REVIEW published: 22 January 2021 doi: 10.3389/fcell.2020.621070 Determinants and Functions of CAFs Secretome During Cancer Progression and Therapy Jenniffer Linares 1† , Juan A. Marín-Jiménez 2† , Jordi Badia-Ramentol 1 and Alexandre Calon 1* 1 Cancer Research Program, Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain, 2 Department of Medical Oncology, Catalan Institute of Oncology (ICO) - L’Hospitalet de Llobregat, Barcelona, Spain Multiple lines of evidence are indicating that cancer development and malignant progression are not exclusively epithelial cancer cell-autonomous processes but may also depend on crosstalk with the surrounding tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are abundantly represented in the TME and are continuously interacting with cancer cells. CAFs are regulating key mechanisms during Edited by: progression to metastasis and response to treatment by enhancing cancer cells survival Marzia Di Donato, University of Campania Luigi and aggressiveness. The latest advances in CAFs biology are pointing to CAFs-secreted Vanvitelli, Italy factors as druggable targets and companion tools for cancer diagnosis and prognosis. Reviewed by: Especially, extensive research conducted in the recent years has underscored the Ferdinando Auricchio, Second University of Naples, Italy potential of several cytokines as actionable biomarkers that are currently evaluated in the Paolo Paoli, clinical setting. In this review, we explore the current understanding of CAFs secretome University of Florence, Italy determinants and functions to discuss their clinical implication in oncology. Fernando Calvo, Consejo Superior de Investigaciones Keywords: cancer-associated fibroblast (CAF), secretome, secreted factors, growth factors, cancer, tumor Científicas (CSIC), Spain microenvironment (TME), metastasis, therapy *Correspondence: Alexandre Calon acalon@imim.es INTRODUCTION † These authors have contributed Cancer-associated fibroblasts (CAFs) are contributing to the production of a wide variety of equally to this work and share first authorship secreted factors impacting tumor progression by directly regulating malignant cancer cells aggressiveness or by indirectly reprogramming tumor immunity and angiogenesis (Sahai et al., Specialty section: 2020). Hence, molecular and functional inter- and intra-tumoral heterogeneity of CAFs has been This article was submitted to a recent focus in oncology research. It is widely accepted that the functional phenotype of CAFs Molecular and Cellular Oncology, is in part determined by the cell of origin, including but not restricted to local resident fibroblasts a section of the journal (D’Arcangelo et al., 2020; Sahai et al., 2020). An alternative hypothesis advocating for CAF being Frontiers in Cell and Developmental a cell state depending on autocrine and paracrine signaling rather than a cell type has also been Biology proposed (Kalluri, 2016). Whether CAFs functional heterogeneity is maintained among different Received: 24 October 2020 solid tumor types or is a constant evolutionary state is still a debated question. However, recent Accepted: 07 December 2020 studies have been investigating the determinants of CAFs secretome and their therapeutic interest Published: 22 January 2021 across different tumor types. Molecular biomarkers predicting the risk of relapse and the potential Citation: benefit from treatments are currently needed for clinical decision-making. In an attempt to reach Linares J, Marín-Jiménez JA, a more comprehensive evaluation of tumors, many CAFs-secreted factors have been included in Badia-Ramentol J and Calon A (2021) gene expression signatures that are considered suitable prognostic tools for clinical diagnosis and Determinants and Functions of CAFs Secretome During Cancer prognostication (Berdiel-Acer et al., 2014; Karlan et al., 2014; Yue et al., 2019). As a matter of fact, Progression and Therapy. recent advances in the understanding of CAFs secretome determinants and functions have brought Front. Cell Dev. Biol. 8:621070. to light the multiple benefits of using CAFs-secreted factors as actionable biomarkers for cancer doi: 10.3389/fcell.2020.621070 diagnosis, treatment, and prognosis. Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions DETERMINANTS OF CAFS SECRETOME different types of stress, such as hypoxia or proteotoxic stress (Dayalan Naidu and Dinkova-Kostova, 2017). Crosstalk With Tumor Cells and TME Complex paracrine signaling through cancer cell-secreted Components factors also regulates CAFs secretome during tumor progression Autocrine- and Paracrine-Secreted Factors (Figure 1) and may depend on genetic alterations occurring Multiple autocrine loops impacting CAFs secretome have been in cancer cells. Indeed, KRAS mutant but not KRAS wild- discovered in the recent years (Figure 1). Among them, members type pancreatic ductal adenocarcinoma (PDAC) cancer cells of the transforming growth factor beta (TGF-beta) superfamily induce CAFs activation through CXCR-2, leading to a nuclear are known to be the main inducers of CAFs activation. Of factor-kappa B (NF-κB)-mediated secretion of pro-tumoral note, CAFs secrete large amounts of TGF-beta isoforms 1, cytokines interleukin (IL)-4, IL-10, and IL-13 (Awaji et al., 2, and 3 (reviewed in Kalluri, 2016). In turn, secreted TGF- 2020). Several additional studies have reported a pro-tumorigenic beta maintains a self-sustained active state -typically regarded crosstalk between cancer cells and CAFs. For instance, kallikrein as myofibroblasts- characterized by alpha smooth muscle actin (KLK)-4 produced by malignant and premalignant prostate (alpha-SMA) expression (Orimo et al., 2005; Kojima et al., lesions can act on normal fibroblasts through the activation of 2010). Other members of the TGF-beta superfamily, such as protease-activated receptor (PAR)-1, which leads to a CAF pro- Nodal, induce pro-tumorigenic phenotypes in fibroblasts from angiogenic secretory phenotype characterized by the increased melanoma and colorectal cancer (CRC) (Li et al., 2019). Likewise, expression of Dickkopf-related protein 1 (DKK-1), growth activin A has showed the ability to induce a secretory phenotype differentiation factor 15 (GDF-15), hepatocyte growth factor in CAFs via the SMAD-2-mediated transcriptional regulation (HGF), IL-8, and vascular endothelial growth factor (VEGF) of genes encoding extracellular matrix (ECM) components, and the decreased expression of insulin-like growth factor- ECM regulators, and soluble factors (Cangkrama et al., 2020). binding protein (IGFBP)-3, monocyte chemoattractant protein 1 Alternatively, CAFs secretome may be maintained during tumor (MCP-1), and platelet-derived growth factor (PDGF)-AA (Kryza progression through enhanced stromal cell-derived factor (SDF)- et al., 2017). In PDAC, S100 calcium-binding protein A11 1 autocrine signaling loops and increased co-expression of (S100A11) secreted by cancer cells activates the surrounding receptors, such as TGF-beta-RI and CXC receptor (CXCR)- fibroblasts through the S100A11–receptor for advanced glycation 4 (Kojima et al., 2010). In this sense, Scherz-Shouval et al. end products (RAGE)–tumor progression locus 2 (TPL2)– reported an increased production of TGF-beta-2 and SDF-1 cyclooxygenase 2 (COX2) pathway to promote metastatic factors by CAFs upon heat shock factor (HSF)-1 cytoplasmic progression (Mitsui et al., 2019). translocation to the nucleus (Scherz-Shouval et al., 2014). HSF- CAFs activation may also occur through paracrine signaling 1 is a transcription factor that mediates the cellular response to with non-cancer cells. For example, cytokines produced by immune cells during the inflammatory process may instruct CAFs function. Among them, IL-1β produced by immune cells in Abbreviations: AKT, protein kinase B; ATRA, all-trans retinoic acid; CCL, C– early hyperplastic lesions activates normal fibroblasts to become C motif chemokine; COX2, cyclooxygenase 2; CXC, C–X–C motif chemokine; CAFs via the NF-κB pathway (Erez et al., 2010). Alternatively, CXCL, CXC ligand; CXCR, CXC receptor; DKK-1, Dickkopf-related protein granulin secretion by macrophages activates resident fibroblasts 1; FGF, fibroblast growth factor; GDF-15, growth differentiation factor 15; GM-CSF, granulocyte-macrophage colony-stimulating factor; HER3, epidermal into tumor-promoting