The authors would like to acknowledge all of the members of the Coller laboratory for helpful input. H.A.C. was the Milton E. Cassel scholar of the Rita Allen Foundation. We acknowledge NIH/NCI 1 R01 CA221296-01A1, NIH 1 R01 AR070245-01A1, Melanoma Research Alliance Team Science Award, Cancer Research Institute Clinical Laboratory Integration Program Award, the Iris Cantor Women&#39;s Health Center/UCLA CTSI NIH Grant UL1TR000124, University of California Cancer Research Coordinating Committee, David Geffen School of Medicine Metabolism Theme Award, the Clinical Translational Science Institute and Jonsson Comprehensive Cancer Center, Innovation Awards from the Broad Stem Cell Research Center (Rose Hills and Ha Gaba), an Award from the UCLA SPORE in Prostate Cancer (National Cancer Institute of the National Institutes of Health under Award Number P50CA092131), an Innovation Award from the Broad Stem Cell Center, the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, the Tumor Cell Biology Training Program (USHHS Ruth L. Kirschstein Institutional National Research Service Award # T32 CA009056), the Dermatology T32 Program at UCLA AR071307, and the UCLA Muscle Cell Biology, Pathophysiology and Therapeutics T32 Training Program 5 T32 AF 65972.

All experiments described were approved by the Animal Care Committee at the University of California, Los Angeles.
NOTE: Select cancer cells and fibroblasts that match the host mice for mouse strain. Select cancer cells and fibroblasts that match the sex of the host mouse. Obtain mice from breeding colonies or purchase them from reputable vendors. Introduce tumors into mice that are ~8-10 weeks of age. Mice with fur will be in the telogen or resting phase of the hair follicle cycle. Plan for a ratio of 0.5 to 3 fibroblasts to cancer cell.
1. Determine the appropriate number of mice to be used for experimentation
<ol>
	<li>Prior to performing the studies, perform a power analysis to determine the appropriate number of mice to use for the studies. Use the following formula to determine sample size for an experiment with the specified power: 
	n = (Z(1-&#945;/2)+Z(1-&#946;)/ES)2 
	If &#945; is the selected level of significance (usually 0.05), then 1-&#945;/2 = 0.975 and Z=1.960. 
	&#946; is the power and if 80% power is desired, then Z=0.84. 
	 
	ES, the effect size = \&#956;1-&#956;0\/&#963; 
	where &#956;0 is the mean under hypothesis H0,&#160;&#956;1 is the mean under hypothesis H1, and&#160;&#963;&#160;is the standard deviation of the outcome of interest22. 
	NOTE: More information on calculating sample size can be found22.</li>
</ol>
2. Generate cancer cells for injection
<ol>
	<li>Determine the relevant growth rate with the following equation: 
	Gr = (ln (N(t)/N(0)))/t 
	 
