Toll like receptor 7 expressed by malignant cells promotes tumor progression and metastasis through the recruitment of myeloid derived suppressor cells

ABSTRACT In non-small cell lung carcinoma (NSCLC), stimulation of toll-like receptor 7 (TLR7), a receptor for single stranded RNA, is linked to tumor progression and resistance to anticancer chemotherapy. However, the mechanism of this effect has been elusive. Here, using a murine model of lung adenocarcinoma, we demonstrate a key role for TLR7 expressed by malignant (rather than by stromal and immune) cells, in the recruitment of myeloid derived suppressor cells (MDSCs), induced after TLR7 stimulation, resulting in accelerated tumor growth and metastasis. In adenocarcinoma patients, high TLR7 expression on malignant cells was associated with poor clinical outcome, as well as with a gene expression signature linked to aggressiveness and metastastic dissemination with high abundance of mRNA encoding intercellular adhesion molecule 1 (ICAM-1), cytokeratins 7 and 19 (KRT-7 and 19), syndecan 4 (SDC4), and p53. In addition, lung tumors expressing high levels of TLR7 have a phenotype of epithelial mesenchymal transition with high expression of vimentin and low abundance of E-cadherin. These data reveal a crucial role for cancer cell-intrinsic TLR7 expression in lung adenocarcinoma progression.


Introduction
Tumors constitute a complex microenvironment, which is composed of genetically altered malignant and non-mutated stromal cells, the latter including fibroblasts, vascular and lymphatic vessels, as well as immune cells 1,2 . Many tumor-infiltrating immune cells including CD4 + and CD8 + T cells, B lymphocytes, or type 1 macrophages (M1) participate in immunosurveillance, meaning that they are commonly associated with good clinical outcome. Other leukocyte subtypes including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and type 2 macrophages (M2) have immunosuppressive functions, meaning that their local presence has a negative clinical impact 1 . The composition of tumor infiltrating immune cells strongly influences the tumor microenvironment and the behavior of the tumor in terms of progression, metastasis and treatment resistance.
Toll-like receptors (TLRs), which recognize ligands from pathogens (pathogen-associated molecular patterns, PAMPs) or dying cells (damaged-associated molecular patterns, DAMPs), are involved in the immune response against pathogens 3 , in thus far that they induce the secretion of inflammatory cytokines and chemokines upon their activation 4 . These receptors are largely expressed by immune cells and epithelial cells but also by several types of tumors cells 5 . The role of TLRs in tumor microenvironment is complex. On one hand, TLRs expressed by immune cells can favor immunosurveillance, because their stimulation favors the maturation and activation of innate and adaptive immune effectors. On the other hand, TLRs expressed by cancer cells may receive stimuli that favor tumor progression 6,7 .
We have previously demonstrated that TLR7, a single stranded RNA receptor that is usually expressed in endosomes of immune cells including plasmacytoid and conventional dendritic cells (pDCs and cDCs), macrophages and B lymphocytes 8,9 , is expressed by malignant cells from NSCLC patients 10 . High expression by cancer cells was associated with poor prognosis, both in early stages that were treated with surgery alone and more advanced stages treated by neoadjuvant chemotherapy. Moreover, in a mouse model of lung cancer, TLR7 stimulation favored tumor growth 11 . The present study has been designed to decipher the mechanisms involved in the pro-tumorigenic and possible pro-metastatic effects of TLR7 stimulation.
Here, we report that the stimulation of TLR7 expressed by malignant cells favors tumor progression and metastasis, increases CCL2 and GM-CSF secretion in the tumor microenvironment, and elicits the recruitment of MDSCs into the tumor. In vivo depletion of MDSCs abolished the TLR7dependent pro-tumorigenic and pro-metastatic effect. Finally, we observed in lung adenocarcinoma patients, that high expression of TLR7 by tumor cells is associated to a pro-metastatic gene expression signature as well as to epithelial-mesenchymal transition (EMT).

