Membrane Extraction Research Articles

Revealing the impact of membrane adsorption on phenol transmembrane mass transfer in aqueous-aqueous membrane extraction

Nanoparticle doping in membranes is an ideal strategy for improving performance of aqueous-aqueous membrane extraction (AAME). However, its derived adsorption has not been investigated regarding performance enhancement/deterioration of phenol transmembrane mass transfer they might bring, especially in porous membranes. Therefore, supercapacitor-activated carbon (YEC-8)-doped (>30 mg/g) and silica-doped membranes (<0.1 mg/g) only with differentiated adsorption capacity were prepared to identify adsorption impact on phenol extraction from saline-wastewater. Surprisingly, membrane adsorption significantly inhibited phenol transmembrane mass transfer regardless of adsorption saturation rather than enhancement as we hypothesized. Compared to PVDF membrane, extraction performance of silica-doped membrane decreased by 24.0% at 2 g/L phenol. For YEC-8-doped membrane, its extraction performance even decreased by 40%-50% when facing multiple phenol concentrations. These results stated the important role of volatilization-diffusion in phenol transmembrane mass transfer in comparison with solution-diffusion. Membrane characterization and Hagen-Poiseuille model analysis demonstrated that membrane pore complexity would induce partial performance degradation. DFT calculation further confirmed that adsorption of gaseous phenol (−1.7292 eV) within membrane pores was primary limiting step in transmembrane mass transfer. Overall, this study elucidates transmembrane mass transfer pathway, process, and mechanism of phenol in AAME with nanoparticle-doped porous membrane for the first time.

Abstract 566: The anticancer effects of polyisoprenylated cysteinyl amide inhibitors involves interaction with the KRAS chaperone calmodulin

Abstract Pancreatic cancer is the third leading cause of cancer-related deaths with a 5-year survival rate of less than 10%. Approximately 90% of reported cases are driven by KRAS mutations. The mutant KRAS acquires its functional localization through interactions with chaperone proteins via its polybasic polyisoprenylated tail. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) mimic this region possibly disrupting the polyisoprenylation-dependent interactions with chaperone proteins. Here, we determine the effect of newly developed PCAIs that were designed to be more water-soluble on cell viability, 3D spheroid invasion, spheroid degeneration and F-actin filaments on MIA PaCa-2 and PANC-1 cells. Of the seven new PCAIs analogs tested, NSL-AB-51 and NSL-AB-45 were the most potent, with EC50 values of 2.5 and 5.6 µM against MIA PaCa-2, and 1.7 and 4.5 µM against PANC-1 cells, respectively. At 5 and 10 µM, NSL-AB-51 decreased the invasion area of 3D spheroids by 98 and 99%, respectively. MIA PaCa-2 spheroids degenerated by 61% after 72 h treatment with 10 µM NSL-AB-51. Treatment with 1.0 μM NSL-AB-51 caused cell rounding and reduction of mean cell areas by 69% and F-actin filaments by 96%. Moreover, to identify the pharmacological target of the PCAIs, NSL-AB-45 was coupled to an epoxy-activated Sepharose resin for affinity trapping of PCAIs-interacting proteins. Affinity-analysis of supernatants and detergent membrane extracts from MIA PaCa-2 and PANC-1 cells revealed single Coomassie band of about 17 kDa that was identified as calmodulin by western blotting analysis. The calmodulin-PCAIs affinity interaction was found to be strongest in the presence of calcium and weakest in the presence of EDTA. These findings reveal that calmodulin is the likely target of PCAIs. Since calmodulin is the chaperone protein responsible for KRAS trafficking and localization to the inner surface of the plasma membrane, this finding strongly suggests, at least in part, an anticancer role for the PCAIs that involve disruption of KRAS functions. Citation Format: Kweku Ofosu-Asante, Jassy Mary Lazarte, Amarender Burra, Nazarius S. Lamango. The anticancer effects of polyisoprenylated cysteinyl amide inhibitors involves interaction with the KRAS chaperone calmodulin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 566.

