Microtissue Size Research Articles

SP7 gene silencing dampens bone marrow stromal cell hypertrophy, but it also dampens chondrogenesis

For bone marrow stromal cells (BMSC) to be useful in cartilage repair their propensity for hypertrophic differentiation must be overcome. A single day of TGF-β1 stimulation activates intrinsic signaling cascades in BMSCs which subsequently drives both chondrogenic and hypertrophic differentiation. TGF-β1 stimulation upregulates SP7, a transcription factor known to contribute to hypertrophic differentiation, and SP7 remains upregulated even if TGF-β1 is subsequently withdrawn from the chondrogenic induction medium. Herein, we stably transduced BMSCs to express an shRNA designed to silence SP7, and assess the capacity of SP7 silencing to mitigate hypertrophy. SP7 silencing dampened both hypertrophic and chondrogenic differentiation processes, resulting in diminished microtissue size, impaired glycosaminoglycan production and reduced chondrogenic and hypertrophic gene expression. Thus, while hypertrophic features were dampened by SP7 silencing, chondrogenic differentation was also compromised. We further investigated the role of SP7 in monolayer osteogenic and adipogenic cultures, finding that SP7 silencing dampened characteristic mineralization and lipid vacuole formation, respectively. Overall, SP7 silencing affects the trilineage differentiation of BMSCs, but is insufficient to decouple BMSC hypertrophy from chondrogenesis. These data highlight the challenge of promoting BMSC chondrogenesis whilst simultaneously reducing hypertrophy in cartilage tissue engineering strategies.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

SummaryBone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

Screening immunotherapy cancer drugs in a scalable, physiologically relevant 3D in vitro tumor microenvironment model

Abstract Interaction of tumor cells with surrounding stromal and immune cells, secreted cytokines, and extracellular matrix proteins, collectively known as the tumor microenvironment (TME), is crucial for cancer progression and metastasis. Here, we present a novel 3D in vitro TME model to assess candidate drugs for cancer therapy. For this study, we prepared lung carcinoma spheroids from GFP-A549 cells co-cultured with cancer-associated fibroblasts (CAFs) and PBMCs. To simulate a pro-inflammatory tumor microenvironment, we activated PBMCs with cytokines and/or anti-CD3/CD28. We assessed the effect of PD-1 inhibitors in combination with cytokines for potential synergistic anti-cancer effects. We then developed a tumor model using melanoma PDX cells labeled with CellTracker to monitor tumor cell viability after exposure to activated PBMCs. We evaluated killing activity and effector function of T cells by measuring tumor microtissue size by automatic stage fluorescence microscopy and release of IL-6, TNFa, IFNγ and GM-CSF with a MAGPIXTM Luminex system over time. Immune cell infiltration and its effects on tumor were assessed by histological analysis using tumor, immune cell and apoptosis markers. Our results show combination treatment with cytokines and anti-PD-1 antibody led to higher secretion of IL-6/TNFa/IFNγ/GM-CSF, reduced tumor size, increased infiltration of the model by CD8+ T cells, stronger expression of Granzyme B resulting in enhanced tumor apoptosis and killing. This confirms our novel 3D in vitro TME model is a promising tool for studying cell-cell interactions and allows for HCS of novel candidates for anti-cancer drugs.

Abstract 5079: Immuno-competent 3d in vitro tumor models as novel platform to assess combinatorial biologics therapy

