ll

Leading Edge

Review
Fibroblasts in cancer: Unity in heterogeneity
Yash Chhabra1,* and Ashani T. Weeraratna1,*
1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Department of Oncology, Sidney Kimmel Cancer

Center, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA

*Correspondence: ychhabr1@jhmi.edu (Y.C.), aweerar1@jhu.edu (A.T.W.)
https://doi.org/10.1016/j.cell.2023.03.016

SUMMARY

Tumor cells do not exist in isolation in vivo, and carcinogenesis depends on the surrounding tumor microen-
vironment (TME), composed of a myriad of cell types and biophysical and biochemical components. Fibro-
blasts are integral in maintaining tissue homeostasis. However, even before a tumor develops, pro-tumori-
genic fibroblasts in close proximity can provide the fertile ‘soil’ to the cancer ‘seed’ and are known as
cancer-associated fibroblasts (CAFs). In response to intrinsic and extrinsic stressors, CAFs reorganize the
TME enabling metastasis, therapeutic resistance, dormancy and reactivation by secreting cellular and acel-
lular factors. In this review, we summarize the recent discoveries on CAF-mediated cancer progression with a
particular focus on fibroblast heterogeneity and plasticity.

INTRODUCTION An Achilles’ heel of current chemo- and immunotherapeutic

The last three decades have generated tremendous knowledge
that has led to defining the ‘‘hallmarks of cancer’’ and ushered
a shift in our approach to understanding tumor biology. Much
of the early research focused on tumor cells and the genetic
and environmental processes promoting their transformation or
driving their malignancy. With breakthroughs in biochemical,
biophysical, and engineering methodologies and technological
explosion in ‘‘-omics’’ fields—and especially with advances in
immunologic approaches to cancer therapy—we have begun
to understand the tumor in its entirety. It is critical to appreciate
the cellular diversity, complex interactions, and turnover rate of
not just the tumor itself but its surrounding microenvironment.
An important and abundant component of both the stromal tis-
sue microenvironment and TME are the fibroblasts that are not
completely characterized. Within a tissue, healthy fibroblasts
play an important role in maintaining homeostasis, such as dur-
ing wound healing and regeneration and in promoting fibrotic
conditions during excessive fibrosis and scarring, as well as can-
cer progression, respectively.1–3 Fibroblasts appear to have a
bimodal effect on cancerous cells, in that they initially prevent
malignant transformation and progression,4–7 but as the disease
progresses, they become misdirected and promote tumor
growth. These tumor-surrounding fibroblasts are known as can-
cer-associated fibroblasts (CAFs). CAFs form a major compo-
nent of the tumor stroma and are primarily associated with tumor
maintenance and promotion via diverse mechanisms,8 including
supporting tumor cell growth by secreting growth factors, che-
mokines, extracellular vesicles (EVs), and extracellular matrix
(ECM) remodeling that regulates tumor proliferation, metastasis,
therapy resistance, immune evasion,1,2,9 and more recently re-
activation from tumor dormancy.10,11 Clinically, activated CAFs
have been associated with worse prognosis, resistance to ther-
apies, and disease recurrence in multiple cancers.12,13
approaches is that a majority of current therapies primarily target
the fast-growing tumor ‘‘seeds’’ but largely ignore the contribu-
tion of fibroblasts or the fertilizing tumor ‘‘soil.’’14,15 Our current
understanding of the impact of fibroblasts in supporting tumor
growth, metastasis, and therapeutic resistance is insufficient to
guide us in establishing treatment modalities and targeting stra-
tegies.16 Therefore, it will be necessary to understand the contri-
bution of the surrounding healthy stroma in malignancy and how
its modification by cancer cells affects the course of the disease.
Advances in transcriptomics and proteomics at single-cell reso-
lution, where a specific CAF population can be associated with a
particular cancer subtype,17 may aid in promoting this under-
standing and predicting responses, stratifying patients, and
personalizing therapy.
This review aims to summarize the premise and promise made
from the recent developments in the field of fibroblast—and in
particular CAF—biology, highlighting commonalities of fibro-
blasts present across different cancer types and factors promot-
ing their differentiation to CAF and other cell types. Finally, we
will focus on the molecular and functional heterogeneity between
CAFs that makes tumors evasive to therapy and highlight the role
of intrinsic processes such as aging and senescence in promot-
ing the transition to CAFs.
NORMAL FIBROBLASTS
Fibroblasts are active in intercellular signaling and are critical in
many developmental processes, physiologic functions, and pa-
thologies.18,19 They are responsible for not only providing struc-
tural integrity but also in regulating tissue homeostasis. Fibro-
blasts are the most abundant cell type in all the connective
tissues and are responsible for generating distinct microarchi-
tecture and providing biochemical cues and biophysical sup-
port.20 Fibroblasts show heterogeneity based on anatomical

