Periodic Reporting for period 2 - BIOCELLPHE (Ultrasensitive BIOsensing platform for multiplex CELLular protein PHEnotyping at single-cell level)
Période du rapport: 2022-04-01 au 2023-09-30
Cancer is a leading cause of death worldwide with approximately 8.2 million deaths each year. The primary cause of death is the metastases that occur upon intravasation of circulating tumor cells (CTCs) into the blood circulation. CTCs can be phenotypically quite heterogeneous, contributing differently to metastasis, therapy resistance, and the subsequent clinical outcome. Therefore, their phenotypic characterization could significantly aid to improve tumor diagnostics, personalized treatment strategies, as well as a means to discover new therapeutic targets. However, the study of CTCs remains a technically challenging due to their extremely low numbers in the bloodstream. In this context, breakthrough technological advances for phenotypic analysis down to single-cell level are urgently needed to realize precision oncology.
BIOCELLPHE provides frontier scientific and technological advancements to generate a breakthrough technology realizing the identification of proteins as diagnostic biomarkers at single-cell level with unmatched sensitivity, multiplexing capabilities and portability. BIOCELLPHE proposes the generation of engineered bacteria able to recognize and bind to specific protein targets on the surface of CTCs responsible for cancer metastasis, thereby triggering the production of chemical signals that can be detected simultaneously, and with extremely high sensitivity by surface-enhanced Raman scattering (SERS). SERS is a powerful analytical technique that employs plasmonic nanoparticles for ultrasensitive chemical analysis achieving single-molecule detection level. These advancements will be implemented toward the generation of an optofluidic lab-on-a-chip (LoC) SERS device enabling ultrasensitive identification and multiplex phenotyping of CTCs. The substantial increase in sensitivity will facilitate the earlier diagnosis of the disease as well as the discovery of new therapeutic targets and biomarkers, driving individualized therapy and precision medicine.
BIOCELLPHE provides frontier scientific and technological advancements to generate a breakthrough technology realizing the identification of proteins as diagnostic biomarkers at single-cell level with unmatched sensitivity, multiplexing capabilities and portability. BIOCELLPHE proposes the generation of engineered bacteria able to recognize and bind to specific protein targets on the surface of CTCs responsible for cancer metastasis, thereby triggering the production of chemical signals that can be detected simultaneously, and with extremely high sensitivity by surface-enhanced Raman scattering (SERS). SERS is a powerful analytical technique that employs plasmonic nanoparticles for ultrasensitive chemical analysis achieving single-molecule detection level. These advancements will be implemented toward the generation of an optofluidic lab-on-a-chip (LoC) SERS device enabling ultrasensitive identification and multiplex phenotyping of CTCs. The substantial increase in sensitivity will facilitate the earlier diagnosis of the disease as well as the discovery of new therapeutic targets and biomarkers, driving individualized therapy and precision medicine.
WP1 involves the development of engineered E. coli pilot strains capable of specific attachment to surface-exposed biomarkers on human circulating tumor cells (CTCs) and producing Raman reporters (RaRs) molecules detectable by Surface Enhanced Raman Spectroscopy (SERS). To this end, the work first focused on the expression on E. coli surface of nanobodies binding to the purified ectodomains of validated biomarkers of clinical relevance in human cancer. Next, the genes encoding these intimin-nanobody fusions were inserted in the chromosome to be constitutively and stably expressed in the OM of bacteria.Then the E. coli strains attaching to tumour cells were further engineered with biosynthetic gene operons for RaR molecules. These operons were inserted in the chromosome of bacteria under the control of promoters. Finally, pilot E. coli strains have been fluorescently labelled by inserting gene constructs constitutively expressing different fluorescent protein reporters.
WP2 involves the generation of a chimeric OM protein able to bind a protein biomarker on the tumor cell surface and produce a signal in the periplasmic space that can be linked to an inner membrane (IM) protein transducer able to elicit a transcriptional response in the bacterium. The main outcomes are: a modular and generalizable trans-envelope signaling platform coupling OM sensors and IM transducers.
WP3 involves the selection of output RaR for multiplex SERS and the generation of biosynthesis pathways of output RaR. The main outcomes are: Shortlisting of RaRs has been carried out by (i) running machine learning programs trained on literature data and (ii) running a retrosynthesis software to assess the difficulties of engineering and optimizing the biosynthesis pathways in E. coli. Five potential RaRs were selected. The biosynthetic pathways producing these 5 RaRs have successfully been engineered in various E. coli strains. These pathways are being integrated in the genome of the BioCellPhe pilote strains and two strains have already shown to produce the targeted RaR.
WP4 involves the fabrication and scale-up of a library of plasmonic nanoparticles with well-defined size and shape, and the fabrication of the plasmonic detector to be implemented in the LoC-SERS and evaluation of the SERS performance. The main outcomes are: Fabrication of a library of gold nanoparticles (nanospheres, nanostars, nanorods and nanooctahedra). Implementation of layer-by-layer and e-beam lithography approaches for the fabrication of plasmonic nanostructured detector. Development of two plasmonic sensing platforms by LBL and template-assisted approaches. Evaluate the SERS performance of both plasmonic sensing platforms by the detection of the selected Raman reporters (Pyocyanin, deoxyviolacein, and violacein).