Intratumoral TLR7 agonist injection favors tumor progression through a direct effect on carcinoma cells
Murine lung adenocarcinoma LLC-luc cells express several TLRs including TLR4, TLR7 and TLR9 ( Figure 1A-C). Expression of TLR7 was also validated by quantitative PCR (data not shown). We have previously demonstrated that intratumoral injection of TLR7 agonist results in increased tumor progression in immunocompetent as well as in immunodeficient mice 11 . However, the precise mechanisms involved in such pro-tumorigenic effects are elusive. To determine if this effect was specific, we analyzed the growth of subcutaneous grafted LLC-luc tumors in wild-type (WT) mice locally injected with TLR4, TLR7 or TLR9 agonists. Bacterial lipopolysaccharide (LPS), an agonist of TLR4, failed to affect tumor growth ( Figure 1A), while CpG, an agonist of TLR9, actually inhibited tumor growth ( Figure 1B). On the contrary, intratumoral injection of the TLR7 agonist CL264 induced a pro-tumorigenic effect ( Figure 1C), in accordance to our previous observations 11 .
The pro-tumorigenic effect of TLR7 stimulation could result from a direct stimulation of TLR7 on tumor cells and/ or from stimulation of immune cells that express TLR7. We determined the level of TLR7 expression of immune cells in the tumor, the spleen and the blood of tumor-bearing mice by flow cytometry after optimization of the gating strategy (Fig. S1). TLR7 was highly expressed by macrophages, pDCs, and to a less extent by cDCs in all compartments including the tumor (Fig. S2). Thus, TLR7 agonists may act both on malignant cells and on tumor-infiltrating myeloid cells to favor tumor progression.
To discriminate the target population of TLR7 agonist, we took advantage of TLR7-deficient mice (TLR7 KO), and generated TLR7-deficient LLC-luc cells (LLC TLR7 KO) by CRISPR/Cas9-mediated gene editing ( Figure 1D). We injected WT (TLR7 expressing) LLC-luc cells into TLR7 KO mice or alternatively TLR7-deficient LLC-luc cells into WT mice, and then evaluated the effects of the TLR7 agonist CL264 on tumor progression. We observed a growth accelerating effect of CL264 in TLR7 KO mice, similar to that observed in WT mice ( Figure 1C). In sharp contrast, the TLR7 agonist failed to stimulate the growth of TLR7-deficient LLC-luc tumors ( Figure 1D). These results clearly demonstrate that the pro-tumorigenic effect of CL264 effect is mediated by an effect on TLR7 expressed by malignant (rather than immune) cells.

Stimulation of TLR7 alterates the intratumoral immune infiltrates
TLR stimulation is known to modify the inflammatory microenvironment of tumors. We analyzed whether the local injection of TLR7 agonist would impact the composition of the immune infiltrate. Immune phenotyping by flow cytometry (for gating strategy see Fig. S1) revealed the percentages of T, B and NK cells, Tregs, macrophages, cDCs, pDCs and MDSCs, including granulocytic and monocytic subpopulations. The immune infiltrate of vehicle-only treated tumors was composed by a majority of myeloid cells including 49% of MDSCs, 15% of cDCs and 5% of macrophages, as well as a by a minority of adaptive immune cells including 10% T cells, 5% NK cells and 3% Tregs (Figure 2A). The local injection of CL264 induced a modification of percentages and absolute cells numbers of MDSCs, with a significant (unpaired Student T test, p < 0.05) increase from 49 ± 7% to 66 ± 10% (Figure 2A-C). CL264 also induced a decrease of cDCs (from 15% ± 1% to 9%± 2%) and of pDCs (from 2% to 1.5%) ( Figure 2D-E). In control tumors, MDSCs were mostly monocytic, and CL264 treatment resulted in the increase of granulocytic MDSCs ( Figure 2F-G). In contrast, CL264 did not affect the frequency of macrophages, NK, T, Tregs, and B cells (Figure 2A, Fig. S3A).

