Dottorato in Fisiopatologia Medica

Contatti

Supervisore

Benedetta Bussolati

Attività di ricerca

Project

Inhibition of tumor angiogenesis by human liver stem cells-derived extracellular vesicles (HLSC-EVs) loaded with anti-tumorigenic and anti-angiogenic miRNAs

1) AIM OF THE PROJECT

The aim of the present project is to select microRNAs which can be shuttled within HLSC-EVs in order to inhibit angiogenic potential of tumor endothelial cells (TECs) and tumor growth.

 2) BACKGROUND

2.0 EXTRACELLULAR VESCICLES AND CANCER

Extra-cellular vescicles (EVs) are small-membrane bound vescicles (50 nm-1um), released by prokaryots, eukaryots and plants. In the past decade, they emerged as a new mechanism of cell-to-cell communication, due to their capability to transfer proteins, lipids and nucleic acids to target cells.

Generally EVs are secreted by all types of cells in basal condition; however, their production is higher in specific cell types (stem cells, progenitor cells and cancer cells) and may increase during cell proliferation and activation, or under stress conditions [1]. Since they are vehicles for cross-talk between cells, they can influence several physiological and pathological functions of both recipient and parent cells. They target specific cells bounding to surface receptors or bioactive lipids, and they deliver their cargo into them. They can release genetic material like transcription factors, DNA and extra-cellular secreted RNA (exRNA), including long-non-coding RNA, mRNA and miRNA that allows them to produce epigenetic changes in target cells both locally and systemically [2]. They have unique molecular profiles acquired from originating cells and they are present in a number of body fluids, including blood and urine. Cancer-derived EVs play critical roles in tumorigenesis: they can activate all cellular mechanisms modified in cancer, such as cell proliferation, migration, invasion, apoptosis and angiogenesis. They can induce metastasis, evasion of host immune defense, chemo-resistance, and promote a pre-metastatic niche promoting tumor formation [3]. On the other hand, EVs secreted by normal cells have their innate therapeutic potential: several studies [2] have previously demonstrated that EVs released by stem cells or progenitor cells may deliver informations to target cells on tissue regeneration or on immune response modulation. Thereby, they can potentially modulate the phenotype of injured tissues, promoting tissue regeneration and differentiation. Several studies have shown an anti-cancer and an anti-angiogenic potential of stem cells-derived EVs, based on the delivery of their miRNAs into target cells. In these studies, a number of miRNAs with anti-tumor and anti-angiogenic properties have been described: let-7 family (et-7a-1, 7a-2, 7a-3, 7b, 7c, 7d, 7e, 7f-1, 7f-2, 7g, 7i, mir-98, and mir-202), miRNA200 family, miRNA15-16 cluster, and miRNA 451 [4,5].

Camussi et al. [6] observed that EVs from human bone-marrow mesenchimal stem cells (MSC) are able to promote the recovery of acute kidney injury (AKI) and cronic kidney disease. A renoprotective action has been described for EVs derived from endothelial progenitor cells (EPC) in a model of ischemia-reperfusion injury, characterized by diffuse endothelial and tubular cell damage. EPC-derived EVs present pro-angiogenic properties, as they can transfer miRNA associated with the PI3K/AKT signaling pathway and proangiogenic miRNA. Once injected into animals with disease, EVs localize within peritubular capillaries and tubular cells, decreasing tissue damage and promoting a rapid recovery from AKI [7]. Camussi et al.[8] also performed a study concerning the emergent promising therapeutic role of EVs secreted by a multipotent human adult liver stem cells (HLSC): they can in fact reprogram in vitro HepG2 hepatoma and primary hepatocellular carcinoma cells (HCC), by inhibition of tumor growth and stimulation of apoptosis, in a dose-dependent manner. The effects in vitro of HLSC-EVs on HepG2 hepatoma cell line and on four HCC lines were evaluated, by testing proliferation and apoptosis. After in vitro experiments, they observed in vivo that 4 weeks after injection of HLSC-EVs in SCID mice with HepG2 tumor formed, cancer size and weight reduced significantly in comparison with control mice. Anti-tumor effects of HLSC-EVs were observed also in lymphoblastoma and glioblastoma [8].

