The authors thank Hanna Voersmann for establishing the 3D organotypic melanoma-spheroid-skin-model and providing excellent protocols. The authors also thank Silke Busch for valuable technical support. The work was supported by BMBF e:Med program "Melanoma Sensitivity" 031A423A.

All human tissue work was performed using approved institutional protocols.
1. Separation of Dermis and Epidermis from Human Skin (Juvenile Foreskin from Circumcision)
<ol>
	<li>Place the foreskin (usually 1 - 2 cm2) into a non-adhesive sterile cell culture dish (&#216; 10 cm) and cover it with 10 - 12 mL of phosphate-buffered saline+ (PBS, 0.5 mM MgCl2, 0.9 mM CaCl2, pH 7.2 = PBS+).Remove all excess (adipose) tissue using sterile scalpel and forceps.</li>
	<li>Cut the skin into 3 mm x 5 mm pieces, wash them with PBS, and transfer them to a non-adhesive sterile cell culture dish (&#216; 6 cm). Cover the tissue pieces with 5 - 7 mL of dispase solution (2 U/mL in PBS). Seal the cell culture dish with paraffin film and incubate for 16 h at 4 &#176;C.</li>
	<li>Retract the epidermal from the dermal part with two sterile forceps. Transfer the dissected tissue pieces to individual non-adhesive cell culture dishes (&#216; 6 cm) and cover each of them with PBS+.</li>
</ol>
2. Isolation of Primary Keratinocytes from the Epidermis
<ol>
	<li>Carefully remove the PBS+ from the cell culture dish using a Pasteur pipette and wash the epidermal tissue pieces (section 1.3) with PBS. Cut the epidermis into smaller pieces (1 mm2) and collect them in a 50-mL conical tube. Add 10 mL 1x Trypsin-EDTA (preheated to 37 &#176;C) and incubate for 5 min in a water bath at 37 &#176;C. Vortex the tissue suspension every 2 min.</li>
	<li>Stop the enzymatic reaction by adding 1 mL fetal calf serum (FCS). Resuspend the cells by pipetting up and down with a 10-mL plastic pipette for 4 min. Try to avoid bubbles and foam formation.</li>
	<li>Pipet the cell suspension into a cell strainer (pore size 100 &#181;m), placed on top of a fresh 50 mL conical tube, and rinse 3x with 5 mL PBS. Centrifuge the cells at 200 x g for 5 min. Resuspend the cell pellet in 2 - 6 mL culture medium containing 1% (v/v) gentamycin. Determine the cell number manually by counting cells in a hemocytometer and seed 6 x 105 cells into a T75 cell culture flask.</li>
</ol>
3. Cultivation of Primary Keratinocytes
<ol>
	<li>Incubate primary keratinocytes isolated in step 2.3 in a cell incubator at 37 &#176;C and 5% CO2 in keratinocyte medium without FCS, to 50 - 70% confluency.</li>
	<li>To passage the keratinocytes carefully remove the medium, add 5 mL PBS-EDTA, and incubate the cells for 10 min at 37 &#176;C. Check if cells have started to detach from the culture flask under a light microscope (4X&#160;magnification: cells will appear rounded but are still attached!). If not, replace old PBS-EDTA with fresh PBS-EDTA and re-incubate for another 10 min at 37 &#176;C.</li>
	<li>Add 5 mL 1x Trypsin-EDTA to the rounded cells still covered with 10 mL PBS-EDTA and re-incubate them for 1 - 3 min at 37 &#176;C. Stop the enzymatic reaction by adding 1 mL FCS and collect the detached cells in a 50-mL conical tube. Centrifuge the cells at 200 x g for 5 min, resuspend the pellet in keratinocyte medium, and seed 6 x 105 cells into a fresh T75 cell culture flask. 
	NOTE: To generate organotypic skin reconstructs, the primary keratinocytes should be used no later than passage 3 - 4.</li>
</ol>
4. Isolation of Primary Fibroblasts from the Dermis
<ol>
	<li>Cut the dermal tissue (section 1.3) into smaller pieces (1 mm2) and transfer them into a 50-mL conical tube. Add 10 mL collagenase-solution (5 U/mL in PBS+) and incubate for 45 min in a water bath at 37 &#176;C. Centrifuge the mixture of tissue pieces and cells at 200 x g for 5 min.</li>
	<li>Wash the cell pellet twice with 10 mL DMEM containing 4.5 g/L glucose and L-Glutamine, but without L-Pyruvate. Finally resuspend the tissue pieces in 2 mL of DMEM containing 1% (v/v) gentamycin and 10% FCS. Transfer them into a T25 cell culture flask and incubate them in a cell incubator at 37 &#176;C and 5% CO2 overnight. 
