Final Report Summary - APTACELLSENS (An aptamer-based multiplex assay to recording molecular signatures of cancer cell fingerprints)
In this research project, a series of DNA aptamers that reveal broad-spectrum interaction patterns with cancer cells derived from solid tumours was proposed to develop a robust multiplex assay format to determine molecular fingerprints of the most prevalent and lethal cancers. It was envisioned that the multiplex aptamer-based assay could allow to get information of the relative expression levels of yet unknown marker proteins present on different cancer cells, enabling the profiling of those cells, which is highly convenient for diagnostic monitoring. A second goal was the identification of the molecular targets of the aptamers by using siRNA libraries, essentially covering the entire human genome and the knock-down of putative target molecules. Loss of aptamer binding in relation to employed siRNA molecules can provide relevant information on cognate aptamer target molecules, and subsequently, they can be studied as potential new biomarkers candidates.
For this, three main objectives were established:
• Objective 1: Aptamer-based cancer fingerprints assay development.
• Objective 2: Assay validation with clinical samples.
• Objective 3: Target identification with siRNA methodology.
During the first part of the project, efforts were focussed in the establishment of the aptamer-based cancer fingerprints assay development. For this purpose, the initial panel of aptamers was tested in a flow cytometry binding assay, resulting in low signal detection. Consequently, the initial schedule was modified with the objective of better characterise the DNA sequences from the SELEX where the initial panel of aptamers was identified. Next generation sequencing (NGS) was then performed for the different selection cycles, and a series of new aptamer candidates were obtained. Based on the total frequency of the obtained sequences in the last round, 16 new sequences have been further characterised together with the 8 sequences from the initial panel. Those sequences were tested for binding to breast cancer cell line (MCF7) and a prostate cancer cell line (PC3) (Figure 1A-B, attached file) with three different techniques: radioactivity binding assay, quantitative PCR and flow cytometry. Some of these new sequences showed binding towards MCF7 cells using these different characterisation techniques, with special emphasis on the consistency, sensitivity and reproducibility in flow cytometry, where the multiplex assay will be developed. Selected aptamers showed targeting to cancer cells derived from solid tumours (such as breast, prostate and lung cancer), while no targeting is exhibited for a lymphoma cell line. Different fluorophores have been tested (Atto dyes and R-phycoerythrin (R-PE)) and quantum dots (QD) in order to improve the sensitivity of the assay and increase the efficiency of the detection. With these promising results it is expected that the establishment of the multiplex analysis will be carried out in the near future, and therefore, can be validated with clinical samples in order to accomplish objective 2.
Moreover, confocal microscopy studies have been performed to monitor the aptamer recognition and internalisation to the cells. The initial panel of aptamers was analysed revealing low internalisation efficiency into breast or prostate cancer cell line in comparison to the control sequence. Those studies are currently being performed with the new set of aptamers to better characterise their binding, internalisation capacity, and cell localisation. This is an important aspect for the potential use of those aptamers for tumour targeting and therapy. Furthermore, preliminary studies of the in vivo targeting of selected aptamers in mouse models reveal higher targeting of those in contrast to a control sequence.
An alternative and attractive application of those aptamers is their use as delivery tools. We had in hand two reporter cell lines (MCF-7 for breast cancer and A549 for lung cancer) that express green fluorescent protein (EGFP) under miR-21 regulation. Those cells have a high endogenous miR-21 level, so expression of EGFP is prevented. For this, the aptamers can be covalently linked with an antimiR (so called aptamiR) for the cell specific delivery of these constructs for silencing of endogenous micro RNA. Those AptamiR chimeras should recognise and internalise into the cells with high specificity due to the aptamer domain, allowing the delivery of antimiR-21 and thus, inducing EGFP expression. AntimiRs derived from the initial set of selected aptamers were tested, showing low delivery efficiency of the antimiR-21 into the target cells, which correlates with low levels of EGFP expression. Within this context, the new selected aptamers that show improved targeting towards solid tumour cancer cells are promising candidates as carriers of antimiR-21 for silencing the endogenous miR-21. Therefore, their potential use as antimiR chimeras is currently under evaluation.
