RESOURCES

Abstract
Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel - tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma – tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Abstract
The role of the extracellular matrix (ECM) in tumor recurrence and metastasis has been gaining attention. Indeed, not only cellular, but also structural proteins influence migratory and invasive capacity of tumor cells, including growth and resistance to drugs. Therefore, new in vitro tumor models that entail improved ECM formation and deposition are needed. Here, we are developed three-dimensional (3D) models of pediatric soft tissue sarcoma (Rhabdomyosarcoma [RMS]) with the two major subgroups, the embryonal (ERMS) and the alveolar (ARMS) form. We applied macromolecular crowding (MMC) technology to monolayer cultures, spheroids, and 3D bioprinted constructs. In all culture models, exposure to MMC significantly increased ECM deposition. Interestingly, bioprinted constructs showed a collagen and fibronectin matrix architecture that was comparable to that of tumor xenografts. Furthermore, the bioprinted model not only showed tumor cell growth inside the structure but also displayed cell clusters leaving the edges of the bioprinted construct, probably emulating a metastatic mechanism. ARMS and ERMS cells reacted differently in the bioprinted structure. Indeed, the characteristic metastatic behavior was much more pronounced in the more aggressive ARMS subtype. This promising approach opens new avenues for studying RMS microenvironment and creating a platform for cancer drug testing including the native tumor ECM.

Abstract
//        James R. Browning 1 , Paige Derr 2 , Kristy Derr 2 , Nicole Doudican 3 , Sam Michael 2 , Samantha R. Lish 1 , Nicholas A. Taylor 3 , James G. Krueger 1 , Marc Ferrer 2 , John A. Carucci 3  and Daniel S. Gareau 1    1   Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York, USA    2   National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA    3   The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA   Correspondence to:     Daniel S. Gareau,  email:    dgareau@rockefeller.edu         Keywords:  squamous cell carcinoma; screening; 3D printing; in vitro model; confocal microscopy     Received:  January 05, 2020      Accepted:  April 03, 2020      Published:  July 07, 2020       ABSTRACT      Cutaneous squamous cell carcinoma (cSCC) causes approximately 10,000 deaths annually in the U. S. Current therapies are largely ineffective against metastatic and locally advanced cSCC. There is a need to identify novel, effective, and less toxic small molecule cSCC therapeutics. We developed a 3-dimensional bioprinted skin (3DBPS) model of cSCC tumors together with a microscopy assay to test chemotherapeutic effects in tissue. The full thickness SCC tissue model was validated using hematoxylin and eosin (H&E) and immunohistochemical histological staining, confocal microscopy, and cDNA microarray analysis. A nondestructive, 3D fluorescence confocal imaging assay with tdTomato-labeled A431 SCC and ZsGreen-labeled keratinocytes was developed to test efficacy and general toxicity of chemotherapeutics. Fluorescence-derived imaging biomarkers indicated that 50% of cancer cells were killed in the tissue after 1?M 5-Fluorouracil 48-hour treatment, compared to a baseline of 12% for untreated controls. The imaging biomarkers also showed that normal keratinocytes were less affected by treatment (11% killed) than the untreated tissue, which had no significant killing effect. Data showed that 5-Fluorouracil selectively killed cSCC cells more than keratinocytes. Our 3DBPS assay platform provides cellular-level measurement of cell viability and can be adapted to achieve nondestructive high-throughput screening (HTS) in bio-fabricated tissues.

Abstract
Three-dimensional (3D) bioprinted culture systems allow to accurately control microenvironment components and analyze their effects at cellular and tissue levels. The main objective of this study was to identify, quantify and localize the effects of physical-chemical communication signals between tumor cells and the surrounding biomaterial stiffness over time, defining how aggressiveness increases in SK-N-BE(2) neuroblastoma (NB) cell line. Biomimetic hydrogels with SK-N-BE(2) cells, methacrylated gelatin and increasing concentrations of methacrylated alginate (AlgMA 0%, 1% and 2%) were used. Young’s modulus was used to define the stiffness of bioprinted hydrogels and NB tumors. Stained sections of paraffin-embedded hydrogels were digitally quantified. Human NB and 1% AlgMA hydrogels presented similar Young´s modulus mean, and orthotopic NB mice tumors were equally similar to 0% and 1% AlgMA hydrogels. Porosity increased over time; cell cluster density decreased over time and with stiffness, and cell cluster occupancy generally increased with time and decreased with stiffness. In addition, cell proliferation, mRNA metabolism and antiapoptotic activity advanced over time and with stiffness. Together, this rheological, optical and digital data show the potential of the 3D in vitro cell model described herein to infer how intercellular space stiffness patterns drive the clinical behavior associated with NB patients.