	NOTE: An example for cells that double in 24 hours is shown 
	Doubling time = ln(2)/growth rate 
	24 hours = ln(2)/growth rate 
	24*growth rate = .693 
	Growth rate = .693/24= .028875</li>
	<li>Determine the number of cancer cells to plate and the amount of time the cells need to be grown prior to injection. 
	NOTE: 0.25-1 million cancer cells are injected for each tumor formed.</li>
	<li>Calculate the number of days required to generate a sufficient number of cells according to the equation 
	N(t)=N(0)egr*t 
	where N(t) is the number of cells at time t, N(0) is the number of cells at time 0, gr is the growth rate, and t is the time. 
	NOTE: An example of these calculations for growing 500,000 cells to generate 106 cells into each of 12 tumors. Based on these calculations, 5 days is sufficient to grow the necessary number of cells: 
	N(t)=N(0)egr*t 
	12 x 106 = 500,000e(gr*t) 
	12 x 106=500,000e(.028875*t) 
	24=e(.028875*x) 
	ln(24)=0.028875x 
	3.178=0.028875x 
	&#8203;X=110 hours= 4.59 days 
	Allow extra time for cells to attach to the plate and for cells that are lost during collection.</li>
	<li>Plate cancer cells. 
	NOTE:&#160;Wear gloves and lab coats when working with cultured cells. 
	NOTE: Perform cell manipulations in a laminar flow hood to minimize the introduction of microbes from the air into the samples. Treat the tissue culture hood with ultraviolet light prior to use. Use sterile technique and only sterile cell culture plasticware and consumables such as tissue culture-treated plates, pipets, and conical tubes.
	<ol>
		<li>Calculate the correct number and size of tissue culture plates based on the number of cells in the vial and the desired cell concentration once plated.</li>
		<li>Label tissue culture plates.</li>
		<li>Prepare a water bath with water at 37 &#176;C.</li>
		<li>Aliquot medium into conical tubes and place in a water bath at 37 &#176;C. Many cancer cells can be grown in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).</li>
		<li>Remove the required number of vials of frozen cells from the liquid nitrogen freezer. 
		NOTE: Use freezer gloves when handling cells in a liquid nitrogen freezer.</li>
		<li>Quickly thaw the cell vials in the water bath while gently shaking. 
		&#8203;NOTE: Add ethanol to gloves while handling vials to maintain sterility.</li>
		<li>Using sterile technique in a tissue culture hood, pipet the cells into a 15 mL conical tube containing 10 mL of DMEM + 10% FBS.</li>
		<li>Centrifuge the cell mixture at 180 x g for 5 minutes.</li>
		<li>Gently remove the supernatant with sterile suction or a sterile 10 mL pipet.</li>
		<li>Gently resuspend the cells in the pellet into 1 mL of DMEM + 10% FBS.</li>
		<li>Pipet the resuspended cells onto a labeled tissue culture plate with a sterile p1000.</li>
		<li>Using sterile 10 mL pipets, pipet medium with serum into tissue culture plates.</li>
		<li>Incubate tissue culture plates in a tissue culture incubator at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Expand cancer cells to generate sufficient cancer cells for injection. 
	NOTE: Monitor cells with light microscopy for confluency. Trypsinize every two to three days or as needed to prevent the cells from reaching 100% confluence. If a large number of cells are required, use hyperflasks (Table of Materials) to grow a large number of cells in a space- and medium-efficient way. Each time the cells need to be passaged, follow this procedure:
	<ol>
		<li>Remove the medium from the plate with sterile suction or a sterile 10 mL pipet.</li>
		<li>Gently wash the plate by pipetting on warm phosphate buffered saline (PBS). Then remove the PBS with sterile suction or a pipet.</li>
		<li>Pipet 5 mL of 1x Trypsin-ethylenediaminetetraacetic acid (EDTA) in PBS for a 10 cm plate onto the cells and incubate for 5 minutes at 37 &#176;C.</li>
		<li>Remove cells from the plate with gentle taps to dislodge the cells from the plate.</li>
		<li>Using a sterile 10 mL pipet, collect the cells into DMEM with 10% FBS serum in conical tubes.</li>
		<li>Centrifuge conical tubes at 180 x g for 5 minutes.</li>
		<li>Remove the supernatant by pouring it off. The cell pellet should be visible. Watch it to make sure it remains in the tube.</li>
		<li>Resuspend the pelleted cells by adding 1 mL of DMEM + 10% FBS and pipetting up and down gently.</li>
		<li>Aliquot resuspended cells onto new tissue culture plates of the appropriate size with DMEM + 10% FBS (10 mL of media is suitable for a 10 cm tissue culture plate).</li>
	</ol>
	</li>
</ol>
3. Generate fibroblasts for injection
NOTE: Primary fibroblasts will senesce after too many doublings/passages. It is important to use primary fibroblasts after a limited number of passages or doublings. Keep track of the number of passages or doublings that the fibroblasts have grown from the mouse or human skin. Use fibroblasts with fewer than 15 passages for primary human dermal fibroblasts. Use fibroblasts with fewer than 9 passages for mouse embryonic fibroblasts. Fibroblasts are altered when they become confluent. Trypsinize the fibroblasts when they are approximately 90% confluent. Fibroblasts will have different properties depending on how they are cultured. Many scientists promote culturing on more physiologically relevant substrates than tissue culture plates such as 3D collagen matrices that more effectively capture tissue-like environments23,24,25.
