Project co–financed by REACT-EU to mitigate the effects of the pandemic crisis

Project Title: „Development of a novel microfluidic device for label-free quantification of prostate cancer-derived extracellular vesicles and analysis of their RNA content (PROCEX)”

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research”

Project No.: 1.1.1.1/20/A/045

Period: 1 May 2021 – 30 November 2023

Project costs: 540 540.54 EUR

Principle Investigator: Dr. biol. Aija Linē

Cooperation partner: SIA “Cellboxlab”

Project summary:

This project aims at developing a radically new technology for non-invasive detection and monitoring of prostate cancer (PC) based on label-free quantification of PC-derived extracellular vesicles (EVs) and analysis of their RNA content. This technology will combine a multi-stage microfluidic system with optical resonators and novel reagents – nanobodies allowing to capture, quantify and analyse the RNA cargo of specific PC-derived EV subpopulations in the blood or urine. The main outcome of the project will be a prototype of the PROCEX device. Its performance will be tested with the clinical samples from well-annotated cohort of PC patients and controls. We expect that this project will lead to the development of novel point-of-care device that has the potential to improve the diagnostics and treatment outcomes in cancer patients. Currently, this technology is at TRL2 and we expect that at the end of the project it will reach TRL4.

 Information published 05.05.2021.

Progress of the project:

1 May 2021 – 31 July 2021

During the reporting period, the kick-off meeting took place and the tasks and expected results for the following 3-months period were defined. In order to produce the EV standards that will be used both for the selection of nanobodies and for the development of the microfluidics device, the mass production of EVs was started. To this end, the prostate cancer cell lines PC3 and LNCaP are grown in a hollow-fiber bioreactor and the cell culture supernatant is collected each second day and used for the isolation of EVs. In order to select the most suitable RNA biomarkers, the previously generated RNAseq data set is now being reanalysed. Furthermore, the first design of the sample mixing module has been generated.

Information published 30.07.2021.

Progress of the project:

1 August 2021 – 31 October 2021

To obtain the EV standards for the selection of nanobodies and development of the microfluidics device, the mass production of EVs from the PC3 and LNCaP cells grown in a hollow-fiber bioreactor was continued. To develop the sample mixing module, 5 different designs were developed and the corresponding 3D moulds were printed. To develop the EV and RNA biosensors, the fabrication of SU-8 waveguides and ZnO cladding was optimised. We have started to work on the functionalisation of the biosensor surfaces with miRNA probes and TIM4 protein.

Information published 29.10.2021.

Progress of the project:

1 November 2021 – 31 January 2022

The mass production of EVs from PC3 and LNCaP cells using the hollow-fiber bioreactor has been completed and the EV standards for the selection of nanobodies and development of the microfluidics device has been established. The obtained EV standards were characterised by nanoparticle tracking analysis, transmission electron microscopy and Western blot analysis. Testing of the selected RNA biomarker candidates have been started. Different designs of the sample mixing module have been tested with two types of magnetic beads. The work on the functionalisation of the biosensor surfaces with miRNA probes and TIM4 protein is in progress.

Information published 31.01.2022.

Progress of the project:

1 February 2022 – 30 April 2022

In order to select nanobodies that recognise specifically prostate cancer-derived EVs, we have started the selection of nanobodies from the yeast surface display libraries. To select the EV-enclosed RNA biomarkers that show the highest diagnostic value, the candidate biomarkers are tested in EVs isolated from plasma and urine from patients with prostate cancer and benign prostatic hyperplasia. The work on different designs of the sample mixing module was continued. A series of experiments were conducted to establish the optimal conditions for the functionalisation of the biosensor surface with TIM4 and RNA probes. Although the TIM4 protein binds well to the ZnO-coated surface, so far we have not been able to verify its capacity to capture EVs, therefore these experiments will be continued.

Information published 29.04.2022.

Progress of the project:

1 May 2022 – 31 July 2022

In this period, we focused mostly on the functionalisation of the surface of biosensors. We replaced the previously used recombinant TIM4 protein with a new one from a different manufacturer and found out that the new TIM4 shows an excellent ability to capture EVs in the ELISA format. Currently, we are testing its ability to capture EVs, when attached on the ZnO-coated slide surface. We have established optimal conditions for attaching RNA-LNA probes to the surface of the slide and currently are optimising hybridisation conditions.

Information published 29.07.2022.

Progress of the project:

1 August 2022 – 31 October 2022

In this period, we continued to work on the selection of nanobodies recognizing PC-derived EVs using the yeast surface display library. In parallel, we continued to work on the functionalization of the biosensors’ surfaces. The surface of the EV biosensor was coated with TIM4 protein and the efficacy of EV capture was tested using labeled EVs. The surface of the RNA biosensor was coated with an RNA-LNA probe and the conditions for hybridization with the complementary miRNA were tested using synthetic miRNA with a fluorescent label. Results of the project were presented at the 17th International Conference on Genomics (ICG-17), Riga and the 1st meeting of the Baltic Society of Extracellular Vesicles in Tartu.

Information published 31.10.2022.

Progress of the project:

1 November 2022 – 31 January 2023

In this period, we resumed testing the RNA biomarker candidates by ddPCR in EVs isolated from patients’ plasma and urine. The selection of nanobodies from the yeast surface display libraries was completed and the production of nanobodies has been started. The work on the functionalization of the biosensors’ surfaces was continued – the protocol for the functionalisation of the slide surface was amended and binding of EVs to the Tim4 was visualised by SEM. In order to develop an optimised protocol for the hybridisation of miRNAs with the corresponding LNA probes, various hybridisation buffers and temperatures were tested.

Information published 31.01.2023.

Progress of the project:

1 February 2023 – 30 April 2023

In this period, we finished testing the RNA biomarker candidates by ddPCR in EVs isolated from patients’ plasma and urine, and currently, we are preparing an article. The results were presented at the Baltic Society for Extracellular Vesicles meeting in Vilnius, 28-29. April. The production of nanobodies was carried on and the mixing module of the microfluidics device was optimised. The previous problems with the functionalisation of the EV biosensor’s surface were solved and the work on determining the limit of detection and limit of quantification is ongoing.

Information published 02.05.2023.

Progress of the project:

1 May 2023 – 31 July 2023

We have prepared a manuscript on the diagnostic value of EV-enclosed RNA biomarkers. Production and testing of nanobodies is completed. The obtained results show that we succeeded in the production of high-affinity nanobodies against CD9 and CD63, whereas nanobodies against prostate-specific antigens apparently are not suitable for capturing EVs, therefore an alternative solution is needed. The work on testing the EV biosensor is ongoing.

Information published 01.08.2022.

Progress of the project:

1 August 2023 – 31 October 2023

CD9 and CD63-positive EV subpopulations were isolated from urine samples of PC patients and RNA biomarkers were tested in these EV subpopulations. Results showed that both EV subpopulations contain PC-derived RNA biomarkers. The final optimisation experiments for the functionalisation of biosensor surfaces were performed and limits of detection and quantification were determined. The microfluidics device was tested with the EV standards.

Information published 31.10.2023.

Progress of the project:

1 November 2023 – 30 November 2023

We performed the last experiments on the testing of the prototype of the microfluidics device with the EV standards and clinical samples. We summarised the results and prepared a publication.  

Information published 30.11.2023.