Current Perspectives on the Crosstalk Between Lung Cancer Stem Cells and Cancer-Associated Fibroblasts

Current Perspectives on the Crosstalk Between Lung Cancer Stem Cells and Cancer-Associated Fibroblasts.

ABSTRACT

Lung cancer, in particular non-small cell lung carcinoma (NSCLC), is the second most common cancer in both men and women and the leading cause of cancer-related deaths worldwide. Its prognosis and diagnosis are determined by several driver mutations and diverse risk factors (e.g. smoking). While immunotherapy has proven effective in some patients, treatment of NSCLC using conventional chemotherapy is largely ineffective. The latter is believed to be due to the existence of a subpopulation of stem-like, highly tumorigenic and chemoresistant cells within the tumor population known as cancer stem cells (CSC). To complicate the situation, CSCs interact with the tumor microenvironment, which include cancer-associated fibroblasts (CAFs), immune cells, endothelial cells, growth factors, cytokines and connective tissue components, which via a dynamic crosstalk, composed of proteins and exosomes, activates the CSC compartment. In this review, we will analyze the crosstalk between CSCs and CAFs, the primary component of the NSCLC microenvironment, at the molecular and extracellular level and contemplate therapies to disrupt this communication.

1 INTRODUCTION

Apart from being one of the most common types of cancer, lung cancer is the leading cause of cancer-related deaths worldwide (Siegel et al., 2016). In 2016 alone, new lung cancer diagnoses increased 14% and mortality related to lung cancer accounted for approximately 1 of every 4 cancer deaths (Siegel et al., 2016). There are 2 main histological subtypes of lung cancer: small cell (SCLC) and NSCLC, the latter being the most frequent (close to 80-85% of all lung cancers) and aggressive (> 5-year survival rate of 10%) (Zang et al., 2017). NSCLC can be further subdivided based on histology into adenocarcinomas (ADC), large cell carcinomas (LCC), bronchioloalveolar carcinomas (BAC) and squamous cell carcinomas (SCC) (Chatziandreou et al., 2015). If identified at an early stage, surgical resection improves prognosis as demonstrated for several cases of tumors in stage I (Harrison et al., 1987); however, early detection tests using standard radiography and sputum cytology have not provided a significant survival benefit. As a result, the use of more sensitive screening modalities (e.g. low-dose CT scanning) are becoming more readily used as they are able to identify small lung nodules three times more than standard early detection tests (Crino et al., 2010). Lung cancer prognosis in general is poor due to its inherent high recurrence frequency, late presentation (stage III/IV), tendency to spread and lack of curative systemic therapy (Park et al., 2016). For NSCLC patients in particular, poor response to chemotherapy due to short duration and infrequency of complete remissions compounds these statistics. The median overall survival rate of advanced NSCLC patients is approximately 10 months (Zang et al., 2017), which is primarily due to fact that patients are commonly diagnosed at an advanced stage since early stage tumor development is usually asymptomatic (Chatziandreou et al., 2015). At the molecular level, NSCLC can be classified based on the presence of common driver mutations, such as AKT1, ALK, BRAF, EGFR, HER2, KRAS, MEK1, and MET (Chatziandreou et al., 2015), with EGFR representing the most common driver mutation (Dogan et al., 2012) (11% to 43% of NSCLC patients) followed by mutations in KRAS (8% to 29% of NSCLC patients). Interestingly, EGFR mutations and ALK arrangements frequently appear in NSCLC patients who have never smoked, and EGFR mutations predominated over KRAS in patients with smoking histories of up to 10 packs per year (Dogan et al., 2012). Nevertheless, smoking is without a doubt an important risk factor, which

2 likewise reduces the efficiency of chemotherapy and aggravates lung cancer prognosis. EGFR is a tyrosine kinase present in most human tissues and which is commonly over-expressed in human cancers such as lung cancer. EGFR-mutated gene have been identified in NSCLC and are associated with poor prognosis. These mutations are somatic, and double mutations in the same allele of the EGFR gene have been observed (Yokoyama et al., 2006). While slightly less common, KRAS-mutant cancers are recognized to be more aggressive (Tomasini et al., 2016). Mutations in both EGFR and KRAS are not necessarily needed for lung cancer pathogenesis and therefore are rarely found concomitantly in the same tumor and are thus inversely correlated. Treatment of lung cancer has been classically based on surgery, radiation therapy to treat or prevent metastasis and radiofrequency ablation to kill cancer cells. Standard of care continues to be based on the use of platinum-based chemotherapies (carboplatin or cisplatin combined with cytotoxic drugs such as docetaxel, paclitaxel, gemcitabine, vinorelbine or pemetrexed) (Rossi and Di Maio, 2016). Recently, immunotherapy-based approaches have been successfully used to treat different types of lung cancer with high cure rates (1- and 2-year survival rates of 42% and 23%, respectively for lung cancer (Keating, 2016)); however, immunotherapy is not effective in all lung cancer patients for reasons that have yet to be determined. Thus, while different targeted therapy drugs are under development and novel therapies and new treatment strategies are showing some promise (reviewed in (Dholaria et al., 2016)), survival rates in general remain extremely low. The reason for these low survival rate statistics is believed to be due, in part, to the existence of a subpopulation of stem-like cancer stem cells (CSCs) or cancer-initiating cells (CICs) within the tumor population that are responsible for the high tumorigenic, metastatic and chemoresistant capacity of this tumor.

