N.J.C was supported by a BBSRC MIBTP CASE Award with Sygnature Discovery Ltd (BB/M01116X/1, 1940003)

The authors have no conflicts of interest to declare.

Main findings and outputs 
This paper provides a detailed protocol to&#160;probe mitochondrial energy metabolism of single 3D spheroids using a series of cancer-derived cell lines with the XFe96 XF Analyzer. A method is developed and described for the rapid cultivation of A549, HepG2/C3A, MCF7, and SK-OV-3 cellular spheroids using cell-repellent technologies for forced aggregation. This protocol addresses many considerations of probing spheroid metabolism with XF technology, including (1) optimizati...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63346">Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis</a>. The Representative Results&#160;section was&#160;updated.
Figure 5 was updated from:
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63346/63346fig05.jpg" /> 
Figure 5: Single or sequential injection of mitochondrial respiratory compounds. Cancer-cell-derived spheroids of MCF-7, HEPG2/C3A, SK-OV-3, and A549 were placed into wells of an XFe96 spheroid microplate in XF RPMI and probed for OCR using the Agilent Seahorse XFe96 analyzer. OCR was measured 5x, after which 2 &#181;g/mL oligomycin (injection Port A: green trace) or 5 &#181;M BAM15 (injection Port A: blue trace or injection port B: green trace) to inhibit the mitochondrial ATP synthase and determine maximal respiratory capacity, respectively. Kinetic OCR data are expressed as % basal (A-D). Maximal respiratory capacity (OCRmax) was calculated as a factor of basal OCR by the equation: OCRmax = OCRBAM15 / OCRbasal. OCRmax was obtained from OCR averages across measurement cycles 8-10 post BAM15 injection with (green bars) and without (blue bars) oligomycin. Data are averages &#177; SEM from 3-8 individual well replicates across the spheroid assay microplate. Abbreviations: OCR = oxygen consumption rate. <a href="https://www.jove.com/files/ftp_upload/63346/63346fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63346/63346fig5v2.jpg" /> 
Figure 5: Single or sequential injection of mitochondrial respiratory compounds. Cancer-cell-derived spheroids of MCF-7, HEPG2/C3A, SK-OV-3, and A549 were placed into wells of an XFe96 spheroid microplate in XF RPMI and probed for OCR using the Agilent Seahorse XFe96 analyzer. OCR was measured 5x, after which 2 &#181;g/mL oligomycin (injection Port A: green trace) or 5 &#181;M BAM15 (injection Port A: blue trace or injection port B: green trace) to inhibit the mitochondrial ATP synthase and determine maximal respiratory capacity, respectively. Kinetic OCR data are expressed as % basal (A-D). Maximal respiratory capacity (OCRmax) was calculated as a factor of basal OCR by the equation: OCRmax = OCRBAM15 / OCRbasal. OCRmax was obtained from OCR averages across measurement cycles 8-10 post BAM15 injection with (green bars) and without (blue bars) oligomycin. Data are averages &#177; SEM from 3-8 individual well replicates across the spheroid assay microplate. Abbreviations: OCR = oxygen consumption rate. <a href="https://www.jove.com/files/ftp_upload/63346/63346fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63346">Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis</a>. The Representative Results&#160;section was&#160;updated.

Erratum: Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids using Extracellular Flux Analysis