Abstract 4251: Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium

Abstract Tumoroids, also known as cancer organoids, are self-assembled three-dimensional cultures of patient-derived tumor cells. Compared to traditional 2D cancer cell lines, tumoroids better maintain key characteristics of the original tumor including the genotype and transcriptome, which lead to better in vitro modeling of clinically relevant functional responses such as drug sensitivity. An under-studied aspect of tumoroid cultures has been their ability to recapitulate dependence on hormone signaling in vitro, with some reports indicating hormone dependence is lost over time in culture. To investigate this, we utilized Gibco™ OncoPro™ Tumoroid Culture Medium and defined tissue-specific supplements, FGF10 and beta-estradiol, that when added to the medium enabled the derivation of endometrial tumoroids from tumor resections. Derivation of tumoroid lines compatible with long-term growth depended on a variety of factors, including tissue quality and time from resection to culture initiation. Each of the 15 samples processed formed tumoroids when initially seeded into culture. 11/15 samples reformed tumoroids through multiple passages (typically 1-2 weeks per passage) over 1-3 months in culture. 7/15 samples met our high criteria for stable tumoroid line growth of &amp;gt;5 passages and reaching &amp;gt;5 cumulative population doublings (from initial number of cells seeded post-resection) in culture. Next-generation sequencing of the established tumoroids and the uncultured tumor material demonstrated a high degree of correlation (pearson’s r&amp;gt;0.9) of genomic mutations in a targeted panel; few differentially expressed genes were detected beyond down-regulation of immune-related genes, presumably because the medium is optimized for outgrowth of cancer epithelial cells, and immune cells do not persist during long-term culture. Endometrial tumoroids were capable of growing embedded in basement membrane extract (BME) or in suspension with dilute BME added and could be cryopreserved and recovered. Endometrial tumoroids retained gene expression levels (both presence and absence) of estrogen receptor and progesterone receptor for up to the 20 passages tested in vitro. A subset of the models was tested and showed increased growth rates in response to increased concentrations of beta-estradiol. Of those tested, 2/3 tumoroid cultures stopped growing within 1-2 weeks after removal of beta-estradiol from the medium, demonstrating dependence on this hormone for growth. Maintenance of hormone receptor expression and dependency should enable in vitro studies to understand the signaling mechanisms and potential ways to modulate them in the context of cancer. Overall, this method provides a toolset for studying endometrial cancer, particularly hormone dependency, and may be more broadly applicable to other gynecological or hormone dependent cancers. Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Pradip Shahi Thakuri, Shyanne Salen, Mark Kennedy, Matt Dallas, David Kuninger. Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4251.

Abstract 636: Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts

Abstract The tumor microenvironment (TME) is well recognized for its role in tumor progression, chemoresistance, and recurrence. The TME consists of various components, such as extracellular matrix (ECM), chemical and mechanical cues, as well as stromal cells and their interactions. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have drawn a lot of attention. It has been reported that CAFs not only directly interact with cancer cells via surface proteins and cytokines, but also influence their behaviors by remodeling the ECM. However, due to their heterogenous characteristics and diverse functions, the influence of tumor-CAF interactions on drug response is not fully understood. Here, we present a coculture model for investigating CAF effects on patient-derived colorectal tumor organoids (CTOs) growth, differentiation, and drug response. It consists of normal colon fibroblasts, CCD18-Co, or patient-derived CAFs seeded with CTOs in a mixed hydrogel containing 90% basement membrane extract (BME) and 10% collagen type I. Media conditions were optimized (1:1 mix of CTO culture media plus reduced media excluding growth factors and inhibitors) to achieve similar organoid sizes in the monoculture and coculture conditions at the onset of drug testing. To support higher throughput investigations, our workflow incorporates a liquid handler for automated CTO seeding, media and drug exchange, and an automated microscope with a robotic arm for multi-plate imaging. Briefly, single cell CTOs and fibroblasts were seeded at a 1:1 ratio (3000 total cells/well) in 96-well plates. After 4 days in culture, anti-cancer drugs were administrated in reduced media to the samples. Brightfield images were acquired on days 0, 3, and 5 after drug administration. The collected images were analyzed with a neural network (NN) to dynamically monitor the growth and viability of individual organoids. The CellTiter-Glo assay was performed on day 5 to quantify the total CTO cell viability. The normalized drug response (NDR) was calculated from the NN processed images to determine the drug response. An immunostaining panel, which includes protein markers such as CK20, Ki67, α-SMA, Vimentin, and FAP, was used to evaluate the fibroblast phenotypes and CTO differentiation. Additionally, the cultures were subjected to fluorescent lifetime imaging microscopy (FLIM), a label-free imaging technique, to measure the metabolic adaptations of each cell population. Preliminary studies revealed a CAF-dependent increase in CTO growth and glycolytic signatures in the co-culture setting; however, minimal effects on drug response to common chemotherapy agents such as 5-fluorouracil and irinotecan were observed. In the future, we will use this integrated pipeline as a screening method to identify new treatment strategies aimed at targeting the stromal microenvironment in colorectal cancer. Citation Format: Yuyuan Zhou, Yu-Kai Huang, Emma J. Fong, Elizabeth Elton, Erin McManus, Brandon Choi, Seungil Kim, Shannon M. Mumenthaler. Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 636.

Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2760.

Abstract 6769: Applications of a novel fibrous extracellular matrix for advanced in vitro cancer models

Abstract Compared to traditional in vitro two-dimensional (2D) planar cell culture system, three-dimensional (3D) mammalian cell culture can better recapitulate physiological environments and the complexity of natural tissues, hence they are crucial in modern biomedical research. Extracellular matrices (ECM) play a key role in in vitro mammalian cell culture systems. The role of the ECM on in vitro 3D cell culture are: 1) to simulate an in vivo-like physiological environment, 2) support cell-cell and cell-ECM interactions, 3) promote cell adhesion, migration, and communication, 4) aid in tissue development, morphogenesis, differentiation, and functioning. Although basal membrane extracts (BME) are the most popular type of ECM supporting 3D modeling in vitro, they often have significant limitations including limited long-term stability, significant batch to batch heterogeneity, restricted cell movement, and relatively high cost for culturing many cancer cell lines. In this investigation, we employ a novel micro-fibrous scaffold that closely mimics natural ECM, providing porous and interconnected networks for 3D culturing of hepatic cells, lung cancer, breast cancer, pancreatic cancer. As revealed by optical and scanning electron microscopy, the scaffold promotes significant cell-to-cell and cell-to-matrix interactions. Moreover, we observed improved cell growth in 3D culture over longer periods than 2D culture systems for all cell types examined. We also evaluated the anticancer efficiency of different drugs including Lapatinib, Cisplatin, Docetaxel, Doxorubicin and observed that 2D cultured cells were more sensitive to drugs treatment in comparison to 3D. Furthermore, ECM was observed to support 3D growth and structure formation with cell pools of primary cells achieved from disassociated human tumors. Tumor organoids were able to form within seven days and ready for anticancer screening within 14 days. We achieved an 80% success rate in developing breast cancer organoids from patient tissues. Additionally, tissue granules were also able to be cultured under the same condition, which allowed us to investigate the invasion of cancer cells into tissue. The primary cells from human tumors formed cell clusters resembling the original tumor tissue's structure. In conclusion, the novel micro-fibrous scaffold, closely mimicking natural extracellular matrixes, promotes significant cell-to-cell and cell-to-matrix interactions, while also facilitating cell growth over extended periods providing a more physiologically relevant platform for evaluating drug responses for various cancer cell types, further showing the importance of 3D culture systems in cancer research. Citation Format: Navneet Kaur, Dazhi Yang, Jamel Ali. Applications of a novel fibrous extracellular matrix for advanced in vitro cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6769.

Fatty Alcohol-Assisted Electro-Driven Liquid Membrane Multi-Stage Extraction Technology for Selective Separation and Purification of 3,4-Dihydroxybenzaldehyde from Salvia miltiorrhiza Bge.

3,4-Dihydroxybenzaldehyde (DHB) is a highly effective water-soluble compound in the traditional Chinese medicine Salvia miltiorrhiza Bge. Which demonstrates clear therapeutic effects against a variety of diseases. Therefore, the development of a selective approach for the separation of 3,4-dihydroxybenzaldehyde is of great significance. In this study, electro-driven extraction combined with multi-stage extraction technology was employed for the first time to separate 3,4-dihydroxybenzaldehyde from S. miltiorrhiza Bge. Decoction (Recovery ≥ 46.09%). Five long-chain fatty alcohols were selected as the supported liquid membrane (SLM) in the electro-driven extraction. Factors influencing extraction efficiency, including pH, voltage, and extraction time, were optimized using a design of experiments. Under the optimal conditions, the linearity of 3,4-dihydroxybenzaldehyde ranged from 0.01 to 100 μg·mL−1), with limits of detection (LOD) and quantification (LOQ) of 5 and 17 ng·mL−1, respectively. The precision of electro-driven liquid membrane extraction for 3,4-dihydroxybenzaldehyde was below 8.42%. This paper reports novel electro-driven liquid membrane extraction for selectively isolating 3,4-dihydroxybenzaldehyde from S. miltiorrhiza Bge.