<ol>
	<li>Use the equation in section 2.1 to determine the growth rate for the fibroblast cells.</li>
	<li>Use the equation in section 2.2 to determine the number of days required to expand the fibroblasts.</li>
	<li>Plate the fibroblasts as described in section 2.3 using the appropriate medium on the appropriate day to ensure cancer cells and fibroblasts will be ready for injection on the same day.</li>
	<li>Expand the fibroblasts in the appropriate medium to generate a sufficient number of fibroblasts needed following the procedures described in section 2.4.</li>
</ol>
4. Shave mice to prepare mice for injection
NOTE: Wear lab coats, hair nets, shoe covers, and gloves when working with mice.
<ol>
	<li>One to two days prior to injection, in accordance with the rules of the Institutional Animal Care Committee, anesthetize the mice. If using isoflurane for anesthesia, use a mixture of isoflurane and O2. Place the mice in an anesthesia chamber and introduce an isoflurane (5%)/ O2 mixture to induce anesthesia. Then, place the nose cone on the anesthetized animal to maintain the surgical plane of anesthesia with a constant flow of isoflurane (2%)/O2 mixture.</li>
	<li>Check the animals to ensure they are in the appropriate surgical plane of anesthesia by pinching the mouse&#39;s toe and confirming the mouse does not move.</li>
	<li>Using an animal clipper with surgical blade #40, gently remove the fur from the appropriate positions on the flanks of the mice by passing the animal clipper over the skin multiple times until the fur is removed.</li>
	<li>Monitor the mice until they recover from anesthesia.</li>
</ol>
5. Prepare cancer cells and fibroblasts for injection
NOTE: Cancer cells and fibroblasts should be injected as soon as possible after collection, preferably within 30 minutes. On the morning of the injection, harvest the cancer cells and fibroblasts separately from tissue culture plates. For each cell type perform the following steps:
<ol>
	<li>Remove the medium from the tissue culture plate with gentle sterile suction or by pipetting off the medium.</li>
	<li>Gently pipet on 5 mL of PBS without disrupting the cells. Swirl the PBS. Gently aspirate the PBS with suction or pipet off the PBS.</li>
	<li>Gently pipet 5 mL of Trypsin-EDTA in PBS onto the cells layer and incubate for 5 minutes at 37 &#176;C.</li>
	<li>Remove cells from the plate with gentle taps to dislodge the cells. Pipet the cells several times with a 10 mL pipet and transfer the cells to a conical tube containing DMEM with 10% FBS.</li>
	<li>Centrifuge the conical tubes at 180 x g for 5 minutes.</li>
	<li>Remove the supernatant by gently pouring it off, retaining the cell pellet. Wash the cell pellet twice with PBS. Each time, pipet 10 mL of PBS onto the cells. Gently mix with a p1000 pipet.</li>
	<li>Centrifuge the cells as in step 5.5. Gently remove the PBS with a pipette and repeat.</li>
	<li>Resuspend the washed, pelleted cells in 1 mL of PBS with a p1000.</li>
	<li>Clean a hemocytometer slide with alcohol and dry.</li>
	<li>Pipet 100 &#181;L of the cell mixture into a tube and add 400 &#181;L of 0.4% Trypan blue for a final Trypan blue concentration of 0.32%.</li>
	<li>Gently fill the chambers of a glass hemocytometer underneath the coverslip by pipetting the cell mixture until the chamber is full.</li>
	<li>Using a microscope, focus on the grid lines of a 10x objective.</li>
	<li>Count the live, unstained cells and blue cells in one set of 16 squares.</li>
	<li>Count all 4 sets of 16 squares.</li>
	<li>Average the cell count for the clear and blue cells from the 4 sets of 16 squares and multiply by 10,000. Multiply by 5 to correct for the 1:5 dilution from the Trypan blue.</li>
	<li>The final counts are the concentration in cells/mL for the viable and dead cells. Multiply by the volume to determine the total number of cancer cells and fibroblasts. Confirm that the fraction of cells that are viable is &#62;80%.</li>
	<li>Confirm that there are a sufficient number of melanoma cells and fibroblasts for all of the injections planned, assuming at least 20% loss of sample during the injections.</li>
	<li>Transfer the required number of cells to a new conical tube and pellet the cells by centrifugation (180 x g for 5 minutes).</li>
	<li>Remove the supernatant with a 10 mL pipet.</li>
	<li>Resuspend the pellet at the required concentration in the appropriate amount of United States Pharmacopeia (USP) GRADE PBS (50 &#181;L per tumor for intradermal injection and 100 &#181;L per tumor for subcutaneous injection).</li>
</ol>
6. Inject cancer cells and fibroblasts into mice