CANCER STEM CELLS

NSCLC is a very heterogeneous tumor in which we can find many types of cells that act differently depending on the mutations they bear and their location within the tumor, more specifically, within the microenvironment. Within the epithelial tumor cell compartment there exists a subpopulation of clinically relevant and highly tumorigenic cells known as CSCs that have been associated with clinical resistance and metastasis. Thus, these cells not only represent a relevant pharmacological target but they have taken center stage and are now

3 being extensively studied in hopes of developing new and more specific anti-cancer therapeutics (Albini et al., 2015; Kim et al., 2005). CSCs are defined by their capacity to self-renew, to give rise to the other tumor cells present within the tumor (e.g. progenitor cells and differentiated cells) and by their exclusive ability to initiate tumors in immunocompromised mice upon serial passage (Nguyen et al., 2012). In addition, CSCs have been directly linked to tumor progression, poor prognosis, disease recurrence, and metastasis (Plaks et al., 2015). These cells are dispersed within the tumor mass and represent only a small percentage of the total number of cancer cells present within the tumor (Albini et al., 2015). While the CSC concept is not new and was first proposed more than 150 years ago by Rudolf Virchow (Virchow, 1881), the identification of CSCs in lung cancer would not occur until 2005 when Kim et al. first discovered that lung cancer contains anatomically and functionally distinct epithelial stem cell populations (Kim et al., 2005), laying the groundwork for understanding the complexity and the heterogeneity of this tumor type at the cellular level. Normal lung somatic stem cells can be found in different locations within the lung (Kim et al., 2005). While it is not known whether lung CSCs arise from resident lung or non-lung infiltrating stem cells or from committed progenitor or more-differentiated epithelial lung cells (e.g. alveolar type II cells), it is believed that CSCs originate from a single cell that has suffered an accumulation of DNA damage events and acquisition of oncogenic mutations, mediating cellular transformation (Martinez-Climent et al., 2006) (Figure 1A). Apart from genetic and epigenetic factors, alterations in the tissue microenvironment (e.g. chronic inflammation) can also facilitate CSC genesis. For example, for some cancers it has been shown that mesenchymal or epithelial cells can suffer a de-differentiation process to become CSCs due to stress applied by the microenvironment (Hanahan and Weinberg, 2011). It has also been shown that an inflammatory microenvironment can contribute to cell plasticity during tumor development, initiating stem-like programs in non-CSC such as intestinal epithelial cells (Schwitalla et al., 2013). Thus, while the exact origin of lung CSCs is still open to debate, what is clear is that the process is likely multifactorial involving internal (Sutherland et al., 2014) (i.e. genetic) and external (i.e. microenvironmental) factors (Shukla et al., 2017). In general, cancer therapy has traditionally focused on treating the tumor as if it were a

4 homogeneous entity. We now realize that the cancer cell populations present within a tumor have different mutations and phenotypes, varying epigenetic signatures, distinct molecular patterns and even different exome sequencing (Zhang et al., 2014), highlighting that tumors are anything but homogenous. For example, a 2013 study by Roberts et al. sequenced 25 spatially distinct regions from seven operable NSCLCs and found that there was intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC (Apoliprotein B mRNA Editing Enzyme Catalytic polypeptide-like) cytidine deaminase activity (Roberts et al., 2013). In addition, the CSC hierarchy/heterogeneity model would suggest that targeting all the cells within the tumor alike would be ineffective and only elimination of the CSC would result in tumor eradication, while failure to do so would inevitably lead to tumor relapse. Thus, dissecting the heterogeneity of CSCs to discover common targetable pathways or signatures may prove more clinically advantageous. Towards this end, numerous studies have identified signaling pathways enriched in CSCs that enhance tumor survival, growth and infiltration (stimulating endothelial proliferation and extracellular matrix components), such as Wnt, SHH, Notch, Activin/Nodal and chemokine signaling (Clevers, 2011). It has also been found that microRNAs are also important in these cells (Bao et al., 2013). Specifically Let-7/miR-200, miR21 (an oncogenic molecule) and miR34a (a tumor suppressor) are involved in different aspects of cancer biology and survival, and are differentially expressed in lung CSCs (Fan et al., 2016). In addition to the intracellular CSC landscape, extrinsic features of the microenvironment surrounding the tumor mutually interact to influence the development and progression of the tumor as well as sustainment of the CSC niche (e.g. promoting proliferation, evading growth suppression, overcoming cell death, modulating cell metabolism) (Hardavella et al., 2016). When CSCs interact with their microenvironment, which includes CAFs, immune cells such as tumor-associated macrophages (TAMs), endothelial cells, connective tissue components, growth factors and cytokines, the overall biology of these cells change. In this Review, we will focus on the interplay and crosstalk between CSCs and CAFs, as an interaction that has been shown to be important in lung cancer (Albini et al., 2015) but has not been extensively reviewed.