Abstract Background: Immunotherapy has revolutionized cancer treatment, but clinical success is still limited to a subset of patients, underlining the need for novel therapeutic targets. Available ex vivo animal platforms for drug screening show limitations due to only partial recapitulation of tumor-intrinsic features and of the interactions of tumors with stromal/immune cells. Here, we review our 3D InSightTM tumor models, a complex in-vitro platform allowing to study the efficacy of immune-targeting agents on tumor infiltration and killing by peripheral blood mononuclear cells (PBMCs) and to acquire insights on the modulatory capacity of immune checkpoint inhibitors. Aim: In the present study we have developed a 3D tumor - immune cell coculture model and evaluated its application to study the effect of immune checkpoint inhibitors on tumor viability, cytokine secretion, and immune cell infiltration. Material and Methods: Lung carcinoma GFP-A549 cells were cultured with dermal fibroblasts and PBMCs in Akura 96/384-well plates. Immune cells were pre-treated using different activation protocols to induce priming and pT-cell exhaustion. We assessed the effect of PD-1 inhibitors in combination with cytokines for potential synergistic anti-cancer effects. Killing activity and effector function of T cells were evaluated by measuring tumor microtissue size by automatic stage fluorescence microscopy and measuring release of IL-6, TNF, IFNγ and GM-CSF with a MAGPIXTM Luminex system over time. Immune tumor infiltration was quantified using fluorescent staining of CD3+ cells and the effect of the immune cell attack on the tumor cells was assessed by IHC staining. Results: Combination treatment with cytokines and anti-PD-1 led to higher release of IL-6/TNF/IFNγ/GM-CSF and increased tumor microtissue infiltration by CD3+ lymphocytes, stronger expression of Granzyme B, resulting in enhanced tumor killing and apoptosis. Conclusion: Our 3D InSightTM tumor models can be successfully employed for optimal high throughput screening of biologics to unveil the efficacy and anti-tumor efficacy of immune-targeting therapeutic agents. Citation Format: Irina Agarkova, Nicole Buschmann, Silvan Strebel, Armin Wolf, Francesca Chiovaro. Immuno-competent 3d in vitro tumor models as novel platform to assess combinatorial biologics therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5079.

Immune-competent 3D InSightTM tumour models as novel platform to assess combinatorial biologics therapy

Abstract Background Immunotherapy has revolutionized cancer treatment, but clinical success is still limited to a subset of patients, underlining the need for novel therapeutic targets. Available ex vivo platforms for drug screening show limitations due to only partial recapitulation of tumor-intrinsic features and of the interactions of tumours with stromal/immune cells. Here, we review our 3D InSightTM tumour models, a complex platform allowing us to study the efficacy of immune-targeting agents on tumour infiltration and killing by peripheral blood mononuclear cells (PBMCs) using clinically relevant models of 3D tumour microtissues from patient-derived xenografts (PDX) with high translational potential. Methods Lung carcinoma GFP-A549 cells were cultured with dermal fibroblasts and PBMCs in Akura 96/384-well plates. To generate a pro-inflammatory tumour microenvironment, PBMCs were activated with cytokines, anti-CD3/CD28 or their combination. We assessed the effect of PD-1 inhibitors in combination with cytokines for potential synergistic anti-cancer effects. 3D InSight™ tumour Microtissue from Melanoma PDX were labeled with CellTracker to monitor for tumour cell viability once exposed to PBMCs. Killing activity and effector function of T cells were evaluated by measuring tumour microtissue size by automatic stage fluorescence microscopy and measuring release of IL-6, TNF, IFNγ and GM-CSF with a MAGPIXTM Luminex system over time. Immune tumour infiltration was quantified and the impact on survival was assessed histologically. Results Combination treatment with cytokines and anti-PD-1 led to higher release of IL-6/TNF/IFNγ/GM-CSF and increased tumour microtissue infiltration by CD8+ T cells, resulting in enhanced tumour killing. Conclusion Our 3D InSightTM tumour models can be successfully employed for optimal high throughput screening of biologics to unveil the efficacy and anti-tumour properties of immune-targeting therapeutic agents. Legal entity responsible for the study InSphero. Funding InSphero AG. Disclosure All authors have declared no conflicts of interest.

Abstract 57: Development of an in vitro 3D model system for testing PD-1/ PDL-1 immune checkpoint inhibitors

