Kohei Shigeta

Aya Matsui

Hiroto Kikuchi

Sebastian Klein

Emilie Mamessier

Ivy X Chen

Shuichi Aoki

Shuji Kitahara

Koetsu Inoue

Ayako Shigeta

Tai Hato

Rakesh R Ramjiawan

Daniel Staiculescu

Dieter Zopf

Lukas Fiebig

Gabriela S Hobbs

Alexander Quaas

Simona Dima

Irinel Popescu

Peigen Huang

Lance L Munn

Mark Cobbold

Lipika Goyal

Andrew X Zhu

Rakesh K Jain

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

Edwin L Steele

Regorafenib combined with PD1 blockade increases CD8 T-cell infiltration by inducing CXCL10 expression in hepatocellular carcinoma

BACKGROUND

Hepatocellular carcinoma (HCC) is a common cancer-related cause of death. 1 2 HCC is an aggressive gastrointestinal cancer with increasing incidence in the USA, but existing therapeutic options have shown limited efficacy. HCCs are highly vascularized tumors, which often develop an arterialized blood supply that feeds tumor progression. [START_REF] Semela | Angiogenesis and hepatocellular carcinoma[END_REF] Sorafenib-a broad tyrosine kinase inhibitor (TKI) with potent antivascular endothelial growth factor receptor (VEGFR)1-3 and platelet-derived growth factor receptor (PDGFR) activity-was the first systemic therapy to show increased overall survival (OS) in patients with advanced HCC in phase III trials. 4 5 However, HCCs become resistant to sorafenib and the OS benefit is limited to approximately 3 months over placebo. Other broad TKIssuch as regorafenib and cabozantinib-and the anti-VEGFR2 antibody ramucirumab (in selected patients with high alphafetoprotein) also showed efficacy in phase III trials in patients with advanced HCC who progressed on sorafenib. [START_REF] Bruix | Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF][START_REF] Abou-Alfa | Cabozantinib in patients with advanced and progressing hepatocellular carcinoma[END_REF][START_REF] Zhu | Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased α-fetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF] Finally, the multitargeted TKI lenvatinib became a firstline therapy in advanced HCC after showing non-inferior survival compared with sorafenib. [START_REF] Kudo | Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial[END_REF] These successes argued for a key role of VEGF/VEGFR-driven angiogenesis in advanced HCC. However, other multitargeted TKIs-such as sunitinib, brivanib-or ramucirumab in unselected patients with HCC did not increase OS despite delaying tumor progression in phase III trials. [START_REF] Kudo | Systemic therapy for hepatocellular carcinoma: 2017 update[END_REF] This argues that the activity seen with the approved drugs may be in part mediated Open access by inhibition of specific targets beyond VEGFRs and specific mechanisms of action.

HCCs usually develop in tissues affected by chronic inflammation and hypoxia due to underlying liver damage. This characteristic feature leads to immunosuppression via multiple mechanisms, including upregulation of the immune checkpoint molecules such as programmed cell death protein 1 (PD1) and its ligand programmed deathligand 1 (PD-L1). [START_REF] Chen | CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice[END_REF][START_REF] Hato | Immune checkpoint blockade in hepatocellular carcinoma: current progress and future directions[END_REF][START_REF] Sia | Identification of an Immunespecific class of hepatocellular carcinoma, based on molecular features[END_REF] Recently, immunotherapy strategies using immune checkpoint blockade (ICB) with anti-PD1 antibodies nivolumab and pembrolizumab alone as well as with nivolumab plus ipilimumab (an anticytotoxic T-lymphocyte antigen 4 antibody) received accelerated approval by the Food and Drug Administration; this was based on phase I/II trial data, which showed responses in 14%-31% of previously treated patients with advanced HCC, some of which were durable. [START_REF] Zhu | Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial[END_REF] [START_REF] El-Khoueiry | Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial[END_REF] However, PD1 blockade alone was insufficient to significantly increase OS in the randomized phase III trials conducted so far.

The limitations of monotherapy approaches in HCC led to the development of anti-VEGFR/PD1 combinations new therapies designed to address treatment resistance mechanisms and achieve synergy by increasing effector T-cell infiltration. [START_REF] Fukumura | Enhancing cancer immunotherapy using antiangiogenics: opportunities and challenges[END_REF] One of the key elements of the efficacy of combinations of anti-VEGF with immunotherapies is maintaining a functional (normalized) vasculature and reducing hypoxia. [START_REF] Fukumura | Enhancing cancer immunotherapy using antiangiogenics: opportunities and challenges[END_REF][START_REF] Hamzah | Vascular normalization in Rgs5-deficient tumours promotes immune destruction[END_REF][START_REF] Jain | Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia[END_REF] We and others have demonstrated that blocking VEGF pathway can transiently normalize the tumor vasculature and increase cytotoxic T lymphocyte (CTL) infiltration, thus facilitating vaccine immunotherapy. [START_REF] Huang | Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy[END_REF] Of note, recent studies showed that antitumor immune responses and vascular normalization can be reciprocally regulated by CD4+ T effector cells in breast cancers. [START_REF] Tian | Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming[END_REF] In HCC, we recently demonstrated that dual VEGFR-2/PD1 blockade using antibodies normalizes tumor vasculature and induces antitumor immunity in mice with underlying liver damage. [START_REF] Shigeta | Dual programmed death receptor-1 and vascular endothelial growth factor receptor-2 blockade promotes vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma[END_REF] The clinical experience is consistent with preclinical findings. The use of a combination of the anti-VEGF antibody bevacizumab with the anti-PD-L1 antibody atezolizumab significantly increased survival outcomes over sorafenib in the randomized phase III IMBRAVE150 trial. [START_REF] Finn | Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma[END_REF] Atezolizumab/bevacizumab objective response rate of 27.3%, including a 5.5% complete response rate and many durable responses, stands as a breakthrough in advanced HCC treatment. Whether anti-VEGFR TKIs-that also target vascular pericytes-can also reprogram the immunosuppressive microenvironment of HCC and enhance ICB efficacy in a similar manner remains unknown. Furthermore, whether combinatorial approaches using multitargeted anti-VEGFR TKIs could address treatment resistance via targeting alternative mechanisms remains unknown. Of note, the initial clinical experience with combination of lenvatinib with the anti-PD1 antibody pembrolizumab showed high response rate and led to a randomized phase III trial. Moreover, a recent phase Ib dose-escalation study evaluated the efficacy of regorafenib at 80-160 mg/day and anti-PD-1 therapy (nivolumab) in refractory gastric or colon cancer. After reducing the regorafenib dose to 80-120 mg/day during the dose-expansion part, study results showed an intriguing objective response rates of 40% overall, including a 36% response rate in microsatellite stable metastatic colorectal cancer, a notoriously 'immunologically cold' ICB-resistant tumor. [START_REF] Fukuoka | Regorafenib plus nivolumab in patients with advanced gastric or colorectal cancer: an open-label, dose-escalation, and Dose-Expansion phase Ib trial (REGONIVO, EPOC1603)[END_REF] Here, we studied the dose-dependent impact of regorafenib, which directly targets VEGFRs, Tie2 and PDGFR but may also have indirect inhibitory activity against signal transducer and activator of transcription 3 (STAT3), an immune-relevant target, in models of HCC with underlying liver damage. 24 25

METHODS

Cells

We used two murine HCC cell lines in this study: RIL-175 (a p53/Hras mutant line syngeneic to C57Bl/6 mouse strain background), [START_REF] Zender | Generation and analysis of genetically defined liver carcinomas derived from bipotential liver progenitors[END_REF] kindly provided by Dr Tim Greten (NIH), and HCA-1, a line derived from a spontaneous HCC in a C3H mouse established in our laboratory. 27 28 Mouse models of orthotopic HCC and liver damage For orthotopic tumor generation, RIL-175 cells were implanted in male mice of C57Bl/6 strain background (wild-type, Rag1 -/-, Cxcr3 -/-, Jackson Labs) and HCA-1 cells in male C3H mice. 29 30 For spontaneous HCC generation, we used Mst1 -/-Mst2 f/-mice (both genders).

Agents and treatments

Regorafenib was provided by Bayer and administered by oral gavage daily at a dose of 5, 10 and 20 mg/kg in 34% 1,2-propandiol (Sigma-Aldrich), 34% PEG400 (Sigma-Aldrich), 12% pluronic F68 (Thermo Fischer Scientific, Waltham, Massachusetts, USA), and 20% water, as per manufacturer's recommendation. Mouse anti-PD1 antibody (clone RMP-014) was purchased from BioXcell (Lebanon, New Hampshire, USA) and Mouse IgG isotype control was purchased from Thermo Fischer Scientific. The STAT3 inhibitor LLL12 was purchased from BioVision (Milpitas, California, USA). Antimouse PD1 antibody or IgG (control) were given intraperitoneally at a dose of 10 mg/kg thrice weekly, as described. [START_REF] Chen | CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice[END_REF] Daily gavage of vehicle and intraperitoneal injection of 10 mg/kg of isotype-matched IgG were given as control treatments for anti-PD1 antibody. For the survival studies, moribund status was used as the end point and moribund was defined as symptoms of prolonged distress, >15% of weight loss compared with the starting date, body condition score >2 and tumor size of >24 mm in diameter.