CL264 stimulates MDSC recruitment by acting on TLR7 expressed by malignant cells
We investigated if the increase of MDSCs within the tumor was a consequence of TLR7 stimulation on cancer or immune cells. The TLR7 agonist CL264 was injected into LLC-luc tumors that were established in WT or TLR7 KO mice.
At baseline levels (without CL264 treatment), WT and TLR7KO mice showed low frequencies of MDSCs, cDCs and macrophages in their blood and spleens (Fig. S4A, B). TLR7 KO mice exhibited a lower proportion of MDSCs, cDCs, NK and T cells and increased proportion of B cells in the spleen and the blood compared to WT mice (Fig. S4A, B). The tumor immune infiltrate was characterized by high frequencies of MDSCs, both in TLR7 KO and in WT mice. Higher proportions of macrophages and T cells and lower proportions of cDCs were detected in the tumors of TLR7 KO compared to WT mice (Fig. S4C).
In tumors growing on TLR7 KO mice ( Figure 2H-N), compared to WT mice (Figure 2A-G) the injection of TLR7 agonist increased the proportion and absolute number of MDSCs ( Figure 2H-J), with a marked shift towards a granulocytic MDSC phenotype ( Figure 2N), concomitant with a slight decreased proportion of cDCs ( Figure 2K), but did not modify the frequency of pDCs ( Figure 2L), macrophages, NK, T, Tregs and B cells (Fig. S3B).
Collectively, these data indicate that CL264 maintains its local immunomodulatory function resulting in an increased presence of MDSCs in WT and TLR7 KO mice.
Mdscs are involved in the pro-tumorigenic effect mediated by TLR7 stimulation To decipher the possible role of MDSCs in the tumor growth stimulatory effect of CL264, we experimentally depleted MDSCs from the system. Tumor bearing mice that were treated with CL264 or vehicle only received intraperitoneal injections of anti-Gr1 monoclonal antibody (mAb) or control isotype every 48 hours ( Figure 3A). During the experiment, less than 0.4% of MDSCs were detectable in the blood of mice, confirming the efficacy of MDSC depletion ( Figure 3B-C). MDSC depletion resulted in decreased tumor growth, both in WT and in TLR7 KO mice ( Figure 3D, G). The TLR7 agonist CL264 stimulated tumor growth ( Figure 3E, H), and this pro-tumorigenic effect was completely lost in the absence of MDSCs, both in WT mice ( Figure 3F) and in TLR7 KO mice ( Figure 3I).
These results demonstrate that the tumor growth exacerbating effect of TLR7 stimulation is mediated by the increased number of MDSCs within the tumor microenvironment.
TLR7 stimulation induced an increased production of CCL2 and GM-CSF within the tumor microenvironment Given the involvement of MDSCs in the pro-tumorigenic effect of TLR7 stimulation, we investigated the mechanisms that might be involved in the local CL264-induced MDSCs expansion. Several cytokines such as IL-6, GM-CSF or CCL2 stimulate the recruitment or proliferation of MDSCs and may be produced by tumor cells. Local injection of the TLR7 agonist CL264 enhanced the ELISA-detectable concentration of CCL2 and GM-CSF within the tumor. The concentrations of CCL2 and GM-CSF were 10407 ± 1326 and 256 ± 15 pg/ml, respectively, in mice treated with CL264, and 7453 ± 1380 and 186 ± 15 pg/ml, respectively, in control mice receiving PBS ( Figure 3J, K). In contrast, CL264 failed to significantly modulate the amount of IL-6 found in the tumor ( Figure 3L). The secretion of CCL2 and GM-CSF by tumor cells was confirmed in vitro, which was found increased in supernatants of CL264-stimulated cells ( Figure 3M, N).
These results suggest that CL264 may stimulate the recruitment of MDSCs via the induction of CCL2 and GM-CSF.

TLR7 agonist does not modify the immunosuppressive capacities of mdscs
To unravel the possible impact of TLR7 stimulation on MDSCs functions, we measured the production of molecules involved in MDSCs-mediated immunosuppressive circuitries. The production of nitrate, H 2 O 2 , and arginase 1 by MDSCs was not affected by CL264 injection. Similarly, the proportion of ROS producing cells was not altered by CL264 (Fig. S5A). In addition, the capacity of MDSCs to suppress T cell proliferation was similar for MDSCs purified from PBS-treated and CL264-treated tumors ( Fig. S5B-C). These results demonstrate that intratumoral injection of TLR7 agonist does not modify the immunosuppressive properties of intratumoral MDSCs.

