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Applications of in situ proximity ligation assays for cancer research and diagnostics
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg. (Kamali-Moghaddam)
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.

In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.

Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.

In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.     

Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.    

A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry. 

 

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2016. , 56 s.
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1241
Nyckelord [en]
Cancer, Diagnostics, Research, Prognostics, Method development, Flow cytometry, Drug delivery
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Molekylär medicin
Identifikatorer
URN: urn:nbn:se:uu:diva-300191ISBN: 978-91-554-9638-8 (tryckt)OAI: oai:DiVA.org:uu-300191DiVA: diva2:951029
Disputation
2016-09-23, B/B22, Husargatan 4, Uppsala, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2016-09-01 Skapad: 2016-08-04 Senast uppdaterad: 2016-09-05
Delarbeten
1. Crosstalk between WNT and Hippo signaling pathways changes upon colon cancer stage and is affected by cell density and loss of or mutated E-cadherin protein
Öppna denna publikation i ny flik eller fönster >>Crosstalk between WNT and Hippo signaling pathways changes upon colon cancer stage and is affected by cell density and loss of or mutated E-cadherin protein
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-300107 (URN)
Tillgänglig från: 2016-08-02 Skapad: 2016-08-02 Senast uppdaterad: 2016-08-26Bibliografiskt granskad
2. Detergent resistant membranes from erythrocytes can form vesicles and be loaded for delivery of pharmaceutics
Öppna denna publikation i ny flik eller fönster >>Detergent resistant membranes from erythrocytes can form vesicles and be loaded for delivery of pharmaceutics
Visa övriga...
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:uu:diva-300090 (URN)
Tillgänglig från: 2016-08-02 Skapad: 2016-08-02 Senast uppdaterad: 2016-08-26
3. Proximity-dependent initiation of hybridization chain reaction
Öppna denna publikation i ny flik eller fönster >>Proximity-dependent initiation of hybridization chain reaction
Visa övriga...
2015 (Engelska)Ingår i: Nature Communications, ISSN 2041-1723, Vol. 6, 7294Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

Nationell ämneskategori
Medicin och hälsovetenskap Kemi
Identifikatorer
urn:nbn:se:uu:diva-255596 (URN)10.1038/ncomms8294 (DOI)000357171100008 ()26065580 (PubMedID)
Forskningsfinansiär
EU, FP7, Sjunde ramprogrammet, 278568, 316929, 259796Stiftelsen för strategisk forskning (SSF)Vetenskapsrådet
Tillgänglig från: 2015-06-17 Skapad: 2015-06-17 Senast uppdaterad: 2016-08-26Bibliografiskt granskad
4. Flow Cytometric Measurement of BCR-ABL Fusion Protein Positive Cells in Blood from Patients with Chronic Myeloid Leukemia
Öppna denna publikation i ny flik eller fönster >>Flow Cytometric Measurement of BCR-ABL Fusion Protein Positive Cells in Blood from Patients with Chronic Myeloid Leukemia
Visa övriga...
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-300087 (URN)
Tillgänglig från: 2016-08-02 Skapad: 2016-08-02 Senast uppdaterad: 2016-08-26
5. Detecting extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
Öppna denna publikation i ny flik eller fönster >>Detecting extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Medicin och hälsovetenskap
Forskningsämne
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-300089 (URN)
Tillgänglig från: 2016-08-02 Skapad: 2016-08-02 Senast uppdaterad: 2016-08-26

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