Advances in in vitro models in biological research have rapidly progressed over the last 20 years. Such models now include organ-on-a-chip modalities, organoids, and 3D microtissue spheroids, all of which have become a common focus to improve the translation between in vitro and in vivo studies. The use of advanced in vitro models, particularly spheroids, spans several research fields, including tissue engineering, stem cell research, cancer, and disease biology1,2,3,4,5,6,7, and safety testing, including genetic toxicology8,9,10, nanomaterials toxicology11,12,13,14, and drug safety and efficacy testing8,15,16,17,18,19.
Normal cell morphology is critical to biological phenotype and activity. Culturing cells into 3D microtissue spheroids allows cells to adopt a morphology, phenotypic function, and architecture, more akin to that observed in vivo but difficult to capture with classical monolayer cell culture techniques. Both in vivo and in vitro, cellular function is directly impacted by the cellular microenvironment, which is not limited to cellular communication and programming (e.g., cell-cell junction formations, opportunities to form cell niches); cell exposure to hormones and growth factors in the immediate environments (e.g., cellular cytokine exposure as part of an inflammatory response); composition of physical and chemical matrices (e.g., whether cells are grown in stiff tissue culture plastic or an elastic tissue environment); and most importantly, how cellular metabolism is impacted by nutrition and access to oxygen as well as the processing of metabolic waste products such as lactic acid.
Metabolic flux analysis is a powerful way to examine cellular metabolism within defined in vitro systems. Specifically, XF technology allows for the analysis of live, real-time changes in cellular bioenergetics of intact cells and tissues. Given that many intracellular metabolic events occur within the order of seconds to minutes, real-time functional approaches are paramount for understanding real-time changes in cellular metabolic flux in intact cells and tissues in vitro.
This paper provides protocols for cultivating cancer-derived cell lines A549 (lung adenocarcinoma), HepG2/C3A (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma), and SK-OV-3 (ovarian adenocarcinoma) as in vitro 3D&#160;spheroid models using forced-aggregation approaches (Figure 1). It also (i) describes in detail how to probe mitochondrial energy metabolism of single 3D spheroids using the Agilent XFe96 XF analyzer, (ii) highlights ways to optimize XF assays using single 3D spheroids, and (iii) discusses important considerations and limitations of probing 3D spheroid metabolism using this approach. Most importantly, this paper describes how datasets are collected that allow the calculation of oxygen consumption rate (OCR) to determine oxidative phosphorylation and thus mitochondrial function in cellular spheroids. Though not analyzed for this protocol, extracellular acidification rate (ECAR) is another parameter that is measured alongside OCR data in XF experiments. However, ECAR is often poorly or incorrectly interpreted from XF datasets. We provide a commentary as to the limitations of calculating ECAR following basic approaches from the technology manufacturer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A549</td>
			<td>ECACC</td>
			<td>&#160;#86012804</td>
			<td>Lung carcinoma cell line</td>
		</tr>
		<tr>
			<td>Agilent Seahorse XF RPMI Medium, pH 7.4</td>
			<td>Agilent Technologies Inc.</td>
			<td>103576-100</td>
			<td>XF assay medium with 1 mM HEPES, without phenol red, sodium bicarbonate, glucose, L-glutamine, and sodium pyruvate</td>
		</tr>
		<tr>
			<td>Agilent Seahorse XFe96 Extracellular Flux Analyzer</td>
			<td>Agilent Technologies Inc.</td>
			<td>-</td>
			<td>Instrument for measuring rates of spheroid oxygen uptake in single spheroids</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Merck Life Science</td>
			<td>A8674</td>
			<td>Mitochondrial respiratory complex III inhibitor</td>
		</tr>
		<tr>
			<td>BAM15</td>
			<td>TOCRIS bio-techne</td>
			<td>5737</td>
			<td>Mitochondrial protnophore uncoupler</td>
		</tr>
		<tr>
			<td>Black-walled microplate</td>
			<td>Greiner Bio-One</td>
			<td>655076</td>
			<td>For fluorescence-based assays</td>
		</tr>
		<tr>
			<td>CELLSTAR cell-repellent surface 96 U well microplates</td>
			<td>Greiner Bio-One</td>
			<td>650970</td>
			<td>Microplates for generating spheroids</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>Assay for the determination of cell viability in 3D microtissue spheroids</td>
		</tr>
		<tr>
			<td>Cultrex Poly-D-Lysine</td>
			<td>R&#38;D Systems a biotechne brand</td>
			<td>3439-100-01</td>
			<td>Molecular cell adhesive for coating XFe96 spheroid microplates to facillitate attachment of spheroids</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Merck Life Sciences</td>
			<td>G8270</td>
			<td>Supplement for cell culture growth and XF assay medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885084</td>