myofibroblasts sustaining metastatic growth factor receptor 3; HGF, hepatocyte growth factor; HIF, hypoxia-inducible growth in PDAC (Nielsen et al., 2016). factor; HspA1A, heat shock 70 kDa protein 1A; ICAM-1, intercellular adhesion In addition to local intra-tumoral autocrine and paracrine molecule 1; IFN, interferon; IGF, insulin-like growth factor; IGF1R, IGF 1 signaling, systemic signaling involving steroid hormones, such receptor; IGFBP, insulin-like growth factor-binding protein; JAK, Janus kinase; as estrogens and androgens, is also able to modulate CAFs JNK, Jun N-terminal kinase; KLK, kallikrein; LIF, leukemia inhibitory factor; M- CSF, macrophage colony-stimulating factor; MAPK, mitogen-activated protein secretome, through binding to their receptors expressed in kinase; MCP-1, monocyte chemoattractant protein 1; MIF, macrophage migration CAFs (Clocchiatti et al., 2018; Rothenberger et al., 2018). inhibitory factor; MMP, matrix metallopeptidase; mTOR, mammalian target of For instance, estrogens (E2) regulate the expression of several rapamycin; MYH-11, myosin 11; NF-κB, nuclear factor-kappa B; NRG, neuregulin; microRNAs (miRNAs) in breast cancer (BC)-derived CAFs OXPHOS, oxidative phosphorylation system; P38 MAPK, p38 mitogen-activated (Vivacqua et al., 2019). In gastric cancer, estrogens stimulate protein kinase; PDGF, platelet-derived growth factor; PDGFRB, platelet-derived growth factor receptor beta; PDPN, podoplanin; PGE2, prostaglandin E2; PI3K, CAFs secretion of IL-6, thereby promoting signal transducer and phosphoinositide 3-kinase; POSTN, periostin; PAR, protease-activated receptor; activator of transcription (STAT)-3 pathway-dependent cancer RAGE, receptor for advanced glycation end products; miRNA, microRNA; mRNA, cells proliferation and invasion (Zhang Y. et al., 2020). Similarly, messenger RNA; lncRNA, long non-coding RNA; S100A11, S100 calcium-binding in prostate cancer (PCa), the activation of CAFs–androgen protein A11; SATB-1, special AT-rich sequence-binding protein-1; SDF, stromal cell-derived factor; SFRP4, secreted frizzled-related protein 4;Shp, Src homology 2- receptor (AR) with dihydrotestosterone modulates the secretion containing protein tyrosine phosphatase; SMAD, acronym for a signal transducer of pro-tumorigenic factors impacting cancer cell growth (Tanner family; SOCS, suppressor of cytokine signaling 1; STAT, signal transducer and et al., 2011). Conversely, AR blockade in CAFs decreases activator of transcription; TAZ, transcriptional coactivator with PDZ-binding the expression of pro-tumorigenic factors, such as insulin-like motif, also known as Wwtr1; TET, ten-eleven translocation enzyme; THBS, growth factor (IGF)-1, fibroblast growth factor (FGF)-7, FGF-10, thrombospondin; TIMP, tissue inhibitors of MMP;TNC, tenascin C; TNF, tumor necrosis factor; TPL2, tumor progression locus 2; VCAM, vascular cell adhesion SDF-1, HGF, and TGF-beta 2 (Yu et al., 2013). protein; VEGF, vascular endothelial growth factor; VEGFA, VEGF type A; YAP, As illustrated by the opposite gene expression programs yes-associated protein. regulated upon FGF and TGF-beta pathway activation Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions FIGURE 1 | Determinants of CAFs secretome during cancer progression. Secreted factors as well as extracellular vesicle (EV)-dependent autocrine and paracrine crosstalk modulate the secretory profile of CAFs. Direct physical interaction between CAFs and epithelial cancer cells or between CAFs and components of the extracellular matrix may additionally regulate CAFs secretome. (Bordignon et al., 2019), CAFs secretome heterogeneity may be gastric cancer, EVs containing miR155, miR193b, and miR210 the result of a delicate balance between autocrine and paracrine prime CAFs to secrete inflammatory chemokines, such as CXC signaling stimulated simultaneously and activating CAFs in ligand (CXCL)-1 and CXCL-8, through the activation of the Janus different ways. It is worth noting that autocrine and paracrine kinase (JAK)/STAT and NF-κB signaling pathways (Naito et al., triggering cues educating CAFs secretory functions may be 2019). Similarly, NF-κB activation in CAFs by EV-derived miR- tumor-dependent or even cancer subtype-dependent. Thus, 1247-3p in metastatic hepatocellular carcinoma (HCC) leads to a the identification of multiple CAFs activation and secretion tumor-promoting secretion of IL-6 and IL-8 (Fang et al., 2018). programs could greatly improve current molecular classification Alternatively, EV-derived miR-210 and miR-155-5p induce a of cancer. pro-angiogenic switch in CAFs through the activation of the JAK-2/STAT-3 pathway in lung cancer and melanoma (Zhou Extracellular Vesicles et al., 2018; Fan et al., 2020). Additional signaling pathways have Extracellular vesicles (EVs) are spherical membrane formations been associated with miRNA-driven secretory stimulation. For comprising exosomes and microvesicles, which can carry instance, EVs containing miR-10b contribute to the enhanced different molecules, such as proteins, DNAs, non-coding RNAs, TGF-beta expression in CAFs through the phosphoinositide and miRNAs/mRNAs. While both are playing a key role in 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of distant intercellular communication, exosomes are derived from rapamycin (mTOR) signaling pathway in CRC (Dai et al., 2018). the endosomal system, and microvesicles are produced by the Finally, EVs containing coding mRNA have also been reported to plasma membrane (reviewed in van Niel et al., 2018). EV- trigger a secretory phenotype in CAFs. In this sense, cancer cell- based intercellular communication between cancer cells and the derived EVs containing mRNA coding for CXCR-4 and IGF-1R tumor microenvironment (TME)–including CAFs (Figure 1)– promote CAFs secretion of growth factors, such as VEGF in acute promotes cancer progression in multiple ways (reviewed in Han myeloid leukemia (Huan et al., 2013). et al., 2019); however, EVs’ ability to induce a secretory phenotype Interestingly, EV-induced CAFs may in turn shed in CAFs still remains an open field of research (Webber additional EVs that will further support tumor growth by et al., 2015). In some cases, tumor cell-derived EVs contain conferring enhanced proliferative and survival capabilities to typical mitogenic factors, such as TGF-beta, which in ovarian cancer cells, thus establishing an EV-mediated bidirectional cancer induces a pro-tumoral secretome leading to increased intercommunication (Savardashtaki et al., 2019; Yang et al., proliferation, motility, and invasiveness of ovarian cancer cells 2019). For instance, CAFs exposed to cancer cell-derived EVs (Giusti et al., 2018). Furthermore, an increasing body of evidence in Hodgkin lymphoma are primed to secrete EVs together with points to the importance of EV-derived miRNA. For instance, in pro-inflammatory cytokines and angiogenic factors (Dörsam Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions et al., 2018). Similar to cancer cell-derived EVs, CAF-derived influence mechano-sensing and should be considered when EVs contain commonly miRNA, which has been described to studying forces instructing CAFs secretory functions (Acerbi promote migration and resistance to treatment in several tumors et al., 2015; Bauer et al., 2020). (Dourado et al., 2019; Hu et al., 2019; Qin et al., 2019; Sun et al., 2019; Wang J.-W. et al., 2019). Besides miRNA, CAF-derived Spatial and Temporal Plasticity EVs containing mitochondrial mRNA can educate cancer Intra-tumor heterogeneity of CAFs functional subtypes and their cells to increase oxidative phosphorylation system (OXPHOS) differential spatial pattern are already a widespread knowledge metabolism, which consequently induces escape from dormancy (Lambrechts et al., 2018; Awaji and Singh, 2019; Neuzillet et al., (Sansone et al., 2017). 