Shuichi Aoki

Koetsu Inoue

Sebastian Klein

Stefan Halvorsen

Jiang Chen

Aya Matsui

Mohammad R Nikmaneshi

Shuji Kitahara

Tai Hato

Xianfeng Chen

Kazumichi Kawakubo

Hadi T Nia

Ivy Chen

Daniel H Schanne

Emilie Mamessier

Kohei Shigeta

Hiroto Kikuchi

Rakesh R Ramjiawan

Tyge Ce Schmidt

Masaaki Iwasaki

Thomas Yau

Theodore S Hong

Alexander Quaas

Patrick S Plum

Simona Dima

Irinel Popescu

Nabeel Bardeesy

Lance L Munn

Mitesh J Borad

Slim Sassi

Rakesh K Jain

Andrew X Zhu

Dr Dan G Duda
email: duda@steele.mgh.harvard.edu

Kohei Shiget

Placental growth factor promotes tumour desmoplasia and treatment resistance in intrahepatic cholangiocarcinoma

Objective Intrahepatic cholangiocarcinoma (ICC)-a rare liver malignancy with limited therapeutic options-is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression. Design We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems. Results PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC.

Conclusion

PlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in micebearing aggressive ICC.

INTRODUCTION

Intrahepatic cholangiocarcinoma (ICC) is a cancer type encompassing diverse epithelial tumours arising in the liver. [START_REF] Bragazzi | New insights into cholangiocarcinoma: multiple stems and related cell lineages of origin[END_REF] ICC has an aggressive phenotype and limited therapeutic options, which makes its prognosis dismal. [START_REF] Razumilava | Cholangiocarcinoma[END_REF] Moreover, ICC incidence is increasing. [START_REF] Adeva | Medical treatment for cholangiocarcinoma[END_REF] Overall survival rate remains extremely poor: 5-year survival rates are 15% for patients with early-stage

Significance of this study

What is already known on this subject? ► Intrahepatic cholangiocarcinoma (ICC) is a rare liver malignancy with limited therapeutic options at advanced stages (chemotherapy). ► ICC is characterised by aggressive progression, desmoplasia and vascular abnormalities.

What are the new findings?

► Placental growth factor (PlGF) is highly expressed in ICCs compared with normal liver tissue, primarily in tumour stromal cells, and is associated with poor outcomes. ► PlGF blockade enriched for more quiescent, less activated cancer-associated fibroblasts, characterised by high gene transcription levels related to the Akt pathway, hypoxia and glycolysis. ► PlGF blockade reduced desmoplasia, decreased tissue stiffness and reopened collapsed tumour vessels in vivo, resulting in improved blood perfusion, a reduction in intratumoral hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing ICC.

How might it impact on clinical practice in the foreseeable future? ► These findings demonstrate a new mechanism by which PlGF mediates ICC desmoplasia and progression. ► Blockade of PlGF is an immediately translatable strategy to reprogramme the desmoplastic and hypoxic ICC microenvironment and inhibit its progression and resistance to standard chemotherapy.

Hepatology

ICC, 6% for those with regional lymph node involvement and 2% for those with metastatic disease. Meanwhile, various genetic alterations in ICC have been identified, leading to multiple clinical trials of molecularly targeted agents for advanced disease. 4 5 However, these approaches benefit only the fraction of patients with appropriate genetic alterations. Systemic chemotherapy using gemcitabine and cisplatin (Gem/Cis) remains the standard of care for all patients with advanced ICC, but the benefits are limited. 3 6 New treatment options are urgently needed.

A defining feature of ICC is the abundant tumour stroma (desmoplasia), which has complex functional interactions with cancer and normal stromal cells. [START_REF] Biffi | Deciphering cancer fibroblasts[END_REF] The desmoplastic stroma consists of cancer-associated fibroblasts (CAFs), which closely surround the neoplastic bile ducts and can provide ICC cells with proliferative and prosurvival/antiapoptotic signals. These CAFs may be activated hepatic stellate cells (HSCs) or portal fibroblasts with a myofibroblast-like phenotype. [START_REF] Cadamuro | Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma[END_REF][START_REF] Karin | The characteristics of activated portal fibroblasts/ myofibroblasts in liver fibrosis[END_REF][START_REF] Fingas | Myofibroblast-derived PDGF-BB promotes hedgehog survival signaling in cholangiocarcinoma cells[END_REF] Activated CAFs produce extracellular matrix (ECM) components and communicate with endothelial, perivascular, immune and cancer cells to induce angiogenesis and immunosuppression, thus promoting tumour progression. [START_REF] Fingas | Myofibroblast-derived PDGF-BB promotes hedgehog survival signaling in cholangiocarcinoma cells[END_REF] In addition, the characteristic vascular abnormalities and tissue hypoxia and desmoplasia also impede drug delivery into the tumours, thereby contributing to resistance to conventional chemotherapy. [START_REF] Jain | Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia[END_REF] Placental growth factor (PlGF), which is a member of the vascular endothelial growth factor (VEGF) family, binds to VEGFR1 but not VEGFR2, as well as to neuropilin-1 and -neuropilin2 (Nrp1 and Nrp2). PlGF binding to VEGFR1 and Nrp1 on endothelial cells can lead to a crosstalk between Nrp1, VEGFR1 and VEGFR2, which consequently enhances VEGF-driven responses. [START_REF] Simons | Mechanisms and regulation of endothelial VEGF receptor signalling[END_REF] However, PlGF can also transmit prosurvival and promigration signals to cancer cells directly through Nrp1 in gastrointestinal cancer. [START_REF] Matkar | Jack of many trades: multifaceted role of neuropilins in pancreatic cancer[END_REF] Conversely, tumour cells can produce PlGF to activate stromal cells and promote angiogenesis, inflammatory and stromal cell recruitment and metastatic progression. [START_REF] Fischer | Anti-PlGF inhibits growth of VEGF(R)-inhibitorresistant tumors without affecting healthy vessels[END_REF][START_REF] Rolny | HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of PlGF[END_REF][START_REF] Heindryckx | The placental growth factor as a target against hepatocellular carcinoma in a diethylnitrosamine-induced mouse model[END_REF][START_REF] Carmeliet | Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions[END_REF] PlGF expression, unlike that of VEGF, is undetectable in healthy conditions but is highly upregulated in pathological conditions, including hypoxia. [START_REF] Green | Placenta growth factor gene expression is induced by hypoxia in fibroblasts: a central role for metal transcription factor-1[END_REF] Therefore, PlGF inhibition is expected to selectively inhibit pathological angiogenesis, without affecting the growth or maintenance of physiological vessels, which is likely to cause fewer side effects. 14 19 In support of this, blockade of PlGF in mouse models has resulted in antitumour effects mediated by changes on myeloid cells. [START_REF] Rolny | HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of PlGF[END_REF] Similarly, Nrp1 expression level is significantly higher in cholangiocarcinoma tissues compared with adjacent normal biliary tissues, as detected by quantitative real-time-PCR analyses and immunohistochemistry (IHC). [START_REF] Zhu | Neuropilin-1 regulated by miR-320 contributes to the growth and metastasis of cholangiocarcinoma cells[END_REF] Nrp1 inhibition also reduced the proliferation and migration of cholangiocarcinoma cells and suppressed tumour growth and lung metastasis in experimental models. [START_REF] Zhu | Neuropilin-1 regulated by miR-320 contributes to the growth and metastasis of cholangiocarcinoma cells[END_REF] Previously, we reported that the PlGF/Nrp1 axis in cancer cells is a driver of paediatric medulloblastoma. [START_REF] Snuderl | Targeting placental growth factor/ neuropilin 1 pathway inhibits growth and spread of medulloblastoma[END_REF] This work has led to a phase I trial in children with brain tumours (NCT02748135).

