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MiR-223-3p inhibits angiogenesis and promotes resistance to cetuximab in head and neck squamous cell carcinoma

MiR-223-3p inhibits angiogenesis and promotes resistance to cetuximab in head and neck squamous... www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 34), pp: 57174-57186 Research Paper MiR-223-3p inhibits angiogenesis and promotes resistance to cetuximab in head and neck squamous cell carcinoma 1,2,5 1 1 1,3,4,5 Alexandre Bozec , Joséphine Zangari , Mathilde Butori-Pepino , Marius Ilie , 3 1 3,4 1,5 1,3,4,5,* Salomé Lalvee , Thierry Juhel , Catherine Butori , Patrick Brest , Paul Hofman 1,5,* and Valérie Vouret-Craviari Université Côte d’Azur, INSERM, CNRS, IRCAN, Nice, France Head and Neck University Institute, Nice, France Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, Nice, France Hospital-Related Biobank (BB-0033-00025), Pasteur Hospital, Nice, France FHU OncoAge, Nice, France These authors contributed equally to this work Correspondence to: Valérie Vouret-Craviari, email: [email protected] Keywords: MiRs, tumors, neutrophils, inflammation, anticancer agents Received: December 21, 2016 Accepted: June 29, 2017 Published: July 11, 2017 Copyright: Bozec et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT MicroRNAs (miRs) participate in tumor growth and dissemination by regulating expression of various target genes. MiR-223-3p is suspected of being involved in head and neck squamous cell carcinoma (HNSCC) growth although its precise role has not been elucidated. In this study, we showed that miR-223-3p is present in biopsies of HNSCC patients and that its presence is correlated with high neutrophil infiltrate. We found that overexpression of miR-223-3p slightly increased proliferation of the CAL27 squamous carcinoma cell line both in vitro and in vivo. Moreover, miR-223-3p induced CAL27 apoptosis in an orthotopic xenograft mouse model, counteracting the proliferative effect and resulting in no impact on overall tumor growth. We analyzed the effect of miR-223-3p overexpression on signaling pathways and showed that it induced pERK2, pAKT and AKT, consistent with an increase in cell proliferation. In addition, we found that miR-223-3p reduced the STAT3 level correlating with increased cell apoptosis and inhibited vasculature formation. In HNSCC tissues, miR- 223-3p expression was inversely correlated to CD31, highlighting the relationship between miR-223 and vessel formation. Finally, we studied the effect of miR-223-3p on response to selected anticancer agents and showed that cells expressing miR-223-3p are more resistant to drugs, notably cetuximab. In conclusion, our study is the rst fi to show the antiangiogenic properties of miR- 223-3p in HNSCC patients and to question whether expression levels of miR-223-3p can be evaluated as an indicator of eligibility for non-treatment of HNSCC patients with cetuximab. of their disease [2]. Patients with HNSCC presented an INTRODUCTION altered cytokine profile compared to healthy controls. In There are approximately 600,000 new cases of head particular, IL-8, IL-6, TNF-α, MCP1 and MIP-1α were and neck squamous cell carcinoma (HNSCC) annually more expressed in the plasma of HNSCC patients [3, 4]. worldwide and HNSCC represents the 6th cause of Neutrophils are important mediators in cancer progression cancer death [1]. Most HNSCC patients are diagnosed and the neutrophils to lymphocytes ratio is an independent with locally-advanced disease and half of them will die predictor of recurrence in HNSCC [5–7]. Recent studies www.impactjournals.com/oncotarget 57174 Oncotarget associated neutrophils with poor clinical outcome in of head and neck origin has been poorly studied. In this HNSCC patients [8, 9]. Peripheral blood neutrophils study, we analyzed the expression of miR-223-3p by in situ from HNSCC patients and healthy donors showed distinct hybridization in 35 tumors from HNSCC patients. As functional differences, among them an increased number shown in Figure 1A, low signal was detected in the normal of immature stages of neutrophils in HNSCC patients [10]. epithelium. On the contrary, T1 and T2 HNSCC exhibited Several studies demonstrated that a high neutrophil a high expression level of miR-223-3p in CK-positive infiltration rate in the tumor was associated with more epithelial cells (Figure 1B). This expression decreased advanced disease and poor prognosis in HNSCC with the size of the tumor (Table 1). Neutrophils being a patients [9, 11]. HNSCC induces recruitment, survival, major source of miR-223-3p, we aimed to characterize the and release of proinflammatory factors such as CCL4 and presence of these cells in HNSCC. As shown in Table 1, IL-8 by neutrophils [11, 12]. Moreover, tumor-infiltrating and illustrated in Figure 1B, some neutrophils are closed neutrophils may be a major source of MMP9, which to miR-223-3p positive cells. As illustrated in Table 1, we can promote cancer cell invasion and metastasis [12]. observed a correlation between high neutrophil infiltration Dumitru et al. reported that neutrophils released soluble and high miR-223-3p expression levels. factors which phosphorylated cortactin in HNSC cells and promoted their migration [13]. Furthermore, these authors Effect of miR-223-3p on in vitro cell proliferation demonstrated that strong cortactin phosphorylation and migration significantly correlated with strong neutrophilic infiltration in tumor tissues from HNSCC patients [13]. Finally, after Since HNSCC expresses high levels of miR-223-3p, neutrophil recruitment in the tumor microenvironment, we aimed to characterize its effect on cell proliferation, HNSC cells can modulate the biology of neutrophils, migration and survival. First, we engineered a head and which in turn may facilitate cancer progression [11]. neck cancer cell line overexpressing miR-223-3p by Deregulation of microRNAs, a group of small transducing CAL27 cells with hsa-miR-223 and luciferase noncoding RNAs, plays a major role in cancer plasmids.RT-qPCR analysis of total RNA isolated from development [14, 15]. Chen et al. recently identified a CAL27 and CAL27 miR-223 cells confirmed that miR- panel of microRNA deregulations that were observed 223-3p was overexpressed in transfected cells (Figure 2A). in HNSCC, including 7 consistently up-regulated It is known that MiRs modulate the transcription of microRNAs (miR-21, miR-7, miR-155, miR-130b, their target genes. Recently it was shown that miR-223 miR-223-3p, miR-34b) [15]. Interestingly, miR-223-3p decreased activation of EGF receptor [22]. Because EGFR plays a critical role in the maturation and biology of plays crucial roles in the biology of head and neck cancer granulocytes [16]. Several studies showed that miR-223-3p cell lines, we verified that miR-223-3p did not inhibit the was up-regulated in tumor tissue and in plasma of cancer EGFR transcription/translation program (Supplementary patients, including esophagus and oral cancer patients Figure 1). Further, we analyzed the effect of miR-223-3p [17, 18]. Furthermore, it has been shown that platelets and on CAL27 cell proliferation and showed that CAL27 macrophages were able to modulate and promote cancer miR-223 cells displayed increased cell proliferation progression through exosome-mediated delivery of miR- as compared to control cells (Figure 2B). However, 223-3p [19–21]. In this context, miR-223-3p may be one expression of miR-223-3p in CAL27 cells had no effect of the main granulocytes-secreted molecular factors able on cell migration, as illustrated in the wound-healing assay to modulate the biology of cancer cells. (Figure 2C). The purpose of this study was to analyze the effects of miR-223-3p on HNSC cells, both in vitro and in vivo. Effect of miR-223-3p on in vivo tumor growth We examined the impact of miR-223-3p expression on migration, proliferation, apoptosis and drug resistance We used an orthotopic xenograft model consisting of properties of HNSC cells and on some key molecular implantation of CAL27 and CAL27-miR-223 cells in the signaling pathways implicated in cancer progression. mouth floor of nude mice to characterize the effect of miR- 223-3p on tumor implantation and tumor growth. One day RESULTS after the injection, we analyzed luciferase activity and kept positive mice for the study, as illustrated in Supplementary Figure 2A and 2B. The mice body-weight follow-up did MiR-223-3p expression in HNSCC and not demonstrate significant differences between the 2 neutrophil infiltration experimental groups of mice (Supplementary Figure 2C). MiR-223-3p is highly expressed in the granulocyte At the end of the experiment, tumors were collected lineage and its deregulation is linked to inflammatory and measured. No significant difference was found, as and tumor lesions. Whereas some reports indicated that illustrated in Figure 3A. miR-223-3p levels are increased in the serum of HNSCC Knowing that miR-223-3p slightly, but consistently, patients, the expression level of mir-223-3p within tumors increases CAL27 proliferation in vitro, we aimed to www.impactjournals.com/oncotarget 57175 Oncotarget characterize its effect ex vivo. Cell proliferation was induced up-regulation of AKT expression and down- evaluated by Ki67 immunostaining on tumor tissue regulation of STAT3 expression. A significant increase in sections collected from mice receiving CAL27 or CAL27 pERK2 and pAKT activity was also observed in CAL27 miR-223 cells. As expected, CAL27 cells were positive for miR-223-3p tumors as compared with CAL27 tumors. Ki67 staining. We observed that overexpression of miR- Growing tumors are hypoxic, and miR-223-3p 223-3p more than doubled Ki67 expression as compared has been reported to attenuate hypoxia-induced vascular with CAL27 mock cells (Figure 3B). To reconcile this remodeling of pulmonary cells [23]. We used carbonic result with our observation that miR-223-3p increases anhydrase 9 (CA9) staining as a read-out of hypoxia neither the tumor engraftment nor tumor size, we analyzed and observed that the hypoxic status of CAL27 and the effect of miR-223-3p on cell apoptosis. Cleaved CAL27 miR-223 tumors was comparable. Indeed, the 2 caspase 3 staining was used to measure the apoptotic index experimental groups showed high levels of hypoxia within of tumor cells. Within CAL27 tumors, some cells were tumors, as indicated by strong nuclear and cytoplasmic stained with anti-cleaved caspase 3 antibody. We observed CA9 staining. Diffusion of CA9 within the cytoplasm that CAL27 miR-223 tumors exhibited a higher apoptotic revealed that hypoxia occurred, but at an undetermined index, as