MYWZ is funded by the The Secretary of State for Business, Energy and Industrial Strategy and the Newton Prize 2020 as a part of the UK&#39;s Official Development Assistance &#34;ODA&#34; and Newton fund. SS is supported by Cancer Research UK Advanced Clinician Scientist fellowship C53575/A29959. SS, FO, and HR receive funding as part of HUNTER, funded through a partnership between Cancer Research UK, Fondazione AIRC, and Fundacion Cientifica de la Asociacion Espanola Contra el Cancer.

1. Cell preparation
<ol>
	<li>Perform all the experiments under sterile conditions in Class II laminar flow microbiological safety cabinet (see Table of Materials).
	<ol>
		<li>Turn on the hood and allow for the stabilization of the airflow.</li>
		<li>Thoroughly spray the interior hood surface with 70% ethanol to eliminate any possible contamination from previous users.</li>
		<li>Prepare 5% of a disinfectant solution in a 500 mL glass beaker. Discard any cell supernatant or cell debris inside the solution.</li>
		<li>Thoroughly clean all the micropipettes and tip boxes with 70% ethanol.</li>
	</ol>
	</li>
	<li>Prepare fresh cell culture media by supplementing Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) high glucose media with 10% heat deactivated fetal bovine serum (FBS), 1% penicillin/streptomycin (100 unit/mL of penicillin and 100 &#181;g/mL of streptomycin), and 2 mM L-glutamine. For the LX2 hepatic stellate cell line, reduce the concentration of the FBS supplement to 2%.</li>
	<li>Warm culture media before initiating the experiment.</li>
	<li>Take Huh7 and Hep3B HCC tumor cell lines and COS7 and LX2 fibroblast cell lines from their storage rack in liquid nitrogen. Rapidly defrost cryopreserved cells.</li>
	<li>Dilute thawed cells with 2 mL of fresh culture media. Centrifuge cells at 200 x g for 4 min at room temperature. Discard the supernatant and resuspend the cell pellet in 1 mL of fresh warm culture media.</li>
	<li>Seed cells in T75 cell culture flask. Incubate the cells in a cell culture incubator in 5% CO2 at 37 &#176;C in 95% humidified conditions until cells reach 60%-70% confluency.</li>
</ol>
2. Cell collection
<ol>
	<li>Aspirate culture media and wash cells three times with phosphate buffer saline (PBS).</li>
	<li>Add 2 mL of pre-warmed 1x Trypsin to detach adherent cells from the bottom of the T75 flasks. Incubate at 37 &#176;C in an incubator for 4 min.</li>
	<li>Inactivate trypsin by adding 4 mL of complete culture media. Collect the cell suspension and centrifuge cells at 200 x g for 4 min at room temperature. Discard the supernatant and resuspend the cells in 4 mL fresh culture media.</li>
</ol>
3. Cell counting
<ol>
	<li>Gently vortex the cell suspension to ensure homogenous distribution of cells in the centrifuge tube.</li>
	<li>Using a 10 &#181;L pipette, mix 10 &#181;L of cell suspension with 10 &#181;L of Trypan blue. Gently pipette the mixture up and down four times to ensure complete staining of the outer cell surface with the dye.</li>
	<li>Count the number of cells using a hemocytometer.
	<ol>
		<li>First, place a coverslip over the hemocytometer counting area before loading the stained cell suspension.</li>
		<li>Place the pipette tip containing the cell suspension into the V groove of the hemocytometer. Gently expel the tip content into the counting slide.</li>
		<li>Leave the slurry to settle for a couple of minutes before fixing it on the microscope stage for cell counting. 
		NOTE: To avoid double-counting, only count the cells on the two sides of the large square.</li>
		<li>Count in cells overlapping the top or the right ruling and avoid those overlapping the bottom or the left ruling.</li>
	</ol>
	</li>
	<li>Calculate the total number of cells. 
	NOTE: Cell number per ml = <img alt="Equation 1" src="/files/ftp_upload/62868/62868eq01.jpg" /></li>
</ol>
4. Collection of fibroblast conditioned media (CM)
<ol>
	<li>Aspirate culture media and wash the LX2 cells three times with PBS.</li>
	<li>Add 2 mL of pre-warmed 1x Trypsin to detach adherent cells from the bottom of the T75 flasks. Incubate the flask at 37 &#176;C in an incubator for 4 min.</li>
	<li>Inactivate trypsin by adding 4 mL of complete culture media. Collect the cell suspension and centrifuge cells at 200 x g for 4 min at room temperature. Count the cells as per step 3.</li>
	<li>Seed 1 x 106 LX2 cells in 10 cm3 dishes for 48 h at 37 &#176;C.</li>
	<li>Collect the fibroblast CM after 48 h. Centrifuge at 200 x g for 4 min at room temperature to pellet any floating cells. Sterile filter the CM using a 0.22 &#181;m filter fixed on the bottom of 20 mL syringes.</li>
	<li>Collect the supernatant CM. Aliquot the CM into 2 mL tubes and store at -80 &#176;C for further applications. 
	&#8203;NOTE: The solution can be stored for 6 months at -80 &#176;C.</li>
</ol>
5. Validating cell densities for perfect spheroids
<ol>
