Organic electrochemical transistors as novel biosensing platforms to study the electrical response of whole blood and plasma.

In this paper, for the first time to the best of our knowledge, organic electrochemical transistors are employed to investigate the electrical response of human blood, plasma and alternative buffer solutions that inhibit red blood cell (RBC) aggregation. Our focus is on selecting a suitable electrolytic platform and the related operating conditions, where the RBC effect on the OECT response can be observed separately from the strong ionic environment of plasma in whole blood. The transient response of whole blood to pulse experiments is characterized by two time constants, which can be related to blood viscosity and to the capacitive coupling between the ionic and electronic components of the overall system. The role of capacitive effects, likely due to enhanced double-layer formation by negatively charged RBCs, is also confirmed by the increase of transconductance which was found in RBC suspensions as compared to the suspending buffer. Overall, the complex behavior found in these experiments provides new insights for the development of innovative blood-based sensing devices for biomedical applications.

The CHO Cell Clustering Response to Pertussis Toxin: History of Its Discovery and Recent Developments in Its Use.

Chinese hamster ovary (CHO) cells respond to pertussis toxin (PT) with a novel clustering pattern, which is dependent on biologically active PT. Since its description in 1983, this cellular response has been refined and used extensively for detection and quantification of PT activity, as well as anti-PT antibodies. There are limitations, however, in the use of this phenomenon as originally described. They are: (1) a subjective, observer-dependent scoring system; (2) the requirement for 16-24 h incubation in order for the response to be clearly detectable; and (3) apparent interference from non-toxin materials. To overcome these limitations, a number of alternative in vitro assays for PT, using CHO cells or other cell types, have been developed and are described elsewhere in this publication. In addressing the challenges associated with the CHO cell assay, we discovered that changes in the electrical impedance-based "normalized cell index" of PT-treated CHO cells obtained with the ACEA xCELLigence instrument enable objective detection/quantification of the PT-induced effect in as little as 3-4 h. To the best of our knowledge, the molecular basis for this intriguing response remains unknown. We present here electron microscopic (EM) images of control and PT-treated cells, which suggest some potential molecular mechanisms.

Adenophora triphylla var. japonica Inhibits Candida Biofilm Formation, Increases Susceptibility to Antifungal Agents and Reduces Infection.

(1) Background: Candida is the most common cause of fungal infections worldwide, but due to the limited option of antifungal therapies, alternative strategies are required. (2) Methods: Adenophora triphylla var. japonica extract was used for the biofilm formation assay using RPMI1640. The combinatorial antifungal assay, the dimorphic transition assay, and the adherence assay were done to see the influence of inhibition of biofilm formation. qRT-PCR analysis were performed to check the gene expression. (3) Results: Adenophora triphylla var. japonica extract inhibited the Candida biofilm formation. Treatment of extract increased the antifungal susceptibility of miconazole from a 37% reduction in fungal growth to 99.05%, and also dose-dependently reduced the dimorphic transition of Candida and the attachment of Candida to HaCaT cells. The extract blocked the expression of hyphal-related genes, extracellular matrix genes, Ras1-cAMP-PKA pathway genes, Cph2-Tec1 pathway gene, and MAP kinase pathway gene. (4) Conclusions: In this study, the treatment of Adenophora triphylla var. japonica extract showed inhibition of fungal biofilm formation, activation of antifungal susceptibility, and reduction of infection. These results suggest that fungal biofilm formation is a good target for the development of antifungal adjuvants, and Adenophora triphylla var. japonica extract should be a good candidate for biofilm-associated fungal infections.

Rapamycin modulates pulmonary pathology in a murine model of Mycobacterium tuberculosis infection.

Tuberculosis (TB) treatment regimens are lengthy, causing non-adherence to treatment. Inadequate treatment can lead to relapse and the development of drug resistance TB. Furthermore, patients often exhibit residual lung damage even after cure, increasing the risk for relapse and development of other chronic respiratory illnesses. Host-directed therapeutics are emerging as an attractive means to augment the success of TB treatment. In this study, we used C3HeB/FeJ mice as an experimental model to investigate the potential role of rapamycin, a mammalian target of rapamycin inhibitor, as an adjunctive therapy candidate during the treatment of Mycobacterium tuberculosis infection with moxifloxacin. We report that administration of rapamycin with or without moxifloxacin reduced infection-induced lung inflammation, and the number and size of caseating necrotic granulomas. Results from this study strengthen the potential use of rapamycin and its analogs as adjunct TB therapy, and importantly underscore the utility of the C3HeB/FeJ mouse model as a preclinical tool for evaluating host-directed therapy candidates for the treatment of TB.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture.