compared with CAL27 tumors (Figure 3C). Thus, time (Figure 3E). Finally, histological analysis of tumor miR-223-3p-increased proliferation was counterbalanced sections stained with hematoxylin and eosin did not reveal by increased apoptosis. This observation could explain necrosis (Supplementary Figure 3B). why tumors from mice receiving CAL27 miR-223 cells were the same size as mice receiving control cells. MiR-223-3p decreases tumor neo-angiogenesis Furthermore, we studied the activity of signaling in vivo pathways known to be involved in cell proliferation and cell death, namely ERK2, AKT and STAT3. Total protein STAT3 was down-regulated in CAL27 miR- extracts from CAL27 and CAL27 miR-223 tumors 223 tumors. Since STAT3 expression is linked to neo- were analyzed by western blot detecting both total and angiogenesis, we characterized more fully the effect active phosphorylated forms of ERK2, AKT and STAT3 of miR-223-3p on tumor vasculature. To do so, we first antibodies (Supplementary Figure 3). Quantification of the analyzed the expression of VEGFR2 by immunostaining. results, shown in Figure 3D, indicated that miR-223-3p As shown in Figure 4A, CAL27 tumors expressed a high Figure 1: miR-223-3p is overexpressed in head and neck cancer. (A) miR-223-3p staining of normal epithelium showed few positive dots (asterisks). (B) Consecutive sections of T2 head and neck tumor stained for miR-223-3p or pan cytokeratin (CK) showed a high positive signal in CK positive transformed epithelial cells. A representative picture of consecutive sections from 8 tumors stained with miR-223-3p probe and CK is shown (magnification 800×), inset 1600×. Arrows highlighted polymormonuclear cells closed to miR-223-3p positive cells. www.impactjournals.com/oncotarget 57176 Oncotarget Table 1: Association of miR-223-3p levels with neutrophil infiltrate and CD31 expression Neutrophil P-value MiR-223-3p staining P-value CD31 expression P-value % high % high Low Medium + High Low Medium + High Negative Positive exp exp * * * Control 6 2 0.042 6 2 0.05 8 0 0.03 # # # Early-stage T 2 20 0.001 2 20 90 0.001 17 5 21 0.2 Advanced- 9 4 0.999 ± 10 3 23 0.99 ± 3 10 77 0.001 ± stage T Statistical analysis: Fisher's exact test. *Control vs. Tumors. Control vs. Early-stage Tumors. ± Control vs. Advanced-stage Tumors. Quantification of neutrophil infiltrate, miR-223-3p staining and CD31 expression from a control group made of 8 healthy people, a group made of 22 HNSCC patients with early-stage (T1 or T2) tumors and a group made of 13 patients with advanced-stage (T3 and T4) tumors was performed by a trained pathologist as following. A sample was considered to display low expression if the percentage of positive cells was from 0 to 10%, median expression (med) if the percentage is between 10 to 50% and high expression if more than 50% of the cells were positive. Negative scoring for CD31 staining corresponds to less than 30% positive cells. Three random fields per tumor were analyzed. This contingency table highlights a correlation between the presence of neutrophils and the expression of miR-223-3p and an inverse correlation between the presence of miR-223-3p and the expression of CD31. level of VEGFR2. This expression level significantly spots were quantified (Supplementary Figure 4B), and the decreased in CAL27 miR-223 tumors. Furthermore, we result confirmed what was shown on Figure 4C, with a quantified micro-vessel density (MVD) based on VEGFR2 3.6-fold higher miR-223-3p presence in biopsies with low staining and confirmed that the expression of miR-223-3p CD31 staining levels. Finally, quantification of neutrophil correlated with less neo-angiogenesis. To confirm this infiltrate, miR-223-3p staining and CD31 expression from interesting result, we performed an independent assay, a control group made of 8 healthy people, a group made based on CD31 staining and quantification of the MVD of 22 HNSCC patients with early-stage (T1 or T2) tumors hot spot (Figure 4B). Both approaches confirmed that and a group made of 13 patients with advanced-stage miR-223-3p decreased neo-angiogenesis. (T3 and T4) tumors highlighted the correlation existing Importantly, this result has been confirmed on tumor between these 3 indicators. As expected, healthy people tissue from HNSCC patients. As shown in Figure 4C showed low neutrophil infiltrate, low miR-223-3p staining and Supplementary Figure 4A, areas of high miR- and low CD31 expression. On the contrary early-stage 223-3p staining correlated with section of low CD31 tumors were characterized by high neutrophil infiltrate, expression, whereas areas of low miR-223-3p staining high miR-223-3p staining and low CD31 expression. This were associated with high CD31 expression. MVD hot result first suggested a correlation between the presence Figure 2: miR-223-3p induced CAL27 proliferation. (A) Characterization of CAL27 miR-223-3p cells. Total RNA was isolated from CAL27-Luci (named CAL27) and CAL27-Luci miR-223-3p (named CAL27 miR-223-3p) cells and RT-PCR analysis was performed. We showed that miR223 is overexpressed in transfected cells. Rnu19 RNA was used as internal control; Cp value for CAL27 cells was 32.60. (B, C) Expression of miR-223-3p in CAL27 cells improved cell proliferation (n = 4), whereas it had no effect on cell migration. (C) Confluent monolayers were scratched with a yellow tip and cell migration was expressed as the percentage of wound recovery ( n = 3). Data are presented as mean ± sem. www.impactjournals.com/oncotarget 57177 Oncotarget of neutrophils and miR-223-3p and second indicated that the presence of miR-223-3p correlated with a decrease in the presence of miR-223-3p dampened the expression of tumor neo-angiogenesis. CD31. Advanced tumors (T3 and T4) demonstrated larger numbers of CD31 positive vessels and weaker levels of MiR-223-3p increases tumor resistance to miR-223-3p. These two later events are correlated with anticancer agents less miR-223-3p staining, highlighting once again the correlation between the presence of neutrophils and MiR-223-3p expression is reported to modulate the presence of miR-223-3p in tumor cells. Finally, chemoresistance. Given that anticancer agents are used comparing miR-223-3p presence and CD31 expression routinely to treat HNSCC, we analyzed the impact of miR- in the group with early-stage tumors, we observed that 223-3p expression on cisplatin, docetaxel, 5-fluorouracil 90% of the patients expressed high levels of miR-223-3p and cetuximab treatment in a survival assay. As shown whereas 21% expressed high levels of CD31. The inverse in Figure 5A, more XTT incorporation was observed in correlation was observed in the group with advanced- CAL27 miR-223 cells versus control CAL27 cells. In stage tumors with 23% of the patients being positive particular, we observed that CAL27 miR-223 cells were with the anti-miR-223-3p probe and 77% being high less sensitive to cisplatin, docetaxel, and 5-fluorouracil CD31 expressers. In conclusion, we showed here that than CAL27 cells. This drug-resistance effect was Figure 3: Effect of miR-223-3p on tumor biology. (A) At day 14, the mice were sacrificed and the tumors were extracted for measurement. Despite the effect of miR-223-3p on cell proliferation, we observed no significant difference between the two experimental groups (n = 17). (B) Cell proliferation (evaluated by Ki67 staining) within tumors from mice injected with CAL27 miR-223-3p increased. Representative results are shown in the panels on the left and the number of proliferating cells (nuclear Ki67 staining) per tumor was determined as indicated in the Materials and Methods section. (C) Similarly, the number of apoptotic cells (evaluated by Cleaved Caspase-3 staining) increased in tumors from mice injected with CAL27 miR-223-3p. Representative pictures are shown on the left. Quantification of the number of apoptotic cells, on the right. (D) Immunoblot analysis of ERK2, AKT, STAT3 and their active phosphorylated forms in tumors from CAL27 and CAL27 miR-223-3p mice. Results showed that STAT3 protein is downregulated in cells that express miR-223-3p. (E) The hypoxic status of the tumors from both CAL27 and CAL27 miR-223-3p injected mice is comparable. CA9 staining was used as a read-out of hypoxia. Representative pictures are shown on the left. Quantification of the number of hypoxic cells, on the right. Data, shown as arbitrary units, are representative of 5 mice per group (mean ± sem).*P ≤ 0.05. Bar = 100 μm. www.impactjournals.com/oncotarget 57178 Oncotarget particularly evident for the anti-EGFR monoclonal the number of colonies and the size of each colony. As antibody cetuximab. This result indicates that expression shown in Figure 5B, we confirmed that miR-223-3p of miR-223-3p increases resistance to anticancer agents. expression increased resistance to anticancer agents. To confirm this observation, we performed an independent In particular, miR-223-3p cells were more resistant to assay, based on clonogenic growth, in which we quantified cetuximab treatment with a twofold increase in colonies Figure 4: miR-223-3p inhibited neoangiogenesis. (A) Tumors from mice injected with CAL27 miR-223-3p showed less VEGFR2 staining and MVD. Representative pictures of VEGFR2-stained MVD are shown on the left. Quantification of both VEGFR2 staining and MVD on the right. Bar = 100 μm. Data, shown as arbitrary units, are representative of 5 mice per group (mean ± sem). **P ≤ 0.01. (B) Neo-angiogenesis characterized by CD31 staining. Representative pictures of CD31 staining are shown on the left, quantification of the number of CD31 positive hot spots per tumor and the number of MVD per hot spot on the right. Bar = 50 μm. Data are representative of 5 mice per group (mean ± sem).