Enabling high throughput drug discovery in 3D cell cultures through a novel bioprinting workflow.

Advanced three dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter RASTRUM with the high throughput screening assay AlphaLISA, we demonstrate the utility of the protocol in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3ß phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

Generation and analysis of 3D cell culture models for drug discovery.

Successful preclinical drug testing relies in part on data generated using in vitro cell culture models that recapitulate the structure and function of tumours and other tissues in vivo. The growing evidence that 3D cell models can more accurately predict the efficacy of drug responses compared to traditionally utilised 2D cell culture systems has led to continuous scientific and technological advances that enable better physiologically representative in vitro modelling of in vivo tissues. This review will provide an overview of the utility of current 3D cell models from a drug screening perspective and explore the future of 3D cell models for drug discovery applications.

N-Alkylisatin-Loaded Liposomes Target the Urokinase Plasminogen Activator System in Breast Cancer.

The urokinase plasminogen activator and its receptor (uPA/uPAR) are biomarkers for metastasis, especially in triple-negative breast cancer. We prepared anti-mitotic N-alkylisatin (N-AI)-loaded liposomes functionalized with the uPA/uPAR targeting ligand, plasminogen activator inhibitor type 2 (PAI-2/SerpinB2), and assessed liposome uptake in vitro and in vivo. Receptor-dependent uptake of PAI-2-functionalized liposomes was significantly higher in the uPA/uPAR overexpressing MDA-MB-231 breast cancer cell line relative to the low uPAR/uPAR expressing MCF-7 breast cancer cell line. Furthermore, N-AI cytotoxicity was enhanced in a receptor-dependent manner. In vivo, PAI-2 N-AI liposomes had a plasma half-life of 5.82 h and showed an increased accumulation at the primary tumor site in an orthotopic MDA-MB-231 BALB/c-Fox1nu/Ausb xenograft mouse model, relative to the non-functionalized liposomes, up to 6 h post-injection. These findings support the further development of N-AI-loaded PAI-2-functionalized liposomes for uPA/uPAR-positive breast cancer, especially against triple-negative breast cancer, for which the prognosis is poor and treatment is limited.

Increased splenic human CD4+:CD8+ T cell ratios, serum human interferon-γ and intestinal human interleukin-17 are associated with clinical graft-versus-host disease in humanized mice.

Graft-versus-host disease (GVHD) is a frequent complication following allogeneic hematopoietic stem cell transplantation (HSCT) with current therapies limited to general immunosuppression. Humanized mouse models of GVHD are emerging as valuable intermediaries to allow translation of findings from allogeneic mouse models to humans to prevent and treat this disease, but such models require further characterization. In this study, humanized mice were generated by injecting immunodeficient non-obese diabetic severe combined immunodeficiency interleukin (IL)-2 receptor γ common chain null (NSG) mice with human peripheral blood mononuclear cells (hPBMCs). Clinical GVHD development was assessed using established scoring criteria (weight loss, posture, activity, fur texture and skin integrity). Differences between humanized NSG mice that developed clinical or subclinical GVHD were then compared. Both groups of mice demonstrated similar frequencies of human leukocyte engraftment. In contrast, mice that developed clinical GVHD demonstrated increased histological damage compared to mice with subclinical GVHD. Furthermore, mice with clinical GVHD exhibited increases in the splenic human CD4+:CD8+ T cell ratio, serum human interferon (IFN)-γ and intestinal human IL-17 expression compared to mice with subclinical GVHD. These cellular and molecular changes could be used as potential markers of disease progression in this preclinical model. This study also provides further insights into GVHD development which may be relevant to human HSCT recipients.

Quantification of ligand density and stoichiometry on the surface of liposomes using single-molecule fluorescence imaging.

Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11 ±â€¯4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic.

Towards clinical translation of ligand-functionalized liposomes in targeted cancer therapy: Challenges and opportunities.