1580 Cell 186, April 13, 2023 ª 2023 Elsevier Inc.


Review
origin, and their types and roles can vary due to tissue composi-
tion (e.g., vascularization, innervation, abundance of fat or mus-
cle), developmental origin (e.g., mesoderm/neural crest in the
skin, epicardium/endothelium in the heart), and the requirement
for tissue-specific functions (e.g., supporting hair follicle forma-
tion in the skin or bone resorption in the synovium).21 These dif-
ferences in the behavior of fibroblasts from different body sites
are a culmination of intrinsic differences and highlights their abil-
ity to respond to stress (mechanical, oxidative) that differs be-
tween regions in the body.22
Fibroblasts and the extracellular matrix
Among other cell types, an important function of fibroblasts is the
production of a tissue-specific basement membrane that serves
as a protective barrier, thus imparting specificity and functionality
to the surrounding epithelium such as in skin, pancreatic islets,
and heart.23–25 This involves synthesizing, as well as degrading,
ECM components such as collagens, fibronectins, laminins,
elastins, proteoglycans, integrins, matrix metalloproteinases
(MMPs), tissue inhibitors of metalloproteinases (TIMPs), and a
host of other ECM proteins in an organ-dependent manner.26,27
ECM by nature is a complex molecular milieu and, although
non-cellular itself, provides cells with the tensile scaffold vital
for appropriate structural assembly into macroscopic structures.
It also serves as a reservoir of growth factors, hormones, and
signaling molecules that integrate positional cues and translate
them into fundamental cellular fates, underlying growth, differen-
tiation, survival, and motility.28,29 ECM is present within all tis-
sues and organs and is essential for tissue morphogenesis,
differentiation, and maintaining homeostasis.30 Fundamentally,
ECM is composed of water, protein, and polysaccharides; how-
ever, the physical, topological, and biochemical composition
of the ECM is markedly heterogeneous and tissue specific,
whereas adhesion, communication, and differentiation remain
its common cellular functions.31 The importance of the fibro-
blast-secreted ECM is also evident in a wide range of syndromes
arising directly from the genetic abnormalities in matrix proteins
in monogenic disorders such as osteogenesis imperfecta,32
Marfan’s syndrome,33 and other genetic disorders of the con-
nective tissue such as Ehlers-Danlos syndrome.34,35 In addition,
a large diversity of acquired human diseases, such as coronary
heart disease, hypertension, chronic obstructive pulmonary dis-
ease, asthma, chronic kidney disease, and neurodegenerative
and autoimmune disorders are directly linked with abnormal
ECM function in their pathogenesis.36
Fibroblast role in wound healing and inflammation
Normal fibroblasts play crucial roles during the process of wound
healing and inflammation. Upon tissue damage, the resident fi-
broblasts proliferate, differentiate, and invade the site of injury.
This occurs in response to platelet clotting, following vessel
injury, resulting in secretion of cytokines (e.g., transforming
growth factor b [TGFb], platelet-derived growth factor [PDGF],
interleukin-1b [IL1b], MMPs, and TIMPs) in the microenviron-
ment that degrade the basement membrane, induce cell prolifer-
ation and migration, and recruit inflammatory cells and fibro-
blasts.27,37 These fibroblasts are referred to as ‘‘activated’’ and
are characterized by de novo expression of a-smooth muscle
ll
actin (aSMA), stress fibers, and fibronectin splice variants,38
which allows ECM synthesis and remodeling at the site of injury.
This deposition of reactive stroma is referred to as desmoplastic
stroma. The induction of aSMA alters cytoskeletal organization,
which increases the contractility between activated fibroblasts—
at this point called myofibroblasts—and the ECM to close the
wound. After wound closure, the activated fibroblasts secrete
matrix proteins and attract epithelial cells to complete the heal-
ing process before undergoing programmed cell death and ulti-
mately a reduction in myofibroblast populations.39,40 As a result,
only quiescent, non-contractile fibroblasts are left at the resolved
wound site. Several parallels have been drawn between wound
healing and tumorigenesis where tumors have been likened to
‘‘wounds that never heal.’’30,41 This is apparent because, unlike
the activated fibroblasts in a normal tissue that undergo
apoptosis, the myofibroblasts in a tumor continue to persist. In
addition, the ability of fibroblasts to significantly influence other
cell types in their vicinity has shed light on their role in inflamma-
tory processes. Fibroblasts can modulate leukocyte activity by
secreting certain signaling molecules at inflammation sites and
can either inhibit immune responses under physiological condi-
tions by removing dead or redundant immune cells or create a
chronic inflammatory environment during fibrosis and cancer
by secreting cytokines that attract immune cells.42–44
Studies have also shown that multiple, functionally distinct
fibroblast subtypes can arise from a single tissue. This is prom-
inent in the dermis, where an unwounded mouse skin was shown
to comprise two distinct fibroblast lineages: papillary (superficial)
dermis and reticular (deeper) dermis that are regulated by
age.19,45 Although papillary fibroblasts undergo rapid prolifera-
tion, reticular fibroblasts actively produce ECM and are respon-
sible for dermal wound repair.46 Other studies have established
striking heterogeneity in the fibroblasts residing in both murine
and human dermis using single-cell RNA sequencing (scRNA-
seq).47 Further investigation comparing fibroblasts in different
wound healing outcomes have established cell-intrinsic differ-
ences that contribute to distinct healing outcomes between
these sites.46,48 This is reflected in the profibrotic dermal fibro-
blasts that promote healing via scarring, whereas those in the
oral mucosa heal with minimal scarring.49 Reciprocal transplan-
tation of these fibroblasts also confirmed that the fibroblasts
responsible for tissue repair in the mouse oral mucosa (defined
by Wnt1 expression) were intrinsically non-fibrotic, whereas
En1-positive fibroblasts from the dorsal dermis were scar pro-
ducing.49,50 This full diversity of fibroblasts within the same organ
is being investigated beyond the skin, where it is also likely to
exist. For example, fibroblasts derived from kidney cortical and
medullary regions exhibit differences in cell culture and in their
responses to mitogenic stimulation.51,52
Fibroblasts across the body also show temporal heterogene-
ity,19,53–57 which is the direct outcome of the interaction with their
immediate microenvironment, including the ECM, crosstalk with
other cell types via paracrine and autocrine signals and response
to biomechanical cues (e.g., tissue stiffness, shear force). Finally,
the fibroblast heterogeneity reflected in a range of phenotypic
states such as quiescence, proliferation, senescence, activa-
tion, migration, differentiation, or aging adds another level of
complexity to their function.
Cell 186, April 13, 2023 1581
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CANCER-ASSOCIATED FIBROBLASTS
CAFs are found in almost all solid tumors; however, their abun-
dance varies between different types of cancers. Multiplex imag-
ing techniques, such as antibody-based staining and nucleic
acid in situ hybridization, have been informative in establishing
spatial location of CAFs within a tumor,58,59 but their origins
remain unclear. Inherent limitations associated with the longitu-
dinal sampling of the same human biopsy samples have made
it nigh impossible to determine the spatiotemporal regulation
of fibroblasts conversion into CAFs. Therefore, cell-line-based
2D and 3D spheroid or organoid models in vitro have been estab-
lished for their ease of use. These models have shed light on few
facets of CAF biology, such as cell communication and crosstalk
with other cell types and matrices, but they fail to recapitulate the
heterogeneous and complex interactions of CAFs.60–62 Other
state-of-the-art methods like liquid-air interface models,63,64
microfluidics technology,65,66 and bio-printed tissues65,66 have
been designed and are being implemented to emulate certain
cancer types. Ultimately, an extensive amount of research in ro-
dent models, utilizing irreversible labeling of cell types in a tissue-
restricted manner, has permitted cell tracing to model disease
progression. Although this is a step in the right direction, manip-
ulating specific fibroblast types in vivo has been technically chal-
lenging due to a lack of specific drivers or owing to overlapping
gene expression or off-target effects of inducing agents.67 More
recently, intravital microscopy in mouse models has aided in lon-
gitudinal assessment of CAFs during tumor progression,68,69 as
well as the interaction between the CAFs and stroma.70,71 The
implementation of scRNA-seq techniques has revealed a previ-
ously unappreciated level of CAF heterogeneity demonstrating
the presence of transcriptionally distinct CAF populations but
also highlighting fibroblast signatures common across different
solid cancers.1 However, a key limitation of this technique is
that there is a potential to induce ectopic gene expression during
cell isolation from tissues, leading to mischaracterization of
certain cell subpopulations.72 Spatial transcriptomics can avoid
this technical artifact by assaying cells in their native tissue
context. Applications of this technique in glioblastoma and
another preliminary study in colorectal cancer have already
started to emerge.73,74 Integrating single-cell and spatial tran-
scriptomics in the future holds promise in determining discrete,
dynamic, and transient cell subpopulations within various tumor
stages. This will allow a more accurate assessment of CAF
origin, their function as well spatial location within a tumor, which
can indicate new therapeutic strategies in the future. Although
the molecular definition of CAFs is still a debated issue, field
experts have suggested a framework that has increased our un-
derstanding of CAF biology;3 nevertheless, the CAFs like normal
fibroblasts are heterogeneous and governed by the same under-
lying conditions (Figure 1). Below, we highlight key characteris-
tics and dynamics of CAFs, with emphasis on their heterogeneity
and plasticity across different cancer types.
Heterogeneity in cancer-associated fibroblasts
Single-cell studies in both human and mouse species undertak-
ing pan-tissue analysis of fibroblasts has suggested the occur-
rence of universal fibroblast states in normal tissues, which ulti-
1582 Cell 186, April 13, 2023
Review
mately serve as reservoirs that yield specialized and activated
fibroblasts in diseased states including cancer.75 Both universal
and specialized fibroblast subtypes were similar between human
and mouse and the heterogeneity was promoted by tissue
type and disease context.75 CAFs can be quiescent or activated
based on their cellular states. The quiescent CAFs often
resemble normal fibroblasts with regards to reduced prolifera-
tion and metabolic capacity.76 On the other hand, activated
CAFs, termed myofibroblasts, are characterized by attributes
not limited to increase in proliferation, ability to migrate, meta-
bolic activity, production of ECM, and growth factors and sus-
ceptible to epigenetic modifications.9 Although it is difficult to
ascertain when the CAFs arise, it is plausible that kinetics of
such a change will be dependent on the anatomical site of origin
since resident fibroblasts exhibit organ-specific transcriptomics
profile77 as well as their positional memory within the tissue,
which imparts a unique gene expression.78,79 This is in addition
to the other surrounding stromal cells that might be in close prox-
imity and can transdifferentiate into CAFs (Figure 2).
The development of immunohistochemical methodologies
and scRNA-seq have redefined classical CAF markers, such
as PDGF receptor-a/b (PDGFRa/b), aSMA, fibroblast-associ-
ated protein (FAP), and fibroblast-specific protein-1 (FSP1), as
subtype-specific.56,58,79–85 For example, elevated expression
of aSMA was reported to be a distinctive marker in activated
CAFs compared to normal fibroblasts, which were later shown
to be present in low abundance in other cancer types. Similarly,
the broadly used marker for both normal and activated fibro-
blasts is FSP1, also called S100A4. However, this protein is
also expressed in several types of immune cells and certain can-
cer cells.80,84–86 Another technical challenge posed during CAF
isolation is the lack of a specific marker since most markers
are shared with other cell types. For example, PDPN is highly ex-
pressed in lymphatic endothelial cells,87 FAP is also expressed
by macrophages,88 and aSMA is mainly expressed by smooth
muscle cells89 and to a lesser extent in pericytes.90 This would
suggest that a set of subtype-specific markers vs. normal fibro-
blast markers are needed to isolate specific CAF population. In
addition to the structural proteins, certain functional markers
have been described for CAFs. Numerous transcriptional fac-
tors, including heat shock transcription factor (HSF1), STAT3,
MYC, and yes-associated protein (YAP) have also been reported
to be involved in defining CAF signature and reprogram-
ming.91–94 It is therefore critical that any interpretation of CAF
function within a tumor takes into consideration their associated
heterogeneity to avoid any reproducibility issues, possibly
arising from the different stages of activation, sources of origin,
tissue location, and molecular characteristics.
Phenotypic heterogeneity
Similar to the normal fibroblasts, CAF identification and isolation
requires a combination of morphological features, biomarkers,
and genetic mutations. As a result, cell populations within a tumor
that lack cancer cell mutations; exhibit an elongated spindle
morphology; are negative for the expression of endothelial,
immune, and epithelial cell markers; and are positive for mesen-
chymal markers (VIM, aSMA, FAP etc.) are defined as CAFs.3,95
However, none of these biomarkers are specific for CAFs, as