	NOTE: The small volume allows the fibroblasts to migrate out of the tissue-debris and forces them to attach to the culture flask.</li>
	<li>The next morning, add another 6 mL DMEM/Gentamycin/FCS and incubate for 2 - 3 days at 37 &#176;C until the cells have reached 80 - 90% confluency.</li>
</ol>
5. Cultivation of Primary Fibroblasts
<ol>
	<li>To passage the fibroblasts, remove the medium and wash adherent cells with PBS. Add 5 mL 1x Trypsin-EDTA and incubate for 3 min at 37 &#176;C.</li>
	<li>Stop the enzymatic reaction by adding 5 mL DMEM/FCS and collect the detached cells in a 50-mL conical tube. Centrifuge the cells at 200 x g for 5 min, resuspend the pellet in DMEM medium, and seed 6 x 105 cells into a fresh T75 cell culture flask. 
	NOTE: To generate organotypic skin reconstructs, the primary fibroblasts should be used no later than passage 4 - 6.</li>
</ol>
6. Generation of 3D Melanoma Spheroids Via the Hanging Drop Method
<ol>
	<li>Culture melanoma cells (e.g., 451-LU, or any melanoma cell line of interest) according to general protocols, using RPMI containing 10% FCS15.
	<ol>
		<li>To generate melanoma spheroids of similar size and quality, wash the melanoma cells in PBS, add 5 mL of 1x Trypsin-EDTA in PBS to the cells in a T175 cell culture flask, and incubate for 3 - 5 min at room temperature (RT). Neutralize the Trypsin by adding 5 mL RPMI/10% FCS. Harvest the cells by centrifugation at 200 x g for 5 min. Re-suspend the cell pellet in RPMI/FCS at a final concentration of 10,000 cells/mL as determined by counting in a hemocytometer.</li>
	</ol>
	</li>
	<li>Spot 40 x 25 &#181;L (= 250 cells) of the cell suspension onto the inner surface of the lid of a sterile non-adhesive cell culture dish (&#216; 10 cm) using an electronic multi-pipette. With a fast but smooth movement, turn the lid around and place it on the respective cell culture dish containing 5 mL PBS.
	<ol>
		<li>Culture &#34;hanging drop dishes&#34; in a cell incubator at 37 &#176;C and 5% CO2 for 10 - 14 days, depending on the cell type used. 
		NOTE: Cells from the MM growth phase typically grow faster and form more solid spheroids in the hanging drop compared to melanoma cells derived from the RGP or VGP. For individual evaluation, observe spheroid growth under a pair of binoculars or under a light microscope (4X&#160;magnification). Usually, spheroids become detectable after 48 h under a pair of binoculars or under a light microscope (4X&#160;magnification). After 10 - 14 days, they are visible without any magnification device.</li>
	</ol>
	</li>
	<li>5 days after the initial drop spotting, add 10&#160;&#181;L of fresh RPMI/10% FCS medium to each drop. Subsequently, exchange 10&#160;&#181;L of medium every other day. The use of an electronic dispenser is very helpful in this step.</li>
	<li>Depending on the cell type, harvest the spheroids (see section 10 for details) after 10 - 15 days by gently rinsing them off the cell culture dish lid with PBS. Collect them in a fresh non-adhesive cell culture dish. 
	NOTE: The cultivation period depends on the tumor growth phase of the melanoma cells when they were initially derived, e.g., spheroids derived from 451-LU cells grow to approximately 500 &#181;m in diameter within 12 days of culturing in the hanging drop15.</li>
</ol>
7. Generation of the Dermal Compartment of Organotypic Full Skin Reconstructs
<ol>
	<li>Generate skin models using 24-well cell microporous membrane inserts (pore size 8 &#181;m) placed in 24-well plates. 
