The fibroblast is a cell type of paramount importance for extracellular matrix (ECM) production and

remodeling in interstitial tissues. Fibroblasts play a major role to produce fibrillar collagens and other
interstitial ECM components.

Fibroblast take active part in matrix remodeling via integrins and release of matrix
metalloproteinases during tissue regeneration events.The transcriptional profile of fibroblasts varies
with the anatomical location.

In tumor biology the activated fibroblasts, oftencalled cancer-associated fibroblasts (CAFs), act in the
realms of the tumor microenvironment (TME) with consequences for tumor growth, formation of
stem cell niches, immunosuppression, metastasis and chemoresistance.

Cancer associated fibroblasts (CAFs) are plump spindle-shaped mesenchymal cells that constitute
the most numerous cells in the tumoral microenvironment. CAFs facilitate the development,
propagation, and invasiveness of tumors.

CAFs exert their proneoplastic activity by secreting and responding to protein mediators. CAFs
actively participate in a collaborative process with neighboring pre-malignant cells and malignant
cells, exchanging growth factors, high-energy metabolites, and other factors in order to aid in the
survival and proliferation of both aberrant cell populations.

• CAFs are derived from multiple cell types and there can be distinct populations of them in
the same stroma.

• CAFs appear to be primarily derived from local stromal fibroblasts. In a mouse xenograft of
pancreatic cancer, up to approximately 25% of fibroblasts in the tumor stroma originated in
the bone marrow.

• Normal and/or tumor epithelial cells may undergo epithelial to mesenchymal transition to
give rise to CAFs. In vitro studies indicate that epithelial cells can transdifferentiate into
myofibroblasts.

• In experimental tumor models, CAF subpopulations may derive from endothelial cells at the
invasive front of malignant tumors. Other possibly less important cells which are progenitors
of CAFs include local mesenchymal cells, transdifferentiated adipocytes, transdifferentiated
smooth muscle cells, and resident stem cells.

CAFs show high expression of alpha-smooth muscle actin (𝛼-SMA), one of the most significant
markers of fibroblasts activation and CAFs differentiation.

𝛼- SMA expression is induced by overexpression of hypoxia- associated microRNA- (miR-) 210, which
converts healthy fibroblasts into CAFs-like cells. 𝛼-SMA is not the only molecular marker useful to
identify CAFs.

In fact, fibroblast activation pro- tein (FAP), fibroblast-specific protein 1 (FSP1), osteonectin, desmin,
platelet-derived growth factor receptors (PDGFR) 𝛼 or 𝛽, neuron-glial antigen-2 (NG2), periostin
(POSTN), podoplanin (PDPN), tenascin-C (TNC), CD90/THY1, or discoidin domain-containing receptor
2 (DDR2) and the mesenchymal cell marker vimentin can be also considered CAF markers.
Desmoplasia( the growth of fibrous or connective tissue) and a lot of signalling pathways in tumours
can induce differentiation of CAFs, which in turn promote tumour progression and metastasis
through the secretion of growth factors and chemokines.

Malignant cells and CAFs communicate via growth factor signaling. The most significant are
transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF).

Cancer cells and CAFs secrete the three known isoforms of TGF-β, (TGF-β1-3), which coordinate CAF
recruitment and the differentiation of normal fibroblasts into myofibroblasts.

While normal fibroblasts have only a nominal effect on tumor growth, this interaction between CAF-
generated SDF-1/CXCR4 and TGF-β results in enhanced propagation and invasiveness of cancer cells.

For simplicity we categorize the biomarkers into membrane proteins, cytoskeletal proteins, cytosolic
protein, and secreted proteins.

Membran protein Integrins are transmembrane receptors that mediate cell interaction with the
extracellular matrix (ECM). Cytoskeletal proteins include αSMA (alpha smooth muscle actin),
Vimentin, FSP-1(Fibroblast specific protein). Moreover, secreted protein include Tenascins,
Osteopontin, Periostin,& Clusterin.