CANCER ASSOCIATED FIBROBLASTS

5 The presence of fibroblasts (i.e. CAFs) in solid tumors with desmoplastic reactions has been known for more than 40 years (Willis, 1960). While their origin is debatable, during tumorigenesis, fibroblasts proliferate, activate and subsequently differentiate into myofibroblasts/CAFs (Figure 1B). In the last decades of the 20th century scientists discovered that apart from specific morphological differences, these tumor-associated cells express a different set of markers compared to local non-tumor-associated fibroblasts, the most common being alpha-smooth muscle actin (α-SMA), a specific marker for myofibroblasts (i.e. differentiated and activated fibroblasts) (Sugimoto et al., 2006). Attempts to describe the unique genetic and molecular pattern of these CAFs have revealed that CAFs are extremely heterogeneous both in the same tumor (subtypes) and when compared to fibroblasts of different origins, such as tissue-resident fibroblasts, vascular smooth muscle cells, pericytes (vascular adventitia), or bone marrow–derived cells (Sugimoto et al., 2006). While they are genetically heterogeneous, the majority of CAFs express α-SMA, as well as vimentin, FSP1 (fibroblast specific protein-1, also known as S100A4) and FAP (Fibroblast Activated Protein) (Sugimoto et al., 2006). In the context of lung cancer, Hoshino et al. also identified a sub-population of podoplanin-positive fibroblasts, originating from vascular adventitial fibroblasts that can enhance lung adenocarcinoma tumor formation (Hoshino et al., 2011), and recently it was shown that podoplanin expression in CAFs predicts poor prognosis in lung cancer patients (Yurugi et al., 2017). At the secretory level, CAFs secrete distinct molecules that are both different and similar to those secreted by normal fibroblasts. For example, like normal fibroblasts, CAFs can secrete typical fibroblast products such as collagen, fibronectin, or laminin, affecting extracellular matrix formation and remodeling (El-Nikhely et al., 2012), but they can uniquely secrete factors such as hepatocyte growth factor, TNF-α, IL-8, IL-1β, IL-6 and TGFβ1, the latter two having been shown to be promoters of EMT, CSC genesis and cancer cell dissemination (Shintani et al., 2013; Shintani et al., 2016). Contrary to what was first believed, we now appreciate that CAFs have an active role in lung cancer progression through paracrine crosstalk with cancer cells and more importantly, with lung CSCs. CAFs not only represent a key cellular component of the CSC niche, but they are believed to coevolve with the tumor and CSCs, migrating, differentiating and secreting factors that influence both surrounding immune cells and CSCs, thus promoting and maintaining

6 certain CSCs features. For example, Chen et al. recently discovered that CAFs, via IGF-II and IGF1-R interactions, can regulate the plasticity of lung cancer stemness via Nanog-dependent mechanisms (Chen et al., 2014) (a transcription factor involved in CSC pluripotency). The authors show that following IGFII/IGF1-R activation, numerous autocrine (LIF and LIFR; TGFβ1 and TGFßR1; Wnt and Frizzled receptor) and paracrine (HGF and HGFR/c-MET; SDF-1 and CXCR4) signaling mediators are induced. They further discuss that CAF secreted HGF, IGF-II, SDF-1, bFGF, Wnt and oncostatin M (OSM) regulate NSCLC CSC “stemness” in a paracrine-specific manner through activation of counterpart EMT, TGFβ1, Wnt, Notch and HH receptor/signaling components and CSC pluripotency factors (Oct3/4, Sox2 and Nanog), elegantly showing that factors secreted by CAFs play a very important role in lung CSC maintenance. Of the many pathways important for CSC biology, the HH signaling pathway has been shown to be important in NSCLC (Abe and Tanaka, 2016), and recently it has been shown that HH inhibition can suppress lung cancer stemness (Zhu et al., 2017). In vertebrates, the HH pathway is activated by three ligands (SHH, IHH and DHH), which influence the morphogenesis of many organs during early development (Abe and Tanaka, 2016). In cancer, CAFs are maintained by paracrine HH signaling pathway activation. Cancer cells (and CSCs) secrete HH factors that activate CAFs. In response, CAFs secrete HH ligands to activate HH signaling pathways in cancer cells and in CSCs, promoting stemness, CSC maintenance and metastatic properties, via downstream GLI transcriptional activators (Abe and Tanaka, 2016). In addition, HH signaling can promote angiogenesis and CAF proliferation through alternate non-canonical routes (Abe and Tanaka, 2016) involving EGFR signaling and TGFß1 (Abe and Tanaka, 2016) secreted by CAFs (Wu et al., 2017). Thus, the HH signaling pathway not only represents an important CAF-CSC crosstalk network, but may represent a feasible CSC pathway to therapeutically target. STAT3 signaling is another well-defined pro-CSC pathway (Bharti et al., 2016) and has been shown to be up-regulated in lung CSCs (Hsu et al., 2011). The family of IL-6 cytokines, including IL-6, OSM, and LIF are the most widely expressed in tumors and are associated with cancer progression (Hibi et al., 1996). These cytokines are known stimulators of STAT3 signaling, and have been shown to promote/activate CSCs and EMT in several cancer entities (Wendt et al., 2014). Shien et al. recently reported that CAFs potentiate the STAT3 signaling