2019). Thus, an effect of CAFs intra-tumoral location upon EV-mediated activation of CAFs can also occur indirectly their secretory function may be expected. Remarkably, fibroblasts through stromal cell mediators present in the TME. Indeed, located next to the invasive front of BC show higher capacity a recent study showed that tumor-associated macrophages to induce cancer cells migration and EMT in comparison with incorporate and transfer cancer cell-derived EVs to CAFs, which those located in the epicenter of the tumor (Gao et al., 2010). allows the formation of a pro-tumorigenic microenvironment In pancreatic cancer, two spatially separated subtypes were (Umakoshi et al., 2019). identified (Figure 2). An inflammatory CAFs subtype (iCAFs), Moreover, EVs content may be changing during tumor distant from cancer cells, showed a secretory phenotype with progression, which could result in a temporal modulation high interleukin and chemokine production [leukemia inhibitory of CAFs secretory phenotypes. Supporting this notion, EVs factor (LIF), IL-6, IL-11, IL-1, CXCL-1] in comparison with derived from primary or metastatic CRC promote different CAF periglandular myofibroblastic CAFs (myoCAFs), specialized in functional profiles, switching from a pro-angiogenic to a more stromal remodeling functions (Öhlund et al., 2017). However, ECM remodeling phenotype (Rai et al., 2019). Therefore, EV- these two subtypes may be the borders of a functional spectrum mediated communication between tumor cells and CAFs may depending on IL-1-R1 expression, conditioned by the balance depend on alterations in the composition of secreted EVs during between TGF-beta (pro-myoCAFs) and IL-1/JAK/STAT (pro- tumor progression. However, additional research is needed to iCAFs) signaling activation (Biffi et al., 2019). An additional confirm this hypothesis. antigen-presenting CAFs (apCAFs) population activating CD4+ T cells was recently identified in PDAC (Figure 2), suggesting Cell–Cell Contact and Mechanical Interactions CAFs subtype-specific immunomodulatory capacity (Elyada CAFs/cancer cells physical interactions can modulate CAFs et al., 2019). Along this line, Neuzillet and colleagues highlighted functions (Yamaguchi and Sakai, 2015), and the high complexity the complexity of CAFs heterogeneity in PDAC, describing at of this interplay has been recently illustrated (Arwert et al., least four different functional and spatially distributed subtypes 2020). For instance, CAFs may sense cancer cell genomic stress depending on periostin (POSTN), myosin 11 (MYH-11), and through cytoplasmic transcytosis and respond by expressing podoplanin (PDPN) expression (Neuzillet et al., 2019). Similarly, interferon (IFN)-β1, leading to an increased production of in BC (Figure 2), a subset of myofibroblastic CAFs (CAF- chemokines, cytokines, and other inflammatory factors [CXCL-1 S1: CD-29Med FAPHi FSP-1Low−Hi αSMAHi PDGFRbMed−Hi and 10, granulocyte-macrophage colony-stimulating factor CAV-1Low ) secreting differentially higher amount of CCL-11, (GM-CSF), macrophage migration inhibitory factor (MIF), CXCL-12, CXCL-13, and CXCL-14 was predominantly detected IL-6, and IL-8, among others] (Arwert et al., 2020). Hetero- close to epithelial tumor cells (Costa et al., 2018). The other cellular gap junction communications have also been described myofibroblastic (αSMAHi ) CAF-S4 subtype was preferentially between CAFs and cancer cells. This mechanism promoting located within the tumor tissue, whereas CAF-S3 (FSP-1Med and epithelial-to-mesenchymal transition (EMT), migration, PDGFRbMed−Hi ) was detected in the juxta tumoral healthy tissue. and invasiveness appears to be rather unidirectional—from CAF-S2 subtype (low or negative expression of stromal markers) CAFs toward cancer cell (Luo et al., 2018); however, a better appeared to be equally distributed in both areas (Costa et al., understanding of the underlying molecular mechanisms 2018). is still required. For greater complexity, temporal evolution of CAFs during Mechano-sensing between CAFs and the surrounding tumor progression makes it difficult to assess the exclusive ECM may equally modulate CAFs secretome. For example, effect of spatial distribution (Kalluri, 2016; D’Arcangelo et al., matrix stiffness and contractile forces have been shown to 2020). Indeed, Nidogen-2, a protein secreted by “vascular CAFs” determine CAFs behavior through different mechano-sensitive in murine BC model, was firstly detected among perivascular pathways. Among them, non-canonical YAP pathway activation cells but relocated within the tumor stroma at later stages of is promoting CAFs ECM remodeling and angiogenic functions tumor progression (Bartoschek et al., 2018). In PDAC patients, (Calvo et al., 2013). Indeed, secretion of pro-angiogenic VEGF-A different phenotypes were detected by CAFs secretome analysis by CAFs depends on factors involved in YAP pathway activation of primary tumors and matched metastatic tissue (Öhlund (Calvo et al., 2015). et al., 2017). In addition, the above-mentioned S1 and S4 CAFs These findings suggest a significant role of physical subsets have been recently found to be enriched in metastatic interactions in determining CAFs secretory phenotype. BC lymph nodes in comparison with matched primary tumor Remarkably, spatial (invasive front or tumor core) as well tissue (Costa et al., 2018; Pelon et al., 2020). In this setting, as temporal (early or late stage cancer) characteristics may CXCL-12 secretion was found to be responsible of CAF-S1 Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions FIGURE 2 | Several CAFs subtypes have been recently described in cancers of distinct origin. CAFs heterogeneity is associated with specific markers and discrete functions. Remarkably, CAFs characteristics may be related to their spatial localization in the tumor. tumor-promoting phenotype, whereas CAF-S4 seemed to be Anti-cancer Therapy as a Determinant of specialized in NOTCH-dependent contractile and remodeling CAFs Secretome functions (Pelon et al., 2020). Similarly, CAFs isolated from Chemotherapy prostate tumors at different stages revealed that CAFs secretome Several studies have addressed the modulatory effect of standard evolves during PCa development. While localized PCa- chemotherapy (CT) on the TME. CT induces shrinkage of cancer derived CAFs are characterized by FGF-7 secretion, CAFs cells compartment resulting in an increased representation of from metastatic PCa showed increased levels of matrix stromal cells in residual tumors (Goto et al., 2017). In this metallopeptidase (MMP)-11 and heat shock 70 kDa protein 1A context, factors secreted by CAFs after CT may significantly (HspA1A) (Eiro et al., 2017). enhance tumor regrowth from residual cancer cells (Hisamitsu As previously mentioned, increasing evidence suggests that et al., 2019). Current evidence points to CT-induced DNA PCa-derived CAFs secretome is modulated by AR signaling (Yu damage being a key mechanism influencing the repertoire of et al., 2013; Cioni et al., 2018). Interestingly, AR expression CAF-secreted factors after treatment (Figure 3). Indeed, CAFs by CAFs evolves during PCa progression (Olapade-Olaopa support tumor regrowth after DNA damage-mediated NF-κB et al., 1999; Gevaert et al., 2018). Indeed, metastatic PCa and signaling activation through secretion of WNT-16B (Sun et al., castration resistance PCa display significantly lower stromal 2012). Alternatively, treatment of lung adenocarcinoma with AR expression than localize tumor- and androgen-dependent cisplatin enhances IL-11 secretion by CAFs, which in turn PCa (Li et al., 2008; Singh et al., 2014). These observations promotes resistance of cancer cells to CT through the STAT-3 suggest that a temporal regulation of CAFs secretome may occur signaling pathway (Tao et al., 2016). Of note, CAF-induced IL- through the modulation of hormone receptors expression during 11/STAT-3 cell–cell survival signaling has been reported in other cancer progression. tumor types (Calon et al., 2012). Thus, CAF-dependent IL-11 Overall, a complex relationship between temporal and resistance mechanism may be of potential relevance in cancers spatial influence may instruct CAFs secretome. However, of distinct origin. Similarly, a direct effect of gemcitabine and it is still unknown whether tumor progression orchestrates 5-FU regimen toward a tumor-supportive secretory phenotype CAFs function and plasticity according to spatial factors of CAFs has been described in PDAC. In that case, higher or, conversely, distinct CAFs spatial subtypes are modeling expression of secreted factors [intercellular adhesion molecule tumor dynamics. 