Patient samples

Formalin-fixed paraffin-embedded HCC tissue and blood samples were obtained with informed consent from a published cohort of deidentified patients who underwent liver resection at the department of Surgery at Fundeni Clinical Institute, Bucharest, Romania. [START_REF] Duda | Potential circulating biomarkers of recurrence after hepatic resection or liver transplantation in hepatocellular carcinoma patients[END_REF] Furthermore,

Open access

we collected blood samples with informed consent from patients treated by regorafenib as standard of care (HCC) or in a phase I clinical trial of regorafenib monotherapy in patients with acute myeloid leukemia (AML). Peripheral blood was collected before regorafenib therapy and after 2 weeks of treatment and processed in a Clinical Laboratory Improvement Amendments-certified facility of the Steele Laboratories at MGH.

Flow cytometry analysis

Prior to immunostaining, cells were washed with the buffer and fixed and permeabilized with FoxP3/Transcription Factor Staining Buffer Set (eBioscience/ Thermo Fischer Scientific) to stain the intracellular markers. Harvested cells were incubated in Dulbecco's Modified Eagle Medium with cell activation cocktail with brefeldin A (Biolegend) for 4 hour at 37°C. The cells were stained with the antibodies of cell surface and intracellular marker in the buffer with brefeldin A.

Antimouse CD16/32 antibody (clone 93, Biolegend, San Diego, California, USA) was added for FcR blockade, and incubated for 5 min at room temperature. After another washing step, antibodies for cell phenotyping were added, and cells were incubated for 40 min at room temperature. The monoclonal antibodies used for flow cytometry analysis were for: CD45 (30-F11), CD3e (145-2 C11), CD4 (RM4-5), CD8a (5H10-1), Foxp3 (FJK-16s), CD39 (Duha59), CXCR3 (CXCR3-173), CD11b (M1/70), Ly6C (HK1.4), Ly6G, CD206 (C068C2), MHC-II (M5/114.15.2), interferon gamma (IFN-γ) (XMG1.2), PD1 (29F.1A12) (eBioscience).

Immunohistochemistry

For analyses of endothelial and perivascular cells, tumor tissues containing the total areas of CD31 (clone DIA-310, Dianova, Germany) positive endothelial cells and αsmooth muscle actin (α-SMA) (Sigma-Aldrich, Saint Louis, Missouri, USA) positive pericytes were identified by scanning tumor sections under ×10 magnification and were counted in five random fields under ×200 magnification. CD8+ lymphocytes were detected using anti-CD8b antibody (eBioscience). Five random fields were selected from the tumor center, which did not include the edge of the tumor. Hypoxia was identified and quantified using immunostaining for anticarbonic anhydrase (CA)-IX (rabbit monoclonal; dilution, 1:100, Abcam). These data were analyzed using ImageJ (US NIH) and Photoshop (Adobe Systems, San Jose, California, USA) software. Analysis was performed using a laser-scanning confocal microscope (Olympus, FV-1000). Anticleaved caspase-3 antibody (Abcam) was used to assess the cell apoptosis in the tumor tissues, and antiphospho-STAT3 antibody (Abcam) for quantitative assessment of p-STAT3. For the latter, immunostained whole slide tumor sections were scanned on a Hamamatsu NanoZoomer S360 slide scanner. For quantification purpose, whole images were analyzed for at least three representative areas of vital tumor which was defined as region-of-interest.

RNA in situ hybridization (RNA-Scope)

The RNA-Scope assay was performed according to manufacturer's instructions. [START_REF] Wang | RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues[END_REF] Briefly, formaldehyde-fixed and paraffin-embedded human and mouse HCC tissue blocks were cut in 5 µm sections, pretreated for 30 min, following digestion and hybridization at 40°C in a HybEZ oven with both dual labeling probes antimurine CXCR3 probe (Mm-CXCR3; 402511-C2) and CXCL10 (Mm-CXCL10; 408921) or human CXCL10 (Hs-CXCL10; 311851), all from Advanced Cell Diagnostics. Counterstaining was performed using hematoxylin for 10 s.

Pharmacokinetic analysis of regorafenib and metabolites

From the orthotopic HCC and liver damage mouse models described above, blood samples were collected at 1, 4, 7 and 24 hours after once-daily oral administration of 5, 10 and 20 mg/kg regorafenib, respectively, for 5 days. Anti-PD1 antibody was given on days 1 and 4 with dose of 10 mg/kg by intraperitoneal injection. Blood samples were drawn from four to five animals per sampling time point. Regorafenib and its active metabolites M-2, M-4 and M-5 were analyzed in plasma samples using a validated bioanalytical liquid chromatography-tandem mass spectrometry. 25 33 Pharmacokinetic (PK) parameters were derived from the area under the plasma concentration versus time curve (AUC) using a non-compartmental analysis.

Statistical analysis

All statistical analyzes were performed using Stata software, V.14.1 (StataCorp, College Station, Texas, USA). Error bars indicate the SEM. Differences were considered significant when p values were <0.05. Quantitative variables were compared using Student's t-test. Analysis of experiments with more than two groups were performed using one-way analysis of variance with Scheffe's correction for multiple comparisons. Wilcoxon signed-rank test was used for analysis of marker changes over time. Based on tumor growth delay data, we estimated that an n>10 mice/group were required to ensure an alpha level of 5% (two-sided) and beta level of 20% (two-sided) for the survival analysis. Median overall survival was estimated using the Kaplan-Meier method. Statistical analyses in the survival experiments were performed by Cox proportional hazard model and HR and 95% CIs were calculated as well.

See additional materials and methods in online supplemental material.

RESULTS

PD1 blockade combined with an intermediate dose of regorafenib induces vessel normalization and increases regorafenib delivery and activity

We first evaluated the dose-dependent effects of regorafenib in combination with anti-PD1 antibodies on HCC vessels in a time-matched manner (after 1 week of treatment). CD31+ surface area-a measure of microvascular density (MVD)was increased after treatment with 10 mg/kg regorafenib and anti-PD1 antibody compared with all the other groups Open access in RIL-175 model (online supplemental figure S1A). Moreover, pericyte coverage was significantly increased, and tissue hypoxia showed a tendency for decrease, after treatment with anti-PD1 antibody with 10 mg/kg but not with 20 mg/ kg of regorafenib (figure 1A,B and online supplemental figure S1B,C).

To further understand the effects of the intermediate dose (10 mg/kg) of regorafenib, we examined its effects alone or in combination with anti-PD1 treatment at a later time-point in the same model (day 12). We found that the significant increase in MVD after 10 mg/kg regorafenib alone and with anti-PD1 treatment compared with control was maintained at this time-point (online supplemental figure S1B). Moreover, the hypoxic tissue area, measured by CA-IX staining, was significantly decreased in the intermediate dose regorafenib and combination treatment groups compared with control (figure 1C,D and online supplemental figure S1D,E). Furthermore, the mature (pericyte-covered) MVD was significantly increased only in the combination group, consistent with both structural and functional vascular normalization in HCC after regorafenib at the 10 mg/kg dose with PD1 blockade (figure 1E,F).

In addition, we evaluated PK parameters in HCCbearing mice after regorafenib administration alone at doses of 5, 10 and 20 mg/kg or in combination with anti-PD1 therapy at the 10 mg/kg regorafenib dose for 5 days to achieve a steady state. We found differential plasma versus tumor PK for regorafenib and anti-PD1 therapy. In plasma, the PK parameters area under the curve within the observation period of 24 hours (AUC 0-

24

) and maximum/peak concentration (C max ) showed a dose-proportional increased for regorafenib (online supplemental table S1). However, while tumor AUC 0-24 and C max also showed a dose-proportional increase for regorafenib, addition of anti-PD1 therapy to regorafenib 10 mg/kg further increased tumor exposure of the drug (online supplemental table S1 and online supplemental figure S1F,G). Consistent with these data, we found an increase in the biologically active metabolite of regorafenib M-4, which occurs prominently in mice, from the lower to higher dose group. While plasma PK of M-4 were not changed by addition of anti-PD1 antibody to regorafenib 10 mg/kg, tumor PK parameters also showed trends for increased M-4 (online supplemental table S1). The concentrations of the active metabolites M-2 and M-5 were below the detection limit of the assay in most samples. Collectively, these results show that an intermediate dose of regorafenib with anti-PD1 blockade is required to promote vascular normalization, which reduces hypoxia and increases regorafenib delivery and activity in the HCC tissue.

Regorafenib at 10 mg/kg dose is required to enhance CTL infiltration and activation when used in combination with PD1 blockade Having demonstrated that regorafenib given at 10 mg/ kg dose with anti-PD1 antibody normalized tumor vessels' structure and function, we next investigated the effects of this combination therapy on antitumor immunity in HCC models. First, we evaluated the intratumoral infiltration and distribution of CD8 T cells in RIL-175 tumors.

Open access

The total number of CD8 T-cells infiltrating inside the tumor, evaluated by IF, was significantly increased after 1 week of treatment in the tumors from mice receiving anti-PD1 therapy, alone or with regorafenib at 5, 10 or 20 mg/kg. However, the CD8 T cell infiltration inside the tumor was significantly higher after 10 mg/kg regorafenib and anti-PD1 antibody compared with all other groups (figure 2A,B).

