INVITED REVIEW ARTICLE
Nagoya J. Med. Sci. 84. 484–496, 2022
doi:10.18999/nagjms.84.3.484

Possible disease-protective roles of fibroblasts in cancer andfibrosis and their therapeutic application
Yukihiro Shiraki, Shinji Mii, Nobutoshi Esaki and Atsushi Enomoto
Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan

ABSTRACT
Cancer and fibrotic diseases are characterized by continuous inflammation, tissue wounds, and injuries.
Cancer is a “wound that does not heal,” and the uncontrolled proliferation of cancer cells disrupts normal
tissue integrity and induces stromal fibroinflammatory reactions. Fibroblasts proliferate extensively in thestroma, playing a major role in the development of these diseases. There has been considerable evidence
that fibroblasts contribute to fibrosis and tissue stiffening and promote disease progression via multiple
mechanisms. However, recent emerging findings, mainly derived from single-cell transcriptomic analysis,
indicated that fibroblasts are functionally heterogeneous, leading to the hypothesis that both diseasepromoting and -restraining fibroblasts exist. We recently showed that a fibroblast population, defined by
the expression of the glycosylphosphatidylinositol-anchored membrane protein Meflin may suppress but
not promote fibrotic response and disease progression in cancer and fibrotic diseases. Although currently
hypothetical, the primary function of Meflin-positive fibroblasts may be tissue repair after injury andcancer initiation occurred. This observation has led to the proposal of a potential therapy that converts
the phenotype of fibroblasts from pro-tumor to anti-tumor. In this short review, we summarize our recent
findings on the function of Meflin in the context of cancer and fibrotic diseases and discuss how we
can utilize this knowledge on fibroblasts in translational medicine. We also discuss several aspects of theinterpretation of survival analysis data, such as Kaplan-Meier analysis, to address the function of specificgenes expressed in fibroblasts.
Keywords: perivascular fibroblasts, mesenchymal stem cells, Meflin, cancer-associated fibroblasts, Immunoglobulin superfamily containing leucine-rich repeat (ISLR)
Abbreviations:
a-SMA: a-smooth muscle actin
PVF: perivascular fibroblast
MSC: mesenchymal stem cell
CAF: cancer-associated fibroblast
ECM: extracellular matrix
PDAC: pancreatic ductal adenocarcinoma
ISLR: immunoglobulin superfamily containing leucine-rich repeat
This is an Open Access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
License. To view the details of this license, please visit (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Received: October 27, 2021; accepted: January 6, 2022
Corresponding Author: Atsushi Enomoto, MD, PhD
Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku,
Nagoya 466-8550, Japan
TEL: +81-52-744-2093, Fax: +81-52-744-2098, E-mail: enomoto@iar.nagoya-u.ac.jp484Disease-protective roles of fibroblasts