1 (ICAM-1), IL-6, GM-CSF, IL-8, CXCL-1] upon treatment has Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions CXCL-12 promotes EMT and invasion in pancreatic cancer (Li D. et al., 2016; Tommelein et al., 2018). Alternatively, RT-activated CAFs may also support intra-tumoral angiogenesis (Hellevik et al., 2013). Interestingly, several studies have reported that the immunoregulation exerted by CAFs remains unaltered after ionizing RT (Hellevik et al., 2013; Gorchs et al., 2015; Berzaghi et al., 2019). Thus, combination with immune-targeted therapies may be an attractive clinical approach for RT-treated patients. Targeted Therapy Originally designed to be specific to cancer cells, targeted therapies may also produce a range of undesirable off-target effects on stromal cells and particularly on CAFs secretome. For instance, specific secretion of HGF and neuregulin (NRG)-1 by fibroblasts upon exposure to vemurafenib -a BRAF inhibitor- was reported in melanoma (Fedorenko et al., 2015). Of note, the activation of MET by HGF has been suggested as a potential mechanism of acquired tyrosine kinase inhibitor (TKI) resistance to gefitinib in epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) (Yano et al., 2008) and to vemurafenib itself in BRAF-mutant melanoma (Wilson et al., 2012). In PCa, HGF and NRG-1 have been identified as non- cell-autonomous drivers of antiandrogen resistance. In this FIGURE 3 | Anti-cancer therapy impacts CAFs secretome. Stromal elements setting, NRG-1/epidermal growth factor receptor 3 (HER-3) develop their own response to systemic chemotherapy, radiotherapy, and signaling axis blockade demonstrated promising tumor growth targeted therapy. As a result, CAFs response to treatment may diminish direct suppression (Zhang Z. et al., 2020). More recently, Hirata and anti-cancer drug efficiency by promoting cancer cells self-renewal and survival. colleagues reported alternative CAF-induced resistance to BRAF inhibition. In this setting, BRAF inhibitor reprograms CAFs by enhancing platelet-derived growth factor receptor (PDGFR) been associated with the activation of stress-associated mitogen- activity, thus increasing ECM production –thrombospondin activated protein kinase (MAPK) signaling pathway [Jun N- (THBS)-1/2, tenascin C (TNC), or POSTN, among other matrix terminal kinase (JNK) and P38 MAPK] (Toste et al., 2016). components– and stiffness to potentiate cancer cells tolerance to Finally, the indirect effect of CT on CAFs secretome may also treatment (Hirata et al., 2015). These data illustrate how CAFs occur due to the dysregulated crosstalk between cancer cells and functions can be affected even by targeted therapies to enable the CAFs. For instance, Nab-paclitaxel treatment increases CXCL- emergence of resistance to treatment. 10 expression in pancreatic cancer cells, leading to lower IL- 6 secretion by CAFs subsequently impairing migration and Epigenetic Regulation of CAFs Secretome invasive capabilities of cancer cells (Feng et al., 2018). CAF phenotype may be driven by epigenetic deregulation rather than by somatic mutations (Qiu et al., 2008; Bianchi-Frias Radiotherapy et al., 2016; Pidsley et al., 2018). DNA methylation, histone Similarly to CT, radiotherapy (RT) has been reported to impact modifications, chromatin remodeling, and non-coding RNA– the secretome of CAFs (Figure 3). Rødningen and colleagues micro RNA (miRNA) and long non-coding RNA (lncRNA)–are showed that ionizing radiation altered the fibroblasts expression the well-described molecular mechanisms behind the epigenetic of genes involved in ECM remodeling as well as Wnt and CAF reprogramming and have been thoroughly reviewed IGF signaling to support cancer stemness and resistance to elsewhere (Marks et al., 2016; Melissari et al., 2020; Pan therapy (Rødningen et al., 2005; Wang Z. et al., 2019). Persistent and Zheng, 2020). Interestingly, several studies support the DNA damage, induction of senescence, and TGF-beta pathway hypothesis that an epigenetic switch would initiate the activation activation rather than cellular death are some of the proposed process leading to a stable CAF cell state with tumor-supportive mechanisms modulating CAFs secretome upon RT (Ansems and secretory phenotype (Albrengues et al., 2015; Kalluri, 2016). Span, 2020). Importantly, a vast body of evidence suggests that For instance, multiple genes coding for secreted factors, such RT enhances paracrine signaling between fibroblasts and cancer as IL-1a, CCL-5, and CCL-26; show differential hypomethylation cells through the IGF and TGF-beta pathways (Barker et al., patterns in CAFs and are consequently overexpressed in 2015; Tommelein et al., 2018). Promotion of EMT, enhanced pancreatic cancer. In this case, paracrine lactate secreted by cancer stemness properties, and increased resistance to therapy PDAC cells leads to ten-eleven translocation (TET) enzyme are among the various effects exerted by RT-activated CAFs activation triggering the epigenomic reprogramming (Bhagat (Arshad et al., 2015; Barker et al., 2015). For instance, IGF-1 et al., 2019). Remarkably, different epigenetic mechanisms activates the IGF-1R/AKT/mTOR survival pathway in CRC, and can merge into the same signaling cascade as happening Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions for the STAT-3 pathway. Indeed, SOCS-1 methylation and chronic hypoxia, is likely to play a relevant role in metabolic downregulation in PDAC-associated CAFs enhance STAT-3- reprogramming of CAFs (Becker et al., 2020). induced IGF-1 expression (Xiao et al., 2016) to promote the survival and proliferation of pancreatic cancer cells (Kopantzev Metastatic Cancer Cells Spreading et al., 2017). Alternatively, LIF-induced methylation of Shp-1 As mentioned above, CAF-secreted factors may also drive promoter abrogates its expression leading to a STAT-3-mediated cancer progression by enhancing the metastatic potential of CAF pro-invasive phenotype (Albrengues et al., 2015). cancer cells (Figure 4). For instance, while TGF-beta activates In conclusion, in the absence of genomic mutations, different genetic programs in a wide range of stroma cells, epigenetic alterations may be seen as key determinants of CAF the main contributor to its pro-tumorigenic influence seems phenotype and could become promising targets for cancer to be an autocrine loop maintaining the secretion of pro- treatment. Importantly, several clinical trials are currently metastatic factors by CAFs (Calon et al., 2012). In addition, exploring the benefits of DNA methylation therapies in CAF-secreted TGF-beta promotes paracrine reprogramming of solid tumors including colorectal (NCT01896856), pancreatic epithelial cancer cells by inducing EMT in tumors of distinct (NCT03264404), or prostate cancer (NCT03572387). Even origin (Zhuang et al., 2015). In CRC, TGF-beta induces IL- though cancer cells are the intended target, the above- 13 expression in fibroblasts, and subsequent TGF-beta/IL-13 mentioned data suggest a potential effect of DNA methylation synergy activates an EMT program in epithelial cells (Scharl et al., therapies over CAFs that will need to be carefully assessed in 2013). Alternatively, Calon and colleagues described TGF-beta- treated patients. activated CAFs secreting IL-6 and IL-11 leading to enhanced STAT-3-dependent survival and spreading of metastatic CRC cells (Calon et al., 2012). Similar observations were made in CAFS SECRETOME AS DETERMINANT OF gastric cancer where CAF-secreted IL-6 induced EMT and TUMOR DEVELOPMENT metastasis through STAT-3 pathway activation (Wu et al., 2017). Alternatively, CAF-secreted vascular cell adhesion protein Cancer Cells Self-Renewal (VCAM)-1 has been shown to stimulate lung cancer cells A vast body of evidence indicates that CAFs secretome may migration and invasion (Zhou et al., 2020). contribute to tumor progression by enhancing cancer stemness Besides their autocrine and paracrine activities regulating (Figure 4). Indeed, CAF-secreted IGF-2 as well as IL-6 and IL- progression to metastasis, CAFs-secreted factors may also 8 produced by CD-10+ GPR-77+ CAFs are promoting cancer support cancer spreading by influencing ECM deposition at both stemness and tumor formation in lung cancer (Chen et al., 2014; the primary and metastatic sites. For example, Chakravarthy Su et al., 2018). Similarly, PCa-derived CAFs with decreased and colleagues described that TGF-beta-activated CAFs could expression of AR promote stemness in cancer cells through IFN- induce the expression of large ECM components related to cancer γ and M-CSF