Periodic Reporting for period 2 - REAP (Revealing drug tolerant persister cells in cancer using contrast enhanced optical coherence and photoacoustic tomography)
Reporting period: 2022-07-01 to 2023-12-31
Cancer treatment faces a major problem: it ultimately stops working for many patients because the tumor becomes resistant. The cellular origin of relapse is often linked to drug tolerant persister (DTP) cells, which survive treatment and can remain for years. Because of their scarcity and heterogeneity, the detection of DTP cells remains a technological challenge of enormous clinical importance. The objective of REAP is to develop two next generation multimodal imaging systems to reveal DTPs. A triple modal two-photon laser scanning optical coherence photoacoustic microscopy system will be built for the in vitro characterization of cancer organoids. Additionally, a dual-modality optical coherence photoacoustic tomography system will be implemented to visualize tumors in vivo in a mouse model. To enable greatly increased sensitivity and specificity, a new type of contrast agent based on biofunctionalized nanoparticles with tailor-made optical properties will be fabricated to specifically label DTPs. For improved imaging performance, several further technological advancements are targeted. Photoacoustic excitation will be realized using innovative microchip lasers addressing the needs for high-energy pulses, high-repetition rate, and multi-wavelength emission. To achieve the required resolution, novel photoacoustic detectors based on integrated optical micro-ring resonator technology will be developed with the potential to completely replace conventional piezoelectric ultrasound transducers. Furthermore, image acquisition speed will be increased by an order of magnitude with the help of an innovative laser source based on photonic integrated circuits at 780 nm. Finally, real-time data handling will be explored along with deep learning-based automatic analysis algorithms. The combined innovation in laser sources, detector technology, nanoparticles, and deep learning-based algorithms will create radically new imaging solutions reaching numerous applications.
The dissemination, exploitation, and communication activities are led by MIL (previously known as LVBT). The Innovation Manager Dr. Julia Ide from MIL is in charge of this work package. During this reporting period, the IPR overview has been regularly updated with the input from the consortium. Additionally, a standardization workshop has been held to familiarize all partners of the consortium with relevant standards and guidelines for REAP (D9.5). In order to increase the exploitation potential, the dissemination and exploitation plan was updated to reflect the current status and knowledge of the project (D9.10). All required deliverables of WP9 have been submitted.
The biology and chemistry related tasks are performed by the Center for Cancer Research (CCR) of MUW, AIT, and USC in WP2 and WP3. WP2 completed task T2.1 “in vitro model of drug induced tumor relapse: repopulation assay” with all its milestones and deliverables. This task included the transcriptional profiling of DTP cells of the model, which had high exploitation power in the selection of the NP targeting strategy. In addition, in vitro biocompatibility testing of the NPs on DTP cells within the frame of this task was an essential feasibility test. In task T2.2 “Establishment of in vitro 3D mammary organoids“, establishment and validation of organoids from metastatic mammary tumors was completed, they will be utilized in whole-body course scan OC-PAT settings of WP8. In addition, cancer associated fibroblast (CAF) cell lines derived from the tumor models have been established, which will contribute to the complex in vitro 3D structures for drug analysis and imaging. Although task T2.3 “Genetic engineering of organoids“, has a slight delay, the established CAFs were successfully engineered to express reporters with complementary fluorescence with the matched organoids, which will allow the simultaneous detection of tumor cells and CAFs via 2-photon microscopy. In tasks T2.4 and 2.5 the “in vitro tumor organoid” and the “in vivo organoid-derived transplantation models”, the complete experimental pipeline is established and optimized, including the type of drugs, the applied concentrations and timings, the expected timings of the drug-induced phenotypic stages (DTP/MRD and repopulation/relapse). The missing step shared for both tasks is the validation of protocols with the final engineered organoids that will allow PAI. In addition, in T2.4 we are optimizing the CAF-organoid co-culture conditions and in T2.5 upon obtaining the ethical approval the live-animal implantation and longitudinal testing of the optical window will be performed.
The biology and chemistry related tasks are performed by the Center for Cancer Research (CCR) of MUW, AIT, and USC in WP2 and WP3. WP2 completed task T2.1 “in vitro model of drug induced tumor relapse: repopulation assay” with all its milestones and deliverables. This task included the transcriptional profiling of DTP cells of the model, which had high exploitation power in the selection of the NP targeting strategy. In addition, in vitro biocompatibility testing of the NPs on DTP cells within the frame of this task was an essential feasibility test. In task T2.2 “Establishment of in vitro 3D mammary organoids“, establishment and validation of organoids from metastatic mammary tumors was completed, they will be utilized in whole-body course scan OC-PAT settings of WP8. In addition, cancer associated fibroblast (CAF) cell lines derived from the tumor models have been established, which will contribute to the complex in vitro 3D structures for drug analysis and imaging. Although task T2.3 “Genetic engineering of organoids“, has a slight delay, the established CAFs were successfully engineered to express reporters with complementary fluorescence with the matched organoids, which will allow the simultaneous detection of tumor cells and CAFs via 2-photon microscopy. In tasks T2.4 and 2.5 the “in vitro tumor organoid” and the “in vivo organoid-derived transplantation models”, the complete experimental pipeline is established and optimized, including the type of drugs, the applied concentrations and timings, the expected timings of the drug-induced phenotypic stages (DTP/MRD and repopulation/relapse). The missing step shared for both tasks is the validation of protocols with the final engineered organoids that will allow PAI. In addition, in T2.4 we are optimizing the CAF-organoid co-culture conditions and in T2.5 upon obtaining the ethical approval the live-animal implantation and longitudinal testing of the optical window will be performed.