The derlin Dfm1 couples retrotranslocation of a folded protein domain to its proteasomal degradation.

Endoplasmic reticulum (ER) proteins are degraded by proteasomes in the cytosol through ER-associated degradation (ERAD). This process involves the retrotranslocation of substrates across the ER membrane, their ubiquitination, and membrane extraction by the Cdc48/Npl4/Ufd1 ATPase complex prior to delivery to proteasomes for degradation. How the presence of a folded luminal domain affects substrate retrotranslocation and this event is coordinated with subsequent ERAD steps remains unknown. Here, using a model substrate with a folded luminal domain, we showed that Cdc48 ATPase activity is sufficient to drive substrate retrotranslocation independently of ERAD membrane components. However, the complete degradation of the folded luminal domain required substrate-tight coupling of retrotranslocation and proteasomal degradation, which was ensured by the derlin Dfm1. Mutations in Dfm1 intramembrane rhomboid-like or cytosolic Cdc48-binding regions resulted in partial degradation of the substrate with accumulation of its folded domain. Our study revealed Dfm1 as a critical regulator of Cdc48-driven retrotranslocation and highlights the importance of coordinating substrate retrotranslocation and degradation during ERAD.

Abstract A015: Multiplexed live-cell imaging for drug responses in patient-derived organoid models of endometrial cancer

Abstract Patient-derived organoid (PDO) models of endometrial cancer are rapidly emerging as a multifunctional research model to recapitulate in vivo cancer progression and therapeutic response. PDO models are generated by reconstituting patient tumor cells in extracellular basement membrane extract (BME) and culturing as three-dimensional domes supplemented with a specialized niche of growth factors. Despite the rise in popularity of PDO models to assess drug response in vitro, there are limited commercially available reagents that are compatible with BME. In addition, most reagents are endpoint assays that require cell lysis, which is problematic because PDOs are a limited resource. Reducing drug response assessment to a single timepoint misses out on valuable kinetic response data, and samples cannot be used for subsequent downstream analyses, such as RNA sequencing or protein assays. Herein we describe a method to assess drug effects in PDO models using kinetic live-cell imaging. Live-cell imaging is a sophisticated form of automated microscopy that, when combined with fluorescent dyes, has the capacity to quantify multiple cellular responses simultaneously. Importantly, live-cell imaging reagents do not perturb cell viability or induce DNA damage, thereby allowing for accurate repeated assessment of PDO response to treatment over time. In proof-of-concept studies, we first established the utility of this approach to assess the effect of cytotoxic and cytostatic agents in PDO models of endometrial cancer. Specific parameters of interest include PDO number, area, cell health and induction of apoptosis. Agents that produced a cytotoxic effect promoted a decrease in organoid area and number over time, concomitant with an increase in apoptosis as measured by Annexin V staining. Ongoing studies are examining the dose-dependent phenotypic effects of cytostatic agents such as progestins in endometrial cancer PDO models. To evaluate the safety of the compounds, we have also conducted experiments using patient-derived organoids of normal endometrium, which was collected from uninvolved regions of the uterus in cytoreductive surgery. We envision that this automated kinetic imaging platform can be expanded to other phenotypic readouts to better understand diverse therapeutic effects of different classes of drugs under investigation for endometrial cancer. Citation Format: Kaitriana E. Colling, Emily L. Symons, Hiruni K. Sumanasiri, Kristina W. Thiel. Multiplexed live-cell imaging for drug responses in patient-derived organoid models of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A015.

The Emerging Role of Cell Membrane-coated Nanomaterials in Cancer Therapy.