Review
they do not exclude for mesenchymal cells (adipocytes or peri-
cytes). Although few reports have identified p53 mutations in
CAFs,96–98 they were later discredited owing to experimental arti-
facts.99 Subsequent studies have confirmed that CAFs indeed lack
any detectable genetic alterations.99–102 Further, relying on cellular
morphology is also not reliable since the tumor cells undergoing
dynamic states of epithelial to mesenchymal transition (EMT) or
phenotype switching (proliferative to invasive state) can also
show elongated morphology similar to fibroblasts. More impor-
tantly, isolation of CAFs from peri-lesional or tumor-surrounding
regions remains challenging since numerous intracellular, extra-
cellular and cell surface proteins are also common to normal fibro-
blasts. This is also confounded by the fact that ex vivo cultures of
isolated tumor and stromal cells including CAFs can undergo
changes based on the substrate, media and environmental condi-
tions and therefore may not remain an accurate representation of
the cell type in vivo. Expectedly, no universal marker of CAFs exists
that suggests significant heterogeneity in the expression of fibro-
ll
Figure 1. Heterogeneity in fibroblast func-
tion during homeostasis and cancer devel-
opment
The heterogeneity principles are common to both
normal fibroblasts and cancer-associated fibro-
blasts (CAFs) and occur at multiple levels; cellular
level depending on the state/function of the fibro-
blast, microenvironment level depending on the
communication with other cellular and acellular
components, regional level depending on the
residing tissue and its function and lastly at the
organismal level encompassing the attributes of
demographics, physiological status, and inter-
sectionality. During normal homeostasis and heal-
ing after an acute injury, the quiescent fibroblasts
become activated (activated fibroblasts) to
mediate repair and eventually perish. In certain
conditions such as desmoplasia, the activated
fibroblasts can establish self-activating and
feedback loop, which gives rise to their pro-
tumorigenic capacities. Intrinsic or extrinsic
stressors enables their transition into CAFs
providing greater proliferative capacity. The prin-
ciples above dictate if the quiescent fibroblast
present in the normal tissues transition into acti-
vated fibroblasts, a phenomenon common to both
wound repair in healthy tissues and transition to
CAFs in a tumor microenvironment. Due to the
inherent plasticity, the proliferative CAFs can qui-
esce and become tumor-restraining, but it remains
unknown if they can completely revert back to their
normal non-tumorigenic state. Within the tumor
microenvironment (TME), CAFs communicate with
each other (via autocrine signaling) and other stro-
mal and tumor cells (in a paracrine manner) through
growth factors, metabolites, extracellular vesicles
(EVs) and chemokines while constantly shaping
and reorganizing the extracellular matrix (ECM) to
promote a tumor permissive environment.
blast markers itself.3 This heterogeneity is
universally observed across tumor types.
Lineage-dependent heterogeneity
Resident fibroblasts within a tumor
mass, by definition, are the most com-
mon source of CAFs.79,103,104 Owing to their high degree of
plasticity, pro-tumorigenic fibroblasts and myofibroblasts get
molded by their surrounding microenvironment, which dic-
tates their function. In response to intrinsic cues and extrinsic
stressors these resident fibroblasts can get activated and
revert from their quiescent state to a proliferative state,
referred to as ‘stromagenesis’, as observed in some tu-
mors.105,106 In that regard, a vast array of cytokines secreted
by the growing tumor and infiltrating immune cells such as
TGFb, several receptor tyrosine kinase (RTK) ligands; PDGF,
fibroblast growth factor (FGF), epidermal growth factor (EGF)
and other inflammatory chemokines, such as interleukin-6
(IL6), IL1b enable this transition to CAFs via nuclear factor
kappa B (NF-kB) and janus kinase (JAK)/signal transducer
and activator of transcription (STAT) pathways, respec-
tively.91,107–109 Contribution from environmental stressors in
the form of ECM stiffness, reactive oxygen species and DNA
damage from therapeutic interventions110,111 and other
Cell 186, April 13, 2023 1583
 ll
sources (UV irradiation) can also give rise to pro-tumorigenic
fibroblasts.112–114 The inherent plasticity of epithelial and
mesenchymal cell types during pathological processes has
become a topic of intense investigation. Numerous cell types
found in the TME or in proximal tissue have been proposed
as candidates for the CAF compartment via processes of
transdifferentiation, endothelial to mesenchymal transition
(EndMT) and epithelial to mesenchymal transition (EMT)
(Figure 2). Some examples of CAF precursors include but
are not limited to bone marrow or adipose-derived mesen-
chymal stem cells (Ad-MSCs), activated pancreatic and he-
patic stellate cells, fibrocytes, pericytes, adipocytes, epithelial
cells as well as peripheral nerves and endothelial cells.115–124
Spatial location and origin of CAFs therefore serve as an
important driver of phenotype leading to considerable intra-
and inter-heterogeneity across the diverse CAF subpopula-
tions, both at the functional and gene expression level.
Consequently, adding to the difficulty in distinguishing CAFs
from other cell types expressing similar markers.
Functional and molecular heterogeneity
The most well recognized of the CAFs reported predominantly
in desmoplastic stroma across different tumors was aSMA
1584 Cell 186, April 13, 2023
Review
Figure 2. Heterogeneity in cancer-associ-
ated fibroblasts occurs at multiple levels
Cancer-associated fibroblasts (CAFs) comprise a
complex and heterogeneous group of cells as a
consequence of its diverse sources of origin.
These cells respond to intrinsic and extrinsic cues
in the tumor microenvironment (TME) that can be
grouped into defined mechanisms permitting CAF
activation. Multiple cell types within the TME can
answer the cancer hijack call in initiating transition
to CAFs through direct activation of resident
fibroblast and stellate cells. Transdifferentiation of
adipocytes, pericytes and smooth muscle cells;
endothelial to mesenchymal transition (EndMT) of
endothelial cells; epithelial to mesenchymal tran-
sition (EMT) of epithelial cells and recruitment and
activation of mesenchymal stem cells are other
mechanisms that can give rise to CAFs. CAFs
show heterogeneity of molecular expression
based on the differentiating markers of its pro-
genitor state. Markers indicated in purple are
shared by both CAF and the cell type of origin,
whereas those indicated in green are unique to the
cell type of origin. In addition, CAFs also show
heterogeneity in their function and features based
on their anatomical localization within the body.
expressing myofibroblasts.125,126 These
myofibroblasts were initially detected
during wound healing and fibrosis and ul-
timately responsible for the concept that
‘tumors are wounds that do not heal’.41
Since then, myofibroblasts that do not
express aSMA (termed proto-myofibro-
blasts) were also reported with function
in specialized normal connective tissue
and early in wound healing.127,128 Overall,
the existence of both myofibroblast CAFs
and non-myofibroblast CAFs appears to
be the most consistent observation across different cancer
types. However, it is the coexistence of different CAF subtypes
within a tumor evident from opposing capabilities such as tumor
restraining vs. tumor promoting that has made it essential to
characterize the CAFs based on their functional heterogeneity.
This has been made possible in recent years by the utilization
of scRNA-seq, which has enabled the redefinition of classical
markers as CAF subtype-specific.
CAFs, like normal fibroblasts are often characterized by
the presence of markers such as vimentin (VIM), but many
other markers have been identified to more specifically identify
activated CAFs. These include aSMA, FAP, FSP1, periostin
(POSTN), PDGFRa/b, podoplanin (PDPN), and integrin-b1
(CD29). However, it should be noted that these markers identify
different cell populations with distinct expression profiles, and
some overlapping, indicating a high level of heterogeneity
among CAFs in the TME.1,3,56,79,108 In addition, the anatomical
site of origin—i.e., cancer type—can have a profound impact
on the CAF functions and ultimately the CAF subtypes since resi-
dent fibroblasts, the main precursors of CAFs, exhibit organ-
specific transcriptomics profile.77 Since numerous local TMEs
can exist in the same tumor, activated fibroblasts can interact
with tumor cells and other stromal cells at several interfaces,