In separate experiments, we tested whether this effect of regorafenib 10 mg/kg/anti-PD1 therapy was maintained at a later time-point (day 12 of treatment). We found that the total number of CD8 T cells measured by flow cytometry in tissue was significantly increased in the combination therapy group as compared with all the other treatment groups (figure 2C), despite the persistence of immunosuppressive myeloid cells (online supplemental figure S2A,B). Furthermore, when we evaluated the intratumoral distribution of the CD8 T cells by IF, we found that their infiltration was significantly increased in the combination group versus other treatment groups (figure 2D,E and online supplemental figure S2C). In addition, combination therapy promoted the activation of CD8 T cells, as measured by IFN-γ expression. Using western blot analysis, we found that overall tissue IFN-γ expression was increased after combination therapy (online supplemental figure S2D); flow cytometry validated that CD8+IFN-γ+activated CTLs were significantly increased only in the combination therapy group (figure 2F).

Regorafenib increases CXCL10 production by HCC cells

To investigate the mechanism mediating the increased intratumoral CTL infiltration after combined regorafenib 10 mg/kg and anti-PD1 treatment in HCC, we examined the transcriptional changes in whole tumor lysate using RNA sequencing analysis after 1 week of treatment (online supplemental dataset S1 and figure S2E). Gene set enrichment analysis (GSEA) using curated gene sets (Gene Ontology) showed significant changes in multiple pathways, including those related to immune response and factors related to CTL trafficking in RIL-175 tumor tissues (figure 3A). We found that an unbiased GSEA using a previously published 'poor prognostic angiogenic gene' signature [START_REF] Tian | Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming[END_REF] showed that these genes were enriched in the control group compared with regorafenib (10 mg/ kg) plus anti-PD1 therapy (online supplemental figure S2F). These data further support the vascular normalization effect which was seen in the time-matched study presented in figure 1.

On the other hand, we found significant upregulation in the gene expression of both the Cxcr3 receptor and its ligand Cxcl10 after combination treatment (compared with control) among the top hits (online supplemental table S2). In addition, we measured protein concentration in the whole tumor tissue (used in the PK experiments) by ELISA and found that CXCL10 showed a non-significant trend for increase after treatment with intermediate dose regorafenib and anti-PD1 antibody (n=4) (online supplemental figure S3A). Of note, we have previously reported RNA sequencing analysis after dual antibody blockade of VEGFR2 and PD-1 antibody using the same orthotopic HCC model, but found no difference in CXCL10 or CXCR3 expression. [START_REF] Shigeta | Dual programmed death receptor-1 and vascular endothelial growth factor receptor-2 blockade promotes vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma[END_REF] These data indicate that upregulation of the CXCL10/CXCR3 axis may be due to the multitargeted (VEGFR-independent) activity of regorafenib.

In the same samples used for the PK study, we also found that regorafenib treatment inhibited phosphorylated (p) STAT3 levels in HCC tissues (online supplemental figure S3B,C). Thus, we evaluated the pSTAT1/pSTAT3 ratio after combination of regorafenib (10 mg/kg)/anti-PD1 treatment. We found an increase in the ratio of STAT1/ STAT3 phosphorylation levels after combination treatment (figure 3B,C). These results indicate that regorafenib treatment can shift the ratio between activated STAT1 and STAT3, potentially leading to increased CXCL10 production. We also used the TCGA database to confirm the correlation between the CXCL10 expression and the other immune-related genes. We found that the CXCR3, STAT1, CD8A and IFNG genes were positively correlated in human HCC tissue (online supplemental figure S3D).

To determine if CXCL10 was produced by murine HCC cells in response to regorafenib treatment, we next cultured RIL-175 cells with various concentration of regorafenib in vitro and measured the CXCL10 levels by ELISA. Regorafenib treatment significantly increased the level of CXCL10 secreted by RIL-175 cells and to a lesser extent in HCA1 cells, with a peak concentration at 1 µM (figure 3D and online supplemental figure S3E). Moreover, we also found that a selective and direct STAT3 inhibitor (LLL12) significantly increased CXCL10 expression in RIL-175 cells (online supplemental figure S3F). Furthermore, we confirmed that the STAT1 activation and STAT3 inhibition were also seen in the RIL-175 cells culture with 1 µM of regorafenib in vitro (figure 3E,F).

To validate whether regorafenib treatment can increase expression of CXCL10 in vivo, and to identify a cell population in HCC expressing CXCL10 and CXCR3, we performed an RNA-Scope in situ hybridization (ISH) dual labeling for both markers. In line with the in vitro findings, we found an increased level of CXCL10 mRNA in the combination group of regorafenib and anti-PD1 antibody in the murine HCC tissue (figure 3G-I). In addition, dual labeling of both CXCL10 and CXCR3 confirmed that TILs in proximity of CXCL10-expressing HCC cells showed expression of CXCR3 mRNA. CXCL10 expression was almost exclusively detected in cancer cells, based on histological analysis performed by a trained liver pathologist (AQ). Moreover, flow cytometric analyses confirmed the increased levels of CXCR3+CD8+ T cells after combined regorafenib/anti-PD1 therapy in the tumor tissue but not in the spleen (online supplemental figure S3G,H).

Finally, we measured the dose-dependent changes in intratumoral CXCL10 protein expression after Open access regorafenib treatment in mice. We found that regorafenib treatment alone at 10 mg/kg and 20 mg/kg increased protein levels of CXCL10 evaluated in whole tumor tissue lysate by western blot analysis as early as 6 hours after treatment (online supplemental figure S3I,J). Moreover, when we evaluated CXCL10 expression

Open access

after prolonged combination treatment with regorafenib and anti-PD1 antibodies (for 1 week in the PK studies), we found increased protein expression level of CXCL10 for the intermediate (10 mg/kg) and the high dose (20 mg/ kg) of regorafenib but not for the low dose (5 mg/kg)

Open access

by western blot analysis in whole tumor tissue lysate (figure 3J,K).

Collectively, these results indicate that CXCL10 expression by HCC cells is increased after regorafenib treatment due to inhibition of STAT3 activity leading to increased intratumoral infiltration CD8+CXCR3+ T cells.

Survival benefit of regorafenib combined with anti-PD1 treatment is regorafenib dose-dependent

We finally tested the efficacy of combining anti-PD1 antibody with regorafenib dose versus each agent alone or control in three different mouse models (orthotopic RIL-175 and HCA-1 grafts, Mst1/Mst2-KO model) with underlying liver damage (figure 4A). In the RIL-175 HCC model, we found that combination treatment showed tumor growth delay but only when regorafenib was used at the dose of 10 mg/ kg with anti-PD1 therapy (figure 4B). Moreover, regorafenib (10 mg/kg) plus anti-PD1 group showed a significant and substantial survival benefit (median survival of 29 days, more than double compared control group in this model) with an HR (HR=0.17; p<0.001) (figure 4C). The low dose (5 mg/kg) of regorafenib plus anti-PD1 therapy showed no significant growth delay and only a non-significant trend for survival benefit (HR=0.47; p=0.089) (online supplemental figure S4A). When we compared the efficacy of the combination Open access treatment of anti-PD1 therapy with regorafenib at 10 mg/ kg versus anti-PD1 therapy with a high dose (20 mg/kg) of regorafenib, we found that only regorafenib at 10 mg/kg significantly improved survival compared with control group (HR=0.24; p=0.004) (online supplemental figure S4B).

We also tested the efficacy of regorafenib combined with anti-PD1 treatment in highly metastatic HCC model with underlying liver damage (HCA-1 grafted in C3H mice). Similar to the RIL-175 model, we found a trend for increased survival for the combination when regorafenib was used at 10 mg/kg (figure 4D); of note, no activity was seen when we combined regorafenib at 20 mg/kg with anti-PD1 therapy (online supplemental figure S4C). Moreover, we tested anti-PD1 therapy with regorafenib at 10 mg/kg against spontaneous HCCs developed in Mst1/ Mst2-deficient mice and found that it significantly delayed tumor growth (figure 4E). We did not detect any overt toxicity (eg, body weight loss) with any of the regimens tested, consistent with prior experience with this drug in mouse models (online supplemental figure S4D). [START_REF] Wilhelm | Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity[END_REF] Finally, when we repeated the survival experiment in an orthotopic RIL-175 model in Rag1 -/-/C57Bl/6 mice, which lack functional T cells, we found no significant benefit for regorafenib 10 mg/kg with or without anti-PD1 antibody (figure 4F).

These results show the safety and efficacy of combining regorafenib at 10 mg/kg with PD1 blockade in mice, and the requirement of functional T cells for the antitumor effects of this combination therapy.

