Periodic Reporting for period 3 - Tumor-LN-oC (Tumor and Lymph Node on Chip for cancer studies)
Okres sprawozdawczy: 2024-05-01 do 2025-10-31
Tumor-LN-oC has developed a robust, automated tumor-lymph node-on-chip platform connecting primary surgically removed human tumors and lymph node (LN) tissue from the same cancer patient. This "biological twin" facilitates the study of primary tumor interactions with lymph nodes.
The main objective pursues by the consortium was to create a Tumor-LN-oC platform to monitor LN metastasis, characterize signaling cues facilitating LN metastasis, and identify spectral and molecular signatures in metastasizing cells. This could lead to new diagnostic tools and therapeutic approaches. Additionally, the platform can serve as a preclinical setting for drug testing for individual lung cancer patients.
Specific objectives include:
1) To introduce novel designs and develop robust, automated microfluidic chips optimized for tumor cell and LN culture enabling the study of their crosstalk,
2) To integrate Quantum Cascade Laser based mid- IR spectroscopy for specific chemical signatures,
3) To molecularly characterize both migrating tumor-derived cells attracted to the LN and the soluble signals driving migration,
4) To demonstrate an advanced image analysis and signal processing platform using deep learning algorithms facilitated by a micro-optics module to monitor in real time the cells migration,
5) To integrate all Tumor-LN-oC technologies in an automated platform prototype incorporating interfaces compatible with existing laboratory equipment.
6) To demonstrate the Tumor-LN-oC TRL5 platform and validate it with real patient samples
7) To establish regulatory pathways and assure regulatory standards and requirements compliance during the development of the Tumor-LN-oC in order to facilitate exploitation and early market entry
The Tumor-LN-oC platform uses a novel multi-compartment microfluidic chip mimicking the tumor microenvironment and its lymphatic connection. Employing mid-IR Photothermal (MIP) spectroscopy and microlens array-based micro-optics, we can generate spectral "fingerprints" of metastasizing cells for diagnostic purposes. Laser-based bioprinting is used to precisely place LN and tumor cells within the chip with high viability. The platform has been developed at TRL5 and validated with real patient samples, considering regulatory pathways to facilitate market entry.
The main objective pursues by the consortium was to create a Tumor-LN-oC platform to monitor LN metastasis, characterize signaling cues facilitating LN metastasis, and identify spectral and molecular signatures in metastasizing cells. This could lead to new diagnostic tools and therapeutic approaches. Additionally, the platform can serve as a preclinical setting for drug testing for individual lung cancer patients.
Specific objectives include:
1) To introduce novel designs and develop robust, automated microfluidic chips optimized for tumor cell and LN culture enabling the study of their crosstalk,
2) To integrate Quantum Cascade Laser based mid- IR spectroscopy for specific chemical signatures,
3) To molecularly characterize both migrating tumor-derived cells attracted to the LN and the soluble signals driving migration,
4) To demonstrate an advanced image analysis and signal processing platform using deep learning algorithms facilitated by a micro-optics module to monitor in real time the cells migration,
5) To integrate all Tumor-LN-oC technologies in an automated platform prototype incorporating interfaces compatible with existing laboratory equipment.
6) To demonstrate the Tumor-LN-oC TRL5 platform and validate it with real patient samples
7) To establish regulatory pathways and assure regulatory standards and requirements compliance during the development of the Tumor-LN-oC in order to facilitate exploitation and early market entry
The Tumor-LN-oC platform uses a novel multi-compartment microfluidic chip mimicking the tumor microenvironment and its lymphatic connection. Employing mid-IR Photothermal (MIP) spectroscopy and microlens array-based micro-optics, we can generate spectral "fingerprints" of metastasizing cells for diagnostic purposes. Laser-based bioprinting is used to precisely place LN and tumor cells within the chip with high viability. The platform has been developed at TRL5 and validated with real patient samples, considering regulatory pathways to facilitate market entry.
Tumor-LN-oC has successfully developed and optimized individual platform components and prepared them for their integration in the final prototype in the last months of the project.
Objective 1: We have designed and fabricated microfluidic chips with artificial cilia for continuous recirculatory flow for culturing tumor and LN cells and tissue samples. The chips were validated for long-term cell culture with cell lines and surgical samples.
Objective 2: Tumor-LN-oC consortium successfully integrated the EC-QCL laser source with the MIP spectroscopy module and completed comprehensive testing and calibration of the system. The integrated MIP system has demonstrated full functionality within the Tumor-LN-oC platform, including successful analysis of tissues and cells in fluidic environments.
Objective 3: We have isolated lymph-like media in the upper section of the Tumor-LN-oC chips; cytokines identified from the patient-derived samples were highly variable and demonstrated patient-specific expression. We have identified “druggable” targets for a more personalized approach in cancer treatment, with findings further implicating Jagged1 as a regulator of both the protein matrix surrounding tumors and cell movement in breast cancer.
Objective 4: The micro-optics module, based on microlens arrays, creates images with an FOV of 8.4 x 6 mm and an optical resolution of 3.9 μm. The high-level control software integrates all system functionalities. Machine learning was used for cell segmentation and motion quantification, enabling real-time monitoring of directional cell motion. Extensive user feedback has resulted in a software package which can be deployed for multiple days to acquire migration data from the microfluidic channels.