	NOTE: Make sure not to use hanging but standing inserts, because they will be placed into a 6-well plate for air-liquid cultivation at a later stage.</li>
	<li>Prepare the gel neutralization solution (GNL)15, as well as the culture media referred to as MM and endothelial growth media (EGM)16,17. To prevent coagulation, store the collagen type I (usually from rat tail, 3.5 - 4 mg/mL in 0.02 N acetic acid) on ice until use, because it starts to gel at RT.</li>
	<li>Re-suspend 1 x 105 fibroblasts per insert in GNL and quickly mix the cell suspension with collagen at a ratio of 1:3 in a final volume of 500 &#181;L/insert. Mix by gently pipetting up and down to avoid bubble formation as bubbles may impair the quality of the skin reconstruct.</li>
	<li>Allow the individual dermal gels to settle by keeping them without medium at RT for 30 min in a sterile hood. Subsequently cover each gel with DMEM containing 4.5 g/L glucose, 1% L-glutamine, 10% FCS, and without L-pyruvate, and incubate overnight at 37 &#176;C.</li>
</ol>
8. Generation of the Epidermal Compartment of Organotypic Full Skin Reconstructs
<ol>
	<li>The next day remove the medium from the dermal gels and equilibrate them with EGM media (10% FCS, 1% PenStrep, 10 mg/mL gentamicin) for 2 h at 37 &#176;C. Withdraw the medium and carefully seed 1 x 105 keratinocytes resuspended in 100 &#181;L EGM on top of the dermal gel.</li>
	<li>Incubate the reconstructs for 1.5 h at 37 &#176;C to allow the keratinocytes to adhere to the dermal compartment. 
	NOTE: During this incubation time, the gels will start to shrink due to fibroblast-induced contraction, and thus, at least partially detach from the insert walls.</li>
	<li>Cover the skin equivalents with approximately 800 &#181;L EGM and carefully remove the residual gel from the insert wall with a small (white) pipette tip.Culture the skin equivalents submerged in EGM for 7 days in a cell incubator at 37 &#176;C, 5% CO2, and change the medium every other day. 
	NOTE: During this time the skin equivalents will shrink significantly.</li>
</ol>
9. Air-liquid Cultivation of Organotypic Full Skin Reconstructs
<ol>
	<li>At day 8, transfer each insert into an individual well of a 6-well plate. Only add 1.2 - 1.4 mL of MM medium to each well, so that the skin reconstruct is supplied with medium from the bottom of the well but is not covered with medium. 
	NOTE: Cultivation at the air-liquid interface allows stratification of the epidermal part and establishment of a full cornified layer (stratum corneum).</li>
	<li>During the next 10 - 17 days, change the medium as stated in section 9.1 every other day. During this period, add drugs or other stimuli as needed to the medium. Carefully remove the full skin reconstruct from the microporous membrane insert using curved tweezers for further analysis, e.g., immunohistochemical analysis (Figure 1).</li>
</ol>
10. Generation of the Organotypic Melanoma Spheroid Skin Models
<ol>
	<li>To integrate the melanoma spheroids into the dermal compartment of the organotypic full skin reconstructs, carefully rinse the spheroids (after step 6.4) off the lid of the hanging drop cell culture dish with PBS. Collect 10 - 20 spheroids/insert in a sterile non-adhesive cell culture dish. Carefully remove excessive PBS with a Pasteur pipette.</li>
	<li>Aspirate the spheroids in the smallest volume possible of EGM. At this point observe and count the spheroids with the naked eye, without any further magnification device. Take 10 - 20 spheroids per skin model/gel, as stated in step 10.1.</li>
	<li>Transfer them to the desired volume of GNL containing fibroblasts (during step 7.3), and mix with the collagen type I. From this step on proceed as described in sections 7.3 - 9.1. 
	NOTE: Tumor spheroids become visible in the dermal gel as white spots (Figure 2)</li>
</ol>

<ol>
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Pathol. 156 (1), 193-200 (2000).</li><li>Sinnberg, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+PI3K-AKT-mTOR+signaling+sensitizes+melanoma+cells+to+cisplatin+and+temozolomide.">Inhibition of PI3K-AKT-mTOR signaling sensitizes melanoma cells to cisplatin and temozolomide.</a> J. Invest. Dermatol. 29 (6), 1500-1515 (2009).</li><li>Niessner, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+farnesyl+transferase+inhibitor+lonafarnib+inhibits+mTOR+signaling+and+enforces+sorafenib-induced+apoptosis+in+melanoma+cells.">The farnesyl transferase inhibitor lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells.</a> J. Invest. Dermatol. 131 (2), 468-479 (2011).</li><li>Foty, R. 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The authors declare that they have no competing financial interests.

The organotypic melanoma spheroid skin model introduced here warrants new insights for a deeper understanding of intra-tumoral and tumor-host interaction, and may provide an advanced screening platform to study molecular mechanisms of tumor development and therapy resistance in the future.