INTRODUCTION
Fibroblasts secrete extracellular matrix (ECM) molecules such as collagen and fibronectinthat are essential to maintain the normal architecture of tissues and organs.1 The ECM producedby fibroblasts is also essential for tissue repair after wounds, injuries, and acute inflammation.
In diseases such as cancer and chronic inflammatory or fibrotic diseases, fibroblasts become
activated and proliferate in the stroma, constituting an essential component of fibroinflammatory
reactions.2,3 Activated fibroblasts, often called myofibroblasts owing to the high expression levelof a-smooth muscle actin (a-SMA) in these cells, produce a large amount of ECM moleculesand proteases as well as inflammatory cytokines and chemokines evoking inflammation.4 To
date, a multitude of studies have investigated the function of fibroblasts that proliferate undervarious disease conditions, leading to the accepted dogma that these cells promote fibrosis andtissue stiffening. Therefore, the development of drugs and tools that tactfully inhibit continuous
activation and proliferation of fibroblasts has been the focus of a substantial amount of research.
Our group has been also interested in the role of fibroblasts in cancer progression.5-9 However,one major obstacle in these studies is the lack of markers specific to fibroblasts that enable us
to distinguish between non-activated and activated fibroblasts.10
Previous studies have highlighted the importance of fibroblast heterogeneity by demonstratingthat the suppression of fibroblast proliferation or their genetic ablation unexpectedly resulted
in the progression of pancreatic ductal adenocarcinoma (PDAC).2,3,11-14 These studies led to thenotion that fibroblasts may both promote cancer (cancer-promoting cancer-associated fibroblasts[pCAFs]) and suppress cancer (cancer-restraining CAFs [rCAFs]).2,3,14 The heterogeneity of CAFs
has been also supported by recently emerging, single-cell transcriptomic analysis, which showedthat the extent of CAF heterogeneity and their cells of origin differ across tumor types. Thissuggests that there is more than one mechanism linking fibroinflammatory reactions to cancer
progression and that CAFs are not simply binary in nature (“good” or “bad”).2,3,10,15 There is aplethora of recently published review articles on CAF diversity and heterogeneity that readers
can refer to for further understanding.2,3,10,15-19
We have previously identified Meflin as a fibroblast marker.3,20-27 Interestingly, our recent studies
on human pathological samples and mouse disease models have suggested that the function of
Meflin-positive (Meflin+) fibroblasts is distinct from that of conventional activated fibroblasts(myofibroblasts and CAFs) in fibrotic disease and cancer conditions. In this review, we briefly
summarize the findings from our own laboratory and other studies on the functions of Meflin
protein and Meflin+ fibroblasts. We also describe a caveat when interpreting the results of the
Kaplan-Meier analysis in studying the molecular function of CAF markers.

MEFLIN IS A MARKER OF MESENCHYMAL STEM CELLS ANDPERIVASCULAR FIBROBLASTS
Meflin is a glycosylphosphatidylinositol-anchored membrane protein encoded by the immunoglobulin superfamily containing leucine-rich repeat (ISLR) gene.20 We previously identified Meflin
as one of the proteins specifically expressed in contact-inhibited fibroblasts.20 Further studies
showed that Meflin was expressed by rare stromal cells that adhere to the abluminal side ofendothelial cells or localize in the perivascular area of micro- or small capillaries in almost allorgans in adult mice (Figure 1). In mouse embryos, Meflin expression was found in immature
mesenchymal cells that constitute the stroma of all tissues and the cartilage primordia of skeletal
tissues. In adult mice, Meflin expression was detected in stromal fibroblasts found in the bone
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Fig. 1

Localization of Meflin+ cells in normal tissues and their proliferation after tissue injury,
cancer initiation or inflammation
Meflin+ cells are PVFs or MSCs found on the abluminal side of endothelial cells, perivascular areas, and adventitia
of capillaries and vessels. They are also sprinkled in the media of small- and mid-sized vessels. The findings
reported by our group and others suggest that these Meflin+ cells proliferate after tissue injury, including cancer
initiation and inflammation. Some Meflin+ cells differentiate into a-SMA+ myofibroblasts or CAFs during diseaseprogression. The factors that induce the differentiation of Meflin+ PVFs/MSCs into myofibroblasts that are negative
or weakly positive for Meflin include TGF-b, stiffness of substrate, hypoxia, and ex vivo culture on plastic.
a-SMA: a-smooth muscle actin
CAF: cancer-associated fibroblast
End: endothelial cells
ECM: extracellular cell matrix
MSC: mesenchymal stem cell
PVF: perivascular fibroblast
TGF-b: transforming growth factor beta

