TLR7 Promotes Tumor Progression, Chemotherapy Resistance, and Poor Clinical Outcomes in Non–Small Cell Lung Cancer Free

The relation between inflammation and cancer is well established. Chronic inflammation in the tumor microenvironment promotes tumorigenesis in many cancers. In such an environment, the transcription factors NF-κB and STAT3 have been found activated in cancer cells (1, 2).

Toll-like receptors (TLR), a family of receptors that recognize various PAMPs and DAMPs (3), are strongly involved in inflammation through the activation of NF-κB. TLRs are expressed by a numerous immune cells and play a key role in the activation of dendritic cells, a prerequisite for the induction of specific antitumor immune responses (4). Given this property, experimental TLR agonists are currently being investigated in clinical trials as adjuvants for the treatment of cancers (5–7). Several studies also demonstrated TLR expression by tumor cells in many solid cancers (8–14), and their implication in tumor progression. TLR2 favors gastric tumorigenesis (15), TLR4 promotes the growth and chemoresistance of epithelial ovarian cancer cells (16), and correlates with tumor grade of head and neck squamous cell carcinoma (17). On the contrary, stimulation of TLR3 leads to tumor regression, by inducing apoptosis of tumor cells (13, 18). More recent studies also demonstrated the influence of TLR4 and MyD88 expression on patient's cancer outcome (19, 20).

Non–small cell lung cancer (NSCLC) is the most frequent lung cancer and one of the leading causes of cancer-related death (21). The therapeutic management of patients with NSCLC from stages I to IIIA, usually consists in surgical resection, often associated (particularly in stages II–IIIA) with preoperative and/or postoperative chemotherapy, using platinum-derived salts combined with a second- or third-generation drug such as gemcitabine, vironelbine, pemetrexed, or taxanes (22). However, the 5-year survival rate in resected patients remains low, around 40%, indicating that the treatment is poorly effective in a high percentage of cases. Indeed, chemoresistance in NSCLC is a well-known phenomenon (22). Several mechanisms of chemoresistance have been described in solid cancers, that can be cell-intrinsic (23) or acquired during the course of the treatment (24). In NSCLC, it has been shown that galectin 1 expressed by tumor cells induces chemoresistance through upregulation of p38MAPK and ERK (25), and that nicotine induces STAT3 phosphorylation, leading to the serine/threonine kinase IKBKE oncogene activation, which leads to chemoresistance (26). However, to date, no biomarker is currently used to predict response to chemotherapy.

Epidemiologic studies have shown that chronic inflammation increases the risk of lung cancer (27, 28). Our previous investigations demonstrated that TLR7, a receptor for ssRNA, is expressed by lung cancer cell lines and primary NSCLC tumor cells and that stimulation of tumor cells with different TLR7 agonists leads to NF-κB activation, increases the expression of antiapoptotic molecules, tumor cell survival, and resistance to chemotherapeutic drugs including cisplatin, carboplatin, doxorubicin, and vironelbine (8). However, TLR7 was not only expressed by tumor cells because we have also observed its expression by tumor-infiltrating immune cells (8), some of them located in the tertiary lymphoid structures that are present in the tumor stroma of patients with NSCLC (29). In the present study, our goals were to determine the in vivo consequences of TLR7 stimulation occurring on both tumor and immune cells, on tumor progression and resistance to chemotherapy, and the impact of TLR7 expression on the clinical outcome of patients.

We show that stimulation of TLR7 in immunodeficient and immunocompetent mice grafted with TLR7-positive tumor cells exerts a protumoral effect and induces resistance to chemotherapy. In large cohorts of surgically treated patients, receiving or not neoadjuvant chemotherapy, we demonstrate that high TLR7 expression correlates with poor prognosis and is associated with lack of response to neoadjuvant chemotherapy.

Collectively these results demonstrate a protumoral role of TLR7 expressed by tumor cells and strongly suggest its implication in the phenomenon of chemoresistance in patients with NSCLC.