NOTE: If approved by the institutional Animal Care Committee, inject two tumors into each mouse, one on each flank. Randomize which mouse will receive which injection on the right and left flanks. Depending on the number of mice to be injected, anesthetize the mice and inject cancer cells and fibroblasts into the mice in batches.
<ol>
	<li>For subcutaneous injection of cancer cells
	<ol>
		<li>Anesthetize mice as described in section 4.1.</li>
		<li>Wipe the shaved skin with alcohol swabs.</li>
		<li>Fill a sterile syringe with the desired volume of cells or cell mixture and cap the syringe with a 27-gauge needle. 
		&#8203;NOTE: Make sure no air bubbles are present. Flick the syringe as needed to remove bubbles. Once cells have been introduced into the syringe, keep the cells within the syringes well-mixed by continuously inverting the syringes to prevent clumping.</li>
		<li>Insert the needle into the subcutaneous region on the mouse&#39;s flank and gently inject the cells into the mouse. Use 100 &#956;L cell suspension per tumor for subcutaneous injection(using volume more than 100 &#956;L per injection is not recommended).</li>
		<li>Monitor the mice until they recover from anesthesia. If needed, provide the mice with extra insulating materials like huts and/or heating pads to help the mice to fully recover.</li>
	</ol>
	</li>
	<li>For intradermal injections of cancer cells
	<ol>
		<li>Anesthetize mice as described in section 4.1.</li>
		<li>Wipe the shaved skin with alcohol swabs.</li>
		<li>Fill a sterile syringe with the desired volume of cells or cell mixture and cap the syringe with a 27-gauge needle. 
		&#8203;NOTE: Make sure no air bubbles are present. Flick the syringe as needed to remove bubbles. Once cells have been introduced into the syringe, keep the cells within the syringes well-mixed by continuously inverting the syringes to prevent clumping.</li>
		<li>Insert a 27-gauge needle into the intradermal region of the skin on the mouse&#39;s flank and gently inject the cells into the intradermal region in the mouse. Use 50&#956;L cell suspension per tumor for intradermal injection (using volume more than 50 &#956;L per injection is not recommended).</li>
		<li>Monitor the mice until they recover from anesthesia. If there are signs that the mice are in distress, consult a veterinarian. If needed, provide the mice with extra insulating materials like huts and/or heating pads to help the mice to fully recover.</li>
	</ol>
	</li>
</ol>
7. Monitor mice during tumor growth
<ol>
	<li>Monitor mice daily for any signs of pain or distress (hunched posture, writhing, vocalization, stretching out along the cage, pressing their abdomen to the bottom of the cage floor, or reluctance to move around the cage) or abscess formation. If there are signs that the mice are in distress, consult a veterinarian.</li>
	<li>Once tumors are formed, measure tumor volume approximately every two to three days. Measure tumor length (the longest side) and width (perpendicular to length) with calipers. 
	NOTE: Best practices are for the same person to perform these measurements throughout the experiment to reduce variability in the data. For measurements, anesthetize mice as described in section 4.1</li>
	<li>Calculate tumor volumes with the formula Volume= 0.5 x (Length x Width2).</li>
</ol>
8. Harvest tumors and measure tumor weights
<ol>
	<li>Euthanize mice when tumor growth reaches an experimental endpoint established by the institution Committee on Animal Care. Place mice in a chamber and euthanize them with slow (20-30% per minute) displacement of chamber air with compressed CO2 for one minute past cessation of respiration.</li>
	<li>Perform cervical dislocation on the mice. Restrain the rodent on a firm, flat surface. Place a sturdy pen or another object against the back of the neck at the base of the skull. Quickly push forward and down with the object restraining the head while pulling backward with the hand holding the base of the tail. Confirm with palpation that the spinal cord is severed. 
	NOTE: Cervical dislocation should be performed by experienced lab personnel.</li>
	<li>For each mouse in the experiment, confirm that the mouse is no longer alive by checking that there is no breathing, no heartbeat and no response to a toe pinch.</li>
	<li>Using a sterile scalpel and surgical scissors, excise the entire tumor from the mouse. With surgical scissors, trim non-tumor tissue from the tumor.</li>
	<li>Weigh the tumor on an analytical balance and record the tumor weight.</li>
</ol>
9. Statistical analysis of tumor volumes and tumor weights
<ol>
	<li>Compare tumor volumes over time in each group with repeated measures analysis of variance (ANOVA). Perform paired analysis if two tumors were injected per mouse.</li>
	<li>Compare tumor weights among the groups with ANOVA using a two-tailed test. Tukey&#39;s post-hoc test can then be used to determine which groups are significantly different from each other. Perform paired analysis if two tumors were injected per mouse.</li>
</ol>