**P ≤ 0.01. (C) CD31 staining is inversely proportional to miR-223-3p staining in human head and neck cancer. Pictures are representative of 35 tumors. Magnification 800×. www.impactjournals.com/oncotarget 57179 Oncotarget as well as colonies with a higher median area (343 DISCUSSION versus 215 mm ), as illustrated in the lower panel and in Supplementary Figure 5. Small non-coding RNAs, referred to as Micro- Based on this in vitro result, we tested the effect RNAs or miRs, are known to regulate the expression of cetuximab in vivo. As expected, cetuximab treatment of target genes at the post transcriptional level. As they inhibited tumor growth by 70% in mice injected with are involved in various biological processes, such as cell CAL27 cells (Figure 5C). In contrast, cetuximab treatment proliferation, metabolism and differentiation as well as did not efficiently decrease CAL27 miR-223 tumor cell death, they are suspected of actively participating in growth, confirming that miR-223-3p promoted resistance tumor growth and propagation [24]. Over the past decade, to cetuximab. many studies using microarray profiling and qRT-PCR Figure 5: miR-223-3p reversed cetuximab cytotoxic effect. (A) CAL27 and CAL27 miR-223-3p cells were treated with the indicated doses of cisplatin, cetuximab, docetaxel and 5-FU for 48 hours and cell proliferation and viability were measured. (B) CAL27 cells expressing the miR-223-3p are resistant to cetuximab. Cells were treated with cetuximab (50 nM, respectively) and the number of clones was determined as indicated in the Materials and Methods section. Representative pictures are shown on the left, quantification of both the surface and the number of the colony on the right. Data are representative of 2 independent experiments. (C) Mir-223-3p- expressing tumors are more resistant to cetuximab. At day 14, the mice were sacrificed and tumors were extracted for measurement. Data are expressed as mean ± sem. *P ≤ 0.01 (n = 10). www.impactjournals.com/oncotarget 57180 Oncotarget analyses aimed to identify miRs differentially expressed Zhang et al. found that miR-223-3p functioned as between malignant HNSCC versus normal tissues. Despite an oncogene in human colorectal cancer cells and never having been analyzed in depth, miR-223 has been demonstrated that reducing miR-223-3p expression consistently described as overexpressed in HNSCC [15]. resulted in decreased cell proliferation, migration and We focused our attention therefore on this particular invasion [32]. This conflicting evidence indicates that, miR, and showed that (i) miR-223-3p was overexpressed depending on the cellular origin of the tumor as well as the in T1 and T2 HNSCC patients, (ii) miR-223-3p slightly nature of the tumor cell transformation, miR-223-3p could increased proliferation of the human CAL27 head and either induce tumor suppression or promote tumor growth. neck cancer cell line in vitro, (iii) miR-223-3p did not To characterize the effect of miR-223-3p more impact CAL27 orthotopic tumor xenograft growth, and precisely, we used an orthotopic xenograft model but did (iv) miR-223-3p decreased tumor neo-angiogenesis and not observe a significant difference between CAL27 and increased tumor resistance to anticancer agents. CAL27 miR-223 tumors. Indeed, the higher proliferation MiR-223-3p expression is principally found index in CAL27 miR-223 tumors as compared to CAL27 in bone marrow with preferential expression in the tumors was counterbalanced by the higher apoptotic myeloid lineage [25]. The provenance of miR-223-3p index, thus resulting in comparable tumor sizes. The in tumor cells remains an open question. At least two pro-apoptotic effect of miR-223-3p has already been hypotheses could be envisaged. First, miR-223-3p, demonstrated in various experimental conditions and on which is localized on the X chromosome, could be on, some types of cells such as endothelial cells, hepatocytes or close to, a fragile site. These fragile sites are known and osteoblasts [34–37]. Immunoblot analysis of CAL27 to predispose to DNA instability and could be amplified miR-223 tumors showed increased pERK2, AKT and during the process of cell transformation, thus leading to pAKT expression as compared with CAL27 tumors. miR-223-3p over-expression. Such a process has been Stimulation of the ERK-AKT pathway in CAL27 miR-223 described for many miRs [26]. Second, miR-223-3p is tumors could explain the increased proliferation index produced by neutrophils and shuttles to the tumor cells found in these tumors. In a recent study analyzing via exosomes [27]. In agreement with this hypothesis, we microRNA expression deregulation in oral squamous cell observed considerable neutrophil infiltration in HNSCC carcinoma, Manikandan et al. identified 5 up-regulated tissue and a correspondence between areas with strong microRNAs including miR-223-3p, which was associated neutrophil infiltration and areas with high miR-223-3p with advanced tumor stage [18]. Moreover, similarly to expression (Figure 1 and Table1). Globally, both our study, the authors found that microRNA deregulation neutrophil infiltration and strong miR-223-3p expression in oral squamous cell carcinoma resulted in activation of in tumor cells were associated with T1 and T2 tumors PI3K/AKT signaling pathway genes. (Table 1). However, this correlation has not been observed We observed that CAL27 miR-223 tumors exhibited with higher tumor size (T3 and T4), a discrepancy that can a significant down-regulation of STAT3 expression be explained by the few cases included in our cohort. In as compared with CAL27 tumors. This result is not a recent study, Liang et al. demonstrated that miR-223- surprising since STAT3 is acknowledged to be one of the 3p delivered by platelet-derived microvesicles promoted miR-223-3p direct target genes. For example, Chen et al. lung cancer cell invasion via targeting tumor suppressor demonstrated that miR-223-3p regulated TLR-triggered EPB41L3 [21]. Of interest, high expression levels of IL-6 and IL-1β production in macrophages by targeting miR-223-3p, in association with high miR-155-5p STAT3 [38]. Such finding concords with our results and low miR-126-3p, constitute a plasma signature showing that CAL27 miR-223 tumors, characterized by a significantly associated with a higher risk for progression low STAT3 expression level, exhibited markedly reduced in adenocarcinoma patients [28]. However, the role of tumor angiogenesis as compared with CAL27 tumors miR-223-3p in cancer progression is controversial and (Figure 5). Similarly, STAT3 decoy oligonucleotide has some studies found that miR-223 inhibited cancer cell been reported to decrease proliferation, migration and invasion and migration [29, 30]. Thus, in the present study, tubule formation of endothelial cells in vitro and to inhibit we decided to explore the exact impact of miR-223-3p on tumor angiogenesis in murine HNSCC xenografts [39]. HNSCC cell biology, both in vitro and in vivo. This evidence indicates that miR-223-3p exhibits an In vitro, we found that miR-223-3p triggered a antiangiogenic effect and that this effect may be attributed, moderate increase in tumor cell proliferation but did not at least in part, to the miR-223-3p-induced down- modify cell migration. Contradictory results have been regulation of STAT3. The antiangiogenic effect of miR- reported in the literature regarding the impact of miR-223- 223-3p is corroborated by observations made on tumor 3p on tumor cell proliferation and migration [29–33].Yang tissue from HNSCC patients, thus indicating that areas et al., for instance, showed that miR-223-3p negatively of high miR-223-3pexpression displayed low CD31 IHC regulated the growth and migration of nasopharyngeal staining, and vice versa (Figure 4C and Table 1). carcinoma cells by reducing expression of the transcription This antiangiogenic effect of miR-223-3p has factor of Maf family members MAFB [29]. In contrast, already been reported by several authors [34, 40]. www.impactjournals.com/oncotarget 57181 Oncotarget Shi et al. showed that miR-223-3p was an antiangiogenic decreased formation of neo-vessels in an orthotopic microRNA that prevented endothelial cell proliferation, xenograft tumor model. This anti-angiogenic effect has at least partially, by targeting β1 integrin [40]. In another been correlated with high expression areas of miR-223-3p recent study, Liu et al. demonstrated that administration in biopsies from HNSCC patients. Furthermore, using the of antagomir-223-3p promoted angiogenesis in rats with xenograft tumor model, we demonstrated that miR-223-3p spinal cord injury [34]. However, there are few data on expression impaired the anti-tumoral effect of cetuximab. the role of microRNAs and, in particular, miR-223-3p, Since we did not observe necrosis in tumor xenografts in tumor angiogenesis. Mathsyaraja et al. showed that (Supplementary Figure 3B), we favor a scenario in which the expression of miR-21, miR-29a, miR-142-3p and the high neutrophil infiltration allows transfer of miR-223 miR-223-3p increased in myeloid cells during tumor to malignant cells, which in turn decreases the production progression in mouse models of breast cancer and of angiogenic factors resulting in hypo vascularization. melanoma metastasis. Furthermore, they demonstrated that Taken together, our results highlight the importance a loss-of-function approach using selective depletion of of studying the expression of miR-223-3p level in tumor the miR-processing enzyme Dicer in mature myeloid cells tissue sections since it may help to predict cetuximab blocks angiogenesis and metastatic tumor growth [41]. To response in patients with HNSCC. our knowledge, the present study is the first to demonstrate the antiangiogenic properties of miR-223-3p in patients MATERIALS AND METHODS with HNSCC. Hence, we examined the impact of miR-223-3p on Cell culture tumor resistance to anticancer agents. In vitro, we found that CAL27 miR-223 cells were less sensitive than CAL27 The human head and neck cancer cell line cells to conventional chemotherapeutic agents commonly CAL27 was supplied by ATCC (CRL-2095). CAL27 used in HNSCC (namely cisplatin, 5-fluorouracil and was first transduced with a viral suspension obtained docetaxel), as well as to the anti-EGFR monoclonal from HEK cells infected with pLenti-Luciferase antibody cetuximab. As the drug resistance effect induced vector. Bioluminescence from luciferase activity was by miR-223-3p was most evident with cetuximab, we quantified using an in vivo imaging system (IVIS, Caliper evaluated the antitumor effect of cetuximab on CAL27 and LifeSciences) according to the manufacturer’s procedure. CAL27 miR-223 tumors implanted orthotopically in the CAL27 Luci cells were then infected with lentiviral mouth floor of nude mice. We confirmed that cetuximab particles for hsa-miR-223 (Cat. #: PMIRH223PA-1) significantly reduced tumor growth of CAL27 tumors. supplied by System Biosciences following the However, when CAL 27 overexpressed miR-223-3p, manufacturer’s instructions. Infection efficiency was cetuximab did not significantly inhibit the tumor growth. measured under a fluorescent microscope one week after This result confirmed our observation in vitro showing the transfection and the GFP positive cells were sorted that miR-223-3p promoted tumor resistance to cetuximab. using a flow cytometer. The sorted cells were used in the There is no other study examining the impact of miR- experiments. 