The mouse strains used include NOD/SCID, C57BL/6, and TLR7-deficient mice (B6.129S1-Tlr7tm1Flv/J strain) and were purchased from Charles River Laboratories. These mice were maintained in the Central Animal facilities of the Centre de Recherche des Cordeliers, in standard pathogen-free conditions. Female mice, 6- to 8-week-old, were used for all experiments. All experiments were performed in accordance with European Union guidelines, and were approved by the Charles Darwin Ethics Committee in Animal Experiment, Paris, France (permit number: Ce5/2010/057).

The human A549 and the murine LL/2 lung adenocarcinoma cell lines were obtained from the American Type Culture Collection (respectively, ATCC #CCL-185 and #CRL-1642). The cells were cultured in DMEM/F12 medium (Invitrogen) containing 10% heat-inactivated FCS (PAA laboratories GmbH), 1% glutamine, non-essentials amino acids, 1% Ultroser G, 1% HEPES, 1% pyruvate 0.2% Normocin, and 1% penicillin-streptomycin (Invitrogen) at 37°C in a humidified atmosphere with 5% CO2. The cumulative culture length of the cells was less than 6 months after resuscitation. Early passage cells were used for all experiments and they were not reauthenticated.

Human and mouse TLR7 agonist loxoribine (InvivoGen), which is a guanosine analog, was used at 40 μg for all in vitro experiments. Human and mouse TLR7 agonist CL264 (InvivoGen), which is a 9-benzyl-8 hydroxyadenine derivative, was used at 10 μg for in vitro experiments and 40 μg for in vivo experiments. TLR7 antagonist IRS661 (Eurofins, MWG Operon) or ODN (Eurofins) control was used at 20 μg concentration for in vitro and in vivo experiments. Cisplatin and carboplatin were obtained from St. Antoine Hospital pharmacy (Paris, France).

LL/2 cells (3 × 10⁵) were cultured in a 6-well plate for 48 hours along with TLR7 agonist (10 μg CL264) followed by the addition of carboplatin (100 μmol/L) for 48 hours. At the end of the experiment (day 3), the cells were stained by Trypan blue for counting of live cells. Annexin V and propidium iodide staining was performed for evaluation of the apoptotic cells.

To induce tumor formation, 1 × 10⁶ cells A549 or LL/2 cells in PBS were grafted subcutaneously on day 0 into mice. The same procedure was followed for control mice injected with PBS. Mice received three intratumoral injections of TLR7 agonist (loxoribine or CL264, 40 μg) and/or IRS661 (20 μg) and ODN (20 μg) control on days 0, 3, and 6 after grafting of tumor cells followed by three injections of cisplatin (100 μmol/L) on days 9, 12, and 15. Tumor growth was measured every 3 or 4 days. The length (l), breath (b), depth (d) of the subcutaneous tumor in mice were measured with a caliper and the tumor volume calculated taking into account all the three values according to the ellipsoid volume calculation formula: tumor volume = 4/3 × π × l/2 × b/2 = 0.5,236 × l × b × d.

Cells were fixed with Fixation/Permeabilization concentrate (eBioscience) diluted 1:4 times in Fixation/Permeabilization diluent and permeabilized using Permeabilization buffer (eBioscience) diluted 1:10 times in distilled water and 2% human serum. Cells were stained with polyclonal PE anti-TLR7 (Imgenex) and PE anti-Rabbit IgG isotype control (Jackson Immunoresearch). To identify the intra-tumoral immune cell populations, membrane stainings were performed with PE anti-CD45 (BD Pharmigen), FITC anti-CD3 (BD Pharmigen), PETR anti-CD4 (Invitrogen), AF700 anti-CD8 (eBioscience), APC anti-CD19 (BD Pharmigen), eF450 anti-CD11b (eBioscience), PE anti-CD11c (eBioscience), and PerCPcy5.5 anti-Gr1 (BD Pharmigen). For the FoxP3 analysis, cells are first stained with the cellular antibodies, permeabilized as previously described, and stained with the primary PE anti-FoxP3 antibody (eBioscience). Staining was assessed with a FACS Fortessa cytometer, and flow cytometry data were analyzed using FACSDiva software (BD Biosciences) and FlowJO (Tree Star) software.