Review
which can regulate their plasticity in a spatial and temporal
manner.
Two distinct subpopulations of CAFs with unique transcrip-
tomic signature, referred to as myofibroblastic CAFs (myCAFs)
and inflammatory CAFs (iCAFs), have been identified in pancre-
atic cancer. The myCAFs are primarily located close to the tumor
mass and have a matrix-producing myofibroblastic phenotype
with high expression of aSMA and low expression of IL6,
whereas iCAFs are located more distally to the tumor edges
and distributed throughout and defined by expression of immu-
nomodulatory molecules such as IL6 and CXCL12.79 These two
subsets were confirmed in a subsequent mouse pancreatic can-
cer study100 and shown to dynamically change from one subset
to the another through interplay of TGFb and IL1 induced
signaling, supporting the existence of different CAFs subtypes
within a tumor at the same time.91 The iCAFs were reported
to share similar transcriptional profiles and signaling pathway
activation with senescent fibroblasts and secretory pheno-
type.129–131 Single-cell transcriptomic analysis has also shown
that myCAFs and iCAFs are driven by different signaling path-
ways and reported in other types of cancer originating from
breast, lung, colon or rectum and ovarian tissues.132–136 Subse-
quently, another study further identified a novel population of
CAFs in late-stage pancreatic cancer with MHC-class II
(MHCII)-related genes and CD74 expression termed antigen-
presenting CAFs (apCAFs). The apCAFs lack the classical co-
stimulatory molecules (e.g., CD40, CD80, and CD86) that are
required to induce activation and clonal expansion of CD4+
T cells after T cell receptor (TCR) ligation, confirming a putative
immune-modulatory function of these CAFs.137 A lack of co-
stimulatory molecules on antigen-presenting cells has been re-
ported to cause T cell anergy and induction of regulatory
T cells (Tregs).138,139 Consistent with these reports, apCAFs acti-
vated CD4+ T cells and promoted their differentiation into Tregs,
directly contributing to tumor immunosuppression.1,137 As
opposed to pancreatic stellate cells being the cell of origin for
myCAFs and iCAFs, apCAFs were shown to be a derivate of
mesothelial cells.133,140 Similarly, existence of apCAFs have
also been reported in breast cancer in addition to the iCAFs
and myCAFs subsets.141 Further analysis of pancreatic cancer
on the basis of stroma density resulted in the discovery of a novel
CAF subtype within the low desmoplastic tumor characterized
by a highly activated glycolytic metabolic state (termed meCAFs)
and associated with increased metastasis but better response to
immunotherapy.142
Single-cell transcriptomics on lung cancer revealed the pres-
ence of 5 distinct clusters where cluster 1 was enriched within tu-
mors and associated with EMT and TGFb signature and had des-
moplastic function similar to myCAFs, whereas cluster 2 was
localized as co-clusters with pericytes and expressed higher
levels of aSMA with a more angiogenic function143 similar to
vCAFs. However, contrary to the immunosuppression function
reported earlier, apCAFs in lung cancer are unique in their ability
to activate effector T cells and are tumor-suppressive in their
function.144
Other scRNA-seq studies in melanoma have uncovered the
existence of three main CAF subsets; CAF-S1, which is similar
to iCAFs and likely engages in immune crosstalk and upregulat-
ll
ing genes involved the recruitment and regulation of immune
cells; CAF-S2, which is similar to myCAFs and shows upregu-
lated genes encoding ECM components that creates a desmo-
plastic matrix and a contractile stromal subset and CAF-S3,
which may represent both pericytes associated with the vascu-
lature (vCAFs) or fibroblasts populations but their precise cellular
identity remains elusive.145 Research on colorectal cancer has
shown that CAFs can be divided into two main subtypes known
as CAF-As and CAF-Bs based on TGFb induced gene expres-
sion. These subtypes can be identified by specific markers,
with CAF-As expressing high levels of MMP2, decorin,
collagen-1a2, and FAP, whereas CAF-Bs express markers
commonly found in myofibroblasts such as aSMA, transgelin,
and PDGFa.83 These observations suggest that CAFs are a
highly diverse group of cells, with varying gene signatures and
protein expressions depending on the type of cancer and even
among patients with the same type of cancer. This variability in
CAF signatures may be useful in developing prognostic factors
and new cancer diagnosis and treatment strategies.
In breast cancer, scRNA-seq identified different CAF subtypes
that were designated based on their gene expression as develop-
ment-associated CAFs (dCAFs) with a signature associated with
differentiation, development and morphogenesis of tissues; ma-
trix-associated CAFs (mCAFs) with a signature associated with
matrisome (ECM) gene set; vascular-associated CAFs (vCAFs)
with a signature associated with angiogenesis and vascular devel-
opment and cycling CAFs (cCAFs) with a signature associated with
proliferation.81 Histological characterization of mammary tumors
revealed distinct cellular sources of CAFs, as vCAFs, mCAFs,
and dCAFs originated from a perivascular location, resident fibro-
blasts, and malignant cells having undergone an EMT, respec-
tively, whereas cCAFs were essentially designated as the prolifer-
ating versions of vCAFs.81 Other studies in breast cancer have
shown the presence of other distinct CAF subpopulations accu-
mulating differentially with regards to breast cancer subtypes
and tumor localization. By analyzing multiple markers, including
FAP, aSMA, and CD29, four different CAF populations (referred
to as CAF-S1 to CAF-S4) were identified.58 These populations
included normal fibroblasts (CAF-S2) devoid of any markers and
similar to those found in healthy tissues; myofibroblasts (CAF-S1
and CAF-S4) based on aSMA expression that are restricted within
the tumor; CAF-S3 was significantly associated with healthy tissue
outside the tumor and CAF-S2 was equally distributed in the two
compartments.58 Genes upregulated in CAF-S1 subset were
involved in cell adhesion, ECM organization, and immune suppres-
sion, whereas CAF-S4 fibroblasts were characterized by muscle
contraction, regulation of actin cytoskeleton and oxidative meta-
bolism58 suggesting that CAF-S1 supports both myCAFs- and
iCAFs-like functions, whereas CAF-S4 is restricted to myCAFs-
like functions. Similar CAF subsets were also reported in ovarian
cancer study where both CAF-S1 and CAF-S4 were myofibroblas-
tic owing to aSMA expression but only CAF-S1 correlated with
iCAFs with regards to immunosuppressive function.146
The increasing complexity in CAF subtypes has been further
brought to light within the same CAF subtype in breast cancer.135
The fibroblast-activated CAFs (CAF-S1) in breast cancer can be
divided into 8 different clusters, with three of them belonging to
the iCAFs subgroup and five of them belonging to the myCAF
Cell 186, April 13, 2023 1585
 ll
subgroup. Using specific pathways and gene signatures for each
cluster, the three subgroups of iCAFs have been named IL-iCAFs
(relating to interleukin signaling), IFNg-iCAF (relating to the Inter-
feron g-related pathway), and detox-iCAFs (relating to detoxifi-
cation pathways). The five subgroups of myCAFs have been
named ecm-myCAFs (relating to ECM proteins), TGFb-myCAFs
(relating to the TGFb-dependent pathway), wound-myCAFs
(relating to wound healing signaling), IFNa/b-myCAFs (relating
to the IFNa/b-related pathway), and acto-myCAFs (relating to
acto-myosin signaling).135 The CAF subtypes across distinct
cancer types are listed in Table 1 and four main CAF subtypes
are described in Figure 3.
Although CAFs remain heterogeneous, few studies aimed at
determining therapy resistance CAF signatures have identified
molecular characteristics of CAF subtypes that are shared across
several cancer types. This has also shed light on how the tissue/
cell of tumor origin affects the functions of CAFs within distinct
TMEs. In that regard, scRNA-seq across ten solid cancers has
revealed six distinct clusters associated with CAFs signatures; my-
ofibroblasts CAFs (CAFmyo), inflammatory CAFs (CAFinfla), and adi-
pogenic CAFs (CAFadi) were the most predominant subtypes along
with three minor components identified based on their origin as
endothelial-to-mesenchymal transition CAF (CAFEndMT), periph-
eral nerve-like CAF (CAFPN), and antigen-presenting CAF
(CAFap).122 In another study, six CAF subtypes were defined,
shared across melanoma, lung and head and neck cancers, with
characteristics associated with immunosuppressive and stem-
ness attributes.173 Similarly, a multiomics approach comparing
pancreatic, skin and breast cancers has identified three conserved
CAF superclusters across species termed steady state-like (PI16 +
DPP4+), mechanoresponsive (POSTN+MGP+), and immunomod-
ulatory CAFs (IL1R1+CXCL12+).174 Interestingly, although steady
state-like and immunomodulatory CAFs originate from normal
mammary fibroblasts, mechanoresponsive CAFs arise during the
malignancy stage and selective disruption of underlying mechan-
ical force or immune checkpoint inhibition therapy resulted in dy-
namic changes in CAF distribution that affected tumor growth.174
A similar observation was also reported where CAF subtypes were
associated with different clinical outcomes allowing identification
of molecular pathways involved in tumor progression and therapy
resistance to immune checkpoint blockade.173 This molecular
level understanding of the pan-cancer attributes as well as hetero-
geneity and plasticity within CAF subpopulations is still devel-
oping, but recent progress being made in defining and character-
izing CAFs will revolutionize the way for CAF-targeting therapeutic
options in combination with immunotherapies.
FIBROBLASTS IN CANCER INITIATION AND
PROGRESSION
Cancer initiation
The effect of the stromal fibroblasts in supporting and promoting
cancer development is well documented and although CAFs are
an outcome of the hijacking signals from tumor cells, it has been
established that fibroblast acquisition of a pro-tumorigenic state
can occur well before a tumor can arise. This is best evident in
the case of mammary fibroblasts in high mammographic breast
density environment,175 during weaning-induced involution of
1586 Cell 186, April 13, 2023
Review
mammary glands,176 and following exposure to low dose radia-
tion.177 As discussed later, intrinsically aged fibroblasts can also
behave like CAFs and can contribute to tumorigenesis. In addi-
tion, a landmark study showed that CAFs alone were sufficient
in driving tumor progression of initiated non-tumorigenic pros-
tate epithelial cells both in vitro and in vivo.178 This study sup-
ports that CAFs and therefore TME could essentially provide
the equivalent of a subsequent mutational hit that is prerequisite
for tumor promotion.179 Further evidence also comes from
occurrence of an altered ECM signature with distinct structural,
mechanical and compositional characteristics prior to any gross
signs of inflammation.180 Presence of pre-symptomatic patho-
logical state can make the surrounding tissue more vulnerable
to cancer and other disease states (Figure 4).
Considering that tumor initiation, which is the outcome of mul-
tiple genetic and epigenetic mutations, occurs directly in the
epithelial cells, it is likely that fibroblasts get activated through
stromagenesis as described earlier. However, it is important to
note that the fibroblast activation within a TME does not imply
that these activated CAFs become cancer promoters from the
onset. The varying degree of CAF activation is evident from the
occurrence of quiescent CAFs, tumor-restraining CAFs, and tu-
mor-promoting CAFs.151 In the early stages of cancer develop-
ment, the tumor-restraining CAFs act as sentinels in close prox-
imity to transformed cells.86,156 This is also bolstered by the fact
that blockade of sonic hedgehog (Shh) signaling or selective
deletion of aSMA expressing CAFs resulted in tumor progression
in PDAC mouse models.168,169,171 Occurrence of tumor-restrain-
ing CAFs have also been reported in breast, bladder, colon, and
intestinal cancers167,170,172,181 suggesting that these CAFs may
be universally present to inhibit tumor initiation.
Cancer progression
CAFs play a complex and multifaceted role during the entire
course of tumor development, from the pre-neoplastic state until
the terminal stage of metastasis (Figure 4). Studies have shown
that tumor cells can educate and recruit subsets of CAFs
residing in close proximity to generate a pro-metastatic
niche.79,182 This is in addition to the effect of tumor genotype
in regulating the functional heterogeneity of both normal fibro-
blasts and CAFs.71,183–185 Other examples highlighting the effect
of genetically altered fibroblasts in tumor promotion come from
mammary epithelial cells that undergo transformation when
co-cultured with fibroblasts overexpressing Wingless-type
MMTV integration site family member 1 (Wnt1).186 Similarly,
inactivation of Phosphatase and tensin homolog (PTEN) in mam-
mary fibroblasts significantly enhanced the malignant transfor-
mation and growth of mammary adenocarcinoma in mice.187
These Pten-inactivated murine fibroblasts also showed a strong
correlation with CAFs in breast cancer patients. Moreover, TGFb
receptor II gene knock-out in FSP1-positive cells promotes pros-
tate intraepithelial neoplasia and fore-stomach squamous cell
carcinoma.188 Further support comes from in vivo experiments
where highly metastatic mouse mammary cancer cells when in-
jected into Fsp1-deficient mouse model exhibit considerably de-
layed and decreased tumor initiation, whereas the co-injection of
FSP1-positive fibroblasts restored tumor formation and pro-
moted metastasis.189 Earlier studies have also shed light on
 Review
Table 1. Functional heterogeneity of CAFs in distinct tumor types
Characteristic markers
Cancer type CAF subtypes (Gene expression) Functions Reference
Pancreatic Cancer myCAFs aSMA, VIM, CTGF, COL1A1, Myofibroblast-like, matrix deposition Öhlunde et al.79
(Pancreatic Ductal COL5A1, COL6A1
Adenocarcinoma) iCAFs IL6, IL1, IL11, LIF Inflammatory infiltration, chemokines and
cytokines secretion, tumor-promoting
CAF-A POSTN Tumor proliferation, metastasis Neuzillet et al.147
CAF-B MYH11 Lymph node metastasis
CAF-C PDPN Immune promotion
myCAFs FAP Reduction of T cell infiltration Fearon, D.T.148
Desmoplastic stroma
myCAFs FAP Tumor aggressiveness Kawase et al. and
Looi et al.149,150
myCAFs aSMA Prevent tumor dissemination Miyai et al.151
CAF-FB1 Cxcl14, Ptn, Igf1, Igfbp4/7, Similar to iCAFs Hosein et al.100
Il6, Ccl2/7, Cxcl12, Pdgfra
CAF-FB2 Ly6a, Ly6c1, Nov, Pi16 Physiological functions of fibroblasts
CAF-FB3 Lrrn4, Gas6, Cav1, Cdh11, Similar to myCAFs
Gpm6a, Msln, Lgals7, Nkain4
iCAFs IL6 Reduction of oxidative stress Elyada et al.137
Secretory-CAFs CXCR2 Pro-tumorigenic secretion Awaji et al.152
iCAFs IL6 Inflammation Biffi et al. and Helms
et al.91,153
apCAFs PDGFRa, SAA3 Tumor growth Djurec et al.6
apCAFs HLA-DRA (MHC II genes), Deactivating CD4 T cell Elyada et al.137
CD74, SAA3, SLPI
meCAFs Increased glycolysis, Higher risk of metastasis and poor prognosis Wang et al.142
PLA2G2A, CRABP2 but better response to immunotherapy
Prrx1-CAFs PRRX1 CAF plasticity Feldmann et al.154
LRRC15-CAFs LRRC15 Poor response to immunotherapy Dominguez et al.133
NetG1-CAFs NETG1 Nutritional support (glutamate/glutamine Francescone et al.155
metabolism), immunosuppression
Cell 186, April 13, 2023 1587

Meflin1-CAFs ISLR Tumor suppression Mizutani et al.156

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ll
1588 Cell 186, April 13, 2023

Table 1. Continued
Characteristic markers
Cancer type CAF subtypes (Gene expression) Functions Reference

ll
Breast Cancer CAF-S1 CD29, FAP, aSMA, PDGFRb, F Tumor proliferation, EMT promotion, migration, Costa et al. and
SP1 and CXCL12 Lymph node metastasis, immune suppression Pelon et al.58,157
CAF-S2 Not reported Not reported
CAF-S3 CD29, FSP1, PDGFRb Unknown (resemble normal fibroblast)
CAF-S4 CD29, FSP1, PDGFRb and aSMA Tumor invasion, Lymph node metastasis
myCAFs aSMA, TAGLN, MYL9, IGFBP3, TNC Tumor proliferation, Migration, Invasion, Sebastian et al.141
Angiogenesis and EMT
iCAFs Ly6c1, CLEC3B, HAS1, DPT, Tumor proliferation, Metastasis, Angiogenesis,
COL14A1 Immune evasion and Chemoresistance
apCAFs CD74, MHC-II genes, KRT18, FSP1 Antigen-present, Immune modulation
CAFs CD10, GPR77 Promote cancer stemness and Su et al.158
chemoresistance
dCAFs SCRG1, SOX9, SOX10 Not reported Bartoschek et al.81
mCAFs FBLN1, PDGFRa, CXCL14 Immune regulation
vCAFs NID2, NOTCH3, EPAS1, COL18A1, Angiogenesis
NR2F2
CAF-S1 FAPhi CD29med-hi SMAhi ECM and Inflammation signature Kieffer et al.135
MCAMlo Restricted to tumor and Lymph nodes
CAF-S2 FAPneg CD29lo SMAneg Normal fibroblast signature
Tumor and normal tissue
CAF-S3 FAPneg CD29med SMAneg Normal fibroblast signature
Tumor and normal tissue
CAF-S4 FAPneg SMAhi CD29hi Perivascular signature
MCAMhi Restricted to tumor and Lymph nodes
detox-iCAF FAPhi CD29med, ANTXR1( ), Detoxification and inflammatory response
GPC3(+), ADH1B, GPX3
IL-iCAF FAPhi CD29med, ANTXR1( ) Growth factor, TNF signaling, and IL pathway
DLK1(+), RGMA, SCARA5
IFNy-iCAF FAPhi CD29med, ANTXR1( ), Response to IFNg and cytokine-mediated
CCL19, CCL5 signaling pathway,
Similar to apCAFs
ecm-myCAF FAPhi CD29med, ANTXR1(+), ECM secretion
acto-myCAF FAPhi CD29med, ANTXR1(+), Acto-myosin contractility
SDC1(+), GGH, PLP2
IFNab-myCAF FAPhi CD29med, ANTXR1(+), IFNa/b signaling
IFIT3, IRF7