The authors have nothing to disclose.

In the experiment in Figure&#160;1, co-introducing human dermal fibroblasts with human A2058 melanoma cells resulted in larger tumors than when the melanoma cells were introduced without co-injected fibroblasts. This difference could be easily detected based on tumor volume and tumor weight. The results are consistent with multiple reports that cancer-associated fibroblasts can promote tumor growth5,6,7</...

Within the tumor microenvironment, one of the most prominent cell type is the cancer-associated fibroblast (CAF)1. These carcinoma-associated fibroblasts can play a tumor-suppressive role2,3. For example, S100A-expressing fibroblasts secrete collagens that can encapsulate carcinogens and protect against carcinoma formation4. Further, depletion of &#945;-smooth muscle actin (SMA)-positive myofibroblasts in pancreatic cancer causes immunosuppression and accelerates pancreatic cancer progression2. CAFs can also co-evolve with cancer cells and promote tumor progression5,6,7,8. Fibroblasts can synthesize and secrete extracellular matrix proteins that create a tumor-promoting environment8. These extracellular matrix proteins can cause mechanical stiffening of the tissue, which is associated with tumor progression9,10. The deposited extracellular matrix can act as a physical barrier that inhibits immune infiltration11. Matrix deposition by CAFs has also been associated with tumor invasion as fibronectin generated by CAFs has been shown to promote tumor invasion12. CAFs promote angiogenesis and recruit immunosuppressive cells to the tumor microenvironment by secreting transforming growth factor-&#946; (TGF- &#946;), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and CXC-chemokine ligand 12 (CXCL12)13,14,15. Because of their central role in promoting tumor growth, cancer-associated fibroblasts are an emerging target for anti-cancer therapy6,16,17,18.
The protocol below describes a method for testing how fibroblasts affect the growth of tumors in a well-established and widely-used mouse model of tumor growth. In order to understand the importance of fibroblasts in the tumor microenvironment, the standard protocol for introducing cancer cells into mice to monitor their growth was modified to include fibroblasts with the cancer cell introduction. The cancer cells can be introduced subcutaneously or intradermally. Intradermal introduction would result in tumors that arise from the skin itself. Xenografts in which cancer cells and fibroblasts are co-injected into mice represent an important methodological tool for dissecting the role of fibroblasts, subpopulations of fibroblasts and protein factors in the ability to promote cancer growth19,20,21. A detailed protocol for co-injection of cancer cells and fibroblasts into mice is provided. This method can be used to compare the presence or absence of fibroblasts, to compare fibroblasts from different sources20, or to compare fibroblasts with and without expression of specific proteins19. After the cancer cells and fibroblasts are introduced, tumor size can be monitored over time. At the end of the experiments, tumors can be dissected and weighed. By monitoring tumor growth over time, the importance of different factors can be dissected.
There are possible alternative approaches for studying the role of fibroblasts in tumor growth. As an example, there are Cre-loxed based models that provide for tissue-specific knockout of genes with drivers expressed preferentially in fibroblasts. Such approaches also provide opportunities to investigate the role of specific genes and pathways in fibroblasts for tumor progression. As compared with Cre-lox-based approaches, the protocol provided would represent a significantly more rapid approach to monitoring the role of fibroblasts because tumor growth would be monitored over just a few weeks. The provided approach is also significantly less expensive because it does not require generating and housing colonies of genetically engineered mice. The protocol provided can be used to rapidly test the effect of knockdown of different genes using shRNAs rather than needing to develop mouse colonies. The provided approach is also more flexible because it would allow for a comparison of different numbers of fibroblasts, different ratios of cancer cells and fibroblasts, knockdown of different genes, and even comparison of fibroblasts from different tissue sites or species. A Cre-lox approach would have the advantage that the fibroblasts are present within the mice in a more physiological context.
The protocol reported here would be valuable for scientists who seek to monitor the effects of fibroblasts on tumor growth rapidly and cost effectively. This protocol is especially valuable for scientists investigating different subsets of fibroblasts or fibroblasts from different sources on tumor growth on tumor growth. If it is important that tumor initiation occurs in a physiological context, then genetically engineered mouse models should be considered.
