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Project Title: „ Testing of patient-derived stem cell extracellular vesicles loaded with drugs in a personalized lung-cancer on-chip platform (LoC4Ev).”

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

Project No.:

Period: 1 February 2021 – 30 November 2023

Project costs: 540 540.53 EUR

Principle Investigator: Dr. biol. Artūrs Ābols

Cooperation partner: SIA “Cellboxlab”

Project summary:

This project aims to develop a personalised NSCLC patient lung cancer on chip (LCoC) and lung on chip (LoC) as a model system where to test the efficacy of drug-loaded patient-derived MSC produced EVs compared to the drug by itself.

Over half of the people diagnosed with lung cancer die within a year of diagnosis and the 5-year survival rate is less than 18%. Non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer cases. Depending on the stage of lung cancer, patients are eligible for certain treatments ranging from surgery to radiation to chemotherapy as well as targeted therapy. Thanks to genetic screening, specific mutations have been identified as a better target treatment for individual patients in the last decade. Also, there are molecular targeted therapies, that are not yet tested in lung cancer but could be applied in NSCLC cases where tumours have certain genetic variation. While there are options for different treatment, there is still a room for improvements in drug delivery, that could increase specificity and therefore decrease necessary concentrations and subsequently side effects of drugs. Extracellular vesicles (EV) have a natural ability to carry functional biomolecules, such as RNA, DNA, proteins and different metabolites in their lumen. This property makes EVs attractive for use in drug delivery. Indeed, several studies are currently underway to develop methods of exploiting EV for use as efficient drug delivery vehicles especially those derived from mesenchymal stem cells (MSC) due to their natural tumour cell tropism. However, current approaches have been tested only in simple in-vitro models or animal models. Next step would be testing these approaches into more advanced – personalised in vitro systems such as organs on chip platform

Information published 01.02.2021.

Progress of the project:

1 February 2021 – 30 April 2021

This reporting period, we started to prepare CMEK permit application for patient sample collection. Agreed on protocols for patient sample collection with doctors. Planning of the protocols for the isolation of lung cancer cells and the formation of organoids from patient surgery samples and the isolation of normal fibroblasts and their reprogramming into induced pluripotent stem cells (iPSC). Protocols for iPSC differentiation into lung epithelium, endothelium, and induced mesenchymal stem cells are also being developed. Lists of reagents for the necessary experiments are compiled. In parallel, the introduction of stem cell-derived extracellular vesicles (ASC52telo) into the first lung cancer on chip (LCOC) prototypes developed using stable cell lines A549 and HUVEC are being tested.

Information published 30.04.2021.

Progress of the project:

1 May 2021 – 31 July 2021

This reporting period we have been working on the TEER electrode design in a computer-aided design suit. Furthermore, we have been evaluating literature on the most optimal thin film electrode thickness and subsequently have been done the initial tests of electrode deposition via thermal evaporation. Additionally, five LOC devices have been fabricated for ensuing biological testing. Next, developed devices were tested with extracellular matrix (ECM) treatment process, functionalisation with HUVEC (Human umbilical vein endothelial cells), HSAEC (Human small airway epithelial cells) and pre-ECM treatment, cell seeding protocol was optimised to get functional models. Next LOC models are cultivated for functional LOC model tests with biosensors and ALI (Air liquid interface) establishment.

Information published 30.07.2021.

Progress of the project:

1 August 2021 – 31 October 2021

This reporting period we have been working on the fabrication of the first version of lung on chip (LOC) devices made from Off-stoichiometry thiol-ene polymer (OSTE) and thermoplastics with thin-film TEER (trans epithelial electric resistance) electrodes. Optimisation of the interconnection between thin-film electrodes and spring-loaded connectors. We are currently testing interconnections made from silver-loaded epoxy that sets in low temperature (60C). Low-temperature reflow soldering paste (138C) is also being tested with the heat applied to the spring-loaded connectors, which are immersed into the paste. Next, we have started to test commercial hiPSC (human induced pluripotent stem cells) differentiation into endothelial cell protocols with STEMCELL Technologies reagents, that will be applied to personalise Loc4Ev devices. LOC devices with integrated TEER biosensor were functionalised with HUVEC (Human umbilical vein endothelial cells) and A549 cells to test TEER functionality. Lecture about organs on a chip including LOC was prepared and presented to the M.D. students.