Abstract Background Immune checkpoint inhibitors provide a new hope for cancer patients not responding to chemotherapy by removing the tumor protection against immune cell attacks. Nevertheless, only a subset of patients responds well to these therapies, and recent studies demonstrated that the mechanisms of action are more complex than expected, often involving multiple cell types of the immune system. Importantly, for a better understanding and assessment of immune cell eliciting cell tumor death, there is a need to have fully human, complex 3D in vitro models. We developed a 3D tumor-immune cell coculture model and show here acquired insights on the modulatory capacity of immune checkpoint inhibitors. Aim In the present study we have developed a 3D tumor - immune cell coculture model and evaluated its application to study the effect of immune checkpoint inhibitors on tumor viability, cytokine secretion, and immune cell infiltration. Material & Methods GFP-labelled A549 tumor cells were aggregated together with human dermal fibroblast to produce 3D tumor microtissues in Akura 96-well and 384-well format. Immune cells were pre-treated using different activation protocols to induce priming and pT-cell exhaustion. 3D tumor-immune cell cocultures were treated for 10 days with Nivolumab, its corresponding isotype and non-treated controls. Tumor viability was measured by GFP-fluorescence over time. Additionally, we have evaluated the effect of nivolumab by measuring the microtissue size by automatic stage fluorescence microscopy. Cytokine levels of IL-2, TNFα, IFNγ and GS-CSM were measured with a MAGPIXTM Luminex system and were monitored over time. The validation of immune cell infiltration and characterization of tumor and immune cell specific markers was based on histological analysis. Results Based on tumor fluorescence we have shown a superior effect of Nivolumab versus its isotype control antibody on tumor viability at the end of the treatment period. The cytokine analysis of supernatants has shown higher levels of IFNγ and GS-CSM in the wells treated with Nivolumab compared to the isotype control. CoCultures with PBMCs have shown stronger activation upon treatment compared to the T-cells alone. The histological analysis has demonstrated a higher number of T-cell infiltration and activation, as well as a stronger induction of apoptosis in the tumor microtissues treated with Nivolumab. Conclusion In summary, our novel in vitro immuno-oncology model system allows high throughput screening of immune check point inhibitors alone and in combination with other anti-cancer drugs for making it efficient for a broad range of tumor diseases. Citation Format: Nicole Buschmann, Silvan Strebel, Francesca Chiovaro, Patrick Guye, Irina Agarkova. Development of an in vitro 3D model system for testing PD-1/ PDL-1 immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 57.

Correction to: Microtissue size and cell-cell communication modulate cell migration in arrayed 3D collagen gels.

The original version of this article unfortunately contained a mistake. One line indicating statistical significance was improperly placed in Fig. 5.

Microtissue size and cell-cell communication modulate cell migration in arrayed 3D collagen gels.

Cells communicate through the extracellular matrix (ECM) in many physiological and pathological processes. This is particularly important during cell migration, where cell communication can alter both the speed and the direction of migration. However, most cell culture systems operate with large volumes relative to cell numbers, creating low cell densities and diluting factors that mediate cell communication. Furthermore, they lack the ability to isolate single cells or small groups of cells. Droplet forming devices allow for an ability to embed single or small groups of cells into small volume segregated 3D environments, increasing the cell density to physiological levels. In this paper we show a microfluidic droplet device for fabricating 3D collagen-based microtissues to study breast cancer cell motility. MDA-MB-231 cells fail to spread and divide in small, thin chambers. Cell migration is also stunted as compared to thick 3D gels. However, larger chambers formed by a thicker devices promote cell spreading, cell division and faster migration. In the large devices, both cell-ECM and cell-cell interactions affect cell motility. Increasing collagen density decreases cell migration and increasing the number of cells per chamber increases cell migration speed. Furthermore, cells appear to sense both the ECM-chamber wall interface as well as other cells. Cells migrate towards the ECM-chamber interface if within roughly 150μm, whereas cells further than 150μm tend to move towards the center of the chamber. Finally, while cells do not show enhanced movement towards the center of mass of a cell cluster, their migration speed is more variable when further away from the cell cluster center of mass. These results show that microfluidic droplet devices can array 3D collagen gels and promote cell spreading, division and migration similar to what is seen in thick 3D collagen gels. Furthermore, they can provide a new avenue to study cell migration and cell-cell communication at physiologically relevant cell densities.

Determination of minimum serum concentration to develop scaffold free micro-tissue

