This research was supported by Natural Science Foundation of Zhejiang Province grants (Z23H290001, LY19H280001); National Natural Science Foundation of China grants (82274364, 81673607, and 81774011); as well as the Public Welfare Research Project of Huzhou Science and Technology Grant (2021GY49, 2018GZ24). We appreciate the great help, technical support, and experimental support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University.

1. Material preparation
<ol>
	<li>Culture 2 x 106 Panc02 cells in 12 mL of DMEM medium (10% fetal bovine serum and 100 U/mL of penicillin-streptomycin), and maintain at 37 &#176;C and 95% humidity in an atmosphere of 5% carbon dioxide and 95% air for 48 h.</li>
	<li>Collect Panc02 cells, centrifuge at 28 x g for 2 min, and then discard the supernatant. Ensure that the sample is of an appropriate size (1 x 107 cells), as otherwise, the following fixation and dehydration steps will not work well.</li>
	<li>Add 1 mL of 2.5% glutaraldehyde as a fixative to fix the fresh Panc02 cells in the 1.5 mL micro-centrifuge tubes overnight at 4 &#176;C. 
	NOTE: The samples need to be centrifuged at 1,006 x g for 5 min in each of the following steps (steps 1.3-1.11).</li>
	<li>Aspirate the fixative, and rinse the samples twice with 0.1 M phosphate-buffered saline (PBS), and then rinse twice with double-distilled water (ddH2O) at room temperature for 10 min each.</li>
	<li>Add 50 &#181;L of a solution containing 1% osmium tetroxide (OsO4) and 1.5% potassium ferrocyanide at a ratio of 1:1 at 4 &#176;C for 1 h. Then, rinse twice with 0.1 M PBS for 10 min each time, and rinse with ddH2O for another 10 min.</li>
	<li>Add 1 mL of 1% thiocarbohydrazide (TCH), incubate at room temperature for 30 min to 1 h, and then rinse with ddH2O four times for 10 min each time.</li>
	<li>Add 50 &#181;L of 1% OsO4, fix at room temperature for 1 h, and then rinse with ddH2O four times for 10 min each time.</li>
	<li>Add 1 mL of 2% uranyl acetate at 4 &#176;C overnight, and then rinse with ddH2O four times for 10 min each time.</li>
	<li>Add 1 mL of Walton solution (0.066 g of lead nitrate dissolved in 10 mL of 0.03 mol/L aspartic acid stock solution) at 60 &#176;C for an additional 1 h of incubation.</li>
	<li>Rinse with ddH2O four times for 10 min each time, and then dehydrate in a graded alcohol series for 10 min each time: 50% alcohol, 70% alcohol, 80% alcohol, and 90% alcohol 1x, and 100% alcohol 2x. Then, dehydrate in 100% acetone twice for 10 min each time. Use 1 mL of each solution for the dehydration.</li>
	<li>Mix 200 &#181;L of acetone with Pon 812 epoxy resin at a ratio of 3:1, 1:1, and 1:3. Soak the sample in resin at room temperature for 2 h, 4 h, and 4 h, respectively, and then take the 100% Pon 812 epoxy resin to impregnate overnight. 
	NOTE: When performing the staining and resin embedding steps, it is important to operate in a fume hood as these are toxic substances. Additionally, lab coats and solvent-resistant gloves must be worn during the experiment.</li>
	<li>Put the specimens into an embedding mold, and then place in an oven at 60 &#176;C for 48 h to polymerize. Use flat embedding29 to reduce the appearance of resin around the sample. This simplifies the specimen installation and decreases the impact of charging artifacts on the subsequent image segmentation. 
	NOTE: To subsequently generate a horizontal cutting surface, a small amount of resin can be added into the mold hole, taking care not to overfill it.</li>
	<li>Prepare silicon wafers by first washing them and then hydrophilizing with a concentrated H2SO4/H2O2 solution for 30 min. Collect serially slice strips with a thickness of 70 nm on the hydrophilized silicon wafers by using an ultramicrotome, and dry them in a 60 &#176;C oven for 10 min. 
	NOTE: Slowly capture the slice as the slice floats to the surface of the wafers to prevent slice loss or deformation.</li>
</ol>
2. Image acquisition and three-dimensional reconstruction
<ol>
	<li>Before mounting a silicon wafer on the stage, wash the sections with distilled water three times, and air-dry them. Cut a conductive carbon tape with the size of 1 cm x 0.5 cm, and stick this between the silicon wafer and SEM specimen stage to complete the sample setup (Figure 2E).</li>