7 in NSCLC via OSM secretion. In addition, this cross-talk could be inhibited with the selective JAK1 inhibitor filgotinib, which effectively suppressed STAT3 activation and OSMR expression. In addition, Hsu et al. showed that cucurbitacin I, a potent STAT3 inhibitor, reduces the tumorigenic ability and radiochemosensitivity of NSCLC-derived CD133-positive cells (Hsu et al., 2011). Thus, inhibiting the CAF-CSC crosstalk at the level of STAT3 signaling may have clinical relevance. EMT activation mediated by interactions between CAFs and cancer cells have been shown for several cancers (Giannoni et al., 2010); however, whether CAF-mediated EMT contributes to stemness and/or cell plasticity in NSCLC is still unknown. CAFs can certainly promote lung cancer EMT and cancer metastasis through the production of a variety of growth factors and proteins such as TGFß1 (a known inducer of EMT) (Shintani et al., 2016), Vascular Endothelial Growth Factor (VEGF-A, an angiogenic factor) and Tenascin-C (O'Connell et al., 2011) (which protects cancer cells against apoptosis). Likewise, through the IL-6/STAT-3/c- Myc pathway NSCLC CAFs can further promote TGFβ1 production (Shintani et al., 2016). While these studies highlight a direct link between CAFs and EMT, the implications reach beyond merely the induction of a migratory and invasive phenotype. For example, EMT transactivators have been associated with the maintenance of stem cell properties, plasticity and cell survival (Sarrio et al., 2012), and EMT induction has been shown to produce de novo breast CSCs (Mani et al., 2008). Thus, while more research is still needed, it would not be surprising that CAFs can also potentiate NSCLC CSCs via EMT-specific mechanisms or even promote non-CSCs to dedifferentiate into CSCs. A schematic summary of this communication is presented in Figure 2.

EXOSOMES AS MEDIATORS OF CSC-CAF CROSSTALK

Exosomes (reviewed in (Weidle et al., 2017)) are spherical nano-vesicles 40-100 nm in diameter, that are secreted by many cells and can be found in most body fluids. They are end products of the recycling endosomal pathway and contain a wide variety of components, which are specific to the origin of the exosomes, including: proteins (e.g. heat shock proteins, cytoskeletal proteins, adhesion molecules, membrane transporter and fusion proteins), lipids, mRNAs, different non-coding RNAs and miRNAs. Exosomes can transfer their constituents and cargo to neighboring or distant cells, with preservation of their function, and induce

8 genetic and epigenetic changes in the recipient cell. It has been noted that exosomes play a crucial role in cancer, not only facilitating the crosstalk between tumor cells and the microenvironment, but also by creating intratumor heterogeneity, mediating immunological responses via cytokine and chemokine production, inducing CAF proliferation and activation, stimulating angiogenesis and contributing to the formation and preparation of the premetastatic niche (Grange et al., 2011; Kobayashi et al., 2014; Minciacchi et al., 2015; Weidle et al., 2017). The role of exosomes in tumorigenesis is a relatively recent concept. The current literature suggests that there is a bidirectional communication between cancer cells and stromal cells mediated by exosomes, resulting in important genetic, epigenetic and phenotypic alterations. With respect to CSCs and NSCLC, the role of exosomes in lung cancer stemness and CAF activation and proliferation has not been fully elucidated; however, since the pluripotent and stem-like state of cancer “stem” cells has been linked to factors within CAF-secreted exosomes in other tumor entities (Hannafon and Ding, 2015), it is likely that lung CAFs communicate with CSCs via exosomes containing CSC-priming factors. For example, in colorectal cancer (CRC), CAFs can directly communicate with CRC CSCs using exosomes. Hu et al. showed that Wnt3a was detectable in both fibroblasts and fibroblast-derived exosomes in CRC, and since Wnt has been implicated in the maintenance of CRC stemness, the authors hypothesize that fibroblast-derived exosomes maintain CRC stemness and prepare CRC CSCs to become more chemoresistant (an inherent CSC property) through Wnt signaling pathway activation (Hannafon and Ding, 2015). Along these lines, fibroblast- derived exosomes were also shown to contain Wnt-11, which drives breast cancer invasion through Wnt-planar cell polarity signaling (Luga et al., 2012). Ye et al. also recently alluded to a partial exosomal signaling-mediated cross-talk between CSCs and the microenvironment in which the microenvironment promoted CSC self-renewal (Ye et al., 2014). Likewise, in a breast cancer model, Shimoda et al. showed that CAF-derived exosomes activated Notch signaling and increased ALDH1 (aldehyde dehydrogenase) expression in recipient breast CSCs (Ye et al., 2014). Thus, as we further our understanding of the cargo contained within CAF-derived exosomes, particularly in NSCLC, we are likely to discover more CSC-priming factors and activators; however, for now we can only extrapolate from other tumor entities with respect to how CAFs can communicate with lung CSCs via exosomes.

9 Given that solid tumors act as “organs”, and CSCs require a specific niche to regulate their stemness and self-renewal, CSCs must also actively communicate with MSCs and endothelial cells to maintain the tumor microenvironment and their niche. Cancer-derived exosomes contain abundant and diverse proteins and signaling factors, including TGF-ß1, TGF-ß2, IL-6, MMP2 and MMP9 (Chowdhury et al., 2015; Heneberg, 2016), factors that have been shown to be important in the transdifferentiation of tissue-resident fibroblast and MSCs to CAFs. Kumar et al. recently demonstrated that prostate and breast CSCs secrete exosomes with characteristics of cancer cell-derived exosomes (Kumar et al., 2015), suggesting that the cargo of CSC exosomes likely also have important roles during tumorigenesis. In fact, it is now accepted that CSCs secrete exosomes that interact with surrounding cancer cells and stromal cells (Grange et al., 2011; Marzesco, 2013). For example, exosomes derived from breast CSCs promote adipose tissue-derived mesenchymal stem cell transdifferentiation into myofibroblast-like cells (Cho et al., 2012). While the mechanism of transdifferentiation of MSCs and tissue-resident fibroblasts into CAFs is still unknown, our understanding of the role of TGF-ß1in this process and the presence of TGF-ß1in CSC-derived exosomes may provide a plausible mechanism of action. Currently, low-dose helical CT-scan is primarily used to detect pulmonary cancer in early stages; however, it is becoming apparent that new, rapid and non-invasive procedures for diagnosis and screening are still needed. Considering the growing relevance of exosomes in different cancers, it is reasonable to assume that they might also be used for early detection or, more importantly, as a prognostic biomarker. Along these lines, a 2016 review by Taverna S. and Giallombardo M et al. nicely summarizes the feasibility of isolating exosomes from NSCLC patient serum samples and characterizing their cargo (e.g. miRNAs) as well as their potential utility/role in clinical practice (Taverna et al., 2016). Regarding the latter, Cazzoli et al. showed earlier on that miRNAs derived from circulating exosomes could indeed be useful in lung adenocarcinoma screening and diagnosis. While at that time the authors stated that “further evaluation is needed to confirm the predictive power of their screening test in higher cohorts of samples”, their published exosome-based technique showed high reproducibility and a lower false positive rate compared to helical CT (Cazzoli et al., 2013). More recently, Qingyun Liu et al. have made significant advances in this area by showing that plasma exosomal miR-23b-3p, miR-10b-5p and miR-21-5p expression provided a significantly