To guarantee the best physiologic and in vivo mimicking conditions of skin reconstructs, the quality of the primary cells is of utmost importance. As stated above, juvenile or pre-natal skin cells show the lowest differentiation grade and are therefore best suitable to generate full-thickness skin equivalents. The first possible quality control is the extent of dermal contraction at day 2 after seeding the keratinocytes and equilibrating the gel to EGM (Protocol sections 8.2 and 8.3). Dermal gels will shrink the most with the best quality of fibroblasts. Also, the integration of too few or too many fibroblasts may impair dermal contraction and consequently attachment of the epidermal to the dermal compartment. This in turn will compromise epidermal differentiation, because this process requires an extensive cross-talk between dermal and epidermal cells.
The quality of primary cells also critically depends on the cell confluency as well as the passage of the cells. If keratinocytes are grown to &#8805;80% confluency they immediately stop proliferating and start to differentiate. It is therefore important to culture them to 40 - 70% confluency during passaging and use them no later than passage 3 - 4. Fibroblasts are less sensitive but should not be used later than passage 4 - 6. Please also note that all primary cells and cell lines used to generate organotypic melanoma spheroid skin models are cultured free of antibiotics, and therefore the contamination risk is high. However, the addition of antibiotics changes the physiology of the individual cells and therefore reduces the quality of the skin equivalents.
In general, tumor spheroid formation is possible from almost all kinds of tumor cell lines and also from tumor cells freshly isolated from patient material. The initial cell number and/or cultivation time for gaining optimal spheroid sizes may vary depending on the cell type used. Up to a size of 150 - 200 &#181;m, all cells included in a spheroid can still be sufficiently supplied with nutrients via simple diffusion. Only spheroids with sizes &#8805;500 &#181;m show a high degree of cellular differentiation and represent typical features of non-vascularized tumor tissue22.
The organotypic melanoma-spheroid-skin-model developed here is particularly suitable to study tumor-host interactions and for melanoma drug testing under in vivo-like conditions15. Still, the environment of melanoma in vivo is even more complex, harboring a variety of tumor associated cell types, including immune cells and endothelial cells. Since the addition of proper primary immune cells faces problems with histocompatibility, these models are not yet able to adequately monitor immune-therapeutic approaches.
Nevertheless, melanoma spheroids can be generated from freshly isolated patient material and once included into the full skin reconstruct, may serve as individual drug screening platforms. Even the integration of pieces of melanoma metastasis into skin reconstructs is conceivable to test drug combinations in a tailored therapeutic approach. Including the organotypic 3D skin-melanoma model in preclinical testing is likely to help ensure that only the most promising novel therapeutic concepts are taken forward into clinical testing, thus reducing the attrition rate of potential new treatments for this disease and increasing the rate of therapeutic success in clinical trials.

The human skin is composed of two distinct compartments that serve different functions in protecting the body from adverse environmental effects1. The lower dermal compartment consists of a fibro-elastic connective tissue. It is composed of loosely connected collagen and elastin fibers synthesized by fibroblasts, serving a mechanical barrier function. The dermis is separated from the upper epidermis by the basal lamina which is produced as an extracellular matrix due to a constant communication between both skin compartments. In contrast to the dermis, the epidermis is a squamous epithelium which mainly consists of keratinocytes and can be differentiated into four layers. The stratum basale consists of undifferentiated basal keratinocytes which constantly derive from skin progenitor cells stratifying through the stages of the stratum spinosum and stratum granulosum into the stratum corneum to protect the body from dehydration and infections2. Melanocytes are aligned at the basal membrane, and communicate through dendritic extensions with multiple keratinocytes. They produce the pigment melanin to protect the skin tissue from the adverse effects of UV radiation, like skin ageing, immunosuppression, inflammation, and induction of non-melanoma skin cancer. UV radiations contribution to the transformation of melanocytes to malignant melanoma, however, is still under debate3.
Melanoma development is differentiated into different tumor progression stages, and characterized by certain genetic, morphologic, and histologic changes4. They originate either de novo or from an innate or acquired nevus due to a local increase of melanocyte proliferation causing benign neoplasia. This precursor lesion may convert into structurally modified dysplastic tissue containing atypic cells, which may continue to the first malignant stage, the radial growth phase (RGP). This early tumor progression phase is characterized by cells radially proliferating within the epidermis, showing few locally invasive cells within the papillary dermis. During the subsequent vertical growth phase (VGP) melanoma cells already show a metastatic and invasive phenotype by breaking through the basal lamina to infiltrate the deeper parts of the dermis as well as the subcutaneous tissue5. Finally, the metastatic melanoma (MM) represents the most aggressive progression stage with metastatic cells systemically spreading throughout the blood- and lymph system to invade distally organs like liver, lung, and brain6.