Objective. Formation of three-dimensional (3D) micro-tissues without scaffolds are widely used not only to define in vivo tissue formation mechanisms but also the development of different tissue-specific drugs. However, depending on high serum and growth factor concentrations, it would be hard to identify major effective biological cues on micro-tissue formation. The aim of the study is to determine the effect of different serum concentrations on Human Umbilicial Vein Endothelial Cells (HUVECs) micro-tissue formation. Methods. Micro-tissue of HUVEC line was formed by using 3D petri dish technique with medium containing 0%, 1%, 5% and 10% fetal bovine serum (FBS). On the 7 th day after micro-tissue formation, live/dead cells analysis was conducted. Micrograph taken on days 1, 3, 5 and 7 th of micro-tissue formation were determined by image analysis with ImageJ. Results. Sizes of micro-tissue formed with 0% FBS on day 1 and 3 determined as 277 ± 12 µm and 279 ± 20 µm, respectively; however, especially on day 7 micro-tissue size significantly decreased to 229 ± 6 µm. When live/dead analysis results were examined, high cell viability was observed in 5% and 10% FBS concentration. Although micro-tissue like structures were observed in 0% and 1% FBS concentrations dead cell ratio considerably increased compared to 5% and 10% FBS concentration. Conclusions. It has been determined that 0% and 1% serum are appropriate for determining the efficacy of biomimetic peptides and different extracellular matrix proteins on micro-tissue formation parameters of HUVEC. High cell viability in micro-tissues was observed with 5% and 10% serum concentrations.

Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment.

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

Abstract 771: Multi-parametric 3D tumor microtissue-based phenotypic compound classification

Abstract Here, we report on the development of homo- and heterotypic ovarian and pancreatic microtissue tumor models for a screening of up to 40 compounds selected from the NCATS Oncology focused library of small molecules. The goal of this study was to develop a high throughput compatible drug screening platform that could leverage the best of high throughput and high content screening assays to generate a pharmacological profile of activities that we hope will help better predict activity in vivo .This platform utilizes 3D multicellular spheroids from ovarian (HEY and SKOV) and pancreatic (Panc-1) cancer and enables the discrimination of tumor-specific efficacy and nonspecific cytotoxicity of a drug candidate with subsequent identification and validation of the molecular mechanism of action (MMOA). The biological responses measured over a 10-day drug exposure period included (i) growth kinetic (microtissue size), (ii) potency (IC50ATP_10_days) and efficacy (max. response ATP and size). The biological response of the compounds was compared between the different cell culture formats tested, 2D, 3D homotypic and 3D heterotypic. The comparison of IC50 values among the different ovarian cancer cell cultures showed that 21 out of the 38 compounds tested were more potent in 3D than in 2D. Within the pancreatic models, 13 out of 20 compounds tested were more potent in 3D than in 2D. Interestingly, most of the compounds which showed stronger potency in 3D were targeted small molecule agents. Comparing drug responses of homo vs heterotypic ovarian tumor model systems, 3 compounds were effective only in the heterotypic model including the WNT inhibitor PNU-74654 and GABA uptake inhibitor (Gat-1) SK&F-89976A. We also evaluated nonspecific cytotoxic effects on the incorporated NIH3T3 fibroblasts by quantifying a secreted reporter over time. The assay system allowed us to discriminate between acute cytotoxic effects (3-day exposure) and sub-chronic toxicity (7-day exposure). Whereas only a very limited number of compounds had acute cytotoxic effects, there were considerable more compounds with sub-chronic cytotoxicity. Based on the single endpoint classifications, we chose an mTOR inhibitor, Torin-2, to further assess molecular mechanism of drug action for all three tumor microtissue models used in this study, applying reverse phase protein array (RPPA) and transcriptome analysis for signaling pathway profiling. Preliminary results of RPPA analysis revealed a clear down-regulation in the cell cycle and PI3K/Akt/mTOR signaling pathway, especially for S6 ribosomal protein phosphorylated at Ser 235, 236, 240 and 244, indicating a strong inhibitory effect on translation and cell growth control. In summary, we present a 3D tumor microtissue-based phenotypic drug discovery testing scheme which allows a multi-parametric endpoint analysis paired with subsequent molecular pathway identification. Citation Format: Madhu A. Lal, Zoe Weydert, Jens Kelm, Marc Ferrer. Multi-parametric 3D tumor microtissue-based phenotypic compound classification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 771.