Regarding the target identification of the aptamers here described (Objective 3), a collaboration with the group of Prof. Silberring at the University of Krakow was established. Efforts have been focused in the optimisation of a pull-down assay to identify the biomolecule towards each aptamer has affinity for. Currently, the methodology has been established by using fluorescently labelled aptamers and the analysis of the pulled proteins by mass spectrometry is ongoing. Validation of those target candidates will be then evaluated using specific siRNAs in order to knock-down their expression and assess the binding behaviour of the aptamers or by knock-in assays, where the protein target is overexpressed. Binding assays with the fluorescently labelled aptamer will be performed then with those modified cells. Once the targets of the used aptamer panel are found, validation of the potential new biomarkers will be performed. Furthermore, with the new selected sequences showing improved binding to the analysed cells, the target identification with siRNA technology will be performed as reported in the proposal.
For this, three main objectives were established:
• Objective 1: Aptamer-based cancer fingerprints assay development.
• Objective 2: Assay validation with clinical samples.
• Objective 3: Target identification with siRNA methodology.
During the first part of the project, efforts were focussed in the establishment of the aptamer-based cancer fingerprints assay development. For this purpose, the initial panel of aptamers was tested in a flow cytometry binding assay, resulting in low signal detection. Consequently, the initial schedule was modified with the objective of better characterise the DNA sequences from the SELEX where the initial panel of aptamers was identified. Next generation sequencing (NGS) was then performed for the different selection cycles, and a series of new aptamer candidates were obtained. Based on the total frequency of the obtained sequences in the last round, 16 new sequences have been further characterised together with the 8 sequences from the initial panel. Those sequences were tested for binding to breast cancer cell line (MCF7) and a prostate cancer cell line (PC3) (Figure 1A-B, attached file) with three different techniques: radioactivity binding assay, quantitative PCR and flow cytometry. Some of these new sequences showed binding towards MCF7 cells using these different characterisation techniques, with special emphasis on the consistency, sensitivity and reproducibility in flow cytometry, where the multiplex assay will be developed. Selected aptamers showed targeting to cancer cells derived from solid tumours (such as breast, prostate and lung cancer), while no targeting is exhibited for a lymphoma cell line. Different fluorophores have been tested (Atto dyes and R-phycoerythrin (R-PE)) and quantum dots (QD) in order to improve the sensitivity of the assay and increase the efficiency of the detection. With these promising results it is expected that the establishment of the multiplex analysis will be carried out in the near future, and therefore, can be validated with clinical samples in order to accomplish objective 2.
Moreover, confocal microscopy studies have been performed to monitor the aptamer recognition and internalisation to the cells. The initial panel of aptamers was analysed revealing low internalisation efficiency into breast or prostate cancer cell line in comparison to the control sequence. Those studies are currently being performed with the new set of aptamers to better characterise their binding, internalisation capacity, and cell localisation. This is an important aspect for the potential use of those aptamers for tumour targeting and therapy. Furthermore, preliminary studies of the in vivo targeting of selected aptamers in mouse models reveal higher targeting of those in contrast to a control sequence.
An alternative and attractive application of those aptamers is their use as delivery tools. We had in hand two reporter cell lines (MCF-7 for breast cancer and A549 for lung cancer) that express green fluorescent protein (EGFP) under miR-21 regulation. Those cells have a high endogenous miR-21 level, so expression of EGFP is prevented. For this, the aptamers can be covalently linked with an antimiR (so called aptamiR) for the cell specific delivery of these constructs for silencing of endogenous micro RNA. Those AptamiR chimeras should recognise and internalise into the cells with high specificity due to the aptamer domain, allowing the delivery of antimiR-21 and thus, inducing EGFP expression. AntimiRs derived from the initial set of selected aptamers were tested, showing low delivery efficiency of the antimiR-21 into the target cells, which correlates with low levels of EGFP expression. Within this context, the new selected aptamers that show improved targeting towards solid tumour cancer cells are promising candidates as carriers of antimiR-21 for silencing the endogenous miR-21. Therefore, their potential use as antimiR chimeras is currently under evaluation.
Regarding the target identification of the aptamers here described (Objective 3), a collaboration with the group of Prof. Silberring at the University of Krakow was established. Efforts have been focused in the optimisation of a pull-down assay to identify the biomolecule towards each aptamer has affinity for. Currently, the methodology has been established by using fluorescently labelled aptamers and the analysis of the pulled proteins by mass spectrometry is ongoing. Validation of those target candidates will be then evaluated using specific siRNAs in order to knock-down their expression and assess the binding behaviour of the aptamers or by knock-in assays, where the protein target is overexpressed. Binding assays with the fluorescently labelled aptamer will be performed then with those modified cells. Once the targets of the used aptamer panel are found, validation of the potential new biomarkers will be performed. Furthermore, with the new selected sequences showing improved binding to the analysed cells, the target identification with siRNA technology will be performed as reported in the proposal.