secretion (Liao et al., 2017). Importantly, cancer progression (Chakravarthy et al., 2018). In this regard, TGF- stemness induced by CAF-secreted factors may involve different beta-activated CAFs may secrete POSTN in order to increase pathways. For instance, PI3K/AKT pathway activation drives proliferation, motility, and invasive properties in head and neck the progression of anal squamous cell carcinoma through IGF- cancer (HNC) cells (Qin et al., 2016). Similarly, CAF-secreted 2 secretion by the PDGFRB+ CAFs population (Cacheux et al., POSTN may also promote tumor progression in CRC through 2019). Similarly, IGF-2-secreting CAFs promote tumor regrowth YAP/TAZ activation in cancer cells (Ma et al., 2020). Of note, and decreased latency after primary resection in CRC (Unger Deng and colleagues showed that the YAP-activated Wnt/β- et al., 2017). Alternatively, NOTCH signaling may be triggered catenin pathway promotes colon tumorigenesis (Deng et al., by CAF-derived CCL-2 to induce stem cell features as observed in 2018). Hence, the overall data suggest a complex paracrine BC cells (Tsuyada et al., 2012). Finally, WNT signaling activation regulation of the Wnt pathway in cancer cells by TGF-beta- by CAF-secreted HGF in colorectal adenocarcinomas or by CAF- activated CAFs through POSTN secretion and YAP activation in secreted POSTN in head and neck squamous cell carcinoma order to enhance tumor progression and metastasis formation. and BC cells may also promote cancer stemness and further metastatic initiation (Vermeulen et al., 2010; Malanchi et al., Immunomodulation 2011; Yu et al., 2018). Somewhat contradictory evidences of CAFs immunoregulatory In addition to cytokine-driven signaling, cancer cells self- functions have been described during cancer progression renewal and aggressiveness may also depend on metabolic cues (Figure 4). While increased abundance of CAFs clearly predicts originating from the TME (reviewed in Reina-Campos et al., immune exclusion and immunotherapy failure, a complete 2017; Li et al., 2020). Specifically, CAFs originating lactate and depletion of CAFs was associated with decreased intra-tumoral glutamine production as well as autophagic alanine secretion are immune infiltration (Özdemir et al., 2014; Chakravarthy et al., sustaining cancer cells metabolism, stemness, and progression 2018). One reason explaining opposite functions of CAFs in to metastasis (Fiaschi et al., 2012; Curry et al., 2013; Sousa modulating immune infiltration may reside in the fact that et al., 2016; Yang L. et al., 2016). The specific mechanisms different CAFs subpopulations with distinct properties are enabling CAFs to deliver distinct nutrients to cancer cells are coexisting inside the tumor. For example, among the four CAF being explored. However, CAFs response to the surrounding subsets identified in BC, only CAF-S1 subtype characterized by constraints, such as the epigenetic reprogramming induced by high FAP expression promoted immunosuppression by secreting Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions FIGURE 4 | CAFs secretome determines cancer progression. CAF-secreted factors regulate cancer development by enhancing directly epithelial cancer cells self-renewal and aggressiveness. Alternatively, a range of CAF-secreted factors may support progression to metastasis by maintaining an immunosuppressive environment or by enhancing pro-tumorigenic angiogenesis. CXCL-12 and enhancing immunosuppressive T regulatory cells Similarly, FAP+ CAF-secreted CCL-2 mediates STAT-3 pathway (Treg ) capacity to block T effector cells proliferation (Costa et al., activation in MDSC to promote liver tumor growth and to 2018). Importantly, FAP+ CAFs are also the main producers suppress T cells proliferation during colorectal carcinogenesis of CXCL-12 in PDAC, and their specific depletion increases (Chun et al., 2015; Yang et al., 2016). Alternatively, CAF-secreted immunological control over tumor growth (Kraman et al., 2010; IL-6 and IL-8 may promote monocytes differentiation into M2 Feig et al., 2013). In this setting, the decreased expression of macrophages and further recruitment in the tumor to diminish CXCL-12 leads to intra-tumoral T cell accumulation, which in natural killer (NK) cells cytotoxicity against CRC cells (Cho turn synergizes with immuno- or chemotherapy to reduce cancer et al., 2018; Zhang et al., 2019). Finally, CAFs may directly cells abundance (Feig et al., 2013; Sherman et al., 2014). interfere with NK anti-tumoral functions through prostaglandin In addition to CXCL-12, a wide range of CAF- E2 (PGE2) secretion or MMPs production decreasing NK produced chemokines has been associated with tumor receptor ligands expression in tumor cells, thus reducing NK immunosuppression. For example, CAF-secreted CCL-5 receptor-dependent cytotoxic activity (Balsamo et al., 2009; Ziani induces CD4+CD25+ Treg cells in BC (Tan et al., 2011; Costa et al., 2017). et al., 2018). Notably, CCL-5 dependent Treg cells recruitment As mentioned earlier, CAFs are major producers of TGF- was associated with lung metastasis in BC mouse model (Tan beta, a key contributor to tumor immunosuppression (Calon et al., 2011). CAFs also participate in immunosuppressive et al., 2015; Batlle and Massagué, 2019). For instance, TGF-beta myeloid cell recruitment and differentiation by secreting SDF-1 produced by CAFs was correlated with CD8+ T cells exclusion, to attract monocytes and to induce their differentiation into which in turn has been associated with resistance to anti- myeloid-derived suppressor cells (MDSCs) contributing to PD-L1 antibody-based immune checkpoint inhibitor therapy cancer immune evasion (Gabrilovich and Nagaraj, 2009; Deng (Mariathasan et al., 2018). In this context, TGF-beta pathway et al., 2017). Alternatively, Wang and colleagues showed that blockade through TGF-beta receptor (TGFBR)-1 inhibition or CAF-secreted POSTN promoted MDSCs infiltration to establish TGF-beta blocking antibody enabled T cells infiltration into a pre-metastatic niche during breast tumor metastasis through the tumor, restoring the anti-tumor immunity induced by AKT and STAT-3 pathway activation (Wang Z. et al., 2016). the anti-PD-L1 antibody in BC and CRC pre-clinical models Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions (Mariathasan et al., 2018; Tauriello et al., 2018). In contrast, vascular formation including TNC production resulting in TGFBR-2 deletion in BC induces MDSCs recruitment to the pro-angiogenic paracrine signaling (Newman et al., 2011; Rupp TME, reducing T cell proliferation and activation (Yang et al., et al., 2016). 2008). Interestingly, TGFBR-2 deletion has been associated with the increased expression of stromal TGF-beta leading Resistance to Anti-cancer Therapy to MDSCs recruitment of indifferent cancers including HNC In addition to modulating tumor progression, CAFs secretome or PCa (Lu et al., 2006; Franco et al., 2011). These data may also promote resistance to systemic and targeted therapies. suggest that the genetic ablation of TGFBR-2 enhances the For instance, CAF-secreted SDF-1 upregulates the expression secondary activation of TGF-beta that may indirectly promote of special AT-rich sequence-binding protein-1 (SATB-1) in intra-tumoral immunosuppression. pancreatic cancer cells, which not only sustains pancreatic tumor growth but also mediates gemcitabine resistance (Wei et al., Angiogenesis 2018). Furthermore, CAF-derived IGF-1 and IGF-2 induce CT Neovascularization is an important process during resistance in pancreatic cancer (Ireland et al., 2016). IGF-2 also tumorigenesis. Interestingly, CAFs are the main source of increases insulin receptor/IGF 1 receptor (IGF1R) axis activation VEGF-A, the most potent pro-angiogenic factor, which binds in cancer cells to enhance resistance to anti-EGFR-targeted to its cognate VEGF receptor (VEGFR)-2 expressed on therapy in cholangiocarcinoma (Vaquero et al., 2018). In the endothelial cells (Figure 4) (Fukumura et al., 1998; Apte et al., same line, fibroblast-secreted HGF activates both the MAPK and 2019). Noma and colleagues showed that TGF-beta pathway PI3K/AKT pathways contributing to BRAF-targeted therapies’ activation in CAFs was essential to induce VEGF secretion primary resistance in melanoma and in a subset of colorectal and (Noma et al., 2008). In addition, Sánchez-Elsner and colleagues