To date, early diagnosis followed by surgery still remains the most effective therapy of malignant melanoma. The prognosis for patients with distant metastasis, however, remains particularly poor7, because classical chemotherapy regimens confer only little survival benefit8,9. However, after decades of stagnation, recent advances in targeted therapies have considerably improved the prognosis of malignant melanoma.
Dysregulation of two major mitogen activated pathways, the RAS-RAF-MEK-ERK and the PI3K-AKT-PTEN signaling pathways, present key drivers of melanoma progression, especially when constitutively activating point mutations of the proto-oncogenes BRAFV600 and NRAS are present10. Accordingly, the invention of targeted kinase inhibitors promised therapeutic benefit for patients suffering from metastatic melanoma. A multitude of clinical trials conducted to this point has not achieved significant benefit for patients with metastatic melanoma. Nearly all responses are partial, with a subpopulation of patients showing primary resistance. Moreover, the acquisition of secondary resistance leading to relapse was observed in the majority of patients11,12.
It becomes clear that analysis of the mutation status alone is not sufficient to develop the most potent therapeutic strategy. New fast and reliable diagnostic tools are necessary to systematically record and analyze the responsiveness of individual cancer cells. The vast majority of currently available data on human melanoma have been obtained from two-dimensional (2D) melanoma cell cultures. Tumor cells, however, grown in 3D allow intercellular crosstalk between differentiated cancer cell subpopulations as well as between cancer cells and the non-transformed surrounding host tissue. Therefore, it would be best to reconstruct the 3D environment in which the melanoma developed, to be used as a preclinical screening model13,14.

<table border="0" cellpadding="0" cellspacing="0" width="625">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">fetal serum albumin (FCS)</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10270106</td>
			<td style="width:167px;">add 10 % to cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25300054</td>
			<td>1X dilute in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">61870010</td>
			<td style="width:167px;">for cultivation of melanoma cell lines</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM&#160;</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">41965062</td>
			<td style="width:167px;">for cultivation of primary fibroblasts</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dispase</td>
			<td style="width:123px;">Gibco life technologies&#160;</td>
			<td>17105041</td>
			<td>2U/ml, dissolve in PBS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Collagen type I</td>
			<td style="width:123px;">BD Biosciences</td>
			<td>354249</td>
			<td style="width:167px;">usually from rat tail, concentration should be 3.5-4 mg/ml</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">24-well inserts Nunclon&#160;</td>
			<td style="width:123px;">Nunc</td>
			<td>140629</td>
			<td style="width:167px;">8 &#181;m pore size; use standing, not hanging inserts!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cell strainer</td>
			<td style="width:123px;">BD Falcon</td>
			<td style="width:119px;">352360</td>
			<td>yellow</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gentamycine</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td>15750060</td>
			<td>dissolve in PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Keralife keratincyte medium</td>
			<td style="width:123px;">Cell Systems</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">do not add FCS or antibiotics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase</td>
			<td style="width:123px;">SERVA</td>
			<td>17454</td>
			<td>NB4 standard grade</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">juvenile human keratinocytes</td>
			<td style="width:123px;">Cell Systems</td>
			<td style="width:119px;">FC-0007</td>
			<td style="width:167px;">alternative to own preparation from juvenile foreskin</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">juvenile human fibroblasts</td>
			<td style="width:123px;">Cell Systems</td>
			<td style="width:119px;">FC-0001</td>
			<td style="width:167px;">alternative to own preparation from juvenile foreskin</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">DMEM 
			[+] 4,5 g/l Glucose, 
			[+] L- Glutamine, 
			[-] L- Pyruvate</td>
			<td style="width:123px;">Gibco life technologies</td>
			<td style="width:119px;">41965</td>
			<td style="width:167px;">mix 1:1 with Ham&#180;s F-12 for MM medium;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; mix 3:1 with Ham&#180;s F-12 for EGM medium</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ham&#180;s F-12 [+] L-Glutamine</td>
			<td style="width:123px;">Gibco life technologies</td>
			<td style="width:119px;">&#160;21765-029</td>
			<td style="width:167px;">add to DMEM for the generation of EGM and MM medium (see lane 15)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EGF&#160;</td>
			<td style="width:123px;">Gibco life technologies</td>
			<td style="width:119px;">&#160;13247-051</td>
			<td style="width:167px;">add 10 ng/ml only for the generation of EGM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Calcium chloride</td>
			<td style="width:123px;">Merck Chemicals</td>
			<td style="width:119px;">2381</td>
			<td style="width:167px;">add 1.9 mM for the generation of EGM and 3.8 mM for MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Selenic acid</td>
			<td style="width:123px;">Alfa Aesar</td>
			<td style="width:119px;">18851</td>
			<td style="width:167px;">add 53 nM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Insulin</td>
			<td style="width:123px;">Lilly</td>
			<td style="width:119px;">HI0219 Hum Pen 3ml 100 E.I./ml</td>
			<td style="width:167px;">add 5 &#181;g/ml for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethanolamine</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">E-0135</td>
			<td style="width:167px;">add 0.1 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">P-Ethanolamine</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-0503</td>