10 improved survival prediction for NSCLC beyond conventional clinical predictors (Liu et al., 2017). While the sum of these studies suggests a putative use of exosomes as predictive biomarkers in NSCLC, no study to date has attempted to study the profile of CSC-derived exosomes. Thus, we may have yet to discover the true biological and prognostic potential of exosomes in NSCLC.

TARGETTING CSCS AND CSC-CAF CROSSTALK

As mentioned above, immunotherapy for NSCLC is “in style”, but it is not yet a perfect solution (D'Errico et al., 2017). Not all patients respond the same way to current immunotherapies, and a significant proportion simply do not respond. Traditional chemotherapy has a worse overall efficacy rate, as cancer cells with acquired resistance inevitably develop to these agents, resulting in treatment failure. These failures can be explained, in part, by intratumoral heterogeneity and the molecular complexity of many cancers, such as genetic mutations, interactions with the microenvironment and the presence of CSCs, the latter of which are known to be extremely radio- and chemoresistant (Chan et al., 2016; Hsu et al., 2011). Thus, there is still a need to develop new and novel NSCLC therapies, such as those that specifically target CSCs. Many different approaches have been explored with respect to targeting CSCs, and include for example, disrupting stem-like pathways that are activated in CSCs, disrupting the CSC niche and the crosstalk with the tumor microenvironment, or directing inhibitors to CSCs by taking advantage of CSCs markers (Figure 3). Every approach, however, has its intrinsic difficulties and obstacles. For example, CSCs exhibit high phenotypic and functional plasticity. As such, they can adapt to inhibitors of specific pathways more easily than non- plastic non-CSCs. For example, a recent study by Sancho et al. showed that metformin sensitive CSCs quickly became resistant to inhibition by this oxidative phosphorylation (OXPHOS) inhibitor by downregulating mitochondrial respiration and upregulating glycolysis to meet their energy requirements (Sancho et al., 2015). In addition, like normal stem cells, CSCs are also quiescent/slow cycling, and therefore it is believed that they are more inherently resistant to chemotherapies that tend to target highly proliferating cells. Furthermore, CSCs appear to evade chemotherapy or radiotherapy cytotoxicity through active mechanisms, such as upregulation of transporters that either efflux or sequester

11 chemotherapies (Visvader and Lindeman, 2012). Along these lines, Miranda et al. showed that CSCs can sequester compounds such as Mitoxantrone in ABCG2-coated intracellular vesicles effectively inhibiting the ability of this antineoplastic cytotoxic drug to induce cellular death (Miranda-Lorenzo et al., 2014). Lastly, targeting/blocking proteins secreted by CAFs or CSC as a means of disrupting the paracrine signaling between these two cells types may be more difficult than previously believed as many of the proteins secreted by these cells are contained within exosomes, as discussed above, making their neutralization difficult with current antibody-based approaches. Nonetheless, progress has been made in the development of inhibitors targetting CSCs and the paracrine signaling networks that exist between CSCs and their microenvironment. CSC cell surface markers. In general lung CSCs have been identified using side population, CD133, CD24, CD44, CD47, CD166 and ALDH1 (Leung et al., 2010; Tirino et al., 2009; Zakaria et al., 2017; Zakaria et al., 2015); however, compared to other types of cancers, lung CSC markers have been poorly defined and explored since lung cancer is considered one of the most genotypic and histologically complex tumors (Giangreco et al., 2007). CD44, for example, is likely the most widely used lung CSC marker, and while CD44 is a cell surface marker that has been used to identify and target CSCs from numerous tumor entities, it can also be expressed on non–CSCs, immune cells, or normal epithelial cells. Thus, similar to what is observed with other CSCs markers such as CD133, CXCR4, EpCAM, there exists no cell surface marker that can uniquely and specifically identify CSCs (Boo et al., 2016). Likewise, CSCs markers may vary in expression between patients with the same malignancy and may exhibit plasticity through the course of the natural history of the disease, as well as through exposures to various therapeutic agents. Nonetheless, researchers have attempted to take advantage of the expression of specific CSC markers on lung CSCs as a means of targeting and eliminating them (Figure 3). In a 2017 study by Liu et al., researchers showed that lung CSCs overexpress the anti-phagocytic “don’t eat me” receptor CD47. Using anti- CD47 antibodies to block CD47 function not only enabled macrophage phagocytosis of lung CSCs, but in immunodeficient mouse xenotransplantation models established with lung cancer cells or lung CSCs, anti-CD47 antibodies inhibited tumor growth and improved survival indicating that that CD47 is a valid CSC target. In fact, anti-CD47 antibody-based therapies is one of the most promising fields of research, as it has been shown to be effective