Here, we examined the impact of PlGF inhibition on the desmoplastic stroma and ICC cell viability and invasion. We also evaluated the impact of genetic or pharmacologic PlGF inhibition on tumour growth and metastatic progression in mice bearing orthotopic ICC in p53-null/Kras-mutant and Idh/Kras-mutant driven tumours, and on tumour response to chemotherapy.

MATERIALS AND METHODS

Mouse models of ICC and treatments

We used two models of orthotopically implanted ICC, using murine ICC cell lines derived from spontaneously arising tumours. 22 23 Primary culture and isolation of CAFs from murine tumours CAFs were isolated from fresh ICC tissues by the outgrowth method, as previously described. 24 25

Single-cell RNA sequencing: droplet encapsulation and library preparation

Single-cell RNAseq (scRNAseq) was performed using a modified version of the DropSeq protocol. [START_REF] Macosko | Highly parallel genome-wide expression profiling of individual cells using Nanoliter droplets[END_REF]

Measurement of Young's modulus (stiffness)

The Young's modulus (stiffness) of tumours was determined by an unconfined compression test. [START_REF] Nia | Solid stress and elastic energy as measures of tumour mechanopathology[END_REF] For additional details, see online supplemental methods and table S1.

RESULTS

PlGF expression is increased with ICC progression in patients

We examined tissues from an archival cohort of patients diagnosed with different disease stages of ICC (N=43). We assigned grading scores based on staining intensity and area (negative, low, moderately high and high) using IHC 28 29 (online supplemental table S2). The vast majority of cases showed moderately high/high expression for both PlGF and Nrp1; only 14% of cases were negative for PlGF expression (online supplemental figure S1A). Moreover, PlGF expression scoring was significantly associated with disease stage (p=0.042) (online supplemental figure S1B) but not with overall survival (not shown).

Since PlGF is a secreted protein, we next used RNA in situ hybridisation to identify the cells expressing PlGF in tissue samples from a clinically annotated cohort of ICC patients (N=52) (online supplemental figure S1C). Nrp1 expression was assessed by IHC. PlGF expression was predominantly found in CAFs and endothelial cells, and rarely in cancer cells; in contrast, Nrp1 was expressed by both ICC cells and CAFs (figure 1A-E and online supplemental figure S1D,E). Expression of PlGF or Nrp1 was rarely detected in surrounding liver tissues.

In addition, we analysed RNA-sequencing data from 28 patient-derived xenograft samples after separating them into mouse and human reads (online supplemental table S3). Median expression of PGF was significantly higher (16-fold) in the mouse stroma compared with the human ICC cells (p<0.01) (figure 1F). Median expression of Nrp1 was fourfold higher in the mouse-derived stromal cells compared with human ICC cells, but below the threshold of p=0.01. Of note, log2RPKM density plots for all genes in mouse and human were largely overlapping. Finally, we examined the levels of blood circulating PlGF in ICC patients enrolled in clinical studies. We found elevated levels of PlGF in patients with advanced disease (online supplemental figure S1F).

These data were in line with analyses of The Cancer Genome Atlas (TCGA) database, [START_REF] Tang | GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses[END_REF] which showed that the PGF gene was expressed at higher levels in human ICC tissues compared with normal liver tissue (online supplemental figure S1G). Furthermore, mining data from published scRNAseq database [START_REF] Ma | Tumor cell biodiversity drives microenvironmental reprogramming in liver cancer[END_REF] showed that PGF was expressed at higher levels in fibroblasts and endothelial cells in human ICC tissues compared with other cell types (online supplemental figure S1H).

Thus, PlGF expression is predominant in tumour stroma and is increased in advanced disease stages, while Nrp1 is broadly expressed in ICC tissues.

Hepatology PlGF is secreted by ICC cells and HSCs and is increased in hypoxic conditions

We next examined PlGF secretion in vitro and the impact of hypoxia, a common feature of ICC. To this end, we examined a panel of available murine ICC cells (425 and SS49), human ICC cells (HUCCT1), and HSCs (one of the CAF precursors in ICC), cultured in normoxic and hypoxic conditions. We measured PlGF protein concentration in culture supernatant using ELISA. All ICC cells and the HSCs secreted detectable levels of PlGF, and the concentration was significantly increased in hypoxic conditions (1%O 2 for 48 hours) and reduced by short-hairpin (sh)-RNA constructs for PlGF (online supplemental figure S1I). We also found that high expression levels of Nrp1-but not of Nrp2 or VEGFR1-were detectable in most murine and human ICC cell lines; these levels were comparable to those seen in human umbilical vein endothelial cells (online supplemental figure S1J).

Thus, PlGF expression is detectable and increased under hypoxic conditions in HSCs and cancer cells, and Nrp1 is expressed across human and mouse ICC lines.

PlGF blockade enriches for a subset of quiescent CAFs characterised by a hypoxia-like phenotype, increased cell glycolysis and Akt-related genes

To examine the role of PlGF/Nrp1 in ICC cells and CAFs, we first generated orthotopic tumours by intrahepatic injection of 425 (p53/Kras-mutant) murine ICC cells. CAFs were isolated from primary cultured tumour tissues using outgrowth and clonal isolation methods. We obtained CAF clones after limited dilution, which were expanded in individual wells of 96-well plates. Then, we validated CAF identity using the mesenchymal cell markers fibroblast activation protein (FAP), platelet-derived growth factor receptor (PDGFR)β and α-smooth muscle actin (SMA) [START_REF] Kalluri | The biology and function of fibroblasts in cancer[END_REF] to distinguish them from other stromal or cancer cells (online supplemental figure S2). In separate experiments, we dissociated the primary ICC tissue and subjected the heterotypic tissue cultures to an antimouse PlGF antibody (5D11D4) or IgG (control). This short term in vitro culture method yielded a high degree of cell viability, allowing scRNAseq analysis. scRNAseq analysis was performed using cells collected after 24 hours of anti-PlGF antibody treatment of the whole tumour lysate, in the absence of hypoxia, to remove the confounding factors related to hypoxia-induced changes. In addition, we performed scRNAseq analyses of 425 ICC cells and CAFs cultured in vitro and used their transcription profile as a reference for the whole tissue culture scRNAseq analyses.

