Organoid Viability Research Articles

ATF6 Promotes Colorectal Cancer Growth and Stemness by Regulating the Wnt Pathway.

ATF6 intervention reduces colorectal cancer cell and organoid viability by interrupting dysregulated Wnt signaling, identifying a novel facilitator and potential therapeutic target in colorectal cancer.

Cryopreservation of human kidney organoids

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1’s efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.

Plexin D1 emerges as a novel target in the development of neural lineage plasticity in treatment-resistant prostate cancer.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) often arises from adenocarcinoma via lineage plasticity in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the specific regulators and targets involved in the transition to NEPC are not well understood. Plexin D1 (PLXND1) is a cellular receptor of the semaphorin (SEMA) family that plays important roles in modulating the cytoskeleton and cell adhesion. Here, we found that PLXND1 was highly expressed and positively correlated with neuroendocrine markers in patients with NEPC. High PLXND1 expression was associated with poorer prognosis in prostate cancer patients. Additionally, PLXND1 was upregulated and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells. Knockdown or knockout of PLXND1 inhibited neural lineage pathways, thereby suppressing NEPC cell proliferation, patient derived xenograft (PDX) tumor organoid viability, and xenograft tumor growth. Mechanistically, the heat shock protein 70 (HSP70) regulated PLXND1 protein stability through degradation, and inhibition of HSP70 decreased PLXND1 expression and NEPC organoid growth. In summary, our findings indicate that PLXND1 could serve as a promising therapeutic target and molecular marker for NEPC.

Enhancing non-small cell lung cancer treatment utilizing natural killer cell-derived extracellular vesicles (NKEVs) as a novel adoptive cellular therapeutic.

e20079 Background: An established mechanism contributing to immune checkpoint inhibitor (ICI) therapy failure is tumor evasion of T-cell responses via downregulation of human leukocyte antigen (HLA). Conversely, the effector function of Natural Killer (NK) cells is enhanced in the absence of HLA expression, making NK-based cellular therapies an attractive option for ICI resistant tumors. NK-derived extracellular vesicles (NKEVs) have the potential to become a personalized adoptive cellular therapeutic that may overcome challenges associated with traditional NK cell-based therapies failing to deliver adequate numbers of cells to the tumor. We sought to investigate the preclinical efficacy of an approach using selective isolation of NK cells and harvesting of NKEVs in NSCLC. Methods: From 10 NSCLC patients, tumor tissue was collected, and blood mononuclear cells (PBMCs) were retrieved pre- and -post surgery. Single-cell RNAseq (scRNAseq) was used to examine the cellular landscape in PBMCs and tumor tissue. NK cells were isolated from PBMCs, expanded in vitro, and NK-derived EVs (NKEV) were collected and the EV RNA and protein cargo was characterized using proteomics and transcriptomics. The functional capabilities of patient derived NKEVs were assayed with patient derived organoids. Results: scRNAseq highlighted significant cellular heterogeneity, particularly in the cancer cell population. Within the tumor, infiltrating immune cells were lymphoid and myeloid in origin. Over 200,000 PBMCs were analyzed with scRNAseq, revealing differences associated with cancer subtype, tumor mutation burden and experimental time point. Exposure to patient NKEV treatment resulted in a 40-45% decrease in organoid viability, significantly lowering the cisplatin dose required to elicit a cytotoxic response. NKEVs derived from healthy controls were less efficient. In Nivolumab-treated PBMC organoid co-culture experiments, NKEV addition increased the proportion of infiltrating CD8+ T cells and CD56+ NK cells compared to PD-1 inhibitors alone, particularly from the pre-tumor resection PBMCs. Conclusions: This work demonstrated that NKEVs can be successfully harvested from patient derived, expanded NK cells, and highlighted their anti-tumor properties in combination with standard-of-care therapies. Additional in-depth immune analysis will be presented. [Table: see text]

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

IntroductionAppendiceal 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.Patients and MethodsPTOs 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.ResultsFrom 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.ConclusionsBromelain 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.

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.

Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform.

Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD. We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days. Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results. Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.

An in vitro three-dimensional (3D) testicular organoid culture system for efficient gonocyte maintenance and propagation using frozen/thawed neonatal bovine testicular tissues

Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of an in vitro culture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for the in vitro maintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 106 cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 μm; the mean number of organoids was 5.57 ± 1.60/105 μm2 on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression of C-KIT but reduced the expression of GFRα1 at day 28 when compared to those without hormone supplements (p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishing in vitro spermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.

