Functional Screen for microRNAs Suppressing Anchorage-Independent Growth in Human Cervical Cancer Cells.

The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus (HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96- and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.

Transforming growth factor-beta1 promotes the migration and invasion of sphere-forming stem-like cell subpopulations in esophageal cancer.

Esophageal cancer is one of the most lethal solid malignancies. Mounting evidence demonstrates that cancer stem cells (CSCs) are able to cause tumor initiation, metastasis and responsible for chemotherapy and radiotherapy failures. As CSCs are thought to be the main reason of therapeutic failure, these cells must be effectively targeted to elicit long-lasting therapeutic responses. We aimed to enrich and identify the esophageal cancer cell subpopulation with stem-like properties and help to develop new target therapy strategies for CSCs. Here, we found esophageal cancer cells KYSE70 and TE1 could form spheres in ultra low attachment surface culture and be serially passaged. Sphere-forming cells could redifferentiate and acquire morphology comparable to parental cells, when return to adherent culture. The sphere-forming cells possessed the key criteria that define CSCs: persistent self-renewal, overexpression of stemness genes (SOX2, ALDH1A1 and KLF4), reduced expression of differentiation marker CK4, chemoresistance, strong invasion and enhanced tumorigenic potential. SB525334, transforming growth factor-beta 1(TGF-ß1) inhibitor, significantly inhibited migration and invasion of sphere-forming stem-like cells and had no effect on sphere-forming ability. In conclusion, esophageal cancer sphere-forming cells from KYSE70 and TE1 cultured in ultra low attachment surface possess cancer stem cell properties, providing a model for CSCs targeted therapy. TGF-ß1 promotes the migration and invasion of sphere-forming stem-like cells, which may guide future studies on therapeutic strategies targeting these cells.

Optimized study of an in vitro 3D culture of preantral follicles in mice.

BACKGROUND: In vitro culture of preantral follicles is a promising technology for fertility preservation. OBJECTIVES: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. METHODS: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E2) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. RESULTS: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. CONCLUSIONS: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

Three dimensional models of dedifferentiated liposarcoma cell lines: scaffold-based and scaffold-free approaches.

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope™, respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.

Cell Viability Assay with 3D Prostate Tumor Spheroids.

WST-8 (Cell Counting Kit 8; CCK-8) is the last generation tetrazolium-based cell viability assay and has recently been accepted as a validated method for measuring the cell viability of 3D in vitro models. Here, we describe how to form 3D prostate tumor spheroids using the polyHEMA technique, apply drug treatments and WST-8 assay to these spheroids, and calculate their cell viability. The advantages of our protocol are the formation of spheroids without adding extracellular matrix components, and the elimination of the critique handling process needed for transferring spheroids. Although this protocol exemplifies the determination of percentage cell viability in PC-3 prostate tumor spheroids, it can be adapted and optimized for other prostate cell lines and other types of cancers.

Assessment of Antitumor and Antiproliferative Efficacy and Detection of Protein-Protein Interactions in Cancer Cells from 3D Tumor Spheroids.

When compared to two-dimensional (2D) cell cultures, 3D spheroids have been considered suitable in vitro models for drug discovery research and other studies of drug activity. Based on different 3D cell culture procedures, we describe procedures we have used to obtain 3D tumor spheroids by both the hanging-drop and ultra-low-attachment plate methods and to analyze the antiproliferative and antitumor efficacy of different chemotherapeutic agents, including a peptidomimetic. We have applied this method to breast and lung cancer cell lines such as BT-474, MCF-7, A549, and Calu-3. We also describe a proximity ligation assay of the cells from the spheroid model to detect protein-protein interactions of EGFR and HER2. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of 3D spheroids using the hanging-drop method Basic Protocol 2: Growth of spheroids using ultra-low-attachment plates Support Protocol 1: Cell viability assay of tumor spheroids Support Protocol 2: Antiproliferative and antitumor study in 3D tumor spheroids Support Protocol 3: Proximity ligation assay on cells derived from 3D spheroids.

Three dimensional in vitro culture systems in anticancer drug discovery targeted on cancer stem cells.

Worldwide, tumors are one of the most common causes of death. Every year 3.7 million new cases occur in Europe and more than 1.9 million patients die (WHO data). Most of the fields of research are focused on developing new therapeutic strategies that will be effective in eliminating the tumor, preventing its remission, and avoiding or reducing the side effects of therapy. In the past, generally classical 2D cell cultures or immunodeficient animal models had been used to cultivate and test drugs on human cancer cell lines. Nowadays, there are increasing interests in three-dimensional (3D) cell cultures, a method with significant differences from flat cultured cells, both considering gene expressions and cell-cell interactions. Various evidence suggests that high tumorigenic properties might be dependent on the occurrence of a small cell population, pointed out to be responsible for metastasis and recurrence. This population is called cancer stem cells (CSCs), hinted to have a lot of similarities with normal stem cells. CSCs are the main reason for chemotherapy failure as well as multi-drug resistance (MDR). CSCs can also interact through the cytokine network, with other cells like the macrophages of the inflammatory system. The big advantage of a 3D culture is the possibility to isolate and investigate the CSCs population surrounded by its environment. This article aims to sum up known 3D cell cultures, especially in the field of CSCs research due to the importance of the tumor's environment on stem cell's markers expression and their development.

Comparison of the formation, adipogenic maturation, and retention of human adipose-derived stem cell spheroids in scaffold-free culture techniques.