This review investigates the revolutionary application of cell membrane-coated nanoparticles (CMNPs) as a promising avenue for cancer therapy within the embryonic landscape of nanotechnology. Nanoparticles, pivotal in cancer treatment, are systematically examined for their diverse physicochemical structures, categorized as organic (lipid-based, protein-based, and polymer-assisted) and inorganic (carbon-based and metal) varieties. A significant focus is placed on CMNPs, which serve as an innovative drug delivery vehicle, overcoming limitations associated with conventional nanoparticle therapies. This manuscript accurately explores the advantages and challenges of various cell membranes, including those derived from cancer cells, red blood cells, platelets, stem cells, and white blood cells. Importance is placed on their roles in enhancing drug delivery precision, immune system circumvention, and targeted recognition. Detailed insights into the crafting of CMNPs are provided, elucidating membrane extraction and fusion techniques, such as sonication, extrusion, co-extrusion, and microfluidic electroporation. Maintaining membrane integrity during extraction and the benefits of coating techniques in augmenting biocompatibility and targeted drug delivery are underscored. This comprehensive resource consolidates the latest advancements in targeted drug delivery, positioning itself at the forefront of nanotechnology and biomedicine research. Encapsulating various methodologies like membrane extrusion, electrospray, and chemical conjugation, this manuscript showcases the expanding toolbox available to researchers in this dynamic field. Focusing on the unique characteristics of CMNPs, this review explores their multifaceted applications in biomedical research, particularly in tumour therapy. It provides an indepth analysis of the biocompatibility of CMNPs, their stability, immune evasion capabilities, targeted drug delivery precision, increased payload capacity, and retained biological functionality. The manuscript outlines current applications and future prospects of CMNPs in targeted chemotherapy, photothermal and photodynamic therapy, immunotherapy, gene therapy, and innovative therapeutic methods. It concludes by highlighting the advantages of CMNPs in tumour therapy and their transformative potential in reshaping the landscape of cancer treatment.

Abstract B008: Modeling radiation sensitivity in patient-derived organoid models of endometrial cancer

Abstract Endometrial cancer is the 4th most common cancer in women, with increasing incidence and deaths each year from 2008 to 2017. The typical treatment for high grade and advanced endometrial cancer includes cytoreductive surgery followed by radiation and/or chemotherapy, depending on stage, grade, and other risk factors. Despite advances in molecular classification of endometrial cancer, there is a paucity of data on the mechanisms of sensitivity and resistance to radiation and chemotherapy. One reason for this lack of understanding is because, clinically, surgery is not typically offered in the setting of recurrence; thus, there are no available specimens to assess how the tumor evolves in response to therapy. Preclinical models can in part overcome this roadblock, yet there are very few preclinical models of endometrial cancer that accurately recapitulate the heterogeneity of human disease. Our objective was to define how endometrial tumors respond to radiation and chemotherapy using patient-derived organoid models. First, we have established a living biorepository that includes over 30 patient-derived organoid models of high risk or advanced endometrial cancer, with available clinical response data. Specifically, tumor specimens were obtained at the time of primary surgery and digested to generate single cell suspensions, which were then resuspended in basement membrane extract (BME), plated as three-dimensional domes, and cultured in specialized media. Organoids with a diameter of &amp;gt;50 µm typically formed in 48-96 hours after initial plating. For therapeutic studies, organoids were plated as BME domes in 96-well plates, followed by exposure to increasing levels of radiation (2-16 Gy) and/or chemotherapy (carboplatin+paclitaxel, 10 nM – 10 µM). Drug effects were monitored using an automated kinetic live-cell imaging platform (Cytation5) in which media was supplemented with fluorescent dyes to multiplex interrogation of cell health and induction of apoptosis. To date, we have observed differential sensitivity to radiation and chemotherapy in patient-derived organoid models of high risk and advanced endometrial cancer as determined by changes in organoid number and size and induction of apoptosis. These studies are the first to examine the therapeutic effects of radiation in endometrial cancer patient-derived organoid models. Ongoing studies are correlating response to therapy in patient-derived organoid models with clinical patient outcomes. We are also performing pre- and post-treatment transcriptomic analyses to define gene signatures of response to radiation and cytotoxic chemotherapy in endometrial cancer organoid models. The ultimate goal is to use these heterogeneous preclinical models to identify pretreatment biomarkers of response that can be translated to a clinical diagnostic test. Citation Format: Sofia Gabrilovich, Kaitriana Powell-Smith, Hiruni Sumanasiri, Emily Symons, Emily Hill, Miles Pufall, Donna Santillan, Kristina W. Thiel. Modeling radiation sensitivity in patient-derived organoid models of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B008.

