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><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&amp;reg; CRL-11147&amp;trade;</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&#039;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&amp;nbsp;</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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Research

JoVE Journal

Cancer Research

4.3K Views.  University of California, Los Angeles. Een protocol om kankercellen en fibroblasten samen te injecteren en de tumorgroei in de loop van de tijd te volgen, wordt verstrekt. Dit protocol kan worden gebruikt om de moleculaire basis voor de rol van fibroblasten als regulatoren van tumorgroei te begrijpen.

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

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Developmental Biology

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Medicine

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 ...

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 ...

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 ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

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

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