Abstract B75: High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds

Abstract Background 3D tumor microtissues or spheroids are widely accepted as a more representative biological model in phenotypic drug discovery. Size and morphology are important determinants to evaluate the biological behavior of 3D spheroids, particularly in development of anti-cancer drugs where monitoring cell growth is a particularly critical endpoint. The increasing number of applications of 3D tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including high-throughput image analysis. In this study, we assessed the growth and morphology changes of HCT116 colorectal cancer spheroids in response to two chemotherapy drugs-gemcitabine and docetaxel- using a high throughput, bright-field spheroid imager (Cell3iMager, SCREEN Holdings Ltd., Inc.). Methods Tumor spheroids consisting of the HCT116 colorectal cancer cell line were aggregated in InSphero's GravityPLUS™ plate (a scaffold-free hanging drop platform). The aggregated spheroids were transferred into a GravityTRAP™ plate prior to dosing. Spheroids were dosed with anti-cancer drugs-gemcitabine and docetaxel (0.8-20nM) at day 0 and re-dosed at day 3. The plates were scanned and spheroid size and morphology were analyzed daily for 7 days. Endpoint analysis of ATP content was carried out at day 7 utilizing a luminescence-based ATP assay (CellTiter-Glo® 2.0 Assay, Promega) Results HCT116 spheroids were measured daily for 7 days to determine dose-response effects of gemcitabine and docetaxel treatment (0.8-20nM) on spheroid area (um2). A size increase was observed for the control and the lowest compound concentration treated spheroids whereas the spheroid growth was inhibited at the higher concentrations tested (>4nM). Among the tested compounds, HCT116 spheroids showed less sensitivity to docetaxel compared to gemcitabine. At day 7 the treatment with 20nM gemcitabine resulted in more than 50% reduction in tumor area (um2) which was positively correlated with the decrease in cell viability, determined by ATP content. Using HCT116 tumor microtissues cultured in InSphero's GravityTRAP™ plates, we demonstrate that the measurement of tumor size with the Cell3iMager could be a suitable endpoint compared to that of ATP content for drug induced cytotoxicity in tumor spheroids, without lysing cells or otherwise interfering with long term culture of spheroids. Conclusions Label-free measurement of changes in 3D microtissue size and morphology using the Cell3iMager enables efficient assessment of phenotypic endpoints without disrupting tumor growth, and can be used to focus selection of therapeutic targets and treatment strategies before costly and tedious testing in animal models. Citation Format: Leena Mol Thuruthippallil, Jens M. Kelm, David Fluri. High-throughput, bright-field imaging of 3D tumor spheroid growth and morphology for therapeutic and efficacy screening: A study using chemotherapeutic compounds. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B75.

Profiling metabolic and bioenergetic properties of 3D tumor microtissues (LB171)

Cellular metabolism and bioenergetics processes are increasingly recognized as critical regulators in the pathogenesis of cancers. Metabolic activity and bioenergetic processes are often characterized using typical 2 dimensional (2D) adherent cell culture systems; however, this approach lacks the 3 dimensional (3D) context and heterogeneity of tumor tissue in vivo, which may have significant effects on metabolic and bioenergetic behavior. A novel and more physiologically relevant model is the use of microtissues (also known as spheroids). Metabolic activity and bioenergetic function were accessed in individual cancer cell line microtissues using a newly designed 96‐well plate for the Seahorse Bioscience XFe96 Extracellular Flux Analyzer. Mitochondrial and glycolytic functions were determined simultaneously using the Cell Mito Stress and Glycolytic Stress Tests, respectively. Several parameters, including: seeding density, microtissue size, and substrate identity and availability were investigated. Further, the potential of the microtissues to switch bioenergetic pathways was determined by stimulating maximal respiration (OCR) and maximal extracellular acidification rate (ECAR). Comparisons of microtissues to typical 2D adherent cell cultures were also performed. Initial comparative data are consistent with a shift from the highly proliferative nature of 2D cultures to a physiological 3D model where growth and metabolic properties of the cell are balanced. Technical aspects of this assay system and implications of this work for future studies in cancer biology will be discussed.

Abstract LB-264: Development of 3D microtissue models to study the activity of novel tumor-targeted immunotherapeutics.