In the whole tissue culture, transcriptional profiling using scRNAseq analysis identified distinct ICC cell and CAF populations (figure 2A). ICC cell clustering was driven primarily by cell cycle related genes and pathways. Since we found no significant difference in proportions after anti-PlGF treatment, we grouped all ICC populations for subsequent analysis. The cell population most impacted by PlGF blockade was a CAF subset (labelled as CAF_2). This population was strongly enriched after treatment with anti-PlGF antibody, with treated cells representing about 80% of the cluster (figure 2B). This is a far greater proportion than what would be expected if cell cluster partitioning in the scRNAseq analysis was not dependent on treatment (p<0.0001; Fisher's exact test).

Initial examination of this CAF_2 population indicated low levels of proliferation/G1 arrest and low expression of collagen type I, PDGFR-β and transforming growth factor (TGF)-β relative to the other CAF populations (online supplemental figure S3). A pathway analysis demonstrated this CAF_2 population exhibits high glycolysis and a hypoxia-like phenotype (online supplemental table S4), despite being cultured in normoxic conditions (figure 2C,D and online supplemental figure S4). Aside from hypoxia and glycolysis related pathways, the CAF_2 population exhibited significant alterations in the AKT pathwayrelated genes when compared with the other CAF populations (figure 2E).

Thus, the scRNAseq analysis identified the CAFs as the major cell population affected by PlGF blockade. The primary transcriptomic disruption induced by PlGF blockade involved pathways related to cell proliferation and hypoxia. These effects are potentially mediated by the AKT pathway in this more quiescent CAF subset. S3; note the significantly higher expression levels in murine vs human PGF, *p<0.05). CAFs, cancerassociated fibroblasts; PlGF, placental growth factor;

Hepatology PlGF promotes a myofibroblast-like phenotype in CAFs via Akt/NF-κB signalling

We next dissected the role of the PlGF/Akt axis using the CAFs isolated from murine ICC tissue. The CAF subsets expressed fibroblast markers (FAP, PDGFR-β) but exhibited a differential activation phenotype and are herein referred to as myofibroblast-like (my)CAFs (highly activated, α-SMA hi ) or quiescent (q)CAFs (figure 3A,B and online supplemental figure S2A,C). Western blotting and qPCR analyses showed that qCAFs expressed lower gene and protein levels of profibrotic markers (sma, acta2 and ctgf) and fibrosis-induced growth factors (tgfb, smad2, ccl3, sdf1a) (online supplemental figure S2B). By bulk RNA sequencing analysis, we found that qCAFs showed a transcriptional enrichment in factors related to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway (figure 3C and online supplemental figure S2D,E). The NF-κB pathway is known as one of the downstream targets of the Akt pathway. Moreover, using Western blotting and IHC, we found that Nrp1 was expressed in qCAFs (figure 3D and online supplemental figure S2F).

Next, we investigated whether PlGF stimulation mediates CAF activation. We found that exposure to recombinant (r) PlGF activated Akt and p65, and induced α-SMA expression in murine CAFs and human HSCs (figure 3E and online supplemental figure S5A,B). Consistent with the scRNAseq cell results, anti-PlGF treatment downregulated Akt/NF-κB pathway activation in both CAFs and HSCs-despite maintenance of high total Akt levels-and inhibited α-SMA and TGF-β expression in a dose-dependent manner (figure 3E and online supplemental figure S5A,B). Moreover, direct inhibition of PI3K/Akt by the inhibitor BKM120 or of NF-kB pathway using the compound QNZ showed decreased α-SMA expression in CAFs and HSCs (figure 3E and online supplemental figure S5A). Furthermore, both PlGF and PI3K inhibition downregulated myofibroblastrelated genes in CAFs (figure 3F). Finally, examination of a publicly available dataset showed a significant direct correlation between PGF and ACTA2 genes in human ICC (online supplemental figure S5C).

These results indicate that PlGF activates Akt/NF-kB pathway and promotes the myofibroblast-like (α-SMA hi ) phenotype in CAFs.

CAFs promote ICC cell invasion via PlGF expression

To further study the paracrine interaction between CAFs and ICC cells mediated by PlGF, we compared the 3-D invasion capabilities using real-time analyses of heterotypic spheroid co-cultures. The co-culture of 425 ICC cells with qCAFs showed

Hepatology

larger spheroid formation than when ICC cells were cultured alone (figure 4A,B). To evaluate ICC cell invasion in real time, we used a time-lapse 3-D culture system with an Olympus IX81 Confocal microscope equipped with the Fluoview software (lens:10X air; slice thickness: 1-5 µm). In brief, spheroids were grown on top of a 1.5% agarose gel under serum-free conditions. When these spheroids were transferred to Matrigel-coated wells, they showed increased invasion into the surrounding matrix. By measuring the invasion area, we found that co-culture significantly enhanced cell invasion area when compared with ICC cells alone; this enhancement was almost completely inhibited by repression of PlGF in the ICC cells or PlGF blockade with antibodies (figure 4C,D and online supplemental movie S1).

We next assessed the direct effect of PlGF inhibition on ICC cell viability and invasiveness. PlGF-knockdown induced a significant but small reduction of proliferation in vitro (p<0.05) in murine ICC cells (425 and SS49), while PlGF blockade had no impact on the size of established spheroids (online supplemental figure S5D,E). However, evaluation of ICC cell invasion in real-time showed that PlGF knockdown or blockade with the antimouse PlGF antibody 5D11D4 significantly suppressed the invasive capabilities of 425 ICC cells, including in the presence of CAF-conditioned media. The invasive ability was rescued by addition of rPlGF to PlGF-knockdown ICC cells (figure 4E-F and online supplemental figure S5F, movie S2).

Using Western blotting, we found that both PlGF knockdown and 5D11D4 treatment decreased the level of phosphorylated Akt in 425 and SS49 murine ICC cells; this effect was prevented by adding rPlGF to the PlGF-knockdown cells (online supplemental figure S5G,H). In addition, knockdown of Nrp1 expression in human ICC cells resulted in a decrease in Akt phosphorylation (online supplemental figure S5I). Finally, stimulation of ICC cells with rPlGF activated Akt in Nrp1 intact cells but not in Nrp1knockdown cells (online supplemental figure S5J).