MDSC are involved in the pro-metastatic effect of TLR7 stimulation
Next, we analyzed the capacity of CL264 to stimulate lung metastasis. NOD/SCID or C57BL/6 mice were subcutaneously grafted with LLC-luc cells, and the apparition of metastasis was imaged over time, by bioluminescence or precisely quantified at necropsy following perfusion with Indina Ink and labelling of the lungs with Feket solution. Either of these methods revealed the capacity of CL264 to stimulate lung metastasis, both in immunodeficient NOD/SCID mice ( Figure 4A) and in immunocompetent C57Bl/6 mice ( Figure 4B).
The number of metastasis was quantified in PBS-and CL264-treated mice. We observed the presence of lung metastases in both groups of mice, but the mice having received three injections of TLR7 agonist had a significantly higher number of metastases with a mean of 9.5 (± 7.5) metastases per lungs for CL264-treated mice, compared to 4.9 (± 4.5) metastases per lungs for the control group ( Figure 4C). Repeated injections of CL264 (every three days throughout the duration of the experiment) led to even higher number of lung metastasis (19.5 (± 11.5) metastasis per lungs) ( Figure 4C). These results indicate that TLR7 stimulation has a marked pro-metastatic effect.
Given the implication of MDSC in metastatic dissemination 12 , we investigated the potential role of MDSCs in the pro-metastatic effects of TLR7 stimulation. MDSC depletion by injection of anti-Gr1 mAbs led to a significant decrease in the number of metastases (mean of 2.5 (± 2) metastases per lungs) in CL264-treated mice compared to control mice that did not receive depleting antibody (mean of 6.5 (± 4) metastases per lungs) ( Figure 4D), showing that TLR7 stimulation increases the number of lung metastases through a mechanism involving MDSCs.

High expression of TLR7 in human lung adenocarcinoma is associated with a metastatic gene expression signature
We further characterized the in vitro impact of CL264 on metastatic properties of malignant cells. The stimulation of LLC cells with CL264 resulted in epithelio-mesenchymal transition gene expression signature, with an increased gene expression of N-cadherin, vimentin, snail1, snail 2 and zeb, and a decreased expression of E-cadherin ( Figure 5A, B).
Immunohistochemistry was then used to determine the expression of E-cadherin, associated to epithelial cell  phenotype, and that of vimentin, expressed by mesenchymal cells, on human malignant cells expressing high or low levels of TLR7. Forty-five adenocarcinoma patients were analyzed for the expression of TLR7, E-cadherin and vimentin (for representative staining patterns see Figure 6A). Patients were divided into two groups that were expressing TLR7 or not on malignant cells. The expression of E-cadherin and vimentin by tumor cells was determined in a semiquantitative fashion. In TLR7 hi patients, the expression of E-cadherin was significantly lower (25.6%± 3.7% of cancer cells expressing E-Cadherin) than in TLR7 low patients (49.3%± 7% of tumor cells expressing E-cadherin) ( Figure 6B). Concomitantly, in TLR7 hi patients, the expression of vimentin was higher (34.6% ± 6.7% of tumor cells expressed vimentin), than in TLR7 low patients (15.3%± 3.6% of tumor cells expressed vimentin) ( Figure 6C). In conclusion, in patients with lung adenocarcinoma, high expression of TLR7 on cancer cells is associated with signs of EMT. Data are mean ± SEM (5 mice/group). *: p < 0,05, **: p < 0,01, ***: p < 0,0001. ns = not significant. Each experiment was repeated three times.
We then determined the physiopathology impact of TLR7 expression on the metastasis process in a large cohort of adenocarcinoma patients. We first determined the prognostic value of TLR7 on a cohort of 154 adenocarcinoma patients (sup Table 1), the malignant cells of whom displayed TLR7 either at a rather low level (< 5%) or were almost completely positive (> 82%, cut off determined in 11 ). We found that high TLR7 expression by tumor cells was associated with poor clinical outcome compared to low TLR7 expression, with a median OS of 45 months, compared to 90 months, respectively ( Figure 7A). Multivariate analysis revealed TLR7 expression as an independent prognostic marker ( Table 1). We then selected twelve patients from this cohort, including six patients having low (< 5%, TLR7 low ) and six having high (100%, TLR7 hi ) TLR7 expression, and performed gene expression analysis using nanostring technology and the pan cancer progression panel. Volcano plot analysis revealed that among 770 genes analyzed, six were significantly overexpressed and five were underexpressed in TLR7 hi compared to TLR7 low patients ( Figure 7B). Unsupervised clustering of differentially expressed genes, revealed a small cluster of three genes including ICAM1, keratin 7 (KRT-7) and keratin 19 (KRT-19) that were overexpressed in TLR7 hi patients. Additionally, NME1 (encoding nucleoside diphosphate kinase 1) and syndecan 4 (SDC4) (heparin sulfate proteoglycan molecule) were found overexpressed in the TLR7 hi group of patients ( Figure 7C). More detailed box-plot analysis of these genes confirmed their overexpression in TLR7 hi patients ( Figure 7D). Interestingly, TP53 was also found highly expressed in TLR7 hi patients ( Figure 7D). We then searched for correlation between the level of gene expression and survival of adenocarcinoma lung cancer patients, using the public software KM plotter 13 . High expression of KRT-7 and KRT-19, as well as that of NME1 and TP53, significantly correlated with poor clinical outcome ( Figure 7E).
Collectively, these data indicate the existence of a gene signature that is associated to TLR7 expression by malignant cells as well as with poor prognosis.

