Pleural Effusion Aspirate for Use in 3D Lung Cancer Modeling and Chemotherapy Screening.

Lung cancer is the leading cause of cancer-related deaths worldwide, yet most currently used in vitro cancer models are confined to traditional 2D cell culture conditions. Recently; however, innovative 3D models such as tumor tissue equivalents (organoids) have been adopted by researchers to recapitulate tissue architecture and physiology in order to improve disease modeling and drug testing. We have hypothesized that 3D lung organoids, incorporating cells directly from patients, will enable personalized disease modeling and tumor cell characterization compared to traditional 2D cultures. Here, we discuss the fabrication of 3D lung cancer organoids using a rare cell source, pleural effusion aspirate. We tracked the phenotypic change that developed in short-term culturing and characterized the cell population within the organoids. We found that isolated patient cells embedded directly into organoids created anatomically relevant structures and displayed lung cancer specific behaviors compared to cultures that first grew in 2D conditions. Additionally, we compared responses of patient cells from pleural effusion aspirates to chemotherapy in 2D and 3D cell culture systems. Our results show that cells in 2D cultures are more sensitive to treatment when compared with 3D organoids. Collectively, we have been able to isolate tumor cells from pleural effusion fluid of lung cancer patients and create organoids that display in vivo like anatomy and drug response. This technology can serve as a more accurate disease model for studying tumor progression and drug development.

Biofabricated 3D in vitro model of fibrosis-induced abnormal hepatoblast/biliary progenitors' expansion of the developing liver.

Congenital disorders of the biliary tract are the primary reason for pediatric liver failure and ultimately for pediatric liver transplant needs. Not all causes of these disorders are well understood, but it is known that liver fibrosis occurs in many of those afflicted. The goal of this study is to develop a simple yet robust model that recapitulates physico-mechanical and cellular aspects of fibrosis mediated via hepatic stellate cells (HSCs) and their effects on biliary progenitor cells. Liver organoids were fabricated by embedding various HSCs, with distinctive abilities to generate mild to severe fibrotic environments, together with undifferentiated liver progenitor cell line, HepaRG, within a collagen I hydrogel. The fibrotic state of each organoid was characterized by examination of extracellular matrix (ECM) remodeling through quantitative image analysis, rheometry, and qPCR. In tandem, the phenotype of the liver progenitor cell and cluster formation was assessed through histology. Activated HSCs (aHSCs) created a more severe fibrotic state, exemplified by a more highly contracted and rigid ECM, as well higher relative expression of TGF-ß, TIMP-1, LOXL2, and COL1A2 as compared to immortalized HSCs (LX-2). Within the more severe fibrotic environment, generated by the aHSCs, higher Notch signaling was associated with an expansion of CK19+ cells as well as the formation of larger, more densely populated cell biliary like-clusters as compared to mild and non-fibrotic controls. The expansion of CK19+ cells, coupled with a severely fibrotic environment, are phenomena found within patients suffering from a variety of congenital liver disorders of the biliary tract. Thus, the model presented here can be utilized as a novel in vitro testing platform to test drugs and identify new targets that could benefit pediatric patients that suffer from the biliary dysgenesis associated with a multitude of congenital liver diseases.

Cell Viability Assays in Three-Dimensional Hydrogels: A Comparative Study of Accuracy.

Three-dimensional (3D) cell culture systems, such as tumor organoids and multicellular tumor spheroids, have been developed in part as a result of major advances in tissue engineering and biofabrication techniques. 3D cell culture offers great capabilities in drug development, screening, testing, and precision medicine owing to its physiological accuracy. However, since the inception of 3D systems, few methods have been reported to successfully analyze cell viability quantitatively within hydrogel constructs. In this study, we describe and compare commercially available viability assays developed for two-dimensional (2D) applications for use in 3D constructs composed of organic, synthetic, or hybrid hydrogel formulations. We utilized Promega's CellTiter-Glo®, CellTiter-Glo 3D, and CellTiter 96® MTS Assay along with Thermo Fisher's PrestoBlue™ assay to determine if these assays can be used accurately in 3D systems. Compared with direct cell viability commonly used in 2D cell culture, our results show cellular health output inaccuracies among each assay in differing hydrogel formulations. Our results should inform researchers of potential errors when using cell viability measurements in 3D cultures and conclude that microscopic imaging should be used, in combination, for validation. Impact statement Three-dimensional (3D) tissue organoids models are a valuable tool not only for studying drug toxicity but also for understanding human embryonic development, intra-tissue morphogenesis, and mechanisms of disease. In cancer organoids, such 3D models may be used for preclinical chemotherapy screening and for understanding cell death and viability mechanisms under physiologically relevant conditions. Cell viability assays are necessary for assessing the effect of biological reagents on cellular health and have been used on in vitro cell cultures for many years. With the increase of 3D systems in cellular biology research to determine therapeutic efficacy, two-dimensional assays that measure cell viability are being used outside their intended use on 3D constructs. In this study, we assess the accuracy of using various commercially available cell viability assays on different 3D hydrogel constructs to help researchers understand expected variability in their experimentation along microscopic imaging validation.