TGFb-myCAF FAPhi CD29med, ANTXR1(+),
LAMP5(+), CST1, TGFB1
Wound-myCAF FAPhi CD29med, ANTXR1(+),
CD9(+), SEMA3C, SFRP4
Review
TGFb signaling pathway and matrisome,
immunoevasion
Assembly of collagen fibrils and wound healing,
anti-PD1 response
(Continued on next page)
 Review
Table 1. Continued
Characteristic markers
Cancer type CAF subtypes (Gene expression) Functions Reference
Colorectal Cancer CAF-A MMP2, DCN, FAP, PDPN ECM remodeling Li et al.83
and COL1A2
CAF-B aSMA, TAGLN, PDGFa, LUM Not reported
Oral Cancer (Oral Squamous CAF-D TGFb EMT, tumor invasion Costea et al.159
Cell Carcinoma) CAF-N Hyaluronan, MMPs Tumor invasion, immunosuppression
Ovarian cancer (High grade pan-CAFs CD49e Not reported Hussain et al.160
serous ovarian carcinoma) Aggressive-CAFs CD49e high Proliferation, migration,
FAP high Lymph node metastasis and therapy resistance
CAF-S1 CD29, FAP, aSMA, FSP1, Tumor proliferation, Immune suppression Givel et al.146
PDGFRb and CXCL12b
CAF-S2 (non-activated) Not reported Not reported
CAF-S3 (non-activated) CD29, FSP1 and PDGFRb Not reported
CAF-S4 CD29, aSMA, FSP1 and Tumor proliferation
PDGFRb
iCAFs IL6, IL10, C1QA/B/C, CFB, Immunomodulation Izar et al.161
CXCL1/2/10/12
Lung Cancer Cluster1 COL10A1 EMT signature Lambrechts et al.143
Cluster2 aSMA Not reported
Cluster4 PLA2G2A Found in the leading edge of tumor
Cluster5 MMP3 Lower myogenesis
Cluster7 CCL2 High mTOR signature
CAF-1 FAP(+) Higher-stage tumors Grout et al.162
CAF-2 FAP(+), aSMA(+) Higher-stage tumors, ECM and contractility,
Immune cell exclusion
CAF-3 MYH11(+), aSMA(+), FAP( ), Low-stage tumors, ECM and contractility,
ADH1B( ) Immune cell exclusion
CAF-4 ADH1B(+) Low-stage tumors, T cell enrichment
HD-CAFs ST8SIA2, WNT7B, SLITRK6, higher matrix reorganizing ability and Hao et al.163
and IGFL3 promotion of tumor cell invasion and growth
LD-CAFs Negative for HD-CAFs markers Low desmoplastic
Cell 186, April 13, 2023 1589

CAF7 FAP(+), PDGFRa( ) Immune suppressive Pellinen et al.164

CAF13 FAP( ), PDGFRa(+) Good prognosis
CAF-I HGFhi, FGF7hi/lo, pSMAD2lo Support tumor cells therapy resistance Hu et al.165
lo hi lo
CAF-II HGF , FGF7 , pSMAD2 Support tumor cells therapy resistance
CAF-III HGFlo, FGF7lo, pSMAD2hi Better clinical response

ll
CAFs CD10, GPR77 Chemoresistance and poor survival Su et al.158
apCAFs MHCII(+), FAP(+), CD45( ) Tumor-suppressive Kerdidani et al.144
(Continued on next page)
1590 Cell 186, April 13, 2023

ll
Table 1. Continued
Characteristic markers
Cancer type CAF subtypes (Gene expression) Functions Reference
Head and Neck Squamous CAF-1 FAP, PDPN, COL1A1, VIM, Lymph node metastasis, promote EMT Puram et al.61
Cell Carcinoma THY1, MMP11, CAV1
CAF-2 FAP, PDPN, JUN, FOS, Not reported
FGF7, TGFbR
CAF3 Not reported Normal fibroblasts
Intrahepatic Cholangiocarcinoma EMT-like CAFs KRT19 Epithelial-like Zhang et al.166
(ICC) myCAFs POSTN, COL5A1 ECM modulation
vCAFs MCAM, IL6 Angiogenesis
apCAFs MHC-II genes, CD74 Antigen presenting, immunomodulation
Oral/pancreatic/Colon/Bladder Restraining-CAFs BMP4, Anti-tumoral effect Gerling et al.; Lee et al.;
Hedgehog signaling Özdemir et al.; Pallangyo
Intestinal cancers IKKb signaling et al.; Rhim et al. and
Shin et al.167–172
VIM vimentin, CTGF connective tissue growth factor, FAPa fibroblast activation protein-a, BMP4 bone morphogenetic protein-4, MCAM melanoma cell adhesion molecule, KRT keratin, POSTN
periostin, COL collagen, MHC major histocompatibility complex, FGF fibroblast growth factor , PDPN podoplanin, MMP matrix metalloprotease, EMT epithelial to mesenchymal transition,
GPR77 complement component 5a receptor 2, HGF hepatocyte growth factor, aSMA a smooth muscle actin, Prrx paired related homeobox, TAGLN transgelin, IL interleukin, TGF transforming
growth factor, PDGF platelet-derived growth factor, PDGFR PDGF receptor, THY1 CD90 cell surface antigen, FSP-1 fibroblast specific protein-1, CXCL chemokine (C-X-C motif) ligand, ADH
alcohol dehydrogenase, CFB complement factor B, MYH11 myosin heavy chain-11, ST8SIA2 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2, WNT7B Wnt family member 7B,
SLITRK6 SLIT and NTRK like family member-6, IGF insulin-like growth factor (IGF), IGFL3 like family member-3, CAV1 caveolin-1, LUM lumican, SEMA3C semaphorin 3C, SFRP4 secreted friz-
zled related protein-4, C1Q complement component 1 Q subcomponent, DCN decorin, LAMP5 lysosomal-associated membrane protein family, member 5, ANTXR1 ANTXR cell adhesion
molecule-1, MYL9 myosin light chain-9, IGFBP IGF binding protein, TNC tenascin, GGH gamma-glutamyl hydrolase, PLP2 proteolipid protein-2, IFIT3 interferon induced protein with tetratri-
copeptide repeats-3, IRF7 interferon regulatory factor-7, CST1 cystatin SN, SDC1 syndecan-1, GPX3 glutathione peroxidase 3, HAS1 hyaluronan synthase-1, DPT dermatopontin, SCRG1 stim-
ulator of chondrogenesis-1, SOX SRY (sex determining region Y)-box, FBLN1 fibulin-1, NR2F2 nuclear receptor subfamily 2, group F, member 2, NID2 nidogen-2, EPAS1 endothelial PAS domain
protein-1, PI16 peptidase inhibitor-16, SLPI Calcineurin-like metallo-phosphoesterase superfamily protein, SAA3 serum amyloid A3, LIF leukemia inhibitory factor, Ptn pleiotrophin, Lrr4 leucine
rich repeat neuronal-4, Gas6 growth arrest specific-6, Cdh11 cadherin-11, Gpm6a glycoprotein M6A, Msln mesothelin, Lgals7 galectin-7, Nkain4 Na+/K+ transporting ATPase interacting-4,
NETG1 netrinG1.

Review

Review
the signaling cascades that operate as a consequence to the
body’s response to cancer, giving way to a tumor-supportive
local microenvironment at distal and metastatic sites.190,191
Further support comes from the role of senescent fibroblasts in
abetting cancer progression from premalignant cells as reported
in skin cancer model where senescent fibroblast-mediated os-
teopontin (OPN) secretion promoted tumorigenesis.192
Cancer growth and stemness
In addition to modulating ECM components, CAFs promote a
conducive environment for tumor proliferation as a result of para-
crine interactions between tumor cells and CAFs. CAFs secrete a
range of growth factors and cytokines that influence the stem-
ness behavior of the epithelium, including hepatocyte growth
factor (HGF), EGF, insulin-like growth factor (IGFs), IGF-binding
protein (IGFBPs), FGF2, OPN, and TGFb. CAFs, either directly
or through EV secretion, additionally provide lipids and metabo-
lites that are taken up by cancer cells for effective prolifera-
tion3,53 in vitro and tumor growth in vivo via activation of their
respective signaling pathways, i.e., integrin/FAK-Src (POSTN),
WNT/b-catenin (HGF and OPN), PI3K/mTOR (CXCL12, HGF,
ll
Figure 3. Functional heterogeneity in can-
cer-associated fibroblasts
Cancer-associated fibroblasts (CAFs) represent
a heterogeneous cellular population within
the tumor microenvironment. Single-cell RNA
sequencing and multiplex imaging have revealed
different CAF subtypes across multiple cancer
types. These CAFs are divided into 4 main groups:
iCAFs (inflammatory CAFs), myCAFs (myofibro-
blastic CAFs), apCAFs (antigen presenting CAFs)
and vCAFs (vascular CAFs). The cellular function,
plasticity and the heterogeneity in the molecular
marker expression associated with these CAFs
are indicated.
and IL22), MAPK (IL6, TGFb, and FGF),
or Hippo (EVs).1,193 These factors also
increase the survival of tumor cells via
upregulation of anti-apoptotic proteins
like B-cell lymphoma-2 (BCL2) and
myeloid cell leukemia-1 (MCL1) or down-
regulation of pro-apoptotic proteins like
BCL2 Associated X protein (BAX).194 In
other situations, fibroblasts succumb to
epigenetic changes (aging, senescence-
induced) and external stressors that ulti-
mately transform them to affect tumor
cell behavior. For example, generation
of reactive oxygen species (ROS) in
an inflammatory TME promotes fibro-
blast activation toward tumor-promoting
functions.3,53
Cancer metastasis
Cancer is characterized by uncontrolled
cellular growth giving rise to a tumor at
the site of origin. The tumor is considered
benign if it lacks the ability to invade other
neighboring. However, when the tumor resists the constraints of
space it starts to spread and infiltrate neighboring normal tissues
becoming malignant.195 This spread to distant sites via angio-
genic, or lymphatic system is called metastasis and is the prom-
inent cause of death from cancer. This multistep process requires
the migration of tumor cells from the primary site, their seeding in
a distal organ, and ultimately outgrowth of those seeded cells. To
do that the tumor cells, must first move through tissues via the
meshwork of matrix.10,196 Moreover, migrating tumor cells have
to maintain survival while penetrating layers of tightly connected
epithelial and endothelial cells and breach several structural and
blood flow barriers.197 During the dissemination across lymphatic
and vascular boundaries, tumor cells make and break contacts
with different proteins and navigate their way before finally
settling at a new site where they either undergo dormancy or
continue to grow and proliferate with decisions dependent on
the age, metabolic health, environmental stressors, and co-mor-
bidities of the host.53,198,199 This progression from an isolated tu-
mor to disseminated metastatic disease is a multistep process,
with each step involving intricate cross talk between the cancer
cells and the surrounding tumor microenvironment (TME) and
Cell 186, April 13, 2023 1591
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Review