			<td>Culture medium for HepG2/C3A spheroids</td>
		</tr>
		<tr>
			<td>EVOS XL Core Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMEX1000</td>
			<td>Phase-contrast imaging microscope</td>
		</tr>
		<tr>
			<td>EZ-PCR Mycoplasma test kit</td>
			<td>Biological Industries</td>
			<td>20-700-20</td>
			<td>Mycoplasma screening in cell cultures</td>
		</tr>
		<tr>
			<td>FIJI Is Just Image J</td>
			<td></td>
			<td></td>
			<td>Analysis of collated images</td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>Merck Life Science</td>
			<td>F7524</td>
			<td>Supplement for cell culture medium</td>
		</tr>
		<tr>
			<td>HepG2/C3A</td>
			<td>ATCC</td>
			<td>&#160;#CRL-10741</td>
			<td>Hepatic carcinoma cell line, a clonal derivative of the parent HepG2 cell line</td>
		</tr>
		<tr>
			<td>Lactate-Glo</td>
			<td>Promega</td>
			<td>J5021</td>
			<td>Assay for measurement of lactate within spheorid culture medium</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM solution)</td>
			<td>Merk Life Sciences</td>
			<td>G7513</td>
			<td>Supplement for cell culture growth and XF assay medium</td>
		</tr>
		<tr>
			<td>M50 Stereo microscope</td>
			<td>Leica Microsytems</td>
			<td>LEICAM50</td>
			<td>Stereo dissection micrscope; used for spheorid handling</td>
		</tr>
		<tr>
			<td>MCF-7</td>
			<td>ECACC</td>
			<td>#86012803</td>
			<td>Breast adenocarcinoma cell line</td>
		</tr>
		<tr>
			<td>Oligomycin from Streptomyces diastatochromogenes</td>
			<td>Merck Life Science</td>
			<td>O4876</td>
			<td>ATP Synthase Inhibitor</td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Antibiotics added to cell culture medium</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay Kit</td>
			<td>Initrogen</td>
			<td>P7589</td>
			<td>Analysis of dsDNA in spehroids</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Merck Life Science</td>
			<td>R8875</td>
			<td>Mitochondrial Respiratory Complex I Inhibitor</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>21875091</td>
			<td>Culture medium for A549, MCF7, and SK-OV-3 spheroids</td>
		</tr>
		<tr>
			<td>Seahorse Analytics</td>
			<td>Agilent Technologies Inc.</td>
			<td>Build 421</td>
			<td>https://seahorseanalytics.agilent.com</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Spheroid FluxPak</td>
			<td>Agilent Technologies Inc.</td>
			<td>102905-100</td>
			<td>Each Seahorse XFe96 Spheroid FluxPak contains: 6 Seahorse XFe96 Spheroid Microplates (102978-100), 6 XFe96 sensor cartridges, and 1 bottle of Seahorse XF Calibrant Solution 500 mL (100840-000)</td>
		</tr>
		<tr>
			<td>Serological pipette: 5, 10, and 25 mL</td>
			<td>Greiner Bio-One</td>
			<td>606107; 607107; 760107</td>
			<td>Consumables for cell culture</td>
		</tr>
		<tr>
			<td>SK-OV-3</td>
			<td>ECACC</td>
			<td>&#160;#HTB-77</td>
			<td>Ovarian adenocarcinoma cell line</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM solution)</td>
			<td>Merck Life Science</td>
			<td>S8636</td>
			<td>Supplement for cell culture growth and XF assay medium</td>
		</tr>
		<tr>
			<td>T75 cm2 cell culture flask</td>
			<td>Greiner Bio-One</td>
			<td>658175</td>
			<td>Tissue culture treated flasks for maintaining cell cultures</td>
		</tr>
		<tr>
			<td>TrypLExpress</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr>
			<td>Wave controller software</td>
			<td>Agilent Technologies Inc.</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide orifice tip</td>
			<td>STARLAB International GmbH</td>
			<td>E1011-8400</td>
			<td>Pipette tips with wide opening for spheroid handling</td>
		</tr>
	</tbody>
</table>

exploring mitochondrial energy metabolism of single 3d microtissue spheroids using extracellular flux analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

To obtain well-formed, compact spheroids, each cell line was optimized individually for seeding density and duration of cultivation (Figure 3). A549, HepG2/C3A, and SK-OV-3 cell lines initially formed loose aggregates that did not progress to round spheroids with clearly defined perimeters until after 7 days in culture. Conversely, MCF-7 cells could form spheroids within 3 days. There was a clear correlation between the initial cell seeding density and spheroid volume after the culture perio...

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Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

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Cancer Research

2.9K Views.  University of Birmingham. These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis. 

Video: Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

We present a protocol for the isolation and freeze-fracturing of extracellular vesicles (EVs) originating from cancerous urothelial cells. The freeze-fracture technique revealed the EVs' diameter and shape and-as a unique feature-the internal organization of the EV membranes. These are of immense importance in understanding how EVs interact with the recipient membranes.

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...