2.1 TUMOR ENDOTHELIAL CELLS AND ANGIOGENESIS

Solid tumor growth and subsequent metastatic process depend on angiogenesis process triggered by chemical signals from tumor cells and microenvironment. Recent studies [9] demonstrated that TECs have their own phenotype, distinct from normal endothelial cells and from tumor cells, and that they are different at molecular and functional levels. This revealed that tumor angiogenesis is not only dependent on the normal endothelial cells, recruited from the surrounding vessels, but also on an intra-tumor embryonic-like vasculo-genesis driven by cancer stem cells having stem cells and progenitor cells properties. In this context, the microenvironment has a pivotal role: normal endothelial cells can be in fact reprogrammed to a proangiogenic phenotype by transferring genetic informations from tumor through apoptotic bodies. Alternatively, proangiogenic mRNAs or miRNAs can be delivered into normal endothelial cells from circulating extracellular vesicles.

TECs expression of genes has been previously investigated. TECs express specific genes, that are not expressed in normal vessels, named tumor endothelial markers (TECs). They present an abnormal genetic profile: the most common mutation is Aneuploidy [10,11]. In renal carcinoma, changes like missing chromosomes, translocations and multiples centromeres were observed [12]. In human haemopoietic tumors, like Multiple Myeloma, TECs shows specific13q14 chromosomal deletion. TECs of renal cancer (and also renal tumor cells) express an embryonic renal transcription factor PAX2, that confers to TECs the resistance apoptosis and proangiogenic properties. PAX2 can also induce in normal endothelial cells a proangiogenic phenotype, similar to that of tumor-derived endothelial cells [13]. TECs show also morphological and functional alterations: tumor blood vessels are irregular and chaotic, pericytes have abnormal shapes and show resistance to senescence in vitro: this is due to their autocrine production of growth factors, like angiopoietin-1 and VEGF-D and angiogenic receptors [14,15,16]. They are more resistant than normal cells to serum starvation and to cytotoxic drugs like vincristine and doxorubicin [17]. A remarkable pathway activated in TECs is Akt: the depletion of Akt determinates apoptosis and abrogates proangiogenic phenotype of TECs in vitro and in vivo [18].

The origin of TECs is still uncertain: they may derive from bone marrow stem or progenitor cells undergoing endothelial differentiation, or from tumor stem cells that produced both tumor cells and TECs. Alternatively, TECs can be targeted by extracellular vescicles of normal endothelial cells or of tumor cells, producing epigenetic alterations [10].

3) EXPERIMENTAL DESIGN

The identification of anti-tumor and/or anti-angiogenic miRNAs will be performed using in vitro methods such as angiogenesis in vitro assay, proliferation, invasion, and apoptosis assays. These models will permit to select the effective anti-angiogenic and/or anti-tumor miRNAs, which will be also tested by in vivo experiments (model of angiogenesis). For investigation of molecular mechnisms involved in miRNAs action I plan to use real time PCR (RT-PCR) and Western blot in order to confirm potential changes of target gene expression. Finally, HLSC-EVs, which demonstrated to have a powerful anti-tumor homing, will be loaded with the selected miRNAs, to create EVs with a more effective anti-tumor and anti-angiogenic action.

3.1 IN VITRO ANALYSIS:

TASK 1) Investigation of the effects of HLSC-EVs on tumor angiogenesis:

Since HLSC-EVs were described as a potent anti-tumor tool [8], I plan to study their effect on TECs angiogenic potential. For this purpose I will perform assays of proliferation, apoptosis and angiogenesis in vitro, described below. Simultaneously I will analyze the expression of miRNA in HLSC-EVs by PCR micro-array method.

EVs isolation: EVs will be obtain from HLSC supernatants cultured overnight in Minimum Essential Medium (MEM) without FBS, by centrifugation and ultracentrifugation.

The number, size and distribution of EVs will be detected using NanoSight.

1) Angiogenesis in vitro assay: TECs (25x10³ cells) transfected 48 hours before will be plated into 24 well-plates coated with growth-factor reduced Matrigel. After incubation of 24 h, phase-contrast images (magnification, ×100) will be recorded and the total length of the network structures will be measured using MicroImage analysis system. The total length per fieldwas calculated in five random fields and expressed as a ratio to the respective control [19].