marrow, skeletal muscle, periosteum, adipose tissue, and immature chondroblasts.20,25 Lineage tracing experiments using a mouse line expressing the tamoxifen-sensitive Cre recombinase CreERT2
under the Meflin gene promoter (Meflin-CreERT2) showed that Meflin+ cells differentiated into
mature white and brown adipocytes, osteocytes, chondrocytes, and skeletal muscles.25,28 Thesefindings suggest that Meflin is a marker of MSCs that exist in almost every organ. Interestingly,
Meflin was not expressed in terminally differentiated cells such as mature adipocytes, chondrocytes, and osteocytes, suggesting that Meflin specifically marked undifferentiated MSCs.20,25 As
described above, most Meflin+ cells were found in the perivascular area of micro- and small
capillaries and the adventitia of muscular vessels, some of which appear to be pericytes (Figure1). These findings are consistent with the previous notion that MSCs exist as pericytes in tissues
or that MSCs contain a subpopulation of pericytes.29,30 Indeed, in situ hybridization (ISH) andthe analysis of a Meflin reporter mouse line showed that the number of Meflin+ cells was much
lower than that of conventional desmin+ pericytes.20,25 Although further detailed analysis should
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be performed, we hypothesize that Meflin marks a subpopulation of pericytes or perivascularfibroblasts (PVFs), some of which are equivalent to tissue-resident MSCs. It is currently unknown,
but possible, that differentiation capacity and lineage commitment of Meflin+ PVFs/MSCs differ
depending on the organ, developmental stage, and pathological context. Of note, Meflin+ cells
were sparsely found in the tunica media of some, but not all, mid-sized muscular vessels (Figure1). The role and significance of these Meflin+ cells in tunica media remain unknown. Throughout
this review, we will describe Meflin+ cells prior to activation and differentiation as Meflin+ PVFs/
MSCs; however, it should be noted that strict discrimination between the terms “PVFs/MSCs”
and “normal tissue-resident fibroblasts” will not be made.

FUNCTION OF MEFLIN IN TISSUE REPAIR AFTER ACUTE TISSUE INJURY
The response of Meflin+ PVFs/MSCs to acute tissue injuries was first investigated using amouse model of acute myocardial infarction (AMI).21 On inducing AMI, Meflin+ PVFs/MSCs
proliferated extensively in the border zone between the intact and necrotic myocardial tissues.
Notably, the proliferation of Meflin+ PVFs/MSCs preceded that of a-SMA+ myofibroblasts in theborder zone after inducing AMI, suggesting that Meflin+ fibroblasts could sense or detect signals
from injured tissues or necrotic cells with a higher sensitivity than conventional myofibroblasts(Figure 1). In addition, most Meflin knockout (KO) mice died because of cardiac rupture after
AMI, and collagen type I production in fibroblasts was significantly affected in Meflin KO
hearts compared with that in wild-type hearts.21 These data suggest that Meflin KO hearts exhibit
increased vulnerability to ischemia or tissue injury.
The role of Meflin in tissue repair after acute tissue injury was also demonstrated by subjecting
Meflin KO mice to bleomycin (BLM)-induced lung injury and fibrosis or dextran sulfate sodium(DSS)-mediated colon inflammation.26,31 The transtracheal administration of BLM induced theproliferation or infiltration of Meflin+ fibroblasts in the lungs. As observed in the AMI model,
Meflin KO mice presented severe lung hemorrhage and died more quickly than wild-type mice
after BLM administration (Nakahara Y and Hashimoto N, unpublished observation); furthermore,they had severe intestinal damage and impaired regeneration after DSS treatment.26,31. These
data further support the view that Meflin is essential for tissue repair after acute tissue injury(Figure 1).
The rapid mobilization of Meflin+ PVFs/MSCs to diseased lesions was also observed in amouse model of pancreatic cancer.3,22 We found that Meflin+ fibroblasts appeared even in small
lesions with acinar-ductal metaplasia, a precancerous lesion in the KPC pancreatic cancer mousemodel, where few a-SMA+ myofibroblasts were found.3 Given that cancer initiation is essentiallyequivalent to repeated wounding and regeneration, the above finding is consistent with the notionthat Meflin+ PVFs/MSCs are involved in acute injury and wound repair (Figure 1). Currently,
the mechanisms by which Meflin+ PVFs/MSCs/fibroblasts infiltrate or get recruited to injured
tissues have not been elucidated.