WP5 is focused on the fabrication, testing and validation of the optofluidic LoC-SERS BIOCELLPHE device. The main outcomes are: Simulation of microdroplet formation, passive sorting of empty droplets and trapping of cell containing droplets in the optofluidic module successfully achieved (T5.1); optimization of recovery parameters from the mass scaled RUBYchip™ from blood (T5.2); optimization of microfluidic modules for microdroplet encapsulation, sorting, trapping and plasmonic detection (T5.2); validation of breast cancer cell labelling with engineered E.Coli in isolation and encapsulation modules (T5.3) and ethical approval of clinical study including first 14 samples from BC patients shipped and under analysis (T5.4).
WP6 involves communication and Dissemination activities, exploitation, and personalized dissemination and exploitation campaign. The main outcomes are: Project web page and logo, dissemination and communication Plan, digital Communication Kit, BIOCELLPHE document templates, and exploitation plan.
WP2 involves the generation of a chimeric OM protein able to bind a protein biomarker on the tumor cell surface and produce a signal in the periplasmic space that can be linked to an inner membrane (IM) protein transducer able to elicit a transcriptional response in the bacterium. The main outcomes are: a modular and generalizable trans-envelope signaling platform coupling OM sensors and IM transducers.
WP3 involves the selection of output RaR for multiplex SERS and the generation of biosynthesis pathways of output RaR. The main outcomes are: Shortlisting of RaRs has been carried out by (i) running machine learning programs trained on literature data and (ii) running a retrosynthesis software to assess the difficulties of engineering and optimizing the biosynthesis pathways in E. coli. Five potential RaRs were selected. The biosynthetic pathways producing these 5 RaRs have successfully been engineered in various E. coli strains. These pathways are being integrated in the genome of the BioCellPhe pilote strains and two strains have already shown to produce the targeted RaR.
WP4 involves the fabrication and scale-up of a library of plasmonic nanoparticles with well-defined size and shape, and the fabrication of the plasmonic detector to be implemented in the LoC-SERS and evaluation of the SERS performance. The main outcomes are: Fabrication of a library of gold nanoparticles (nanospheres, nanostars, nanorods and nanooctahedra). Implementation of layer-by-layer and e-beam lithography approaches for the fabrication of plasmonic nanostructured detector. Development of two plasmonic sensing platforms by LBL and template-assisted approaches. Evaluate the SERS performance of both plasmonic sensing platforms by the detection of the selected Raman reporters (Pyocyanin, deoxyviolacein, and violacein).
WP5 is focused on the fabrication, testing and validation of the optofluidic LoC-SERS BIOCELLPHE device. The main outcomes are: Simulation of microdroplet formation, passive sorting of empty droplets and trapping of cell containing droplets in the optofluidic module successfully achieved (T5.1); optimization of recovery parameters from the mass scaled RUBYchip™ from blood (T5.2); optimization of microfluidic modules for microdroplet encapsulation, sorting, trapping and plasmonic detection (T5.2); validation of breast cancer cell labelling with engineered E.Coli in isolation and encapsulation modules (T5.3) and ethical approval of clinical study including first 14 samples from BC patients shipped and under analysis (T5.4).
WP6 involves communication and Dissemination activities, exploitation, and personalized dissemination and exploitation campaign. The main outcomes are: Project web page and logo, dissemination and communication Plan, digital Communication Kit, BIOCELLPHE document templates, and exploitation plan.
Single-cell level measurement of proteins can provide valuable insight into mechanisms involved in disease and can potentially transform personalized medicine. Conventional methods for analytical characterization of surface biomarkers at single cell level still face important limitations claiming for radically new approaches oriented to enhance sensitivity, multiplexing capabilities, low cost and portability. BIOCELLPHE will result in a robust device, suitable for low-cost implementation, with ultrasensitive and multiplexing capabilities, moving forward protein phenotyping at single-cell level far beyond what had previously been considered the upper limit of feasibility.
BIOCELLPHE initiated a radically new line of research and technology to unsolved diagnostic and healthcare challenge; Protein profiling of living single cells (i.e. CTCs) for tumor diagnostics. The impact of BIOCELLPHE will not only be that of a successful technology, but also the new generated knowledge in diverse areas: Core advancements in synthetic biology, mathematical simulation for programming new biological processes, as well as novel SERS-active optofluidic systems that will open the door for new opportunities where all invigorating parts implicated will profit. BIOCELLPHE will also stimulate other research activities such as: Artificial intelligence to program cell behavior, SERS-based biodetection, plasmonics, advanced microfluidics, new hardware and software technology for optical monitoring.
BIOCELLPHE initiated a radically new line of research and technology to unsolved diagnostic and healthcare challenge; Protein profiling of living single cells (i.e. CTCs) for tumor diagnostics. The impact of BIOCELLPHE will not only be that of a successful technology, but also the new generated knowledge in diverse areas: Core advancements in synthetic biology, mathematical simulation for programming new biological processes, as well as novel SERS-active optofluidic systems that will open the door for new opportunities where all invigorating parts implicated will profit. BIOCELLPHE will also stimulate other research activities such as: Artificial intelligence to program cell behavior, SERS-based biodetection, plasmonics, advanced microfluidics, new hardware and software technology for optical monitoring.