	<li>Aspirate culture media and wash the HCC cell lines three times with PBS.</li>
	<li>Add 2 mL of pre-warmed 1x Trypsin to detach adherent cells from the bottom of the T75 flasks. Incubate at 37 &#176;C in an incubator for 4 min.</li>
	<li>Inactivate trypsin by adding 4 mL of complete culture media. Collect the cell suspension and centrifuge cells at 200 x g for 4 min at room temperature.</li>
	<li>Count the cells as per step 3.</li>
	<li>Pipette different densities from the tumor cell lines (12000, 6000, 3000, 1500, 1000, 750, 500, 250, and 125 cells) in 20 &#181;L of media on the interior surface of a 10 cm3 lid of a Petri dish.</li>
	<li>Add 10 mL of sterile PBS to the bottom of the dish to provide humid conditions for the process of spheroid formation.</li>
	<li>Invert the lid of the 10 cm3 dish to allow the media, including the cell suspension, to hang over a humid environment. Leave the hanging droplets for 3 days.</li>
	<li>Take images of the spheroids at 50x magnification using an inverted microscope 3 days after hanging the original droplets.</li>
</ol>
6. Heterotypic tumor/stromal spheroids
<ol>
	<li>Suspend 1500 Huh7 HCC cells with 1500 COS7 mammalian fibroblast cells (1:1 ratio) in hanging droplets to form spheres. Add 10 mL of sterile PBS to the bottom of the dish to provide humid conditions for the spheroids.</li>
	<li>Invert the lid of the 10 cm3 dish to allow the media, including the cell suspension, to hang over a humid environment. Leave the hanging droplets for 3 days.</li>
	<li>Take images of the spheroids using an inverted microscope from day 3 until day 10 of culture.
	<ol>
		<li>Put the 10 cm3 dish on the microscope stage. Adjust the magnification of the microscope at 50x for all spheroids.</li>
		<li>Open the microscope software on the attached computer and adjust its focus to have a clear image of every spheroid. Use the Capture Tool on the microscope software to save the acquired pictures.</li>
	</ol>
	</li>
</ol>
7. Homotypic Hep3B spheroids in LX2 CM
<ol>
	<li>Suspend 3000 Hep3B HCC cells in the hanging droplets to form spheres. Add 10 mL of sterile PBS to the bottom of the dish to provide humid conditions for the spheroids.</li>
	<li>Invert the lid of the 10 cm3 dish to allow the media, including the cell suspension, to hang over a humid environment. Leave the hanging droplets for 3 days.</li>
	<li>Transfer Hep3B spheroids into 20 &#181;L of fresh CM from LX2 cells in hanging droplets. 
	NOTE: Use sterile autoclaved unfiltered 200 &#181;L pipette tips in the process of spheroid transfer to avoid any disruption or injury to the formed spheroids. This is also to eliminate any residual cells that remain unattached to the main single spheroid.
	<ol>
		<li>Use an autoclaved 20 &#181;L pipette for the transfer process. Adjust the pipette volume to 2 &#181;L. Attach the pipette tip to the pipette.</li>
		<li>Invert the lid of the 10 cm3 dish on which the spheroids were formed. Fix the lid on the stage of a light microscope. Adjust the fine focus of the microscope to make each spheroid visible.</li>
		<li>Carefully empty the air from the micropipette by pressing the plunger button. Insert the pipette tip in the droplet, including the spheroid, to be transferred. Get very close to the spheroid without touching it with the tip.</li>
		<li>Gently release the pressure on the plunger button to allow the suction of the spheroid into the micropipette tip in 2 &#181;L media.</li>
		<li>Transfer the spheroid into a new droplet hanging on a new 10 cm3 dish, having new media/conditioned media/treatment. 
		NOTE: Ensure that all the spheroids are successfully transferred to the new 10 cm3 dish using the light microscope.</li>
	</ol>
	</li>
	<li>Take images of the spheroids at 50x magnification using an inverted microscope from the day of transfer (day 3) until day 7 of culture in LX2 CM.</li>
</ol>
8. Calculation of spheroid volume
<ol>
	<li>Assign a unique numeric identifier for each spheroid so that images from matched spheroids can be captured daily.</li>
	<li>Analyze images of the growing spheroids using an image analysis software package.
	<ol>
		<li>Open each spheroid image within the software package. Using the Freehand selection tool and outline each spheroid. From the Analysis dropdown button, select Set Measurement, and then Area. Press OK.</li>
		<li>Manually draw a circle around each spheroid. Once the sphere is circled, press Ctrl + M to allow the program to calculate the spheroid area in Pixels. Convert the area of the spheroid into a volume. 
		NOTE: Volume of spheroidmm3&#160;=0.09403 &#215; <img alt="Equation 2" src="/files/ftp_upload/62868/62868eq02.jpg" /></li>
		<li>Calculate the change in spheroid volume relative to its volume on the first day of image capture. 
		NOTE: This is to normalize the spheroid volume to the starting volume and improve accuracy given the natural variation in the starting size.</li>
	</ol>
	</li>
</ol>