	<li>Set the FE-SEM accelerating voltage parameter to 2 kV with a working distance of 4 mm (Figure 3B and Table 1). 
	NOTE: In order to improve the signal-to-noise ratio and reduce the noise in the image, observe at low magnifications whenever possible.As the multiple increases, the scanning range of the SEM decreases, leading to an increase in charge accumulation on the images.</li>
	<li>Click on the H/L icon in the top menu bar. First, at low magnification, orient the first section of the target slice strips, and then click again on the H/L option (Figure 3A) to switch to high-magnification mode; collect an appropriate image for the structure of interest by adjusting the image brightness, contrast, and magnification.</li>
	<li>Select Project View &#62; Open Data, and import the image files to be analyzed into the software.</li>
	<li>Align the pictures by clicking on Align Slices &#62; Edit (Figure 4B), and adjust the Intensity Range value in the lower-left menu bar by modifying the image transparency. Here, use a semi-automatic alignment strategy; specifically, through automatic alignment, make the pictures overlap with a previous picture, and then perform manual fine-tuning. 
	NOTE: In this work, during the process of alignment, the previous image was used as a reference, and the move and rotate operations were utilized to maximally overlap the target structure of the two images. Clicking on Align Current Slice Pair can help to align the images automatically, but misplacement may occur.</li>
	<li>Select the Extract Subvolume module, and clip the aligned data sets to fit the size of the overlapping portion of the whole stack.</li>
	<li>Under the Segmentation subsection (Figure 4C), select Resample &#62; Segmentation &#62; Save. Choose the threshold of the Magic Wand and the size of Brush tool to select the correct range. Here again, adopt a semi-automatic segmentation method by first using the Magic Wand to automatically select a suitable large area, and then use the Brush to accurately describe the details. 
	NOTE: The Magic Wand can be applied to segment images based on the obvious differences in the pixel intensity existing between the structure of interest and the surrounding structures by altering the Masking value and using the Draw Limit Line. It is incapable of distinguishing the charging artifacts generated during image acquisition. Therefore, it may result in incorrect segmentation for the target regions. The Brush tool can be used to segment manually to accurately reconstruct the biological structures.</li>
	<li>Under Segmentation &#62; Selection, click on the + icon to add the selected area (Figure 4C).</li>
	<li>Repeat the above step (2.8) to select the same target structure in different images until the selection is complete for reconstructing the microstructures of interest. 
	NOTE: During the mitochondrial remodeling processes, the selection of the target object must be performed from the picture in the middle of a group of sequential images. If the required area is selected from the first or last picture, the reconstruction result will not be able to present a complete 3D visualization.</li>
	<li>After completing the regional segmentation, generate the image file according to the best results for the size of the object and the picture resolution. Click on Crop Editor, and then enter the number 6 in the Virtual Slider box (Figure 4C).</li>
	<li>In the drop-down menu on the left, right-click on the gray area under the Project subsection, and select Generate Surface &#62; Create &#62; Apply. In the generated file, use the Surface View module to create the surface structure and 3D representation.</li>
</ol>
3. Quantification
<ol>
	<li>Click on Remove Small Spots &#62; Apply (Figure 4D). Under the Project subsection, right-click on the gray area, and select Label Analysis &#62; Apply (Figure 4D).</li>
	<li>Customize the name of the column, and choose a measure group. Select Volume3d and Length3d from the Native Measurements box.</li>
	<li>Click on the Apply button in the lower-left corner of the screen. Copy the data, and graph in the statistical software, such as GraphPad.</li>
</ol>