Combination therapy of regorafenib with anti-PD1 antibody promotes CTL infiltration via CXCL10/CXCR3 axis

To demonstrate that intratumoral CD8 T-cell infiltration after combination therapy of regorafenib with anti-PD1 antibody was mediated by CXCL10/CXCR3 axis, we next evaluated the effects of combined regorafenib (10 mg/ kg)/anti-PD1 antibody therapy on RIL-175 HCC growing in Cxcr3-deficient (Cxcr3 -/-) versus Cxcr3-proficient (Cxcr3 +/+ ) C57Bl/6 mice. In time-matched experiments, we found that the number of CTL infiltrating HCC tissue was significantly decreased in Cxcr3 -/-mice compared with Cxcr3 +/+ mice after 1 week of combination treatment (figure 5A,B). Of note, despite this decrease, combination therapy induced a small increase in intratumoral CD8+ T cells even in Cxcr3 -/-mice, likely mediated by vascular normalization. Moreover, when we evaluated the efficacy of regorafenib at 10 mg/kg with anti-PD1 antibody treatment in Cxcr3 -/-mice in survival experiments, we found no additional survival benefit for this combination over anti-PD1 therapy alone (figure 5C); these data are in contrast to the benefits seen with regorafenib at 10 mg/kg with anti-PD1 antibody treatment in Cxcr3 +/+ tumor-bearing mice (figure 4C).

These results demonstrate that CXCR3 is a mediator of intratumoral CD8 T-cell infiltration-along with vascular normalization-and is critical for the added survival benefit after combination of regorafenib at 10 mg/kg with anti-PD1 therapy in HCC models.

CXCL10 is detectable in human HCC, is measurable in blood samples from patients with cancer and is a potential biomarker of regorafenib's immuno-modulatory activity We next performed RNA-ISH analysis of human tumor tissues and ELISA in blood samples from a published cohort of resectable patients with HCC. [START_REF] Duda | Potential circulating biomarkers of recurrence after hepatic resection or liver transplantation in hepatocellular carcinoma patients[END_REF] Furthermore, we collected blood samples from patients treated by regorafenib as standard of care (HCC); in addition, we examined the effect of regorafenib on blood CXCL10 levels in a clinical trial in a different disease-AML (NCT03042689). In line with preclinical data, we found that CXCL10 Open access expression was detectable primarily in human HCC cells; >60% (30/48) untreated (surgically resected) tumors showed positivity for CXCL10 expression in HCC cells (figure 6A-D). Seven cases showed elevated levels of CXCL10 expression, with 13%-17% of tumor cells showing positivity for CXCL10 mRNA (figure 6A-D). Moreover, CXCL10 was detectable in the blood concentration of all patients with HCC (median: 617.9 pg/mL, n=20) and AML (median: 547.4 pg/mL, n=8). Finally, we found an increased concentration of plasma CXCL10 in both patients with advanced HCC (n=1) and AML (n=7) after 2 weeks of treatment (figure 6E,F). These data indicate that CXCL10 is objectively measurable and its concentration can increase in the blood of patients with HCC-supporting its future examination as a potential biomarker of the immunomodulatory activity of regorafenib treatment.

DISCUSSION/CONCLUSION

In this study, we demonstrate the impact of regorafenib dosing when combined with ICB on vascular structure and function, antitumor immunity, tumor growth and survival in HCC models in mice. A significant survival benefit was seen only in the group where PD1 blockade was combined with regorafenib at the intermediate regorafenib dose (10 mg/kg), which both normalized the vasculature and increased CXCL10 expression in HCC cells. Both effects were critical for achieving efficacy: use of a higher dose (20 mg/kg) of regorafenib in combination with anti-PD1 antibodies did not show a benefit despite increased regorafenib concentration and STAT3 inhibition as well as increased CXCL10 expression in the HCC tissue. This result is consistent with our published report using sorafenib and may be due to excessive pruning of vessels. [START_REF] Chen | CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice[END_REF] On the other hand, use of a low dose of regorafenib (5 mg/kg) normalized the vessels but did not inhibit STAT3 activity or induce CXCL10 expression and failed to show a benefit when combined with anti-PD1 therapy. While the PK/PD of regorafenib is expected to be different in human patients with HCC, the parameters measured in HCC-bearing mice with liver damage provide proof-of-principle data on the relevance of judicious dosing of regorafenib to increase the efficacy of concomitant anti-PD1 immunotherapy (figure 6). These results are in line with predictions from mathematical models of dual VEGF/PD1 inhibition, [START_REF] Mpekris | Combining microenvironment normalization strategies to improve cancer immunotherapy[END_REF] as well as with emerging clinical data with combined use of lower dosed of regorafenib with anti-PD1 therapy in other gastrointestinal cancers. [START_REF] Fukuoka | Regorafenib plus nivolumab in patients with advanced gastric or colorectal cancer: an open-label, dose-escalation, and Dose-Expansion phase Ib trial (REGONIVO, EPOC1603)[END_REF] Moreover, we unraveled the pathophysiological, cellular and molecular mechanisms underlying the benefits achieved with the intermediate dose of regorafenib and anti-PD1 therapy. First, we found that combination therapy was more effective than regorafenib or anti-PD-1 therapy alone for normalizing the structure and the function of the abnormal HCC vasculature, that is, increased pericyte covered vessels, decreased hypoxia and increased drug uptake. This supports our recent finding that anti-PD1 therapy has vascular normalizing activity when combined with anti-VEGFR therapy in HCC. [START_REF] Shigeta | Dual programmed death receptor-1 and vascular endothelial growth factor receptor-2 blockade promotes vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma[END_REF] Second, using genetically engineered mouse models and transcriptional analyzes, we showed that the benefits of combined regorafenib/anti-PD1 therapy were mediated by T cells. We and others have shown that vascular normalization can enhance CD8 T-cell infiltration in tumors, including in HCC models. 16 21 Here, we show that regorafenib can increase CXCL10 expression in cancer cells in vitro and Open access in vivo, likely owing to its multitargeted activity. This effect further enhances intratumoral CXCR3+CD8+ T-cell infiltration in HCC. Through these two mechanisms, judicious dosing of regorafenib could enhance the benefits of ICB treatment in the 'immunologically cold' and ICBresistant HCCs.

CXCL10 expression is stimulated by IFN-γ on downstream STAT1 activation. [START_REF] Jackson | Stat1 and NF-κB inhibitors diminish basal interferon-stimulated gene expression and improve the productive infection of oncolytic HSV in MPNST cells[END_REF] STAT1 and STAT3 have opposite effects in antitumor immune response. Previous studies have shown the reciprocal regulatory mechanism of STAT1 and STAT3 using the transgenic animals and concluded that their activation may be cross-regulated. 37 38 Here, we found that intratumoral CD8 T-cell infiltration was promoted by CXCL10 upregulation in cancer cells after STAT3 inhibition by regorafenib and was facilitated in the context of PD1 blockade. STAT3 is a pathway known to promote cell survival, proliferation and immune tolerance, [START_REF] Regis | Ups and downs: the STAT1:STAT3 seesaw of Interferon and gp130 receptor signalling[END_REF] and may mediate cellular immune responses in HCC after multitargeted TKI treatment. [START_REF] Avella | Regression of established hepatocellular carcinoma is induced by chemoimmunotherapy in an orthotopic murine model[END_REF] Moreover, CXCR3 is expressed on a fraction of CTLs both in mouse and human HCC, as shown in our study and in the study by Zheng et al. [START_REF] Zheng | Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing[END_REF] A recent study demonstrated that intratumoral activity of the CXCR3 chemokine system is required for the efficacy of anti-PD1 therapy. [START_REF] Chow | Intratumoral activity of the CXCR3 chemokine system is required for the efficacy of anti-PD-1 therapy[END_REF] Here, we show that CXCR3 expression in CTLs was critical for the survival benefit seen with regorafenib/anti-PD1 therapy, which was not seen in HCC-bearing mice deficient for CXCR3.

Previous studies have proposed blood circulating CXCL10 as a potential biomarker of response to pegylated IFN-antiviral treatment in patients with HCV, but its role in the migration of effector T cells to the infected liver remains unclear. [START_REF] Butera | Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C[END_REF] We found that CXCL10 is often expressed by cancer cells in human HCC tissue specimens. We also evaluated the concentration of CXCL10 in blood of patients with HCC and AML and we found that the CXCL10 is detectable in all patients and is increased after regorafenib treatment.

Despite our use of three different murine HCC models to confirm the activity of regorafenib combined with anti-PD-1 antibody, clinical translation of our results will be challenging. Our models do not account for the significant heterogeneity seen with HCC in patients, and thus the effects of the regorafenib/anti-PD1 therapy will need to be studied across different subtypes and etiologies. Moreover, evaluation of CXCL10 and microenvironmental changes, and their role in patients with HCC, will need to be performed in larger studies. Such analyses would be most impactful if performed at the time of radiological response or resistance to regorafenib/anti-PD1 therapy. These limitations notwithstanding, these data provide clear evidence that CXCL10 is expressed by cancer cells and suggest that plasma CXCL10 changes should be further evaluated as a potential biomarker of regorafenib's immunomodulatory effects, particularly in patients receiving concurrent immunotherapies.

In conclusion, we show here that regorafenib treatment can significantly enhance PD1 blockade effects in a dosedependent manner in HCC models. The benefit was due to the activity of the two agents both on normalization of HCC vasculature and stimulation of anti-tumor immunity. Combination treatment inhibited STAT3 activity and increased the expression of CXCL10 chemokine, which increased both tumor penetration of activated CD8 T cells and survival. This concept is highly relevant for the future of development of combination treatment strategies in patients with advanced HCC using drugs (multitargeted TKIs or specific VEGFR and STAT3 inhibitors) with known pharmacology and toxicity profiles. Furthermore, the regorafenib-specific mechanism of action on tumor immune microenvironment is relevant in HCC treatment, and potentially in other cancers that metastasize to the liver.

