility of TX-245 in targeting mutations in the ER-LBD. Citation Format: Karla Parra, Suryavathi Viswanadhapalli, Tanner Reese, Xihui Liu, JunHao Liu, Zexuan Liu, Tae-Kyung Lee, Chia Yuan Chen, Kara Kassees, Jung-Mo Ahn, Ratna Vadlamudi, Ganesh Raj, Carlos Roggero. Targeting the mutant estrogen receptor in metastatic breast cancer [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 3440.

Abstract 3081: KDM1A/LSD1 inhibition enhances estrogen receptor beta mediated tumor suppression in ovarian cancer

Abstract Introduction: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers. Recent studies suggest that OCa cells express estrogen receptor beta (ERb), which functions as a tumor suppressor. However, ERb expression decreases during tumor progression and under the selection pressure of chemotherapy; this decrease occurs via epigenetic mechanisms and is inversely correlated with OCa progression. Lysine-specific histone demethylase 1A (KDM1A/LSD1), an epigenetic modifier, is highly overexpressed in OCa. However, it is not known if KDM1A regulates ERb expression and its corresponding tumor suppressor functions. In this study, we explored the hypotheses that KDM1A inhibits ERb expression and function and that ERb-mediated tumor suppression in OCa is enhanced by KDM1A inhibition. Methods: Transduction of KDM1A-specific shRNA or gRNA was used to generate KDM1A knockdown (KD) or knockout (KO) cells, respectively. Cell viability, survival, apoptosis, and invasion assays were used to determine the impact of KDM1A knockdown or inhibitor therapy on ERb agonist response in established and patient-derived OCa cells. Mechanistic studies were conducted using RNA-seq, discovery proteomics, chromatin immunoprecipitation (ChIP), co-immunoprecipitation (co-IP), proximity ligation, ERE-Luc reporter, RT-qPCR, and western blot analysis. The efficacy of the combination of KDM1A inhibitor and ERb agonist therapy was investigated using patient-derived explant (PDeX) models. In vivo efficacy of KDM1A inhibitor and ERb agonist was determined using orthotopic OCa and patient-derived xenograft (PDX) murine models. Results: Analysis of TCGA datasets revealed a negative correlation between ERb and KDM1A expression in OCa patients. ChIP experiments showed that KDM1A is enriched at the ERb 0N promoter and that KDM1A KD, KO, or inhibition specifically upregulates the expression of ERb isoform 1. Co-IP and proximity ligation assays demonstrated that KDM1A interacts with ERb. Combining the ERb agonist LY500307 with KDM1A inhibitor NCD38 decreased the cell viability, survival, and invasion of both established and patient-derived primary OCa cells while increasing apoptosis. KDM1A KD- or inhibition potentiated the efficacy of ERb agonist in inducing ERb target gene expression via altered histone methylation marks. RNA-seq and proteomic analysis demonstrated that KDM1A KO and ERb agonist treatment increased the expression of apoptotic genes and downregulated cell cycle, EMT, and DNA repair genes. Importantly, combination therapy significantly reduced OCa cell proliferation in PDeX models and reduced in vivo tumor growth in orthotopic and PDX models. Conclusions: Our findings demonstrate that KDM1A inhibition enhances ERb expression and tumor suppressive functions, suggesting that combination therapy of a KDM1A inhibitor and ERb agonist may be a promising therapeutic option for treating OCa. Citation Format: Prabhakar P. Venkata, Sridharan Jayamohan, Yi He, Salvador Alejo, Jessica D. Johnson, Uday P. Pratap, Suryavathi Viswanadhapalli, Susan Weintraub, Rajeshwar R. Tekmal, Ratna K. Vadlamudi, Edward Kost, Gangadhara R. Sareddy. KDM1A/LSD1 inhibition enhances estrogen receptor beta mediated tumor suppression in ovarian cancer [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 3081.

Abstract 4122: TIL-containing patient-derived explant cultures reveal role of metformin on antigen presenting cell activation