Taken together, these results show that CAFs stimulate the invasive 3-D growth of ICC cells, which can be inhibited by PlGF blockade.

Genetic or pharmacological PlGF inhibition in ICC delays tumour progression in mice

We next investigated the in vivo role of PlGF in tumour progression using orthotopic ICC models generated by intrahepatic injection of cells derived from tumours spontaneously arising in p53 KO /Kras G12D mice (425 cells) and Idh2 R172K /Kras G12D mice (SS49 cells). These ICC models exhibited excessive ECM accumulation and abundance of α-SMApositive myofibroblast-like CAFs surrounding vascular structures associated with neoplastic ducts. Moreover, they displayed a hypoxic tumour microenvironment and similar clinical symptoms to human ICC, such as abundant bloody ascites and frequent metastases to distant organs.

We first evaluated the effect of PlGF expression knockdown in cancer cells using shRNA constructs for PlGF (sh-PlGF) vs a scrambled sh-RNA (sh-control) (online supplemental figure 1G and figure 5C), with or without anti-mouse PlGF antibody (5D11D4) vs IgG control. Measurement of plasma PlGF concentration confirmed the near complete blockade of circulating PlGF in the mice treated with 25 mg/kg 5D11D4 (online supplemental figure S6A). We found that genetic ablation of PlGF expression in ICC cells significantly delayed the initial growth of 425 ICC tumours in SCID mice, and that complete pharmacologic blockade with 5D11D4 significantly delayed the growth of established 425 ICCs (figure 5A,B). Moreover, anti-PlGF treatment of established sh-PlGF tumours further inhibited ICC growth compared with all other groups (figure 5A,B).

To investigate the mechanism of benefit of PlGF inhibition, we conducted a time-matched study in which all mice were sacrificed on day 11 after the start of treatment. We found that anti-PlGF treatment significantly decreased the cell proliferation index in orthotopic ICC tumour tissues, assessed by Ki67 staining (figure 5C,D). Furthermore, PlGF inhibition prevented the formation of bloody ascites and significantly reduced regional (lymph node) and distant (lung) metastasis (figure 5E-G). These results were confirmed in the SS49 model of ICC, in which PlGF blockade significantly delayed tumour growth (online supplemental figure S6B).

Finally, to interrogate the specific roles of stroma-derived versus tumour-derived PlGF, we orthotopically implanted 425 ICC cells in PlGF-proficient vs PlGF-deficient C57Bl/6 mice. Tumour formation was reduced after implantation of ICC cells with decreased

Hepatology

PlGF expression and when stromal cells were deficient in PlGF, and completely prevented when both tumour and stromal cell PlGF expression was inhibited (online supplemental figure S6C).

Thus, PlGF expression plays a critical role in the initial ICC growth, and stroma-derived PlGF is mediating tumour formation and progression in two genomically distinct murine ICC models.

PlGF blockade reduces tumour desmoplasia and tissue stiffness, and results in increased blood perfusion in ICC

Next, we studied the functional impact of PlGF inhibition on relevant components of the tumour microenvironment. Analysis of ICC tissues showed a high vascular density, but many of the tumour vessels were collapsed (figure 6A), similar to human ICC (figure 1E and online supplemental figure 6D).

Here, vessel collapse was associated with desmoplasia (ie, high density of myofibroblast-like (α-SMA+) CAFs and collagen I expression (figure 6B-E, online supplemental figures S6E and S7), and increased tissue stiffness (figure 6F). PlGF blockadebut not PlGF knockdown in ICC cells-decreased the expression of collagen I, as well as tissue stiffness, resulting in the opening of blood vessels (figure 6A-D and online supplemental figure S6E). A significant reduction in α-SMA required both PlGF knockdown in ICC cells and antibody blockade (figure 6E). Opening of collapsed blood vessels resulted in improved blood perfusion and significantly reduced intratumorous hypoxia, reflected by reduced hif1 transcription after treatment (figure 6G-H). There were no significant differences in tumour-associated macrophage numbers or polarisation (online supplemental figure S8).

These results indicate that tumour stromal cells are a key factor in PlGF-mediated desmoplasia by deposition of collagen I in murine ICC.

Hepatology Anti-PlGF treatment enhances the efficacy of standard chemotherapy against ICC in mice

Having revealed the impact of PlGF blockade on vascular function and desmoplasia, we examined the impact of PlGF blockade on ICC response to a standard chemotherapy regimen. We compared the efficacy of anti-PlGF therapy when combined gemcitabine and cisplatin (Gem/Cis) versus each therapy alone or control IgG in the orthotopic 425 ICC model. We found that combination therapy (Gem/Cis plus anti-PlGF antibody) for 3 weeks (ie, until mice in control group were sacrificed) significantly enhanced tumour growth delay (figure 7A,B). This benefit was associated with significant decreases in bloody ascites and pleural effusions (online supplemental figure S9A,B). Moreover, when treatment was given continually, combination therapy significantly increased overall survival compared with each treatment alone, with an HR of 0.04 vs control (p<0.0001) and an HR=0.31 vs Gem/Cis (p=0.018) (figure 7C).

We next examined whether PlGF blockade impacted ICC cell sensitivity to chemotherapy. In vitro evaluation of cytotoxicity using an MTT assay showed that exposure to Gem induced a dosedependent cell killing in 425 cells. However, the cytotoxicity of Gem was not affected by concomitant PlGF or NF-kB blockade in ICC cells (figure 7D and online supplemental figure S9C).

Collectively, these results show that PlGF blockade can substantially enhance the efficacy of chemotherapy in ICC in vivo via tumour stroma-mediated mechanisms.

DISCUSSION

ICCs are characterised by aggressive (invasive) growth and an abundant stroma (desmoplasia), mediated by several molecular pathways and a hypoxic tumour microenvironment. [START_REF] Kawahara | Enhanced expression of thrombospondin-1 and hypovascularity in human cholangiocarcinoma[END_REF] PlGF can promote the formation of an abnormal vasculature and increased hypoxia via elevated matrix deposition and increased tissue stiffness-all of which are determinants for tumour aggressiveness and resistance to treatment. 14 18 34 35 We report here that PlGF and Nrp1 are broadly expressed in human ICC, particularly in the stroma, and that PlGF expression levels are elevated by hypoxia and in advanced disease. We found that PlGF is highly expressed by CAFs in ICC, promoting desmoplasia by the induction of CAF activation and invasive tumour growth through CAF/ICC cell interactions (figure 7E).