A retrospective series of 352 NSCLC stage I-III patients who underwent primary surgery (without neoadjuvant chemotherapy) and who were operated between 2001 and 2005 was obtained from Hôtel-Dieu hospital (Paris, France). A retrospective cohort of 210 stage III NSCLC patients who received neoadjuvant chemotherapy and who were operated between 2000 and 2007 was obtained from Hôtel-Dieu hospital. Pathologic staging of lung cancer was reviewed and classified according to the new tumor–node–metastasis (TNM) classification 2009 (30), and histologic types were determined according to the classification of the World Health Organization (WHO; ref. 31).

Lung tumor samples were analyzed with the agreement of the French ethic committee (agreement 2008-133 and 2012 06-12) in accordance with article L.1121-1 of French law.

Tumor samples were fixed in neutral-buffered 10% formalin solution and paraffin embedded. TLR7 staining was performed as previously described (8) using TLR7-specific polyclonal antibody (ENZO Lifesciences) at 10 μg/mL. Positive staining was identified as a clear red perinuclear staining of the tumor cells. The expression level of TLR7 for each patient was determined as the average percentage of TLR7 expression by tumor cells for 10 fields at ×20 magnification under a light microscope (Nikon eclipse, 80i), by three independent readers (S. Chatterjee, D. Damotte, and I. Cremer), one of them being a pathologist (D. Damotte).

Statistical analysis for in vivo experiments was performed using GraphPad Prism 5 (GraphPad Software) and using the R software (RFoundation for Statistical Computing; www.r-project.org). All mice experiments were analyzed using ANCOVA method (P > 0.001 for all parameters tested including tumor growth over time, tumor growth in different groups of mice, and interaction between tumor growth over time in the fourth groups of mice). Post hoc analysis with Bonferroni corrections were applied for pair-wise comparisons of tumor growth over time in different groups of mice. Tumor volumes at the endpoint of each experiment were compared using a nonparametric Mann–Whitney test with Bonferroni corrections for pair-wise comparisons. Mann–Whitney tests were also used to compare percentages and numbers of immune cells infiltrating the tumors in mice.

Overall survival (OS) curves were estimated by the Kaplan–Meier method and compared by the log-rank test. Survival comparison was adjusted for either imbalanced or prognostic baseline covariates using a Cox model. Groups of patients were obtained using the cutoff at minimal P value. Because the P values obtained might be overestimated, OS log-rank P values were corrected using the formula proposed by Altman and colleagues (32). The OS was defined from the date of the surgery until the date of death or the last day of the patient's visit to the hospital. Univariate and multivariate analysis with the Cox proportional hazards regression model was carried out to identify possible factors that could influence the survival of the patients. Parameters identified in univariate analysis as possibly influencing outcome (P ≤ 0.05) were introduced in a multivariate Cox proportional hazards regression model.

Comparisons of the mean and the median TLR7 expression in tumor cells in responders and nonresponders were performed using a t test and a Mann–Whitney test, respectively.

A Wilcoxon matched-pairs signed rank test was used to compare TLR7 expression in lymph nodes and in primary tumors for the same patients after neoadjuvant chemotherapy. Correlations were obtained by the Spearman test. All P values were two tailed.

P ≤ 0.05 was considered statistically significant for all experiments.

We grafted human A549 cell lines in NOD/SCID mice and evidenced TLR7 expression by subcutaneous tumors using IHC (Fig. 1A). Injections of the TLR7 agonist loxoribine resulted in a marked increase in tumor volume as compared with control PBS (Fig. 1B and C). The injection of cisplatin resulted in slower tumor growth. However, when the mice received a combination of loxoribine and cisplatin, tumor progression was restored to control levels, abolishing partially the positive effect of cisplatin on tumor regression (Fig. 1B and C). We obtained similar results when mice received vironelbine instead of cisplatin (data not shown).