FO is a director and shareholder of Fibrofind limited.

The context in which the experimental cell lines grow influences their gene expression profile, pathway analysis, and functional criteria. For instance, in breast cancer cells, the coordination between different oncogenic pathways is retained only if cancer cells are grown in 3D conformation27. Deregulated genes in 3D melanoma and breast cancer spheroids, but not in the monolayer cells, are more relevant to the in vivo human tumor28,29. For example, &#946;1 integrin levels in mammary epithelial cells and tumor counterparts were lower than levels grown in a 2D format29. Furthermore, fibroblasts in 3D structures tend to migrate distinctly in terms of morphology and speed compared to those cultured on plastic30. In addition, the mechanical stiffness of the growth substrate activates specific pathways that encourage the malignant transformation of normal epithelial cells via deregulation of the extracellular signal-regulated kinase (ERK)/Rho in epithelial cells31. These factors favor the transition from conventional 2D culture to 3D spheroid models, as 3D cultures are more in keeping with human disease.
The current study used conventional laboratory tools and supplies to produce a 3D tumor spheroid model. The formed spheroids responded to the proliferation signals coming from fibroblasts, as shown by their significantly increased growth. This model belongs to the multicellular tumor spheroids category. There are three other categories of 3D tumor spheroids, including tumorospheres32, tissue-derived tumor spheres33,34, and organotypic multicellular spheroids35. In multicellular tumor spheroids, tumor cells are suspended in low-adhesion conditions to allow them to aggregate together to form spheres without touching the bottom of the culture vessel36. Several approaches have been employed to deliver this anchorage-independent technique, ranging from rotating systems, liquid overlay techniques, and uncoated ultra-low attachment U-shaped plates37. In HCC, most of the in vitro spheroid cultures use the ultra-low attachment 24- or 96-well plates to form rounded spheroids18,19,20,21,22. This technique is, however, not cost-effective and doesn&#39;t exclude any accidental cell-plastic contact. Other systems use trans-wells23 or liver slices12 to produce liver micro-tissues. Using human tissues in translational work is the gold standard but not always available or accessible by many research groups. The current approach benefited from the hanging droplets theory. Cell suspension is added so that the tumor spheroids are formed in an accessible liquid-air interface to form single rounded spheres38. Advantages of using this technique are the lack of any cell-plastic contact and the ease of studying the autocrine and paracrine crosstalk between tumor and fibroblast cells. This model was further validated in another study deciphering the importance of the non-parenchymal TREM2 in protecting the liver from HCC development39. This work showed that the volume of both Hep3B and PLC/PRF5 spheroids was higher in control LX2 CM versus TREM2-overexpressing LX2 CM in a Wnt-dependent manner39.
In the current study, fibroblasts were mixed with tumor cells before spheroid formation to investigate the proliferative impact of this co-culture. Others have added fibroblasts, endothelial cells, and immune cells with tumor cell suspension after forming a tumor spheroid to study stromal cell migration into the tumor40,41. The current heterotypic spheroid model showed a similar growth pattern to the previously reported models and the original in vivo tumor; spheroids show exponential growth followed by a delayed growth phase, most probably due to the depletion of nutrients and enlargement of the necrotic core42. Instead of adding growth factors and mitogens to help spheroid formation, this study used a more physiological approach by having these growth factors from direct contact with fibroblasts or their secretome in the fibroblast CM. It is essential to mention that LX2 cells can be further activated by treating with TGF&#946;1 or PDGF43. LX2 cells have been treated with 10 ng/mL TGF&#946;1 for 48 h before removing the media for different experimental settings. LX2 cells were washed three times with PBS (to exclude any direct TGF&#946;1 effect), and then fresh media was added for another 24 h before collecting the CM. The CM collected from TGF&#946;1-stimulated LX2 induced the Hep3B spheroids&#39; growth at a higher rate than the CM from control LX2 (data not shown). This flexible system can lend itself to co-cultures of different cancer cells plus/minus fibroblasts, as well as patient-derived lines. It can also offer a medium-throughput screening assay for drugs that could be quickly adopted and inserted into a translational drug discovery pipeline to inform dosing for in vivo studies.
A limitation of the current study is the use of the immortalized cell lines in spheroid formation rather than freshly isolated HCC tumor cells and cancer-associated fibroblasts. Another limitation is comparing the homotypic Hep3B spheroid&#39;s growth between CM from LX2 and in fresh media rather than CM from other non-fibroblast cell lines. The latter limitation could be addressed by the genetic modification of a gene of interest in the fibroblasts followed by applying the CM from wild type versus genetically engineered fibroblast to homotypic spheroids.

