Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

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Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

International Journal of Molecular Sciences ◽

10.3390/ijms21134804 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4804

Author(s):

Vincent van Duinen ◽

Wendy Stam ◽

Eva Mulder ◽

Farbod Famili ◽

Arie Reijerkerk ◽

...

Keyword(s):

Drug Screening ◽

Cell Formation ◽

Cell Types ◽

Culture Conditions ◽

In Vitro Assays ◽

Vegf Receptor ◽

Screening Assay ◽

Angiogenesis Assay

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

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Patient-derived organoids for personalized drug screening in intrahepatic cholangiocarcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.4_suppl.581 ◽

2020 ◽

Vol 38 (4_suppl) ◽

pp. 581-581

Author(s):

Ricardo J. Antonia ◽

Kan Toriguchi ◽

Eveliina Karelehto ◽

Dania Annuar ◽

Luika Timmerman ◽

...

Keyword(s):

High Throughput ◽

Drug Screening ◽

Intrahepatic Cholangiocarcinoma ◽

Three Dimensional ◽

Small Samples ◽

Patient Specific ◽

Mtor Inhibition ◽

Drug Responses

581 Background: Despite standard treatment with gemcitabine and cisplatin, median survival for unresectable Intrahepatic Cholangiocarcinoma (ICC) is < 1 year. Clearly, novel therapeutic strategies are urgently needed. The paucity of targetable mutations in ICC and the as yet unproven benefit of genetically targeted drugs led us to ask whether a reliable clinical benefit may be revealed by patient-specific therapeutic testing in novel models of ICC. Here we describe our ability to establish patient-derived three-dimensional organoid cultures (PDO) that enable individualized identification of active single agents or drug combinations in surrogate models of ICC. Methods: To model patient-specific drug responses, we used the freshly resected ICCs from small samples of single patient tumors to generate PDXs and PDOs, small spheroidal clusters of tumor cells grown in vitro. We have employed a high-throughput drug screening platform using AI-enhanced robotics (Yamaha Motor Corporation) to identify and distribute single, uniformly sized PDOs into 384-well ultra-low adherent plates. This is coupled with a TECAN D300e drug dispenser that rapidly delivers nanoliter volumes of a 34-drug panel, thereby facilitating rapid, reliable drug response analyses. Results: Our data show that PDOs retain characteristic genomic and histological features of the patients’ tumors. Drug responses were specific to each patient tumor, but PDOs from all patients responded to a greater or lesser degree to mTOR inhibition, suggesting that this pathway is important in ICC. The responses of PDO to the mTOR inhibitor Sapanisertib (INK128), was recapitulated in the same patient’s PDX. Further, INK128 was synergistic with gemcitabine in patient 970 PDOs as well as in vivo in PDX also from patient 970. Conclusions: As it is believed that PDX can predict patient responses to drugs, our results suggest that PDO may also predict patient drug responses. The establishment of PDO may allow economical patient-specific, high throughput drug screens that could ultimately inform clinical practice. [Table: see text]

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A Novel Multiplex Drug Screening Assay for Identification of Potential Compounds with Anti-Myeloma Activity from a Library of 1120 Drugs.

Blood ◽

10.1182/blood.v108.11.3403.3403 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3403-3403

Author(s):

Rentian Feng ◽

Anna Lokshin ◽

Elieser Gorelik ◽

Suzanne Lentzsch

Keyword(s):