A number of CAF subtypes have been defined within tumor stroma. Pioneer work has defined two
major types of fibroblasts in pancreatic cancer, inflammatory CAF (iCAFs) and myofibroblastic CAFs
(myCAFs), and four major subclasses of CAFs in breast cancer (CAF-S1-S4), distinguished by different
levels of αSMA and fibroblasts activation protein (FAP) expression.

CAFcultures generated from primary pancreatic adenocarcinomas displayed no somatic mutations or

evidence of p53 mutations, indicating that CAFs display normal allelotypes. It ismore generally
accepted that epigenetic changes contribute to the CAF phenotype.

Gastric cancer CAFs, which were propagated in culture or present in transgenic murine models,
demonstrated a global loss of DNA methylation. Using a novel epigenetic profiling tool, methylation-
specific digital karyotyping, the epigenomes of epithelial, myoepithelial, and stromal fibroblasts all
showed distinct epigenetic changes during breast tumorigenesis

The absence of an abnormally activated tumour microenvironment, both genetic and epigenetic
mutations in cancer cells are not sufficient to sustain cancer progression.

Necessary step for cancer initia- tion and progression is represented by CAFs differentiation, which
can either occur at the early phase of cancer or surprisingly precede the genetic alterations of
epithelial cells, triggering the malignant transformation of adjacent cells.

CAFs dif-ferentiation can be induced by tumour cell-derived trans- forming growth factor 𝛽 (TGF-𝛽),
epidermal growth factor (EGF), PDGF𝛼, PDGF𝛽, basic fibroblast growth factor (bFGF, also known as
FGF2), interleukin 6 (IL-6), and interleukin 1𝛽 (IL-1𝛽) .

Reactive oxygen species (ROS) to regulate the metabolic reprogramming of both cancer cells and
CAFs, supporting the adaptation to oxidative stress that triggers CAFs differentiation, tumorigenesis,
and chemoresistance. In the tumour microenvironment, cancer cells produce high levels of ROS
deriving from mitochondrial dysfunction. This condition is associated with early step of
carcinogenesis, and can occur hypoxia.

The increase of ROS levels in tumour environment also induces the proinflammatory transcriptional
factor NF𝜅B activity in fibroblasts, leading to a CAF-like phenotype.

CAFs secrete higher levels of H2O2 compared with normal cells, suggesting that extracellular H 2O2
could lead to stroma remodelling. The higher H2O2 production by CAFs is due to an impaired
TGF-𝛽 signalling leading to the suppression of the antioxidant enzyme glutathione peroxidase 1
(GPx1).

Indeed, treatment of NFs with CAF-conditioned medium or exogenous H 2O2 leads to the acquisition
of an oxidative CAF-like state.

• Multiple compounds that inhibit CAFs and tumor CAF communication are in pre-clinical and
clinical development. These agents can be categorized by their specific target.

• Class 1 agents inhibit signal transduction pathways involved in CAF and tumor/CAF
communication; since these pathways are involved in a variety of cell types, these agents’
activities are not restricted to CAFs and tumor/CAF communication.

• Class 2 agents more uniquely target CAFs and CAF products; many of these agents have been
exhaustively studied in the past although newer agents that fall into this category are
currently being investigated. Some examples of these agents include inhibitors of MMPs,
cathepsin, serine proteases, and tenascin-C.

 Cancer-associated fibroblasts (CAFs) are a specific but heterogenous population of cells that
reside in the tumor microenvironment and play a significant role in the neoplastic process.

 CAFs can derive from the activation of resident fibroblasts or other precursor cells
represented by bone marrow derived mesenchymal stem cells, epithelial cells, carcinoma
cells, endothelial cells, pericytes, smooth muscle cells, adipocytes, fibrocytes, stellate cells in
pancreas and liver, myoepithelial cells in breast, and pericryptal myofi- broblasts of the
gastrointestinal tract.

 CAFs participated in tumorigenesis, provide a nurturing environment for neoplastic cells,

promote malignant cell growth and invasiveness by remodelling the local extracellular
matrix, they have pro-inflammatory and proangiogenic properties.

 Multiple compounds that inhibit CAFs and tumor CAF categorized by their specific target.