The global incidence and mortality from liver cancer have continued to increase, despite advances in treatments for liver disease and most other types of cancer. In 2018, liver cancer surpassed colorectal and stomach cancer to become the second&#160;most common cause of cancer-related death globally1. In 2020, there were more than 9,00,000 new diagnoses, accounting for 4.7% of total cancer cases worldwide1. This is particularly disappointing, given that the significant risk factors for the development of HCC, the most common form of liver cancer, are well characterised2. Cirrhosis is the most common risk factor for the development of HCC, with 80% of cases developing on the background of established cirrhosis2. Chronic liver diseases, which progress to cirrhosis, and consequently HCC, include Hepatitis B virus (HBV), Hepatitis C virus (HCV), alcohol-related liver disease (ARLD), non-alcoholic fatty liver diseases (NAFLD) - the latter attributed to obesity and type 2 diabetes mellitus (T2DM)2,3. The current management protocols for HCC are stage-dependent and limited for those with advanced cancer, who most often have a poor outcome4. There have been significant advances using kinase inhibitors and, more recently, immune-oncology treatments, although realistically, benefit only a minority of patients with advanced liver cancers5. Moreover, there is concern that HCCs arising in patients with NAFLD - the most rapidly growing underlying cause, accounting for more than 50% of newly diagnosed HCC cases in western nations, may be more resistant to Programmed death 1 (PD1) checkpoint inhibitor therapy6.
There has been a massive investment in clinical trials for patients with HCC, including significant improvements in clinical trials and their endpoints7. After a decade of failures, these investments have started to change the opportunities for patients. However, the reality is that the overall proportion of responders remains relatively poor, with patients recruited into trials often poorly representing those cared for in the clinics. The danger is that the advances are costly and benefit the few rather than the many. As more candidate therapies emerge for single-use or combination, it is essential to have preclinical models more predictive of in vivo responses. These are likely to be models that incorporate additional factors contributing to the variability seen in patient responses that better reflect human HCC heterogeneity and pathological complexity8. Systems that recreate the in vivo pathophysiological conditions of HCC are needed to help understand the biology of tumor evolution, growth, and progression. The existing experimental models of chronic liver diseases and HCC usually fall under three main categories: in vivo animal-based models (reviewed in9), in vitro cultures10, and ex vivo11,12 models. Animal-based approaches are extensively used to study chronic liver diseases, including HCC; however, the genetic variability, high running costs, and the different immune systems between species are among the main limitations for applying such models9. While some ex vivo models provide an excellent tool for focusing on human tissues compared to other in vitro cell line models, tissue availability and the limited experimental time course obstruct their utilization on a large scale.
On the other hand, the in vitro cell line models remain a good option for scientists working with limited resources, with a lesser need to have a constant supply of fresh human tissues10. These models also provide a tool that can be used as a first screen to help with target validation of drug selection before proceeding to more complex in vivo models. The recent modification of the traditional 2D monolayer cultures into 3D cultures has improved the efficacy of these in vitro models13,14.
3D in vitro models can recapitulate critical features seen in human normal and pathological conditions. Under physiological conditions, signal transduction is initiated through cellular crosstalk and interaction with other connective tissue molecules, namely, the extracellular matrix (ECM) proteins, forming a 3D interaction network15,16. The tumor evolves in a 3D spherical form during malignant transformation, for which oxygen and nutrients are readily abundant in non-tumor/tumor interphase. At the same time, hypoxic conditions predominate at the tumor core. This heterogeneity in nutrient availability results in the activation of spatially distinct signaling and metabolic pathways that regulate tumorigenesis. These conditions are poorly recapitulated in the conventional 2D monolayer cultures14, in which cells grow on stiff culture plastic in a physiologically irrelevant fashion. Cancer cells also communicate with other non-parenchymal cells, the primary source of ECM, growth, and invasion signaling within the tumor microenvironment. Unlike 2D cultures, 3D in vitro models can provide a more suitable platform to study this tumor-stromal interaction17.