There are several possible approaches for performing these experiments. Immune-competent mice can be used as hosts, which would allow for investigation of fibroblast-immune cell interactions. For immune-competent mouse models, mouse cancer cells and mouse embryonic fibroblasts (MEFs) must be injected. The use of MEFs also allows the investigator to take advantage of the wide range of knockout mouse strains to test the presence or absence of a gene of interest. Alternatively, immune-deficient mice can be used to test the role of human fibroblasts in promoting the growth of tumors in mice that are derived from human cancer cells. Introduction of the cancer cells can be performed subcutaneously or orthotopically. For melanoma, as described below, the tumor-fibroblast mixture can be injected intradermally for orthotopic injection that more closely simulates the location within the skin where a melanoma would develop.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>26G Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>Fisher Scientific</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal clipper miniARCO with surgical blade #40</td>
			<td>WAHL Professional</td>
			<td>8787-450A</td>
			<td></td>
		</tr>
		<tr>
			<td>Athymic nude mice (NU/J)</td>
			<td>The Jackson labs</td>
			<td>002019</td>
			<td>These mice are immunocompromised and can be used for experiments in which human cells are introduced. Immunocompetent mice can also be used if mouse cancer cells and fibroblasts will be introduced.</td>
		</tr>
		<tr>
			<td>Cancer cells</td>
			<td>ATCC</td>
			<td>ATCC&#174; CRL-11147&#8482;</td>
			<td>This is the catalog number for a primary human melanoma cell line. Other cancer cell types can also be used.</td>
		</tr>
		<tr>
			<td>Cell Culture Multi Flasks</td>
			<td>Fisher Scientific</td>
			<td>14-826-95</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for conical tubes capable of reaching 180 x g</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Fisher Scientific</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblasts&#160;</td>
			<td>ATCC</td>
			<td>PCS-201-012</td>
			<td>We isolate fibroblasts from skin in our lab. This is a catalog number for an adult primary human dermal fibroblast cell line. MEFs and fibroblasts derived from other sites can also be used.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC 11695-6776-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS USP grade for injection into mice</td>
			<td>Fisher Scientific</td>
			<td>50-751-7476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 10 ml serological pipet</td>
			<td>Celltreat</td>
			<td>667210B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 5 ml serological pipet</td>
			<td>Celltreat</td>
			<td>229005B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50 ml centrifuge tubes</td>
			<td>Genesee Scientific</td>
			<td>28-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringe Filters pore size 0.2 microns</td>
			<td>Fisher Scientific</td>
			<td>09-740-61A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tissue culture-grade Trypsin-EDTA</td>
			<td>Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tissue-culture grade PBS</td>
			<td>Fisher Scientific</td>
			<td>50-751-7476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterle 25 ml serological pipet</td>
			<td>Celltreat</td>
			<td>667225B</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 100 x 20 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-202</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 150 x 20 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-203</td>
			<td></td>
		</tr>
		<tr>
			<td>TC treated 60 x 15 mm dishes</td>
			<td>Genesee Scientific</td>
			<td>25-260</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A2058 human melanoma cells and primary human dermal fibroblasts were cultured under sterile conditions. Cells were collected and washed three times with PBS. Immunodeficient mice (NU/J - Foxn1 nude strain) were injected subcutaneously on one flank with 0.25 million A2058 melanoma cells alone. On the other flank, mice were injected with a mixture of 0.25 million A2058 melanoma cells and 0.75 million fibroblasts. Cells were injected into 12 immune-deficient mice. Injections into left and ri...

Watch this Scientific Journal Video about A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth at JoVE.com

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

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 ...

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4.2K Views.  University of California, Los Angeles. 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.

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

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

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 ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

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

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

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

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

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...