Discussion
Our present data indicate that TLR7 stimulation induces protumorigenic and pro-metastatic effects in a preclinical model of lung cancer while providing two salient insights. First, our data indicate that TLR7 must be expressed by the cancer cells (rather than by immune cells) so that the local presence of a TLR7 agonist can stimulate tumor growth. Second, we reveal the direct involvement of MDSCs in both the tumor growth-promoting and pro-metastatic effects of TLR7 stimulation. The presence of excess amount of a TLR7 agonist within the tumor causes an expansion of the number of MDSCs yet fails to enhance their immunosuppressive capacity. Our results also suggest that MDSC recruitment is mediated via a higher CCL2 and GM-CSF production in the tumor microenvironment after TLR7 stimulation. In addition, we demonstrated that high TLR7 expression by tumor cells from adenocarcinoma patients is associated to signs of EMT, as well as to a pro-metastatic gene expression signature. In particular, we observed a higher expression of KRT-7, KRT-19 and TP53, which are associated to the metastasis process and to a poor prognosis.
MDSC, a cell type with marked immunosuppressive properties 34 , has been linked to tumor progression in various murine models of cancer [35][36][37] . In cancer patients, the number of circulating MDSCs significantly increases compared to healthy subjects 38 . Our study, however, is the first demonstration in a cancer model that TLR stimulation leads to local MDSCs expansion, accounting for enhanced tumor growth and metastasis.
TLR7 stimulation has previously mostly reported to stimulate anticancer immunosurveillance, for instance through direct stimulation of TLRs on antigens presenting cells, leading to their maturation 39,40 . However, in recent years, TLR7 expression by tumor cells has been associated with a pro-tumoral effect in pancreatic adenocarcinoma 25 and in NSCLC 10,11 . We found that stimulation of TLR7 expressed by adenocarcinoma cells modulated the immune infiltrate, leading to a significant expansion in MDSCs, associated with an increased secretion of CCL2 and GM-CSF in the tumor microenvironment. Both these cytokines are known to play a key role in the recruitment of MDSCs 38,41,42 . Indeed, the absence of CCL2 production by cancer cells considerably reduces the recruitment of MDSCs into tumors 42 . Importantly, TLR7 stimulation had a pro-metastatic effect, while MDSC depletion drastically reduced this number of metastasis. In melanoma, MDSCs are associated with tumor progression, signs of EMT such as decreased E-Cadherin expression and increased migratory capacities of malignant cells 36 . Similarly, MDSCs may contribute to metastatic dissemination of different types of cancer, including NSCLC 43 . Finally, Srivastava's team demonstrated that when MDSCs were depleted, the size of tumors decreased and cell migration was reduced 44 . In line with the involvement of EMT in the metastatic process, we found that TLR7 expression in lung adenocarcinoma was associated with EMT. We observed that a high expression of TLR7 was associated with low expression of E-cadherin and overexpression of vimentin. EMT is a process in which cells lose adhesive properties because of down-regulating E-cadherin expression and rearrange their cytoskeleton through enhanced vimentin expression 45 . These changes in cancer cells play a critical role in metastasis development.
In adenocarcinoma patients, high TLR7 expression was associated with gene expression signature possibly linked to metastasis, with overexpression of KRT-7, KRT-19 and SDC4. These three genes have already been ascribed with pro-metastatic properties in different types of cancers. Indeed, KRT-7 is established as a diagnostic marker in a variety of malignancies including biliary duct, bladder, breast, cervix, kidney, liver, lung adenocarcinoma, ovary and pancreas cancer 46 . In patients with lung adenocarcinoma, strong expression of KRT-7 was associated with a poor overall survival 47 . Keratin 19 is a diagnostic marker in some tumors, such as in biliary duct, bladder, breast, cervix, colon, lung adenocarcinoma, ovary, pancreas, pleura, prostate, stomach and uterus cancer 46 . In addition, KRT-19-positive hepatocellular carcinomas highly express invasion-related/metastasis-related markers 48 . Finally, SDC4 plays a role in the development and metastatic occurrence in renal cell carcinoma 49 .
Strong expression of TLR7 in patients with lung cancer is associated with poor survival prognosis but also with a prometastatic gene signature. As a result, it would be interesting to explore the molecular etiology of this pathogenic TLR7 overexpression. Ligation of TLR7 by agonists can stimulate TLR7 expression in a feedforward loop 50 , which implies that high TLR7 abundance might be a sign of the natural stimulation of this receptor by ligands present in the tumor microenvironment. The molecular nature and source of such natural TLR7 agonists remains to be clarified. Moreover, it will be important to develop and characterize TLR7 antagonists or inhibitors of TLR7-elicited signals that might have immunostimulatory and anticancer effects.