12 in many different tumor entities (Cioffi et al., 2015; Kaur et al., 2016; Lee et al., 2014) and currently several Phase I clinical trials are evaluating anti-CD47 monoclonal antibodies (e.g. Hu5F9-G4 and CC-90002) in treating patients with hematological malignancies and advanced solid tumors (ClinicalTrials.gov Identifier: NCT02678338 and NCT02216409). Likewise, CD133 targeted antibodies alone, conjugated or biofunctionalized on the surface of nanoparticles are also being tested against CSCs, but currently such therapies are still at the pre-clinical level (Huang et al., 2016; Prasad et al., 2015). Taken together, while some advances have been made (Naujokat, 2014), we are still far from capitalizing on CSC markers as an efficient means to identify, isolate and/or target these cells. Novel anti-CSC molecules. In the search for additional and novel CSC targets, miRNA or siRNA libraries as well as compound high-throughput screening have become areas of attention and increasing focus (Boo et al., 2016; Subedi et al., 2016; Yang et al., 2016). The application of high throughput screens to identify miRNAs that target CSC properties have revealed interesting findings. For example, miR-27a (Miao et al., 2013), miR-145 (Hu et al., 2014) and miR-34a (Basak et al., 2013) have been shown to modulate/regulate stem-like properties of lung CSCs. miR145, for example, has been shown to be a tumor suppressor and Hu et al. demonstrated that the repressive effects of miR-145 on CSC properties and EMT were mediated via the regulation of the pluripotency-associated gene Oct4 (Hu et al., 2014). miR-34a targets CD44, a cell surface marker used to enrich lung CSCs, and Jang et al. using nano-vesicle-mediated systemic delivery of miR-34a, inhibited CSC activity by repressing CD44 (Jang et al., 2016). Compound high throughput screens have also advanced the identification of selective inhibitors of CSCs (Gupta et al., 2009). For example, salinomycin, a class of compounds that had previously not been considered as anti-cancer drugs, reduced breast cancer tumor growth and lung metastases, possibly via directly targeting breast CSCs. While these approaches certainly open up new avenues of research for the future development of novel and anti-CSC therapies, and there are many small- and macro-molecules in the pipeline that have received patent protection from 2012-2015 (reviewed in (Kharkar, 2017)), these discoveries are still far from reaching patients in the clinical setting. More promising are the latest advances in the clinical development of new anti-CSC treatment strategies, focusing on targeting pathways known to control stem-cell self-renewal, proliferation, survival and differentiation. Agents that block the HH, Notch or Wnt signaling

13 pathways (pathways that intervene in tissue homeostasis, self-renewal and pluripotency) are notable examples as many CSCs, including lung CSCs, upregulate and rely on these archetypal developmental signaling pathways (O'Flaherty et al., 2012; Takebe et al., 2015). While we appreciate that there is a crosstalk and biological overlap between these pathways, and this adds to the complexity of cellular responses to external stimuli and poses challenges for the development of investigational drugs, these overlaps could be advantageous as multiple “stem” cascades could be potentially inhibited by directly targeting just one pathway. Thus, new treatment strategies targeting these pathways to control stem-cell replication, survival and differentiation are under development and extensively reviewed in refs. (Dholaria et al., 2016; Takebe et al., 2015) (Figure 3). Examples include, among others: Napabucasin (BBI608), considered a first-in-class cancer stemness inhibitor, functions by inhibiting the STAT3 pathway, and has been evaluated in NSCLC patients in combination with paclitaxel (ClinicalTrials.gov Identifier: NCT01325441). Demcizumab (VS-6063), a humanized IgG2 anti-DLL4 (delta-like ligand 4) antibody that suppresses Notch signaling, has shown interesting results when used in with carboplatin and pemetrexate in lung adenocarcinoma (ClinicalTrials.gov Identifier: NCT01189968). Likewise, Tarextumab (OMP-59R5), a human anti-Notch 2/3 receptor monoclonal antibody, is under evaluation for lung cancer (ClinicalTrials.gov Identifier: NCT01189968). CAFs and the CSC niche. Tumors are made up of malignant cells together with inflammatory cells such as TAMs, endothelial cells, adipocytes, and CAFs. Although some CSCs conceivably do not require a dedicated niche, others will be dependent on a specific set of extrinsic/paracrine interactions with their microenvironment. Thus, targeting the CSC niche could also be a viable therapeutic option to indirectly target CSCs (Figure 3). A multitude of studies have been published examining the cross-talk between TAMs and CSCs and the therapeutic benefit resulting from the disruption of this interaction (reviewed in (Sainz et al., 2016)); however, fewer studies have explored the potential of targeting/inhibiting the crosstalk that exists between CSCs and CAFs. One reason may be due to unfortunate result of translating to the clinic the study by Tuveson and colleagues using IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the HH cellular signaling pathway. In their 2009 study, Olive et al. showed that IPI-926 increased intratumoral vascular density and delivery of gemcitabine, resulting in transient disease stabilization in genetically-