glioblastoma cancer cells (Straussman et al., 2012). demonstrated a synergistic cooperation between SMADs and Regarding hormonotherapy, CAFs-secreted factors contribute hypoxia-inducible factor (HIF)-1 alpha proteins to drive VEGF to estrogen receptor alpha (ER-α) phosphorylation in BC cells, expression, suggesting a potential link between TGF-beta thereby promoting tamoxifen resistance (Pontiggia et al., 2012). pathway and hypoxia to promote tumor angiogenesis (Sánchez- In addition, Li and colleagues reported that letrozole, an Elsner et al., 2001). Nevertheless, there is controversial evidence aromatase inhibitor lowering estrogen production, had opposite about the role of TGF-beta in tumor angiogenesis suggesting functional effects on CAFs secretome (Li K. et al., 2016). a differential expression of TGF-beta ligands during different Letrozole reduces CCL-2, CCL-5, and CXCL-1 expression in phases of angiogenesis (Pardali et al., 2010). For instance, Liu CAFs, possibly contributing to its efficacy against BC cells. and colleagues showed that TGFBR-1 inhibitor and VEGF Conversely, letrozole also increases CAFs secretion of POSTN, a synergistically induced tumor angiogenesis through α5-integrin factor involved in BC progression and metastasis (Kyutoku et al., upregulation (Liu et al., 2009). In contrast, TGF-beta-TGFBR-1 2011; Li K. et al., 2016). In PCa, Zhang and colleagues, described signaling has been involved in the promotion of BC angiogenesis that CAF-secreted NRG-1 activates the HER-3 signaling pathway through MMP-9 upregulation and through CAF-secreted leading to resistance to antiandrogen therapy (Zhang Z. et al., TGF-beta and SDF-1 in HCCs (Safina et al., 2007; Yang J. et al., 2020). Notably, NRG-1 expression in PCa-derived CAFs is 2016). increased after antiandrogen therapy that may contribute to In addition to promoting VEGF-dependent angiogenesis, potentiate resistance to treatment (Zhang Z. et al., 2020). CAFs may enhance tumor angiogenesis through additional Consequently, the presence in the tumor of a range of secreted paracrine signaling between stromal and cancer cells (Orimo factors involved in resistance to therapy may be the result et al., 2005; Unterleuthner et al., 2020). For instance, CAF- of CAFs’ own response to anti-cancer treatment. Indeed, CT derived CXCL-12 leads to CXCL-8 secretion by PDAC induces senescence-associated secretory phenotype in fibroblasts, cells inducing new vessel formation by endothelial cells typically characterized by the increased secretion of cytokines, (Matsuo et al., 2009). Alternatively, CAF-secreted galectin- chemokines, and growth factors previously associated with tumor 1 induces VEGF-A production by BC cells to promote progression and treatment resistance (Demaria et al., 2017). tumor angiogenesis (Tang et al., 2016). FGF has also been More specifically, Sun and colleagues demonstrated that primary recognized as a potent pro-angiogenic factor involved in tumor prostate fibroblasts increased WNT-16B expression in response angiogenesis (Compagni et al., 2000). Correspondingly, Pietras to CT. In this setting, WNT-16B enhanced prostate tumor growth and colleagues demonstrated that cervical carcinoma-derived and diminished CT cytotoxicity against cancer cells (Sun et al., CAFs are secreting FGF-7 and FGF-2, inducing both epithelial 2012). Finally, previously mentioned IL-11 secreted by CAFs proliferation and tumor angiogenesis (Pietras et al., 2008). upon cisplatin-based CT induced STAT-3 pathway activation and Finally, CAFs not only are directly promoting tumor chemoresistance in lung cancer cells (Tao et al., 2016). angiogenesis by secreting pro-angiogenic factors but also act indirectly by participating in ECM remodeling (Figure 4) (De Palma et al., 2017). For example, CAFs may support PROGNOSTIC AND PREDICTIVE VALUE vascularization through biomechanical activity and ECM OF CAFS SECRETOME stiffness promoted by MMP activity (Bordeleau et al., 2017; Sewell-Loftin et al., 2017). Importantly, CAFs are the most The identification of tumor features predicting prognosis, risk important suppliers of ECM-associated proteins essential to of relapse, and benefit from treatment is absolutely essential Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions for clinical decision-making in oncology. In this sense, a better (Yang et al., 2020). Finally, increased THBS-2 predicted decreased understanding of the TME has provided multiple prognostic OS in CRC patients (Wang X. et al., 2016). and diagnostic biomarkers in cancers of distinct origin (Bremnes CAF-derived exosomes transferring miRNA (Au Yeung et al., et al., 2011). For instance, CAFs biomarkers (Ha et al., 2014; 2016; Fang et al., 2019; Qin et al., 2019) and lncRNA (Qu et al., Dourado et al., 2018), tumor:stroma ratio quantification (Kemi 2016) to tumor cells have been reported to promote treatment et al., 2018; Vangangelt et al., 2018), and stromal gene expression resistance in different tumor types and were also evaluated as profiling (Finak et al., 2008; Frings et al., 2013; Calon et al., potential predictive biomarkers. In this context, serum detection 2015) are powerful tools predicting clinical outcome. However, of plasma exosomal miR-196a levels correlated with tumor size, addressing the exclusive impact of CAFs secretome on cancer lymph node metastasis, advanced tumor stage, and poor OS while prognosis remains a major challenge, and to our knowledge, there accurately discriminating chemoresistant and sensitive patients is no example of CAF-secreted biomarkers currently applied to in HNC (Qin et al., 2019). Of note, suppressing or interfering the clinical setting. with CAF-derived exosomes transference to cancer cells has been Cytokines and chemokines have been proposed as prognostic proposed as a novel therapeutic approach (Li et al., 2017). factors in different types of cancer. Yet, their pleiotropic nature Similarly to soluble factors, many ECM-related products are and multiple cellular origin as well as their distinct releasing contributing to the predictive power of stromal-originating gene patterns make their use as biomarkers especially challenging. signatures (Andriani et al., 2018; Yuzhalin et al., 2018; Jiang Circulating IL-6 has been associated with cancer progression et al., 2019). ECM remodeling enzymes, such as MMPs and tissue and poor prognosis in melanoma, BC, and gastrointestinal inhibitors of MMP (TIMP), which are not exclusively but mainly tumors, among others (Salgado et al., 2003; Hoejberg et al., released by CAFs, have also been suggested to predict cancer 2012; Vainer et al., 2018), but only few studies have assessed progression and response to treatment (Liu et al., 2019). Among IL-6 cellular origin. In bladder cancer patients for instance, them, Eiró et al. assessed the potential influence over patients’ CAF-derived IL-6 and ACTA-2 (αSMA coding gene) co- prognosis of TIMP-2 expression by CAFs at the tumor center upregulation correlated with poor survival (Goulet et al., 2019). and the invasive front of early stage BC. TIMP-2 resulted to be In esophageal adenocarcinoma, although IL-6 serum levels did a potent poor outcome predictor at both locations (Eiró et al., not correlate with patients’ outcome, the expression of ADAM- 2015). Many members of the MMP family promoting tumor 12, a surrogate marker for IL-6-producing CAFs, predicted invasion through ECM degradation are also impacting cancer poor prognosis after neoadjuvant chemoradiation (Ebbing et al., prognosis. For instance, increased MMP-1 expression by CAFs 2019). CXCL chemokines are additional examples of promising was associated with high risk of relapse in stage II CRC, and prognosis biomarkers associated with CAFs functions. Indeed, CAFs expression of MMP-9,−11, and−13 correlated with shorter CAF-derived CXCL-14 expression was correlated with shorter relapse-free survival in BC (González et al., 2009; Eiro et al., recurrence-free survival in estrogen receptor negative, triple 2019). Remarkably, the relevance of MMPs for prognosis might negative, and basal-like BC (Sjöberg et al., 2016), as well be cancer specific, as illustrated by MMP-2, which predicts poor as in additional tumor types (reviewed in Westrich et al., OS and PFS in NSCLC but associates with better survival in 2020). Similarly, CXCL-1 and CXCL-8 positivity in CAFs was patients with high