FUNCTION OF MEFLIN IN FIBROSIS
The role of Meflin in fibrotic diseases was first investigated in a mouse model of chronic heart
failure induced by transverse aortic constriction (TAC).21 In the normal heart, Meflin is expressedby PVFs in the interstitium of the ventricular walls and the pericardium and endocardium.
A lineage-trace experiment using a mouse line expressing Cre recombinase under the Meflin
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Fig. 2 Hypothesis on the role of Meflin+ PVFs/MSCs or fibroblasts in fibrotic diseases
Meflin is expressed by quiescent PVFs/MSCs in normal tissues, but also in cells that proliferate in the earlystages of inflammatory and fibrotic diseases. Tissue injuries or inflammatory lesions are either resolved or become
chronic, depending on the underlying etiology and the initial cause of the disease. Our data suggest that Meflinfunctions include the augmentation of BMP7 signaling and suppression of Lox-mediated crosslinking of theextracellular matrix. Furthermore, some a-SMA+ myofibroblasts derive from Meflin+ PVFs/MSCs, but there areother sources of cells that give rise to a-SMA+ myofibroblasts.
a-SMA: a-smooth muscle actin
BMP: bone morphogenetic factor
CAF: cancer-associated fibroblast
ECM: extracellular cell matrix
MSC: mesenchymal stem cell
PVF: perivascular fibroblast(ISLR) promoter (Meflin-Cre) showed proliferation of Meflin-lineage cells in the fibrotic area.
These cells were positive for myofibroblast markers such as a-SMA and periostin, suggestingthat Meflin+ cells were one of the origins of myofibroblasts in cardiac fibrosis (Figure 1, 2).
We also identified factors that induced the differentiation of Meflin+ PVFs/MSCs into a-SMA+
myofibroblasts using cultured MSCs and cardiac fibroblasts. These factors included transforming
growth factor-b (TGF-b), substrate stiffness, hypoxic conditions, and long-term culture or passages on plastic (Figure 1). Notably, significant downregulation of Meflin expression in cultured
fibroblasts was observed when a-SMA expression was increased by TGF-b stimulation, leading to
the speculation that Meflin+ PVFs/MSCs gave rise to Meflin-/low/a-SMA+ myofibroblasts in fibrosis
conditions (Figure 1). However, the exact contribution of Meflin+ PVFs/MSCs in generating
a-SMA+ myofibroblast populations has not been elucidated, and our recent data suggest that it
is less than we had expected and differs among diseases (Figures 1 and 2). Lineage tracing ina mouse model of renal fibrosis showed that Meflin+ PVFs/MSCs gave rise to an approximately
10% of all a-SMA+ myofibroblasts. Additionally, Meflin lineage cells comprised two types offibroblasts that exhibited (1) high or (2) low/negative a-SMA expression (Minatoguchi and Saito
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et al, unpublished observation). Although the data obtained from such lineage trace experiments
using the CreERT2 system do not generally reflect the entire lineage potential of cells of origin, we
entertain the possibility that the above finding is one of the mechanisms that underlie fibroblast
heterogeneity in disease conditions.
Interestingly, Meflin KO hearts exhibited accelerated fibrosis and severe diastolic dysfunction
after TAC induction compared to wild-type hearts.21 This was accompanied by increased stiffnessof the cardiac tissue and poor prognosis in Meflin KO mice. Notably, ejection fraction (EF), a
marker of global systolic function, was comparable between wild-type and Meflin KO hearts after
TAC induction, indicating that Meflin KO mouse hearts after TAC induction may be a model ofhuman heart failure with preserved EF (HFpEF).21 These data suggest that Meflin plays a rolein suppressing cardiac fibrosis.
A search for Meflin ligands identified bone morphogenetic protein 7 (BMP7).21 Our biochemical assay showed that Meflin bound to BMP7 to augment its signaling in cultured cells.21,24 Given
that BMP7 has an anti-fibrotic function by counteracting TGF-b signaling,32 it was hypothesizedthat the primary function of Meflin may be TGF-b and fibrosis suppression in physiological anddisease conditions (Figures 1 and 2). The anti-fibrotic role of Meflin was also shown in cultured
fibroblasts isolated from the lungs of wild-type and Meflin KO mice.26 Lung fibroblasts from
Meflin KO mice exhibited a significant increase in a-SMA and collagen expression comparedto wild-type fibroblasts when stimulated with TGF-b.
The possible role of Meflin in cellular senescence remains unknown; however, recent findings
have shown that lung fibroblasts isolated from Meflin KO mice acquired an accelerated senescencephenotype compared to those from wild-type mice when they were stimulated with TGF-b.26 In
addition, the quantification of Meflin mRNA in mouse hearts showed that its expression was
significantly lower in aged mice than in young mice.21 Thus, the role of Meflin in aging andage-dependent physiological decline of organ function should be a focus of future research.