Abstract
Bioprinting technologies are powerful new bioengineering tools that can spatially reproduce multiple microenvironmental cues in a highly controlled, tunable, and precise manner. In this study, microvalve bioprinting technology was successfully used to print in close proximity endothelial and tumor cells at higher concentrations than previously thought possible, while preserving their viability. We propose that the resulting multicellular models, bioprinted in a controlled extracellular matrix microenvironment, are well-suited to study endothelial and cancer cell crosstalk within a cancer niche. As proof of concept, microvalve bioprinting was applied to the bioengineering of a simplified glioblastoma model in which biological processes involved in tumor expansion, such as tumor cell invasion patterns, cell proliferation, and senescence could be easily visualized and quantified. In this model, U251 glioblastoma cells and primary human umbilical vein endothelial cells (HUVECs) exhibited good printability and high viability after printing. U251 cells formed physiologically relevant clusters and invasion margins, while HUVECs generated vascular-like networks when primary fibroblasts were added to the model. An oxidative stress mimicking the one encountered within a tumor microenvironment during radiotherapy or genotoxic chemotherapy was shown to both diminish endothelial cells proliferation and to increase their senescence. Results also suggested that stressed glioblastoma cells may alter normal endothelial cell proliferation but not impact their senescence. This data demonstrates the potential of microvalve bioprinting to fabricate in vitro models that can help decipher endothelial and tumor cell crosstalk, within controlled and modulable microenvironments, and can then be used to address critical questions in the context of cancer recurrence.

Abstract
Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.

Abstract
The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.

Abstract
The tumor extracellular matrix (ECM) plays a vital role in tumor progression and drug resistance. Previous studies have shown that breast tissue-derived matrices could be an important biomaterial to recreate the complexity of the tumor ECM. We have developed a method for decellularizing and delipidating a porcine breast tissue (TDM) compatible with hydrogel formation. The addition of gelatin methacrylamide and alginate allows this TDM to be bioprinted by itself with good printability, shape fidelity, and cytocompatibility. Furthermore, this bioink has been tuned to more closely recreate the breast tumor by incorporating collagen type I (Col1). Breast cancer cells (BCCs) proliferate in both TDM bioinks forming cell clusters and spheroids. The addition of Col1 improves the printability of the bioink as well as increases BCC proliferation and reduces doxorubicin sensitivity due to a downregulation of HSP90. TDM bioinks also allow a precise three-dimensional printing of scaffolds containing BCCs and stromal cells and could be used to fabricate artificial tumors. Taken together, we have proven that these novel bioinks are good candidates for biofabricating breast cancer models.

Abstract
Three dimensional (3D) bioprinting technology has been making a progressive advancement in the field of tissue engineering to produce tissue constructs that mimic the shape, framework, and microenvironment of an organ. The technology has not only paved the way to organ development but has been widely studied for its application in drug and cosmetic testing using 3D bioprinted constructs. However, not much has been explored on the utilization of bioprinting technology for the development of tumor models to test anti-cancer drug efficacy. The conventional methodology involves a two dimensional (2D) monolayer model to test cellular drug response which has multiple limitations owing to its inability to mimic the natural tissue environment. The choice of bioink for 3D bioprinting is critical as cell morphology and proliferation depend greatly on the property of bioink. In this study, we developed a multicomponent bioink composed of alginate, diethylaminoethyl cellulose, gelatin, and collagen peptide to generate a 3D bioprinted construct. The bioink has been characterised and validated for its printability, shape fidelity and biocompatibility to be used for generating tumor models. Further, a bioprinted tumor model was developed using lung cancer cell line and the efficacy of 3D printed construct for drug screening application was established.

Abstract
Metastatic, castration-resistant prostate cancer (mCRPC) is an advanced prostate cancer with limited therapeutic options and poor patient outcomes. To investigate whether multitargeted kinase inhibitors (KIs) represent an opportunity for mCRPC drug development, we applied machine learning{textendash}based functional screening and identified two KIs, PP121 and SC-1, which demonstrated strong suppression of CRPC growth in vitro and in vivo. Furthermore, we show the marked ability of these KIs to improve on standard-of-care chemotherapy in both tumor response and survival, suggesting that combining multitargeted KIs with chemotherapy represents a promising avenue for mCRPC treatment. Overall, our findings demonstrate the application of a multidisciplinary strategy that blends bench science with machine-learning approaches for rapidly identifying KIs that result in desired phenotypic effects.Castration-resistant prostate cancer (CRPC) is an advanced subtype of prostate cancer with limited therapeutic options. Here, we applied a systems-based modeling approach called kinome regularization (KiR) to identify multitargeted kinase inhibitors (KIs) that abrogate CRPC growth. Two predicted KIs, PP121 and SC-1, suppressed CRPC growth in two-dimensional in vitro experiments and in vivo subcutaneous xenografts. An ex vivo bone mimetic environment and in vivo tibia xenografts revealed resistance to these KIs in bone. Combining PP121 or SC-1 with docetaxel, standard-of-care chemotherapy for late-stage CRPC, significantly reduced tibia tumor growth in vivo, decreased growth factor signaling, and vastly extended overall survival, compared to either docetaxel monotherapy. These results highlight the utility of computational modeling in forming physiologically relevant predictions and provide evidence for the role of multitargeted KIs as chemosensitizers for late-stage, metastatic CRPC.All study data are included in the article and/or supporting information.