Figure 1

 1 Figure 1 Dose-dependent effects of regorafenib on vascular normalization when used in combination with programmed cell death protein 1 (PD1) blockade in RIL-175 hepatocellular carcinoma (HCC) model. (A, B) Mice with established (4-5 mm in diameter) HCCs were treated with anti-PD1 therapy (P) alone or in combination with 5 mg/kg (R5), 10 mg/kg (R10) or 20 mg/kg (R20) regorafenib versus control (C) (n=8 mice per group, 1-week treatment). We used immunofluorescence (IF) for evaluation of tumor vessels by CD31 and α-smooth muscle actin (α-SMA) immunostaining. Quantitative analysis of IF data showed increased pericyte coverage only after R10+P treatment (plots in B). Representative IF of tumor sections after staining for CD31 (for endothelial cells in green) and α-SMA (for pericytes in red) and DAPI counterstaining (in blue). Five random fields from the tumor center (defined as no inclusion of the edge of the tumor) were evaluated for each sample. Data represent mean values. Scale bars=500 µm. (C) Representative IF confocal microscopy images of tumor vessels using and CD31 staining (green) and tissue hypoxia using CA-IX staining (red) of whole liver sections; scale bars=2 mm. (D) Tissue hypoxia was significantly reduced by 10 mg/kg regorafenib treatment alone or with anti-PD1 blockade (n=5 mice, 12 days of treatment, 5 images/tumor sample). (E) Representative IF confocal microscopy images of mature tumor vessels using CD31 staining (green) and α-SMA staining (red) of RIL-175 tumors; scale bars, 100 µm. (F) CD31+α-SMA+ mature vessel density was significantly increased in the combination treatment group (n=5 mice, 12 days of treatment, images/tumor sample). Data represent mean±SEM. *P<0.05; **p<0.01; ***p<0.001.

Figure 2

 2 Figure 2 Intermediate dose of regorafenib with antiprogrammed cell death protein 1 (anti-PD1) therapy increases intratumoral CD8+ cytotoxic T lymphocyte (CTL) infiltration and activation in RIL-175 model. (A) Mice with established (4-5 mm in diameter) hepatocellular carcinomas (HCCs) were treated with anti-PD1 therapy (P) alone or in combination with 5 mg/kg (R5), 10 mg/kg (R10) or 20 mg/kg (R20) regorafenib versus control (C) (n=8 mice per group, 1 week treatment). We used immunofluorescence (IF) for evaluation of T-cell infiltration by CD8 immunostaining. Representative IF of tumor sections after staining for CD8 (for T cells in red) and DAPI counterstaining (in blue). Five random fields from the tumor center (defined as no inclusion of the edge of the tumor) were evaluated for each sample. Data represent mean values. Scale bars=500 µm. (B) Quantitative analysis of IF data showed a significant increase in CD8+ T cells after R10+ P treatment compared with all other groups. (C) Regorafenib (10 mg/ kg daily, R10) combined with anti-PD-1 therapy (P) significantly increased the number of CD8+ T cells per gram RIL-175 tumor tissue measured by flow cytometry versus control or R10-treated mice (n=11-13 mice, 12 days of treatment). (D) Representative IF confocal microscopy images of CD8 T-cell distribution (in red) in whole RIL-175 tumor tissue sections; CD31 staining of vessels in green and DAPI counterstaining in blue; scale bars=2 mm. (E) Quantification of intratumoral CTL infiltration in RIL-175 HCCs showing a more substantial increase in intratumoral CTL infiltration in R10/P-treated group, which was significantly higher than each treatment alone or control (n=5 mice, 12 days of treatment, 5 random fields from the tumor center (defined as no inclusion of the edge of the tumor) per sample). (F) R10/P combination therapy significantly increased the number of CD8+ interferon gamma (IFN-γ)+CTLs per gram of RIL-175 tumor tissue measured by flow cytometry versus each treatment alone or control (n=10-13 samples, 12 days of treatment). *P<0.05; **p<0.01; ***p<0.001.

Figure 3

 3 Figure 3 Combined treatment with regorafenib with antiprogrammed cell death protein 1 (anti-PD1) antibodies inhibits signal transducer and activator of transcription (STAT) activation and increases CXCL10 production by hepatocellular carcinoma (HCC) cells. (A) Changes induced by combination of regorafenib (10 mg/kg) with anti-PD1 antibody in immune-related pathways from the gene set enrichment analysis (GSEA) using curated gene sets in RIL-175 HCC model (see also online supplemental dataset S1 and figure S2E). (B, C) Changes induced by combination of regorafenib (10 mg/kg) with anti-PD1 antibody in STAT3 and STAT1 activity (B), showing an increased ratio of phosphorylated (p)-STAT1/p-STAT3 (normalized to total STAT1 and STAT3, respectively) by western blot analysis of whole RIL-175 HCC tissue (C) (n=4 mice). (D) Treatment with regorafenib increases CXCL10 expression by RIL-175 HCC cells in vitro after 36 hours, in a dose-dependent manner (n=6). Culture medium alone was used as a negative control (NC). (E, F) Changes induced by regorafenib (1 µM) in STAT3 and STAT1 activity (E), showing an increased ratio of p-STAT1/p-STAT3 (normalized to total STAT1 and STAT3, respectively) by western blot analysis of RIL-175 HCC cells (F). (G, H) Representative in situ hybridization (ISH) pictures of CXCL10 (red dots, arrow heads) and CXCR3 (green dots) on RIL-175 HCC tissue from the control group (G) versus combination therapy (RP) group (H). Scale bars=50 µm (G, H). (I) Combination therapy significantly increases in the number of HCC cells expressing CXCL10 compared with control. Data represent mean±SEM. (J, K) Combination therapy increases CXCL10 protein expression in whole RIL-175 tumor tissue 6 hours and 12 hours after in vivo when regorafenib treatment was used at doses of 10 mg/kg (R10) and 20 mg/kg (R20) (n=3) by western blot analysis (J); quantitative data of CXCL10/β-actin ratios are shown in (K). C, control; P, anti-PD1 antibody; R, regorafenib 10 mg/kg; R10-P, regorafenib 10 mg/kg+anti-PD1 antibody; n=3-4 per group. *P<0.05; **p<0.01; ***p<0.001.

Figure 4

 4 Figure 4 Intermediate dose of regorafenib shows efficacy when combined with antiprogrammed cell death protein 1 (anti-PD1) blockade in the RIL-175 mouse model of advanced hepatocellular carcinoma (HCC). (A) Example of tumor implantation and treatment schedule for survival studies in HCC models. (B, C) Survival experiment data from RIL-175 orthotopic mouse model. Tumor growth was significantly delayed in the group treated with a combination of regorafenib 10 mg/kg and anti-PD1 antibody (aPD1) compared with the other groups (n=10-13 mice) (B); moreover, this combination significantly prolonged overall survival (C). (D) Survival experiment data from HCA-1 highly metastatic orthotopic mouse model. Mice treated with combination with R10+P show a non-significant trend for increased survival versus control (HR=0.48, p=0.075) (n=11-13 mice). (E) Tumor growth is delayed after combination of R10+P in a spontaneous HCC model using Mst1 -/-Mst2 F/-mice (n=6-9 mice). (F) Treatment with regorafenib (10 mg/kg daily, R10) alone or in combination with anti-PD-1 therapy shows no benefit in survival (Kaplan-Meier survival distributions) compared with control. *P<0.05, **p<0.01, ***p<0.001. C, control; P, anti-PD1; R10, regorafenib 10 mg/kg daily; R10+P, regorafenib 10 mg/kg+anti-PD1. Error bars, SEM.

Figure 5

 5 Figure 5 Increased intratumoral infiltration by CD8+ cytotoxic T lymphocytes (CTLs) after regorafenib plus antiprogrammed cell death protein 1 (anti-PD1) treatment is mediated by CXCL10/CXCR3 axis. (A, B) Representative immunofluorescence (IF) confocal microscopy images of whole liver sections (upper panel, low magnification, scale bars=2 mm; lower panel, high magnification, scale bars=50 µm) showing increased intratumoral localization of CD8+ CTLs co-localized with areas of cell apoptosis in orthotopic RIL-175 tumors in Cxcr3 +/+ /C57Bl/6 mice (upper panel) compared with orthotopic tumors grown in Cxcr3 -/-/ C57Bl/6 mice (lower panel) (A) after 1 week of regorafenib/ anti-PD1 combination treatment. (B) Quantification of IF data showing that intratumoral CTL infiltration after regorafenib/ anti-PD1 combination treatment is prevented when tumors are grown in Cxcr3 -/-/C57Bl/6 mice (n=5 samples, 5 random areas/samples): C, control; R10+P, regorafenib 10 mg/ kg+anti-PD1 antibody. (C) The added survival benefit of regorafenib with anti-PD1 blockade over anti-PD1 therapy alone is compromised in Cxcr3 -/-/C57Bl/6 mice bearing hepatocellular carcinoma (HCC) (n=9-10 mice). C, control; P, anti-PD1 antibody; R10, regorafenib 10 mg/kg; R10+P, regorafenib 10 mg/kg+anti-PD1 antibody. Data represent mean±SEM. *P<0.05; **p<0.01; ***p<0.001.