Information published 30.10.2021.

Progress of the project:

1 November 2021 – 31 January 2022

During this reporting period, we worked on the integration of O2 biosensors into LOC developed from Off-stoichiometry thiol-ene polymer (OSTE) and thermoplastics. The sensors were tested in both fluids such as 1xPBS and cell culture medium and in gas mixtures in cell culture incubators under normoxia and hypoxia. The commercial hiPSC (human-induced pluripotent stem cell) differentiation protocol into endothelial cells with STEMCELL Technologies reagents was optimised, that will be used to personalize Loc4Ev devices. Currently we are working to optimize the cisplatin packaging into mesenchymal stem cell derived extracellular vesicles methodology.

Information published 31.01.2022.

Progress of the project

1 February 2022 – 30 April 2022

Throughout the reporting period we have optimised the design of the device to improve the OSTED filling in the sidewalls of the device, which has led to improved fabrication yield of TEER devices. An alternative material to the mold material has been selected, which has significantly reduced particle contamination onto the thin-film TEER electrodes, therefore the resistance variation from channel to channel is now <10% for the current protocol, tested across multiple devices. Furthermore, an improved device design has allowed incorporation of 3 oxygen measurement ports per chip as opposed to initial design of 2 ports per chip. Experiments with the oxygen sensing in the chips are now being biologically validated. Work has been completed on optimizing cisplatin packaging in mesenchymal stem cell secreted extracellular vesicles. In parallel, testing of cisplatin-filled extracellular vesicles in lung cancer on a chip and lung on chip made up of commercial primary cells and stable cell lines was initiated. Results are compared between EVs without cisplatin and cisplatin without EVs. In addition, during this period we participated in the publication of the project topic in an online interview organized by the Riga Technical University Student Council in the studio entitled “What if?”.

Information published 29.04.2022.

Progress of the project:

1 May 2021 – 31 July 2021

TEER containing devices were tested by growing commercial primary and stable cell lines during lung on chip and lung cancer on chip establishment and during establishing ALI. Oxygen sensor placement outside the device within custom jig has been designed. HPLC methodology has been optimised to measure cisplatin within EVs. Next, EV staining by SYTOX and uptake experiments have been also optimised. In addition, during this period we participated and presented research project topics within conversation festival LAMPA at Cēsis, Latvia to the general public.

Information published 29.07.2022.

Progress of the project:

1 August 2022 – 31 October 2022

During this report period several NSCLC patient tissue, urine and blood samples were collected, NSCLC organoids were developed and normal fibroblasts isolated from operation samples. Additionally, somatic cells from urine samples and PBMC from blood samples were collected. Cisplatin containing and empty extracellular vesicles as a control were produced by using MSC cells. Extracellular vesicles were quantified and characterised.

Throughout the period, continued experimental work on TEER electrode setup was done. A source of signal noise was identified and significantly reduced through a novel experimental setup. An alternative O2 measurement has been designed and currently is prototyped, which would allow to measure O2 levels without additions of oxygen port.

Results of the project were presented at two international conferences with oral presentations as an invited speaker. Additionally, short video was made and published about organ on a chip technology developed by LBMC and CellboxLabs in popular scientific language in “Aculiecinieks”.

Information published 31.10.2022.

Progress of the project:

1 November 2022 – 31 January 2023

During this report period several additional NSCLC patient tissue, urine and blood samples were collected, NSCLC organoids were developed and normal fibroblasts isolated from operation samples. Additionally, somatic cells from urine samples and PBMC from blood samples were collected. Cisplatin measurement method was established to quantify it in EVs. Additionally, normal fibroblast reprogramming into iPSC was started.

Throughout the period, work on chip design optimization and fabrication protocol was done, in order to increase the channel yield. Further TEER measurement setup was optimized to ensure consistent conditions during TEER measurements. Experiments and design process with a novel O2 measurement setup is on-going.

Results of the project were presented at international conference with poster presentation. Additionally, project authors participated in a radio interview:  “Zināmais nezināmajā” – Pirmsklīniskie pētījumi – orgāni uz čipa un laboratoriju peles.

Information published 31.01.2023.