In murine ICC models, PlGF blockade suppressed orthotopic tumour growth, in part by decreasing Akt activation and reducing the proliferation and invasion of ICC cells. Interestingly, we found that PlGF blockade had the greatest impact on subsets of CAFs exhibiting high Akt activation. Activation of the Akt/NF-κb signalling promoted a myofibroblast-like phenotype in murine CAFs with increased expression patterns associated with cell proliferation, hypoxia and glycolysis. Akt/NF-κb signalling has been associated with an activated phenotype of CAFs in breast, pancreatic, prostate and ovarian cancers. 36 37 The inhibition of this pathway induced a significant reduction of chemokine and cytokine stimulation in CAFs. [START_REF] Rolny | HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of PlGF[END_REF] Here, we show that inhibiting PlGF or Akt and NF-kB activity reduced the myofibroblast-like phenotype in murine CAFs and HSCs, one of the precursors of CAFs in human ICC.

CAFs are the major source of matrix components in ICC. 8 However, recent reports identified several subsets of CAFs based on transcriptional signatures and highlighted the benefits of targeting those that provide support to cancer cells during tumour progression. 7-9 31 38-41 We provide here new mechanistic insights into the antidesmoplastic effect of PlGF blockade. Specifically, myofibroblast-like (α-SMA hi ) CAFs secrete multiple profibrogenic factors, including PlGF and TGF-β, and induced morphological activation and proliferation in close interaction with cancer cells. 10 42 43 The abundance of myofibroblast-like CAFs and CAFmediated desmoplasia increases tissue stiffness, which induces collapse of tumour vessels and intratumorous hypoxia, similar to pancreatic cancer. [START_REF] Chauhan | Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by decompressing tumour blood vessels[END_REF] The myofibroblast CAF phenotype may be regulated by several pathways, including Rho GTPase, c-Jun N-terminal kinase and Hedgehog. 10 42 44 In evaluating CAF clones derived from ICC with differential activation, we found differential expression in markers such as TGF-β and α-SMA. PlGF exposure increased

Hepatology

Akt and p65 activation, as well as myofibroblast-like expression of α-SMA and TGF-β in more quiescent CAFs. Conversely, blockade of PlGF prevented these effects.

Previous studies have investigated anti-PlGF therapy in liver cancers, including cirrhosis, hepatocellular carcinoma and cholangiocarcinoma models. 16 34 45 These studies invoked mechanisms of benefit related to antiangiogenic/antivascular (vascular pruning) effects, as well as effects on inflammation (on the infiltration of tumour-associated macrophages), mediated by VEGFR1. [START_REF] Van De Veire | Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease[END_REF] Here, we are reporting a distinct mechanism of benefit for PlGF inhibition in ICC, involving decreased cancer cell invasion and myofibroblast-like activation in CAFs. These effects resulted in decreased desmoplasia and hypoxiaby opening of blood vessels as opposed to vessel pruning-as demonstrated in preclinical mouse models of ICC. This effect potentiated the tumour growth delay in two genomically distinct mouse models and increased overall survival from a treatment of Gem/Cis, the standard therapy for advanced ICC patients.

Future prospective clinical studies will be necessary to establish whether these effects of PlGF blockade will translate into a benefit in the hypovascular human ICC. 33 47 However, previous studies have established that anti-VEGFR approaches have no efficacy in biliary tract cancers, including when combined with chemotherapy in randomised clinical trials of agents targeting VEGFR1. [START_REF] Goyal | Chemotherapy and antiangiogenics in biliary tract cancer[END_REF] Moreover, as reviewed in refs. 49 50, all prior attempts to improve on the efficacy of chemotherapy have failed in randomised trials over the last decade, leaving Gem/Cis chemotherapy as the only proven efficacious option currently available for biliary tract cancers, as per ABC-02 trial data.

Recent reports converge towards an interplay between tumour hypoxia, fibrosis and immune evasion as determinants of tumour progression and treatment resistance in ICC subsets. [START_REF] Chen | Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice[END_REF][START_REF] Murphy | Total neoadjuvant therapy with Folfirinox in combination with losartan followed by chemoradiotherapy for locally advanced pancreatic cancer: a phase 2 clinical trial[END_REF][START_REF] Job | Identification of four immune subtypes characterized by distinct composition and functions of tumor microenvironment in intrahepatic cholangiocarcinoma[END_REF] Our findings have clinical implications for other emerging therapies, such as radiotherapy or immune checkpoint blockade, which have shown promise in ICC. 54 55 For example, programmed cell death 1 (PD-1) blockade has shown efficacy in microsatellite instable carcinomas, including ICC. [START_REF] Czink | Successful immune checkpoint blockade in a patient with advanced stage microsatellite-unstable biliary tract cancer[END_REF] PD-ligand 1 is expressed in 33%-46% of resected human ICC samples and is associated with poor prognosis. 57 58 Therefore, determining whether reprogramming of the ICC microenvironment by PlGF blockade could also enhance the efficacy of radiotherapy or immunotherapy in ICC is warranted.

In summary, we demonstrate here that PlGF is a mediator of ICC progression and is a potential therapeutic target. Antibody blockade of PlGF in ICC models reprogrammed the desmoplastic microenvironment, resulting in enhanced efficacy of standard chemotherapy. These results reveal a new and actionable approach for a novel combination therapy in this disease with a dismal prognosis.

Figure 1

 1 Figure 1 Expression of PlGF in human intrahepatic cholangiocarcinoma (ICC). (A) Representative in situ hybridisation (PlGF-ISH, red dots) staining of PlGF in ICC human tissue. (B) Same image of (A) showing the overlay of classifying cells according to cell type (red, PlGF-positive CAF; yellow, PlGF-negative CAF; grey, PlGF-negative ICC cell). (C) Representative serial section showing PDGFRβ staining (arrow highlights positive cell) to identify fibroblasts. (D) Representative serial section showing AE/AE3 staining (arrow highlights positive cell) to identify cancer cells. (E) Representative serial section showing CD31 staining (DAB, arrow highlights positive cell) of endothelial cells (scale bar 250 µm). (F) Differential levels of the Nrp1 and PGF gene expression calculated by subtracting logRPKM for human (ICC cells) from mouse (stromal cells) in a collection of 22 patient-derived xenograft (PDX) ICC tissues (online supplemental tableS3; note the significantly higher expression levels in murine vs human PGF, *p<0.05). CAFs, cancerassociated fibroblasts; PlGF, placental growth factor;

Figure 2

 2 Figure 2 Single cell RNA sequencing analysis of gene expression changes after PlGF blockade. (A) tSNE plot (each dot represents a cell) demonstrating different populations of CAFs and ICC cells based on transcriptional profiles in whole tumour lysate from orthotopic 425 ICC model cultured in vitro with anti-PlGF antibody. The CAF_2 (arrow) cluster was strongly enriched after PlGF blockade. (B) Expression of the PGF gene (encoding for PlGF) across CAF and ICC cell clusters. (C) Proportion plot demonstrating representation of treated and control cells in each cluster. (C-E) The cluster of cells affected by PlGF blockade showed a gene expression pattern associated with increased expression in genes related to hypoxia (hyp) (C) glycolysis (glyc) (D) and Akt pathways (path) (E). *P<0.05; **p<0.001; ***p<0.0001. CAF, cancer-associated fibroblast; ICC, intrahepatic cholangiocarcinoma; PIGF, placental growth factor; tSNE, t-distributed stochastic neighbor embedding.