Figure 4. Role of fibroblasts in carcinogenesis

The phenotype and functions of fibroblasts are in dynamic evolution along with tumor initiation and progression. During early stages of carcinogenesis, pro-
tumorigenic fibroblasts can drive tumor initiation and promote proliferation of mutated cancer cells. These activated fibroblasts in close proximity to the tumor
cells can then transition to cancer-associated fibroblasts (CAFs). During this initial phase of cancer development, the heterogeneous cancer-associated
fibroblast (CAF) subpopulations, referred to as tumor-restraining CAFs, can potentially protect normal tissue against cancer invasiveness through the various
indicated mechanisms. However, in advanced-stage disease, tumor cells and other stromal cells reprogram the CAFs via continuous crosstalk to promote a
tumor permissive niche. This allows tumor cells to proliferate and grow through dynamic processes that includes ECM remodeling, growth factors, cytokines and
extracellular vesicles secretion, metabolic reprogramming and creating an immunosuppressive tumor microenvironment (TME). The mechanical coupling be-
tween CAFs and cancer cells, permits local invasion and regional dissemination of tumor cells from the primary tumor site. As the tumor evolves, CAFs reorganize
the TME by promoting angiogenesis and epithelial-to-mesenchymal transition of tumor cells enabling distal metastasis (shown here with lung as an example)
following intra- and extravasation. These disseminated tumor cells also overcome immune surveillance by resident immune cells, and direct immune sup-
pression. At the metastatic site, the disseminated tumor cells, in the face of contextual signals, either enter or exit dormancy. This is governed by a plethora of
microenvironmental cues, encompassing the cellular, molecular, and physical factors converge to induce either stress-related or mitogenic signals to tumor cells.
Upon reawakening, proliferating cancer cells then colonize the metastatic niche forming macrometastases, co-opting and recruiting local stromal cells to further
support cell growth by indicated mechanisms. Subsequently, fibroblasts and CAFs continue to play important roles across the continuum of tumor progression
and metastasis to visceral sites.

acellular extracellular matrix (ECM).200–202 Numerous studies
have now shown that TME regulates this tumor behavior from
initiation to progression by influencing cancer cell invasion in a
qualitative and quantitative manner.
In addition to the local tumor growth, CAFs have been shown
to play an integral role in cancer invasion across the epithelial
and endothelial basement membranes, which typically involves
coordinated adhesive and proteolytic activities.3 CAF-mediated
stromagenesis drives increased cancer invasiveness at the pri-
mary tumor and facilitates metastasis to distant organs. Further,
recent studies have shown that CAFs like cancer cells can also
disseminate via circulation95 to distant metastatic sites as indi-
vidual CAFs or in clusters with cancer cells, suggesting that
they have even more complex roles in metastasis.203 Crosstalk
between CAFs and cancer cells causes changes in the ECM
components and structural scaffolding within the TME as well
as induces EMT in cancer cells imparting cell motility and
increasing invasive capacity.204 Beyond tumor invasion, CAFs