To demonstrate that the protumoral effect of the agonist was mediated by TLR7, we used the IRS661 immunoregulatory RNA sequence, an antagonist previously described to specifically block TLR7-mediated inflammation (33). Treatment of A549 cells with IRS661 specifically resulted in a decrease of TLR7 expression (Fig. 1D), whereas it did not affect the expression of intracellular TLR3, TLR8, and TLR9 (Supplementary Fig. S1). We grafted NOD/SCID mice with A549 cells and injected either loxoribine, IRS661, or control ODN, alone or in combination, followed or not by cisplatin. We observed a decrease in the tumor volume in mice injected with loxoribine in combination with IRS661 compared with mice treated with loxoribine in combination to control ODN, suggesting an antagonistic effect conferred by TLR7 blockade (Fig. 1E and 1F). However, we did not observe any significant difference in tumor volume in mice having received IRS661 alone or control ODN. This result shows that TLR7 antagonist has an effect only when coinjected with the agonist. Injection of cisplatin induces a strong reduction of tumor growth. The injection of IRS661 had no significant effect, injected alone or with cisplatin.

We previously demonstrated that some intratumoral immune cells express TLR7 (8). Therefore, to analyze the effects of TLR7 stimulation on both tumor and immune cells, we performed a series of experiments in immunodeficient NOD/SCID mice, in immunocompetent WT C57BL/6 or in TLR7-deficient mice grafted with syngeneic LL/2 cells, which also express TLR7 (Supplementary Fig. S2A). For this series of experiments, we used CL264, which is a strong agonist for TLR7 in murine tumor cells (data not shown). We first demonstrated that CL264 induces in vitro chemoresistance in murine LL/2 cells (Supplementary Fig. S2B). We then grafted LL/2 cells in NOD/SCID mice and confirmed by IHC that subcutaneous tumors express TLR7 (Fig. 2A). CL264 injections resulted in increased tumor growth as compared with control PBS (Fig. 2B and 2C), demonstrating a protumoral effect of TLR7 stimulation. In addition, cisplatin injections induced tumor regression, which was abolished when the mice received CL264 before cisplatin treatment. This demonstrates induction of chemoresistance by CL264 in immunodeficient mice. Similar results were obtained in immunocompetent wild-type C57/BL6 mice (Fig. 2D and 2E) and in TLR7-deficient C57/BL6 mice (Fig. 2F and 2G). In the group of mice receiving CL264 injections, tumor growth was 2-fold increased as compared with control group receiving PBS. The injection of 40 μmol/L cisplatin alone resulted in inhibition of tumor growth. However, we observed no inhibitory effect of cisplatin when the mice had received TLR7 agonist both in wild-type and in TLR7-deficient mice. In fact, the combination of cisplatin with CL264 resulted in increased tumor growth as compared with control PBS. Similar results were obtained using imiquimod instead of CL264 as TLR7 agonist (data not shown). In addition, we observed that the CL264 induced resistance to cisplatin treatment occurred in wild-type mice having received doses of cisplatin from 100 μmol/L up to 300 μmol/L (Supplementary Fig. S3). We also injected the TLR7 antagonist IRS661 alone or in combination with the CL264, in wild-type mice, and observed similar results than in NOD/SCID mice. The coinjection of CL264 with IRS661 induced a strong decrease in tumor volume, whereas the injection of IRS661 alone resulted in same tumor volumes as observed in the control mice receiving control ODN (Supplementary Fig. S4).

Altogether, these results demonstrate that stimulation of TLR7-expressing tumor cells leads to increased tumor survival and resistance to chemotherapy, through a direct effect on tumor cells, and that the coinjection of TLR7 agonist and IRS661 antagonist induces strong reduction of the tumor.