Abstract B048: Targeting PPARG to block obesity-associated pancreatic cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer-related death in the United States and one of the few cancers for which incidence is increasing. This trend in increased incidence has been attributed in part to obesity. Obesity is associated with increased risk of pancreatic cancer and advanced disease stage in patients, concordant with rodent studies showing that obesity accelerates tumorigenesis and metastasis. Although studies have largely focused on the role of inflammation in obesity-associated pancreatic cancer, the cancer cell-autonomous mechanisms by which lipid availability in obesity impacts tumorigenesis remain quite poorly understood. Given the abundant evidence for dysregulated lipid metabolism in tumors, I hypothesized that cancer cells are primed to exploit exogenous lipids in obesity, accelerating disease progression. By mapping lipid-protein interactions in tumor and metastasis-enriched RNA-seq networks, I identified the nuclear hormone receptor PPARG as a potential mediator of obesity-associated lipid signaling in pancreatic cancer. PPARG is an attractive prospective mediator of obesity-associated PDA progression because it is a metabolic regulator activated by a broad set of lipid ligands, suggesting a dual role in lipid sensing and metabolic control. PPARG is also positioned at an interesting junction in PDA etiology; PPARG agonists are used to treat diabetes (a PDA risk factor) and have previously been studied for anti-cancer effects. However, PPARG agonists have seen little clinical success in and recent work has suggested that their use may even increase bladder cancer risk. PPARG is overexpressed in pancreatic tumors where it is associated with poor prognosis, further casting doubt on its anti-tumorigenic function. In my own studies, I found that PPARG agonists had no effect on tumor viability, while the PPARG antagonist T0070907 reduced organoid viability in vitro. Modeling the convergent effects of PPARG and obesity, I supplemented organoids with a mixture of lipids in the presence of PPARG antagonist and found that lipids boosted the viability of both tumor and metastatic organoids dependent on PPARG activity in vitro. These results suggest that PPARG can mediate the pro-tumorigenic function of exogenous lipids. As the incidence of both obesity and pancreatic cancer continue to rise, defining the role of PPARG in PDA could have significant implications for the development of new strategies to treat or prevent obesity-associated cancer. Citation Format: L. Paige Ferguson. Targeting PPARG to block obesity-associated pancreatic cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B048.

Abstract B090: Targeting the unfolded protein response enhances sensitivity to chemotherapy

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is currently the 3rd leading cause of cancer-related deaths in North American with ~10% survival 5 years after diagnosis. A contributing factor to this low survival rate is chemotherapy resistance. Major risk factors for PDAC, including smoking and chronic pancreatitis, promote activation of the unfolded protein response (UPR). The UPR reduces protein load to alleviate stress, and cancer cells can utilize the UPR to promote cell survival. We previously showed loss of Activating Transcription Factor 3 (ATF3), a mediator of the UPR, restricted early events in PDAC progression. The goal of this study was to determine if ATF3 and the UPR promote resistance to chemotherapy in PDAC. We hypothesized that cancer cells activate the UPR and ATF3 to reduce cell stress and promote chemotherapeutic resistance. Methods: Murine-derived organoids (MDOs) were developed from pancreatic tissue expressing KRASG12D with (Ptf1acreERT/+KRASG12D/+) or without Atf3 (Atf3-/-Ptf1acreERT/+KRASG12D; referred to as APK) expression, and patient-derived organoids (PDOs) were examined for mediators of the UPR. Transcriptomic changes related to ATF3 loss was determined by RNA-sequencing of Ptf1acreERT/+KRASG12D/+ and APK organoids. UPR activation was examined by qRT-PRC in MDOs and PDOs with and without gemcitabine treatment, and related to organoid viability and phenotype. PDOs viability was examined following treatment with gemcitabine +/- UPR inhibitors. Results: RNA-seq analysis of Ptf1acreERT/+KRASG12D/+ and APK MDOs identified drug metabolism cytochrome P450, metabolism of xenobiotics by cytochrome P450, and platinum drug resistance as key pathways affected by the loss of ATF3 suggesting reduced UPR activation impacts drug metabolism in KRASG12D expressing cells. Ptf1acreERT/+KRASG12D/+ and APK MDOs showed a differential response in cell viability and UPR activation to gemcitabine treatment suggesting an importance of ATF3 in gemcitabine resistance. Consistent with this role, treatment of PDOs with gemcitabine resulted in UPR activation in a patient-specific fashion, particularly regarding increased expression of ATF3 and spliced XBP1. In lines showing enhanced UPR activation to gemcitabine, combinatorial treatment with UPR inhibitors showed increased cell death compared to gemcitabine alone. Conclusions: This study shows the UPR and ATF3 are rapidly activated in response to gemcitabine, but in a patient-specific fashion. These results suggest cancer cells activate the UPR to increase resistance to chemotherapies, and a subset of patients may benefit from combined gemcitabine treatment with UPR inhibitors. Since the tumor microenvironment is believed to be a major contributing factor to chemotherapy resistance, future experiments will include the tumor microenvironment interactions and UPR inhibition. Citation Format: Mickenzie B. Martin, Teresa Borello, Fatemeh Mousavi, Nelson Dusetti, Christopher L. Pin. Targeting the unfolded protein response enhances sensitivity to chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B090.

