Journal of Cancer Research and Practice 4 (2017) 123e126 Contents lists available at ScienceDirect Journal of Cancer Research and Practice journal homepage: http://www.journals.elsevier.com/journal-of-cancerresearch-and-practice Review Article The novel roles of stromal ﬁbroblasts in metronomic chemotherapy: Focusing on cancer stemness and immunity Wen-Ying Liao a, Tze-Sian Chan b, Kelvin K. Tsai a, b, c, * a Laboratory for Tumor Aggressiveness and Stemness, National Institute of Cancer Research, National Health Research Institutes, Tainan City, Taiwan Laboratory of Advanced Molecular Therapeutics, Division of Gastroenterology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan c Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan b a r t i c l e i n f o a b s t r a c t Article history: Received 6 June 2017 Received in revised form 26 July 2017 Accepted 4 August 2017 Available online 8 August 2017 Metronomic chemotherapy involves the administration of cytotoxic chemotherapy at reduced doses administered at regular and frequent intervals. Low-dose metronomic (LDM) chemotherapy represents an alternative to standard maximum tolerated dose (MTD) chemotherapy as it is less toxic and offers additional beneﬁcial biological effects; such effects include inhibition of tumor neovascularization and reduced recruitment of immune-suppressive cells. In desmoplastic cancers such as breast and pancreatic cancers, carcinoma-associated ﬁbroblasts (CAFs) in the tumor stroma constitute an important cellular target of systemic chemotherapy, and the treatment-modulated CAFs may deleteriously inﬂuence treatment efﬁcacy. Herein, we reviewed the novel roles of CAFs in metronomic chemotherapy in desmoplastic cancers. We discuss the differential effects of MTD- and LDM-chemotherapy on the heterotypic interactions among CAFs and cells in the other cancer compartments, emphasizing the roles of cancer stem cells and myeloid-derived suppressor cells. The novel mechanistic roles of CAFs in cancer therapy provide an additional rationale for the clinical development of LDM chemotherapy. © 2017 Taiwan Oncology Society. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Carcinoma-associated ﬁbroblasts Metronomic chemotherapy Cancer stemness Myeloid-derived suppressor cells 1. Introduction 1.1. The importance of cancer-associated ﬁbroblasts (CAFs) in tumor microenvironment (TME) In 1889, Stephan Paget ﬁrst broached the “seed and soil” concept to explain the importance of the TME for cancer metastasis.1 The “soil” of tumor is composed of ﬁbroblasts, immune cells, surrounding blood vessels, signaling molecules, and extracellular matrix (ECM) which nourishes cancer cells. The TME is a dynamic milieu,2 and a profound crosstalk exists between stromal cells and immune cells. For instance, it is widely accepted that CAFs in the TME of desmoplastic cancers secrete amounts of growth factors, ECM components and matrix metalloproteinases (MMPs) for supporting cancer survival or proliferation. When CAFs become activated, they secrete many mesenchymal-speciﬁc proteins such as * Corresponding author. Graduate Institute of Clinical Medicine, Taipei Medical University, 250 Wuxing St., Xinyi Dist., Taipei City 11031, Taiwan. E-mail address: [email protected] (K.K. Tsai). Peer review under responsibility of Taiwan Oncology Society. ﬁbroblast-speciﬁc protein (FSP-1), ﬁbroblast-activating protein (FAP), vimentin, a-smooth muscle actin (a-SMA), cytokines, chemokines (e.g., CXCL18, CXCL12) and growth factors (e.g., VEGF, TGFb, EGF, PDGF).3,4 Some of the CAF-derived factors play important roles in tumorigenesis and cancer metastasis. For example, the CXCL12 (SDF-1)-CXCR4 signaling axis has been involved in bone and breast metastasis.5,6 On the other side, chemokines such as CCchemokine ligand 2 (CCL2) and CCL5 derived from CAFs may alter the composition of macrophage in the TME and immune cells recruitment into tumor, including regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSCs).7 In terms of CAFocentric