Figure 3

 3 Figure 3 PlGF promotes a myofibroblast-like phenotype in CAFs via increased NF-kB/Akt activation. (A, B) RNAseq analysis of two CAF clones (A) comparing high α-SMAexpressing myCAF versus low α-SMAexpressing qCAF versus 425 cells (B) (see online supplemental dataset 1). (C) GSEA shows enrichment in NF-kB pathway-related genes in qCAF cells versus control (425 ICC cells). (D) High protein expression of Nrp1 but not Nrp2 or VEGFR1 in CAFs. (E) Recombinant PlGF further activates NF-kB/Akt axis and increases α-SMA expression in qCAFs, while PlGF blockade using 5D11D4 (5D11) treatment downregulates NF-kB/Akt pathway and α-SMA expression. Moreover, inhibition of PI3K using the compoundBKM120 and NF-kB with the compound QNZ also inhibited activation of NF-kB and α-SMA expression. (F) Both PlGF blockade and PI3K inhibition reduced the expression of profibrotic genes in qCAFs (analysed in triplicate). CAF, cancer-associated fibroblast; PIGF, placental growth factor.

Figure 4

 4 Figure 4 Impact of PlGF inhibition on ICC cell invasion. (A-D) Representative images of in vitro 3-D coculture of 425 ICC cells with GFP-expressing qCAFs versus ICC cells alone (A); CAFs increased the spheroid formation compared with 425 ICC cells alone, and this effect was suppressed by PlGF blockade with 5D11D4 (5D11) antibody (B); Scale bar, 160 µm. In addition, 3-D co-culture of ICC cells with CAFs (C) further increased ICC cell invasion, and PlGF blockade with 5D11 antibody inhibited this effect (D). (See online supplemental movie S1). (E, F) Analysis of ICC cells alone in 3-D (spheroid) invasion assays (E) showed that PlGF knockdown or anti-PlGF treatment using 5D11D4 (5D11) inhibited 425 ICC cell invasion in Matrigel (F); this effect was rescued by addition of recombinant (r)PlGF exposure in PlGF knockdown cells (see online supplemental movie S2). All experiments were performed in triplicate; p values from Tukey's test. CAF, cancerassociated fibroblast; ICC, intrahepatic cholangiocarcinoma; PlGF, placental growth factor.

Figure 5

 5 Figure 5 Impact of genetic inhibition of PlGF in ICC cells on ICC growth and/or pharmacologic blockade of PlGF on established tumour progression. (A, B) PlGF knockdown in ICC cells delayed tumour onset, while complete pharmacological blockade significantly delayed established ICC growth in the orthotopic 425 model (individual growth curves in (A) and average tumour size with SD in surviving mice in (B)). (C, D) Representative IHC for Ki-67 as a marker of cell proliferation in tumour sections from the four treatment groups (C); PlGF blockade-but not PlGF knockdown in ICC cells-significantly reduced in vivo ICC cell proliferation in size-matched orthotopic 425 tumours (D). (E) PlGF blockade significantly reduced the occurrence of bloody ascites. (F, G) Combined genetic and pharmacologic inhibition of PlGF reduced metastasis to the lymph nodes (F) and lungs (G). P values from Mann-Whitney U test (A, B), Tukey's test (D, G) and Fisher's test (E, F). In vivo studies were performed in duplicate; n=10 mice per group. Scale bars, 100 µm in (C). ICC, intrahepatic cholangiocarcinoma; IHC, immunohistochemistry; PlGF, placental growth factor.

Figure 6

 6 Figure 6 PlGF inhibition reduces ICC desmoplasia and improves tumour blood perfusion by opening collapsed vessels. (A) PlGF inhibition induced opening of collapsed blood vessels (representative IHC of vessels in inset). (B) Representative immunofluorescence (IF) for collagen I and CK19 (upper panel) and α-SMA and CK19 (lower panel) in tumours after PlGF inhibition. (C-E) Quantification of IF data showing that PlGF blockade and PlGF knockdown in ICC cells reduced CK19-positive cell area (C) and collagen I expression (D); α-SMA+ 'myofibroblast-like' CAF density was reduced in the PlGF knockdown groups, most substantially after PlGF blockade (E). (F) PlGF blockade significantly decreased tumour stiffness. (G) Representative OCT of patent blood vessels in ICC with or without PlGF blockade. (H) Anti-PlGF treatment significantly reduced intratumour hypoxia, reflected by reduced hif1 transcription. P values from Tukey's test (A, C-E) and Mann-Whitney U teat (F, H). Scale bars, 500 µm in (C). ANOVA, analysis of variance; ICC, intrahepatic cholangiocarcinoma; IHC, immunohistochemistry; OCT, optical coherence tomography; PlGF, placental growth factor.

Figure 7

 7 Figure 7 PlGF blockade enhances the anti-tumour effects of conventional chemotherapy and increases survival. (A, B) The combination of anti-PlGF antibody 5D11D4 (5D11) with conventional gemcitabine plus cisplatin (GC) chemotherapy for 3 weeks significantly delayed tumour growth compared with GC or 5D11 alone (individual growth curves in A) and average tumour size with SD in surviving mice in B). (B) Kaplan-Meier survival distributions after continuous treatment with GC, 5D11 or their combination vs control in mice with established ICC. In vivo studies were performed in duplicate; n=10 mice per group. (D) Addition of 5D11 did not further reduce the viability of 425 cells after gemcitabine (Gem) treatment in vitro. P values from Mann-Whitney U test (B) and log-rank test (D). (E) Proposed mechanism of action of PlGF blockade in ICC: PlGF is produced primarily by CAFs and affects both cancer and stromal cells in an autocrine and paracrine manner in ICC. PlGF inhibition reduces ICC cell invasion and tumour desmoplasia and stiffness, which enhances blood perfusion and the efficacy of chemotherapy. CAF, cancer-associated fibroblast; ICC, intrahepatic cholangiocarcinoma; PlGF, placental growth factor.