The ultimate goal of REAP can be broken down into individual objectives in each technology involved in the project.
• Contrast agent: biofunctionalized contrast agent will be developed to target the breast cancer cells. The contrast agent will feature tunable absorption peaks specifically designed to enhance the contrast in photoacoustic imaging (PAI).
• Photoacoustic Detector: micro-ring resonator (MRR) based detector with ≤50 μm diameter and ≥160 MHz bandwidth will be developed. Together with the interrogation laser developed in this project, we target at sub-Pa noise equivalent pressure (NEP). MRR arrays will also be developed for real time PAT imaging.
• Laser: triple wavelength photoacoustic microscopy (PAM) excitation laser will be developed with high repetition rate (100 kHz), high energy (1 μJ), low cost (5 k€), and compact size (15 × 15 × 5 cm3). An optical parametric oscillator (OPO) will be developed for PAT excitation with exceptionally high repetition rate (≥ 1 kHz) and energy (≥ 10 mJ). Two dual-purpose lasers will be developed for both all optical detection photoacoustic interrogation and optical coherence tomography (OCT) at 780 nm and 1310 nm with wide bandwidth (40 nm and 100 nm, respectively), narrow linewidth and stable phase.
• System: horizontal 2PLS-OC-PAM system will be developed with subcellular resolution, deep penetration (≥1 mm from one side and ≥2 mm with sample rotation), multiple contrast channels (fluorescence contrast for two- photon laser scanning microscopy (2PLSM), scattering contrast for OCT, absorption contrast for PAM), and fast imaging speed. A high-resolution OC-PAT system will be developed using planar MRR array for localized tumor imaging and image-guided biopsy. A rectangular-MRR-array-based photoacoustic tomography (PAT) system will be developed for deep tissue (≥2 cm penetration depth) imaging and metastasis screening.
• Algorithm: real time image reconstruction algorithms will be developed for all the imaging modalities involved. Needle tracing algorithm will be developed for image-guided biopsy. 3D visualization and quantification algorithms will be developed to perform quantitative analysis of the tumor and the DTP cells.
• Contrast agent: biofunctionalized contrast agent will be developed to target the breast cancer cells. The contrast agent will feature tunable absorption peaks specifically designed to enhance the contrast in photoacoustic imaging (PAI).
• Photoacoustic Detector: micro-ring resonator (MRR) based detector with ≤50 μm diameter and ≥160 MHz bandwidth will be developed. Together with the interrogation laser developed in this project, we target at sub-Pa noise equivalent pressure (NEP). MRR arrays will also be developed for real time PAT imaging.
• Laser: triple wavelength photoacoustic microscopy (PAM) excitation laser will be developed with high repetition rate (100 kHz), high energy (1 μJ), low cost (5 k€), and compact size (15 × 15 × 5 cm3). An optical parametric oscillator (OPO) will be developed for PAT excitation with exceptionally high repetition rate (≥ 1 kHz) and energy (≥ 10 mJ). Two dual-purpose lasers will be developed for both all optical detection photoacoustic interrogation and optical coherence tomography (OCT) at 780 nm and 1310 nm with wide bandwidth (40 nm and 100 nm, respectively), narrow linewidth and stable phase.
• System: horizontal 2PLS-OC-PAM system will be developed with subcellular resolution, deep penetration (≥1 mm from one side and ≥2 mm with sample rotation), multiple contrast channels (fluorescence contrast for two- photon laser scanning microscopy (2PLSM), scattering contrast for OCT, absorption contrast for PAM), and fast imaging speed. A high-resolution OC-PAT system will be developed using planar MRR array for localized tumor imaging and image-guided biopsy. A rectangular-MRR-array-based photoacoustic tomography (PAT) system will be developed for deep tissue (≥2 cm penetration depth) imaging and metastasis screening.
• Algorithm: real time image reconstruction algorithms will be developed for all the imaging modalities involved. Needle tracing algorithm will be developed for image-guided biopsy. 3D visualization and quantification algorithms will be developed to perform quantitative analysis of the tumor and the DTP cells.