1592 Cell 186, April 13, 2023


Review
can also play a vital role in the colonization of distant organs in
metastatic disease, by creating a niche for tumor cells. CAFs
have been shown to provide stromal support for disseminated
cancer cells through a combination of environmental factors,
including POSTN and tenascin-C (TNC) proteins.205 In addition,
TGFb is an established inducer of EMT through paracrine
signaling, allowing premalignant cells to acquire mesenchymal
properties for invasion and metastasis.197 Further, CAF expres-
sion of caveolin-1 was shown to promote matrix remodeling,
invasiveness, and increased metastases in mice injected with
breast adenocarcinoma cells.206 Further, anisotropic fibronectin
organization orchestrated by CAFs also provides directions for
cell migration.207 Additionally, in some cases CAFs have been
shown to migrate with metastasizing cells and remodel the
ECM, as well as the metastatic site’s milieu, to sustain growth
and survival of the tumor cells54 (Figure 4).
CAF regulation of ECM
In the early stages of tumor growth, the host tissue architecture
gets distorted from the aberrant accumulation of ECM compo-
nents. CAFs are the most prominent ECM-producing cells in
TME that together with immune cells contribute to desmoplasia
and matrix-remodeling via MMPs. In PDAC for instance, the
accumulation of thick desmoplastic stroma that also contains
high amounts of hyaluronan, promotes tumor growth in mice
and correlates significantly with poor prognosis in patients.28,208
In addition to altered biochemical signals provided by the des-
moplastic TME, the production and re-assembly of ECM,
including linear collagen structures, alters the physical proper-
ties and biomechanical activity of the microenvironment.209
Desmoplasia induces tumor stiffness via processes closely
attributed to the over-activation of lysyl oxidase (LOX), an
enzyme that crosslinks collagen and other ECM compo-
nents.210,211 The increased tumor stiffness promotes integrin-
based focal adhesion assembly and increases the formation of
adhesion complexes in both cancer and stromal cells, thus
creating an increasingly pro-tumorigenic microenvironment.
Increased integrin activity in CAFs transduces mechanical forces
that further change the orientation of collagen and fibronectin
fibers, promoting cancer growth and invasion.200,207,212 In gen-
eral, the generation of physical forces and stiffness-dependent
cytoskeletal rearrangements are tightly linked to the dysregula-
tion of YAP1 transcriptional co-activator in fibroblasts and can-
cer cells, which leads to transcriptional programs to further
potentiate CAF activation and cancer cell growth.92 Excessive
stroma acts as a barrier to drug delivery and results in excessive
interstitial fluid pressure caused by compression of the blood
and lymphatic vessels.213 Further, ECM-altering properties of
CAFs allow prolonged secretion of inflammatory cytokines
such as IL8, IL6 and leukemia inhibitory factor (LIF) by tumor
cells, which in turn sustains the pulsatile TGFb and STAT3
signaling as reported in the pancreatic cancer TME.214–217 These
events further accelerate proliferation and phenotypic switch of
CAFs into a tumor-promoting phenotype.
CAF regulation of angiogenesis
CAFs secrete several angiogenic molecules like VEGF, PDGF,
CXCL12, or HGF that can promote proliferation and recruitment
ll
of endothelial cells and pericytes tumor to drive invasion and
metastasis.218–222 Autocrine signaling through PDGF and VEGF
further promotes the production of pro-angiogenic molecules
IL6, and IL8.223–226 Elevated levels of WNT2 selectively in colon
CAFs has been shown to increase angiogenesis by secreting
pro-angiogenic molecules and ECM.227 Additionally, chemokine
production from CAFs has also been shown to induce vasculo-
genic mimicry by actions of TGFb and CXCL12.228 CAFs ex-
pressing connective tissue growth factor (CTGF) were shown to
increase the micro-vessel density and tumor growth activity in
a prostate cancer xenograft model.229 CAFs mediated ECM syn-
thesis and remodeling also generates solid stress resulting in the
vascular and lymphatic vessel compression, reduction in perfu-
sion rates and increased hypoxia. This generation of hypoxic
TME by CAFs gives rise to aggressive clones of tumor cells by
formation of new vasculature and mediating an angiogenic
switch in addition to generating an immunosuppressive environ-
ment.230–232 However, chronic hypoxia has been shown to
dampen the ECM remodeling by head and neck and vulval
CAFs and revert their phenotype.233 In addition to the growth fac-
tors, angiogenesis is also promoted by degradation of ECM com-
ponents and basement membrane driven by CAFs secreted
MMPs.234,235 Finally, CAFs can enhance vascular growth addi-
tionally through biomechanical forces and involving the Rho/
ROCK pathway and YAP signaling.236
CAF regulation of anti-tumor immunity
The role of CAFs in immunomodulation of cancer tissue serves
two primary purposes: CAFs facilitate tumorigenesis by inducing
a chronic inflammatory state for cancer cells, and by establishing
an immunosuppressive TME to maintain survival. This is made
possible by secretion of immunomodulating cytokines such as
TGFb, IL6, CXCL1, and CXCL12.134–137 Additionally, CAFs can
express molecules such as netrinG1,155 and signal to immuno-
suppressive populations, such as neutrophils and myeloid-
derived suppressor cells (MDSCs), which can further promote
conversion into CAFs.237–239 A direct outcome of crosstalk be-
tween tumor cells and CAFs through TGFb signaling is the reduc-
tion in cytotoxic T cells but increase in infiltration of Tregs as well
as the recruitment and enhanced differentiation of macrophages
into tumor-associated macrophages (TAMs) resulting in immuno-
therapy failure.240–242 CAFs work closely with TAMs in providing
metastatic niches for tumor cells, by facilitating the uptake of tu-
mor derived EVs by TAMs and migrating to the metastatic site to
prepare for tumor growth.243–245 Finally, CAFs may directly inter-
fere with anti-tumoral role of natural killer (NK) cells through pros-
taglandin E2 (PGE2) secretion or MMPs-mediated decrease in
NK activating receptor ligands in tumor cells, thus reducing NK
receptor-dependent cytotoxic activity.246,247
CAF regulation of cancer cell metabolism
Metabolic adaptations in tumors require access to amino acids,
nucleotides, lipids to assist with their growth and metastatic
needs. This is also reflected in metabolic heterogeneity within
the tumor owing to dynamic EMT (or phenotype switch) pro-
cesses, as some tumor cells demand higher nutrients and switch
to alternative carbon sources such as glutamine.248 CAFs also
contribute to metabolic heterogeneity based on their spatial
Cell 186, April 13, 2023 1593
 ll
location within the tumor as reported in CAFs from melanoma,
breast, colon, and pancreatic cancers.76,249–251 This also en-
compasses the synergistic crosstalk between CAFs and other
cell types within the TME, to meet constant energy requirements
for tumor growth, invasion, and metastasis, can also be met by
autophagy, lipid secretion and ECM remodeling.
CAF regulation of therapeutic resistance
One of the most intriguing aspects of CAFs is their contribu-
tion to therapeutic resistance in several different cancers.
Several studies have indicated that CAFs promote chemoresist-
ance via different mechanisms such as secretion of cytokine
and miRNAs, increase in cancer stem cells (CSCs), and
decreased drug bioavailability via production of a stromal
barrier.71,110,111,216,252–256 In breast, colorectal and pancreatic tu-
mors, CAFs promote the resistance to chemotherapeutic agents
like gemcitabine and doxorubicin and to combination chemo-
therapy like doxorubicin, 5-fluorouracil and cisplatin through the
production of IL6, IL17A, PDGF, and insulin like growth factor
(IGF) that activate the NF-kB and ERK1/2 pathways in tumor
cells.194,257–260 CAFs are also a source of extracellular vesicles
(EVs) that contain regulatory microRNAs, which can in turn pro-
mote resistance to cisplatin and paclitaxel in cancer cells by
downregulating p21 expression and ferroptosis.261,262
FIBROBLASTS IN CANCER DORMANCY AND
REAWAKENING
Subsequent to metastatic dissemination, cancer cells can
remain latent and stay undetected for years before emerging
as a proliferative and aggressive disease.263 There is now
emerging evidence that connects fibroblasts to tumor dormancy
but precedent from pancreatic cancer studies supports that
depletion of CAFs results in a more aggressive pheno-
type.168,169,171 It is therefore reasonable to assume that there
also exists a subpopulation of CAFs associated with the entry
into or awakening from cancer dormancy. CAFs by nature
secrete a milieu of soluble and insoluble factors, the same can
be responsible for initiating dormancy or their reawakening in a
tissue-dependent manner. In that regard, several cytokines
TGFb, IFNb, IL8, have been documented to contribute to main-
tenance of cancer cells’ quiescence at distal metastatic sites.1,11
In addition, owing to their high capacity of ECM synthesis and
remodeling, tumor stiffness can also regulate cancer cell
dormancy by constricting growth of tumor cells especially as
they invade from tissues of variable or contrasting stiffness.264
This has been shown using a 3D fibrin gel system in melanoma
cells265 and an ECM-mimicking semi-solid Matrigel in lung
adenocarcinoma cells266 resulting in an induced dormancy
state. Part of the dormancy program is the gain of drug-resis-
tance mechanisms as they await further signals from the sur-
rounding microenvironment before switching to a proliferative
state and spreading to distal sites. In addition to ECM remodel-
ing, its composition can also determine the fate of the dormant
cancer cells as both type I collagen and fibronectin have been
shown to promote proliferation via b1-integrin induced PI3K/
AKT and FAK activation.267,268 Similarly, TNC, which is increas-
ingly in the metastatic niche, could inhibit pro-dormancy cellular
1594 Cell 186, April 13, 2023
Review
reprogramming by sequestering TGFb in an inactive state.269,270
Further evidence of tumor reawakening comes from normal aged
lung fibroblasts271 and activated fibroblast -mediated produc-
tion of POSTN at the micrometastatic site, which promotes not
just colonization but also maintenance of stem-cell like proper-
ties in the cancer cells, necessary for tumor initiation and
growth.11 However, considering only a few invasive cancer cells
reach distal sites, a question remains regarding the phenotypic
and secretory behavior of these CAFs at distal sites: are they
programmed by cancer cells or an outcome of epigenetic
changes in the surrounding fibroblasts at the metastatic niche.
Interestingly, an earlier study has suggested the possibility that
tumor cells can carry stromal cells during metastasis.203
AGING AND FIBROBLASTS
Genomic instability is a shared hallmark of aging and cancer272
and age-mediated accumulation of alterations in normal epithe-
lium can initiate carcinogenesis and induce malignancy.273,274
Whereas several somatic mutations are detected in cancer cells,
no somatic genetic alterations have been reported in fibro-
blasts,99–102 suggesting a strong contribution from epigenetic
changes. More importantly, recognizing the role of intrinsic/
healthy aging in fibroblast biology is especially critical now
more than ever, considering aging itself has important implica-
tions across every stage of tumorigenesis; from initiation to
metastasis, anti-tumor immunity, chemoresistance and cancer
dormancy to reawakening.1,53 It is therefore not surprising that
aging in addition to the individual’s physiological state can expe-
dite the transition of normal fibroblasts to an activated pro-
tumorigenic state and eventually to CAFs. In that regard, aging
is enforced by the accumulation of senescent fibroblasts at the
tissue level, which ultimately results in the functional impairment
and eventually predisposition to emergence of malignancies
among other diseases. This is evident across most cancer types
where intrinsic aging in conjunction with the individual’s genetic
background and extrinsic stressors, like UV irradiation, smoking
history, and socioeconomic background increases susceptibility
to cancer21,275 (Figure 5).
Increasing age is inversely proportional to fibroblast abun-
dance as well as ECM integrity.276–278 Analyses of dermal fibro-
blasts from young and old mice has revealed age-mediated
variability in wound healing, also evident in elderly patients.279
In addition, numerous studies have highlighted that age is asso-
ciated with characteristic cellular attributes associated with ge-
netic damage, telomere attrition, and inflammation.1,272,280 In
that regard, scRNA-seq analysis of mouse dermis has revealed
that aged fibroblasts exhibit transcriptional noise while partially
losing their identity and gaining adipogenic traits.281 This was
also supported in human dermal fibroblasts where aged fibro-
blasts showed partial loss in their cell identity while exhibiting
differential secretory, mesenchymal, and pro-inflammatory func-
tional annotations as compared to young fibroblasts.282 Impor-
tantly, secreted factors and ECM modulation mediated by these
intrinsically aged dermal fibroblasts supported melanoma
progression.255,276,283
Previous evidence has indicated that senescent non-malig-
nant cells exhibit SASP acquisition following a wide range of
 ll
Review

Figure 5. Aging, cancer and fibroblasts

Fibroblasts have been shown to play tumor-promoting roles in cancer during aging. This can occur when fibroblasts become senescent, or prematurely aged, due
to replicative stress or oxidative stress and DNA damage, or in response to therapeutic interventions or when fibroblasts age normally (epigenetic changes).
Secreted changes that occur during senescence such as the acquisition of senescence-associated secretory phenotype (SASP) as well as the aged fibroblasts
have been shown to promote tumor progression and therapy resistance by modulating the immune microenvironment, driving multiple changes including pro-
angiogenesis, ECM remodeling, metabolic changes that also impact the outgrowth of tumor cells at metastatic sites. However, an important distinguishing factor
between senescent fibroblasts and aged CAFs is that, whereas senescence promotes growth arrest, CAFs continue to proliferate. It remains unknown if aged
CAFs resemble quiescent senescent CAFs or can proliferate via crosstalk with the surrounding tumor cells.

cellular stressors, including persistent DNA damage, epigenomic
perturbations, therapy-induced toxicity, and oxidative stress.284
Although senescence is a context-dependent phenomenon, it is
characterized by cell-cycle arrest, a flattened morphology
in vitro, heterochromatin changes, and expression of senes-
cence-associated b-galactosidase (SA b-gal).284–286 An impor-
tant aspect of senescence is the acquisition of senescence-asso-
ciated secretory phenotype (SASP) resulting in production of a
range of secreted factors. In both young and healthy tissues,
SASP plays a critical role in directing and recruiting immune cells
for clearance of senescent fibroblast cells and terminating inflam-
mation.53,272 However, with age the senescent fibroblasts ac-
quire SASP, which disrupts this balance making them pro-tumor-
igenic.287,288 It is also important to note that SASP factors are not
unique to senescent cells but also expressed in non-senescent,
proliferating cells such as CAFs.284 This would suggest that aging
induced senescence and SASP may serve as a precursor in at-
taining the CAF phenotype (aged CAFs) earlier in aged vs. young
TME and that these aged CAFs would also lack any tumor-re-
straining function since they are primed to be pro-tumorigenic.
Hence, these aged-CAFs can expedite tumor progression mainly
by the secretion of SASP factors, which can cause chronic
inflammation, promote angiogenesis, and enhance immunosup-
pressive activity,289,290 features of an aged TME53 (Figure 5).
However, contrary to their dividing counterparts in a young
TME, it is unknown if aged CAFs retain sufficient plasticity to