223-3p on cetuximab resistance, which represents a Cells were cultured at 37°C in controlled atmosphere critical issue in HNSCC. However, it has previously been (5% CO2 and 95% air) with Dulbecco’s Modified reported that miR-223 was able to reverse tumor resistance Eagles Medium, (Life Technologies) supplemented with of EGFR tyrosine kinase inhibitors (TKIs) [42, 43]. For 10% heat-inactivated fetal calf serum with penicillin/ example, Han et al. demonstrated that downregulation streptomycin. Prior to injecting the mice, cells were of miR-223-3p promoted resistance of non-small cell trypsinized and prepared in Ringer lactate solution at lung cancer cells to erlotinib, an EGFR TKI, through 1 × 10 cells/ml. For the proliferation assay, cells were activation of the IGF1R/PI3K/AKT pathway [43]. These plated at 1 × 10 cells/well in 24-well plates (BD Falcon) conflicting results may be explained by the fact that, in our at day 0 in growing medium. Cells were detached by HNSCC model, pERK2, AKT and pAKT levels increased trypsin treatment and counted each day. For the wound in CAL27 miR-223 tumors. This miR-223-3p-induced healing assay, cells were plated at 1 × 10 cells/well in a activation of the downstream effectors of the EGFR 6-well plate. At confluency, a wound was made using a signaling pathway may explain the resistance to cetuximab yellow tip and the first image was taken ( t = 0) to measure observed in our study. The molecular mechanisms leading the distance separating the two rims. At the indicated to this activation occurred downstream of the interaction times, the distance between the two rims was assayed and between EGFR and its ligands since we showed that the percentage of recovery was calculated. EGFR expression was not modified by miR-223-3p transfection (Supplementary Figure 1). Patients and samples In summary, we demonstrated that overexpression of miR-223-3p in the human head and neck cell line Surgically-removed tumors embedded in paraffin- resulted in decreased expression of STAT3 but also in wax blocks from 35 cases of HNSCC were retrieved www.impactjournals.com/oncotarget 57182 Oncotarget from the archives of the Laboratory of Clinical and as previously described [45]. Relative changes in gene Experimental Pathology (Pasteur Hospital) at the expression were reported as fold changes compared with University of Nice, Nice, France. All tumor specimens the control CAL27 cell line. were collected, stored, and used with the informed consent Protein extracts were resolved by SDS-PAGE and of the patients (Hospital-Integrated Biobank BB-0033- transferred onto a polyvinylidene difluoride membrane 00025, Pasteur Hospital, Nice, France). The study was (Immobilon-P; Millipore). After saturation, membranes approved by the Ethics Committee of the University of were incubated overnight with the indicated antibody, Nice-SophiaAntipolis and performed according to the washed in Tris-NaCl buffer (TN) supplemented three guidelines of the Declaration of Helsinki. The cases, times with 0.1% Triton X-100 (TNT), and finally recruited between 2005 and 2010, included squamous cell with TN buffer. Membranes were then incubated with carcinomas taken exclusively from metastasis-free patients. the secondary anti-rabbit or anti-mouse horseradish Cases were included in this study only if a follow-up of at peroxidase-conjugated antibody. Bound antibodies were least 5 years was obtained, and clinical data were available. revealed using an ECL system (Pierce) and the signal was Mean age at surgery was 61 years (range: 39 – 71) and quantified with a Pxi Camera (Ozyme, FR).Phospho-ERK 21 patients were male. Selected patients displayed similar (#9101), ERK (#9102), phospho-AKT (#9271), AKT health status and absence of concurrent chronic illnesses, (#9272),Phospho-STAT3 (#9131), STAT3 (#9139) and or tumors elsewhere. The primary sites of the carcinomas EGFR (#2085) antibodies were from Cell Signaling and were: oropharynx (18), hypopharynx (9), and larynx the anti-ACTB (Actin, clone AC40) antibody was from (8). Tumor stage (primary tumor) according to the 2009 Sigma Aldrich. American Joint Committee on Cancer staging system was T1 (12), T2 (10,) T3 (8) and T4 (5). Of note, eighty per Histology and immunohistochemical analysis cent of the carcinomas were keratinizing, with a range of grades: grade I (9), grade II (15), and grade III (11). For histopathology, paraffin-embedded tissues were sectioned (4 μm-thick slices), stained with hematoxylin and eosin and evaluated blind by three pathologists (CB, MI, PH). Animal strains and xenograft orthotopic model To investigate the cell-specific distribution of miRNA of HNSCC in normal and head and neck tumors, in situ hybridization This study was approved by the Institutional Care and was performed using 5ʹ- and 3ʹ-end digoxigenin (DIG)- Use Committee of the University of Nice-Sophia Antipolis. labeled LNA-modified DNA oligonucleotides (LNAs) Animal protocols were approved by the committee for complementary to the mature miRNA (Exiqon A/S, Research and Ethics of the PACA region (CIEPAL azur, Denmark). In this study, the global expression of miR-223- #PEA 12-153) and followed the European directive 3p was examined (LNA-scrambled as a negative control). 2010/63/UE. NMRI nude mice (nu/nu) were supplied by Scoring of miR-223-3p, neutrophil infiltrate and CD31 Janvier laboratories (Le Genest-St-Ile, France). A hundred expression were done by the pathologists at the invasive μl of ringer lactate solution containing 1 × 10 CAL27 front of the tumors. Luci +/− miR-223 were injected into the mouth floor of Immunohistochemical (IHC) staining for KI67, seven-week-old female mice as described previously [44]. cleaved caspase3, CA9, VEGFR2 and CD31 was On day one, cell implantation was verified by IVIS performed using tumor sections. Antigen retrieval was imaging and groups were stratified (8 to 10 animals per performed by boiling sections for 10 min in citrate buffer group). For mice receiving treatment, cetuximab (Erbitux, (pH 6.0) and cooling at RT°, followed by blocking of Merck Serono, Darmstadt, Germany) 5 mg/kg, cisplatin endogenous peroxidase activity with 0.3% H O for 2 2 (Milan, Amsterdam, Netherlands) and docetaxel (Accord 30 min. The sections were blocked with 2.5% horse serum Healthcare, Lille, France) 20 mg/kg were diluted in NaCl in TBS solution for 30 min in a humid chamber prior to 0.9% prior to the injection via the intraperitoneal route incubation with optimal dilutions of anti-Ki67 (Epitomics, (100 μl per injection) at days +3 and +9. Before sacrifice 1/300), anti-cleaved-caspase-3 (Imgenex, 1/500), anti- (day 14), luciferase activity was scored. CA9 (Abcam, 1/500), anti-VEGFR2 (Cell Signaling, 1/400), and anti-CD31 (M0823 Dako, clone JC70A, 1/50) overnight at 4°C. Positive cells were detected using an Quantitative real time PCR and immunoblotting ImmPRESS HRP anti-rabbit detection kit or directly with Total RNA was isolated from cells using the an anti-Streptavidin-Alexa-594 antibody. The immune AllPrep DNA/RNA/Protein Mini Qiagen kit (Qiagen) complexes were visualized using a Peroxidase Substrate following the manufacturer’s instruction, and 1 µg was DAB kit (Vector) according to the manufacturer’s reversed transcribed using the high-capacity cDNA RT kit protocol, and slides were counterstained with hematoxylin. (Applied Biosystems). RT-qPCR analysis was performed Blind quantification of brown staining was done as on a StepOne Real-Time PCR system using TaqMan PCR follows: Ki67 and cleaved caspase-3 tumor nuclear Master Mix (Applied Biosystems; Life Technology), staining corresponded to no cells (1), 1 to 10 % (2), 10 www.impactjournals.com/oncotarget 57183 Oncotarget to 30% (3), 30 to 50% (4) and > 50% (5). Membranous VV-C collected and analyzed the data. CB, MI and PH and cytoplasmic staining of CA9 corresponded to less participated in subject recruitment and in the Tissue Bank. than 80% (1) or 80 to 100 % of the cells (2). The final PB and PH gave technical support and conceptual advice. score corresponded to a mean of 5 fields per tumor AB, PH and VV-C wrote the manuscript. with 5 to 10 tumors per group. Tumor angiogenesis was quantified using both VEGFR2 and CD31 staining. ACKNOWLEDGMENTS VEGFR2 staining was scored following two criteria: the percentage of positive cells with no cells (1), 1 to 5 % (2), The authors wish to thank Marine Pedro, Katia 5 to 30% (3) and > 30 % (4) and the number of aligned Zahaf (LPCE, Nice) and Marie Bazin (IRCAN) for positive cells (considered as microvascular density, MVD) their expert technical assistance. Dr Nathalie Ebran with a single cell (1), 2 to 3 cells (2) and more than 4 (Centre Antoine Lacassagne) kindly provided luciferase aligned cells (4). MVD was also measured following expressing CAL27 cells. We acknowledge the IRCAN’s the method described by Weidner at al. [46]. In brief, Animal Core facility. scanned slides (NanoZoomer 2.0 HT from Hamamatsu) were initially evaluated at 50× magnification in order CONFLICTS OF INTEREST to identify and count areas with higher vascular density characterized as hot spots. In addition, 3 representative We have no conflicts of interest. hot spots measuring 70000 μm were selected. Images of the selected fields were analyzed at 400× magnification. FUNDING Any brown endothelial cell clusters (≥ 3 cells) that were clearly separated from adjacent microvessels, tumor cells This work was supported by grants from the and other connective tissues were considered as countable “Institut National de la Santé et de la Recherche microvessels. The total number of vessels counted in Médicale”, the “Association pour la Recherche contre le each case was divided by the number of hot spots, thus Cancer” (ARC Grants # SL220110603478), the PACA providing the mean MVD for that case. Cancéropole, the “Centre National de la Recherche Scientifique” and the French Government (National Cell survival and clonogenic assay Research Agency, ANR through the “Investments for the 4 Future” LABEX SIGNALIFE: program reference #ANR- For cell survival assay, 1 × 10 CAL27 +/− miR-223 11-LABX-0028-01. cells were seeded on a 96-well plate and, where notified, pre-treated with the indicated dose of cisplatin, cetuximab, docetaxel and fluorouracil (5-FU) for 48 hrs. XTT was added REFERENCES to each well and its cleavage to formazan was followed every 15 minutes over a 10 hr period. Formazan dye was 1. Simard EP, Torre LA, Jemal A. International trends in head quantified using a scanning multi-well spectrophotometer, and neck cancer incidence rates: differences by country, sex as indicated by the manufacturer (Roche, # 11465015001). and anatomic site. 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MiR-223-3p inhibits angiogenesis and promotes resistance to cetuximab in head and neck squamous cell carcinoma