Abstract Globally, breast cancer is among the most diagnosed cancer types for women. Current and upcoming breast cancer therapies are being investigated in combination with compounds that stimulate an immune response, but whether the therapeutic agents themselves have unexpected immunomodulatory effects is often overlooked. Here, we have developed a method to grow 3D cultures of intact fragments of patient-derived tissue (Patient-Derived Explant Cultures; PDECs) to assess the preclinical potential of studying human tumor cells and immune cells simultaneously ex vivo Single cell sequencing, flow cytometry, gene expression profiling and cytokine profiling data show that the tumor immunocontexture is conserved in PDECs and that these resident immune cells respond to distinct immune stimulus We performed gene expression profiling, flow cytometry, and cytokine profiling of drug-treated human explants and found that metformin has antitumor potential through the activation of antigen presenting cells. We further validated in vitro that metformin-mediated APC activation is largely through mitochondrial respiration inhibition irrespective of the presence of tumor cells. Our PDEC platform highlights the preclinical potential of ex vivo explants by simultaneously offering information of tumor and immune cell toxicity and mechanism. Citation Format: Rita J. Turpin, Ruixian Liu, Pauliina Munne, Aino Peura, Jenna Rannikko, Gino Philips, Natasha Salmelin, Elina Hurskainen, Ilida Suleymanova, Minna Mutka, Tuomo Meretoja, Johanna Mattson, Satu Mustjoki, Päivi Saavalainen, Diether Lambrechts, Jeroen Pouwels, Maija Hollmén, Juha Klefström. TIL-containing patient-derived explant cultures reveal role of metformin on antigen presenting cell activation. [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 4122.

Breast cancer patient-derived explant cultures recapitulate in vivo drug responses.

Assessment of drug sensitivity in tumor tissue ex vivo may significantly contribute to functional diagnostics to guide personalized treatment of cancer. Tumor organoid- and explant-cultures have become attractive tools towards this goal, although culturing conditions for breast cancer (BC) tissue have been among the most challenging to develop. Validation of possibilities to detect concordant responses in individual tumors and their respective cultures ex vivo is still needed. Here we employed BC patient-derived xenografts (PDXs) with distinct drug sensitivity, to evaluate different conditions for tissue dissociation, culturing and monitoring of treatment efficacy ex vivo, aiming to recapitulate the in vivo drug responses. The common challenge of discriminating between tumor and normal cells in the cultured tissue was also addressed. Following conventional enzymatic dissociation of BC tissue, the tumor cells stayed within the non-disrupted tissue fragments, while the single cells represented mostly normal host cells. By culturing such fragments as explants, viable tumor tissue could be maintained and treated ex vivo, providing representative indications on efficacy of the tested treatment. Thus, drug sensitivity profiles, including acquired chemoresistance seen in the PDXs, were recapitulated in the respective explants. To detect the concordant responses, however, the effect monitoring had to be harmonized with the characteristics of the cultured tissue. In conclusion, we present the feasibility of BC explants ex vivo to capture differences in drug sensitivity of individual tumors. The established protocols will aid in setting up an analogous platform for BC patient biopsies with the aim to facilitate functional precision medicine.

P089 Colonic explant lactate concentration and inflammatory protein secretion in ulcerative colitis

Abstract Background Lactate, previously thought of as a waste product of cellular metabolism, is now known to act as an immunoregulator with pleiotropic effects. In murine models lactate has been shown to suppress colonic inflammation &amp; restore colonic homeostasis. We aimed to investigate if lactate concentration in tissue cultured media (TCM) from ulcerative colitis (UC) patient-derived explants is associated with TCM concentration of inflammatory proteins known to be involved in UC pathogenesis. Methods UC patients were prospectively recruited in 2 cohorts: discovery (2018 – 2019) and validation (2020 - 2021). Endoscopic biopsies were collected from the sigmoid colon and TCM generated as per previously described methods. Total protein content of biopsies was quantified to allow normalisation of secretions. Patients demographics, baseline characteristics and disease behaviour were characterised. TCM secreted lactate was quantified using a colorimetric L-Lactate assay (Abcam, UK). Secreted inflammatory protein profiles were analysed via 54 V-plex ELISA (Meso Scale Diagnostics, USA). Results 52 patients were recruited (discovery cohort n=28, replication cohort n=24), baseline characteristics are described in Figure 1. Median follow up time was significantly longer in discovery compared with validation cohort p=0.02. In the discovery cohort TCM lactate concentration was inversely correlated with CRP and CRP/Albumin ratio, however, this correlation was not replicated in the validation cohort (Figure 2). Baseline explant TCM lactate concentration did not correlate with TCM CRP concentration. Baseline explant TCM lactate concentration was correlated with IL-2 and IL-31 concentration in both discovery and validation cohorts (Figures 3 &amp; 4). Multivariate analysis, including prior biologic exposure, clinical and endoscopic Mayo scores demonstrated TCM lactate concentration to be independently associated with IL-2 concentration in discovery and validation cohorts, p&amp;lt;0.001 respectively. A similar multivariate analysis demonstrated TCM lactate concentration to be independently associated with IL-31 concentration in the discovery (p&amp;lt;0.001), however, not validation cohort. Conclusion TCM lactate concentration is directly correlated with IL-2 &amp; IL-31 in UC explants. The proposed immunoregulatory properties of lactate are in keeping with its positive correlation with TCM IL-2 concentration given reduced IL-2 secretion has previously been associated with UC disease progression. Further studies are required to define the effects of lactate in the UC tissue microenvironment.