2) Proliferation assay: transfected TECs will be seeded at 2000 cells/well into 96-well plates in EndoGro complete with 10% FBS. DNA synthesis will be detected as incorporation of 5-bromo-2′-deoxyuridine (BrdU) into cellular DNA by using an ELISA kit according to the protocol suggested by the manufacturer. Briefly, 10 μM BrdU will be add to transfected or untreated TECs and incubated for 18 h. BrdU incorporated into the DNA will be detected by using an anti-BrdU peroxidase-conjugated mAb and visualized with a soluble chromogenic substrate. Optical density will be measured with an ELISA reader [19].

3) Invasion assay: The effect of miRNAs on TECs invasion will be detected by the Transwell assay. Transfected TECs and control TECs will be resuspended in EngoGro (serum free) and will be plated into the upper compartment of an invasion chamber (50 × 10³cells per well), containing a polycarbonate membrane with an 8 μm pore size which will be coated with a layer of growth factor reduced Matrigel. FBS (10%) will be used as the attractant and added to the lower well. After 48 h of incubation, the invasive cells migrated through the Martigel layer to the complete medium in the lower compartment. The invasive cells will be stained with Mayer and the number of invaded cells will be counted [19].

4) Apoptosis assay: Dead cells will be quantified by Annexin V and Dead cells assay. Transfected and control TECs (2x10⁴-1x10⁵ per samples) will be prepared in different eppendorf with 100 ul of 1% BSA and 1% FBS medium, and will be incubated with 100 ul of Annexin Reagent for 20 minutes at RT. Four populations of cells will be distinguished by Muse cell Analyzer: non-apoptotic cells, early apoptotic cells, late stage apoptotic and dead cells and mostly nuclear debris [19].

TASK 2) Identification of miRNAs with anti-tumor and/or anti-angiogenic potential:

Based on literature data [4,5], I will select miRNAs with described anti-tumor and/or anti-angiogenic effects. I will study the expression of these miRNAs in TECs and I will take in consideration for my study, only those miRNAs which will not be expressed in TECs.

1) Transfection of TECs: TECs (0,4-0,6 x10⁵ cells) will be plated in 24 well-plates with 0,5 ml of EndoGro complete medium at Day 0. At the Day 1 transfection with selected miRNAs will be perform using HiPerfect Transfection Reagents. Selected miRNAs and HiPerfect in diverse concentrations will be co-incubated for 5-10 miutes at RT, in order to allow the formation of transfection complexes. After that this mix will be added to TECs and the cells will be incubated under normal condition. 24 hours after the growth medium will be changed and next day transfected cells will be prepared for the sequential tests [19].

TECs will be transfected with selected miRNAs and in vitro assays described before will be performed, to check their viability and angiogenic potential. For study possible molecular mechanisms involved in miRNAs effects on TECs I will use PCR, Western blot and ELISA technics:

3) RT-PCR array for pathway-focused gene expression analysis: expression of target genes for selected miRNA will be analyzed in transfected TECs to demonstrate specific molecular mechanisms.

4)Western blot and ELISA: for genes whose expression will be changed significantly after TECs transfection with selected miRNAs I will perform Western blot analysis or ELISA to demonstrate the alterations of expression on protein level.

TASK 3) Loading of HLSC-EVs with selected anti-tumor and anti-angiogenic miRNAs to enhance anti-angiogenic effects:

HLSC-EVs will be loaded with selected most effective anti-angiogenic miRNAs, which are not expressed in native HLSC-EVs. Their anti-tumor and anti-angiogenic potential will be then tested on TECs by described in vitro experiments. Among several methods for EV loading, the most promising method is the Electroporation [20]. Briefly, EVs will be re-suspended in specific electroporation buffer and mixed with miRNA. Then electroporation will be performed in 0.4 cm cuvettes with aluminum electrodes using a Bio-Rad Gene Pulser I or II with capacitance extender set at 400 V and 125 μF. At the end, all electroporation cuvettes will be incubated on ice for 30 minutes [21]. All samples of electroporated EVs and target cells (TECs) stimulated with these EVs, will be analyzed by RT-PCR to test effective miRNAs transfer.