concept, CAFs play essential roles in the crosstalk between various components in TME.8 1.2. Chemotherapy-activated CAFs support cancer progression Chemotherapy remains the standard treatment for unresectable cancer. Conventional chemotherapy is usually given in its maximum tolerated dose (MTD) to maximize the effect to resistant cancer cells. However, growing evidence has demonstrated that chemotherapeutic agents target both the tumor and the associated neighboring stroma. Treatment-altered TME can paradoxically http://dx.doi.org/10.1016/j.jcrpr.2017.08.001 2311-3006/© 2017 Taiwan Oncology Society. Publishing services by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). 124 W.-Y. Liao et al. / Journal of Cancer Research and Practice 4 (2017) 123e126 promote tumor progression. In a study undertaken to question the candidate biomarkers associated with breast cancer treatment resistance, factors such as CXCL2, MMP1, IL8, RARRES1, FGF1, and CXCR7 were highly induced by chemotherapy in CAFs.9 WNT16B is another important secreted protein derived from the stroma after cytotoxic chemotherapy treatment, and it promotes cell resistance to chemotherapy.10,11 Conventional chemotherapy can also activate CAFs to support tumor cellular hierarchy through IL-17A, and induce remodeling of the TME.12 From this, CAFs which have been “educated” through the process of chemotherapy are accomplices during aggressive tumor progression. 1.3. Metronomic chemotherapy prevents the effects of therapyactivated CAFs Metronomic chemotherapy, which is deﬁned as repeated administration of anti-neoplastic drugs at low doses frequently and without long drug-free period, renders a “cytotoxic” conventional chemotherapy into “therapeutic” by additional mechanisms, including inhibition of angiogenesis and stimulation of the immune system.13e15 In our recent work, we demonstrated that MTD chemotherapy could lead to accumulation of CAFs in the tumor stroma and induce oncogenic functions of CAFs in pancreatic ductal adenocarcinoma (PDAC) through the stromal-epithelial ELR-motifpositive (ELRþ)-chemokine/CXCR2 signaling axis. The paracrine signaling resulted in tumor stemness, neovascularization, tumorassociated macrophages (TAMs) inﬁltration, and tumor aggressiveness.16 A relevant study using the CYTOF platform and SPADE analysis showed that a signiﬁcant enrichment of MDSC subpopulation was found in breast cancer PDX mice models with MTDcapecitabine regimen.17 On the contrary, low-dose metronomic (LDM) chemotherapy regimen prevented therapy-induced activation of CAFs and attenuated ELRþ-chemokines production. Additionally, it reduced the recruitment of TAMs and MDSCs and attenuated cancer aggression. Therefore, understanding how LDM chemotherapy may enhance host immune response is an important subject that needs to be addressed. 1.4. The impact of therapy-activated CAFs on cancer stem cells Tumors are highly heterogeneous structures which contain a distinct subset of cancer cells termed cancer stem cells (CSCs). These cells are able to self-renew and generate the diverse cell types in the tumor.18 CSCs are intrinsically resistant to therapy, and their proportion increases following systemic treatment, facilitating tumor relapse.19 The homeostasis of CSCs is regulated by neighboring signals provided by surrounding stromal cells of the TME. The copious crosstalk among CSCs and cells in the TME is mediated by soluble paracrine factors from the different types of stromal cells.20e22 In keeping with this paradigm, inﬂammatory mediators, such as interleukin (IL)-6, and IL-8, have been found to be substantially involved in the regulation of CSCs, contributing to cancer invasion and metastasis.23e25 When the TME becomes injured and damaged by systemic chemotherapy, for example, CAFs will respond and become a resistant stromal cell type that contributes greatly in therapy resistance, mediated by enhancing tumor stemness. Conventional chemotherapy may activate CAFs to maintain colorectal CSCs which lead to tumor progression.12 Our previous report showed that traditional cytotoxic chemotherapy induced secretion of ELRþ-chemokines, which signal through CXCR-2 on carcinoma cells to trigger their phenotypic conversion into CSCs and promoted their invasive behaviors, leading to paradoxical tumor aggression following therapy. Interestingly, the effects can be tempered by changing the administration scheme to metronomic chemotherapy.16 These studies illustrated the importance of stroma in cancer therapy, and how its impact on treatment resistance could be induced by promoting tumor stemness. 