			<td style="width:167px;">add 0.1 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Holo-Transferrine</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T-0665</td>
			<td style="width:167px;">add 5 &#181;g/ml for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Triiodthyronine</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-6397</td>
			<td style="width:167px;">add 20 pM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hydrocortisone</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">H-088816</td>
			<td style="width:167px;">add 0.4 &#181;g/ml for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Progesterone</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8783</td>
			<td style="width:167px;">add 10 nM only for the generation of EGM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hepes</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">H-3784</td>
			<td style="width:167px;">add 15 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Serine</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">S-4311</td>
			<td style="width:167px;">add 1 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cholinchloride</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">C-7017</td>
			<td style="width:167px;">add 0.64 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">dFCS</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">F-0392 Lot 086K0361</td>
			<td style="width:167px;">add 2 % for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Adenine</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">A-9795</td>
			<td style="width:167px;">add 0.18 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">L-Glutamine</td>
			<td style="width:123px;">PromoCell</td>
			<td style="width:119px;">C-42209</td>
			<td style="width:167px;">add 7.25 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Strontiumchloride</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">255521</td>
			<td style="width:167px;">add 1 mM for the generation of EGM and MM medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti cytokeratin 10 antibody</td>
			<td style="width:123px;">Dako</td>
			<td style="width:119px;">M7002</td>
			<td>1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti cytokeratin 14 antibody</td>
			<td style="width:123px;">Santa Cruz</td>
			<td style="width:119px;">sc-53253</td>
			<td>1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;anti laminin 5 antibody</td>
			<td style="width:123px;">Santa Cruz</td>
			<td style="width:119px;">sc-32794</td>
			<td>1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti filaggrin antibody</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">MA5-13440</td>
			<td>1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti involucrin antibody</td>
			<td style="width:123px;">Acris Antibodies</td>
			<td style="width:119px;">AM33368PU</td>
			<td>1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">secondary polyclonal goat anti-mouse IgG FITC labeled</td>
			<td style="width:123px;">LifeSpan Biosciences</td>
			<td>LS-C153907</td>
			<td>1:50</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">secondary polyclonal goat anti-mouse IgG Cy3 labeled</td>
			<td style="width:123px;">Jackson Immuno Research</td>
			<td style="width:119px;">115-165-003</td>
			<td>1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:119px;">10236276001</td>
			<td>1:100</td>
		</tr>
	</tbody>
</table>

a 3d organotypic melanoma spheroid skin model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Successful treatment of melanoma metastasis can be influenced by the cross-talk between tumor cells as well as between tumor and non-transformed host cells. The purpose of developing organotypic models of cancer in vitro is to provide suitable preclinical test systems that recapitulate the 3D organization and complexity of human melanoma in vivo. This allows the study of the therapeutic impact on the tumor within an organotypic environment and the adverse effects on the surrounding primary tissue in parallel.
To develop the best organotypic skin models, the quality of the primary cells is crucial. It is advantageous to use juvenile primary fibroblasts and keratinocytes, because they are typically less differentiated compared to adult primary skin cells. Juvenile skin cells can either be isolated from juvenile foreskin as described in the protocol sections 1 - 5, but can also be purchased from companies as pre-natal primary fibroblasts and keratinocytes. If purchasing, it is necessary to order cells from different donors to avoid donor-specific results, e.g., for drug sensitivity. The whole protocol is displayed as a scheme in Figure 3.
Quality control of 3D full-skin equivalents requires immunohistochemical analysis. A first impression can be obtained by Hematoxylin-Eosin (H&#38;E) staining of paraffin embedded sections (3 &#181;m). Detailed analysis of the quality of epidermal differentiation and formation of the basal lamina between the dermis and epidermis requires immunohistochemical analysis using specific antibodies against an epidermal stratification marker. This allows distinguishing between undifferentiated, highly proliferative cells located close to the basal membrane and highly differentiated and keratinized cells at the stratum corneum through the formation of distinct epidermal layers in between. As shown by immune-histological staining (Figure 4), differentiation of keratinocytes throughout the epidermis could be achieved similar to normal skin: mainly the undifferentiated cells from the lower epidermal layers (stratum basale and stratum spinosum) stain positive for keratin 14, while the more differentiated cells from the supra-basal layers (stratum granulosum and stratum corneum) stain positive for keratin 10 and involucrin. Accordingly, filaggrin staining could only be observed in highly differentiated cells of the stratum corneum. Most importantly, laminin 5 staining reveals that a basal lamina was generated to physiologically connect the epidermal to the dermal compartment of the artificial skin reconstruct. This proves that a communicating organotypic microenvironment has been generated to host melanoma cells or spheroids for physiologic and pathophysiologic analysis.