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www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 34), pp: 57174-57186 Research Paper MiR-223-3p inhibits angiogenesis and promotes resistance to cetuximab in head and neck squamous cell carcinoma 1,2,5 1 1 1,3,4,5 Alexandre Bozec , Joséphine Zangari , Mathilde Butori-Pepino , Marius Ilie , 3 1 3,4 1,5 1,3,4,5,* Salomé Lalvee , Thierry Juhel , Catherine Butori , Patrick Brest , Paul Hofman 1,5,* and Valérie Vouret-Craviari Université Côte d’Azur, INSERM, CNRS, IRCAN, Nice, France Head and Neck University Institute, Nice, France Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, Nice, France Hospital-Related Biobank (BB-0033-00025), Pasteur Hospital, Nice, France FHU OncoAge, Nice, France These authors contributed equally to this work Correspondence to: Valérie Vouret-Craviari, email: [email protected] Keywords: MiRs, tumors, neutrophils, inflammation, anticancer agents Received: December 21, 2016 Accepted: June 29, 2017 Published: July 11, 2017 Copyright: Bozec et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT MicroRNAs (miRs) participate in tumor growth and dissemination by regulating expression of various target genes. MiR-223-3p is suspected of being involved in head and neck squamous cell carcinoma (HNSCC) growth although its precise role has not been elucidated. In this study, we showed that miR-223-3p is present in biopsies of HNSCC patients and that its presence is correlated with high neutrophil infiltrate. We found that overexpression of miR-223-3p slightly increased proliferation of the CAL27 squamous carcinoma cell line both in vitro and in vivo. Moreover, miR-223-3p induced CAL27 apoptosis in an orthotopic xenograft mouse model, counteracting the proliferative effect and resulting in no impact on overall tumor growth. We analyzed the effect of miR-223-3p overexpression on signaling pathways and showed that it induced pERK2, pAKT and AKT, consistent with an increase in cell proliferation. In addition, we found that miR-223-3p reduced the STAT3 level correlating with increased cell apoptosis and inhibited vasculature formation. In HNSCC tissues, miR- 223-3p expression was inversely correlated to CD31, highlighting the relationship between miR-223 and vessel formation. Finally, we studied the effect of miR-223-3p on response to selected anticancer agents and showed that cells expressing miR-223-3p are more resistant to drugs, notably cetuximab. In conclusion, our study is the rst fi to show the antiangiogenic properties of miR- 223-3p in HNSCC patients and to question whether expression levels of miR-223-3p can be evaluated as an indicator of eligibility for non-treatment of HNSCC patients with cetuximab. of their disease [2]. Patients with HNSCC presented an INTRODUCTION altered cytokine profile compared to healthy controls. In There are approximately 600,000 new cases of head particular, IL-8, IL-6, TNF-α, MCP1 and MIP-1α were and neck squamous cell carcinoma (HNSCC) annually more expressed in the plasma of HNSCC patients [3, 4]. worldwide and HNSCC represents the 6th cause of Neutrophils are important mediators in cancer progression cancer death [1]. Most HNSCC patients are diagnosed and the neutrophils to lymphocytes ratio is an independent with locally-advanced disease and half of them will die predictor of recurrence in HNSCC [5–7]. Recent studies www.impactjournals.com/oncotarget 57174 Oncotarget associated neutrophils with poor clinical outcome in of head and neck origin has been poorly studied. In this HNSCC patients [8, 9]. Peripheral blood neutrophils study, we analyzed the expression of miR-223-3p by in situ from HNSCC patients and healthy donors showed distinct hybridization in 35 tumors from HNSCC patients. As functional differences, among them an increased number shown in Figure 1A, low signal was detected in the normal of immature stages of neutrophils in HNSCC patients [10]. epithelium. On the contrary, T1 and T2 HNSCC exhibited Several studies demonstrated that a high neutrophil a high expression level of miR-223-3p in CK-positive infiltration rate in the tumor was associated with more epithelial cells (Figure 1B). This expression decreased advanced disease and poor prognosis in HNSCC with the size of the tumor (Table 1). Neutrophils being a patients [9, 11]. HNSCC induces recruitment, survival, major source of miR-223-3p, we aimed to characterize the and release of proinflammatory factors such as CCL4 and presence of these cells in HNSCC. As shown in Table 1, IL-8 by neutrophils [11, 12]. Moreover, tumor-infiltrating and illustrated in Figure 1B, some neutrophils are closed neutrophils may be a major source of MMP9, which to miR-223-3p positive cells. As illustrated in Table 1, we can promote cancer cell invasion and metastasis [12]. observed a correlation between high neutrophil infiltration Dumitru et al. reported that neutrophils released soluble and high miR-223-3p expression levels. factors which phosphorylated cortactin in HNSC cells and promoted their migration [13]. Furthermore, these authors Effect of miR-223-3p on in vitro cell proliferation demonstrated that strong cortactin phosphorylation and migration significantly correlated with strong neutrophilic infiltration in tumor tissues from HNSCC patients [13]. Finally, after Since HNSCC expresses high levels of miR-223-3p, neutrophil recruitment in the tumor microenvironment, we aimed to characterize its effect on cell proliferation, HNSC cells can modulate the biology of neutrophils, migration and survival. First, we engineered a head and which in turn may facilitate cancer progression [11]. neck cancer cell line overexpressing miR-223-3p by Deregulation of microRNAs, a group of small transducing CAL27 cells with hsa-miR-223 and luciferase noncoding RNAs, plays a major role in cancer plasmids.RT-qPCR analysis of total RNA isolated from development [14, 15]. Chen et al. recently identified a CAL27 and CAL27 miR-223 cells confirmed that miR- panel of microRNA deregulations that were observed 223-3p was overexpressed in transfected cells (Figure 2A). in HNSCC, including 7 consistently up-regulated It is known that MiRs modulate the transcription of microRNAs (miR-21, miR-7, miR-155, miR-130b, their target genes. Recently it was shown that miR-223 miR-223-3p, miR-34b) [15]. Interestingly, miR-223-3p decreased activation of EGF receptor [22]. Because EGFR plays a critical role in the maturation and biology of plays crucial roles in the biology of head and neck cancer granulocytes [16]. Several studies showed that miR-223-3p cell lines, we verified that miR-223-3p did not inhibit the was up-regulated in tumor tissue and in plasma of cancer EGFR transcription/translation program (Supplementary patients, including esophagus and oral cancer patients Figure 1). Further, we analyzed the effect of miR-223-3p [17, 18]. Furthermore, it has been shown that platelets and on CAL27 cell proliferation and showed that CAL27 macrophages were able to modulate and promote cancer miR-223 cells displayed increased cell proliferation progression through exosome-mediated delivery of miR- as compared to control cells (Figure 2B). However, 223-3p [19–21]. In this context, miR-223-3p may be one expression of miR-223-3p in CAL27 cells had no effect of the main granulocytes-secreted molecular factors able on cell migration, as illustrated in the wound-healing assay to modulate the biology of cancer cells. (Figure 2C). The purpose of this study was to analyze the effects of miR-223-3p on HNSC cells, both in vitro and in vivo. Effect of miR-223-3p on in vivo tumor growth We examined the impact of miR-223-3p expression on migration, proliferation, apoptosis and drug resistance We used an orthotopic xenograft model consisting of properties of HNSC cells and on some key molecular implantation of CAL27 and CAL27-miR-223 cells in the signaling pathways implicated in cancer progression. mouth floor of nude mice to characterize the effect of miR- 223-3p on tumor implantation and tumor growth. One day RESULTS after the injection, we analyzed luciferase activity and kept positive mice for the study, as illustrated in Supplementary Figure 2A and 2B. The mice body-weight follow-up did MiR-223-3p expression in HNSCC and not demonstrate significant differences between the 2 neutrophil infiltration experimental groups of mice (Supplementary Figure 2C). MiR-223-3p is highly expressed in the granulocyte At the end of the experiment, tumors were collected lineage and its deregulation is linked to inflammatory and measured. No significant difference was found, as and tumor lesions. Whereas some reports indicated that illustrated in Figure 3A. miR-223-3p levels are increased in the serum of HNSCC Knowing that miR-223-3p slightly, but consistently, patients, the expression level of mir-223-3p within tumors increases CAL27 proliferation in vitro, we aimed to www.impactjournals.com/oncotarget 57175 Oncotarget characterize its effect ex vivo. Cell proliferation was induced up-regulation of AKT expression and down- evaluated by Ki67 immunostaining on tumor tissue regulation of STAT3 expression. A significant increase in sections collected from mice receiving CAL27 or CAL27 pERK2 and pAKT activity was also observed in CAL27 miR-223 cells. As expected, CAL27 cells were positive for miR-223-3p tumors as compared with CAL27 tumors. Ki67 staining. We observed that overexpression of miR- Growing tumors are hypoxic, and miR-223-3p 223-3p more than doubled Ki67 expression as compared has been reported to attenuate hypoxia-induced vascular with CAL27 mock cells (Figure 3B). To reconcile this remodeling of pulmonary cells [23]. We used carbonic result with our observation that miR-223-3p increases anhydrase 9 (CA9) staining as a read-out of hypoxia neither the tumor engraftment nor tumor size, we analyzed and observed that the hypoxic status of CAL27 and the effect of miR-223-3p on cell apoptosis. Cleaved CAL27 miR-223 tumors was comparable. Indeed, the 2 caspase 3 staining was used to measure the apoptotic index experimental groups showed high levels of hypoxia within of tumor cells. Within CAL27 tumors, some cells were tumors, as indicated by strong nuclear and cytoplasmic stained with anti-cleaved caspase 3 antibody. We observed CA9 staining. Diffusion of CA9 within the cytoplasm that CAL27 miR-223 tumors exhibited a higher apoptotic revealed that hypoxia occurred, but at an undetermined index, as compared with CAL27 tumors (Figure 3C). Thus, time (Figure 3E). Finally, histological analysis of tumor