Cell lines
LLC-Luc cells (gift from Vincenzo di Bartolo, Institut Pasteur, Paris) were obtained from LLC cells (Lewis Lung Carcinoma, ATCC CRL-1642) that were stably transfected with firefly luciferase. LLC-Luc cells were cultured in DMEM+ GlutaMax (Gibco) with 10% heat-inactivated FCS, 1% penicilin/streptomycin mixture, and 0.5mg/ml hygromycin B. The cumulative culture length of the cells was fewer than 6 months after resuscitation. Early passage cells were used for all experiments and they were not reauthenticated.

Mice
Wild-type C57Bl/6J mice, mice deficient in TLR7 (B6.129S1-Tlr7tm1Flv/J strain), and NOD/SCID mice were purchased from Charles River Laboratories. All the animals were house kept at CEF (Centre d'Explorations Fonctionnelles of Centre de Recherche des Cordeliers, Agreement no. A75-06-12). They were maintained in a constant temperature and humidity in light controlled room with a 12 hours light cycle. They had free access to food (SAFE Laboratory) and tap water. All experiments were conducted in 6-8 week-old female mice in accordance with the institutional guidelines and the recommendations for the care and use of laboratory animals put forward by the Directive 2010/63/ EU revising Directive 86/609/EEC on the protection of animals used for scientific purposes (project has been approved by a user establishment's ethics committee and the Project Authorization: number Ce5/2010/057 and 6629).

Tumor growth and metastasis
Cancer cells (1 × 10 6 in 100μl PBS/injection) were injected into the flank of recipient mice. Mice then received intra-tumor injections of LPS (40 μg/injection), CL264 (40 μg/injection) or CpG (30 μg/ injection) (Invivogen) at day 0, 3 and 6 after tumor grafting. Tumor volume (length (l), breath (b), depth (d)) was measured every 2-3 days using caliper, and was calculated taking into account the three values according to the ellipsoid volume calculationformula:tumorvolume=4/3×π×l/2×b/2=0.5236×l×b×d. For metastasis detection, mice were injected (i.p.) with 225mg/kg body weight D-luciférine K+ (Interchim), then were anaesthetized using isofluran, 10 minutes before imaging for bioluminescence using an IVIS imaging system (Lumina II). Mice were sacrificed between days 22 and 24. The rib cage was excised and 1 ml of India ink (7.5% solution) was injected into the trachea. The lungs were harvested and washed with 1X PBS. Separation of lobes and connectives tissues elimination was performed. The lungs were immersed and stored in 1ml of Feket solution (300ml of 70% ethanol, 30ml of 37% formaldehyde and 5 ml of glacial acetic acid). The number of metastatic foci was counted.
Staining was assessed with a FACS Fortessa cytometer, and flow cytometry data were analyzed using FACSDiva or FlowJO software. Kaplan-Meier survival curve for overall survival (OS) for the 154 lung adenocarcinoma patients, divided in two groups according to the stratification of TLR7 expression. Optimal cutoff of 82% was previously determined 11 . Patients are divided into TLR7 hi (> 82% expression of TLR7) and TLR7 low group (< 5% TLR7 expression). p-value was determined by log-rank test (A). Gene expression analysis was performed in 12 adenocarcinoma patients including 6 TLR7 hi and 6 TLR7 low , using nanostring technology. Volcano plot comparison of differentially expressed genes between TLR7 hi and TLR7 low group of patients. The horizontal dimension is the variation of median gene expression (log2 (ratio)) between the 2 groups, and the vertical axis represents the Mann-Whitney test p-value between TLR7 hi and TLR7 low patients. In red are represented genes that are over-or under-expressed in TLR7 hi patients, with a fold-change superior to 2 (11 genes among 770 studied) (B). Hierarchical clustering of the 11 differentially expressed genes in TLR7 hi compared to TLR7 low groups. Individual patients are oriented in columns and expression level for each gene is oriented in rows (C). Box plot representation of ICAM1, KRT-19, KRT-7, SCD4, NME1, and P53 gene expression in TLR7 hi and in TLR7 low patients (D). Kaplan-Meier survival curve for overall survival (OS) for adenocarcinoma patients divided in two groups according to the stratification of ICAM1, KRT-19, KRT-7, SCD4, NME1, and P53 gene expression, using online survival analysis software KM plotter. p value was determined by log-rank test (E).