Vascularized tissue on mesh-assisted platform (VT-MAP): a novel approach for diverse organoid size culture and tailored cancer drug response analysis.

This study presents the vascularized tissue on mesh-assisted platform (VT-MAP), a novel microfluidic in vitro model that uses an open microfluidic principle for cultivating vascularized organoids. Addressing the gap in 3D high-throughput platforms for drug response analysis, the VT-MAP can host tumor clusters of various sizes, allowing for precise, size-dependent drug interaction assessments. Key features include capability for forming versatile co-culture conditions (EC, fibroblasts and colon cancer organoids) that enhance tumor organoid viability and a perfusable vessel network that ensures efficient drug delivery and maintenance of organoid health. The VT-MAP enables the culture and analysis of organoids across a diverse size spectrum, from tens of microns to several millimeters. The VT-MAP addresses the inconsistencies in traditional organoid testing related to organoid size, which significantly impacts drug response and viability. Its ability to handle various organoid sizes leads to results that more accurately reflect patient-derived xenograft (PDX) models and differ markedly from traditional in vitro well plate-based methods. We introduce a novel image analysis algorithm that allows for quantitative analysis of organoid size-dependent drug responses, marking a significant step forward in replicating PDX models. The PDX sample from a positive responder exhibited a significant reduction in cell viability across all organoid sizes when exposed to chemotherapeutic agents (5-FU, oxaliplatin, and irinotecan), as expected for cytotoxic drugs. In sharp contrast, PDX samples of a negative responder showed little to no change in viability in smaller clusters and only a slight reduction in larger clusters. This differential response, accurately replicated in the VT-MAP, underscores its ability to generate data that align with PDX models and in vivo findings. Its capacity to handle various organoid sizes leads to results that more accurately reflect PDX models and differ markedly from traditional in vitro methods. The platform's distinct advantage lies in demonstrating how organoid size can critically influence drug response, revealing insights into cancer biology previously unattainable with conventional techniques.

Establishment of Patient-Derived Organoid Model from Hydrothorax and Ascites

Organoids are miniaturized organs cultured in vitro, exhibiting self-renewal and self-organization capabilities. In comparison to traditional 2D cell cultures, organoids can reproduce the spatial structure and functionality resembling corresponding tissues or organs, providing highly physiologically relevant in vitro systems. Consequently, they can be utilized for simulating disease occurrence, testing the tumor drug sensitivity and co-culturing with other cells. This study aims to establish a Patient-Derived Organoid (PDO) model using hydrothorax and ascites samples. To ensure cell viability, it is essential to store the collected malignant fluid at 4°C. Upon arrival at the laboratory, the fluid should be divided into centrifuge tubes for subsequent centrifugation, allowing the collection of cell precipitates. These cell precipitates need to be washed multiple times with DPBS until the supernatant appears clear. If the pellet contains red blood cells, they should be lysed, followed by cell counting. Depending on the desired seeding density, cells should be resuspended in Matrigel and plated as domes in a culture plate. The plate should then be inverted and placed in a 37°C incubator for 30 minutes to allow the Matrigel to solidify. Once solidified, complete culture medium should be added, and the culture plate should be placed in a 37°C incubator for further incubation. Organoid growth can be monitored using microscopy to assess their development and progression. Once the organoids have reached a stable growth state, immunohistochemistry can be performed to identify specific cell types or markers within the organoids. Furthermore, the drug responsiveness of the organoids can be evaluated through cell viability assays, enabling researchers to determine the effects of different drugs or organoid viability and function.