For the purpose of melanoma drug screening, single melanoma cells can also be integrated into the dermis of full skin equivalents to allow de novo melanoma nest formation18,19. Therefore, melanoma cells are combined with primary fibroblasts at a ratio of 5:1, centrifuged together at 200 x g for 5 min and resuspended in GNL prior to mixing with collagen. As a result, the melanoma cell nests will spontaneously form in the dermal compartment. According to our experience, only cells of the metastatic growth phase form proper nests, compared to melanoma cells of the RGP or VGP15. One major drawback of these types of models is the fact that the number and size of melanoma nests formed cannot be predicted, and may vary between individual skin reconstructs, independently of any treatment. For example, 1,000 cells seeded into the dermal compartment may gain 10 nests consisting of 100 cells or 100 nests consisting of 10 cells each (Figure 5). These biologic variables present with three deficiencies: first, the number and size of melanoma nests formed are unpredictable; second, the metastases in vivo are usually larger than melanoma nests and exhibit a more complex intra-tumoral diversity; and third, due to the limited life-span of tumor-nest models, treatment is initiated early, and consequently rather inhibits tumor outgrowth instead of causing regression of existing tumor nests.
To overcome these limitations organotypic melanoma spheroid skin models can be generated. By culturing 250 metastatic melanoma cells in a hanging drop for 14 days20, spheroids are reproducibly generated consisting of viable melanoma cells presenting a compact structure with a final diameter of approximately 500 &#181;m mimicking non vascularized tumor nodes, micro-metastasis, or inter-capillary micro regions of solid tumors21,22. In general, any melanoma cell line is suitable for the generation of spheroids via the hanging drop method; however, cells derived from more advanced metastatic tumor stages form more solid spheroids compared to cell lines derived from early progression stages, e.g., the RGP.
For some cells, it is advantageous for proper spheroid formation to enhance the viscosity of the hanging drop culture medium. This can be achieved by the addition of 10 - 50% methyl-cellulose to the culture medium. For the methyl-cellulose stock solution, autoclave 1.2 g methyl-cellulose together with a magnetic stir bar in a 100 mL glass bottle. Add 100 mL preheated (60 &#176;C) medium and stir for 20 min at room temperature, and another 1 - 2 h at 4 &#176;C. Centrifuge the stock solution for 2 h at 5,000 x g and store the viscous supernatant at 4 &#176;C until use.
Proper validation of full skin melanoma spheroid models is provided by the fact that a defined number of melanoma spheroids can - at least statistically - be integrated into the dermal fibroblast/collagen I scaffold at day 1 of the skin model construction, allowing them to co-develop during epidermal differentiation for 25 - 27 more days. The yield of spheroids can be analyzed directly after seeding, because spheroids appear as white spots within the transparent dermal gel and can be seen without any magnification device (Figure 2). As a result, a 3D skin model is generated that harbors mature melanoma spheroids, which had been cultured in vitro for a total of approximately 42 days, showing the highest level of intra-tumoral cell differentiation15.