Solvent-free parallel artificial liquid membrane extraction for drugs of abuse in plasma samples using LC-MS/MS

BackgroundParallel artificial liquid membrane extraction (PALME) is a 96-well plate setup variant of liquid-phase microextraction. Basic or acidic analytes are extracted in neutral form from the sample, through a supported liquid membrane (SLM), and into aqueous acceptor. PALME is already considered a green extraction technique, but in the current conceptual work, we sought to make it even greener by replacing the use of organic solvents with essential oils (EO). PALME was combined with LC-MS/MS for analysis of plasma samples and multiple drugs of abuse with toxicological relevance (amphetamines, phenethylamines, synthetic cathinones, designer benzodiazepines, ayahuasca alkaloids, lysergic acid diethylamide, and ketamine). ResultsFourteen EO were compared to organic solvents frequently used in PALME. The EO termed smart & sassy yielded the best analyte recovery for all drugs studied and was thus selected as SLM. Then, factorial screening and Box-Behnken were employed to optimize the technique. The extraction time, concentration of base, sample volume, and percentage of trioctylamine significantly impacted analyte recovery. The optimum values were defined as 120 min, 10 mmol/L of NaOH, 150 μL, and 0%, respectively. Once optimized, validation parameters were 1–100 ng mL-1 as linear range, accuracy ±16.4%, precision >83%, 1 ng mL-1 as limit of quantitation, 0.1–0.75 ng mL-1 as limit of detection, matrix effect <20%, and recovery 20–106%. Additionally, EO purchased from different production batches were tested and achieved acceptable reproducibility. Data were in compliance with requirements set by internationally accepted validation guidelines and the applicability of the technique was proven using authentic samples. SignificanceIn this study, the use of an EO provided a solvent-free sample preparation technique suited to extract different classes of drugs of abuse from plasma samples, dismissing the use of hazardous organic solvents. The method also provided excellent sample clean-up, thus being a simple and efficient tool for toxicological applications that is in agreement with the principles of sustainable chemistry.

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 ß (HERß-1)-pathway, with HERß-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

The healing power of amniotic membrane extract eye drops. A 100 year old man saved from an Ocular Perforation.

The healing power of amniotic membrane extract eye drops. A 100 year old man saved from an Ocular Perforation.

Development of Polarity-Reversed Endometrial Epithelial Organoids.

The uterine epithelium is composed of a single layer of hormone responsive polarized epithelial cells that line the lumen and form tubular glands. Endometrial epithelial organoids (EEO) can be generated from uterine epithelia and recapitulate cell composition and hormone responses in vitro. As such, the development of EEO represents a major advance for facilitating mechanistic studies in vitro. However, a major limitation for the use of EEO cultured in basement membrane extract and other hydrogels is the inner location of apical membrane, thereby hindering direct access to the apical surface of the epithelium to study interactions with the embryo or infectious agents such as viruses and bacteria. Here, a straightforward strategy was developed that successfully reverses the polarity of EEO. The result is an apical-out organoid that preserves a distinct apical-basolateral orientation and remains responsive to ovarian steroid hormones. Our investigations highlight the utility of polarity-reversed EEO to study interactions with E. coli and blastocysts. This method of generating apical-out EEO lays the foundation for developing new in vitro functional assays, particularly regarding epithelial interactions with embryos during pregnancy or other luminal constituents in a pathological or diseased state.

Targeted chemotherapy via HER2-based chimeric antigen receptor (CAR) engineered T-cell membrane coated polymeric nanoparticles