Objective 5: All modules were integrated into a functional TRL5 Tumor–LN-on-Chip prototype, with demonstrated troubleshooting, stable fluidic control and long term live cell culture across multiple chip configurations and cell/tissue types. The platform supported real-time imaging of single cell migration, maintained viable cultures under flow, and enabled mid-infrared photothermal spectroscopy in microfluidic chips.
Objective 6: Tumor-LN-oC platform underwent extensive optimization to support metastasis diagnosis and molecular characterization of migrating tumor cells. Cells cultured on the microfluidic platform remained significantly healthier than those in traditional Transwell (TW) systems. These results confirm that tumour slices cultured under continuous flow on the Tumor-LN-oC generate sufficient, high-quality mRNA for downstream analysis of migrating versus non migrating tumour cells.
Objective 7: The regulatory roadmap for metastasis diagnosis and drug testing applications was generated, identifying applicable guidelines and technical standards. 2 meeting with the EMA’s innovation task force were held. A comprehensive regulatory whitepaper summarizes lessons learned and outlining the standards, guidelines, and strategic pathways relevant to future market authorization. The exploitation and commercialization plan provides a foundation for future exploitation and continued cooperation of partners.
Objective 1: We have designed and fabricated microfluidic chips with artificial cilia for continuous recirculatory flow for culturing tumor and LN cells and tissue samples. The chips were validated for long-term cell culture with cell lines and surgical samples.
Objective 2: Tumor-LN-oC consortium successfully integrated the EC-QCL laser source with the MIP spectroscopy module and completed comprehensive testing and calibration of the system. The integrated MIP system has demonstrated full functionality within the Tumor-LN-oC platform, including successful analysis of tissues and cells in fluidic environments.
Objective 3: We have isolated lymph-like media in the upper section of the Tumor-LN-oC chips; cytokines identified from the patient-derived samples were highly variable and demonstrated patient-specific expression. We have identified “druggable” targets for a more personalized approach in cancer treatment, with findings further implicating Jagged1 as a regulator of both the protein matrix surrounding tumors and cell movement in breast cancer.
Objective 4: The micro-optics module, based on microlens arrays, creates images with an FOV of 8.4 x 6 mm and an optical resolution of 3.9 μm. The high-level control software integrates all system functionalities. Machine learning was used for cell segmentation and motion quantification, enabling real-time monitoring of directional cell motion. Extensive user feedback has resulted in a software package which can be deployed for multiple days to acquire migration data from the microfluidic channels.
Objective 5: All modules were integrated into a functional TRL5 Tumor–LN-on-Chip prototype, with demonstrated troubleshooting, stable fluidic control and long term live cell culture across multiple chip configurations and cell/tissue types. The platform supported real-time imaging of single cell migration, maintained viable cultures under flow, and enabled mid-infrared photothermal spectroscopy in microfluidic chips.
Objective 6: Tumor-LN-oC platform underwent extensive optimization to support metastasis diagnosis and molecular characterization of migrating tumor cells. Cells cultured on the microfluidic platform remained significantly healthier than those in traditional Transwell (TW) systems. These results confirm that tumour slices cultured under continuous flow on the Tumor-LN-oC generate sufficient, high-quality mRNA for downstream analysis of migrating versus non migrating tumour cells.
Objective 7: The regulatory roadmap for metastasis diagnosis and drug testing applications was generated, identifying applicable guidelines and technical standards. 2 meeting with the EMA’s innovation task force were held. A comprehensive regulatory whitepaper summarizes lessons learned and outlining the standards, guidelines, and strategic pathways relevant to future market authorization. The exploitation and commercialization plan provides a foundation for future exploitation and continued cooperation of partners.
The Tumor-LN-oC project has introduced the first artificial model realistically mimicking tumor and LN crosstalk, offering a novel personalized paradigm (biological twin) with no existing alternatives. It is an automated platform for studying tumor biology and personalized approaches in biomarker and drug target identification and preclinical testing of therapeutic schemes, which has been tested and validated at TRL5. The integrated Tumor-LN-oC platform addresses several unmet needs and will achieve the following impacts:
1. Verifiable progress in the application of Organ-on-Chip technologies for in-vitro research;
2. Reduction of the need for animal and clinical testing;
3. Lowering of barriers for application of Organ-on-Chip technology;
4. Improved competitiveness and attractiveness of the European biomedical and healthcare sector
5. Increased awareness and knowledge about medical regulatory policies and requirements, especially by academics and SMEs.
User-friendly software and interfaces with standard lab equipment ensure accessibility to non-experts in microfluidics, broadening the user base. Innovations in microfluidics, cell culture, tissue engineering, real-time detection, and monitoring significantly advance these interdisciplinary fields. Integrating the chip with spectroscopic detection tools and image analysis technologies further enhance the platform's value.
1. Verifiable progress in the application of Organ-on-Chip technologies for in-vitro research;
2. Reduction of the need for animal and clinical testing;
3. Lowering of barriers for application of Organ-on-Chip technology;
4. Improved competitiveness and attractiveness of the European biomedical and healthcare sector
5. Increased awareness and knowledge about medical regulatory policies and requirements, especially by academics and SMEs.
User-friendly software and interfaces with standard lab equipment ensure accessibility to non-experts in microfluidics, broadening the user base. Innovations in microfluidics, cell culture, tissue engineering, real-time detection, and monitoring significantly advance these interdisciplinary fields. Integrating the chip with spectroscopic detection tools and image analysis technologies further enhance the platform's value.