Figure 6

 6 Figure 6 CXCL10 expression of in hepatocellular carcinoma (HCC) tissues and concentration in blood samples from patients with cancer. (A-C) CXCL10 expression by RNA-in situ hybridization (ISH) in human HCC samples; staining was scores 'high' (13%-17%) (A), medium (1%-11%) (B) or low (<1%) (C) fractions of HCC tumor cells with CXCL10 ISH positivity. Scale bars=500 µm. (D) Distribution of CXCL10 mRNA counts using ISH. (D) Concentration of CXCL10 in blood samples collected from HCC (n=20) and acute myeloid leukemia (AML) (n=8) patients prior to treatment. Detectable CXCL10 levels in all patients, with no significant difference between the two groups. (F) Increase of CXCL10 after 2 weeks of regorafenib treatment in patients with HCC (n=1) and AML (n=7). (G) Schematic diagram of the proposed mechanisms of benefit when using the appropriate dose of regorafenib combined with antiprogrammed cell death protein 1 (anti-PD1) therapy. Judicious dosing of regorafenib in combination with anti-PD1 antibodies can normalize HCC vessels, which results in increased drug delivery and synergy with antitumor immunity stimulated by concomitant anti-PD1 therapy. The increased CD8+ cytotoxic T lymphocyte (CTL) penetration and activation mediates this synergistic activity and is facilitated by CXCL10 expression by the cancer cells on inhibition of STAT3 phosphorylation by regorafenib.

Shigeta K, et al. J Immunother Cancer 2020;8:e001435. doi:10.1136/jitc-2020-001435

Acknowledgements The authors would like to thank Mark Duquette, Anna Khachatryan, Sylvie Roberge and Alexandra Florin for outstanding technical support.

Contributors KS, AM and HK contributed equally: they designed and performed experiments, analyzed the data and wrote the manuscript. SK, EM, AQ, KS, SA, KI, RRR, DS, TH and AS performed experiments and analyzed the data. IC and KS analyzed the RNA sequencing data. LF, DZ and KS performed the PK/PD studies and analyzed the data. SD, IP and PH provided reagents and analyzed the data. MC, LLM and RKJ analyzed the data, provided reagents and edited the manuscript. LG and AXZ provided reagents and edited the manuscript. DGD designed the experiments, analyzed the data, obtained funding and wrote the manuscript. All authors approved the final version of the manuscript.

Funding This study was supported by sponsored research agreements with Bayer Pharmaceuticals (to DGD), which also provided the study drug. DGD's work was also supported through NIH grants P01-CA080124 (to RKJ and DGD), R41-CA213678 and Proton Beam/Federal Share Program (to DGD). RKJ's work was supported through NIH grants R35-CA197743, R01-CA208205 and U01-CA224173, and by the National Foundation for Cancer Research, Harvard Ludwig Cancer Center, Jane's Trust Foundation and Advanced Medical Research Foundation. SD and IP were supported by a Norway-Romania joint project (grant 4SEE P4/30.06.2014). KS received a Postdoctoral Fellowship from Uehara Memorial Foundation, TH received a Postdoctoral Fellowship from Astellas Foundation for Research on Metabolic Disorders, Japan and EM received a grant from the Philippe Foundation and the Cancéropôle PACA.

Disclaimer The content is solely the responsibility of the authors and does not represent the official views of Harvard University and its affiliated academic healthcare centers, of Bayer Pharma or of the NIH.

Competing interests

LG reports personal fees from Agios Pharmaceuticals, Alentis Therapeutics, QED Therapeutics, H3 Biomedicine, Taiho Pharmaceuticals, Debiopharm, Incyte Corporation, SIRTEX and AstraZeneca. AXZ is a consultant/ advisory board member for Bayer. RKJ received honorarium from Amgen and Open access consultant fees from Chugai, Ophthotech, Merck, SPARC, SynDevRx and XTuit. Dr Jain owns equity in XTuit, Enlight, SPARC, SynDevRx and Accurius Therapeutics and serves as a paid member of the boards of trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund and Tekla World Healthcare Fund. He is a member of the scientific advisory board of Accurius Therapeutics. DZ, LF and the spouse of LLM are Bayer employees. MC is an AstraZeneca employee. DGD received consultant fees from Bayer, Simcere, Surface Oncology and Bristol Myers Squibb, and research grants from Bayer, Exelixis and Bristol Myers Squibb. No potential conflicts of interest were disclosed by other authors.

Patient consent for publication Not required.

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement Data are available on reasonable request from the corresponding author DGD. The authors used deidentified participant data at Fundeni Clinical Institute, Bucharest, Romania and Massachusetts General Hospital, Boston, USA.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.


Alain Marcati
email: alain.marcati@ensiacet.fr

Laurent Prat
email: laurent.prat@ensiacet.fr

Christophe A Serra
email: serrac@ecpm.u-strasbg.fr

Josiane Tasselli

Pascal Dubreuil

J Tasseli

Fast Built and Designed Microdevices for Early-Stage Liquid-Liquid System Studies

come   L'archive ouverte pluridisciplinaire

Introduction

For chemical engineering, controlling liquid-liquid flows in a microchannel is a major challenge. One main application for this kind of systems is to acquire data to identify kinetic [1], thermal [2] or mass transfer [3] models. Furthermore, the microscale is also directly used to generate objects at a controlled dimension. Applications can be found in crystallization [4] or polymerization [5]. For the experimentation, first stage is to develop a microfluidic device design which often presents different parts in terms of operations (mixing, droplets generation, residence time for the reaction activation, phases separation). A lot of iterations between design and manufacturing could be needed to obtain a complete operating device. It is then of interest to be able to generate fast built and cheap systems to explore the earlystage designs.

Liquid-liquid flows in microchannel are governed by many parameters such as the nature of the fluids, the injection mode, the geometry and the surface characteristics of the channels. As interfacial effects are a key factor, lots of works have been made on injection geometry and different materials have been used for microfluidic devices. Many regimes of droplet generation have been studied by using microfluidic flow focusing in twodimension [6]. Regular flows are obtained with simple cross-junction [7]. T-junction is also used to form droplets [8]. Water-in-oil emulsions are more often studied than oil-in-water in these devices except in microchannel emulsions [9]. These different geometries are developed to increase the shear rate on the dispersed phase. But, the interactions between liquids and walls are also an important parameter for the droplet generation. In particular, the continuous phase should be wetting the surface and the dispersed one non-wetting [10]. Intrinsic fluid properties such as viscosity, density or interfacial tension and flow conditions are responsible of the droplet break-up. To explain this phenomenon, investigations have been made on dimensionless numbers: Capillary, Reynolds or Weber numbers [11][12][13]. It is possible to modify these fluids properties and help droplet generation and stabilization with the use of surfactants or viscous additives [14,15]. Different techniques of wall treatment can also be applied on surfaces to make them hydrophilic or hydrophobic: siliconization, silanization or polymer coating for glass or silicon [16]; UV/ozone treatment for elastomers [17].But the simpler is to adapt material to the kind of emulsions needed. Consequently, manufacturing techniques are often adapted to the material used. Plastic (PDMS, PU, PMMA…) devices are made by photolithography [18]. A silicon mask with the desired design is first created. Then polymer is moulded on this mask and cured with temperature; a flat polymer layer is then sealed to the polymer replica by plasma oxidation. PDMS is a cheap material and allows rapid prototyping by replica moulding but it is not suitable for oil-in-water droplets. Although PDMS can be treated to become hydrophilic, it is not long-term available [19,20]. Silicon or glass devices are fabricated in two main steps: channels formation by dry [21] or wet [22] etching, device capping by anodic or diffusion bonding [START_REF] Enikov | Microsystems Mechanical Design[END_REF]. Glass or silicon microstructuring is a reproducible and reliable technique but it requires specific equipment and use restrictive operating conditions. Glass and silicon devices are suitable for oil-in water droplets generation but are very expensive. Based on the work of Hisamoto [START_REF] Hisamoto | [END_REF] that embedded functionalized capillaries in a PDMS network for analytical purposes, we propose to use untreated fused silica capillaries embedded in Plexiglas plates to generate oil-in-water droplets. Nisisako [16] used PMMA milled plates to generate water in oil droplets and Xu [14] added a quartzose capillary in the PMMA network to provide aqueous phase. In order to approach silicon-glass devices, the droplets flow directly in the fused silica capillaries, Plexiglas plates are just a support for capillaries.

This paper proposes a comparison between two different microfluidic devices: a silicon-glass microchip and a device based on fused silica capillaries assembly. The objectives of this paper are (i) to describe a fast technique to build capillaries based microsystems; (ii) to present results obtained with this device in term of droplets generation, (iii) to show that such a device can be used as a precursor of silicon-glass devices.