3D Technique to Visualize Mitochondrial Cristae in Pancreatic Cancer Cells

The authors declare no conflicts of interest.

The method presented here is a useful step-by-step guide for applying the 3D reconstruction technique, which involves applying electron microscopy and image processing technology to the stacking and segmentation of 2D tomographic images generated from serial ultrathin sections. This protocol highlights a limitation of 2D images that can be addressed by 3D visualization of the organelle ultrastructure, which has the advantages of strong reproducibility of the structures at a high-resolution level and higher accuracy. More...

Mitochondria are one of the most important organelles in the cell. They serve as the central hub of cellular bioenergetics and metabolism1,2 and play a critical role in cancer3. Pancreatic cancer (PC) is one of the most difficult cancers4 to treat due to its rapid spread and high mortality rate. Mitochondrial dysfunction, which is mainly caused by changes in the mitochondrial morphology3,5,6,7, has been linked to the disease mechanisms underlying PC8. Mitochondria are also highly dynamic, which is reflected by the frequent and dynamic changes in their network connectivity and cristae structure9. The reshaping of the cristae structure can directly impact the mitochondrial function and cellular state10,11, which are significantly altered during tumor cell growth, metastasis, and tumor microenvironment changes12,13.
In recent years, scientists have studied this organelle using EM observation14,15,16,17; for example, researchers have analyzed the mitochondrial dynamics using 3D reconstruction techniques6,7,18,19. The general concept and method for the 3D reconstruction of electron microscopy images were formally established as early as 196820 and involved combining electron microscopy, electron diffraction, and computer image processing to reconstruct the T4 phage tail. Up until now, electron microscopy 3D imaging technology has made significant advancements in terms of the image resolution21, degree of automation22, and processing volume23 and has been used on an increasingly broad scale in biological research, from the tissue level to the organelle ultrastructure level at the nanometer scale24. In recent years, electron microscopy 3D imaging has also become a promising technology for a wide range of applications25,26,27.
The growing attention on mitochondrial cristae particularly illustrates the essential requirements for ultrastructural volume imaging. Transmission electron microscopy (TEM) has been used to visualize samples collected on a copper grid (400 mesh)28, with the electron beam passing through the section. However, due to the limited range of the copper grid, it is impossible to fully image continuous slices of the same sample29. This complicates the study of target structures during TEM imaging. Additionally, TEM relies on time-consuming and error-prone manual tasks, including cutting and collecting multiple slices and imaging them sequentially21, so it is not adapted for ultrastructural reconstructions of large-volume samples23. At present, the high-resolution reconstruction of large-volume sample imaging is achieved through the use of specialized equipment, such as the TEM camera array (TEMCA)30 or two second-generation TEMCA systems (TEMCA2)31, which enable automated high-throughput imaging in a short time. However, this type of imaging does not have the advantage of being easy to obtain and universal due to the requirement for customized equipment.
Compared with TEM, the method for automatically generating thousands of serial volumetric images for large areas based on SEM32,33 enhances the efficiency and reliability of serial imaging and delivers higher z-resolutions34. For example, serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) have both made it possible to achieve the 3D reconstruction of ultrastructure with high speed, efficiency, and resolution35,36. However, it is inevitable that the block surface is mechanically shaved off by the diamond knife of the SBF-SEM or by milling with the focused ion beam of FIB-SEM33,37. Due to the destructiveness of the two methods to the samples, it is not possible to reconstruct the same target structure again for further analysis38,39,40. In addition, few studies have attempted to reconstruct the 3D organelle ultrastructure of cancer cells using EM to observe pathological changes12. For these reasons, in order to further elucidate the pathological mechanisms of cancer cells, such as pancreatic cancer cells, we propose a novel technology for the 3D reconstruction of serial section images using an ultramicrotome and a field emission scanning electron microscope (FE-SEM) to analyze the mitochondrial ultrastructure at the cristae level; with this technology, high-resolution data can be acquired using an efficient and accessible method. The serial ultrathin sections made using an ultramicrotome can be semi-permanently stored in a grid case and reimaged multiple times, even after several years41. FE-SEM is highly valued as a tool in various research fields due to its ability to provide high-resolution imaging, high magnification, and versatility42. In an attempt to display the fine structure of organelles in 3D, the technique for producing serial 2D image stacks with useful resolution using back-scattered electrons produced by FE-SEM43,44 can also be used to achieve high-throughput and multi-scale imaging of target regions or their associated structures without special equipment45. The generation of charge artifacts directly affects the quality of the acquired images, so it is particularly important to keep the dwell time short.
Thus, the present study elaborates on the experimental procedures employed in this SEM technique to reconstruct the 3D structure of mitochondrial cristae46. Specifically, we show the process developed to achieve the semi-automatic segmentation of mitochondrial regions and digitize the 3D reconstruction using Amira software, which also involves making slice samples using the conventional OTO specimen preparation method44,47, completing the section collection using ultramicrotome slicing, and acquiring sequential 2D data by FE-SEM.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(1S,3R)-RSL3</td>
			<td>MCE</td>
			<td>HY-100218A</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>SIGMA</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>Amira</td>
			<td>Visage Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspartic acid</td>
			<td>MCE</td>
			<td>HY-42068</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#8217;s medium</td>
			<td>Gibco</td>
			<td>11995115</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrostatin-1</td>
			<td>MCE</td>
			<td>HY-100579</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437010</td>
			<td></td>
		</tr>
		<tr>
			<td>Field emission scanning electron microscope</td>
			<td>HITACHI</td>
			<td>SU8010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Alfa Aesar</td>
			<td>A10500.22</td>
			<td></td>
		</tr>
		<tr>
			<td>Lead nitrate</td>
			<td>SANTA CRUZ</td>
			<td>sc-211724</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium Tetroxide</td>
			<td>SANTA CRUZ</td>
			<td>sc-206008B</td>
			<td></td>
		</tr>
		<tr>
			<td>Panc02</td>
			<td>European Collection of Authenticated Cell Cultures&#160;</td>
			<td>98102213</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Biosharp</td>
			<td>BL505A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Biosharp</td>
			<td>BL302A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pon 812 Epoxy resin</td>
			<td>SPI CHEM</td>
			<td>GS02660</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrocyanide</td>
			<td>Macklin</td>
			<td>P816416</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiocarbohydrazide</td>
			<td>Merck</td>
			<td>223220</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>LEICA</td>
			<td>EMUC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>RHAWN</td>
			<td>R032929</td>
			<td>2020.2</td>
		</tr>
	</tbody>
</table>

a three-dimensional technique for the visualization of mitochondrial ultrastructural changes in pancreatic cancer cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