Progress of the project

1 February 2023 – 30 April 2023

During this period additional OOC devices were fabricated. Cisplatin packaging into MSC derived EVs protocol was established. MSC derived extracellular vesicles with and without cisplatin were tested on Lung and lung cancer on a chip. Cytotoxicity and barrier integrity was measured. In parallel 3 patient normal fibroblasts were reprogrammed into iPSC, normal karyotype was confirmed, and normal lung organoids were developed, while from 6 patients lung cancer organoids were successfully developed. Additionally, during this time project results were presented at two international conferences with poster and oral presentation.

Information published 02.05.2023.

Progress of the project:

1 May 2023 – 31 July 2023

In the given period, five more OOC devices were constructed. Development of induced MSC (iMSC) and endothelial cells was accomplished from iPSCs sourced from two patients. The iMSCs were verified through specific markers and differentiation. For one patient, iMSC EVs and EVs with cisplatin were generated. The cytotoxicity of these generated EVs was evaluated on the patient’s healthy lung organoids as well as lung cancer organoids. Furthermore, the findings of the project were communicated at the MPS World Summit 2023 through a poster presentation aimed at the scientific community. The project was also brought to the attention of the local public through a radio broadcast titled “Prāts izglābs pasauli”.

Information published 01.08.2023.

Progress of the project:

1 August 2023 – 31 October 2023

Throughout the duration of this project, we have achieved significant milestones in our experiments. We successfully developed in total 10 normal fibroblast lines and lung cancer organoids from tissue samples of 9 patients with non-small cell lung cancer (NSCLC). Additionally, we collected and cryopreserved urine somatic cells and peripheral blood mononuclear cells (PBMCs) from blood samples.

A notable achievement was the reprogramming of induced pluripotent stem cells (iPSCs) from 4 patients. These iPSC lines were meticulously characterized and confirmed to maintain a normal phenotype. Utilizing these iPSCs, we differentiated them into endothelial cells, normal lung organoids, and iPSC-derived mesenchymal stem cells (MSCs), ensuring comprehensive characterization and confirmation of each line.

A key part of our research involved testing extracellular vesicles (EVs) derived from iPSC-MSCs of NSCLC patients. These EVs, packed with cisplatin and compared with both cisplatin alone and cisplatin-naïve EVs, were evaluated for their cytotoxic effects and induction of apoptosis in lung cancer and normal lung organoid models.

Throughout the project, we developed and utilized chip-on-a-chip COC/OSTE organ-on-a-chip (OOC) devices, along with the successful development and validation of trans-epithelial electrical resistance (TEER) and oxygen (O2) biosensors.

Our team also developed lung cancer and normal lung models on a chip, refining protocols using stable and commercially available primary cell lines, including air-liquid interface (ALI) cultures. In total, we executed more than 80 experiments in this area.

Towards the project’s conclusion, we focused on the production and thorough characterization of cisplatin-enclosed EVs and cisplatin-naïve EVs derived from two patient iPSC-MSC cell lines. These EVs were employed in experimental setups involving lung-on-a-chip and lung cancer-on-a-chip models derived from patient cell samples, primarily to assess cytotoxicity. The results highlighted the need for protocol optimization based on patient-derived samples. However, it is important to note that we successfully achieved the methodological milestones and objectives as initially planned in the project.

Information published 30.10.2023.

Progress of the project:

1 November 2023 – 30 November 2023

Over the course of this project, our team has demonstrated exceptional dedication and success. We have meticulously prepared and submitted two comprehensive research articles, highlighting the significant outcomes of our project. In addition to these publications, we have compiled and submitted detailed reports covering all aspects of the project, culminating in the successful submission of the project’s final report.

Our team’s efforts and findings were prominently showcased at 9 international conferences, where we engaged the scientific community through poster presentations, oral presentations, and were honoured with two opportunities to present as invited speakers. This level of engagement not only emphasized the project’s impact but also facilitated valuable exchanges with peers in the field.

Furthermore, our commitment to public dissemination was evident in our active participation in 17 events aimed at the broader community. These events allowed us to engage with the public and disseminate our findings beyond the scientific community, enhancing the project’s visibility and impact.

In summary, the successful conclusion of this project is a testament to our team’s hard work and dedication. We have not only met but significantly exceeded the set milestones, as is thoroughly documented in our final project report. This achievement reflects our commitment to advancing research and contributing valuable knowledge to our field.

Information published 30.11.2023.