Cytokine detection by ELISA
Mice were sacrificed 10 days after cancer cell injection and tumors were excised, cut into pieces of 1 mm 3 and placed in RPMI medium supplemented with 1% penicilin/streptomycin and 1% Glutamine for 24h at 37°C. Supernatants were then collected and stored at −80°C.
100000 LL/2 cells were cultured in a 12 well plate for 12 or 24 hours along with TLR7 agonist (2.5 µg/mL CL264), supernatants were collected and stored at −80°C.
T cells were purified from spleens using the Pan T cell Isolation Kit II (Miltenyi Biotech). Following centrifugation, purified T cells were labeled with CFSE (10 6

Immunohistochemistry
Human tumor samples were fixed in neutral-buffered 10% formalin solution and paraffin embedded. TLR7 staining and quantification was performed as previously described 11 using TLR7-specific polyclonal antibody (ENZO Lifesciences) at 10 mg/mL.
The expression of E-cadherin (Dako M3613, 1/100 dilution) and vimentin (Cells Signaling 5741, 1/100) by tumor cells was performed as follows. Serial 5-μm tissue sections were deparaffinized, rehydrated, and pretreated in 10 mM citrate buffer, pH 6, for antigen retrieval. Sections were incubated with hydrogen peroxide for 15 minutes, then blocked in 5% human serum for 30 minutes at room temperature. The slides were then incubated with a primary antibody (diluted in Dako real solution, antibody diluent) anti-E-cadherin (Dako M3613, 1/100 dilution) for one hour at room temperature or anti-vimentin (Cells Signaling 5741, 1/100) overnight. Slides were then incubated for 30 minutes at room temperature with the secondary antibody (anti-rabbit coupled to biotin, 1/200, JIR 715-066-150), followed by 30 minutes incubation with Twenty µm of FFPE sections were obtained from paraffin blocks. Deparaffination was performed by adding 1 mL 100% clearene for 5 min at room temperature. After centrifugation the pellet was washed twice with 1 mL 100% ethanol. Protease digestion was performed by adding digestion buffer and protease form RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (AM1975), then samples were incubated 3h at 50°C and for 15 min at 80°C for RNA isolation. Gene expression analysis was performed on 100ng RNA, using nCounter XT gene expression assays from nanosting (pancancer progression panel), following the recommendations of the manufacturer.
CRISPR/CAS9 mediated tlr7 gene editing LLC-luc cells were transiently transfected with a plasmid coding for Tlr7-targeting gRNA and Cas9 protein fused to a red fluorescent protein (RFP) (Sigma Aldrich, St. Louis, MO, US) by means of lipofectamin 2000 (Thermo Fisher scientific, Waltham, MA, US) according to the manufacturer recommendation. The following day, RFP expressing cells were selected by single cell sort using a BD FACS Aria Fusion (Becton Dickinson, Franklin Lakes, NJ, US). Following TLR7-deficient clones were then selected.

Statistical analysis
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) and the package survival. Significance threshold for p values was set to 0.05. All mice experiments were analyzed using twoway ANOVA method. Post hoc analyses 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 pairwise 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. 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. Unsupervised hierarchical clustering was performed with Euclidian distance and Ward linkage criterion. Comparisons of the expression of vimentin and E-cadherin in TLR7 hi and TLR7 low patients were performed using a Mann-Whitney test.