Abstract C104: Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC

Abstract Aberrant expression of the transcription factor MYC is found in most cancers including non-small cell lung cancer (NSCLC) and has critical roles in a wide range of oncogenic processes including immune evasion and EGFR inhibitor resistance. Despite MYC’s therapeutic attractiveness, potent inhibitors of MYC activity have been elusive, due to its intrinsically disordered protein structure and tightly autoregulated expression. We have developed NSCLC-specific programmable epigenomic mRNA therapy, termed a MYC Omega epigenomic controller (NSCLC MYC-OEC) that is designed to target the insulated genomic domain of MYC. We have previously shown that NSCLC MYC-OEC effectively downregulates MYC expression pre-transcriptionally and have demonstrated that NSCLC MYC-OEC inhibits NSCLC patient-derived organoid viability in vitro and abrogates xenograft tumor growth in vivo. To identify combination treatments with NSCLC MYC-OEC, we retrospectively evaluated baseline MYC expression and real-world progression-free survival (PFS) in a cohort of 283 NSCLC patients treated with pembrolizumab, an anti-PD1 monoclonal antibody. This analysis revealed that NSCLC patients with MYC-overexpressing tumors exhibited shorter PFS when treated with pembrolizumab. Moreover, analysis of PD1 or PD-L1 transcript levels in tumors from 13,230 lung adenocarcinoma patients revealed a significant positive correlation between PD1 or PD-L1 and MYC mRNA levels, consistent with a role for MYC in regulation of the immune response. In vivo evaluation of NSCLC MYC-OEC activity and immune checkpoint immunotherapy (ICI) treatment of preclinical Lewis lung carcinoma syngeneic tumor models demonstrated that the combination of NSCLC MYC-OEC with anti-PD-L1 antibody enhanced inhibition of tumor growth and prolonged survival over administration when compared to either NSCLC MYC-OEC or anti-PD-L1 antibody monotherapy. Retrospective analysis of a cohort of 301 NSCLC patients also showed that patients with MYC-overexpressing tumors exhibited significantly shorter PFS when treated with osimertinib, a third generation EGFR inhibitor. To further understand this, we investigated the effects of NSCLC MYC-OEC and osimertinib treatment alone or in combination in EGFR mutant cells, H1975 and PC9. While NSCLC MYC-OEC and osimertinib monotherapy treatments resulted in downregulation of MYC protein expression in both cell lines, combination treatment led to a significantly greater decrease in MYC expression. Furthermore, the combination synergistically reduced H1975 and PC9 cell viability in vitro and led to a significantly greater inhibition of H1975 xenograft growth relative to each single agent in vivo. Together, these data provide preclinical proof-of-concept in NSCLC for development of OEC-mediated pre-transcriptional epigenomic inhibition of MYC expression in combination with immune checkpoint and EGFR inhibitors. Furthermore, these findings suggest that NSCLC MYC-OEC combination with ICI or EGFR-targeted therapy may improve patient outcomes. Citation Format: Defne Yarar, Eugine Lee, Padraich Flahardy, Cameron Vergato, Graeme Hodgson, Thomas McCauley. Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C104.

Stimulatory Anticancer Effect of Resveratrol Mediated by G Protein-Coupled Estrogen Receptor in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most high-risk cancers; however, it has been suggested that estrogen signaling in CRC could have a protective effect. Therefore, we focused on the function of the G protein-coupled estrogen receptor (GPER) among the estrogen receptors in CRC. In this study, we investigated the therapeutic effect of resveratrol via GPER in CRC (RKO and WiDr) cells, CRC cell-derived xenograft models, and organoids (30T and 33T). Resveratrol significantly suppressed cell viability and proliferation in highly GPER-expressing RKO cells compared to that in low GPER-expressing WiDr cells. In xenograft models, resveratrol also delayed tumor growth and exhibited a high survival rate depending on GPER expression in RKO-derived tumors. Furthermore, resveratrol significantly inhibited the viability of organoids with high GPER expression. Additionally, the anticancer effect of resveratrol on CRC showed that resveratrol rapidly responded to GPER, while increasing the expression of p-ERK and Bax and cleaving PARP proteins.