Cell 186, April 13, 2023 1595

 ll
regain the ability to proliferate due to crosstalk with tumor cells
and surrounding ECM. But it is highly likely that the aged CAFs
by virtue can act as quiescent CAFs and maintain and propagate
senescence (and SASP) as well awaken dormant tumor cells at
distal sites and impart therapy resistance, an attribute particularly
common in aged patients.271 A recent study has identified two
aging CAF gene signatures in low grade glioma associated with
distinct prognosis and TME characteristics.291 It remains to be
assessed if a shared aged CAFs signature exists, which is appli-
cable to other cancer types. Future research and technological
advances will make it plausible to elucidate biomarkers for pre-
dicting prognosis and therapy response.
FIBROBLAST PLASTICITY
It is established that CAFs show distinct functional states sug-
gesting that the CAF subtypes are cell states rather than end-
points in differentiation.1 More importantly, this plasticity
observed in CAFs is not unique, but an extension of the same
behavior evident in normal fibroblasts.79,91,137,292 This has
been evident in dermal fibroblasts that change from reticular to
papillary permitting ECM production or proliferation, respec-
tively and eventually tumor invasion.46,293 This plasticity has
also been reported during lung fibrosis294 and is subjected to
epigenetic regulation and dynamic state transitions.57,295,296
There is also remarkable diversity in the tissue microenviron-
ments that contain fibroblasts, and these niches can change
dramatically following injury, as is the case during reprogram-
ming of myofibroblasts, into adipocytes,.297 In addition to these
parameters, the plasticity in CAFs also occurs in response to
crosstalk with tumor cells or as collateral during therapeutic in-
terventions.298 This was shown in PDAC mouse model where
CAF reprogramming in the presence of JAK inhibitor shifted
the iCAFs to myCAFs subtype.91 Other studies have shown
that KRAS mutation and p53 mutational status in PDAC can
also influence CAFs resulting in enhanced desmoplastic stroma
and ECM remodeling.171,299
It is increasingly recognized that sufficient plasticity also exists
among some CAF subsets and is even more pronounced during
tumor progression.122,300,301 For example, in vitro experiments
have shown that iCAFs can be converted to myCAFs when
cultured in a monolayer.79 Independent of the switch mecha-
nism, the induction of iCAF leads to elevated production of cyto-
kines and chemokines, including VEGFA, PDGF, IL6, tumor
necrosis factor (TNF), NF-kB, HGF, CXCL12.1 Furthermore, the
responsiveness of matrix production by fibroblasts and the
aSMA promoter to a range of extracellular cues, including sub-
strate stiffness, supports the idea that the aSMA-high, matrix-
producing-high state is reversible.302,303 Considering the hetero-
geneity and plasticity of CAFs, it remains to be seen alteration of
this behavior can be made beneficial by converting their tumor-
promotion behavior to tumor restraining.
THERAPEUTIC TARGETING OF CAFs
Recent advances in CAF biology have led to the development of
new therapeutic strategies targeting the tumor stroma, particu-
larly the CAFs.16,296,304 Novel avenues are actively being
1596 Cell 186, April 13, 2023
Review
explored to target CAFs alone or in combination with existing
chemo- or immuno-therapies. These strategies (Table 2) can
be stratified as: (a) using cell surface markers to eliminate or limit
the presence of CAFs, (b) switching CAFs to a dormant state,
(c) antagonizing or sequestering the CAF secretome
(d) targeting the signaling pathways that activate CAFs,
(e) targeting the downstream elements that are crucial for CAF
function, and (f) targeting ECM proteins. Although various pre-
clinical and clinical studies in targeting CAFs are ongoing, the re-
sults have been inconsistent, suggesting that caution should be
exercised when using CAF-directed therapies.
SUMMARY AND FUTURE OUTLOOK
The ability of tumor cells to defy the basic cellular principles and
norms that apply to tissue homeostasis but grow uncontrollably is
well recognized. However, as numerous therapeutic modalities
and regimens have demonstrated, approaches to eliminate the
tumor "seed’’, or discard the ECM ‘‘soil’’ have been discouraging.
It has become apparent that the CAF component within the TME
is actively involved in tumor initiation, dissemination, drug resis-
tance and even extending to tumor dormancy and reawakening.
This requires a better understanding of the heterogeneous nature
and complexity within the CAF populations. The identity and pro-
portion of CAF subtypes also differ across normal, inflammatory,
premalignant, and malignant states; however, it is important to
note that both normal fibroblasts and CAFs are governed by
the same set of principles regarding the tissue state, local and
regional variation and most importantly the host attributes of in-
tersectionality (age, sex, ethnicity to name a few).
Although elegant lineage tracing studies have discovered the
origin of fibroblasts in normal or fibrotic tissues, not enough is
known about the origin of CAFs. Technological advances in sin-
gle-cell resolution and bio-fabricated 3D models have unveiled
incredible insights into cellular heterogeneity and interactions
with other cell types, particularly an unexpected diversity
amongst the fibroblasts at the level of molecular marker expres-
sion. New strategies to stratify fibroblasts that are guided by their
functional diversity rather than the expression of specific marker
genes are essential to understand and compare fibroblast het-
erogeneity in different organ systems and across different spe-
cies. We are only beginning to understand the functional rele-
vance of this dynamic and spatiotemporal heterogeneity, for
organ function in homeostasis and cancer biology, and the chal-
lenges are numerous. Finally, translating the discoveries in animal
models to the human setting is a difficult hurdle. Rodent models
are impressively establishing the trajectory of certain cell types
toward a pathological phenotype, but this is difficult to replicate
in human tissues. Together, CAFs have emerged as dynamic
players in the TME. Although challenges remain, the field of
CAF biology is on the cusp of the next big discovery that will facil-
itate tailored targeting of cancer types or stages, if not alone, then
as combination strategy to optimize clinical benefit.
ACKNOWLEDGMENTS
We apologize for omitting relevant works and citations due to space con-
straints. A.T.W. is also supported by P01CA114046 (NCI), U01CA227550
 Review
Table 2. Strategies directed against CAFs
Drug Cancer type Clinical Stage Mode of action (pathways/molecules) Reference
aFAP-PE38 Breast Preclinical Blocks FAPa Fang et al.305
Sibrotuzumab Phase I
Pan-FGFR inhibitors Urothelial, Phase 1 Inhibits CAF activation Formisano et al.; Guagnano et al.;
(Erdafitinib, BAY1163877, NSCLC, esophageal, Phase 2 Kim et al.; Palakurthi et al. and
BGJ398) cholangiocarcinoma Perera et al.306–310
Breast, HCC,
Navitoclax Cholangiocarcinoma Preclinical BCL2 inhibitor to initiate cell death Mertens et al.311
PT630 Lung Preclinical FAP deletion Santos et al.312
ProAgio Breast, Preclinical Integrin avb3 (reduces collagen, decreases Sharma et al. and Turaga et al.313,314
Pancreatic CAF secretome)
HA-PTX Breast, solid tumors Preclinical MMP (suppresses angiogenesis, ECM Chiappori et al.; Heo et al. and Yu
S-3304 Phase I degradation) et al.315–317
Astragaloside IV Gastric Preclinical miR-214 and miR-301a (reduce oncogenic Wang et al.318
SOX2, NANOG)
Fasudil PDAC Preclinical Rho/ROCK inhibitor Huo et al. and Whatcott et al.319,320
SCLC
NT157 Colorectal Preclinical IGF1R and STAT3 inhibitor Sanchez-Lopez et al.321
sTRAIL LPD Solid tumors Preclinical Induces fibroblast reprogramming Miao et al.322
Dasatinib Lung Preclinical PDGFR inhibitor Haubeiss et al.323
Vitamin D receptor ligand PDAC Preclinical Reprograms pancreatic stellate cells to Sherman et al.324
(Paricalcitol) quiescence
EB1089, LE135 Squamous cell carcinoma Preclinical Increases apoptosis by blocking Vitamin D Chan et al.325
Receptor/RARb
AC1MMYR2 Breast, gastric, glioblastoma Preclinical microRNA-21 maturation inhibitor Ren et al.326
Ruxolitinib Head and neck, lung, breast Phase 2 JAK/STAT pathway and DNA Albrengues et al.295
methyltransferase activity inhibitor
Minnelide PDAC Preclinical TGFb signaling inhibitor Dauer et al.327
All-trans-retinoic acid (ATRA) Breast Phase 1 Downregulates actomyosin contractility via Chronopoulos et al.328
PDAC, Prostate MLC2
Reprograms CAFs to quiescence
HA-PTX/MATT LY2157299 Breast, ovary Preclinical Blocks CAF activation by suppressing Lv et al. and Zhang et al.329,330
TGFb signaling and inhibiting TGFb-R1
Cell 186, April 13, 2023 1597

GKT137831 Head and neck, colorectal Preclinical Transdifferentiation to CAFs by Hanley et al.331
inhibiting NOX4
Artemisinin derivative Breast Preclinical Suppresses TGFb pathway Yao et al.332
Tranilast Lung Preclinical Suppresses proliferation and TGFb release Ohshio et al.333
Pirfenidone Pancreatic, NSCLC Phase 1 Suppresses proliferation and downregulate Mediavilla-Varela et al.334

ll
LY210976 Breast, liver Preclinical TGFb, PDGF and collagen synthesis
Bevacizumab Pleural mesothelioma Phase 3 Inhibits angiogenesis via VEGF Zalcman et al.335
(Continued on next page)
1598 Cell 186, April 13, 2023

ll
Table 2. Continued
Drug Cancer type Clinical Stage Mode of action (pathways/molecules) Reference
Metformin Ovarian Preclinical NF-kB inhibitor and inhibit IL6 secretion Xu et al.336
Imatinib Cervical Preclinical PDGFR inhbitor Pietras et al.226
Sonidegib TNBC Phase I Hedgehog pathway inhibitor Cazet et al.214
Talabostat Colorectal Phase 2 FAP activity inhibitor Narra et al.337
Pasireotide PDAC Phase 1 MTOR/4E-BP1 protein synthesis inhibitor Duluc et al.338
Losartan Breast, stomach Preclinical Collagen 1 synthesis inhibitor Diop-Frimpong et al.339
AMD3100 Gastric Preclinical CXCL12/CXCR4 signaling inhibitor Izumi et al.340
5-Fluorouracil Lung Blocks CAF recruitment Ma et al.341
JNJ-40346527 Lung, breast, colon Preclinical Blocks PMN-MDSC recruitment via CSF1R Kumar et al.342
SB225002 and CXCR2 respectively
Disulfiram/Copper Nasopharynx Preclinical Induces cytotoxic effects in CAFs through Li et al.343
ROS/MAPK and ferroptosis pathway
MORAb-004 Colorectal Phase 2 Blocks CD248 receptor-ligand interaction Diaz et al.344
131
I-m81C6 Brain Phase 2 Delays tumor growth by blocking tenascin Reardon et al.345
Anti-GPR77 Breast Preclinical Improve chemosensitivity Su et al.158
IPI-926 + Gemcitabine Pancreatic Preclinical Depletes CAF by reducing stroma Ko et al. and Olive et al.346,347
Phase 1 (discontinued)
Tocilizumab Gastric Preclinical Reduces IL6 Ham et al.348
PEGPH20 Pancreatic Phase 2 Increases drug delivery by targeting Hingorani et al. and Thompson et al.349,350
hyaluronan
ATRA all-trans-retinoic acid, CXCL12 CXC motif ligand 12, CXCR4 CXC motif chemokine receptor 4, IGF1R insulin-like growth factor type 1 receptor, IL6 interleukin-6, FAP fibroblast activation
protein, JAK Janus kinase, MLC2 myosin light chain 2, mTOR mammalian target of rapamycin, NF-kB nuclear factor kB, NSCLC non-small-cell lung cancer, PDAC pancreatic ductal adenocar-
cinoma, PDGF platelet-derived growth factor, PDGFR PDGF receptor, ROCK Rho kinase, TNBC triple negative breast cancer, STAT3 signal transducer and activator of transcription 3, TGF-b
transforming growth factor-b, BCL2 B-cell lymphoma 2, CSF1R Colony Stimulating Factor 1 Receptor, CXCR2 C-X-C Motif Chemokine Receptor 2, PMN-MDSC Polymorphonuclear Myeloid-
derived suppressor cells.

Review

Review
(NCI), R01CA232256 (NCI), R01CA207935 (NCI), a Bloomberg Distinguished
Professorship and the EV McCollum Endowed Chair.
DECLARATION OF INTERESTS
The authors declare no competing interest.
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