OBJECTIVES: For clinical applications of cell-based therapies, a large quantity of human pluripotent stem cells (hPSCs) produced in standardized and scalable culture processes is required. Currently, microcarrier-free suspension culture shows potential for large-scale expansion of hPSCs; however, hPSCs tend to aggregate during culturing leading to a negative effect on cell yield. To overcome this problem, we developed a novel protocol to effectively control the sizes of cell aggregates and enhance the cell proliferation during the expansion of hPSCs in suspension. MATERIALS AND METHODS: hPSCs were expanded in suspension culture supplemented with polyvinyl alcohol (PVA) and dextran sulphate (DS), and 3D suspension culture of hPSCs formed cell aggregates under static or dynamic conditions. The sizes of cell aggregates and the cell proliferation as well as the pluripotency of hPSCs after expansion were assessed using cell counting, size analysis, real-time quantitative polymerase chain reaction, flow cytometry analysis, immunofluorescence staining, embryoid body formation, teratoma formation and transcriptome sequencing. RESULTS: Our results demonstrated that the addition of DS alone effectively prevented hPSC aggregation, while the addition of PVA significantly enhanced hPSC proliferation. The combination of PVA and DS not only promoted cell proliferation of hPSCs but also produced uniform and size-controlled cell aggregates. Moreover, hPSCs treated with PVA, or DS or a combination, maintained the pluripotency and were capable of differentiating into all three germ layers. mRNA-seq analysis demonstrated that the combination of PVA and DS significantly promoted hPSC proliferation and prevented cell aggregation through improving energy metabolism-related processes, regulating cell growth, cell proliferation and cell division, as well as reducing the adhesion among hPSC aggregates by affecting expression of genes related to cell adhesion. CONCLUSIONS: Our results represent a significant step towards developing a simple and robust approach for the expansion of hPSCs in large scale.

Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System.

The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.

Facile and green approach towards biomass-derived hydrogel powders with hierarchical micro-nanostructures for ultrafast hemostasis.

Although a series of biomass-derived hemostats has been developed, the desire for green-prepared hemostatic materials with biosafety has not decreased. Herein, we constructed porous carboxymethyl chitosan/sodium alginate/Ca(OH)2 powders (PCSCPs) with suitable adaptability for instant control of irregular hemorrhage via a facile and green approach. By one-pot chemical crosslinking of carboxymethyl chitosan and sodium alginate, hydrogels were formed and immediately ionically cross-linked along with the generation of Ca(OH)2 to prepare PCSCPs. As hydrogel powders, PCSCPs with abundant hydrophilic carboxymethyl groups and porous hierarchically micro-nanostructures displayed a high water absorption ratio of over 1600%. The PCSCPs were confirmed with favorable hemocompatibility, non-cytotoxic effects and excellent degradability. Hemostasis assays in vitro showed that PCSCPs possessed an outstanding property of platelet activation and red blood cell aggregation. The PCSCPs effectively shortened the hemostatic time and blood loss to ca. 50% in rodent bleeding models compared with medical gauze and commercial chitosan-based hemostats. Furthermore, a mouse subcutaneous implantation model demonstrated an ignorable inflammation response and potential tissue repair capability of PCSCPs. It's believed that green-prepared and biomass-derived PCSCPs are feasible biomedical hemostatic materials in view of engineering and provide a promising platform to design hemostats in prehospital management and clinical settings.

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-ß2), 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.

Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells.

Several studies have shown how different cell lines can influence the differentiation of stem cells through co-culture systems. The House Ear Institute-Organ of Corti 1 (HEI-OC1) is considered an important cell line for in vitro auditory research. However, it is unknown if HEI-OC1 cells can promote the differentiation of embryonic stem cells (ESCs). In this study, we investigated whether co-culture of ESCs with HEI-OC1 cells promotes differentiation. To this end, we developed a co-culture system of mouse ESCs with HEI-OC1 cells. Dissociated or embryonic bodies (EBs) of ESCs were introduced to a conditioned and inactivated confluent layer of HEI-OC1 cells for 14 days. The dissociated ESCs coalesced into an EB-like form that was smaller than the co-cultured EBs. Contact co-culture generated cells expressing several otic progenitor markers as well as hair cell specific markers. ESCs and EBs were also cultured in non-contact setup but using conditioned medium from HEI-OC1 cells, indicating that soluble factors alone could have a similar effect. The ESCs did not form into aggregates but were still Myo7a-positive, while the EBs degenerated. However, in the fully differentiated EBs, evidence to prove mature differentiation of inner ear hair cell was still rudimentary. Nevertheless, these results suggest that cellular interactions between ESCs and HEI-OC1 cells may both stimulate ESC differentiation.

EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic breast cancer subtypes lacking targeted therapy. Our recent work demonstrated that circulating tumor cell (CTC) clusters and polyclonal metastasis of TNBC are driven by aggregation of CD44+ cancer stem cells (CSC) and associated with an unfavorable prognosis, such as low overall survival. However, there is no existing therapeutic that can specifically block CTC or CSC cluster formation. Methods: Using patient-derived xenograft (PDX) models, we established an ex vivo tumor cell clustering assay for a pilot screening of blockade antibodies. After identifying EGFR as a target candidate, we modulated the gene expression and inhibited its kinase activity to determine its functional importance in tumor cell clustering and therapeutic inhibition of lung metastasis. We also examined the molecular regulation network of EGFR and a potential connection to CSC marker CD44 and microRNAs, which regulate CTC clustering. Results: We report here that EGFR inhibition successfully blocks circulating CSC (cCSC) clustering and lung metastasis of TNBC. EGFR enhances CD44-mediated tumor cell aggregation and CD44 stabilizes EGFR. Importantly, blocking EGFR by a novel anti-EGFR monoclonal antibody (clone LA1) effectively blocked cell aggregation in vitro and reduced lung metastasis in vivo. Furthermore, our data demonstrated that the tumor suppressor microRNA-30c serves as another negative regulator of cCSC clustering and lung metastasis by targeting CD44 as well as its downstream effector EGFR. Conclusion: Our studies identify a novel anti-EGFR therapeutic strategy to inhibit cCSC aggregation and therefore abolish cCSC cluster-mediated metastasis of TNBC.

Gastruloids generated without exogenous Wnt activation develop anterior neural tissues.

When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

The lectin Orysata induces phosphatase-mediated and carbohydrate-independent aggregation of insect cells.

Lectins, or carbohydrate-binding proteins, can cause agglutination of particular cells. This process is mediated by the interaction of the carbohydrate-binding domain with sugar structures on the cell surface, and this binding can be inhibited by pre-incubation of the lectin with its specific sugars. However, when incubated with insect cells, Orysata, a mannose-binding lectin from rice, caused aggregation of the cells, independent from carbohydrate binding activity. This phenomenon was observed for multiple insect cell lines, confirming the robustness of this phenotype. While the carbohydrate-dependent agglutination of red blood cells happens within minutes, the carbohydrate-independent aggregation of insect cells requires longer incubation times. Further analysis with the galactose-binding lectins SSA and Jacalin, validated the robustness of this lectin-induced, carbohydrate-independent aggregation in different insect cell lines. Since proteomic analysis revealed no changes in the proteome after treatment with the lectins, this cell aggregation is likely caused by the (in) activation or re-organization of the existing surface proteins. The use of inhibitors of phosphorylation and dephosphorylation, staurosporine (STS) and a phosphatase inhibitor (PPI) cocktail, pointed to dephosphorylation as a key mechanism in the lectin-induced, carbohydrate-independent aggregation of insect cells. Similar to contact inhibition, cell proliferation in cell aggregates was decreased. Analysis of the marker for cell proliferation, cyclin E, confirmed that aggregated cells enter a quiescent state. The current data offer a new perspective on the mechanism by which lectins execute their activities, specifically through lectin-induced phosphatase-mediated cell aggregation and proliferation inhibition, independent from their carbohydrate-binding activity.

Antifibrotics Modify B-Cell-induced Fibroblast Migration and Activation in Patients with Idiopathic Pulmonary Fibrosis.

B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and ß-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell-secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell-mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.

Sulphur mustard induces progressive toxicity and demyelination in brain cell aggregate culture.

Sulphur mustard (H; bis(2-chloroethyl) sulphide) is a vesicant chemical warfare (CW) agent that has been well documented as causing acute injury to the skin, eyes and respiratory system. Although a great deal of research effort has been expended to understand how H exerts these effects, its mechanism of action is still poorly understood. At high exposures, H also causes systemic toxicity with chronic and long-term effects to the immune, cardiovascular and central nervous systems, and these aspects of H poisoning are much less studied and comprehended. Rat aggregate cultures comprised of multiple brain cell types were exposed to H and followed for four weeks post-exposure to assess neurotoxicity. Toxicity (LDH, caspase-3 and aggregate diameter) was progressive with time post-exposure. In addition, statistically significant changes in neurofilament heavy chain (NFH), glial fibrillary acidic protein (GFAP), Akt phosphorylation, IL-6, GRO-KC and TNF-α were noted that were time- and concentration-dependent. Myelin basic protein, CNPase and vascular endothelial growth factor (VEGF) were found to be especially sensitive to H exposure in a time- and concentration-dependent fashion, with levels falling to ∼50 % of control values at ∼10 µM H by 8 days post-exposure. Demyelination and VEGF inhibition may be causal in the long-term neuropsychological illnesses that have been documented in casualties exposed to high concentrations of H, and may also play a role in the peripheral neuropathy that has been observed in some of these individuals.