The USP46 complex deubiquitylates LRP6 to promote Wnt/β-catenin signaling

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

Fisetin induces the upregulation of AKAP12 mRNA and anti-angiogenesis in a patient-derived organoid xenograft model

Colorectal cancer (CRC) is associated with high incidence and mortality rates. Targeted therapies for CRC cause various adverse effects, necessitating the development of novel approaches to control CRC progression. In this milieu, we investigated the anti-CRC effects of fisetin, a natural plant flavonoid. Cytotoxicity was performed in CRC patient-derived organoids (30 T and 33 T). Fisetin-induced tumor growth was evaluated in a CRC patient-derived organoid xenograft (PDOX) model. RNA sequencing, immunohistochemistry, and western blotting were performed subsequently. Fisetin significantly decreased organoid viability in a dose-dependent manner. In the PDOX model, fisetin significantly delayed tumor growth, showing a decrease in Ki-67 expression and the induction of apoptosis. In tumor tissues, four genes were identified as differentially expressed between the control and fisetin-treated groups. Among these, A-kinase anchoring protein 12 (AKAP12) level was significantly increased by fisetin treatment (fold change > 2, p < 0.05). Notably, fisetin significantly inhibited vascular endothelial growth factor (VEGF) and epithelial cell adhesion molecule (EpCAM) via upregulation of AKAP12. Our results demonstrate the upregulation of AKAP12 mRNA and inhibition of angiogenesis by fisetin as a therapeutic strategy against CRC.

Natural nanogels crosslinked with S-benzyl-L-cysteine exhibit potent antibacterial activity

Biofilm-forming bacteria E. coli and P. aeruginosa have both exhibited resistance against multiple antibiotics in clinical settings. To find a solution, researchers have turned to antibacterial structurally modified from natural materials that are harmless to the human body. Among these is DNA, a natural polymer composed of deoxyribose that when treated with HCl exposes its aldehyde groups and produces DNA-HCl. Here, we crosslinked these aldehyde groups with the primary amines in S-benzyl-L-cysteine (SBLC) using a Schiff reaction to obtain DNA-HCl-SBLC. We additionally treated alginate acid (AA) with EDAC, obtaining AA-EDAC, and substituting it with SBLC to produce AA-SBLC. We incorporated the above reactions with an emulsification process to produce nanogels (NGs) that were verified to be spherical and possessing benzene rings successfully grafted onto DNA-HCl and AA-EDAC. These natural NGs were proven to be negatively charged through zeta potential analysis and presented low cytotoxicity toward normal cells in cell organoid viability assays. These SBLC-modified polymers provided better inhibition of bacterial growth than those without modification. Moreover, after incubation with SBLC-modified NGs, bacteria expressed intracellular recA or pvdA in a dose-dependent manner, which was consistent with SEM data from damaged bacteria. Out of four tested NGs, DNA-HCl-SBLC NGs suppressed P. aeruginosa-induced sepsis most effectively and extended the lifespan of C. elegans. This study provides an alternative clinical solution to antibiotics-resistant biofilm strains.

Abstract B008: Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in prostate cancer