Improved Antitumor Activity of the Fluoropyrimidine Polymer CF10 in Preclinical Colorectal Cancer Models through Distinct Mechanistic and Pharmacologic Properties.

Chemotherapy regimens that include 5-fluorouracil (5-FU) are central to colorectal cancer treatment; however, risk/benefit concerns limit 5-FU's use, necessitating development of improved fluoropyrimidine (FP) drugs. In our study, we evaluated a second-generation nanoscale FP polymer, CF10, for improved antitumor activity. CF10 was more potent than the prototype FP polymer F10 and much more potent than 5-FU in multiple colorectal cancer cell lines including HCT-116, LS174T, SW480, and T84D. CF10 displayed improved stability to exonuclease degradation relative to F10 and reduced susceptibility to thymidine antagonism due to extension of the polymer with arabinosyl cytidine. In colorectal cancer cells, CF10 strongly inhibited thymidylate synthase (TS), induced Top1 cleavage complex formation and caused replication stress, while similar concentrations of 5-FU were ineffective. CF10 was well tolerated in vivo and invoked a reduced inflammatory response relative to 5-FU. Blood chemistry parameters in CF10-treated mice were within normal limits. In vivo, CF10 displayed antitumor activity in several colorectal cancer flank tumor models including HCT-116, HT-29, and CT-26. CF10's antitumor activity was associated with increased plasma levels of FP deoxynucleotide metabolites relative to 5-FU. CF10 significantly reduced tumor growth and improved survival (84.5 days vs. 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer model that spontaneously metastasized to liver. Improved survival in the orthotopic model correlated with localization of a fluorescent CF10 conjugate to tumor. Together, our preclinical data support an early-phase clinical trial of CF10 for treatment of colorectal cancer.

Manipulating the Tumor Microenvironment in Tumor Organoids Induces Phenotypic Changes and Chemoresistance.

Tumors comprised a tightly surrounded tumor microenvironment, made up of non-cellular extracellular matrix (ECM) and stromal cells. Although treatment response is often attributed to tumor heterogeneity, progression and malignancy are profoundly influenced by tumor cell interactions with the surrounding ECM. Here, we used a tumor organoid model, consisting of hepatic stellate cells (HSCs) embedded in collagen type 1 (Col1) and colorectal cancer cell (HCT-116) spheroids, to determine the relationship between the ECM architecture, cancer cell malignancy, and chemoresistance. Exogenous transforming growth factor beta (TGF-ß) used to activate the HSCs increased the remodeling and bundling of Col1 in the ECM around the cancer spheroid. A dense ECM architecture inhibited tumor cell growth, reversed their mesenchymal phenotype, preserved stem cell population, and reduced chemotherapy response. Overall, our results demonstrate that controlled biofabrication and manipulation of the ECM in tumor organoids results enables studying tumor cell-ECM interactions and better understand tumor cell response to chemotherapies.

Simulating the human colorectal cancer microenvironment in 3D tumor-stroma co-cultures in vitro and in vivo.

The tumor microenvironment (TME) plays a significant role in cancer progression and thus modeling it will advance our understanding of cancer growth dynamics and response to therapies. Most in vitro models are not exposed to intact body physiology, and at the same time, fail to recapitulate the extensive features of the tumor stroma. Conversely, animal models do not accurately capture the human tumor architecture. We address these deficiencies with biofabricated colorectal cancer (CRC) tissue equivalents, which are built to replicate architectural features of biopsied CRC tissue. Our data shows that tumor-stroma co-cultures consisting of aligned extracellular matrix (ECM) fibers and ordered micro-architecture induced an epithelial phenotype in CRC cells while disordered ECM drove a mesenchymal phenotype, similar to well and poorly differentiated tumors, respectively. Importantly, co-cultures studied in vitro, and upon implantation in mice, revealed similar tumor growth dynamics and retention of architectural features for 28 days. Altogether, these results are the first demonstration of replicating human tumor ECM architecture in ex vivo and in vivo cultures.