FUNCTION OF MEFLIN IN CANCER PROGRESSION
Our recent study showed that Meflin was a marker of pancreatic stellate cells (PSCs), which
are resident fibroblasts of the pancreas with a high capacity to restore retinol and retinyl esters
in their cytoplasm.22,33 Consistent with the notion that PSCs are a cell of origin of CAFs in
PDAC,33 Meflin+ PSCs proliferate in the stroma of both human PDAC and tumors developedin the KPC PDAC mouse model.22 A lineage tracing study using Meflin-CreERT2 mice bearing
a subcutaneous mT5 pancreatic cancer xenograft showed the possibility that Meflin+ PSCs gave
rise to CAFs that were highly positive for a-SMA. The contribution of Meflin lineage cells to
CAFs has also been shown in an autochthonous mouse model of colorectal cancer.34 Multiplex
ISH assay showed that there were at least two subsets of CAFs in the stroma of human PDAC:
Meflin-high and a-SMA-low CAFs and Meflin-low and a-SMA-high CAFs22 (Figure 3). We
further found that the numbers of Meflin+ CAFs differed across patients with PDAC, suggesting
intertumoral heterogeneity and diversity of CAFs. Interestingly, patients with a high number ofMeflin+ CAFs showed better prognosis than those with a low number of Meflin + CAFs, andMeflin KO mice developed more advanced PDAC than wild-type mice when they were crossedwith the KPC PDAC model.22 Moreover, exogenous expression of Meflin in CAFs retarded tumor
progression in a subcutaneous tumor model.22 Thus, our findings suggest that Meflin+ PSCs and
CAFs play a role in suppressing tumor progression but give rise to Meflinlow/– and a-SMA+
CAFs. Another recent study from our group suggested that Meflin was biochemically bound tolysyl oxidase (Lox), which crosslinked collagen or elastin to form stiff tissues with increased
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Yukihiro Shiraki et altensile strength and integrity, thereby inhibiting its enzymatic activity.35 Given the role of Lox incancer progression,36 we hypothesized that Meflin suppressed cancer progression by inhibiting Lox
activity and augmenting BMP7 signaling, as described earlier (Figure 3). The tumor-suppressiverole of Meflin+ CAFs was also demonstrated in our recent study, which showed that Meflincompeted with another CAF marker protein, Gremlin 1.24 Gremlin 1 is a negative regulator of
BMP signaling in the tumor microenvironment of colorectal cancer.24 The study also showed thetherapeutic potential of the anti-Gremlin 1 antibody that neutralized the activity of Gremlin 1
and adeno-associated virus-mediated transduction of the Meflin gene to modulate CAF function
in treating patients with metastatic colorectal cancer.

