Periodic Reporting for period 3 - FanCY (Flow and Deformation of Cancer tumours near Yielding)
Periodo di rendicontazione: 2022-04-01 al 2023-09-30
The fight against cancer is long-standing, with the focus of intense research aimed at suppressing cell invasion into surrounding tissues (based on a molecular basis). Often, primary tumours do not kill patients, but secondary tumours do. These so-called metastatic tumour cells disassociate from a primary tumour and, ultimately, prove fatal. Cancer cell migration (associated with cancer spread) into the surrounding extracellular matrices (ECM) and tumor-microenvironment (TME) is a critical step at the early stages of cancer metastasis. Currently, we do not understand the fundamentals of the mechanical pathways and mechanisms of the metastasis of cancer, hampering medical intervention.
The project will have an important impact on our understanding of the physics and biomechanics of cancers. The mechanical properties of the invaded cells are key to metastasis. This work will provide new platforms for cancer cell invasion studies and drug screening applications. The outcomes will also lead to the design of a novel strategy to study cell migration and invasion in the presence of external forces, facilitating cell metastasis and treatment based on mechanical pathways. The outcomes will significantly aid the treatment of cancer in the near future by bridging the gap between chemical and mechanical pathways of cancer metastasis.
As specified in the proposal, we have focused on the mechanical pathway of metastasis based on micro/nano-engineering approaches in the first period of the project. The overall aim of the project is to understand when, how, and why cancer tumor cells detach and disassociate from a primary tumor. In addition, we aim to illustrate the interplay between tumour cell rheology, confinement and cellular motion and pinpoint the physical factors determining invasion behaviour in solid tumours. To obtain these objectives, it is important to create good cellular models (e.g. cancer spheroids) for rheological and mechanical characterizations.
All research activities are underway as anticipated in the project. First, we developed several novel microfluidics to create well-controlled spheroids models. In addition, we have developed several new platforms to investigate the response of cancer spheroids in the presence of different TME and external forces.
The project will have an important impact on our understanding of the physics and biomechanics of cancers. The mechanical properties of the invaded cells are key to metastasis. This work will provide new platforms for cancer cell invasion studies and drug screening applications. The outcomes will also lead to the design of a novel strategy to study cell migration and invasion in the presence of external forces, facilitating cell metastasis and treatment based on mechanical pathways. The outcomes will significantly aid the treatment of cancer in the near future by bridging the gap between chemical and mechanical pathways of cancer metastasis.
As specified in the proposal, we have focused on the mechanical pathway of metastasis based on micro/nano-engineering approaches in the first period of the project. The overall aim of the project is to understand when, how, and why cancer tumor cells detach and disassociate from a primary tumor. In addition, we aim to illustrate the interplay between tumour cell rheology, confinement and cellular motion and pinpoint the physical factors determining invasion behaviour in solid tumours. To obtain these objectives, it is important to create good cellular models (e.g. cancer spheroids) for rheological and mechanical characterizations.
All research activities are underway as anticipated in the project. First, we developed several novel microfluidics to create well-controlled spheroids models. In addition, we have developed several new platforms to investigate the response of cancer spheroids in the presence of different TME and external forces.
The first step of the project, as specified in the proposal, has focussed on the preparation of cancer spheroids and microfluidics fabrications. The beginning of 2019, has been used to screen and select suitable researchers for this ERC project. During their first year, these Ph.D. students have followed several courses, made an extensive literature overview of their research topics, and started with carrying out their first experiments and modeling. To this end, a new protocol for the formation of cancer spheroids has been developed and micro-scale platforms were prepared and tested for the cancer invasion and spheroid deformation. Currently, we are preparing a journal paper about this work. In addition, we published a perspective article on Spheroid mechanics and implications for cell invasion (published in Advances in Physics: X). Furthermore, we are also preparing an article on using programable micro-planforms for cancer cell invasion. Finally, we are working to use microfluidics platforms for the rheological characterization of cancer spheroids. In the meantime, we have submitted another perspective article on the application of microfluidics on 3D collective cell migration (in Trends in Cancer). Finally, we have created biomimetic ECM for cancer cell invasion with controlled porosity and stiffness for cancer migration studies (associated with key invasion biomarkers such as epithelial-mesenchymal transition-EMT).