grade BC (Leinonen et al., 2008; Niemiec et al., significantly associated with poor prognosis in gastric cancer 2013). patients (Naito et al., 2019), whereas CXCL-8,−10, and−11 CAFs Along this line, other CAF-secreted ECM components, expression correlated with resistance to neoadjuvant CT and such as collagens, have been recently recognized as important poor prognosis in BC (Xu et al., 2020). contributors to cancer progression and as potential liquid As previously mentioned, CAFs-secreted growth factors, such biopsy biomarkers in different tumor types (Giussani et al., as HGF or IGFs, are involved in resistance to targeted therapy 2019). Serum level of PRO-C3 –a biomarker of collagen III and may be of use to predict response to treatment. Indeed, production– is a promising example predicting poor OS in HGF expression was correlated with innate resistance to BRAF PDAC patients and may be a non-invasive actionable biomarker inhibition, and increased HGF plasma levels predicted worse for desmoplasia-targeting therapies (Willumsen et al., 2019). overall survival (OS) and progression-free survival (PFS) in Interestingly and probably due to TGF-beta capacity to stimulate BRAF-mutant melanoma patients (Straussman et al., 2012; collagens production by CAFs, serum levels of collagen fragments Wilson et al., 2012). Alternatively, CAF-secreted IGF-binding have been associated with response to TGF-beta-targeted therapy proteins have been proposed as a potential therapeutic target and (Nissen et al., 2019). prognostic biomarker in pancreatic cancer and other tumor types Overall, these exciting findings are calling for further (Thomas and Radhakrishnan, 2020). validation of CAF-derived biomarkers in order to improve the Additional CAFs-secreted proteins, such as VCAM-1, THBS- standard of care and decision-making in oncology. However, 2, and POSTN, involved in cancer progression were recently it is worth noting that methodological variability will be evaluated as potential actionable biomarkers. In lung cancer, the especially relevant when transferring biomarkers from bench to soluble fraction of VCAM-1 predicted relapse and lower OS, bedside. Indeed, evaluating either a single or a panel of factors whereas high POSTN expression was associated with shorter PFS through distinct techniques may complicate the validation of in ovarian cancer patients (Ryner et al., 2015; Zhou et al., 2020). CAFs secretome-based prognostic and predictive tools in the Of note, a recent meta-analysis confirmed the value of POSTN clinical setting (Paulsson and Micke, 2014). Spatial and temporal as a biomarker predicting poor outcome in different solid tumors heterogeneity (see the Determinants of CAFs Secretome section) Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions may be an additional issue that could be overcome by liquid (NCT04258150, NCT02767557), or liver cancer (NCT04338685). biopsy, a non-invasive method allowing real-time evaluation Among CAF-secreted molecules, ECM components have also of CAF-secreted biomarkers circulating in the bloodstream raised special interest as therapeutic targets. Yet, clinical trials (Herrera et al., 2019). failed to demonstrate the clinical benefit from COL-3, MMP-2, and MMP-9 inhibitors in patients (Chu et al., 2007). Similarly, PEGPH20, a PEGylated recombinant human hyaluronidase, did CAF-SECRETED FACTORS AS A not add benefit to standard CT in PDAC patients (Tempero et al., THERAPEUTIC TARGET 2020). Off-target effects and indirect resistance to treatment Targeting the CAFs Secretome Regulating involving CAFs functions may explain the lack of benefit of Cancer Cells Proliferation current stroma-targeting therapies. For example, Casanovas Since CAFs complete depletion or blockade of fibroblast- and colleagues described that VEGFR blockade induces tumor rich tumor stroma formation resulted in decreased anti- hypoxia and paradoxically triggers pro-angiogenic factors tumor immune infiltration and more aggressive tumors, recent production, such as FGF family members (Casanovas et al., strategies have rather focused on the regulation of CAFs 2005). Likewise, CAFs derived from anti-VEGF-resistant tumors originating paracrine and autocrine signaling (Özdemir et al., are secreting PDGF-C in order to reactivate tumor angiogenesis 2014; Rhim et al., 2014). For instance, CAFs reprogramming by (Crawford et al., 2009). A potential solution may come from vitamin A and vitamin D was shown to inhibit tumor-supportive multi-targeted therapies, such as nintedanib, a triple angiokinase secretome associated with cancer progression (Froeling et al., inhibitor blocking VEGFR, FGFR, and PDGFR. Nintedanib 2011; Sherman et al., 2014). Remarkably, gemcitabine and either reduces the CAFs expression of IL-6, IL-8, VEGF, and VCAM- vitamin A or vitamin D regimen resulted in significant tumor 1 as well as OPN and showed important clinical benefit in burden reduction in PDAC pre-clinical models (Sherman et al., combination with CT in NSCLC patients (Hilberg et al., 2008; 2014; Carapuça et al., 2016). Hence, a phase II trial is currently Reck et al., 2014; Yamanaka et al., 2020). evaluating the combination of CT and vitamin D in PDAC patients (NCT03415854). Yet, a previous phase II trial did not show any survival benefit with high doses of vitamin D3 Overcoming CAFs-Induced compared with standard doses in combination with CT in Immunosuppression metastatic CRC patients (Ng et al., 2019). Several preclinical studies have recently underscored the Importantly, vitamin A and vitamin D reprograming relevance of TGFBR-1 targeting in order to activate an anti- strategies are both associated with TGF-beta –a key autocrine tumor immune response. TGFBR-1 inhibitor (galunisertib) and and paracrine mediator of CAFs signaling– pathway inhibition. anti-PD-L1 combination treatment increased intra-tumoral T Indeed, vitamin D receptor ligands decrease fibroblast activation cell infiltration and activation, thereby promoting a potent by TGF-beta (Ding et al., 2013), whereas all-trans retinoic anti-tumor response in CRC and BC pre-clinical models. acid (ATRA) –the active metabolite of vitamin A– inhibits the Characteristically, this effect was associated with a reduction fibroblasts capacity to release active TGF-beta, thus impeding of fibroblasts activation and an increased anti-tumor immune autocrine TGF-beta activation (Sarper et al., 2016). Of note, genes expression (Holmgaard et al., 2018; Mariathasan et al., ATRA reprogramed fibroblasts are in addition reducing Wnt–β- 2018; Tauriello et al., 2018). Curiously, anti-PD-L1 treatment catenin signaling in the surrounding cancer cells through SFRP-4 alone enhanced the expression of TGF-beta activated CAFs secretion (Froeling et al., 2011). biomarkers, suggesting that TGF-beta pathway inhibition is TGF-beta has emerged as a potential therapeutic target in essential to enhance anti-PD-L1 therapy (Holmgaard et al., oncology. Notably, TGF-beta inhibitor in combination with 2018). In this context, two phase I trials are being conducted to gemcitabine improved the OS in locally advanced and metastatic evaluate the benefit of galunisertib in combination with immune PDAC patients (Melisi et al., 2018). A phase I/II trial is currently checkpoint inhibitors (anti-PD-1, anti-PD-L1) in refractory testing the ability of TGF-beta inhibitor to restore the sensitivity metastatic cancer patients (NCT02423343, NCT02734160). In to CT in patients with TGF-beta-activated program in metastatic contrast, Zhao and colleagues showed that another TGFBR-1 CRC resistant to CT (NCT03470350). The analysis of the TGF- inhibitor (TEW-7197) failed to increase anti-PD-L1 treatment beta-activated program in CAFs provided several additional efficacy in melanoma mouse model. TEW-7197 promoted therapeutic targets. Among them, IL-6 and IL-11 are secreted fibroblasts proliferation and diminished PD-L1 expression in interleukins activating STAT-3-dependent survival and spreading cancer cells through MMP-9 secretion by CAFs. Interestingly, the of metastatic CRC cancer cells (Calon et al., 2012). IL-11 or IL- authors reported increased TGFBR-2 expression upon treatment 6 inhibitors displayed potent anti-cancer activity in pre-clinical (Zhao et al., 2018). In this context, previously mentioned TGFBR- models of cancer of distinct origins (Coward et al., 2011; Putoczki 2-induced