Fig. 3 Hypothesis on the role of Meflin+ PVF/MSCs or fibroblasts in cancer
As in fibrotic and inflammatory diseases, Meflin+ PVFs/MSCs are the source of CAFs in cancer. We hypothesizethat Meflin+ PVFs/MSCs proliferate and constitute rCAFs in the early stages of cancer but give rise to a-SMA+
CAFs during cancer progression. Am80 administration increases the number of Meflin+ rCAFs, which leads to adecrease in tumor stiffness and increase in tumor vessel area and tumor sensitivity to chemotherapeutics.
a-SMA: a-smooth muscle actin
BMP: bone morphogenetic factor
CAF: cancer-associated fibroblast
ECM: extracellular cell matrix
MSC: mesenchymal stem cell
PVF: perivascular fibroblast

The role of Meflinlow/– and a-SMA+ CAFs remains controversial. A previous study showedthat the genetic depletion of a-SMA+ CAFs resulted in the acceleration of PDAC progression in
mice,12 whereas a number of studies demonstrated a significant correlation between the numberof a-SMA+ CAFs and poor outcomes in many types of human cancers.3 Our data showed that
a-SMA was weakly expressed in Meflin+ CAFs21 and that the depletion of a-SMA+ CAFs resulted
in the depletion of Meflin+ CAFs. Thus, it would be inaccurate to neatly divide CAFs into pCAFs
and rCAFs, which may confound the interpretation of experimental results in preclinical models
and clinical samples. Most importantly, one should distinguish between the functions of a-SMA
protein and the roles of a-SMA+ CAFs, which are often confounded by researchers.
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CAF DIVERSITY: A DIFFERENT PHENOTYPE OF THE SAME CELLS?
Recent advances in single-cell transcriptomic analysis have enabled a deeper understandingof CAF heterogeneity across many tumor types, which led to the identification of a number ofgenes expressed in CAFs defining different potential CAF subsets.15 Readers can refer to several
review articles describing in details the proposed CAF classification, which are not included inthis review.2,10,14,15,37 One of the major problems in the current CAF classification is that it isbased on arbitrary descriptions that are primarily rooted in existing knowledge on CAF marker
functions and their correlation with the outcome in animal models and patients. Furthermore,
the biological significance of each CAF subset has not necessarily been experimentally proven.
We and other authors have proposed a simple CAF classification model, in which CAFs wereclassified according to their functions in cancer progression.2,3,14,18 Based on our previous datathat showed the forced expression of exogenous Meflin in CAFs retarded their pro-tumor function,22,24,35 we proposed a model in which each CAF expressed both proteins with anti-tumor
functions and those with pro-tumor functions, and the role of each CAF was determined by thebalance between the levels of these proteins (Figure 4). For example, analysis of publicly available data from single-cell analysis of CAFs and immunohistochemical staining for various CAF
markers showed that Meflin was co-expressed with CAF marker proteins that promoted cancer

Fig. 4 Hypothesis on the mechanism of fibroblast heterogeneity
The most plausible hypothesis based on a number of previous studies on the diversity and heterogeneity offibroblasts is that they have different origin or derived from various lineages during disease development. However,
it is possible that the function of fibroblasts could be determined by the relative amounts and balance of proteins
with cancer-restraining functions and those with cancer-promoting functions. The involvement of liquid factors
derived from other cells, including cancer and inflammatory cells, and epigenetic mechanisms should also be
considered as regulators of the conversion and plasticity between rCAFs and pCAFs. Most importantly, one
should discriminate the function of a CAF subset expressing a particular protein and the function of that protein
in terms of whether they are pro-tumor or anti-tumor.
a-SMA: a-smooth muscle actin
CAF: cancer-associated fibroblast
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progression, such as fibroblast activation protein-a.22 This notion was further corroborated byrecent studies that showed that CAF expression of collagen type I alpha 1, which was reported
to be expressed in some pCAFs, played a role in suppressing cancer progression by affecting
the immune response to tumors and mechanistically restraining tumor spread.38,39 This was provenby an induction of tumor development in mouse lines in which the gene for collagen type Ialpha 1 was conditionally deleted in a-SMA+ cells or hepatic stellate cells. Those experiments
showed that collagen type I alpha 1 protein was tumor suppressive. However, the role of collagentype I alpha 1+ CAFs remains unknown and should be carefully determined by the net balanceof the expression of cancer-promoting and -restraining proteins. Likewise, our data showed that
Meflin was a protein with a tumor-suppressive role, whereas the role of Meflin+ CAFs was notnecessarily tumor suppressive. Our previous study showed that the genetic depletion of Meflin+
CAFs resulted in the development of poorly differentiated tumors and a decrease in a-SMA+
CAFs but did not change tumor volume in a subcutaneous tumor transplantation model.22