Device Manufacturing

Silicon-glass device

Silicon-glass devices are fabricated with two major steps: plasma etching to define both the microchannel and the inlet and outlet holes through the silicon wafer and anodic bonding to seal the channel with the glass wafer. Deep Reactive Ion Etching (DRIE) is commonly used for the fabrication of silicon microstructures when high aspect ratios up to 30:1 and vertical sidewalls are required. For the fabrication of a microchannel with a precise depth, a single photoresist layer is usually necessary to mask the silicon wafer during etching. For the microstructuration of microchannels with various depths on a same silicon wafer, it is necessary to use more complex masking layers. For our application, we needed to etch channels of 10µm, 60µm and 100µm depth, the two smallest emerging into the widest one. To do that we used a technological process developed at IMM [21] (Huber 2003 et al). This process necessitates to deposit four masking layers (thermal and PECVD silicon oxides SiO 2 , a chromium layer and AZ4562 photoresist layer), to perform three photolithographies, three dry etching of the oxides, two wet etching and three deep silicon etching. A schematized description is reported on figure 1. The different steps are described here: after deposit of a 0.5µm thermal oxide on the silicon wafer, the three channels are patterned; deposit of 0.1µm of evaporated chromium layer followed by 1µm PECVD silicon oxide (figure 1a); after the 60µm and 100µm channels opening by PECVD SiO 2 dry etching, a first DRIE of 40µm-depth is performed (figure 1b); after AZ4562 resist removing, a second DRIE of 50µm-depth is made; after PECVD SiO2 and chromium removing, the last DRIE of 10µmdepth is performed (figure 1c). As a result all the channels are structured.

Figure 2 shows a MEB view of a 60µm wide and deep silicon channel emerging into a 100µm wide and deep channel. There are two possibilities to define the inlet and outlet of the channels: either perform holes through the cap glass wafer by drilling, or perform through wafer holes into the silicon wafer by DRIE. The first method is easier but not appropriate for glass wafers of 500µm-thick. So we opted for dry etching of holes of 1mm-diameter through silicon. At the end of the process the glass wafer (Pyrex 7740 ® from Corning) and the structured silicon wafer are sealed by anodic bonding.

Capillary device

In this device, capillaries are imprisoned and glued in two Plexiglas support plates. The lower plate is, first etched thanks to a computer numerical controlled milling machine and a micro drill which width is closed to the outer diameter of capillaries. T or cross junctions, as generating areas, are then created on the centre of the plate by computer-aided design. Additional channels of larger dimension are machined far around the generating area: they will receive the excess of glue and prevent the clogging of the junction while capping the device. The upper plate is flat and provides confinement to the device. Capillary tube lengths (at least 20 centimeters) are adjusted to that of the channels; an additional length is needed to make the connections with the syringes and prevent capillary breaking when bending is important. The cutting has to be straight which is achievable with a simple ceramic cutter; 100µm 60µm 10µm 60µm 60µm 100µm imperfect cutting can play a negative role on the droplet generation. It is checked with a microscope before using the capillary in the device. For each tube, in order to eliminate the protective coating, the side used for the generating zone is stripped by burning and cleaned with ethanol. This provides a window where phenomena can be observed. The setting of the capillaries in the channels is described on figure 3. It starts with the lateral one (figure 1a). Its outlet reaches the end of the etched channel. Once the positioning is right, quick setting glue is used to fix the tube with an adhesive dot on the edge of the plate.

The first longitudinal tube is then added to the device (figure 1b). The end of the tube coincides with the output of the dispersed-phase tube. The walls of the respective tubes can touch each other. Once the tube is precisely set, another adhesive dot freezes the distance between the two outlets. The last tube is finally set on the plate (figure 1c). In order to have a symmetrical device, the main and the continuous phase tube are at the same distance from the centre of the dispersed phase one. As all the capillaries are fixed to the plate, a layer of low setting glue is spread in the channels (except in the drop generating site or the enlargement area to avoid obstructing the tubes) in order to decrease the dead-volumes. The glue should not overpass the channels. During the time the glue is thickening, it is possible to adjust the placement of the capillaries tubes laterally to make them touch each other. Precision of the T is strongly dependant on that of the capillary cutting. When the glue is hardened, the cap is ready to be fixed. Its dimensions are slightly inferior to the lower plate to favour the bonding. The gluing process is described on figure 4. The same low setting glue is used: a few clusters are set all around the larger channels. The cap is settled and both plates are pressed against each other by hand. The pressure applied has to make the glue spread in the larger channels and around the T but not reach the generating site. The pressure is kept a few minutes until the glue is starting to harden. It is then, possible to add a glue layer all around the junctions of the two plates to increase the tightness of the device. The complete manufacturing takes less than a day before starting experiments. The key step of the fabrication is the cap bonding: there must be enough glue to bring tightness to the device but glue has to be cautiously spread to avoid capillary clogging. Figure 4 presents two examples of devices built following this protocol with different generating junctions: T (a) or Cross (b) By using the same gluing technique, it is possible to connect capillaries. An enlargement (respectively a reduction) of the main tube diameter can be made by adding a second capillary of different diameter end to end with it (figure 5a). This junction requires a good precision of capillary cutting and also needs confinement so it has to locate between the two Plexiglas plates. As it is difficult to handle with short-length capillaries, the contact is then made between the larger channel and the edge of the plate.

The enlargement can be useful to transform a plug flow to a droplet flow In order to connect different devices made of capillaries, a larger capillary can be used. Standard capillary is 360 microns outer diameter. If 360 microns capillaries are stripped of their coating, they can be inserted into a larger capillary with an inner diameter of 320 microns. Only a few centimetres of larger capillary are needed and it is better if the both small capillaries are inserted symmetrically as shown on figure 5b. Once both small capillaries are put in contact they are fixed with the larger one with two dots of quick setting glue.

The capillary technique presented here does not pretend to be completely reproducible. Capillaries are indeed cut and placed in the channels by hand, it is thus nearly impossible to reproduce exactly the same device twice.

Another key point is the reliability of such devices; a clear attention has to be paid to which solvents are used in these devices: for example, acetone will damage Plexiglas plates. Some solvents may dissolve the glue used to seal the device or create block connections. The commercial glues used for the fabrication are resistant to temperature in the range of -30 to 70°C and Plexiglas can afford up to 80°C. If extended temperature range (up to 350°C) is needed, it is possible to thermostat the capillary at the outside of the device. In terms of mechanical resistance, the capillary device can be fed at 10

(b) (a)

ml.h -1 with 100 microns square section capillaries on a length of 50 centimeters, which corresponds roughly to 4,5 bars, the devices may then work without leakage up to 5 bars. The fabrication of such devices is pretty fast and it only uses cheap materials. As a wide collection of capillaries is available through the market, the capillary device is then modular: with the same milled plates, it is possible to produce many different devices with varying inner diameters; it is also possible to change the diameter of the mill to adapt it to the capillary outer diameter. The connection technique presented above extends further the possibilities of such devices beyond the simple droplet generation.

Experimental material

The capillary tubes used have either a square section (side 100 µm) or a round section: standard 360 microns outer diameter and larger ones. They are made of fused silica and coated with a protective polyimide layer. The quick setting glue is cyanolite glue. The low setting one is a bi-component araldite. The plates are made of Lexan Plexiglas or polycarbonate. The fluids are stocked into 1 to 20 ml syringes placed on syringe pumps. They are injected at constant flow through polyethylene tubing. The connection with the capillaries is then assured with fittings without dead-volumes (figure 4b). To protect Si-Glass microreactors and make the connection with syringes, a case has been designed. The case is composed of two Plexiglas round plates maintained by screws. The inferior plate is bored and threaded: each hole corresponds to each input or output of the microreactor. The same polyethylene tubing is then used to connect syringes to headless nanoports mounted with viton joints. These nanoports are screwed in the inferior plates until the microreactor touches the upper plate. Openings are made on the upper plate in order to check if the microreactor is well-centred on the case. Viton joints bring the tightness to the system. The dispersed phase is silicone oil (20 cSt viscosity) and the continuous phase is demineralised water. As the channels have a small width and consequently, the droplets velocities are very high, the observation is made with a fast CCD Camera coupled with a microscope (Nikon SMZ-10). Images acquisition rate is 1800 frames per second. Exposition time (0.55 ms) is chosen in order to have enough light for contrast distinction and short exposition for the droplets edges sharpness. A 100W spot lamp is used to provide necessary light. The images are acquired with transmission light at the exit of the capillary device whereas they are acquired just after droplet generation with reflexion light with Si-Glass microreactors.

Results and discussion

This part describes results obtained with silicon-glass microreactors and capillary devices while generating oil droplets in water. Both devices are compared and an empirical model of droplet size is proposed.

Silicon-glass device

First fluid admittance in the Si-Glass microreactors can be difficult, so before first use, they are kept in water for a few hours. Because of microscope limitations and difficulty to introduce fluids, it was difficult to handle with 10 microns microchannels, then only reactors with 60 microns and 100 microns microchannels are presented in this article. Before starting droplets generation, it was important to determine the range of flow rates in each device. In order to find these operating conditions, the oil flow rate (F o ) is set at 100 µL.h -1 , while water flow (F w ) rate is changed. For 60-microns microchannels, oil flow rate varies from 10 to 200 µL.h -1 and water flow rate from 100 to 1500 µL.h -1 . For 100-microns microchannels, oil flow rate is set between 50 to 500 µL.h -1 while water flow rate between 500 to 4000 µL.h -1 . More important water flow rates can be delivered through the channels but there is a strong observation limitation due to reflection light. At high flow rates, the interface between water and oil becomes less and less sharp up to having difficulty to distinguish oil droplets. At first use, flow becomes stable after 15-20 minutes and slug regime is observed. The time of flow stabilization is lower when just a parameter is changed. Frames are acquired just after the generation site. Slugs appear to be monodisperse (slug length variation < 5%). As expected, when the water flow rate increases, the droplet size decreases and the distance between droplets increases. Dispersed phase flow rate has also an impact on slugs' length. Figure 6 presents data on 60x60 microchannels: reducing oil flow rate from 50 to 10 µL.h -1 involves a 10% decrease of slug length. Whatever the size of the channels, a potency law fits well the plug length in function of water flow or either Ca number. The exponent is within de range of 0.15 to 0.25 whatever the conditions or the channels' diameter. The Si-Glass microreactors have a good reproducibility. Figure 7 shows the data obtained for two different plates at the same water and oil (100 µL.h -1 ) flow rates. The difference on the slug lengths and slug velocities is pretty thin.