1.5. The relationship between therapy-activated CAFs and tumor immunity Immuno-oncology is a new focus in the ﬁeld of novel drug development. Immuno-checkpoint inhibitors hold great promise as a treatment for cancer. The most impressive effect of immune checkpoint blockade is its success regarding prolonging patient survival and inducing long-lasting tumor regression.26e28 A metaanalysis of 1861 patients with advanced melanoma who received ipilimumab, an anti-CTLA-4 monoclonal antibody, followed up for 3e10 years displayed approximately 20% long-term overall survival.27 However, other types of cancer such as PDAC respond poorly to immunotherapies, suggesting alternative mechanisms of resistance such as tumor microenvironment-driven immune suppression. CCL2 chemokine nitration resulted in the trapping of tumor-speciﬁc T cell in the tumor-stroma, contributing to tumor evasion from T cell surveillance.29 CAFs also mediated immune suppression through CXCL12/CXCR4 axis by decreasing the number of T cells in the tumor immune microenviroment.30,31 Consistently, existing studies have illustrated the importance of host immune responses as key regulators to cancer therapy.32 Thus, the way to overcome immunotherapy resistance in the tumor and improve the efﬁcacy of immune-checkpoint inhibitor remains an urgent issue. 1.6. Therapy-activated CAFs and myeloid-derived suppressor cells In an immunosuppressive tumor microenvironment, MDSCs play important roles in helping the tumor cell escape immune surveillance by promoting T-cell dysfunction through a variety of mechanisms, including oxidative stress via inducible nitric oxide synthase and nutrient depletion mediated by arginase production.33,34 MDSCs can also secrete S100A8/9 to enhance cancer cell survival,35 and matrix metalloproteinases (MMPs) such as MMP14, MMP13 and MMP2 to promote tumor cell invasion.36 They also secrete IL6 to induce CSCs through STAT3 and NOTCH signaling.37 MDSCs can be phenotypically and morphologically divided into the monocytic (Mo-MDSCs; CD11bþLy6ChiL6Glow) and the granulocytic (Gr-MDSCs; CD11bþLy6ClowL6Ghi) subsets in mice. MoMDSCs mainly express CD11b and the chemokine receptor CCR2, and expand in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or CCL2.38,39 On one hand, a study using a murine liver tumor model indicated that FAP-expressing CAFs were a major source of CCL2, which enhanced the recruitment of MDSCs through FAP-STAT3-CCL2 signaling,40 on the other hand, the trafﬁcking of Gr-MDSCs to the tumor relies primarily on their expression of CXCR2.41,42 In humans, LinHLADR-CD33þCD11bþMDSCs isolated from the blood of patients with cancers are found to be immunosuppressive, and their levels increase especially in patients with high metastatic tumor burden.43 However, little is known about the crosstalk between CAFs and MDSCs after chemotherapy treatment. It is generally understood that MTD chemotherapy-treated CAFs can drive oncogenic activities and induce ELRþ-chemokine production. However, whether CAFs in chemotherapy-treated desmoplastic tumors could contribute to immune surveillance, and if so, whether this can be avoided by tuning the way to metronomic chemotherapy remain key questions to be addressed in the future. 2. Conclusion and future directions To date, a number of clinical trials have supported either LDM W.