miR-223-3p-increased proliferation was counterbalanced sections stained with hematoxylin and eosin did not reveal by increased apoptosis. This observation could explain necrosis (Supplementary Figure 3B). why tumors from mice receiving CAL27 miR-223 cells were the same size as mice receiving control cells. MiR-223-3p decreases tumor neo-angiogenesis Furthermore, we studied the activity of signaling in vivo pathways known to be involved in cell proliferation and cell death, namely ERK2, AKT and STAT3. Total protein STAT3 was down-regulated in CAL27 miR- extracts from CAL27 and CAL27 miR-223 tumors 223 tumors. Since STAT3 expression is linked to neo- were analyzed by western blot detecting both total and angiogenesis, we characterized more fully the effect active phosphorylated forms of ERK2, AKT and STAT3 of miR-223-3p on tumor vasculature. To do so, we first antibodies (Supplementary Figure 3). Quantification of the analyzed the expression of VEGFR2 by immunostaining. results, shown in Figure 3D, indicated that miR-223-3p As shown in Figure 4A, CAL27 tumors expressed a high Figure 1: miR-223-3p is overexpressed in head and neck cancer. (A) miR-223-3p staining of normal epithelium showed few positive dots (asterisks). (B) Consecutive sections of T2 head and neck tumor stained for miR-223-3p or pan cytokeratin (CK) showed a high positive signal in CK positive transformed epithelial cells. A representative picture of consecutive sections from 8 tumors stained with miR-223-3p probe and CK is shown (magnification 800×), inset 1600×. Arrows highlighted polymormonuclear cells closed to miR-223-3p positive cells. www.impactjournals.com/oncotarget 57176 Oncotarget Table 1: Association of miR-223-3p levels with neutrophil infiltrate and CD31 expression Neutrophil P-value MiR-223-3p staining P-value CD31 expression P-value % high % high Low Medium + High Low Medium + High Negative Positive exp exp * * * Control 6 2 0.042 6 2 0.05 8 0 0.03 # # # Early-stage T 2 20 0.001 2 20 90 0.001 17 5 21 0.2 Advanced- 9 4 0.999 ± 10 3 23 0.99 ± 3 10 77 0.001 ± stage T Statistical analysis: Fisher's exact test. *Control vs. Tumors. Control vs. Early-stage Tumors. ± Control vs. Advanced-stage Tumors. Quantification of neutrophil infiltrate, miR-223-3p staining and CD31 expression from a control group made of 8 healthy people, a group made of 22 HNSCC patients with early-stage (T1 or T2) tumors and a group made of 13 patients with advanced-stage (T3 and T4) tumors was performed by a trained pathologist as following. A sample was considered to display low expression if the percentage of positive cells was from 0 to 10%, median expression (med) if the percentage is between 10 to 50% and high expression if more than 50% of the cells were positive. Negative scoring for CD31 staining corresponds to less than 30% positive cells. Three random fields per tumor were analyzed. This contingency table highlights a correlation between the presence of neutrophils and the expression of miR-223-3p and an inverse correlation between the presence of miR-223-3p and the expression of CD31. level of VEGFR2. This expression level significantly spots were quantified (Supplementary Figure 4B), and the decreased in CAL27 miR-223 tumors. Furthermore, we result confirmed what was shown on Figure 4C, with a quantified micro-vessel density (MVD) based on VEGFR2 3.6-fold higher miR-223-3p presence in biopsies with low staining and confirmed that the expression of miR-223-3p CD31 staining levels. Finally, quantification of neutrophil correlated with less neo-angiogenesis. To confirm this infiltrate, miR-223-3p staining and CD31 expression from interesting result, we performed an independent assay, a control group made of 8 healthy people, a group made based on CD31 staining and quantification of the MVD of 22 HNSCC patients with early-stage (T1 or T2) tumors hot spot (Figure 4B). Both approaches confirmed that and a group made of 13 patients with advanced-stage miR-223-3p decreased neo-angiogenesis. (T3 and T4) tumors highlighted the correlation existing Importantly, this result has been confirmed on tumor between these 3 indicators. As expected, healthy people tissue from HNSCC patients. As shown in Figure 4C showed low neutrophil infiltrate, low miR-223-3p staining and Supplementary Figure 4A, areas of high miR- and low CD31 expression. On the contrary early-stage 223-3p staining correlated with section of low CD31 tumors were characterized by high neutrophil infiltrate, expression, whereas areas of low miR-223-3p staining high miR-223-3p staining and low CD31 expression. This were associated with high CD31 expression. MVD hot result first suggested a correlation between the presence Figure 2: miR-223-3p induced CAL27 proliferation. (A) Characterization of CAL27 miR-223-3p cells. Total RNA was isolated from CAL27-Luci (named CAL27) and CAL27-Luci miR-223-3p (named CAL27 miR-223-3p) cells and RT-PCR analysis was performed. We showed that miR223 is overexpressed in transfected cells. Rnu19 RNA was used as internal control; Cp value for CAL27 cells was 32.60. (B, C) Expression of miR-223-3p in CAL27 cells improved cell proliferation (n = 4), whereas it had no effect on cell migration. (C) Confluent monolayers were scratched with a yellow tip and cell migration was expressed as the percentage of wound recovery ( n = 3). Data are presented as mean ± sem. www.impactjournals.com/oncotarget 57177 Oncotarget of neutrophils and miR-223-3p and second indicated that the presence of miR-223-3p correlated with a decrease in the presence of miR-223-3p dampened the expression of tumor neo-angiogenesis. CD31. Advanced tumors (T3 and T4) demonstrated larger numbers of CD31 positive vessels and weaker levels of MiR-223-3p increases tumor resistance to miR-223-3p. These two later events are correlated with anticancer agents less miR-223-3p staining, highlighting once again the correlation between the presence of neutrophils and MiR-223-3p expression is reported to modulate the presence of miR-223-3p in tumor cells. Finally, chemoresistance. Given that anticancer agents are used comparing miR-223-3p presence and CD31 expression routinely to treat HNSCC, we analyzed the impact of miR- in the group with early-stage tumors, we observed that 223-3p expression on cisplatin, docetaxel, 5-fluorouracil 90% of the patients expressed high levels of miR-223-3p and cetuximab treatment in a survival assay. As shown whereas 21% expressed high levels of CD31. The inverse in Figure 5A, more XTT incorporation was observed in correlation was observed in the group with advanced- CAL27 miR-223 cells versus control CAL27 cells. In stage tumors with 23% of the patients being positive particular, we observed that CAL27 miR-223 cells were with the anti-miR-223-3p probe and 77% being high less sensitive to cisplatin, docetaxel, and 5-fluorouracil CD31 expressers. In conclusion, we showed here that than CAL27 cells. This drug-resistance effect was Figure 3: Effect of miR-223-3p on tumor biology. (A) At day 14, the mice were sacrificed and the tumors were extracted for measurement. Despite the effect of miR-223-3p on cell proliferation, we observed no significant difference between the two experimental groups (n = 17). (B) Cell proliferation (evaluated by Ki67 staining) within tumors from mice injected with CAL27 miR-223-3p increased. Representative results are shown in the panels on the left and the number of proliferating cells (nuclear Ki67 staining) per tumor was determined as indicated in the Materials and Methods section. (C) Similarly, the number of apoptotic cells (evaluated by Cleaved Caspase-3 staining) increased in tumors from mice injected with CAL27 miR-223-3p. Representative pictures are shown on the left. Quantification of the number of apoptotic cells, on the right. (D) Immunoblot analysis of ERK2, AKT, STAT3 and their active phosphorylated forms in tumors from CAL27 and CAL27 miR-223-3p mice. Results showed that STAT3 protein is downregulated in cells that express miR-223-3p. (E) The hypoxic status of the tumors from both CAL27 and CAL27 miR-223-3p injected mice is comparable. CA9 staining was used as a read-out of hypoxia. Representative pictures are shown on the left. Quantification of the number of hypoxic cells, on the right. Data, shown as arbitrary units, are representative of 5 mice per group (mean ± sem).*P ≤ 0.05. Bar = 100 μm. www.impactjournals.com/oncotarget 57178 Oncotarget particularly evident for the anti-EGFR monoclonal the number of colonies and the size of each colony. As antibody cetuximab. This result indicates that expression shown in Figure 5B, we confirmed that miR-223-3p of miR-223-3p increases resistance to anticancer agents. expression increased resistance to anticancer agents. To confirm this observation, we performed an independent In particular, miR-223-3p cells were more resistant to assay, based on clonogenic growth, in which we quantified cetuximab treatment with a twofold increase in colonies Figure 4: miR-223-3p inhibited neoangiogenesis. (A) Tumors from mice injected with CAL27 miR-223-3p showed less VEGFR2 staining and MVD. Representative pictures of VEGFR2-stained MVD are shown on the left. Quantification of both VEGFR2 staining and MVD on the right. Bar = 100 μm. Data, shown as arbitrary units, are representative of 5 mice per group (mean ± sem). **P ≤ 0.01. (B) Neo-angiogenesis characterized by CD31 staining. Representative pictures of CD31 staining are shown on the left, quantification of the number of CD31 positive hot spots per tumor and the number of MVD per hot spot on the right. Bar = 50 μm. Data are representative of 5 mice per group (mean ± sem).