Multiple Myeloma ◽

Growth Factors ◽

Drug Screening ◽

Myeloma Cell ◽

Screening Assay ◽

Drug Screening Assay ◽

Rpmi 8226 ◽

Stimulation Of

Abstract The majority of drug screening assays are aimed at selection of compounds that affect proliferation or survival of myeloma cells. However, this approach might fail to identify compounds with a potent therapeutic activity that are unable to directly inhibit tumor cell proliferation in vitro but might have potent anti-tumor activity in vivo by targeting the microenvironment of the myeloma cell. For this purpose we used a Multiplex drug-screening assay (MDSA) to identify compounds with potential anti-myeloma activity from a library of 1120 compounds provided by the Multiple Myeloma Research Foundation (MMRF). MDSA is based on use of the Luminex technology (LabMAP Multianalyte Profiling), and testing various myeloma producing factors (MPFs), such as cytokines, chemokines and growth factors that are important for myeloma cell proliferation and survival. The multiple myeloma cell lines MM1.S, RPMI-8226, and IM9 were tested for their capacity to secrete the full set of 31 cytokines, chemokines and growth factors. RPMI-8226 was selected for MDSA due to its high capacity to secrete MPFs (IL-8, VEGF, MCP-1, MIP-1α, MIP-1β, IP10, RANTES and SIL-6R). RPMI-8226 cells were treated with 10 10−6M of each compound (first screening phase) and 1 10−6M (secondary screening), and supernatants from 72-hour cultures were analyzed. The criterion of effective drugs for each cytokine was set up as the ability to inhibit or stimulate MPFs (exceed +/− 1.5 mean value of non-treated control). The resulting data on the drugs were graded by the degree to which they caused inhibition or stimulation of all MPFs (greater than 50% and greater than 90%). A total of 205 of the 1,120 candidates were picked out from the first screening at 10 10−6M. Results from the second analysis (at 1 10−6M) indicated that 14 compounds achieved inhibition of all MPFs and dequalinium dichloride manifested the strongest inhibition of all MPFs. Forty drugs were able to selectively inhibit certain MPFs at levels that exceeded 50% and 14 drugs inhibited MPFs by 90%. With respect to stimulation of cytokine secretion, a total of 39 compounds demonstrated selective stimulation of some MPFs and three drugs (amethopterin (R, S), etoposide, and lasalocid sodium salt) induced stimulation at the level of 90% or greater. Overall, MDSA is a powerful high throughput screening assay to analyze compounds with inhibitory or stimulatory effects on cytokines, chemokines and growth factors that are involved in the pathogenesis of multiple myeloma. Potent compounds identified in this study warrant further investigation for their anti-myeloma effects in vitro and in vivo.

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Mesenchymal-like glioma cells are enriched in the gelatin methacrylate hydrogels

10.1101/2021.03.24.436751 ◽

2021 ◽

Author(s):

Nameeta Shah ◽

Pavan M. Hallur ◽

Raksha A. Ganesh ◽

Pranali Sonpatki ◽

Divya Naik ◽

...

Keyword(s):

Therapeutic Response ◽

Immune Cell ◽

Three Dimensional ◽

Glioma Cells ◽

Cellular Phenotype ◽

3D Scaffold ◽

Promising Alternative ◽

Cells Cultured

AbstractGlioblastoma is the most lethal primary malignant brain tumor in adults. Simplified two-dimensional (2D) cell culture and neurospheres in vitro models fail to recapitulate the complexity of the tumor microenvironment, limiting its ability to predict therapeutic response. Three-dimensional (3D) scaffold-based models have emerged as a promising alternative for addressing these concerns. One such 3D system is gelatin methacrylate (GelMA) hydrogels, which can be used for modeling the glioblastoma microenvironment. We characterized the phenotype of patient-derived glioma cells cultured in GelMA hydrogels (3D-GMH) for their tumorigenic properties using invasion and chemoresponse assays. In addition, we used integrated single-cell and spatial transcriptome analysis to compare cells cultured in 3D-GMH to cells in vivo. Finally, we assessed tumor-immune cell interactions with a macrophage infiltration assay and a cytokine array. We show that cells cultured in 3D-GMH develop a mesenchymal-like cellular phenotype found in perivascular and hypoxic regions present in the core of the tumor, and recruit macrophages by secreting cytokines in contrast to the cells grown as neurospheres that match the phenotype of cells of the infiltrative edge of the tumor.