14 engineered mouse models of pancreatic cancer (Olive et al., 2009); however, on January 27, 2012, Infinity Pharmaceuticals stopped the IPI-926-03 trial after an interim analysis found that pancreatic cancer patients in the gemcitabine + IPI-926 arm showed reduced overall survival compared to gemcitabine alone. While these results suggested that targeting the tumor-associated stromal tissue (i.e. CAFs) could be counterproductive in cancer patients, a new trial using vismodegib (formerly known as GDC-0449) has confirmed therapeutic effectiveness and response, and vismodegib is now being evaluated in multiple clinical trials against different tumor types, including lung cancer (ClinicalTrials.gov Identifier: NCT02465060). Thus, in the case of NSCLC, targeting CAFs with vismodegib, preventing myofibroblast activation and proliferation or selectively targeting CSC-CAF crosstalk factors may represent alternative or adjunct approaches to indirectly targeting lung CSCs. The effect of vismodegib on CSC and CAF cross-talk has already been shown for mammary gland tumors. Valenti et al. recently showed that CSC and CAF cross-talk is driven by combined activation of WNT/β-catenin and HGF/MET signaling. Specifically, CSCs secrete SHH, which regulates CAFs via paracrine activation of HH signaling, resulting in the secretion of factors by CAFs that promote CSC properties. In vivo treatment of tumors with the vismodegib reduced CAF and CSC expansion, resulting in an overall delay of tumor formation (Valenti et al., 2017). Likewise, in CRC, TGF-β signaling induces myofibroblasts to secrete HGF, reciprocally driving CRC proliferation and CSC expansion through cMET- dependent signaling (Gibbons et al., 2013). Moreover, HGF can maintain CSC function by activating the Wnt pathway, which induces CSC features and tumorigenic capacity in differentiated cancer cells that otherwise had limited tumorigenic capacity (Todaro et al., 2014). Selective targeting of TGF-β-mediated myofibroblast activation with TGF-β inhibitors or HGF/MET signaling in lung CSCs with ATP-competitive inhibitors of MET, AXL and FGFR1-3 receptors (i.e. S49076 (Clemenson et al., 2017; Rodon et al., 2017)) would be predicted to interfere with myofibroblast proliferation, activation and secretion of pro-CSC factors, potentially debilitating lung CSC or preventing the generation of CSCs from the non- CSC compartment (Dawood et al., 2014).

FUTURE PERSPECTIVES AND CONCLUSIONS While we are making gains, and appreciate the important role that CAFs play in lung cancer

15 and the direct impact they have on lung CSCs, we are still far from fully understanding this unique cellular relationship. Indeed, compared to other tumor entities such as CRC, breast and pancreatic cancer, few papers have been published dissecting the specific cross-talk between lung CSCs and CAFs. This is likely due to the biologically complex nature of lung cancer at the genetic and histological level. In Figure 4, we present a working model of the communication that exists between CAFs and CSCs. Nevertheless, more studies are still critically needed to complete this model and fully dissect the role CAFs play in lung CSC- mediated tumorigenesis. Since lung cancer continues to be the most common cause of cancer- related mortality worldwide, anti-CSC research is not only necessary, but may reveal new avenues of therapeutic intervention to treat this extremely aggressive and highly chemoresistant tumor. It is true that the development of CSC pathway or cross-talk inhibitors requires a deeper understanding of key nodes in the stem-cell signaling network, but with invested research efforts focused on this subpopulation of cells, such inhibitors may not be too far from becoming a clinical reality. Lastly, it is also very likely that the cross-talk between lung CSCs and CAFs plays an important role in shaping the immune evasion tumor landscape. To date little is known regarding Programmed death-ligand 1 (PD-L1) or Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression on CSCs, but as we increase our knowledge of lung CSCs, the CSC niche and the communication that exists between CSCs and the cells of the tumor stroma, we will surely discover that the expression of immune checkpoints inhibitors in lung cancer is affected by CSC-CAF crosstalk. Thus, as immunotherapy becomes a permanent part of the treatment arsenal for lung cancer, we should not loose site of CAFs and CSCs as critical players in tumor immune evasion.

ACKNOWLEDGEMENTS We would like to acknowledge the Introduction to Biomedical Research (18533) course offered by the Universidad Autónoma de Madrid School of Medicine for the opportunity to research and develop this comprehensive review, in particular the course director Dr. Maria del Carmen Iglesias de la Cruz.

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Figure legends

Figure 1. The origin of lung CSCs and CAFs. Shown are the putative origins of both (A) lung CSCs and (B) CAFs. Within the lung or in other tissues, normal stem cell (NSC), 26 mesenchymal stem cells (MSC), lung epithelial cells and/or myofibroblasts can be directly involved in this trans-differentiation process as a consequence of DNA damage events and/or the accumulation of mutations in EGFR, AKT1, BRAF HER2, KRAS or the epigenetic regulation of miRNAs such as miR21 or miR34. Likewise, chronic inflammation, microenvironmental stress conditions and extrinsic factors, such as smoking, can also play an indirect role in promoting the evolution of CAF or CSCs.