To decipher the effect of TLR7 stimulation by CL264 on the recruitment of immune cells into the tumor microenvironment, we analyzed the composition of the immune infiltrates. The presence of CD45⁺ immune cells including T cells, B cells, macrophages, dendritic cells, granulocytes, and myeloid-derived suppressor cells was observed in the tumor microenvironment. The injection of CL264 resulted in a significant increase in the myeloid compartment, whereas the lymphocyte compartment was not modified significantly. The injection of CL264 induced an increase in percentages and absolute numbers of dendritic cells (6%–13%, P = 0.008; 2.34 × 10⁶–6.9 × 10⁶ cells, P = 0.016), macrophages (25%–40%, P = 0.008; 0.46 × 10⁶–0.99 × 10⁶ cells, P = 0.016), granulocytes (0.69%–1.5%, P = 0.032; 0.017 × 10⁶–0.048 × 10⁶ cells, P = 0.032), and myeloid-derived suppressor cells (16.1%–30.1%, P = 0.008; 0.32 × 10⁶–0.77 × 10⁶ cells, P = 0.008) in the tumors (Supplementary Fig. S5).

To determine the relevance of TLR7 expression in patients with NSCLC, we quantified TLR7-expressing tumor cells in a cohort of 352 untreated patients with stages I to III disease (Supplementary Table S1). We found a heterogeneity of TLR7-positive tumor cells among different patients, ranging from 0% to 100% (Fig. 3A). Twenty seven percent of the patients had no expression of TLR7 on their tumor cells, and 33% patients had more than 80% of tumor cells expressing TLR7 (Fig. 3B).

We determined whether such expression of TLR7 on tumor cells had an impact on the clinical outcome of the patients. We divided the patients in two groups based on the TLR7 distribution. The optimal cutoff, found at 82%, was determined taking into consideration the least significant P value (Supplementary Fig. S6), and was validated by the AUC method. We found a significant worse outcome (P = 0.0021) among patients who had more than 82% tumor cells expressing TLR7 compared with the patients expressing less than 82% (Fig. 3C). The mean OS was 36 months for the TLR7 >82% group and increased to 72 months for the TLR7 ≤82% group. Univariate and multivariate proportional hazard Cox analyses (Table 1) revealed that among parameters tested (gender, age, smoking history, histologic type, and pathologic stage), the pathologic stage and TLR7 are strong and independent predictors of survival for resected NSCLC patients.

We quantified the percentages of TLR7-expressing tumor cells in a cohort of 210 stage III patients treated with chemotherapy before the surgery (Supplementary Table S2). We observed a heterogeneous distribution of TLR7 expression among different patients, ranging from 0% to 100%. Twenty eight percent of the patients had no expression of TLR7 on their tumor cells and 30% patients had more than 80% of tumor cells expressing TLR7 (Fig. 4A). We also observed that high TLR7 expression (more than 81% of tumor cells expressing TLR7 determined as the optimal cutoff for this cohort, using the minimal P value approach; Supplementary Fig. S7) confers poor clinical outcome (P = 0.0032; Fig. 4B). The mean survival was 17 months for the TLR7 > 81% group and increased up to 34 months for the TLR7 ≤81% group.

Having observed that TLR7 triggering conferred chemoresistance both in vitro and in mice, we searched whether the percentage of TLR7-expressing tumor cells was associated with response to neoadjuvant chemotherapy. At the end of the treatment, the global response, called “downstaging,” was estimated by pathologists to determine whether the tumor had regressed or not in response to chemotherapy. Among the 166 patients for whom this information was available, tumor regression was observed only in 55 patients. These patients, considered as responders, were stage III before the treatment and stage I or II after chemotherapy. As expected, the patients for whom no downstaging was observed have a poor clinical outcome as compared with the others (Supplementary Fig. S8). We observed strong differences in the mean and the median percentages of TLR7-expressing tumor cells when we compared patients who responded or not to the chemotherapy. The mean of TLR7-expressing cells was 27% and 50% for the responders and the nonresponders, respectively (P = 0.0004) and the median TLR7 was 3% and 50%, respectively (P = 0.0003; Fig. 4C). In addition, pathologic examination of the resected lung and nodal dissection after treatment also allowed the evaluation of pathologic lymph node and tumor response and assessment of possible downstaging. For few patients (n = 27), we compared the percentages of TLR7-expressing tumor cells in the metastatic lymph node and in the primary lung tumor. We observed a strong positive correlation between TLR7 expression on tumor cells in lymph node and lung of the same patient (r² = 0.5084, P = 0.0009; Supplementary Fig. S9). Again, a significant difference in the percentage of TLR7-expressing cells was observed in the responders and the nonresponder patients both in tumor and lymph node samples (Supplementary Fig. S10). We also showed significant differences in global, tumor, and lymph node downstaging in patients having more than 81% of tumor cells expressing TLR7 compared with patients having less than 81% of TLR7 expression (Supplementary Table S2). We compared TLR7 expression in responders and nonresponder patients treated with gemcitabine or with vironelbine, in combination with cisplatin, and observed similar levels of mean and median TLR7 expression among the responders, whatever the type of chemotherapy (Fig. 5).

