Stroma NCL inhibition increases pericyte coverage of tumor blood vessels.

mPDAC orthopic model characterization.

N6L inhibits PDAC proliferation and migration

Supplemental Figure 3. A) iRGD-TAMRA (red) binding after 15 min at 4 degree in normal cells (Low integrins-NRP1) and in human and mice PDAC cells (High integrins-NRP1). Scale bars 200μm B) qRT-PCR of miR-21-5p expression after treatment of PANC1, BxPC3 and PL-45 cells with TPN-21 at the dose of 100nM after 48h. C) Representation of 3D-model treatment course with TPN-21. For qPCR each miRNA sample was normalized on the basis of its 18s content. Error bars, mean {plus minus} s.d *P = 0.01-0.05; **P = 0.001-0.01; ***P < 0.001; ****P < 0.0001 N.S., not significant, two-tailed t-test; n = 3 biological replicates.

Abstract Purpose: Since drug responses vary between patients, it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. In this study, we tested whether RNA-based therapeutics were suitable for personalized medicine by using patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models. Experimental Design: We performed microRNA (miRNA) profiling of PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients. To deliver personalized RNA-based-therapy targeting oncogenic miRNAs that form part of this common PDAC miRNA over-expression signature, we packaged antimiR oligonucleotides against one of these miRNAs in tumor-penetrating nanocomplexes (TPN) targeting cell surface proteins on PDAC tumors. Results: As a validation for our pre-clinical strategy, the therapeutic potential of one of our nano-drugs, TPN-21, was first shown to decrease tumor cell growth and survival in PDO avatars for individual patients, then in their PDX avatars. Conclusions: This general approach appears suitable for co-clinical validation of personalized RNA medicine and paves the way to prospectively identify patients with eligible miRNA profiles for personalized RNA-based therapy. Clin Cancer Res; 24(7); 1734–47. ©2018 AACR.

Supplemental Figure 2. A) Relative expression Left panel: Mean of fluorescence and right panel: qPCR validation of miR-21-5p expression level in the set of cell lines, 3D-models and organoid compared to normal controls. B) qPCR analysis of PDCD4 and PTEN expression levels in the PANC1 stable cell lines (Lenti-21) inhibiting miR- 21 activity (pLenti-III-miR-off). C) qRT-PCR of miR-21-5p expression after lipofectamine transfection of PANC1, BxPC3 and PL-45 cells with anti-miR-21 inhibitor (mirVana ®) at the dose of 50nM after 48h. For qPCR each miRNA sample was normalized on the basis of it's 18s content and on the basis of GAPDH for mRNAs.

NCL blocking antibody and Ncl deletion affects PDAC cell proliferation.

Supplemental Figure 1. A) Left panel: Visualization of the miRNA profile of normal pancreas samples (n=3 control). Right panel: Visualization of the miRNA profile of PDAC PDX tumor samples (n=27 cases). Relative expression is shown as mean of fluorescence (MFI). miRNAs profiling was done through Firefly Circulating miRNA Assay and normalized using two miRNAs that are not significantly deregulated between all samples miR-22-3p and miR-30b-5p defined by the geNorm-like 37 algorithm. B) Visualization of miRNA profiles of PDAC cell lines (n=4), PDAC 3Dmodel (n=2) and Patient-derived-organoid (PDO 286 and PDO 281) compared to normal pancreatic cell and organoid (n=2 control). miRNAs profiling was done via Firefly Circulating miRNA Assay and normalized using miR-181b-5p, miR-103-3p, miR-30b-5p defined by the geNorm-like algorithm. Normalized miRNA signal intensities are presented as fold-change (log10 of the ratio between a probe value to the average of all the other samples for that probe). Green represents highly expressed miRNAs and red represents lowly expressed miRNAs.

Supplemental Figure 4. A) qPCR analysis of miR-21-5p expression level after 4 treatments with TPN-21 in PDO 286. B) Relative tumor burden after (4 I.V injection of PBS (n=6) TPN-control n=6 or TPN-21 n=5 (5mg/kg). For qPCR each miRNA sample was normalized on the basis of its 18s content.

Proteome array analysis of angiogenic molecules secreted by ECs.

Supplemental table 1A List of miRNAs, miRBase accession number and mature sequence of 46 selected-miRNAs used to build the Firefly 46plex circulating custom panel. There miRNAs have previously been linked to pancreatic cancer or to KRAS.