3D models are widely used in the HCC field, and they vary in the way the micro-tissue is formed18,19,20,21,22,23. Most of these models used either the ultra-low binding plates18,19,20,21,22 or trans-wells23 in the process of spheroid formation. The protocol described introduces the hanging droplet technique as an alternative, plastic-free, and cost-effective in vitro 3D tumor spheroid model. This may facilitate assessing the paracrine and autocrine roles of fibroblasts on the proliferation of the tumor cells in a 3D format.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x Trypsin</td>
			<td>Sigma Aldrich</td>
			<td>59429C</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biorender</td>
			<td>Biorender.com</td>
			<td>Online</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II laminar flow BioMAT2 hood</td>
			<td>Medair Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Biosera</td>
			<td>M-D1107</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel sofware</td>
			<td>Microsoft office 365</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad prism sofware</td>
			<td>GraphPad software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat deactivated fetal bovine serum (FBS)</td>
			<td>life tech</td>
			<td>105000064</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>Fiji</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G7513-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin (100 unit/ml penicillin and 100 &#956;g/ml streptomycin)</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 90 mm, triple vented</td>
			<td>Greiner</td>
			<td>633175</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Virkon&#8482; Broad Spectrum Disinfectant, 5kg Bucket</td>
			<td>Rely+On&#8482;</td>
			<td>SCI-129999</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziess inverted microscope</td>
			<td>Ziess</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Cells cultured in a multi-layered 3D format more accurately reflect the complexity of the tumor microenvironment than conventional 2D cultures24,25. Previously, many studies have supplemented the spheroids culture media with different mitogens and growth factors26 to initiate spheroid formation. In this study, however, the addition of fibroblasts, or their CM, provides essential mitogens and growth factors to accelerate spheroid growth.
Figure 1&#160;depicts data from a pilot study in which Huh7 human HCC cells were seeded in a descending seeding density starting from 12,000 down to 125 cells in 20 &#181;L fresh media for 3 days. A seeding density of 12,000 cells yielded spheroids with an asymmetric shape, which was not corrected even after halving the cell seeding density. However, spheroids created from 3000 cells appeared more rounded (Figure 1, upper row). Further reduction of cell concentration neither succeeded in forming single spheres (125, 250, 500 cell-density spheres) nor had a regular spherical-like appearance (1000 and 1500 cell-density spheroids). It is worth mentioning that many small spheres were formed at a density of 500 tumor cells; however, only one small spheroid was captured at 50x magnification (Figure 1, upper row). The same optimized protocol was applied to the COS7 primate kidney fibroblast cell line (Figure 1, middle row). Suspending 125, 250, and 500 COS7 fibroblasts in 20 &#181;L hanging droplets resulted in one rounded spheroid with multiple smaller spheroids, while higher cell densities (1000, 1500, and 3000) formed single semi-rounded spheroids. Similar to the Huh7 tumor spheroids, higher cell suspensions (6,000 and 12,000 cells/sphere) resulted in the formation of irregular cell aggregates, and hence higher cell concentrations were discarded (Figure 1, middle row). Low densities of Huh7/COS7 heterotypic cell suspensions (125, 250, and 500 cells/sphere) generated a single spheroid with multiple floating or semi-attached spheroids (Figure 1, bottom row). 1000- and 1500-cell heterotypic spheroids were semi-rounded, while a seeding density of 3000 cells (1500 per cell type) gave a rounded 3D spheroid. As noted earlier, higher cell densities resulted in the formation of aggregates rather than well-defined spheroids (Figure 1, bottom row). In conclusion, 3000 cell-density spheroids were rounded, similar to human HCC tumors, and were adapted for further experiments. Culturing the cell suspensions as hanging droplets for 3 days was sufficient to promote spheroid formation.
After optimizing the best cell concentration and time point for spheroid formation in the current context, the proliferative impact of co-culturing tumor and fibroblast cell lines was assessed longitudinally. Figures 2 and Figure 3 show Huh7/COS7 heterotypic spheroids (3000 total cell numbers per spheroid) monitored from day 3 until day 10. Homotypic Huh7 and COS7 spheroids (1500 cells each) served as controls. Starting from day 4, heterotypic spheroids grew in an ideal round-like shape compared to homotypic spheroids (Figure 2). The initial volume of the heterotypic spheroid was larger than that of each homotypic spheroid. The growth of spheroids was calculated as the change in their volume relative to initial volume at the day of spheroid formation to avoid normal variation in spheroid volume (day 3, Figure 3). Heterotypic spheroids initially showed a rapid growth phase starting from day 4 up to day 7 followed by a slower phase of growth at day 8 (Figures 2, upper row, and Figure 3). The spheroid volume decreased on days 9 and 10, possibly reflecting the depletion of nutrients or a hypoxic core and cell death.