We performed univariate and multivariate proportional hazard Cox analyses on the 166 patients (Table 2). We observed that in univariate analyses among all clinical parameters tested, the number of cycles of chemotherapy, the global downstaging, and the TLR7 expression were strong predictors of survival for patients with NSCLC. However, in multivariate analyses, tumor TLR7 expression is not an independent prognostic marker.

Altogether, these results underline a major role for TLR7 in conferring resistance to chemotherapy consisting of platinum salt combined with gemcitabine or vironelbine.

In the present study, we demonstrated that a high expression of TLR7 confers poor OS and impacts response to treatment in patients treated with neoadjuvant chemotherapy. To our knowledge, this is the first time that the prognostic and predictive values of TLR7 expression in a large cohort of patients with NSCLC before and after treatment with neoadjuvant chemotherapy have been studied.

The strong impact of TLR7 in promoting tumor cell survival and resistance to treatment has been validated in murine models. Indeed, experiments performed in wild-type or TLR7-deficient C57BL/6 and in NOD/SCID mice revealed that injection of TLR7 agonist promoted high tumor growth and resistance to treatment. Among the mechanisms that could confer resistance to chemotherapeutic drugs, we previously observed an upregulation of Bcl-2 in lung tumor cell lines stimulated with TLR7 agonists and also in patients with NSCLC (8). Our results are in accordance with a link between chemoresistance and chronic inflammation driven by TLR7 stimulation. In vivo, several mechanisms could explain the protumoral effects of TLR7 agonists, either a direct stimulation of tumor cell proliferation and/or an indirect effect implying the stroma, composed of matrix proteins, fibroblasts, and tumor-infiltrating immune cells. In our study, blocking of TLR7 expression and/or signaling by the inhibitor IRS661 reversed the protumoral effect of TLR7 agonist in vivo and resulted in strong inhibition of tumor growth. However, when the IRS661 antagonist is injected without TLR7 agonist, no antitumoral effect was observed, which suggests that IRS661 in combination with TLR7 agonists could interfere with other signaling pathways involved in tumor growth. It could be hypothesized that the inhibition of TLR7 expression on tumor cells could downregulate the inflammatory processes involving TLR7 signaling, resulting in inhibition of tumor growth. Several studies already demonstrated that this inhibitor strongly reduces local inflammation. Indeed it was showed that IRS661 blocked TLR7-mediated IFNα production by plasmacytoid DC in a systemic lupus erythematous model (33), and a reduced imiquimiod-induced release of IL6 and TNFα cytokines by monocytes (34). In addition, it has been recently shown that inhibition of TLR7 in vivo is protective against pancreatic tumor progression (35). Experiments performed in immunocompetent mice revealed that injection of TLR7 agonist promoted increased tumor growth and resistance to treatment. Analysis of tumor-infiltrating immune cells indicated that CL264 treatment induced a significant increase of the myeloid cell compartment, namely dendritic cells, macrophages, and MDSCs, whereas it had no impact on lymphocyte recruitment.