Abstract
Radium 223 (223Ra) is an α-emitter approved for the treatment of bone metastatic prostate cancer (PCa), which exerts direct cytotoxicity towards PCa cells near the bone interface, whereas cells positioned in the core respond poorly, due to short α-particle penetrance. β1 integrin (β1I) interference has been shown to increase radiosensitivity and significantly enhance external beam radiation efficiency. We hypothesized that targeting β1I would improve 223Ra outcome. We tested the effect of combining 223Ra and anti-β1I antibody treatment in PC3 and C4-2B PCa cell models expressing high and low β1I levels, respectively. In vivo tumor growth was evaluated through bioluminescence. Cellular and molecular determinants of response were analyzed by ex vivo three-dimensional imaging of bone lesions, proteomic analysis and further confirmed by computational modeling and in vitro functional analysis in tissue-engineered bone mimetic systems. Interference with β1I combined with 223Ra reduced PC3 cell growth in bone and significantly improved overall mouse survival, while no change was achieved in C4-2B tumors. Anti-β1I treatment decreased PC3 tumor cell mitosis index and spatially expanded 223Ra lethal effects two-fold, in vivo and in silico. Regression was paralleled by decreased expression of radio-resistance mediators. Targeting β1I significantly improves 223Ra outcome and points towards combinatorial application in PCa tumors with high β1I expression.

Abstract
The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.

Abstract
Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible.

Abstract
Abstract The composition and intercellular interactions of tumor cells in the tissues dictate the biochemical and metabolic properties of the tumor microenvironment. The metabolic rewiring has a profound impact on the properties of the microenvironment, to an extent that monitoring such perturbations could harbor diagnostic and therapeutic relevance. A growing interest in these phenomena has inspired the development of novel technologies with sufficient sensitivity and resolution to monitor metabolic alterations in the tumor microenvironment. In this context, surface-enhanced Raman scattering (SERS) can be used for the label-free detection and imaging of diverse molecules of interest among extracellular components. Herein, the application of nanostructured plasmonic substrates comprising Au nanoparticles, self-assembled as ordered superlattices, to the precise SERS detection of selected tumor metabolites, is presented. The potential of this technology is first demonstrated through the analysis of kynurenine, a secreted immunomodulatory derivative of the tumor metabolism and the related molecules tryptophan and purine derivatives. SERS facilitates the unambiguous identification of trace metabolites and allows the multiplex detection of their characteristic fingerprints under different conditions. Finally, the effective plasmonic SERS substrate is combined with a hydrogel-based three-dimensional cancer model, which recreates the tumor microenvironment, for the real-time imaging of metabolite alterations and cytotoxic effects on tumor cells.

Abstract
Polydopamine can form biocompatible particles that convert light into heat. Recently, a protocol has been optimized to synthesize polydopamine/protein hybrid nanoparticles that retain the biological function of proteins, and combine it with the stimuli-induced heat generation of polydopamine. We have utilized this novel system to form polydopamine particles, containing transferrin (PDA/Tf). Mouse melanoma cells, which strongly express the transferrin receptor, were exposed to PDA/Tf nanoparticles (NPs) and, subsequently, were irradiated with a UV laser. The cell death rate was monitored in real-time. When irradiated, the melanoma cells exposed to PDA/Tf NPs underwent apoptosis, faster than the control cells, pointing towards the ability of PDA/Tf to mediate UV-light-induced cell death. The system was also validated in an organotypic, 3D-printed tumor spheroid model, comprising mouse melanoma cells, and the exposure and subsequent irradiation with UV-light, yielded similar results to the 2D cell culture. The process of apoptosis was found to be targeted and mediated by the lysosomal membrane permeabilization. Therefore, the herein presented polydopamine/protein NPs constitute a versatile and stable system for cancer cell-targeting and photothermal apoptosis induction.