Cell membrane-derived nanoparticles (NPs) have recently gained popularity due to their desirable features in drug delivery such as mimicking properties of native cells, impeding systemic clearance, and altering foreign body responses. Besides NP technology, adoptive immunotherapy has emerged due to its promise in cancer specificity and therapeutic efficacy. In this research, we developed a biomimetic drug carrier based on chimeric antigen receptor (CAR) transduced T-cell membranes. For that purpose, anti-HER2 CAR-T cells were engineered via lentiviral transduction of anti-HER2 CAR coding lentiviral plasmids. Anti-HER2 CAR-T cells were characterized by their specific activities against the HER2 antigen and used for cell membrane extraction. Anti-cancer drug Cisplatin-loaded poly (D, l-lactide-co-glycolic acid) (PLGA) NPs were coated with anti-human epidermal growth factor receptor 2 (HER2)-specific CAR engineered T-cell membranes. Anti-HER2 CAR-T-cell membrane-coated PLGA NPs (CAR-T-MNPs) were characterized and confirmed via fluorescent microscopy and flow cytometry. Membrane-coated NPs showed a sustained drug release over the course of 21 days in physiological conditions. Cisplatin-loaded CAR-T-MNPs also inhibited the growth of multiple HER2+ cancer cells in vitro. In addition, in vitro uptake studies revealed that CAR-T-MNPs showed an increased uptake by A549 cells. These results were also confirmed via in vivo biodistribution and therapeutic studies using a subcutaneous lung cancer model in nude mice. CAR-T-MNPs localized preferentially at tumor areas compared to those of other studied groups and consisted of a significant reduction in tumor growth in tumor-bearing mice. In Conclusion, the new CAR modified cell membrane-coated NP drug-delivery platform has demonstrated its efficacy both in vitro and in vivo. Therefore, CAR engineered membrane-coated NP system could be a promising cell-mimicking drug carrier that could improve therapeutic outcomes of lung cancer treatments.

The effect of extracellular matrix on the precision medicine utility of pancreatic cancer patient-derived organoids.

The use of patient-derived organoids (PDOs) to characterize therapeutic sensitivity and resistance is a promising precision medicine approach, and its potential to inform clinical decisions is now being tested in several large multiinstitutional clinical trials. PDOs are cultivated in the extracellular matrix from basement membrane extracts (BMEs) that are most commonly acquired commercially. Each clinical site utilizes distinct BME lots and may be restricted due to the availability of commercial BME sources. However, the effect of different sources of BMEs on organoid drug response is unknown. Here, we tested the effect of BME source on proliferation, drug response, and gene expression in mouse and human pancreatic ductal adenocarcinoma (PDA) organoids. Both human and mouse organoids displayed increased proliferation in Matrigel compared with Cultrex and UltiMatrix. However, we observed no substantial effect on drug response when organoids were cultured in Matrigel, Cultrex, or UltiMatrix. We also did not observe major shifts in gene expression across the different BME sources, and PDOs maintained their classical or basal-like designation. Overall, we found that the BME source (Matrigel, Cultrex, UltiMatrix) does not shift PDO dose-response curves or drug testing results, indicating that PDO pharmacotyping is a robust approach for precision medicine.

High-Temporal-Resolution In Situ Sensor for Oceanic CO2 Isotope Measurement Enabling Multidimensional Isotope Tracing Analysis (R13C, R18O, and R17O) via Laser Absorption Spectroscopy.

Combined in situ analysis of oceanic CO2 concentrations and diverse C and O isotope characteristics can offer a unique perspective with multiple isotopic tracing dimensions for identifying marine biogeochemical processes. Applying this strategy in marine environments is urgently required, yet it faces inherent challenges in terms of existing analytical methods and instruments, e.g., a lack of in situ sensors, limited detectable isotope variety, and low-temporal-resolution data. Here, we report an underwater in situ dissolved CO2 isotope sensor based on mid-infrared tunable diode laser absorption spectroscopy (MIR-TDLAS) and membrane extraction technology. Through the proposed targeted strategies, the sensor is capable of providing high-temporal-resolution in situ measurement of all monosubstituted isotopes of dissolved CO2 (16O13C16O, 18O12C16O, and 17O12C16O) at marine background concentrations. The sensor is demonstrated to provide comparable precision to that of isotope ratio mass spectrometry. At 400 ppmv, the precision for R13C, R18O, and R17O could achieve 0.084, 0.042, and 0.013‰, respectively, for a 1 s integration time. By enabling a high-frequency in situ analysis in fixed-point time-series field deployment, a 17O anomaly with strong regularity is observed, which is not obvious in 18O and 13C, and therefore, the superiority of the proposed multidimensional in situ isotope tracing strategy is demonstrated. The developed sensor has great potential to open up new prospects for advancing marine carbon research.

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.