Abstract Introduction and Objective Several studies have shown that cancer-associated fibroblasts (CAFs), the most abundant component in prostate cancer (PCa) microenvironment, promote resistance to androgen deprivation therapies, and metastatic development. The aim of this study is to elucidate the mechanisms of CAF-induced therapy resistance through the establishment of in vitro 3D co-cultures. Methods In this study, the androgen-dependent PNPCa patient-derived xenograft (PDX) model (derived from soft tissue metastasis), and the androgen-independent LAPC9 PDX model (bone metastasis) are used. Epithelial and stromal cells (human and mouse origin, respectively) are separated through magnetic-associated cell sorting to generate PDX-derived organoids and fibroblasts. Fibroblast RNA and protein expression is assessed through RT-qPCR, Western Blot, and Immunofluorescence. Direct 3D co-cultures are obtained by combining fluorescently labelled organoids and fibroblasts in ultra-low attachment plates in defined media. In indirect co-cultures, organoids are cultured in Spherical Plates (Kugelmeiers) with fibroblasts on transwell inserts. Organoid viability is measured after 9 days by CellTiter-Glo 3D Assay. Results PDX-derived fibroblasts express typical CAF genes (e.g. α-smooth muscle actin and Tenascin C), and androgen receptor (AR), both at RNA and protein level. AR protein expression (as well as RNA expression of the AR target gene Fkbp5) is increased in CAFs upon treatment with dyhydrotestosterone (DHT) 10nM, while co-treatment with the AR inhibitor Enzalutamide 10mM abrogates this effect, suggestive of functional AR signaling. These CAFs can organize into tumor/CAF organoid 3D structures upon combination with PDX-derived tumor epithelial cells, providing an in vitro functional model to study tumor-stromal interactions. In indirect (transwell) co-cultures, CAFs increase the viability of the androgen dependent PNPCa organoids, but not that of androgen independent LAPC9. This growth-promoting effect is observed even upon culturing of PNPCa organoids in non-optimal growth conditions, at low DHT concentrations (DHT 0.5nM and 0.25nM versus the standard DHT 1nM). The factors mediating this process will be identified by gene expression profiling of CAFs and organoids in transwell co-cultures and/or monoculture. Conclusions Our data indicate that PCa metastatic cells modify the properties of neighbor stromal cells to support tumorigenesis, since PDX-derived fibroblasts, originally part of the mouse dermal stroma, display CAF features and active AR signaling. 3D co-cultures represent a useful tool to study the tumor-stromal interactions. Our transwell co-culture experiments suggest that CAFs play an important role in supporting the viability and growth of early metastatic androgen dependent PCa cells (PNPCa) by secreting pro-tumorigenic factors. The possibility that CAFs exert different effects depending on the tumor stage setting (early vs advanced) will be subsequently investigated. Citation Format: Francesco Bonollo, Natalie Sampson, George Thalmann, Sofia Karkampouna, Marianna Kruithof-de Julio. Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance 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 B008.

Abstract 170: Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens

Abstract Introduction: Glioblastoma is resistant to standard treatment, leading to tumor recurrence and early death of patients. Using organoid models, we aim to explore the mechanisms of resistance to cancer therapies and identify novel therapeutic vulnerabilities, including stress protecting melanoma-associated antigens (MAGEs). MAGEs are normally restricted to expression in testis but are abnormally activated in cancer and are associated with therapy resistance. Methods: Organoids have been established from fresh tumor biopsies and are stored in Gliobank, a Slovenian collection of patient tumor samples with corresponding clinical data. Organoids were first characterized and compared with the corresponding parental tumor tissue by immunofluorescence and qPCR of selected markers. Organoids were then treated with a combination of irradiation, the chemotherapeutic agent temozolomide and/or a chemokine receptor antagonist, and response to therapy was assessed by immunofluorescence, qPCR, cell viability and invasion assays, multiplex ELISA and proximity ligation assay. Results: Organoids recapitulated the gene expression profile of parental glioblastoma tissues, including expression of genes related to cancer stem cells, DNA damage response, cell cycle progression, epithelial- to- mesenchymal transition, and cytokine signaling. In addition, the organoids preserved the cellular composition of the parental glioblastoma tissues, consisting of glioblastoma (stem) cells, macrophages, microglia, lymphocytes, and endothelial cells. Standard treatment decreased viability and invasion of organoids in only 2 of 6 patients, suggesting that organoids recapitulate tumor therapy resistance. The increased expression of MDM2 and CDKN1A in organoids after treatment with irradiation and temozolomide suggests that the p53 pathway and DNA damage response mechanisms may contribute to the therapy resistance. Next, we detected high abundance of secreted cytokines in the culture medium of the organoids, including CXCL12. However, treatment of the organoids with the CXCR4 antagonist Plerixafor, which blocked the interactions between CXCL12 and CXCR4 in the organoids, had no effect on organoid viability and invasion, suggesting resistance of glioblastoma to this immunotherapy. Since high expression of several type I MAGEs correlates with poor prognosis of glioma patients based on TCGA database analysis, we investigate the role of MAGEs in therapy resistance of glioblastoma organoids. Conclusions: Patient-derived tumor organoids provide a valuable tool to 1) identify novel therapeutic vulnerabilities in the context of the tumor microenvironment, 2) evaluate the effect of novel candidate treatments including immunotherapy, and 3) discover novel markers of therapy resistance. Citation Format: Barbara Breznik, Bernarda Majc, Anamarija Habič, Gloria Krapež, Andrej Porčnik, Jernej Mlakar, Tamara Lah Turnšek, Metka Novak, Klementina Fon Tacer. Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens [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 170.

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 ...