A CAVEAT IN EVALUATING THE FUNCTION OF GENES EXPRESSEDIN CAFS USING KAPLAN-MEIER ANALYSIS
One question that we are frequently asked is: Why is it that a simple comparison of theoutcomes in cancer patients stratified based on Meflin expression levels sometimes shows that

Fig. 5 A trap in interpretation of the data obtained from Kaplan-Meier analysis
Tumors are diverse in terms of the extent of fibroinflammatory and fibrotic reactions in the stroma, even in thesame type of cancer. Less invasive tumors, which exhibit favorable prognosis, tend to have fewer stromal reactions
with limited numbers of rCAFs and pCAFs, whereas more invasive tumors are accompanied by greater stromal
reactions with large numbers of rCAFs and pCAFs. Therefore, simple interpretation of Kaplan-Meier analysis
based on the expression level of proteins or genes expressed in rCAFs does not accurately depict their roles incancer development (a). Correct conclusions may be made when tumors with the same extent of stromal reactions
are analyzed for the expression of genes or proteins specifically in rCAFs (b).
CAF: cancer-associated fibroblast
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the Meflin-high group exhibits poorer outcomes than the Meflin-low group? (Figure 5). We
believe that to conclude that Meflin is a bad gene that promotes tumor progression from such ananalysis is an oversimplification. Because tumors with more desmoplastic and fibroinflammatory
reactions, which are accompanied by increased infiltration of Meflin+ CAFs, are more invasive
and show worse outcomes than those with less desmoplastic reactions, the comparison of all
the patients with minimally invasive tumors, including carcinoma in situ, to those with highlyinvasive tumors, will lead to misunderstanding the function of genes expressed in CAFs (Figure5a). For example, the analysis of all non-small cell lung cancer patients deposited in The Cancer
Genome Atlas database showed that patients with an increased number of Meflin+ CAFs had
worse prognosis than those with lower number of Meflin+ CAFs.40 However, analysis of patientswith stage III non-small cell lung cancer who presumably exhibited comparable stromal reactions
tended to show more favorable outcomes in patients with high Meflin+ CAFs than in thosewith low Meflin+ CAFs40 (Figure 5b). Furthermore, the number of Meflin+ CAFs correlates wellwith the favorable response of patients with non-small cell lung cancer to immune checkpoint
inhibitors.41 Thus, it is suggested that simple interpretation of Kaplan-Meier analysis based on
the expression level of CAF marker genes in whole tumor tissue samples does not necessarily
lead to a correct understanding of the functions of these genes. We believe that the same holds
true when interpreting the function of genes expressed in cancer and immune cells. Thus, it isimportant to remember that Kaplan-Meier analyses are suitable to identify markers that predictpatient outcome and compare different arms of treatment or cohorts in clinical studies but do
not necessarily reveal the function of genes of interest in terms of whether they are pro-tumor
or anti-tumor. Meflin expression could be a marker that predicts worse outcomes in patientswith cancer, but the functions of Meflin and Meflin+ cells should be examined by multifaceted
approaches, including studies on animal models and cultured cells.