Figure 8 Reproducibility of droplet generation on different microreactors in terms of slug flow and velocity (upper graph)

The upper graph of the figure represents the measured velocity function of the imposed superficial velocity. The different measured velocities are very close to the imposed one (dotted line); it seems then that there is no slippery of the slug in the continuous phase. On the 100x100 microchannels, it is possible to test the influence of the dispersed phase channel diameter on the slug length. Data are shown on figure 8. Results are pretty interesting because for the same oil flow rate there is a slug length decrease of 20% by changing a 100 microns channel to a 60 microns. Distance between slugs is also reduced while using a 60 microns channel. Si-Glass microreactors are very simple of use and provide interesting results and good reproducibility. Nevertheless, they are pretty expensive and can be long to fabricate. It can then be interesting to have reactors much simple to produce and with comparable results in order to make preliminary tests and design conception. As fused silica is closed to glass and silicon, capillaries devices have been constructed on this purpose.

Capillary device

Before starting droplets generation, the air imprisoned in the device while bonding the plates, has to be removed. The presence of air in the device can block the flow or provokes serious turbulences. The aim of this article is not to present a detailed flow chart but to show the feasibility of droplets generation in this kind of device. As capillaries have the same diameter and square section, the same operating conditions are used. Despite the important dead-volumes, oil does not seem to enter in contact with the walls of the plates. A thin oil line follows the water flow line until the inlet of the output tube where the droplet break-up takes place. The water wets well the capillary walls while oil is relatively non-wetting. The flow becomes stable after a few minutes and droplets are monodisperse (variation < 5%). The measures are at constant oil flow rate (F o = 200 µL.h -1 ) and water flow rate is varied from F w = 500 µL.h -1 to F w = 4000 µL.h -1 . As expected, when the water flow rate increases, the droplet size decreases and the distance between droplets increases. It is noticeable on the frames of figure 9. The range of slug lengths decreases from 185 µm to 125 µm. An increment in water flow rate may further reduce droplet length but it also reduces the sharpness of the droplets edge and makes difficult the length determination. In terms of droplets velocities, the range is from 20 to 120 mm.s -1 . In this device also, there does not seem to be a droplet slip velocity by comparison with the applied velocities.

Devices Comparison

The capillary used for the hand-made devices have a 100 microns square section. The dimensions are thus comparable to a 100 microns microchannel on the silicon-glass microreactor. A comparison of the slug length is presented on figure 11: a T junction is used in both cases for the droplet generation. A variation of 30 to 40 microns slug length is noticeable between the capillary device and the siliconglass chip. The difference seems to reduce at higher flow rate. Nevertheless both curves have quite the same shape. As the same flow rates, injection mode and diameter are used, it seems that this difference is due to the material. The other difference between both devices is the presence of dead-volumes around the generation area (figure 5a), but their main effect is to slightly reduce the shear rate of the continuous phase on the dispersed phase. Dead volumes should consequently have a negative impact on rather than helping the droplet generation.

Empirical model

Based on the precedent considerations, main parameters influencing the droplet generation [14,15,25] are listed: channels geometry, intrinsic fluid properties and flow rates. As it is easier to handle with dimensionless numbers, slug length is divided by the hydraulic diameter. Dimensionless numbers: Capillary number, that measures the magnitude of viscous to surface tension forces and Reynolds number, that measures the magnitude of inertial to viscous forces, are also worth entering the model. A potential law is used as it fits well the experimental data (figure 4). The resulting model is developed on equation 1.

ε δ γ β α 2 1 2 1 2 1 1 Re Re × × × ×         × = Ca Ca d d A d L (1)
L is the length of the plug and d the hydraulic diameter of the channel. The capillary number is written Ca = µQ/Sσ with µ as the viscosity of the fluid, Q the flow rate, S the section of the channel, σ the interfacial tension between continuous and dispersed phases and Reynolds number Re = ρQd/Sµ with ρ the density of the fluid. A, α, β, γ, δ, and ε are constants. 1 refers to the continuous phase, 2 to the dispersed phase.

The model has been applied to the data presented previously on Si-Glass and capillary devices. The model parameters are identified by fitting the experimental values. Results are shown on table 1. The comparison of the models and the data is represented on figure 12. Experimental data are placed on abscissa and calculated values on ordinate. The straight line symbolizes the perfect correspondence between models and experimental data; dotted lines a deviation of 10%. Calculated values for Si-glass devices (black squares) and capillary devices (grey dots) are closed to the experimental data. Models can thus be validated.

Figure 12 Comparison of the model with the data gathered for the capillary device ( ) and the silicon-glass chip (▪) in the range of slug aspect ratio between 1.1 and to 2.2

The only difference between the two models is on the constant A: it is higher for the Si-Glass device and this constant has approximately the same relative deviation than the variation of slug length in favour of capillary devices noticeable on figure 11. The exponents are common to both devices. This proves that is possible to build a model that could characterize and make the link between two microdevices using the same geometry and same fluids but different materials. This similarity between the two devices is interesting: it offers the possibility to make preliminary tests on a capillary device before designing a silicon glass microchip. Capillary devices are indeed far less cost effective and faster to build. They can provide for example, data on the design or the residence time needed for special applications. The silicon glass device, more reproducible, will then be directly designed and functional for the desired application.

With the capillary device, it is also possible to connect different capillary blocks in order to apply further actions on droplets or provide more residence time. The connection described on figure 3b has been tested and three different blocks have been connected without leakage or perturbation on the twophase flow.

Conclusion

This paper presents two different techniques to create microfluidic devices. Both devices are used to generate oil-in-water droplets without surfactant or wall treatment and present comparable results in terms of droplet size and velocity. The first device is a silicon glass microchip obtained by dry etching and anodic bonding. Its fabrication is complex and requires specific equipment and is cost effective but is very precise and reproducible. On the other hand, a simple and rather fast technique is used to create a device based on fused silica capillaries, Plexiglas plates and commercial glues. This device has the advantage to be cheap and modular. It is then a practical tool to make a preliminary study on droplet generation before designing a more complex device. As connections are possible between capillary devices, this droplet generator can be considered as the first operating unit in a chemical engineering approach. The connection between blocks will provide further microfluidic units such as reactive chamber, residence time or separation systems. Thanks to the similarity of both devices, the capillary device can be defined as a precursor of silicon-glass chips

Figure 1

 1 Figure 1 Cross-sectional schematic view of the multilevel silicon depths and widths process. (a) Patterning of the 10µm, 60µm and 100µm-wide channels. (b) First 40µm-deep etching for the 100µm-wide channel. (c) DRIE etching of 50µm and 10µm-deep respectively to obtain the three desired channel depths.

Figure 2

 2 Figure 2 MEB view of a 60µm wide and deep silicon channel emerging into a 100µm wide and deep channel.

Figure 3

 3 Figure 3 Steps of capillary placement in the etched channels for a T-junction: (a) dispersed phase supply, (b) continuous phase supply, (c) two-phase flow capillary. Capillaries are in full lines and channesl walls in dotted lines

Figure 4

 4 Figure 4 Schematic view of the sealing process: when pressing both plates, the glue (grey) spreads out of the cap (bold square) and into the larger channel (dotted lines) preserving the T-Junction.

Figure 5

 5 Figure 5 Examples of devices built: (a) T junction, (b) cross junction. On the bottom right, there is a zoom on the generation area

Figure 6

 6 Figure 6 Examples of connections between capillaries: (a) diameter enlargement, (b) 2-block connection

Figure 7

 7 Figure 7 Slug length function of water flow rate at different oil flow rates: 50 µL.h -1 ( ) and 10 µL.h -1 (-)

Figure 9

 9 Figure 9Slug length function of water flow rate at the same oil flow rates (100 µL.h -1 ) but with different dispersed channel size: 100 microns ( ) and 60 microns(-)

Figure 10

 10 Figure 10 (a) Slug length function of water flow rate at constant oil flow rate (200 µL.h -1 ), slug velocity function of superficial velocity (upper graph) and flow visualisation at different water flow rates (b).

Figure 11

 11 Figure 11 Slug length in function of water flow rate at the same oil flow rates (200 µL.h -1 ), injection mode (T junction) and channel size (100 microns) for the capillary device ( ) and the silicon-glass chip (▪)

Table 1

 1 Constants of the model obtained for a T junction in capillary and Si-Glass devices.

Acknowledgement

Authors acknowledge the French Ministry of Education and Research for having funded this work through the grant ANR n° NT05-1_45715.