Scientists previously thought that fibroblasts are a homogeneous group of connective tissue cells, but new research shows that there are several different types and that they have important roles in diseases such as breast cancer. By using specific biomarkers to sort cells, researchers are now beginning to understand how distinct types of fibroblasts contribute to the development and complexity of cancer.
Breast cancer is a very common type of cancer, affecting millions of women each year, but its complex nature still remains somewhat of a mystery. The complexity results from genetic variation, the diversity in the types and stages of breast cancer and the ability of tumours to adapt and to resist treatment.
“Despite the immense variation in the types of breast cancer, almost all originate in the terminal duct lobular unit, a special glandular area of the breast where milk is produced. Why this is so remains a mystery. But according to our research, fibroblasts may be key to understanding this very focal origin of breast cancer,” explains one of the lead authors, Mikkel Morsing Bagger, Postdoctoral Fellow, Division of Translational Cancer Research, Lund University, Sweden.
“Somewhat surprisingly, we identified a particular lobular fibroblast that supports the growth of tumour cells,” adds Mikkel Morsing Bagger.
Several types discovered
Fibroblasts are connective tissue cells essential for the body’s functions and are present everywhere in the human body. They are crucial for many functions, including wound healing and maintaining the structural integrity of the skin and other organs by producing collagen, which provides tissue strength and also produces other important matrix components in the surrounding tissue.
“Until recently, fibroblasts were considered one cell type. Their importance was virtually overlooked, and they were regarded as a relatively passive and uniform group of cells without much variation,” explains Mikkel Morsing Bagger.
The new research reveals, however, that both the normal breast and breast tumours harbour two types of fibroblasts: lobular and interlobular, of which only the lobular fibroblasts promote the growth of tumour cells.
“Comparing subtypes provides deeper understanding of the changes that occur in fibroblasts when cancer develops and may enable us to develop more effective treatments in the future,” says Mikkel Morsing Bagger.
Current analysis kills the cells
The study is far from the first to identify potential differences between types of fibroblasts. In recent years, new technologies have expanded the opportunities for examining the differences. The approaches include microarrays, sequencing and especially single-cell RNA sequencing technology – a method for analysing the gene expression of individual cells.
“Single-cell RNA sequencing has provided increasingly detailed insight into the cells’ possible function and differences, opening our eyes to the transcriptional heterogeneity of fibroblasts and that they probably comprise several different types, even within a single tissue such as the breast,” explains Mikkel Morsing Bagger.
The RNA profile provides an overview of the genes expressed at a given time point, but the technology also has its limitations.
“Since you kill the cells to extract the RNA, you cannot study the cell afterwards and confirm whether the observed differences in the RNA profiles translate into functional differences and whether these affect the growth of tumour cells or the properties of other cells,” says Mikkel Morsing Bagger.
“This is where our study stands out: we used a biomarker to separate two types of living fibroblast cells, which then turned out to differ not only in their RNA profiles but also in their essential cellular functions,” adds Mikkel Morsing Bagger.
Fibroblasts are altered in a tumour
The researchers identified the biomarker by analysing normal breast glandular tissue, which has two units: smaller lobules, which produce milk during lactation, and larger ducts that transport the milk to the nipple.
“The structure is somewhat like a bunch of grapes, with the smaller lobules resembling grapes and the larger ducts resembling the stems. We previously identified a distinct fibroblast subtype in lobules expresses a certain protein, CD105 or endoglin. We call these the lobular fibroblasts, and those around the larger ducts are called interlobular fibroblasts,” explains Mikkel Morsing Bagger.
The glandular and the connective tissue change during cancer development, often to the extent that lobules and ducts can no longer be distinguished. But the CD105 biomarker enabled the researchers to trace the two types of fibroblasts in the tumour’s connective tissue.
“The fibroblasts undergo changes in a tumour, although not due to mutations as in tumour cells, and they are called cancer-associated fibroblasts (CAFs). So we addressed which changes lobular fibroblasts may undergo as they become CAFs – for example, do they still support tumour cell growth?” says Mikkel Morsing Bagger.
myCAFs and iCAFs
To enable comparisons, the researchers obtained breast tumour tissue from the Department of Pathology of Rigshospitalet in Copenhagen and isolated the fibroblasts according to their expression of CD105 by using fluorescence-activated cell sorting, a technique for sorting living cells based on their expression of cell surface proteins.
“This analysis showed that lobular fibroblasts were altered significantly,” explains Mikkel Morsing Bagger.
The CD105-expressing fibroblasts in breast tumours turned out to resemble myofibroblast cancer–associated fibroblasts (myCAFs), a type of specialised cells with a key role in repairing tissue by contributing to building the extracellular matrix.
“Parts of their lobular-like identity were preserved in the tumours, whereas others had changed, but most importantly, the myCAFs still supported the growth of the tumour cells in the same way as their normal counterparts, the lobular fibroblasts from normal tissue,” says Mikkel Morsing Bagger.
Likewise, interlobular-like fibroblasts in breast tumours had undergone significant changes and expressed genes that are typically found in or affect immune cells and inflammation. Hence, these cells were referred to as inflammatory CAFs (iCAFs).
“But neither iCAFs nor their normal counterpart, the interlobular fibroblasts, seem to play any role in developing tumours, which was very surprising,” adds Mikkel Morsing Bagger.
Invaluable progress
When researchers measure how fibroblasts affect the growth of tumour cells, they make xenografts: that is, transplant fibroblasts and tumour cells into the mammary glands of mice. The tumour size can then be measured using a caliper, and the growth can be followed over time.
“We typically have to use many fibroblasts for this, and this is difficult because they have a limited lifespan in the laboratory. To solve this, we inserted a telomerase gene into the cells that extends their lifespan,” explains Mikkel Morsing Bagger.
A major breakthrough was succeeding in establishing cell lines from breast tumour fibroblasts, even from each of the iCAF and myCAF subtypes.
“This is a major step forward. There are no equivalents today. In general, cell lines are invaluable when researchers investigate the function of cells, thereby enabling them to reveal the mechanisms by which particular types of fibroblasts support the growth of tumour cells – in the hope of being able to inhibit the tumour,” says Mikkel Morsing Bagger.
The researchers also argue that CAFs derive from local fibroblasts rather than from bone marrow–derived mesenchymal stem cells as demonstrated in mice, where such cells are able to migrate to tumours and facilitate metastasis.
“If this is true, finding and inhibiting such cells would be crucial. However, our experiments show that, unlike in mice, there are very few fibroblasts from bone tissue in human breast tumours, if any. This suggests that the normal fibroblasts in the breast tissue change and become CAFs,” explains Mikkel Morsing Bagger.
Fibroblast profile reveals poor prognosis
The new experiments clearly show that iCAFs develop from interlobular fibroblasts, and myCAFs originate from lobular fibroblasts.
“Hitherto, this has been a matter of speculation. Now, for the first time, we have shown that the lobule, in which most breast tumours arise, contains a fibroblast subtype that apparently supports the growth of the tumour cells,” says Mikkel Morsing Bagger.
The distinction between myCAFs and iCAFs and their origin from lobular and interlobular fibroblasts further underscores the diverse roles of fibroblasts in breast cancer. The researchers found specific RNA profiles associated with poor prognosis.
“However, this needs to be substantiated with more data, but since we have access to material and clinical data from one of the world’s largest unique studies of breast cancer, the Sweden Cancerome Analysis Network – Breast (SCAN-B) Initiative , we hope to be able to continue studying the importance of fibroblasts in breast cancer,” concludes Mikkel Morsing Bagger.
