Cancer associated fibroblasts: how do they contribute to cancer progression?

Author: AcceGen R&D Team

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Cancer associated fibroblasts (CAFs), or Carcinoma associated fibroblasts, can contribute to cancer progression through multiple mechanisms, including the secretion of soluble factors and extracellular matrix, physical interactions with cancer cells, the regulation of angiogenesis, immunity and metabolism, and resistance to chemoprevention according to many present researches. Therefore, CAFs are attracting increasing attention as a therapeutic target in cancer.

What is CAFs?

Tumors are multicellular tissues consisting of not only tumor cells but also resident cells in the local stroma. Fibroblasts are a significant portion to the cell composition of most solid tumors, including prostate cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, and pancreatic cancer. CAF cells, which mainly originate from local tissue fibroblasts, are a heterogeneous population of activated fibroblasts found within the tumor microenvironment (TME) [1]. CAFs produce nutrients and metabolites which tumor use for energy production, proliferation, invasion, and migration.

How to isolate and culture CAFs?

Tumor tissues were harvested within 30 minutes after resection and kept in DMEM with 10% FBS and penicillin-streptomycin on ice for immediate transportation to the laboratory. Tissues were washed three times with PBS containing 100 U/ml penicillin and 100 μg/ml streptomycin, then minced into small pieces and digested for 8 h at 37℃ in DMEM containing 10% FBS and 0.5 mg/ml collagenase I. After centrifugation and washing with PBS, cells pellets were resuspended in the DMEM with 10% FBS and transferred into 100-mm tissue-culture dishes. CAFs were routinely maintained in DMEM containing 10% FBS at 37℃ in humidified atmosphere containing 5% CO₂ [2].   Especially, Yoray Sharon et al. developed a method based on Fluorescence-Activated Cell Sorting (FACS), which can utilize PDGFRα or α-SMA as a surface marker to isolate pure populations of normal fibroblasts (NFs) and CAFs from fresh mouse and human tissue. And isolated CAFs can be cultured to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle during CAFs culture [3].

Characterization of CAFs

CAFs present a spindle-shaped morphology with an elongated, thin nucleus, which is slightly thicker than that of non-activated fibroblasts [1]. CAFs show many similarities to myofibroblasts, referred to as activated fibroblasts, in physiological wound healing and even more to myofibroblasts in pathological fibrosis. NFs constitutively express vimentin and fibroblast-specific protein 1 (FSP1) [4]. Activated fibroblasts highly express a number of markers, such as α-SMA, PDGFRα and FAP, which have been widely used to isolate CAF populations [5].   Figure 1 demonstrates characterization of CAFs and NFs. Representative images of H&E staining of frozen sections from breast tumor tissue and its corresponding normal mammary tissue are shown (Fig.1A and B). The primary cultures of CAFs and NFs were respectively isolated from above tissue. And the expression of the fibroblast marker Fibronectin (FN) by immunofluorescence staining was then tested. Whether CAFs or NFs were universally positive for FN (Fig.1C and D). Furthermore, CAFs displayed a high level of α-SMA, a biomarker of the activated fibroblast, in contrast to NFs (Fig.1E and F) [2].

Figure 1. Characterization of CAFs and normal fibroblasts (NFs) [2]

CAFs Functions

Figure 2. CAFs interaction with cancer cells [6]

The roles of CAFs in the progression of various types of cancers are well established. CAFs can affect cancer progression and metastasis through pleiotropic mechanisms. As shown in figure 2, the development of CAFs is initiated by the cancer and particularly affected by extracellular matrix (ECM) and immune cells. CAFs regulate the growth, survival, and invasion of cancer cells by contacting directly with the cancer cells, by secreting cytokines, growth factors and extracellular vesicles, and by altering the organization and composition of the ECM. Furthermore, CAFs influence immune cell response and tumor angiogenesis [6].

Figure 3. Summary of CAFs functions and the relative mechanisms [7]

According to Figure 3, the biological functions of CAFs are indicated in dark blue text boxes, while the processes and mechanisms resulting in the control of function are indicated in light blue, green, purple and grey text boxes. And mechanisms boxes are connected to functions boxes by lines. Both the secretion of soluble factors and matrix remodelling promote cancer invasion. Soluble secreted factors also contribute to tumor growth and changes in the immune microenvironment, which is also influenced by the altered metabolic state of the tumor [7].

Targeting CAFs for Clinical Benefit

Table 1. Current CAFs clinical trial activity [7]

CAFs can serve as a target, which could be manipulated for therapeutic benefit in cancer patients. Now many clinical trials involving CAFs-targeting agents are combined with existing therapies, which could yield greater clinical benefit than either conventional therapies or T cells to the tumor. In some cases, researchers are developing new strategies to target fibroblasts specifically, such as FAP ligands coupled to cytotoxic drugs. In other cases, crosstalk between cancer cells and fibroblasts is targeted, such as Hedgehog pathway inhibition. In addition, existing drugs are found to affect the functions of CAFs strongly and are repurposed as anti-stromal ones. For example, losartan is mainly used in the treatment of high blood pressure but also in the regulation of the tumor ECM. Table 1 outlines ongoing clinical trial activity in these areas.

Conclusions

Researches on CAFs are at an exciting and critical stage. Numerous experiments reveal a tumor-promoting role of CAFs. In order to identify CAFs-specific drug targets with strong potential benefits for cancer patients, we still require more knowledge on CAFs and their role in different cancer types.

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Reference

[1] Ishii G, Ochiai A, Neri S: Phenotypic and functional heterogeneity of cancer-associated fibroblast within the tumor microenvironment. Adv Drug Deliv Rev, 2016: 186–196. [2] Liuyang Zhao, Yan Suna, Yixuan Hou, et al: MiRNA expression analysis of cancer-associated fibroblasts and normal fibroblasts in breast cancer. The International Journal of Biochemistry & Cell Biology, 2012 (44): 2051-2059. [3] Yoray Sharon, Lina Alon, Sarah Glanz, et al.: Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS). Journal of Visualized Experiments. 2013 (71):1-6. [4] Raghu Kalluri and Michael Zeisberg: Fibroblasts in cancer. NATURE REVIEWS | CANCER, 2006(6):392-401.  [5] Martin Nurmik, Pit Ullmann, Fabien Rodriguez, et al: In search of definitions: Cancer-associated fibroblasts and their markers. Int. J. Cancer 2020 (146): 895–905. [6] Mei Qi Kwa, Kate M. Herum, Cord Brakebusch: Cancer-associated fibroblasts: how do they contribute to metastasis? Clinical & Experimental Metastasis, 2019 (36): 71–86. [7] Erik Sahai, Igor Astsaturov, Edna Cukierman, et al.: A framework for advancing our understanding of cancer- associated fibroblasts. Nature 2020 (20) : 174-186