Periodic Reporting for period 3 - INTERCELLMED (SENSING CELL-CELL INTERACTION HETEROGENEITY IN 3D TUMOR MODELS:TOWARDS PRECISION MEDICINE)
Okres sprawozdawczy: 2021-02-01 do 2022-04-30
Precision oncology and personalized cancer treatment aim to determine an optimal therapy for each patient. However, translation of results of drug testing performed on patient‐derived tumor samples to the clinic is highly inefficient, partly because these models (e.g. xenografts, tumor tissue slices, and 2D cell cultures) do not sufficiently reflect the heterogeneity and complexity of human tumors. Therefore, there is an urgent need for novel patient-derived tumor models and novel functional testing capable of improving the care of cancer patients through better therapy monitoring, new drug development and rational treatment planning. The main goal of INTERCELLMED is to develop a platform for next-generation chemosensitivity assay producing 3D cell cultures that mimic the complex tumour-stroma interactions while measuring the changes of key analytes that are involved in regulation of crucial physiological mechanisms, such as K+, pH and O2 levels. The first objective of INTERCELLMED is to develop new protocols for synthesis of particles-based optical sensors sensitive to changes of K+, pH and oxygen. The second objective of INTERCELLMED is to create a biocompatible 3D matrix integrating K+, pH and O2 sensing elements and capable to induce formation of tumour-like structures (i.e. tumoroids) by using a rapid, inexpensive and reproducible synthesis method. The third objective is to validate the 3D sensing scaffold systems for pharmacological studies of anticancer therapy, revealing how K+, pH and O2 changes in and around cells correlate with cell response to drugs. The validation is focused on pancreatic cancer that is one of the most heterogeneous cancers, resulting in high resistance to therapy, with a dismal five-year survival rate of only 7 percent. Our approach is very important for the society because the development of a platform for sensing the tumour microenvironment and cell:cell interactions in 3D in vitro patient-derived tumour models is considered a crucial prerequisite for successful personalized drug development and treatment planning.
So far, most effort has been focused on the synthesis and characterization of new silica-based ratiometric optical sensors for measuring pH, O2 and K+ analytes with average diameter 1.3 µm and a very uniform size distribution. Notably, we developed new functionalization strategies to stably bind the reporter dyes and the reference dyes to the silica particles while maintaining the high sensitivity of the probes. We have also studied, in more detail, the influence of surface charge modification of silica microparticles on pH sensitivity. The success in synthesizing solid silica microparticles motivated us to move on to synthesize silica microrods which has larger aspect ratio compared to spherical particles. This variation in aspect ratio will be further used as an additional parameter in differentiating different types of sensing particles during fluorescence microscopy of 3D cell cultures. We have also focused on developing new procedures for efficiently grow tumor and stromal cells in biocompatible matrixes (e.g. hydrogels) and we also compared several protocols for ideal embedment of our sensing particles into the 3D cell-seeded matrixes. In another study, we developed a new model to observe what happens when two different cell populations (i.e. tumor and stromal) are free to move and to communicate with each other, moving closer or further away, and to form the complex structures that we find inside the PDAC. Notably, we used ad hoc made algorithms to precisely track cells during drug treatment gaining new insights about the microscopic interactions that regulate the very first steps of tumor developments, when tumor and stromal cells are still very distinct entities.
The project INTERCELLMED is now proceeding accordingly to the planned activities and to date very promising results in the field of developing new methodologies for quantifying cell:cell interactions in tumour-stromal co-cultures during drug treatment have been achieved going beyond the state of the art. We are also advancing the field of 3D in vitro tumour models either by developing new silica microparticles that can measure pH and O2 changes by means of fluorescence time lapse microscopy or by setting new protocols that allow to integrate the sensing particles inside biocompatible matrices to be used for 3D co-cultures of tumour and stromal cells. We are additionally progressing with computational analyses by implementing proper mathematical models for calibrating the sensing particles and precisely quantifying local changes of analytes in the cell cultures over time. These studies are crucial in order to validate, within the second portion of the project, the sensing platforms for spatio-temporal mapping K+, pH and O2 gradients in 3D in vitro tumour models and for inferring the altered metabolism and heterogeneity during drug testing.