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Targeting mesenchymal stromal cells plasticity to reroute acute myeloid leukemia course

Blood ◽

10.1182/blood.2020009845 ◽

2021 ◽

Author(s):

Giulia Borella ◽

Ambra Da Ros ◽

Giulia Borile ◽

Elena Porcù ◽

Claudia Tregnago ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Synergistic Effects ◽

Three Dimensional ◽

Cell Interaction ◽

Acute Myeloid

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but still >30% of patients relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the mesenchymal stromal cells (MSCs) role in the leukemic niche to define its contribution to the mechanisms of leukemia escape. We generated humanized three-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation, and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when co-cultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features, at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSCs selective CaV1.2 channel blocker drug, Lercanidipine, is able to impair leukemia progression in 3D niche both in vitro and when implanted in vivo, if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

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Molecular Mechanism of Bushen Jianpi Inhibition of Postoperative Recurrence and Metastasis of Hepatocellular Carcinoma

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2021.3018 ◽

2021 ◽

Vol 17 (1) ◽

pp. 53-63

Author(s):

Zhangjie Zhou ◽

Shujuan Fu ◽

Yun Li ◽

Zujun Que ◽

Xinhua Liu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Bile Acid ◽

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Liver Cancer ◽

Immune Cell ◽

Cancer Recurrence ◽

Bile Acid Metabolism

Compared with western medicine, traditional Chinese medicine can better regulate the internal environment and inhibit liver cancer recurrence and metastasis. Bushen Jianpi Recipe (BSJPR) is a traditional Chinese medicine for tonifying the kidney and invigorating the spleen. It has also been used to treat tumors and other related diseases. Here we explore the efficacy of BSJPR inhibition of hepatocellular carcinoma (HCC) in vivo and in vitro . We hypothesize that BSJPR reduces intrahepatic cholestasis and inflammation and increases expression of the bile acid receptor and downstream targets. This study aims to test this hypothesis and determine whether the inhibitory effect of BSJPR on liver cancer recurrence and metastasis is related to bile acid metabolism. We also observed changes in immune cell expression, suggesting that regulation of the immune microenvironment could inhibit the recurrence and metastasis of HCC. These findings provide a basis for the treatment of HCC and new ideas for follow-up studies of BSJPR.

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Bi-functional oxidized dextran–based hydrogel inducing microtumors: An in vitro three-dimensional lung tumor model for drug toxicity assays

Journal of Tissue Engineering ◽

10.1177/2041731417718391 ◽

2017 ◽

Vol 8 ◽

pp. 204173141771839 ◽

Cited By ~ 2

Author(s):

Dhaval Kedaria ◽

Rajesh Vasita

Keyword(s):

Drug Screening ◽

Drug Toxicity ◽

Disease Modeling ◽

Lung Tumor ◽

Three Dimensional ◽

Two Dimensional ◽

Chitosan Hydrogel ◽

Oxidized Dextran

Cancer is a serious death causing disease having 8.2 million deaths in 2012. In the last decade, only about 10% of chemotherapeutic compounds showed productivity in drug screening. Two-dimensional culture assays are the most common in vitro drug screening models, which do not precisely model the in vivo condition for reliable preclinical drug screening. Three-dimensional scaffold–based cell cultures perhaps mimic tumor microenvironment and recapitulate physiologically more relevant tumor. This study was carried out to develop bi-functional oxidized dextran–based cell instructive hydrogel that provides three-dimensional environment to cancer cells for inducing microtumor. Oxidized dextran was blended with thiolated chitosan to fabricate an in situ self-gelable hydrogel (modified dextran–chitosan) in a one-step process. The hydrogels characterization revealed cross-linked network structure with highly porous structure and water absorption. The modified dextran–chitosan hydrogel showed reduced hydrophobicity and has reduced protein absorption, which resulted in changing the A549 cell adhesiveness, and encouraged them to form microtumor. The cells were proliferated in clusters having spherical morphology with randomly oriented stress fiber and large nucleus. Further microtumors were studied for hypoxia where reactive oxygen species generation demonstrated 15-fold increase as compared to monolayer culture. Drug-sensitivity results showed that microtumors generated on modified dextran–chitosan hydrogel showed resistance to doxorubicin with having 33%–58% increased growth than two-dimensional monolayer model at concentrations of 25–100 µM. In summary, the modified dextran–chitosan scaffold can provide surface chemistry that induces three-dimensional microtumors with physiologically relevant properties to in vivo tumor including growth, morphology, extracellular matrix production, hypoxic phenotype, and drug response. This model can be potentially utilized for drug toxicity studies and cancer disease modeling to understand tumor phenotype and progression.