secondary activation of TGF-beta could potentially et al., 2013; Song et al., 2014). Siltuximab, an IL-6 inhibitor, did contribute to CAFs activation and to combination treatment not show benefit as monotherapy in CT-pretreated castration- failure (Lu et al., 2006; Franco et al., 2011). resistance PCa patients (Dorff et al., 2010). However, additional Bintrafusp alfa, a bi-functional fusion protein composed IL-6 pathway inhibitors are currently tested in combination of an anti-PD-L1 antibody and a TGF-beta “trap,” was with CT in patients with breast (NCT03135171), pancreatic recently designed to potentiate immune checkpoint and Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions TGF-beta inhibitors regimen. Bintrafusp alfa induces tumor but does not advocate for their translation into the clinical setting. regression and decreased spontaneous metastasis in a CRC Comprehensive -omics analyses are currently being used for mouse model by activating CD8+ T cells and NK cells an extensive characterization of CAFs secretome in order to while abrogating CAFs activation in the tumor (Lan et al., discover and validate robust biomarkers and novel targets within 2018). Clinical benefit of bintrafusp alfa is currently under the stroma (Principe et al., 2018). In this sense, liquid biopsy evaluation in several cancer patients’ trials including NSCLC appears to be a promising method for real-time evaluation of the (NCT03840902), BC (NCT03579472), HNSCC (NCT04247282), different components of CAFs secretome, allowing the detection and CRC (NCT03436563). of CAF-derived soluble factors, CAF-derived exosomes, and even Remarkably, previously mentioned ATRA-mediated CAFs circulating CAFs as potential biomarkers (Herrera et al., 2019). reprograming increases T cells infiltration in PDAC while Recent findings suggest a clear impact of both systemic blocking CAFs autocrine TGF-beta activation (Ene-Obong et al., and targeted anti-cancer therapies upon CAFs secretome that 2013; Sarper et al., 2016). In view of the above-mentioned data will need to be carefully assessed in view of patients’ response on TGF-beta inhibition, pancreatic cancer patients who will be to treatment (Figure 3). Understanding the impact of CAFs treated with ATRA (NCT04241276) may as well benefit from secretome on treatment resistance after therapy exposure will combination regimen with immune checkpoint inhibitors. provide original tools to monitor patient’s response. The Finally, CAF-secreted factors, such as FN and TNC, have implementation of clinical criteria evaluating components of been targeted for antibody-based delivery of immune enhancers the CAFs secretome will help in refining patients’ selection for specifically to the tumor site in order to concentrate their suitable therapies and improve oncological outcomes. effect on cancer tissue and decrease treatment-associated toxicity While CAFs contribution over tumor development is still a on healthy tissue (Pasche and Neri, 2012). For example, L19- matter of research, CAFs secretome has already demonstrated its TNF and L19-IL-2 immunocytokines conjugate an antibody- potential as a target for original therapeutic strategies in a wide recognizing FN with cytokines (TNF-α and IL-2, respectively) range of cancers (Figure 4). However, a better understanding (Neri, 2019). Preclinical data already suggested the robust of CAFs secretome has also underscored the importance of anti-cancer potential of L19-based immunocytokines (Lieverse defining the cell-type specific response to secreted factors. et al., 2020), and phase II and III trials are currently For instance, TGF-beta is a pro-metastatic cytokine currently testing the clinical benefit of L19-TNF-a and L19-IL-2 in targeted in multiple clinical trials (NCT04031872, NCT02452008, patients with soft tissue sarcoma (NCT03420014) or melanoma NCT03834662, NCT04574583). Yet, TGF-beta is also considered (NCT02938299, NCT03567889). as a tumor suppressor due to its cytostatic effect on cancer cells (Akhurst and Hata, 2012). Another example is IL-11, a CONCLUSIONS pro-metastatic factor that can be successfully targeted by IL- 11 signaling antagonist to reduce cancer progression (Putoczki From a general perspective, CAFs should be considered et al., 2013). Until recently, IL-11 was better known for its as a heterogeneous and dynamic stromal component which capacity to stimulate platelet production, and for decades, cancer evolution to distinct functional subpopulations is paralleling patients have been treated with rhIL-11 to overcome CT-induced the tumorigenic process. Spatio-temporally regulated factors thrombocytopenia (Isaacs et al., 1997; Cairo et al., 2005; Wu et al., and crosstalk with other TME components drive this co- 2012). These findings underscore the importance of carefully evolution from local tissue resident fibroblasts and other assessing every cell-type specific response to secreted factors cell types to distinct CAFs subtypes. Indeed, the intra- before therapeutic translation into the clinical setting. tumoral location effect over CAFs function is determined by CAFs may not be understood and targeted as a unique family spatial distribution of tumor cell-derived factors (Figure 1). anymore, and a detailed definition of CAFs secretome is still Among them, autocrine or paracrine communications and EVs an unmet need. For instance, CAFs and senescent fibroblasts interchange have been described to define CAF phenotypes. coexisting within the TME are able to secrete equivalent factors. However, the influence of other cell populations–infiltrating Hence, CAFs secretome and senescence-associated secretory immune cells, endothelial cells–, and biomechanical stress (ECM phenotype (SASP) may have overlapping tumor-promoting stiffness) also differentially distributed within the TME may effects (Sahai et al., 2020). play an important role in modulating CAFs behavior. Among Different treatment strategies, such as targeting a specific other potential functions associated with CAFs, there is solid functional subtype of CAFs, reprogramming CAFs backwards evidence of several well-defined cell sub-specializations with to a tumor-suppressor phenotype, or even switching between either contractile and ECM remodeling functions or secretory distinct functional subtypes, are currently being addressed. and immunomodulating functions (Figure 2). Along this line, a major challenge in precisely defining CAFs The potential role of CAFs secretome for predicting patient’s heterogeneity and therapeutically targeting CAFs secretome outcome and response to treatment has also been intensively will reside in a better comprehension of CAFs spatiotemporal investigated. Overall findings illustrate the potential benefit of evolution during tumor progression. using CAFs secretome biomarkers to improve patient’s selection Overall, it is reasonable to believe that increasing our and treatment follow-up. Notwithstanding their potential understanding of CAFs inter- and intra-tumoral heterogeneity as actionable biomarkers, current knowledge is still mostly will be key to fully grasp CAFs secretome diverseness and providing descriptive information of individual secreted factors potentiate anti-cancer therapy. Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 January 2021 | Volume 8 | Article 621070 Linares et al. CAFs Secretome Determinants and Functions AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS JL, JM-J, and AC participated to the manuscript preparation and This work has been supported by grants to AC from Instituto revision (writing, reviewing, discussing, and editing). JB-R made de Salud Carlos III with FEDER/FSE co-financing (CP16/00151; substantial contribution to the manuscript revision. All listed PI17/00211), to JL from the Spanish Association against Cancer authors approved this manuscript for publication. (AECC; Junior Clinician fellowship). REFERENCES in colon cancer stimulates cancer associated fibroblast-mediated prometastatic activin A signaling. Sci. Rep. 10:7606. doi: 10.1038/s41598-020-64239-2 Acerbi, I., Cassereau, L., Dean, I., Shi, Q., Au, A., Park, C., et al. (2015). Human Becker, L. M., O’Connell, J. T., Vo, A. P., Cain, M. P., Tampe, D., Bizarro, breast cancer invasion and aggression correlates with ECM stiffening and L., et al. (2020). 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