The development of therapeutic resistance to targeted anticancer therapies remains a significant clinical problem, with intratumoral heterogeneity playing a key role. In this context, improving the therapeutic outcome through simultaneous targeting of multiple tumor cell subtypes within a heterogeneous tumor is a promising approach. Liposomes have emerged as useful drug carriers that can reduce systemic toxicity and increase drug delivery to the tumor site. While clinically used liposomal drug formulations show marked therapeutic advantages over free drug formulations, ligand-functionalized liposomes that can target multiple tumor cell subtypes may further improve the therapeutic efficacy by facilitating drug delivery to a broader population of tumor cells making up the heterogeneous tumor tissue. Ligand-directed liposomes enable the so-called active targeting of cell receptors via surface-attached ligands that direct drug uptake into tumor cells or tumor-associated stromal cells, and so can increase the selectivity of drug delivery. Despite promising preclinical results demonstrating improved targeting and anti-tumor effects of ligand-directed liposomes, there has been limited translation of this approach to the clinic. Key challenges for translation include the lack of established methods to scale up production and comprehensively characterize ligand-functionalized liposome formulations, as well as the inadequate recapitulation of in vivo tumors in the preclinical models currently used to evaluate their performance. Herein, we discuss the utility of recent ligand-directed liposome approaches, with a focus on dual-ligand liposomes, for the treatment of solid tumors and examine the drawbacks limiting their progression to clinical adoption.

Translationally controlled tumour protein TCTP is induced early in human colorectal tumours and contributes to the resistance of HCT116 colon cancer cells to 5-FU and oxaliplatin.

BACKGROUND: Translationally controlled tumour protein TCTP is an anti-apoptotic protein frequently overexpressed in cancers, where high levels are often associated with poor patient outcome. TCTP may be involved in protecting cancer cells against the cytotoxic action of anti-cancer drugs. Here we study the early increase of TCTP levels in human colorectal cancer (CRC) and the regulation of TCTP expression in HCT116 colon cancer cells, in response to treatment with the anti-cancer drugs 5-FU and oxaliplatin. METHODS: Using immunohistochemistry, we assessed TCTP levels in surgical samples from adenomas and adenocarcinomas of the colon, compared to normal colon tissue. We also studied the regulation of TCTP in HCT116 colon cancer cells in response to 5-FU and oxaliplatin by western blotting. TCTP mRNA levels were assessed by RT-qPCR. We used mTOR kinase inhibitors to demonstrate mTOR-dependent translational regulation of TCTP under these conditions. Employing the Real-Time Cell Analysis (RTCA) System and the MTS assay, we investigated the effect of TCTP-knockdown on the sensitivity of HCT116 cells to the anti-cancer drugs 5-FU and oxaliplatin. RESULTS: 1. TCTP levels are significantly increased in colon adenomas and adenocarcinomas, compared to normal colon tissue. 2. TCTP protein levels are about 4-fold upregulated in HCT116 colon cancer cells, in response to 5-FU and oxaliplatin treatment, whereas TCTP mRNA levels are down regulated. 3. mTOR kinase inhibitors prevented the up-regulation of TCTP protein, indicating that TCTP is translationally regulated through the mTOR complex 1 signalling pathway under these conditions. 4. Using two cellular assay systems, we demonstrated that TCTP-knockdown sensitises HCT116 cells to the cytotoxicity caused by 5-FU and oxaliplatin. CONCLUSIONS: Our results demonstrate that TCTP levels increase significantly in the early stages of CRC development. In colon cancer cells, expression of this protein is largely upregulated during treatment with the DNA-damaging anti-cancer drugs 5-FU and oxaliplatin, as part of the cellular stress response. TCTP may thus contribute to the development of anti-cancer drug resistance. These findings indicate that TCTP might be suitable as a biomarker and that combinatorial treatment using 5-FU/oxaliplatin, together with mTOR kinase inhibitors, could be a route to preventing the development of resistance to these drugs.

N-alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer cell lines.

The search for novel anticancer therapeutics with the ability to overcome multi-drug resistance (MDR) mechanisms is of high priority. A class of molecules that show potential in overcoming MDR are the N-alkylated isatins. In particular 5,7-dibromo-N-alkylisatins are potent microtubule destabilizing agents that act to depolymerize microtubules, induce apoptosis and inhibit primary tumor growth in vivo. In this study we evaluated the ability of four dibrominated N-alkylisatin derivatives and the parent compound, 5,7-dibromoisatin, to circumvent MDR. All of the isatin-based compounds examined retained potency against the MDR cell lines; U937VbR and MES-SA/Dx5 and displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines. We show that one mechanism by which the isatin-based compounds overcome MDR is by circumventing P-glycoprotein (P-gp) mediated drug efflux. Thus, as the isatin-based compounds are not susceptible to extrusion from P-gp overexpressing tumor cells, they represent a promising alternative strategy as a stand-alone or combination therapy for treating MDR cancer.