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

Summary

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.

Abstract

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.

Introduction

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 role^2,3. For example, S100A-expressing fibroblasts secrete collagens that can encapsulate carcinogens and protect against carcinoma formation⁴. Further, depletion of α-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 progression^5,6,7,8. 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 progression^9,10. 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-β (TGF- β), 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 therapy^6,16,17,18.

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 growth^19,20,21. 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.

Protocol

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…

Representative Results

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…

Discussion

In the experiment in Figure 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 growth^5,6,7</…

Materials

26G Needles	Fisher Scientific	14-826-10
Alcohol swabs	Fisher Scientific	326895
Animal clipper miniARCO with surgical blade #40	WAHL Professional	8787-450A
Athymic nude mice (NU/J)	The Jackson labs	002019	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.
Cancer cells	ATCC	ATCC® CRL-11147™	This is the catalog number for a primary human melanoma cell line. Other cancer cell types can also be used.
Cell Culture Multi Flasks	Fisher Scientific	14-826-95
Centrifuge for conical tubes capable of reaching 180 x g	Fisher Scientific	14-432-22
Countess Cell Counting Chamber	Fisher Scientific	C10228
Dulbecco's Modified Eagle Medium	Fisher Scientific	11965-118
Fetal bovine serum	Fisher Scientific	MT35010CV
Fibroblasts	ATCC	PCS-201-012	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.
Isoflurane	Henry Schein Animal Health	NDC 11695-6776-2
PBS USP grade for injection into mice	Fisher Scientific	50-751-7476
Sterile 10 ml serological pipet	Celltreat	667210B
Sterile 5 ml serological pipet	Celltreat	229005B
Sterile 50 ml centrifuge tubes	Genesee Scientific	28-108
Sterile Syringe Filters pore size 0.2 microns	Fisher Scientific	09-740-61A
Sterile tissue culture-grade Trypsin-EDTA	Fisher Scientific	15400054
Sterile tissue-culture grade PBS	Fisher Scientific	50-751-7476
Sterle 25 ml serological pipet	Celltreat	667225B
TC treated 100 x 20 mm dishes	Genesee Scientific	25-202
TC treated 150 x 20 mm dishes	Genesee Scientific	25-203
TC treated 60 x 15 mm dishes	Genesee Scientific	25-260
Trypan blue	Fisher Scientific	C10228