-Y. Liao et al. / Journal of Cancer Research and Practice 4 (2017) 123e126 125 Fig. 1. The schematic displays the proposed mechanisms underlying the pro-oncogenic functions of therapy-activated CAFs. chemotherapy alone or in combination with targeted therapeutics or anti-angiogenic drugs to be an effective approach in cancer treatment.44,45 For instance, in patients with breast cancer, it has been estimated that LDM chemotherapy yielded an average response rate of 39%, with an average overall clinical beneﬁt of 57%. Recently, a large randomized phase III trial, the CAIRO3 trial, provided solid support for the clinical beneﬁts of maintenance LDM chemotherapy in metastatic colorectal cancer.46 In our recent study, we have undertaken elegant molecular and in vivo studies to demonstrate the role of therapy-modulated CAFs on the treatment outcome of desmoplastic cancers. Speciﬁcally, MTDchemotherapy-treated CAFs were proﬁcient in promoting tumor aggression, invasiveness and progression by bolstering cancer stemness through the CAF-speciﬁc NF-kB/STAT1-ELRþ-chemokineCXCR2 paracrinal signaling. The same paracrine signaling process also led to increased angiogenesis and immunosuppressive macrophage inﬁltrations following MTD chemotherapy, which further promoted tumor progression (see Fig. 1). By contrast, LDM chemotherapy can temper the inadvertent activation of CAFs and improve cancer treatment outcomes. Our study also raises the possibility of the role of MDSCs in chemotherapy-induced stromal changes and treatment outcome in desmoplastic cancer. ELRþ-chemokine, including CXCL1, CXCL2, CXCL5 and CXCL8, have been shown to be elevated in various types of solid cancers, including breast cancer,35,36 pancreatic cancer,42 colorectal cancer47 or sarcomas.41 Since genetic deletion or pharmacological inhibition of CXCR2 or its ligands blunt the tumortrafﬁcking of Gr-MDSCs and enhanced the anti-tumor efﬁcacy of PD1 therapy for several types of malignancies,35,41,42 we posit that the ELRþ-chemokines secreted by MTD-chemotherapy-treated CAFs in desmoplastic cancers would induce the trafﬁcking of CXCR2-positive Gr-MDSCs into the tumor stroma, contributing to the immune-suppressive microenvironments of the tumor following the therapy, thereby promoting tumor progression and resistance to immune-modulatory agents. If so, the therapyinduced MDSCs recruitment and immune suppression can be prevented or at least tempered by adopting the LDM chemotherapy regimens, which are associated with attenuated ELRþ-chemokine production from CAFs. In this regard, we presume that LDM chemotherapy may have a better synergistic effect with immunemodulatory agents such as immune checkpoint blockade therapy than the traditional MTD chemotherapy in desmoplastic cancers. Further proof-of-principle preclinical studies and clinical trials are warranted to support this new therapeutic concept, which may provide a novel avenue to enhance the treatment outcomes of patients with desmoplastic cancers. Conﬂict of interest The authors declare no conﬂicts of interest in this study. Acknowledgments This work was supported in part by grants MOST 102-2628-B400-MY3, MOST 104-2314-B-400-022-MY3 and MOST 103-2314-B400-019 from Ministry of Science and Technology, Taiwan, and NHRI CA-103-SP-01 and NHRI-014-A1-CASP01-014 from National Health Research Institutes, Taiwan (K.K. Tsai). References 1. Paget S. The distribution of secondary growths in cancer of the breast. Cancer Metastasis Rev. 1989;8:98e101. 2. Swartz MA, Iida N, Roberts EW, et al. Tumor microenvironment complexity: emerging roles in cancer therapy. Cancer Res. 2012;72:2473e2480. €berg E, Frings O, et al. Cancer-associated ﬁbroblasts expressing 3. Augsten M, Sjo CXCL14 rely upon NOS1-derived nitric oxide signaling for their tumorsupporting properties. Cancer Res. 2014;74:2999e3010. 4. Karagiannis GS, Poutahidis T, Erdman SE, Kirsch R, Riddell RH, Diamandis EP. Cancer-associated ﬁbroblasts drive the progression of metastasis through both paracrine and mechanical pressure on cancer tissue. 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