H&#38;E staining of the skin melanoma spheroid model reveals the histological appearance and cellular distribution of melanoma spheroids to be very similar to the one of non-vascularized human melanoma skin metastases in vivo15 (Figure 6). Two subpopulations of melanoma cells are clearly distinguishable under these conditions: a peripheral proliferating subpopulation and a central subpopulation mainly consisting of shrunken, apoptotic or necrotic cells, forming the so-called &#34;necrotic&#34; center. Immunohistochemically living and proliferating subpopulations can be detected using antibodies against the proliferation marker KI-67, whereas cells of the necrotic center can be visualized by TUNEL-staining15. This particular distribution of tumor cell subpopulations is warranted by the spheroid size (&#8805;500 &#181;m), resulting from a lack of nutrients and oxygen in the central part where catabolic waste accumulates. Following the protocol provided here will allow the generation of a reliable and reproducible organotypic human full-thickness skin model with embedded human melanoma spheroids that mimic human melanoma skin metastasis. Applications of this model include drug testing, screening of toxins, influence of cosmetic compounds or laser therapy on melanoma outgrowth, and treatment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57500/57500fig1.jpg" /> 
Figure 1: Cultivation of 3D organotypic skin reconstructs submerged with medium and at the air-liquid interface. At day 0 primary keratinocytes are seeded on top of the dermal compartment consisting of primary fibroblasts embedded into a collagen type I matrix. 3D skin reconstructs stay cultivated submerged with EGM for 7 days, detach from the insert wall, and start shrinking. At day 8 the inserts are transferred to 6-wells and cultivated at the air-liquid interface to allow epidermal stratification. <a href="/files/ftp_upload/57500/57500fig1large.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/57500/57500fig2.jpg" /> 
Figure 2: Melanoma spheroids embedded into the dermal compartment appear as white spots. While preparing the dermal compartment of the 3D skin reconstruct, a defined number of melanoma spheroids can be added to the fibroblast collagen type I mix. Once the dermal gel has settled, melanoma spheroids become visible as white spots.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57500/57500fig3.jpg" /> 
Figure 3: Scheme of 3D organotypic skin model construction. Remove adipose tissue from the skin sample and cut it into smaller pieces. Incubation with dispase solution overnight at 4 &#176;C facilitates the separation of the epidermis from the dermis. Isolated primary fibroblasts and keratinocytes should be cultivated separately and used between passage 4 - 6 and 3 - 4, respectively. Subsequently, the generation of the 3D skin model can proceed as described in the protocol. <a href="/files/ftp_upload/57500/57500fig3large.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/57500/57500fig4.jpg" /> 
Figure 4: 3D organotypic skin reconstructs show a differentiation level similar to normal human skin. Paraffin sections of skin equivalents (A) compared to normal human skin (B) were stained for the expression of keratins 14 (red: &#955;ex 554 nm; &#955;em 568 nm) and 10, involucrin (green: &#955;ex 490 nm; &#955;em 525 nm), filaggrin (green: &#955;ex 490 nm; &#955;em 525 nm), and laminin 5 (green: &#955;ex 490 nm; &#955;em 525 nm), and analyzed with a confocal fluorescence microscope. Cell nuclei were visualized by DAPI staining (blue: &#955;ex 340 nm; &#955;em 488 nm). Immunohistochemical examination of the 3D full-thickness of skin equivalents revealed proper epidermal stratification forming distinct layers of the epidermis as seen in normal human skin. While cells from the lower epidermal layers stained positive for keratin 14, the more differentiated cells from the supra-basal layer showed keratin 10 and involucrin staining. Highly differentiated cells close to the stratum corneum expressed filaggrin. Laminin 5 staining shows that a basal lamina is generated to physiologically connect the epidermal to the dermal compartment (lowest panel). This figure has been taken from Voersmann et al.15 with permission. <a href="/files/ftp_upload/57500/57500fig4large.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/57500/57500fig5.jpg" /> 
Figure 5: The number and size of spontaneously formed melanoma nests cannot be predicted. De novo melanoma nest formation in the dermal compartment of full skin equivalents can be achieved by mixing a defined number of melanoma cells with primary fibroblasts to embed both cell types in the collagen type I matrix. The number and size of spontaneously formed melanoma nests can only be analyzed from a mature 3D skin reconstruct after about 21 days. As depicted from two samples (A) and (B), the numbers and sizes of the melanoma nests may vary between individual skin reconstructs. As a consequence, it is difficult to validate these models and to predict the therapeutic impact. <a href="/files/ftp_upload/57500/57500fig5large.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/57500/57500fig6.jpg" /> 
Figure 6: Melanoma spheroids integrated into skin equivalents recapitulate key features of human cutaneous melanoma metastasis. H&#38;E stained paraffin sections of tumor spheroids embedded into skin equivalents revealed spheroids to share key features with non-vascularized human cutaneous melanoma metastases in vivo. Two subpopulations of cells are clearly discernible: a peripheral living subpopulation and central subpopulation mainly consisting of shrunken, apoptotic or necrotic cells, forming the &#34;necrotic&#34; center. This distribution of tumor cell subpopulations is guaranteed by the spheroid size. This figure has been modified from Voersmann et al.15 with permission. <a href="/files/ftp_upload/57500/57500fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A 3D Organotypic Melanoma Spheroid Skin Model at JoVE.com

A 3D Organotypic Melanoma Spheroid Skin Model

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

a-3d-organotypic-melanoma-spheroid-skin-model

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JoVE Journal

Cancer Research

15.6K Views.  TU-Dresden. Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Video: A 3D Organotypic Melanoma Spheroid Skin Model

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 ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...