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Three-Dimensional Culture System of Cancer Cells Combined with Biomaterials for Drug Screening

Cancers ◽

10.3390/cancers12102754 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2754 ◽

Cited By ~ 2

Author(s):

Teruki Nii ◽

Kimiko Makino ◽

Yasuhiko Tabata

Keyword(s):

Cell Culture ◽

Cancer Cells ◽

Drug Screening ◽

Culture System ◽

Three Dimensional ◽

3D Culture ◽

New Drugs ◽

Biological Research

Anticancer drug screening is one of the most important research and development processes to develop new drugs for cancer treatment. However, there is a problem resulting in gaps between the in vitro drug screening and preclinical or clinical study. This is mainly because the condition of cancer cell culture is quite different from that in vivo. As a trial to mimic the in vivo cancer environment, there has been some research on a three-dimensional (3D) culture system by making use of biomaterials. The 3D culture technologies enable us to give cancer cells an in vitro environment close to the in vivo condition. Cancer cells modified to replicate the in vivo cancer environment will promote the biological research or drug discovery of cancers. This review introduces the in vitro research of 3D cell culture systems with biomaterials in addition to a brief summary of the cancer environment.

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Gelatin methacrylate hydrogels culture model for glioblastoma cells enriches for mesenchymal-like state and models interactions with immune cells

Scientific Reports ◽

10.1038/s41598-021-97059-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nameeta Shah ◽

Pavan M. Hallur ◽

Raksha A. Ganesh ◽

Pranali Sonpatki ◽

Divya Naik ◽

...

Keyword(s):

Immune Cell ◽

Three Dimensional ◽

Glioma Cells ◽

Glioblastoma Cells ◽

Treatment Resistant ◽

3D Scaffold ◽

Promising Alternative ◽

Cells Cultured

AbstractGlioblastoma is the most lethal primary malignant brain tumor in adults. Simplified two-dimensional (2D) cell culture and neurospheres in vitro models fail to recapitulate the complexity of the tumor microenvironment, limiting its ability to predict therapeutic response. Three-dimensional (3D) scaffold-based models have emerged as a promising alternative for addressing these concerns. One such 3D system is gelatin methacrylate (GelMA) hydrogels, and we aimed to understand the suitability of using this system to mimic treatment-resistant glioblastoma cells that reside in specific niches. We characterized the phenotype of patient-derived glioma cells cultured in GelMA hydrogels (3D-GMH) for their tumorigenic properties using invasion and chemoresponse assays. In addition, we used integrated single-cell and spatial transcriptome analysis to compare cells cultured in 3D-GMH to neoplastic cells in vivo. Finally, we assessed tumor-immune cell interactions with a macrophage infiltration assay and a cytokine array. We show that the 3D-GMH system enriches treatment-resistant mesenchymal cells that are not represented in neurosphere cultures. Cells cultured in 3D-GMH resemble a mesenchymal-like cellular phenotype found in perivascular and hypoxic regions and recruit macrophages by secreting cytokines, a hallmark of the mesenchymal phenotype. Our 3D-GMH model effectively mimics the phenotype of glioma cells that are found in the perivascular and hypoxic niches of the glioblastoma core in situ, in contrast to the neurosphere cultures that enrich cells of the infiltrative edge of the tumor. This contrast highlights the need for due diligence in selecting an appropriate model when designing a study‘s objectives.

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A mesh microelectrode array for non-invasive electrophysiology within neural organoids

10.1101/2020.09.02.279125 ◽

2020 ◽

Cited By ~ 1

Author(s):

Matthew McDonald ◽

David Sebinger ◽

Lisa Brauns ◽

Laura Gonzalez-Cano ◽

Yotam Menuchin-Lasowski ◽

...

Keyword(s):

Microelectrode Array ◽

Single Cells ◽

Three Dimensional ◽

Microelectrode Arrays ◽

Polymer Scaffolds ◽

Neural Models ◽

New Class ◽

Non Invasive

AbstractOrganoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is best measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes, their cultivation for at least nine months, and could measure spontaneous electrical activity within organoids. Our concept should enable a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.

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