Name: Cancer-Associated Fibroblasts from Mouse Mammary Tumors as Tools for Molecular and Computational Studies (Video) | JoVE Journal
Uploaded: 2025-07-03T13:30:00.000+00:00
Duration: 9 min 1 s
Description: Discover how cancer-associated fibroblasts drive breast cancer progression using molecular, in vitro, in vivo, and computational approaches. This method reveals STAT3-mediated mechanisms and therapeutic targets.

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5 minutes

This work was supported by the Italian Cancer Research Association (AIRC, IG16930 and IG24851 to V.P.); the Italian Ministry of University and Research (MIUR PRIN 2017 and 2022 to V.P.); the Truus and Gerrit van Riemsdijk Foundation, Liechtenstein, donation to V.P.; Piedmont Region (Deflect). A.S. was supported by an Italian Cancer Research Foundation (FIRC) post-doctoral fellowship, and L.A. was supported by Fondazione Umberto Veronesi.

The authors declare no competing interests.

In this study, we present optimized protocols for the immortalization of murine CAFs and the characterization of their pro-tumorigenic activities both in vitro and in vivo. Rather than selectively isolating specific CAF subtypes based on marker expression, we chose to retain the entire cell population to preserve heterogeneity. The cells were functionally characterized as a whole, and their gene expression profiles were analyzed to capture their diversity. Immortalizing CAFs was crucial for this researc...

Breast cancer (BC) is the most common malignancy affecting women and is characterized by significant molecular heterogeneity, with distinct molecular profiles guiding diagnosis, treatment decisions, and prognosis. The tumor microenvironment (TME) is also highly heterogeneous and plays a critical role in tumor progression, immunity, and treatment outcomes.
Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the TME and key regulators of tumor development. They contribute to extracellular matrix deposition and remodeling, angiogenesis, immune evasion, and drug resistance1,2,3,4,5,6. CAFs influence tumor cells through direct cell-cell contact, the release of extracellular vesicles7, and the secretion of various soluble factors2,5 to promote tumor growth, invasion, and therapy resistance8,9,10. Indeed, stroma-related gene signatures have predictive value for BC progression and therapy response and are associated with resistance to chemotherapy11,12,13,14. However, despite substantial evidence suggesting CAFs as an attractive therapeutic target, clinical trials targeting these cells have largely failed15. One major challenge is the limited understanding of the molecular mechanisms by which CAFs modulate tumor behavior. Recent advances in single-cell and spatial transcriptomic analyses have provided deeper insights into the complexity of CAFs in BC16,17,18,19,20,21, enabling the identification of distinct CAF subtypes with specific functional programs16,22 and further highlighting their complexity and plasticity23.
Genetically engineered mouse models (GEMMs), in which oncogenes or tumor suppressor genes are over-expressed or inactivated, are well-established models for studying human cancer24,25,26,27. Compared to syngeneic models, which involve grafting tumor cells in wild-type mice23,28, GEMMs more accurately mimic the multistep progression of BC and the interactions between cancer cells and the TME. Manipulating CAFs&#39; activities in GEMMs has, however, been challenging due to their heterogeneity, which makes it difficult to target all CAF subtypes without off-target effects on other stromal or epithelial components5. Combining GEMMs with complementary approaches, such as in vitro CAF manipulation, can help generate cellular models for loss-of-function experiments in both in vitro and in vivo settings. However, developing stable cellular models to explore the functional roles of specific genes remains challenging due to the rapid onset of senescence.
Here, we describe a robust platform for stable loss-of-function experiments in CAFs, followed by functional in vitro and in vivo analysis and transcriptional profiling of the engineered cells; this approach offers a comprehensive strategy for dissecting the functional relevance of CAF-expressed genes in breast cancer, with advantages in specificity, reproducibility, and adaptability over existing methods. This approach is also complemented by a novel web-based tool for exploring transcriptomes of laser-capture microdissected stromal and epithelial compartments from human breast tumors, enabling cross-species comparisons and facilitating hypothesis generation from human data.

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cancer-associated fibroblasts from mouse mammary tumors as tools for molecular and computational studies

Cancer-Associated Fibroblasts from Mouse Mammary Tumors as Tools for Molecular and Computational Studies

A compelling body of research has confirmed the critical role of the tumor microenvironment (TME) in the growth and aggressiveness of breast cancer (BC). Cancer-associated fibroblasts (CAF), the predominant cell component of the TME, significantly influences its behavior through complex crosstalk with cancer and other stromal cells in response to tumor-derived activating signals. Murine models are extensively used to investigate the mechanisms underlying CAF's role in BC progression, as they allow for the evaluation of the endogenous TME and the assessment of immune system involvement using syngeneic xenograft studies. Here, we present a set of methods for culturing BC-derived CAFs, manipulating gene expression, and evaluating their pro-tumorigenic functions using both in vitro and in vivo approaches. We also describe protocols for CAF gene expression profiling and transcriptomic analysis, alongside a computational procedure for inferring transcriptional signatures in primary tumors using publicly available bulk RNA-seq data. Additionally, we introduce a user-friendly, web-based tool for analyzing the transcriptomes of stromal and epithelial components from laser capture micro-dissected BC tumors. Ultimately, these methods can provide valuable insights into the molecular mechanisms driving CAF-tumor interactions and hold potential for identifying novel therapeutic targets.

In vitro assessment of CAFs' activities 
The contribution of mammary tumor-derived cancer-associated fibroblasts (CAFs) to the aggressive features of breast cancer (BC) was studied using immortalized CAFs (iCAFs) generated according to step 3. iCAFs were super-activated as described in step 6, then starved in sDMEM to collect their CM, which was used to treat 4T1 cells for 48 h (see step 7.1). This treatment was followed by in vitro functional assays. <strong class="...

This protocol outlines methods to culture, manipulate, and analyze murine CAFs, including in vitro/in vivo functional assays, transcriptomics, and computational analysis. Moreover, it describes a web-based platform developed to analyze the publicly available transcriptomes of laser capture micro-dissected stromal and epithelial components from a collection of human breast cancer tumors.

A compelling body of research has confirmed the critical role of the tumor microenvironment (TME) in the growth and aggressiveness of breast cancer (BC). ...

cancer-associated-fibroblasts-from-mouse-mammary-tumors-as-tools-for

Research

JoVE Journal

Cancer Research

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Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

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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.

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

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

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This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Discover how cancer-associated fibroblasts drive breast cancer progression using molecular, in vitro, in vivo, and computational approaches. This method reveals STAT3-mediated mechanisms and therapeutic targets.

Cancer-Associated Fibroblasts from Mouse Mammary Tumors as Tools for Molecular and Computational Studies

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