Abstract IL-2 therapy can result in durable responses in a proportion of cancer patients, but the treatment is associated with significant toxicity. To improve the liabilities associated with IL-2 therapy, we recently developed a novel monomeric tumor-targeted immunocytokine (CEA-IL2v), which completely lacks binding to CD25 (IL-2Rα) but retains binding to IL-2Rβγ. When tested in classical 2D tumor/lymphocyte co-culture systems, CEA-IL2v induces concentration-dependent proliferation and activation of NK, CD4 and CD8 T cells, but differently from IL-2, does not lead to preferential activation of Tregs and induces lower levels Fas-induced apoptosis. To further explore the properties of CEA-IL2v, including the assessment of antibody targeting and stimulation of immune-cell activation and infiltration, we developed a novel 3D heterotypic microtissues model consisting of tumor cells, fibroblasts and lymphocytes. To address the penetration and targeting, heterotypic 3D microtissues consisting of CEA-expressing human tumor cells and fibroblasts were incubated with tumor-targeted IL-2v (CEA-IL2v), stroma-targeted IL-2v (FAP-IL2v), and the corresponding untargeted control (DP47-IL2v). CEA-IL2v successfully penetrated into microtissues and specifically bound to CEA-positive tumor cells, FAP-IL2v was specifically retained by FAP-expressing fibroblasts whereas the DP47-IL2v was not detected in any of the components. The activity of the immunocytokines was further evaluated by addition of human lymphocytes to 3D microtissues. CEA-IL2v and DP47-IL2v activated different lymphocyte subsets, stimulated their infiltration and selectively promoted the elimination of tumor cells as shown by (a) reduction of microtissue size, (b) increase of LDH release, (c) elimination of CEA-positive tumor cells, (d) increase of lymphocytes infiltrating into the microtissues and (e) increase of cytokine and chemokine levels secreted in the supernatants, including MIP-1β, IL-6 and IFN-γ. Taken together, the heterotypic tumor/fibroblast/lymphocyte 3D microtissue model presented here offers the possibility to monitor antibody penetration and targeting to tumor and stroma components, to study the interaction of tumor cells with immune cells in a system that more closely resembles the in vivo tumor microenvironment and thus provides an in vitro platform for optimal immunotherapy development. The study also points out that the tumor-targeted CEA-IL2v immunocytokine is an elegant tool for expansion and activation of immune effector cells, which may successfully be applied for cancer immunotherapy. Citation Format: Inja Waldhauer, Laura Morra, Steffi Lehmann, Irina Agarkova, Philine Zumstein, Jens M. Kelm, Pablo Umana, Christian Klein, Marina Bacac. Development of 3D microtissue models to study the activity of novel tumor-targeted immunotherapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-264. doi:10.1158/1538-7445.AM2013-LB-264

Microtissue size and hypoxia in HTS with 3D cultures

The three microenvironmental factors that characterize 3D cultures include: first, chemical and/or biochemical composition, second, spatial and temporal dimensions, and third, force and/or substrate physical properties. Although these factors have been studied individually, their interdependence and synergistic interactions have not been well appreciated. We make this case by illustrating how microtissue size (spatial) and hypoxia (chemical) can be used in the formation of physiologically more relevant constructs (or not) for cell-based high-throughput screening (HTS) in drug discovery. We further show how transcriptomic and/or proteomic results from heterogeneously sized microtissues and scaffold architectures that deliberately control hypoxia can misrepresent and represent in vivo conditions, respectively. We offer guidance, depending on HTS objectives, for rational 3D culture platform choice for better emulation of in vivo conditions.

Encapsulated Arrays of Self-Assembled Microtissues: An Alternative to Spherical Microcapsules

Micro-encapsulation and immuno-isolation of allogenic and xenogenic tissues and cells is a promising method for the treatment of a variety of metabolic disorders. Many years have been spent optimizing spherical microcapsules, yet micro-encapsulation has not achieved its full clinical potential. As an alternative to spherical microcapsules, this study presents an alginate-encapsulated array of self-assembled three-dimensional (3D) microtissues. Monodispersed HepG2 cells were seeded onto a micro-molded agarose gel. Cells settled to the bottom of the mold recesses and self-assembled 3D microtissues (n = 822) within 24 h. This array of densely packed microtissues was encapsulated in situ using alginate. When separated from the agarose micro-mold, the encapsulated array had HepG2 microtissues in close proximity to its surface. This surface could be further modified by a simple dipping process. Microtissue size, viability, and albumin secretion were all controllable by the number of cells seeded onto the original agarose micro-mold, and microtissue shape and spacing were controllable by the design of the micro-mold. This approach to encapsulation and the use of self-assembled/self-packing 3D microtissues offers new design possibilities that may help to address certain limitations of conventional microcapsules.

Design of Artificial Myocardial Microtissues

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.