**P ≤ 0.01. (C) CD31 staining is inversely proportional to miR-223-3p staining in human head and neck cancer. Pictures are representative of 35 tumors. Magnification 800×. www.impactjournals.com/oncotarget 57179 Oncotarget as well as colonies with a higher median area (343 DISCUSSION versus 215 mm ), as illustrated in the lower panel and in Supplementary Figure 5. Small non-coding RNAs, referred to as Micro- Based on this in vitro result, we tested the effect RNAs or miRs, are known to regulate the expression of cetuximab in vivo. As expected, cetuximab treatment of target genes at the post transcriptional level. As they inhibited tumor growth by 70% in mice injected with are involved in various biological processes, such as cell CAL27 cells (Figure 5C). In contrast, cetuximab treatment proliferation, metabolism and differentiation as well as did not efficiently decrease CAL27 miR-223 tumor cell death, they are suspected of actively participating in growth, confirming that miR-223-3p promoted resistance tumor growth and propagation [24]. Over the past decade, to cetuximab. many studies using microarray profiling and qRT-PCR Figure 5: miR-223-3p reversed cetuximab cytotoxic effect. (A) CAL27 and CAL27 miR-223-3p cells were treated with the indicated doses of cisplatin, cetuximab, docetaxel and 5-FU for 48 hours and cell proliferation and viability were measured. (B) CAL27 cells expressing the miR-223-3p are resistant to cetuximab. Cells were treated with cetuximab (50 nM, respectively) and the number of clones was determined as indicated in the Materials and Methods section. Representative pictures are shown on the left, quantification of both the surface and the number of the colony on the right. Data are representative of 2 independent experiments. (C) Mir-223-3p- expressing tumors are more resistant to cetuximab. At day 14, the mice were sacrificed and tumors were extracted for measurement. Data are expressed as mean ± sem. *P ≤ 0.01 (n = 10). www.impactjournals.com/oncotarget 57180 Oncotarget analyses aimed to identify miRs differentially expressed Zhang et al. found that miR-223-3p functioned as between malignant HNSCC versus normal tissues. Despite an oncogene in human colorectal cancer cells and never having been analyzed in depth, miR-223 has been demonstrated that reducing miR-223-3p expression consistently described as overexpressed in HNSCC [15]. resulted in decreased cell proliferation, migration and We focused our attention therefore on this particular invasion [32]. This conflicting evidence indicates that, miR, and showed that (i) miR-223-3p was overexpressed depending on the cellular origin of the tumor as well as the in T1 and T2 HNSCC patients, (ii) miR-223-3p slightly nature of the tumor cell transformation, miR-223-3p could increased proliferation of the human CAL27 head and either induce tumor suppression or promote tumor growth. neck cancer cell line in vitro, (iii) miR-223-3p did not To characterize the effect of miR-223-3p more impact CAL27 orthotopic tumor xenograft growth, and precisely, we used an orthotopic xenograft model but did (iv) miR-223-3p decreased tumor neo-angiogenesis and not observe a significant difference between CAL27 and increased tumor resistance to anticancer agents. CAL27 miR-223 tumors. Indeed, the higher proliferation MiR-223-3p expression is principally found index in CAL27 miR-223 tumors as compared to CAL27 in bone marrow with preferential expression in the tumors was counterbalanced by the higher apoptotic myeloid lineage [25]. The provenance of miR-223-3p index, thus resulting in comparable tumor sizes. The in tumor cells remains an open question. At least two pro-apoptotic effect of miR-223-3p has already been hypotheses could be envisaged. First, miR-223-3p, demonstrated in various experimental conditions and on which is localized on the X chromosome, could be on, some types of cells such as endothelial cells, hepatocytes or close to, a fragile site. These fragile sites are known and osteoblasts [34–37]. Immunoblot analysis of CAL27 to predispose to DNA instability and could be amplified miR-223 tumors showed increased pERK2, AKT and during the process of cell transformation, thus leading to pAKT expression as compared with CAL27 tumors. miR-223-3p over-expression. Such a process has been Stimulation of the ERK-AKT pathway in CAL27 miR-223 described for many miRs [26]. Second, miR-223-3p is tumors could explain the increased proliferation index produced by neutrophils and shuttles to the tumor cells found in these tumors. In a recent study analyzing via exosomes [27]. In agreement with this hypothesis, we microRNA expression deregulation in oral squamous cell observed considerable neutrophil infiltration in HNSCC carcinoma, Manikandan et al. identified 5 up-regulated tissue and a correspondence between areas with strong microRNAs including miR-223-3p, which was associated neutrophil infiltration and areas with high miR-223-3p with advanced tumor stage [18]. Moreover, similarly to expression (Figure 1 and Table1). Globally, both our study, the authors found that microRNA deregulation neutrophil infiltration and strong miR-223-3p expression in oral squamous cell carcinoma resulted in activation of in tumor cells were associated with T1 and T2 tumors PI3K/AKT signaling pathway genes. (Table 1). However, this correlation has not been observed We observed that CAL27 miR-223 tumors exhibited with higher tumor size (T3 and T4), a discrepancy that can a significant down-regulation of STAT3 expression be explained by the few cases included in our cohort. In as compared with CAL27 tumors. This result is not a recent study, Liang et al. demonstrated that miR-223- surprising since STAT3 is acknowledged to be one of the 3p delivered by platelet-derived microvesicles promoted miR-223-3p direct target genes. For example, Chen et al. lung cancer cell invasion via targeting tumor suppressor demonstrated that miR-223-3p regulated TLR-triggered EPB41L3 [21]. Of interest, high expression levels of IL-6 and IL-1β production in macrophages by targeting miR-223-3p, in association with high miR-155-5p STAT3 [38]. Such finding concords with our results and low miR-126-3p, constitute a plasma signature showing that CAL27 miR-223 tumors, characterized by a significantly associated with a higher risk for progression low STAT3 expression level, exhibited markedly reduced in adenocarcinoma patients [28]. However, the role of tumor angiogenesis as compared with CAL27 tumors miR-223-3p in cancer progression is controversial and (Figure 5). Similarly, STAT3 decoy oligonucleotide has some studies found that miR-223 inhibited cancer cell been reported to decrease proliferation, migration and invasion and migration [29, 30]. Thus, in the present study, tubule formation of endothelial cells in vitro and to inhibit we decided to explore the exact impact of miR-223-3p on tumor angiogenesis in murine HNSCC xenografts [39]. HNSCC cell biology, both in vitro and in vivo. This evidence indicates that miR-223-3p exhibits an In vitro, we found that miR-223-3p triggered a antiangiogenic effect and that this effect may be attributed, moderate increase in tumor cell proliferation but did not at least in part, to the miR-223-3p-induced down- modify cell migration. Contradictory results have been regulation of STAT3. The antiangiogenic effect of miR- reported in the literature regarding the impact of miR-223- 223-3p is corroborated by observations made on tumor 3p on tumor cell proliferation and migration [29–33].Yang tissue from HNSCC patients, thus indicating that areas et al., for instance, showed that miR-223-3p negatively of high miR-223-3pexpression displayed low CD31 IHC regulated the growth and migration of nasopharyngeal staining, and vice versa (Figure 4C and Table 1). carcinoma cells by reducing expression of the transcription This antiangiogenic effect of miR-223-3p has factor of Maf family members MAFB [29]. In contrast, already been reported by several authors [34, 40]. www.impactjournals.com/oncotarget 57181 Oncotarget Shi et al. showed that miR-223-3p was an antiangiogenic decreased formation of neo-vessels in an orthotopic microRNA that prevented endothelial cell proliferation, xenograft tumor model. This anti-angiogenic effect has at least partially, by targeting β1 integrin [40]. In another been correlated with high expression areas of miR-223-3p recent study, Liu et al. demonstrated that administration in biopsies from HNSCC patients. Furthermore, using the of antagomir-223-3p promoted angiogenesis in rats with xenograft tumor model, we demonstrated that miR-223-3p spinal cord injury [34]. However, there are few data on expression impaired the anti-tumoral effect of cetuximab. the role of microRNAs and, in particular, miR-223-3p, Since we did not observe necrosis in tumor xenografts in tumor angiogenesis. Mathsyaraja et al. showed that (Supplementary Figure 3B), we favor a scenario in which the expression of miR-21, miR-29a, miR-142-3p and the high neutrophil infiltration allows transfer of miR-223 miR-223-3p increased in myeloid cells during tumor to malignant cells, which in turn decreases the production progression in mouse models of breast cancer and of angiogenic factors resulting in hypo vascularization. melanoma metastasis. Furthermore, they demonstrated that Taken together, our results highlight the importance a loss-of-function approach using selective depletion of of studying the expression of miR-223-3p level in tumor the miR-processing enzyme Dicer in mature myeloid cells tissue sections since it may help to predict cetuximab blocks angiogenesis and metastatic tumor growth [41]. To response in patients with HNSCC. our knowledge, the present study is the first to demonstrate the antiangiogenic properties of miR-223-3p in patients MATERIALS AND METHODS with HNSCC. Hence, we examined the impact of miR-223-3p on Cell culture tumor resistance to anticancer agents. In vitro, we found that CAL27 miR-223 cells were less sensitive than CAL27 The human head and neck cancer cell line cells to conventional chemotherapeutic agents commonly CAL27 was supplied by ATCC (CRL-2095). CAL27 used in HNSCC (namely cisplatin, 5-fluorouracil and was first transduced with a viral suspension obtained docetaxel), as well as to the anti-EGFR monoclonal from HEK cells infected with pLenti-Luciferase antibody cetuximab. As the drug resistance effect induced vector. Bioluminescence from luciferase activity was by miR-223-3p was most evident with cetuximab, we quantified using an in vivo imaging system (IVIS, Caliper evaluated the antitumor effect of cetuximab on CAL27 and LifeSciences) according to the manufacturer’s procedure. CAL27 miR-223 tumors implanted orthotopically in the CAL27 Luci cells were then infected with lentiviral mouth floor of nude mice. We confirmed that cetuximab particles for hsa-miR-223 (Cat. #: PMIRH223PA-1) significantly reduced tumor growth of CAL27 tumors. supplied by System Biosciences following the However, when CAL 27 overexpressed miR-223-3p, manufacturer’s instructions. Infection efficiency was cetuximab did not significantly inhibit the tumor growth. measured under a fluorescent microscope one week after This result confirmed our observation in vitro showing the transfection and the GFP positive cells were sorted that miR-223-3p promoted tumor resistance to cetuximab. using a flow cytometer. The sorted cells were used in the There is no other study examining the impact of miR- experiments. 