TECs will be stimulated with engeneered HLSC-EVs during 24 hours and then they will be tested with in vitro methods described previously. Engeneered HLSC-EVs that will show the most potent effects on tumor angiogenesis inhibition, will be used for the following in vivo experiments.

3.2 IN VIVO ANALYSIS

TASK 1) Evaluation of engeneered HLSC-EVs effect on tumor angiogenesis in vivo:

To evaluate the effects of loaded HLSC-EVs on TECs in vivo, TECs stimulated 24 hour with these EVs will be injected subcutaneously into SCID mice within Matrigel suspension. Brieftly, TECs (1x10⁶ cells/injection) will be incubated with loaded HLSC-EVs (1x10¹⁰ EVs per 1x10⁶ of TECs) during 24 hours. Then, mice (8 weeks old) will be injected subcutaneously with 0.5 ml of ice-cold Matrigel. Equivalent quantity of non-stimulated TECs will be used as control. The Matrigel plugs will be excised after 10 days and fixed in 4% paraformaldehyde for 4 h. Matrigel-containing paraffin sections (5–8 μm thick) will be stained by trichrome stain method [22]. The vessel lumen area (mean size per square millimeter) and quantity of erythrocyte-containing vessels will be determined using computerized image analysis software MicroImage analysis system [19].

4) EXPECTED RESULTS

First, my project is devoted to demonstrate the influence of HLSC-EVs on angiogenesis and to investigate microRNA potentially able to reduce pro-angiogenic properties of TECs. Then, my study will involve the expression of these miRNAs in HLSC-EVs, also trying to “exclude” them in order to enrich HLSC-EVs with various anti-angiogenic and anti-tumor miRNA not present in EVs. Moreover, my aim is to develop an effective method for loading EVs with miRNAs, enriching HLSC-EVs with selected miRNAs to enhance their anti-angiogenic effects on TECs. Finally, their effects on TECs, both in in vitro and in vivo models, will be tested.

The achievement of an anti-angiogenic effect on TECs by loading miRNA within HLSC-EV, could be of great interest in cancer research, as tumor angiogenesis shows molecular mechanisms different from those involved in normal angiogenesis, and it is not currently well investigated. Moreover, I expect to have new insights on the EVs capability of being tools to deliver miRNA into target cells, exploiting their physiological innate properties.

 

Articles 

“Extracellular vesicles from human liver stem cells inhibit tumor angiogenesis” 2018 T. Lopatina, C. Grange, V. Fonsato, M. Tapparo, A. Brossa, S. Fallo, A. Pitino, MB. Herrera-Sanchez, S. Kholia, G. Camussi, B. Bussolati  

 Attended lectures 

Come scrivere e pubblicare un lavoro scientifico -Riccarda Granata

New perspectives on C.difficile infections-Francesco De Rosa

Forme monogneiche di ipertensione arteriosa- Paolo Mulatero

Essential hypertension: pathogenesis, diagnosiss and treatment- Franco Veglio

Infezioni virali nei pazienti sottoposti a trapianto-Rossana Cavallo

Cellule staminali e insufficienza epatica acuta-Renato Romagnoli

Trapianto di rene: aspetti immunologici e attualità e prospettive delle terapia immunosoppressiva- Luigi Biancone

 

Corso di Ricerche bibliografiche - 8 hours total - 8,15,18 maggio

 

D-DAY 2018: The spare part: reflections on kidney transplantation-Andrea Pietrabissa 13/09/2018

D-DAY 2018: Reprogramming of Metabolic Pathways by PI 3-Kinase and AKT Signalling in Cancer- Alex Toker 13/09/2018

 

Attended Seminars 

 "Epigenetic and trascrptional landscape of ageing stem cells" 04/04/2018 Dott.ssa Sabrina Bertinetti

"Preclinical PET/CT Imaging: research, applications and advantages" 10/04/2018 Doc. Olivia Kelada

 "Innovations and new technologies in cell biology: 3D Bioprinting and Dynamic Cell Cultures" 25/06/12018 Dr. Mario Barlocco

 "Triggering endogenous mechanisms of cardiac regeneration via paracrine action. A role for the stem cell secretome" 03/07/2018 Prof. Sveva Bollini