In contrast, homotypic Huh7 and COS7 spheroids grew at a much slower rate (Figure 2, middle and bottom rows; Figure 3). Homotypic spheroids exhibited a relatively static growth curve until the fifth day of culture (two days after spheroid formation). Starting from day 6, the homotypic spheroids began to show a gradual increase in their growth curve, albeit at a significantly lower rate than that of the heterotypic spheroids (Figure 3). In conclusion, tumor/fibroblast heterotypic spheroids grow at a higher rate than homotypic spheroids suggesting that the direct contact of tumor cells and fibroblasts increases the size of the tumor spheroids.
Finally, to validate the above findings and to study the paracrine impact of mesenchymal cells on the proliferation of HCC spheroids in a liver-related context, 3-day-old homotypic Hep3B HCC spheroids were grown in regular fresh media or CM from LX2 hepatic stellate cells (Figure 4 and Figure 5) for an additional 4 days. At first glance, 3000 Hep3B cells formed perfectly rounded spheroids after 3 days (Figure 4). Hep3B spheroids showed continual proliferation in fresh media from day 3 till day 7 (Figure 4, upper row). The growth rate was enhanced when Hep3B spheroids were maintained in LX2 CM (Figure 4, lower row). This media-dependent conversion in Hep3B spheroid growth exhibited statistical significance from day 4 up to the end of the experiment (Figure 5), suggesting a fibroblast-driven proliferation of tumor spheroids.
In conclusion, this study has successfully modified the existing 3D spheroid cultures to exploit the crosstalk between HCC tumor cells and different fibroblast cell lines and investigate the proliferative significance of this direct and indirect cellular interaction (Figure 6). Further characterization of the formed spheroids is needed to improve how tumor cells interact with the surrounding microenvironment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62868/62868fig01.jpg" /> 
Figure 1: Optimization of the optimal cell density for spheroid formation. Columns represent different cell seeding densities, and rows represent spheroids formed from Huh7 cells (top row), COS7 cells (middle row), and Huh7/COS7 heterotypic cells (bottom row). Images represent spheroids formed after suspending 125, 250, 500, 1000, 1500, 3000, 6000, and 12000 cells (from left to right) in 20 &#181;L media for 3 days. The pilot study included two spheroids per condition, and the images were taken at 50x magnification, scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62868/62868fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62868/62868fig02.jpg" /> 
Figure 2: Heterotypic versus homotypic spheroid growth. Columns represent the day at which spheroid images were taken, and rows show representative images for spheroids formed from Huh7 cells (top row), COS7 cells (middle row), and Huh7/COS7 cells (bottom row). The images represent the spheroids formed after suspending 3000 cells from each condition (homotypic Huh7, COS7, or the Huh7/COS7 heterotypic spheroids) of different time points from left to right. The experiment included 10 spheroids per condition, and the images were taken at 50x magnification, scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62868/62868fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62868/62868fig03.jpg" /> 
Figure 3: Longitudinal analysis of heterotypic versus homotypic spheroid growth. The graph shows the growth curve of heterotypic Huh7/COS7 spheroids versus homotypic Huh7 and COS7 spheroids. Data are presented as mean &#177; s.e.m; n = 10 independent spheroids. ** p &#60; 0.01; ****p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/62868/62868fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62868/62868fig04.jpg" /> 
Figure 4: Growth of Homotypic Hep3B spheroids in LX2 CM. Columns represent the day at which spheroid images were captured, and rows show representative images of Hep3B spheroids cultured in fresh DMEM culture media (top row) or LX2 CM (bottom row). The experiment included seven spheroids per condition (fresh media or LX2 CM), and images were taken at 50x magnification, scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62868/62868fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62868/62868fig05.jpg" /> 
Figure 5: Longitudinal analysis of the homotypic Hep3B spheroid growth. The graph shows the growth curve of Hep3B spheroids in fresh DMEM culture media or LX2 CM. Data are presented as mean &#177; s.e.m; n = 7 independent spheroids. ****p &#60; 0.001. <a href="https://www.jove.com/files/ftp_upload/62868/62868fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62868/62868fig06.jpg" /> 
Figure 6: Schematic representation of the spheroid formation process. The cell suspension is pipetted on the inner lid of 10 cm3 Petri-dish. The lid is inverted and kept for 3 days to allow for homotypic or heterotypic spheroid formation. Spheroid images are taken at 50x magnification. The figure is created using a web-based science illustration tool (see Table of Materials). <a href="https://www.jove.com/files/ftp_upload/62868/62868fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma at JoVE.com