223-3p on cetuximab resistance, which represents a Cells were cultured at 37°C in controlled atmosphere critical issue in HNSCC. However, it has previously been (5% CO2 and 95% air) with Dulbecco’s Modified reported that miR-223 was able to reverse tumor resistance Eagles Medium, (Life Technologies) supplemented with of EGFR tyrosine kinase inhibitors (TKIs) [42, 43]. For 10% heat-inactivated fetal calf serum with penicillin/ example, Han et al. demonstrated that downregulation streptomycin. Prior to injecting the mice, cells were of miR-223-3p promoted resistance of non-small cell trypsinized and prepared in Ringer lactate solution at lung cancer cells to erlotinib, an EGFR TKI, through 1 × 10 cells/ml. For the proliferation assay, cells were activation of the IGF1R/PI3K/AKT pathway [43]. These plated at 1 × 10 cells/well in 24-well plates (BD Falcon) conflicting results may be explained by the fact that, in our at day 0 in growing medium. Cells were detached by HNSCC model, pERK2, AKT and pAKT levels increased trypsin treatment and counted each day. For the wound in CAL27 miR-223 tumors. This miR-223-3p-induced healing assay, cells were plated at 1 × 10 cells/well in a activation of the downstream effectors of the EGFR 6-well plate. At confluency, a wound was made using a signaling pathway may explain the resistance to cetuximab yellow tip and the first image was taken ( t = 0) to measure observed in our study. The molecular mechanisms leading the distance separating the two rims. At the indicated to this activation occurred downstream of the interaction times, the distance between the two rims was assayed and between EGFR and its ligands since we showed that the percentage of recovery was calculated. EGFR expression was not modified by miR-223-3p transfection (Supplementary Figure 1). Patients and samples In summary, we demonstrated that overexpression of miR-223-3p in the human head and neck cell line Surgically-removed tumors embedded in paraffin- resulted in decreased expression of STAT3 but also in wax blocks from 35 cases of HNSCC were retrieved www.impactjournals.com/oncotarget 57182 Oncotarget from the archives of the Laboratory of Clinical and as previously described [45]. Relative changes in gene Experimental Pathology (Pasteur Hospital) at the expression were reported as fold changes compared with University of Nice, Nice, France. All tumor specimens the control CAL27 cell line. were collected, stored, and used with the informed consent Protein extracts were resolved by SDS-PAGE and of the patients (Hospital-Integrated Biobank BB-0033- transferred onto a polyvinylidene difluoride membrane 00025, Pasteur Hospital, Nice, France). The study was (Immobilon-P; Millipore). After saturation, membranes approved by the Ethics Committee of the University of were incubated overnight with the indicated antibody, Nice-SophiaAntipolis and performed according to the washed in Tris-NaCl buffer (TN) supplemented three guidelines of the Declaration of Helsinki. The cases, times with 0.1% Triton X-100 (TNT), and finally recruited between 2005 and 2010, included squamous cell with TN buffer. Membranes were then incubated with carcinomas taken exclusively from metastasis-free patients. the secondary anti-rabbit or anti-mouse horseradish Cases were included in this study only if a follow-up of at peroxidase-conjugated antibody. Bound antibodies were least 5 years was obtained, and clinical data were available. revealed using an ECL system (Pierce) and the signal was Mean age at surgery was 61 years (range: 39 – 71) and quantified with a Pxi Camera (Ozyme, FR).Phospho-ERK 21 patients were male. Selected patients displayed similar (#9101), ERK (#9102), phospho-AKT (#9271), AKT health status and absence of concurrent chronic illnesses, (#9272),Phospho-STAT3 (#9131), STAT3 (#9139) and or tumors elsewhere. The primary sites of the carcinomas EGFR (#2085) antibodies were from Cell Signaling and were: oropharynx (18), hypopharynx (9), and larynx the anti-ACTB (Actin, clone AC40) antibody was from (8). Tumor stage (primary tumor) according to the 2009 Sigma Aldrich. American Joint Committee on Cancer staging system was T1 (12), T2 (10,) T3 (8) and T4 (5). Of note, eighty per Histology and immunohistochemical analysis cent of the carcinomas were keratinizing, with a range of grades: grade I (9), grade II (15), and grade III (11). For histopathology, paraffin-embedded tissues were sectioned (4 μm-thick slices), stained with hematoxylin and eosin and evaluated blind by three pathologists (CB, MI, PH). Animal strains and xenograft orthotopic model To investigate the cell-specific distribution of miRNA of HNSCC in normal and head and neck tumors, in situ hybridization This study was approved by the Institutional Care and was performed using 5ʹ- and 3ʹ-end digoxigenin (DIG)- Use Committee of the University of Nice-Sophia Antipolis. labeled LNA-modified DNA oligonucleotides (LNAs) Animal protocols were approved by the committee for complementary to the mature miRNA (Exiqon A/S, Research and Ethics of the PACA region (CIEPAL azur, Denmark). In this study, the global expression of miR-223- #PEA 12-153) and followed the European directive 3p was examined (LNA-scrambled as a negative control). 2010/63/UE. NMRI nude mice (nu/nu) were supplied by Scoring of miR-223-3p, neutrophil infiltrate and CD31 Janvier laboratories (Le Genest-St-Ile, France). A hundred expression were done by the pathologists at the invasive μl of ringer lactate solution containing 1 × 10 CAL27 front of the tumors. Luci +/− miR-223 were injected into the mouth floor of Immunohistochemical (IHC) staining for KI67, seven-week-old female mice as described previously [44]. cleaved caspase3, CA9, VEGFR2 and CD31 was On day one, cell implantation was verified by IVIS performed using tumor sections. Antigen retrieval was imaging and groups were stratified (8 to 10 animals per performed by boiling sections for 10 min in citrate buffer group). For mice receiving treatment, cetuximab (Erbitux, (pH 6.0) and cooling at RT°, followed by blocking of Merck Serono, Darmstadt, Germany) 5 mg/kg, cisplatin endogenous peroxidase activity with 0.3% H O for 2 2 (Milan, Amsterdam, Netherlands) and docetaxel (Accord 30 min. The sections were blocked with 2.5% horse serum Healthcare, Lille, France) 20 mg/kg were diluted in NaCl in TBS solution for 30 min in a humid chamber prior to 0.9% prior to the injection via the intraperitoneal route incubation with optimal dilutions of anti-Ki67 (Epitomics, (100 μl per injection) at days +3 and +9. Before sacrifice 1/300), anti-cleaved-caspase-3 (Imgenex, 1/500), anti- (day 14), luciferase activity was scored. CA9 (Abcam, 1/500), anti-VEGFR2 (Cell Signaling, 1/400), and anti-CD31 (M0823 Dako, clone JC70A, 1/50) overnight at 4°C. Positive cells were detected using an Quantitative real time PCR and immunoblotting ImmPRESS HRP anti-rabbit detection kit or directly with Total RNA was isolated from cells using the an anti-Streptavidin-Alexa-594 antibody. The immune AllPrep DNA/RNA/Protein Mini Qiagen kit (Qiagen) complexes were visualized using a Peroxidase Substrate following the manufacturer’s instruction, and 1 µg was DAB kit (Vector) according to the manufacturer’s reversed transcribed using the high-capacity cDNA RT kit protocol, and slides were counterstained with hematoxylin. (Applied Biosystems). RT-qPCR analysis was performed Blind quantification of brown staining was done as on a StepOne Real-Time PCR system using TaqMan PCR follows: Ki67 and cleaved caspase-3 tumor nuclear Master Mix (Applied Biosystems; Life Technology), staining corresponded to no cells (1), 1 to 10 % (2), 10 www.impactjournals.com/oncotarget 57183 Oncotarget to 30% (3), 30 to 50% (4) and > 50% (5). Membranous VV-C collected and analyzed the data. CB, MI and PH and cytoplasmic staining of CA9 corresponded to less participated in subject recruitment and in the Tissue Bank. than 80% (1) or 80 to 100 % of the cells (2). The final PB and PH gave technical support and conceptual advice. score corresponded to a mean of 5 fields per tumor AB, PH and VV-C wrote the manuscript. with 5 to 10 tumors per group. Tumor angiogenesis was quantified using both VEGFR2 and CD31 staining. ACKNOWLEDGMENTS VEGFR2 staining was scored following two criteria: the percentage of positive cells with no cells (1), 1 to 5 % (2), The authors wish to thank Marine Pedro, Katia 5 to 30% (3) and > 30 % (4) and the number of aligned Zahaf (LPCE, Nice) and Marie Bazin (IRCAN) for positive cells (considered as microvascular density, MVD) their expert technical assistance. Dr Nathalie Ebran with a single cell (1), 2 to 3 cells (2) and more than 4 (Centre Antoine Lacassagne) kindly provided luciferase aligned cells (4). MVD was also measured following expressing CAL27 cells. We acknowledge the IRCAN’s the method described by Weidner at al. [46]. In brief, Animal Core facility. scanned slides (NanoZoomer 2.0 HT from Hamamatsu) were initially evaluated at 50× magnification in order CONFLICTS OF INTEREST to identify and count areas with higher vascular density characterized as hot spots. In addition, 3 representative We have no conflicts of interest. hot spots measuring 70000 μm were selected. Images of the selected fields were analyzed at 400× magnification. FUNDING Any brown endothelial cell clusters (≥ 3 cells) that were clearly separated from adjacent microvessels, tumor cells This work was supported by grants from the and other connective tissues were considered as countable “Institut National de la Santé et de la Recherche microvessels. The total number of vessels counted in Médicale”, the “Association pour la Recherche contre le each case was divided by the number of hot spots, thus Cancer” (ARC Grants # SL220110603478), the PACA providing the mean MVD for that case. Cancéropole, the “Centre National de la Recherche Scientifique” and the French Government (National Cell survival and clonogenic assay Research Agency, ANR through the “Investments for the 4 Future” LABEX SIGNALIFE: program reference #ANR- For cell survival assay, 1 × 10 CAL27 +/− miR-223 11-LABX-0028-01. cells were seeded on a 96-well plate and, where notified, pre-treated with the indicated dose of cisplatin, cetuximab, docetaxel and fluorouracil (5-FU) for 48 hrs. XTT was added REFERENCES to each well and its cleavage to formazan was followed every 15 minutes over a 10 hr period. Formazan dye was 1. Simard EP, Torre LA, Jemal A. International trends in head quantified using a scanning multi-well spectrophotometer, and neck cancer incidence rates: differences by country, sex as indicated by the manufacturer (Roche, # 11465015001). and anatomic site. 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Oncotarget – Pubmed Central

Published: Jul 11, 2017

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