Acknowledgements

We thank Mark Duquette, Anna Khachatryan, Sylvie Roberge (MGH Boston) and Alexandra Florin (University of Muenster, Germany) for outstanding technical support, and Sergey Kozin and Cyril Benes (MGH Boston) for useful discussions.

of Research and Innovation, CCCDI-UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0797/66PCCDI, within PNCDI III. SA's research was supported by a Postdoctoral Fellowship from Cholangiocarcinoma Research Foundation, TH received a Postdoctoral Fellowship from Astellas Foundation for Research on Metabolic Disorders, Japan, DS received a Postdoctoral Fellowship from Humboldt Foundation, KS received a Postdoctoral Fellowship from Uehara Memorial Foundation, and EM received a grant from the Philippe Foundation and the Cancéropôle PACA.

Competing interests IC is an employee of STIMIT. TY has served in a consulting or advisory role for Bristol Myers Squibb. RKJ received honorarium from Amgen and consultant fees from Chugai, Ophthotech, Merck, SPARC, SynDevRx. RKJ owns equity in Accurius, Enlight, SPARC, and SynDevRx, and serves on the Boards of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. AXZ is a consultant/advisory board member for Bayer. DGD received consultant fees from Bayer, Simcere, Surface Oncology and BMS and research grants from Bayer, Exelixis and BMS. No reagents or support from these companies was used for this study.

Funding DD's work was supported through NIH grants P01-CA080124, R41-CA213678 and Proton Beam/Federal Share Programme and Department of DefenseDefence grants #W81XWH-19-1-0284 and W81XWH-19-1-0482. RKJ's work was supported through NIH grants P01-CA080124, R35-CA197743, R01-CA208205 and U01-CA224173, and by the National Foundation for Cancer Research, Harvard Ludwig Cancer Centre, Advanced Medical Research Foundation and Jane's Trust Foundation. IP's work was supported by the Romanian Ministry

Data availability statement Data are available on reasonable request. Data are available on reasonable request from the corresponding author DGD. The

Author affiliations


Décrire les habitudes d'utilisation du smartphone chez les étudiants infirmiers. Évaluer la dépendance au smartphone, l'insuffisance de convergence subjective (fatigue oculaire) des étudiants infirmiers. Décrire les connaissances des étudiants sur l'usage du smartphone.

Introduction 1 Objectifs 2

  Département Universitaire de Sciences Infirmières, Université de Limoges (2) : Inserm U1094, IRD U270, Univ. Limoges, CHU Limoges, EpiMaCT -Epidémiologie des maladies chroniques en zone tropicale, Institut d'Epidémiologie et de Neurologie Tropicale, OmegaHealth, Limoges, France

	Méthode Etude mixte transversale monocentrique exhaustive. Données quantitatives : Score CISSV15 -insuffisance de convergence ; PUMPQ -dépendance au smartphone ; données d'utilisation du smartphone et de santé visuelle. Données qualitatives : 12 entretiens semi-directifs. (1) : "Quand on ne l'a pas, on 3 se sent vide. On ressent la dépendance. C'est un besoin..." ESI 3

4 Résultats 86 % des étudiants présentent une addiction au smartphone Utilisation quotidienne moyenne : 6h15 Utilisation hebdomadaire moyenne : 44h15 Réseaux sociaux +++ 50 étudiants infirmiers 19,6 ± 1,8 ans, 92% femme 58% 1ere année -42% 2ième année 66 % des étudiants présentent une fatigue visuelle 5,9/10 Score de connaissance sur les effets nocifs du smartphone :

	Dernière consultation santé visuelle : 1,7 ± 1,6 ans Professionnel consulté : •38 (76%) ophtalmologiste, •18 (36%) opticien, •5 (10%) orthoptiste.	"Dès qu'on m'enlève mon téléphone ou qu'on me contrarie en me disant de ne pas être dessus, je vais m'énerver, me contrarier et être ensuite de mauvaise humeur" ESI 6
	Discussion	5
	Références	588
	bibliographiques :

Habitudes d'utilisation du smartphone chez les étudiants infirmiers : une étude mixte

A. Thebault *(1), J.Toniolo* (1,2), F. Girard (1), S. Hantz (1), P.Beloni (1,2).


S Coutin

J-M Dienot

R Ruscassie

I Ramos

Caractérisation fréquentielle du vieillissement de matériaux diélectriques

La problématique étudiée ici concerne les matériaux diélectriques utilisés dans les systèmes d'isolation électrique de bobinages statoriques présents dans les machines électriques.

Pour améliorer la durée de vie de ces équipements, il est intéressant de caractériser le vieillissement des isolants, en fonction des contraintes qu'ils subissent (électrique, thermique, chimique…). Une approche possible consiste à suivre l'évolution de l'impédance du bobinage sur une large bande de fréquences [1]. Nous présentons aussi des mesures de champ électriques pouvant fournir des informations concernant un changement au sein du bobinage et des matériaux qui le constitue [2].

Introduction

Les bobinages statoriques alimentés par des convertisseurs fonctionnant avec des semi-conducteurs à grand gap font face à des contraintes plus sévères que celles rencontrées jusqu'alors. Les puissances commutées et les fréquences de commutation pouvant être atteintes justifient toutefois l'utilisation de ces semi-conducteurs. Cependant, ces nouvelles contraintes imposées peuvent provoquer un vieillissement prématuré des bobinages [3].

L'impédance de ces bobinages peut être modélisée par une représentation circuit composée de différentes cellules RLC [1]. Cette modélisation permet d'étudier l'évolution fréquentielle de l'impédance en simulation où les phénomènes de vieillissement peuvent être pris en compte.

L'objectif est donc d'obtenir par des caractérisations électriques large bande des bobinages, différentes informations à corréler avec les phénomènes de vieillissement. Ces caractérisations peuvent être réalisées par des mesures de paramètres S (scattering parameters) transformées en impédances et complétées par des mesures de champ proche par l'intermédiaire de sondes dédiées [2].

Modélisation et structures des bobinages

Une structure simplifiée de bobinage peut être utilisée selon les phénomènes à observer ou les applications visées.

Sur la figure 1, une modélisation 2D du bobinage est proposée. Elle tient compte des effets capacitifs inter-spires (Ctt) et entre conducteurs et noyau (Ctc). Ces capacités dépendent de l'état de l'isolant qui va être modifié par les phénomènes de vieillissement [4].

La modélisation numérique 3D est une autre approche possible. Sur la figure 2, un modèle simplifié est proposé, constitué de 2 plaques de cuivre séparé par un échantillon de résine. Il permet d'obtenir des informations aux interfaces cuivre/résine et en volume concernant le matériau diélectrique. Cette configuration peut se retrouver dans un bobinage à conducteurs rectangulaires. Les avantages d'une telle structure sont multiples. L'analyse des phénomènes observés est simplifiée, en simulation et expérimentalement. Il est aussi possible d'instrumenter ce type d'échantillon au plus proche des zones étudiées, en insérant par exemple des outils de mesure directement dans les plaques de cuivre.