In the cohort of resected patients not treated with neoadjuvant chemotherapy, the level of TLR7 expression and the pathologic stage were found to have a significant impact on the outcome of the patients. Multivariate analysis revealed TLR7 as a strong prognostic marker. Among TLR family members or signaling molecules involved in TLR pathways, TLR4 and MyD88 have also been shown to be associated with poor prognosis factors in colorectal (19) and in epithelial ovarian cancers (20). MyD88 expression in these cancer cells has also been demonstrated to be a predicting factor of poor response to paclitaxel chemotherapy (36).

The fact that high tumor TLR7 expression confers poor clinical outcome to patients suggests that endogenous ligands are present in the tumor microenvironment. Such ligands could be DAMPs, such as RNA released from locally dying cells or miRNA (miR) released the by tumor cells themselves in exosomes. Several sequences of ssRNA have been shown to stimulate human TLR7 in macrophages (37). Moreover, it has been recently demonstrated that miR-21 and miR-29a present in exosomes secreted by lung tumor cells trigger TLR7-mediated inflammatory responses (38).

We demonstrated here that TLR7 could also be a predictive marker for response to chemotherapy. The analysis of pathologic specimens indicates that most of the patients who did not respond to treatment expressed high levels of TLR7 compared with the patients who display good response. This observation strongly suggests that high TLR7 expression confers resistance to chemotherapy in patients, which is in accordance with the results from our in vitro and in vivo experiments. No major difference was found in the expression of TLR7 among the patients based on the type of chemotherapy administered, cisplatin plus vironelbine or gemcitabine, suggesting a broad effect of TLR7 on different chemotherapeutic treatments. NSCLC is described as highly chemoresistant type of cancer (22), and several biomarkers conferring a resistance in patients have already been described including DNA-repair enzymes (39). However, none of them is currently used for patient's management. Therefore, TLR7 could be a new interesting prognostic and predictive marker for chemoresistance in NSCLC.

Interestingly, we observed a good correlation of TLR7 expression on tumor cells present in the metastatic lymph nodes before and after chemotherapy and in primary tumors after chemotherapy (data not shown). This suggests that mediastinal lymph nodes can be used as a representative sample for TLR7 expression in the primary tumor. Our results imply that TLR7 can be a useful predictive marker for the identification of patients with high risk of resistance to treatment. Indeed, we suggest that the level of TLR7 expression on the tumor cells should be taken into consideration before administration of chemotherapy. Thus, our results bring novelty in the comprehension of mechanisms conferring resistance to chemotherapy treatment, and could have a strong clinical application in the therapeutic management of patients with NSCLC.

No potential conflicts of interest were disclosed.

Conception and design: S. Chatterjee, J. Cherfils-Vicini, C. Sautes-Fridman, I. Cremer

Development of methodology: S. Chatterjee, I. Cremer

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Chatterjee, D. Damotte, K. Iribarren, M. Alifano, A. Lupo, J. Cherfils-Vicini, M. Younes

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Chatterjee, L. Crozet, D. Damotte, K. Iribarren, C. Schramm, M. Alifano, J. Goc, S. Katsahian, M.-C. Dieu-Nosjean, W.-H. Fridman, I. Cremer

Writing, review, and/or revision of the manuscript: S. Chatterjee, A. Lupo, J. Cherfils-Vicini, J. Goc, M.-C. Dieu-Nosjean, W.-H. Fridman, C. Sautes-Fridman, I. Cremer

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Damotte, I. Cremer

Study supervision: I. Cremer

The authors thank P. Bonjour for technical assistance, M. Bovet for help in clinical data collection, and A.C. Joly (Hôpital St Antoine, Paris, France) for the gift of chemotherapeutic drugs.

This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Fondation ARC pour la recherche sur le cancer, the Fondation de France (00012068), Université Pierre et Marie Curie, Université Paris-Descartes, Institut National du Cancer (2011-1-PLBIO-06-INSERM 6-1, PLBIO09-088-IDF-KROEMER), CARPEM (Cancer Research for Personalized Medicine), and Labex Immuno-Oncology (LAXE62_9UMS872 FRIDMAN).

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