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

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 ...

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4.9K Views.  Minia University. 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.

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

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

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 ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is typically performed via transfemoral access (TFA). However, transradial access (TRA) is preferred in coronary artery interventions due to decreased complications and mortality. Whether the advantages of TRA can be applied to TACE required investigation.
Patients receiving TRA TACE at a single center were retrospectively enrolled for study. Procedural details, technical success, radial artery occlusion (RAO) rate, and access site-related bleeding complications were evaluated. From October 2017 to October 2018, 112 patients underwent 160 TRA TACE procedures. The overall technical success rate was 95.0% (152/160). The rate of crossover from TRA to TFA was 1.9%. No access site-related bleeding complications were found in any cases. Asymptomatic RA occlusion occurred in three patients (2.7%). Compared with TFA, TRA can increase safety and patient satisfaction while decreasing access site-related bleeding complications. Moreover, TRA interventions can benefit patients with advanced age, obesity, or a high risk of bleeding complications.

Transarterial chemoembolization (TACE) is the standard therapy for patients in the intermediate stage of hepatocellular carcinoma and is typically performed through femoral artery access. Compared with transfemoral access, transradial access (TRA) can decrease the rate of bleeding complications and improve patient tolerance. A method is presented here to perform transarterial chemoembolization via radial artery access.

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

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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 ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...