Supplementary Materials and Methods

Supplemental Figure 5. A) Representatives PDO 286 images and metabolic activity measured from MTT assay after repeated TPN-21 or gemcitabine treatments (100nM) or both treatments. Scale bars 1000μm. B) Quantification of organoid size after treatments. Organoids size was measure through ImageJ software on a total of ⩾ 35 spheroids and are presented in arbitrary units (UA). Error bars, mean {plus minus} s.d *P = 0.01- 0.05; **P = 0.001-0.01; ***P < 0.001; ****P < 0.0001 N.S., not significant, two-tailed ttest; n = 3 biological replicates.

Supplemental table 1B List of PDX pancreatic samples. Information regarding KRAS-mutation and gemcitabine resistance profile. S for sensitive, R for Resistant.

AbstractPurpose: miRNA-155 is an oncogenic miRNA highly expressed in B-cell malignancies, particularly in the non–germinal center B-cell or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL), where it is considered a potential diagnostic and prognostic biomarker. Thus, miR-155 inhibition represents an important therapeutic strategy for B-cell lymphomas. In this study, we tested the efficacy and pharmacodynamic activity of an oligonucleotide inhibitor of miR-155, cobomarsen, in ABC-DLBCL cell lines and in corresponding xenograft mouse models. In addition, we assessed the therapeutic efficacy and safety of cobomarsen in a patient diagnosed with aggressive ABC-DLBCL. Experimental Design: Preclinical studies included the delivery of cobomarsen to highly miR-155–expressing ABC-DLBCL cell lines to assess any phenotypic changes, as well as intravenous injections of cobomarsen in NSG mice carrying ABC-DLBCL xenografts, to study tumor growth and pharmacodynamics of the compound over time. To begin to test its safety and therapeutic efficacy, a patient was recruited who underwent five cycles of cobomarsen treatment. Results: Cobomarsen decreased cell proliferation and induced apoptosis in ABC-DLBCL cell lines. Intravenous administration of cobomarsen in a xenograft NSG mouse model of ABC-DLBCL reduced tumor volume, triggered apoptosis, and derepressed direct miR-155 target genes. Finally, the compound reduced and stabilized tumor growth without any toxic effects for the patient. Conclusions: Our findings support the potential therapeutic application of cobomarsen in ABC-DLBCL and other types of lymphoma with elevated miR-155 expression.

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules.

Retinal vascular diseases (RVD) have been identified as a major cause of blindness worldwide. These pathologies, including the wet form of age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy are currently treated by intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents. However, repeated intravitreal injections can lead to ocular complications and resistance to these treatments. Thus, there is a need to find new targeted therapies. Nucleolin regulates the endothelial cell (EC) activation and angiogenesis. In previous studies, we designed a pseudopeptide, N6L, that binds the nucleolin and blocks the tumor angiogenesis. In this study, the effect of N6L was investigated in two experimental models of retinopathies including oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). We found that in mouse OIR, intraperitoneal injection of N6L is delivered to activated ECs and induced a 50% reduction of pathological neovascularization. The anti-angiogenic effect of N6L has been tested in CNV model in which the systemic injection of N6L induced a 33% reduction of angiogenesis. This effect is comparable to those obtained with VEGF-trap, a standard of care drug for RVD. Interestingly, with preventive and curative treatments, neoangiogenesis is inhibited by 59%. Our results have potential interest in the development of new therapies targeting other molecules than VEGF for RVD.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease marked by poor response to virtually all available treatments. Clinical trials of chemo, targeted therapies, and radiation, have resulted in minimal advances, and survival after diagnosis is generally less than a year. In the pursuit of new avenues for therapeutic development for this lethal disease, a number of studies have implicated the role of tumor-promoting microRNAs with altered expression PDAC tissues. Here, using in vitro 3D organoids we explore the strategy of combining photodynamic therapy (PDT) with a novel microRNA therapeutic platform to target miR21, an established onco-miR that targets multiple tumor suppressors. This approach is motivated by recent clinical studies establishing the technical feasibility of light delivery to the pancreas, combined by evidence that PDT may disrupt or loosen stroma and potentially increase delivery of anti-miR agents. To evaluate this hypothesis, we use in vitro co-culture models of PDAC cells and stromal fibroblasts that recapitulate the dense fibrotic stroma of PDAC tumors to evaluate response to treatment in verteporfin PDT and anti-miR combination and monotherapy treatment arms. Using stably-transfected miR-21 sponge expressing we also perform mechanistic studies to investigate the role of miR 21 in response to PDT and chemotherapy in 3D cultures with and without stromal partners.