Mini-TrpRS is essential for IFNγ-induced monocyte-derived giant cell formation.

Truncated tryptophanyl-tRNA synthetase (mini-TrpRS), like any other aminoacyl-tRNA synthetases, canonically functions as a protein synthesis enzyme. Here we provide evidence for an additional signaling role of mini-TrpRS in the formation of monocyte-derived multinuclear giant cells (MGCs). Interferon-gamma (IFNγ) readily induced monocyte aggregation leading to MGC formation with paralleled marked upregulation of mini-TrpRS. Small interfering (si)RNA, targeting mini-TrpRS in the presence of IFNγ prevented monocyte aggregation. Moreover, blockade of mini-TrpRS, either by siRNA, or the cognate amino acid and decoy substrate D-Tryptophan to prevent mini-TrpRS signaling, resulted in a marked reduction in expression of the purinergic receptor P2X 7 (P2RX7) in monocytes activated by IFNγ. Our findings identify mini-TrpRS as a critical signaling molecule in a mechanism by which IFNγ initiates monocyte-derived giant cell formation.

Investigating PEGDA and GelMA Microgel Models for Sustained 3D Heterotypic Dermal Papilla and Keratinocyte Co-Cultures.

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Mesothelin blockage by Amatuximab suppresses cell invasiveness, enhances gemcitabine sensitivity and regulates cancer cell stemness in mesothelin-positive pancreatic cancer cells.

BACKGROUND: Mesothelin is a 40-kDa glycoprotein that is highly overexpressed in various types of cancers, however molecular mechanism of mesothelin has not been well-known. Amatuximab is a chimeric monoclonal IgG1/k antibody targeting mesothelin. We recently demonstrated that the combine therapy of Amatuximab and gemcitabine was effective for peritonitis of pancreatic cancer in mouse model. METHODS: We discover the role and potential mechanism of mesothelin blockage by Amatuximab in human pancreatic cells both expressing high or low level of mesothelin in vitro experiment and peritonitis mouse model of pancreatic cancer. RESULTS: Mesothelin blockage by Amatuximab lead to suppression of invasiveness and migration capacity in AsPC-1 and Capan-2 (high mesothelin expression) and reduce levels of pMET expression. The combination of Amatuximab and gemcitabine suppressed proliferation of AsPC-1 and Capan-2 more strongly than gemcitabine alone. These phenomena were not observed in Panc-1 and MIA Paca-2 (Mesothelin low expression). We previously demonstrated that Amatuximab reduced the peritoneal mass in mouse AsPC-1 peritonitis model and induced sherbet-like cancer cell aggregates, which were vanished by gemcitabine. In this study, we showed that the cancer stem cell related molecule such as ALDH1, CD44, c-MET, as well as proliferation related molecules, were suppressed in sherbet-like aggregates, but once sherbet-like aggregates attached to peritoneum, they expressed these molecules strongly without the morphological changes. CONCLUSIONS: Our work suggested that Amatuximab inhibits the adhesion of cancer cells to peritoneum and suppresses the stemness and viability of those, that lead to enhance the sensitivity for gemcitabine.

Sulfur sequestration promotes multicellularity during nutrient limitation.

The behaviour of Dictyostelium discoideum depends on nutrients1. When sufficient food is present these amoebae exist in a unicellular state, but upon starvation they aggregate into a multicellular organism2,3. This biology makes D. discoideum an ideal model for investigating how fundamental metabolism commands cell differentiation and function. Here we show that reactive oxygen species-generated as a consequence of nutrient limitation-lead to the sequestration of cysteine in the antioxidant glutathione. This sequestration limits the use of the sulfur atom of cysteine in processes that contribute to mitochondrial metabolism and cellular proliferation, such as protein translation and the activity of enzymes that contain an iron-sulfur cluster. The regulated sequestration of sulfur maintains D. discoideum in a nonproliferating state that paves the way for multicellular development. This mechanism of signalling through reactive oxygen species highlights oxygen and sulfur as simple signalling molecules that dictate cell fate in an early eukaryote, with implications for responses to nutrient fluctuations in multicellular eukaryotes.

Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer.

Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 µM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 µM) and Imatinib (10 µM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.