La difficulté réside cependant dans la transposition des résultats à des structures plus complexes.

Mise en oeuvre et résultats

Un banc permettant de piloter des machines électriques à hautes fréquences a été développé dans [2]. Le convertisseur mis en place est composé de MOSFET SiC pouvant atteindre des commutations à des fréquences de plusieurs centaines de kHz.

Ce banc comporte aussi un jeu de capteurs permettant de caractériser électriquement l'équipement testé, notamment des sondes de champ intégrées sur PCB.

Le système d'acquisition de données qui a été développé permet une caractérisation fréquentielle largebande (1 GHz).

Des premiers résultats permettent d'observer les variations de signature fréquentielle liées à la présence de défauts sur une phase statorique d'un moteur. Sur la figure 3, nous voyons ces déviations aux alentours de 700kHz et 20MHz où la mesure de champ présente des pics dont la fréquence varie en fonction des défauts. Ces défauts ont été introduits en implantant des capacités parasites de différentes valeurs entre une phase et le neutre d'un moteur. L'objet de notre étude est maintenant d'obtenir ce type de résultat sur nos nouveaux prototypes de bobinages, ceci après avoir imposé des cycles de vieillissement électrique ou thermique.

Conclusion

Les différents outils de simulation et d'expérimentation mis en place précédemment vont permettre d'étudier les liens existant entre signature fréquentielle et vieillissement des matériaux diélectriques au sein d'enroulements statoriques. C'est aussi une base de travail pour le développement d'autres moyens de mesure non-intrusive adapté à de nouveaux bobinages.

Sur ces enroulements statoriques, des campagnes de vieillissement électrique, thermique ou encore chimique vont être menées pour pouvoir corréler le plus finement possible l'évolution des caractéristiques fréquentielles aux phénomènes physiques correspondant à la dégradation de l'isolant.

Bibliographie

Figure 1 :

 1 Figure 1 : Modèle 2D d'un bobinage [1]

Figure 2 :

 2 Figure 2 : Modèle 3D d'un échantillon cuivre-résine-cuivre

[ 1 ]

 1 J.I. Ramos Chavez, « Design of high frequency operating mechatronic systems: tools and methods of characterization of electromagnetic couplings between electromechanic converters and power electronics converters » Thèse, INP Toulouse, 2016 [2] J-M. Larbaig, «Study of the electromagnetic stresses induced by SiC components in a power conversion chain for high frequency optimization» Thèse, UPPA, 2021 [3] M. Kilper, S. Fickel, H. Naumoski and K. Hameyer, "Effects of Fast Switching Semiconductors Operating Variable Speed Low Voltage Machines," 2019 9th International Electric Drives Production Conference (EDPC), 2019, pp. 1-7, doi: 10.1109/EDPC48408.2019.9011884 [4] F. Perisse, P. Werynski and D. Roger, "High frequency behavior of AC machine: application of turn insulation aging diagnostic," Conference Record of the 2006 IEEE International Symposium on Electrical Insulation, 2006, , pp. 555-559, doi: 10.1109, /ELINSL.2006.1665379 .1665379

Figure 3 :

 3 Figure 3 : Mesure de champ électrique avec simulation d'un défaut sur une phase d'un bobinage [2]


Andrea Remo

Ines Simeone

Massimo Pancione

Pietro Parcesepe

Pascal Finetti

Halima Bensmail

Luigi Cerulo

Vittorio Colantuoni

Daniel Birnbaum

Franco Bonetti

Systems biology analysis of gene expression data and gene network reverse-engineering approaches reveal NFAT5 as a candidate biomarker in Inflammatory Breast Cancer

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

P O S T E R P R E S E N T A T I O N Open Access

Systems biology analysis of gene expression data and gene network reverse-engineering approaches reveal NFAT5 as a candidate biomarker in Inflammatory Breast Cancer Andrea Remo 1 † , Ines Simeone 2 † , Massimo Pancione 3 † , Pietro Parcesepe 4 , Pascal Finetti 5 , Halima Bensmail 2 , Luigi Cerulo 3 , Vittorio Colantuoni 3 , Daniel Birnbaum 5 , Franco Bonetti 4 , Francois Bertucci 5 , Erminia Manfrin 4 , Michele Ceccarelli 2* † From Breast Cancer Immunotherapy Symposium (BRECIS), part of the Sidra Symposia Series, held in partnership with the Society for Immunotherapy of Cancer Doha, Qatar. 13-14 April 2015

Inflammatory Breast Cancer (IBC) is the most aggressive and highly metastatic form of breast cancer [START_REF] Levine | Inflammatory breast cancer: the experience of the Surveillance, Epidemiology and End Results (SEER) Program[END_REF][START_REF] Cristofanilli | Inflammatory breast cancer (IBC) and patterns of recurrence: understanding the biology of a unique disease[END_REF][START_REF] Tarpin | Defining the molecular biology of inflammatory breast cancer[END_REF]. In a recent study [START_REF] Manfrin | Comparison between invasive breast cancer with extensive peritumoral vascular invasion and inflammatory breast carcinoma. A clinical pathological study of 161 cases[END_REF], we analysed breast cancer with peritumoral neoplastic lymphovascular invasion (ePVI) in comparison with inflammatory breast cancer, showing that ePVI breast cancer have more clinicopathologic affinity than differences with the most aggressive cancer in the breast. Here, we aim to identify potential master regulators (MRs) that drive the expression pattern in IBC.

Transcriptomic (i.e., mRNA) data from 197 breast tumours were used for this analysis (GEO GSE23720) [START_REF] Bekhouche | High-resolution comparative genomic hybridization of inflammatory breast cancer and identification of candidate genes[END_REF]. All tumours were classified as "IBC" (n=63) or "nIBC" (n=134). To identify novel MRs that drive the IBC phenotype, all expression data were analysed using a network-based strategy (ARACNe [START_REF] Margolin | ARACNE: an algorithm for the reconstruction of gene † Contributed equally 2 Qatar Computing Research Institute (QCRI), Qatar Foundation, Doha, Qatar Full list of author information is available at the end of the article Remo et al[END_REF]) and Master Regulator Analysis (MRA) [7]. We chose to perform invivo IHC analysis, in two independent cohorts of IBCs (n = 39), nIBCs (n = 82) and normal breast tissues (n = 15), for the top significant Master Regulators: MGA, CTNNB1 and NFAT5. Biological validation confirmed that NFAT5 expression was higher in IBC than in nIBC (70% vs. 20%) and that the majority of NFAT5-positive IBC samples displayed NFAT5 nuclear expression in comparison with nIBC samples (89% vs. 12%).

We provide evidence that NFAT5 transcription factor could constitute a novel IBC biomarker that could help to identify the most aggressive forms of BC into routine clinical practice.