While three-dimensional spheroids outperform traditional two-dimensional monolayer culture for human adipose-derived stem cells (hASCs), there is not a consensus on the most successful method for enhancing their adipogenic differentiation and minimizing the loss of physiologically relevant, fatty spheroids during culture. To this end, we compared three culture methods, namely, elastin-like polypeptide-polyethyleneimine (ELP-PEI) coated surfaces, ultra-low attachment static culture, and suspension culture for their ability to form and retain productive hASC spheroids. The ELP-PEI coatings used the ELP conjugated to two molecular weights of PEI (800 or 25,000 g/mol). FTIR spectroscopy, atomic force microscopy, and contact angle goniometry revealed that the ELP-PEI coatings had similar chemical structures, surface topography, and hydrophobicity. Time-lapse microscopy showed that increasing the PEI molecular weight resulted in smaller spheroids. Measurement of triglyceride content showed that the three methods induced comparable differentiation of hASCs toward the adipogenic lineage. DNA content and morphometric analysis revealed merging of spheroids to form larger spheroids in the ultra-low attachment static culture and suspension culture methods. In contrast, the retention of hASC spheroid sizes and numbers with a regular spheroid size (~100 µm) were best atop the ELP-PEI800 coatings. Overall, this research shows that the spheroid culture atop the ELP-PEI coatings is a suitable cell culture model for future studies involving long-term, three-dimensional culture of mature adipocytes derived from hASCs.

A Simple Three-Dimensional In Vitro Culture Mimicking the In Vivo-Like Cell Behavior of Bladder Patient-Derived Xenograft Models.

Patient-derived xenograft (PDX) models allow for personalized drug selection and the identification of drug resistance mechanisms in cancer cells. However, PDX models present technical disadvantages, such as long engraftment time, low success rate, and high maintenance cost. On the other hand, tumor spheroids are emerging as an in vitro alternative model that can maintain the phenotype of cancer cells long enough to perform all assays and predict a patient's outcome. The present work aimed to describe a simple, reproducible, and low-cost 3D in vitro culture method to generate bladder tumor spheroids using human cells from PDX mice. Cancer cells from PDX BL0293 and BL0808 models, previously established from advanced bladder cancer, were cultured in 96-well round-bottom ultra-low attachment (ULA) plates with 5% Matrigel and generated regular and round-shaped spheroids (roundness > 0.8) with a diameter larger than 400 µm and a hypoxic core (a feature related to drug resistance in solid tumors). The responses of the tumor spheroids to the antineoplastic drugs cisplatin, gemcitabine, and their combination were similar to tumor responses in in vivo studies with PDX BL0293 and BL0808 mice. Therefore, the in vitro 3D model using PDX tumor spheroids appears as a valuable tool that may predict the outcome of in vivo drug-screening assays and represents a low-cost strategy for such purpose.

Effect of propofol on pyroptosis of lung cancer A549 cells by NLRP3/ASC/caspase-1 pathway / 中国医科大学学报

Objective To investigate the effect of propofol on pyroptosis and A549 cells via the NLRP3/ASC/caspase-1 pathway.Methods Establish a three-dimensional culture model of A549 tumor cells using ultra-low attachment plates,A549 cells were cultured using ultra-low adsorption culture plates to establish a three-dimensional culture model.The CCK-8 method was used to detect the effect of propofol on A549 cell proliferation;the inflammatory factors interleukin(IL)-18,IL-1β,and IL-6 were detected in the A549 lung cancer cell supernatants using enzyme-linked immunosorbent assays;western blotting was used to detect the expression levels of pyrolysis-asso-ciated proteins NLRP3,ASC,caspase-1,GSDMD-N,and IL-1β in A549 lung cancer cells in each group.Results Compared to the blank control group,the survival rate of A549 cells in low,medium,and high concentrations of propofol in each group decreased in turn(P＜0.05);the levels of inflammatory factors IL-18,IL-1β,and IL-6 in the A549 cell supernatant,and scorch related protein NLRP3,ASC,caspase-1,GSDMD-N,and IL-1β increased with the increased propofol concentrations(P＜0.05).Conclusion The three-dimensional culture model of lung cancer A549 cells was successfully established using the ultra-low adsorption culture method.Propofol can promote cell apoptosis and inhibit the pyroptosis of A549 lung cancer cells via activating the NLRP3/ASC/caspase-1 pathway.

The Generation of Three-Dimensional Head and Neck Cancer Models for Drug Discovery in 384-Well Ultra-Low Attachment Microplates.

The poor success rate of cancer drug discovery has prompted efforts to develop more physiologically relevant cellular models for early preclinical cancer lead discovery assays. For solid tumors, this would dictate the implementation of three-dimensional (3D) tumor models that more accurately recapitulate human solid tumor architecture and biology. A number of anchorage-dependent and anchorage-independent in vitro 3D cancer models have been developed together with homogeneous assay methods and high content imaging approaches to assess tumor spheroid morphology, growth, and viability. However, several significant technical challenges have restricted the implementation of some 3D models in HTS. We describe a method that uses 384-well U-bottomed ultra-low attachment (ULA) microplates to produce head and neck tumor spheroids for cancer drug discovery assays. The production of multicellular head and neck cancer spheroids in 384-well ULA-plates occurs in situ, does not impose an inordinate tissue culture burden for HTS, is readily compatible with automation and homogeneous assay detection methods, and produces high-quality uniform-sized spheroids that can be utilized in cancer drug cytotoxicity assays within days rather than weeks.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better. Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation. Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300-500 µm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 µg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43). Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.