During cell culture (Figure 1A), we first divided the pancreatic cancer cells into a control group cultured with complete culture medium, a (1S,3R)-RSL348 (RSL3, a ferroptosis activator, 100 nM) group, and an RSL3 (100 nM) plus ferrostatin-149 (Fer-1, a ferroptosis inhibitor, 100 nM) group. Through the above experimental steps, the scanning electron microscope acquired 38 (Supplementary&#160;Figure 1), 43 (Supplementary...

Watch this Scientific Journal Video about A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells at JoVE.com

Author Spotlight: A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells  

This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

This project aims to review the anti-cancer effect of drugs by changing the structure at the subcellular nano-scale level, and establishing the link between organelle morphology and the cancer pathological mechanism. This technology combines traditional electron microscopy technology with three dimensional reconstruction technology without special equipment. Compared to other techniques, it can improve imaging efficiency and the reconstruction flexibility, and the real life reconstruction of target structures at any time.So it's highly easy and universal. This technology of three dimensional reconstruction of the continuous ultra-thin slides can repeatedly restore the real stereochemical without specific expensive equipment. It can be used for qualitative and quantitative analysis of all organelles at any time.It realizes the easy operation of observation.

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1.5K Views.  School of Pharmaceutical Science of Zhejiang Chinese Medical University. This project aims to review the anti-cancer effect of drugs by changing the structure at the subcellular nano-scale level, and establishing the link between organelle morphology and the cancer pathological mechanism. This technology combines traditional electron microscopy technology with three dimensional reconstruction technology without special equipment. Compared to other techniques, it can improve imaging efficiency and the reconstruction flexibility, and the real life reconstruction of ...

Video: Author Spotlight: A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells  

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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 ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Robotic Pancreatoduodenectomy for Pancreatic Head Cancer: a Case Report of a Standardized Technique

Robotic pancreatoduodenectomy (RPD) for pancreatic cancer is a challenging procedure. Aberrant vasculature may increase the technical difficulty. Several studies have described the safety of RPD in case of a replaced or aberrant right hepatic artery, but detailed video descriptions of the approach are lacking. This case report describes a step-&#173;by-&#173;step technical video in case of a replaced right hepatic artery. A 58-year-old woman presented with an incidental finding of a 1.7 cm pancreatic head mass. RPD was performed using the da Vinci Xi system and involves a robotic-assisted pancreatico- and hepatico-jejunostomy and open gastro-jejunostomy at the specimen extraction site. The operation time was 410 min with 220 mL of blood loss. The patient had an uncomplicated postoperative course and was discharged after 5 days. Pathology revealed a pancreatic head cancer. RPD is a feasible and safe procedure in case of a replaced hepatic artery when performed in selected patients in high-volume centers by experienced surgeons.

Robotic pancreatoduodenctomy (RPD) has been highly standardized in recent years and may be used in selected patients with pancreatic head cancer, including those with a replaced right hepatic artery. This case report describes a standardized and reproducible technique for RPD, which includes the approach of the Dutch LAELAPS-3 training program to an aberrant vasculature.

Robotic pancreatoduodenectomy (RPD) for pancreatic cancer is a challenging procedure. Aberrant vasculature may increase the technical difficulty. Several ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary gland secretion, circadian rhythm, macular degeneration, immune function, and cardiac physiology. Murine surgical sympathectomy is a method for studying the effects of adrenergic innervation by allowing for complete, unilateral adrenergic ablation while avoiding the need for repeated pharmacologic intervention and the associated side effects. However, surgical sympathectomy in mice is technically challenging because of the small size of the superior cervical ganglion. This study describes a surgical technique for reliably identifying and resecting the superior cervical ganglion to ablate the sympathetic nervous system. The successful identification and removal of the ganglion are validated by imaging the fluorescent sympathetic ganglia using a transgenic mouse, identifying post-resection Horner's syndrome, staining for adrenergic markers in the resected ganglia, and observing diminished adrenergic immunofluorescence in the target organs following sympathectomy. This model enables future studies of cancer progression as well as other physiological processes regulated by the sympathetic nervous system.

The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion.

Growing evidence suggests that the sympathetic nervous system plays an important role in cancer progression. Adrenergic innervation regulates salivary ...