MODULATION (REPROGRAMMING) OF FIBROBLAST FUNCTION IN CANCER
The failure of preclinical and clinical studies to target pathways that regulate the proliferation of CAFs or deplete them have led to the idea that CAFs have a role in restraining cancer
progression as described above.2,3,11-13,19 Our recent line of studies showed that Meflin may be
one of the proteins that conferred anti-tumor functions to CAFs.3,22,24 Another approach targeting CAFs that recently emerged was to reprogram or engineer pro-tumor CAFs such that they
become anti-tumor. A pioneering study by Sherman et al showed that the administration of thevitamin D analog calcipotriol increased the sensitivity of pancreatic cancer to chemotherapy in
an autochthonous pancreatic cancer mouse model.42 This effect of calcipotriol was mediated bytranscriptomic changes, such as alterations in the expression of genes involved in the regulationof inflammation and the extracellular matrix, in CAFs or activated PSCs that proliferate in thestroma of pancreatic cancer. These observations led to several clinical trials that investigatedthe effect of combinations of calcipotriol and other conventional chemotherapies or immune
checkpoint inhibitors in patients with PDAC (https://clinicaltrials.gov).
Our group recently screened a chemical library of nuclear receptor ligands, which led to thediscovery of Am80 as a compound that significantly upregulated Meflin expression in CAFs35(Figure 3). Oral administration of Am80 resulted in a significant increase in Meflin expression
but suppressed a-SMA expression in CAFs in a mouse model of pancreatic cancer. Consistent
with the finding that Meflin suppresses fibrosis by augmenting BMP7 signaling and inhibiting
the activity of Lox, tumors administered Am80 were softer than control tumors, which was
accompanied by an increase in vessel area in the developed tumors. Furthermore, Am80 adNagoya J. Med. Sci. 84. 484–496, 2022

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ministration resulted in increased intratumor concentrations of gemcitabine and its efficacy in apancreatic cancer mouse model. These effects of Am80 were not observed in Meflin KO mice,suggesting that Am80 exerted its anti-tumor effect by upregulating Meflin in CAFs34 (Figure 3).
Based on these observations, our colleagues (Dr Mitsuhiro Fujishiro, Tokyo University and Dr
Hiroki Kawashima, Nagoya University) have started an investigator-led clinical trial to investigate
the safety and tolerability of Am80 (generic name: tamibarotene; developmental code: MIKE1) in combination with the conventional tumoricidal drugs gemcitabine and nab-paclitaxel inpatients with unresectable PDAC and determine the recommended dose and explore its efficacy(ClinicalTrials.gov identifier: NCT05064618).43

CONCLUSION AND PERSPECTIVESIn this short review, we briefly described the pathological roles of Meflin in PVFs, MSCs,
and CAFs in various disease conditions. The precise molecular function of Meflin remains to
be understood, but we hypothesize that Meflin plays a role in inhibiting fibrotic reactions byaugmenting BMP7 signaling and suppressing Lox activity. The involvement of Meflin in theregeneration of skeletal tissues and myoblast differentiation has also been evaluated in otherstudies but it was not covered in this review.44,45
As described earlier, the function of the Meflin protein and that of Meflin+ cells should bedetermined separately to better understand their specific roles in disease progression. It is also
important to assess whether diverse fibroblasts are derived from different lineages or represent
different faces of the same fibroblasts with plasticity. Furthermore, identification of transcription
factors and epigenetic mechanisms that regulate fibroblast heterogeneity and plasticity is vital tothis field of research. We believe that the accumulation of studies on the functions of fibroblast
markers will lead to a deeper understanding of fibroblast heterogeneity and the development oftherapies for cancer and fibrotic diseases.

ACKNOWLEDGMENTS
We thank all of the members of the Department of Pathology, Nagoya University Graduate
School of Medicine, and our collaborators for their helpful input and discussions on the studies
described in this review article. This work was supported by the Naito Foundation (to AE), a
Grant-in-Aid for Scientific Research (B) (grant no 18H02638 to AE), and commissioned by the
Ministry of Education, Culture, Sports, Science and Technology of Japan; Nagoya University
Hospital Funding for Clinical Research (to AE); AMED-CREST (Japan Agency for Medical
Research and Development, Core Research for Evolutional Science and Technology, grant nos
21gm0810007h0106 and 21gm1210009s0103 to AE), and the Project for Cancer Research andTherapeutic Evolution (P-CREATE) from AMED (grant no 21cm0106377h0002 to AE).

DISCLOSURE STATEMENT
The authors declare no competing interests.

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Disease-protective roles of fibroblasts

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doi:10.18999/nagjms.84.3.484