Bioengineered Tumor Organoids.

Recent advances in biofabrication technologies, such as cell culture systems, and biomaterials have led to the development of three-dimensional (3D) cell culture platforms, such as tumor organoids. Tumor organoids are more physiologically accurate to the in vivo system, which they are intended to model, compared with traditional 2D cancer cell culture systems. Tumor organoids can mimic pathological and physical characteristics of tumors as well as maintain genetic stability of the cancer cells. Furthermore tumor organoids have advantage over animal models, being made from human cells and easily controlled in the laboratory to attain the desired tissue characteristics. In this section, we describe general tumor organoid technologies, the importance of the tumor microenvironment (TME) in model culture systems, and the use of tumor organoids in drug development and precision medicine. Organoid technologies continue to develop rapidly for applications in academic, clinical, and pharmaceutical settings.

Dysregulated Pyrimidine Biosynthesis Contributes to 5-FU Resistance in SCLC Patient-Derived Organoids but Response to a Novel Polymeric Fluoropyrimidine, CF10.

Chemo-immunotherapy is central to the treatment of small cell lung cancer (SCLC). Despite modest progress made with the addition of immunotherapy, current cytotoxic regimens display minimal survival benefit and new treatments are needed. Thymidylate synthase (TS) is a well-validated anti-cancer drug target, but conventional TS inhibitors display limited clinical efficacy in refractory or recurrent SCLC. We performed RNA-Seq analysis to identify gene expression changes in SCLC biopsy samples to provide mechanistic insight into the potential utility of targeting pyrimidine biosynthesis to treat SCLC. We identified systematic dysregulation of pyrimidine biosynthesis, including elevated TYMS expression that likely contributes to the lack of efficacy for current TS inhibitors in SCLC. We also identified E2F1-3 upregulation in SCLC as a potential driver of TYMS expression that may contribute to tumor aggressiveness. To test if TS inhibition could be a viable strategy for SCLC treatment, we developed patient-derived organoids (PDOs) from human SCLC biopsy samples and used these to evaluate both conventional fluoropyrimidine drugs (e.g., 5-fluorouracil), platinum-based drugs, and CF10, a novel fluoropyrimidine polymer with enhanced TS inhibition activity. PDOs were relatively resistant to 5-FU and while moderately sensitive to the front-line agent cisplatin, were relatively more sensitive to CF10. Our studies demonstrate dysregulated pyrimidine biosynthesis contributes to drug resistance in SCLC and indicate that a novel approach to target these pathways may improve outcomes.

Improved potency of F10 relative to 5-fluorouracil in colorectal cancer cells with p53 mutations.

AIM: Resistance to fluoropyrimidine drugs (FPs) is a major cause of mortality in colorectal cancer (CRC). We assessed the potency advantage of the polymeric FP F10 relative to 5-fluorouracil (5FU) in four human CRC cell lines that differ only in TP53 mutational status to determine how p53 mutations affect drug response and whether F10 is likely to improve outcomes. METHODS: HCT-116 human CRC cells (p53+/+) and three isogenic variants (p53-/-, R248W/+, R248W/-) were assessed for drug response. Resistance factors were derived from cell viability data and used to establish the relative potency advantage for F10. Rescue studies with exogenous uridine/thymidine determined if cytotoxicity resulted from DNA-directed processes. RESULTS: Significant resistance to 5-FU resulted from p53-loss or from gain-of-function (GOF) mutation (R248W) and was greatest when GOF mutation was coupled with loss of wild-type p53. F10 is much more potent than 5-FU (137-314-fold depending on TP53 mutational status). F10 and 5-FU induce apoptosis by DNA- and RNA-directed mechanisms, respectively, and only F10 shows a modest enhancement in cytotoxicity upon co-treatment with leucovorin. CONCLUSION: TP53 mutational status affects inherent sensitivity to FPs, with p53 GOF mutations most deleterious. F10 is much more effective than 5-FU regardless of TP53 mutations and has potential to be effective to CRC that is resistant to 5-FU due, in part, to TP53 mutations.6,7.

Exploring the activity of a polyazine bridged Ru(ii)-Pt(ii) supramolecule in F98 rat malignant glioma cells.

The mixed-metal supramolecular complex, [(Ph2phen)2Ru(dpp)PtCl2]2+, displays significant DNA modification, cell growth inhibition, and toxicity towards F98 malignant glioma cells following visible light irradiation. The design of this complex affords superior cellular uptake and antiproliferative activity compared to the classic chemotherapeutic agent, cisplatin.