Name: a mouse model to investigate the role of cancer-associated fibroblasts in tumor growth
Uploaded: 2020-12-22T13:00:00
Description: Watch this Scientific Journal Video about A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth at JoVE.com

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...

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 ...

This protocol can be used to understand the molecular basis for the role of fibroblast as regulators of tumor growth. The protocol described can provide more insight into functional role of cancer-associated fibroblasts in tumor growth and progression. This technique can help to develop new therapies for melanoma.While our study is focused on melanoma, this technique can be extended to other types of cancer as well. Begin by placing the bottle containing DMEM supplemented with 10%FBS in a water bath at 37 degrees Celsius. Once the medium is warm, bring the bottle into the biosafety cabinet and aliquot 10 milliliters of the medium into 15 milliliter conical tubes.Remove the required number of vials of frozen cells from the liquid nitrogen freezer and quickly thaw them in a water bath while gently swirling under sterile conditions. Pipet the cells into the tubes containing DMEM and centrifuge the cells at 180 x g for five minutes. Gently remove the supernatant with sterile suction or a pipette and resuspend the cell pellet in DMEM containing 10%FBS.Then transfer the cells into a labeled tissue culture plate. Using sterile 10 milliliter pipettes, pipette medium with serum into the plate wells and incubate the plate and a tissue culture incubator at 37 degrees Celsius with 5%carbon dioxide. Remove the medium from the incubated tissue culture plate containing cells and gently add 10 milliliters of PBS without disrupting the cells.Swirl the plate. Then gently aspirate the PBS. Next, pipet five milliliters of trypsin-EDTA in PBS solution onto the cells and incubate for five minutes at 37 degrees Celsius.Dislodge the cells from the plate with gentle taps and pipet them into a conical tube containing DMEM with 10%FBS. Centrifuge the tubes at 180 x g for five minutes and remove the supernatant by aspiration or pipetting. Wash the cell pellet twice with PBS and remove the PBS by centrifugation after each wash.Resuspend the washed cell pellet in one milliliter of PBS. Next, clean the hemocytometer slide with alcohol and dry it. Then add 10 microliters of trypan blue to 10 microliters of cell mixture into a fresh tube.Gently fill the hemocytometer glass chambers underneath the cover slip by pipetting the cell mixture. Focus on the grid lines of a 10x objective under a microscope and count the blue cells and live unstained cells in one set of 16 squares. Then count all four sets of 16 squares.Average the count of unstained cells and multiply it by 10, 000. Then multiply by the appropriate dilution factor to obtain the final concentration in cells per milliliter. After confirming that there are sufficient melanoma cells and fibroblasts for all of the injections planned, assuming at least 20%loss of sample during the injections, transfer the required number of cells to a new conical tube and pellet them by centrifugation.Then discard the supernatant, resuspend the cell pellet of the required concentration in an appropriate amount of USP-grade PBS and place the cells on ice until further use. After anesthetizing the mouse, pinch its toe to ensure it's in the appropriate surgical plane of anesthesia. Next, wipe the skin of the mice with alcohol swabs.Fill a sterile syringe with the desired volume of cells or cell mixture without air bubbles and cap it with a 27 gauge needle. Insert the needle into the intradermal region of the skin on the animal's flank and gently inject the cells inside. Monitor the mice until they recover from an anesthesia and provide them with extra insulating materials if required.Approximately every three days after tumor formation, measure the tumor length and width using calibers and calculate its volume. After euthanizing the mouse, excise the entire tumor using a sterile scalpel and surgical scissors. Trim the non-tumor tissue with surgical scissors.Then weigh the tumor on an analytical balance. The plot of tumor volume over time shows the comparison of melanoma growth in the presence and the absence of co-injected fibroblasts. The tumor volumes were monitored on 12, 14, 16, 19, and 21 days post-injection.The images of the excise tumors upon the euthanasia show that the melanoma and fibroblast tumors are larger than the melanoma tumors alone. The presence of fibroblasts result in significantly larger tumors as evidenced by the final weight of tumors at the end of the experiment on day 21. Cancer-associated fibroblasts promote tumor growth by creating a tumor promoting microenvironment.This protocol can provide more insight into the functional role of CAF in tumor growth and progression.

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