Figure 2. A model of the known molecular pathways involved in CAF-CSC crosstalk. (A) Cancer associated fibroblasts (CAFs) are tumor environmental cells that originate from a variety of sources (stromal fibroblasts, adventitia cells, bone marrow stem cells, etc.) and express markers that set them apart from ordinary fibroblasts, such as α-smooth muscle actin (α-SMA) or fibroblast activation protein (FAP). In the complex tumor microenvironment, CAFs promote the upregulation of several “stem” pathways in CSCs by secreting a milieu of different molecules and factors. (B) Of the most relevant in NSCLC are the Hedgehog (HH) ligands that have been shown to be necessary for the maintenance of CSC properties (via HH ligands and GLI transcriptional factors), but CSCs can also secrete HH ligands to promote CAF activation, proliferation and angiogenesis by means of non-canonical signals. (C) CAF paracrine signaling is also necessary to maintain CSC plasticity and pluripotency (through Nanog pathway activation via CAF-secreted IGF-II), the activation of known “stemness” routes such as WNT or Notch (due to HGF or WNT factors), EMT (by way of JAK1/STAT3 signaling through IL-6 and OSM), and the promotion of cancer metastasis and CSC genesis (via secreted TGFβ-1).

Figure 3. Pharmaceutical agents and their targets in intrinsic CSC pathways, cell- surface markers and microenvironment components. (A) While CSCs are inherently radio and chemoresistant, in part due to the expression of transporters that actively pump chemotherapeutics out of the cell, different experimental and clinical approaches have been contemplated to target, neutralize or eliminate CSCs in NSCLC. (B) First, overexpressed genes or pathways give way to new CSCs and these pathways are also important for their continuous maintenance, survival and proliferation. These CSC pathways could be targeted and inhibited by different agents, such as GSIs and Demcizumab (Notch inhibition),

27 Napabucasin and paclitaxel (STAT inhibition), Vismodegib (HH inhibition) or Vanituctumab or a BRAF inhibitor (Wnt inhibition). Wnt is also relevant because it is over-activated due to HGF secreted by myofibroblasts, and secreted Wnt has been shown to enable the dedifferentiation of differentiated cancer cells into CSCs. In theory, the inhibition of all these pathways would considerably reduce the CSC population within the tumor. (C) Secondly, CSCs can also be directly targeted based on the expression of intracellular and extracellular markers. Targeting these cell surface markers with monoclonal antibodies (against cell surface CSC markers such as CD44, CD47, or CD133) or targeting intracellular enzymes, such as ALDH-1 with diethylaminobenzaldehyde (DEAB), could facilitate the elimination or neutralization of existing CSCs. (D) Lastly, the microenvironment and the CSC niche represents a new putative CSC target. Research has shown that targeting CAFs using Akt or SHH inhibitors, or other components of the CSC niche, could severely impact CSC properties in the tumor.

Figure 4. Summary of the communication between CSCs and CAFs. During tumor and microenvironment/stroma establishment, CAFs facilitate extracellular matrix remodeling (collagen, fibronectin, laminin), stiffness and vascularization via cytokines, metalloproteins, and growth factors. CSCs on the other hand, mediate tumor growth and progression via their inherent stem and self-renewal properties. The latter is facilitated and strongly dependent on signals received from the microenvironment. CAFs and CSC maintain a dynamic and complex crosstalk mediated by secreted factors or exosomes containing proteins, lipids or RNAs. These free or encapsulated factors can trigger and control important and critical stem pathways, such as Nanog (pluripotency) and Hedgehog signaling, the sum of which participate in tumor progression, survival, metastasis, disease recurrence, and therapeutic resistance. These cells can additionally interact with Th2 lymphocytes, endothelial cells, epithelial cells and M2 macrophages M2, also present in the tumor microenvironment. Dissecting this complex crosstalk and deciphering all the mechanisms used by CSCs to communicate with tumor cells could have clinical implications at the level of treating NSCLC.

28 Current Perspectives on the Crosstalk Between Lung Cancer Stem Cells and Cancer-Associated Fibroblasts.

Cristina Alguacil Núñez1†, Inés Ferrer Ortiz1†, Elena García Verdú1†, Pilar López Pirez1†, Irene Maria Llorente Cortijo1†, Bruno Sainz, Jr. 1,2,3*

1Department of Biochemistry, Cancer Stem Cell and Tumor Microenvironment Group, Universidad Autónoma de Madrid (UAM), Madrid, Spain. 2Department of Cancer Biology, Instituto de Investigaciones Biomédicas “Alberto Sols” (IIBM), CSIC-UAM, Madrid, Spain. 3Chronic Diseases and Cancer Area - Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.

†These authors contributed equally to this work

*Corresponding author: Bruno Sainz, Jr., UAM, Calle del Arzobispo Morcillo 4, Madrid, Spain 28029. Phone: +34 91-497-3385, Email: [email protected]

Word count: Abstract: 163 words Text: 5691 words (without references)

Keywords: Lung cancer, non-small cell lung carcinoma, Cancer Stem Cells, Microenvironment, Cancer-Associated Fibroblasts, Exosomes, CSC-specific therapies, Epithelial Mesenchymal Transition.

Abbreviations (by alphabetical order): Anaplastic Lymphoma Kinase, ALK; Cancer- Associated Fibroblast, CAF; Cancer Stem Cell, CSC; Colorectal Cancer, CRC; Epithelial-to- mesenchymal transition, EMT; Epidermal Growth Factor Receptor, EGFR; Hedgehog, HH; Hepatocyte Growth Factor, HGF; Insulin-like Growth Factor, IGF; Kirsten rat sarcoma viral oncogene homolog GTPase, KRAS; Mesenchymal Stem Cell, MSC; Non-Small Cell Lung Cancer, NSCLC; Stromal cell-derived factor 1, SDF-1.

1 Current Perspectives on the Crosstalk Between Lung Cancer Stem Cells and Cancer-Associated Fibroblasts.