We have purchased all relevant and necessary equipment at the beginning of the project (inverted fluorescence microscope with Hamamatsu Orca Flash camera, Micro-tester cell scale for spheroids deformation) and upgraded our optical tweezers systems for cancer cell manipulations and characterization.
Finally, several collaborative publications have been obtained via close collaboration with Leiden University Medical Center (LUMC) and Erasmus-MC colleagues by using novel microfluidics for cell migration studies, cell and micro-patterned interactions, and correlation of cytoskeletal elements to the cell membrane properties.
We have purchased all relevant and necessary equipment at the beginning of the project (inverted fluorescence microscope with Hamamatsu Orca Flash camera, Micro-tester cell scale for spheroids deformation) and upgraded our optical tweezers systems for cancer cell manipulations and characterization.
Finally, several collaborative publications have been obtained via close collaboration with Leiden University Medical Center (LUMC) and Erasmus-MC colleagues by using novel microfluidics for cell migration studies, cell and micro-patterned interactions, and correlation of cytoskeletal elements to the cell membrane properties.
Metastasis of tumors is regarded as the largest contributor to cancer-related deaths, but physiologically relevant tumor models for cancer cell metastasis researches are still lacking. Although the two-dimensional and three-dimensional tumor models are widely studied, they still exist drawbacks in recapitulating the complex and heterogeneous tumor microenvironment, especially in mimicking precise spatial organization of spheroids and stroma, and the dynamic release of cytokines.
Cellular interaction and adhesion determine the dynamics of cancer cell invasion (associated with EMT features) through complex tumor micro-environment (TME), but it has remained inaccessible due to the lack of proper in–situ analysis techniques for quantification of EMT in presence of physical forces and complexity of the TME feature. We have studied the EMT features in lung cancer spheroids (made from A549 cell lines) coupled with TGF-beta and ECM stiffness and in–situ analysis by video imaging, revealing cell migration modes and strategies under different TME conditions. In this work, we developed a new microbuckets-hydrogel (Mb-H) micro platform for cancer cell invasion. It can be integrated with spatial controlled release of cytokine transforming growth factor-beta (TGFβ) and further programmed with multiple functions to better mimic the complex in vivo microenvironment. Based on this micro platform, multi-cancer cell spheroids were formed, the guiding relationship of single-cell migration and collective cell migration with epithelial-mesenchymal transition (EMT) were demonstrated, and the temporal-spatial controlled manipulation of 3D invasive migration of cancer cells was realized. This programmable and adaptable micro platform addresses the current limitation of constructing an artificial 3D microenvironment by combining microfluidics and soft matter technology to supply an ingenious strategy for 3D cancer cell invasion researches. It takes a new step towards mimicking dynamically changing tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, and drug screening to allow for improved personalized treatments of cancer patients. Moreover, it opens a new avenue for recreating 3D in vivo microenvironment and will inspire more future research in this arena.
In addition, we have studied the role of co-culture and interstitial flow (IF) on cancer cell migration and invasion of cancer spheroids, which provide mechanistic insights about in vivo conditions on cancer cell invasion coupled with TME key features (e.g. ECM stiffness, TGF-beta, and shear forces). We showed that TME features can be mimicked precisely and robustly in laboratory conditions (in vitro conditions) by combining microfluidics and soft matter technologies. This work does not only provide fundamental insights into the cancer cell invasion through complex TME but also shows how to describe cell migration mode through complex TME and combine different techniques to characterize TME features with cancer cell migration/invasion.
Cellular interaction and adhesion determine the dynamics of cancer cell invasion (associated with EMT features) through complex tumor micro-environment (TME), but it has remained inaccessible due to the lack of proper in–situ analysis techniques for quantification of EMT in presence of physical forces and complexity of the TME feature. We have studied the EMT features in lung cancer spheroids (made from A549 cell lines) coupled with TGF-beta and ECM stiffness and in–situ analysis by video imaging, revealing cell migration modes and strategies under different TME conditions. In this work, we developed a new microbuckets-hydrogel (Mb-H) micro platform for cancer cell invasion. It can be integrated with spatial controlled release of cytokine transforming growth factor-beta (TGFβ) and further programmed with multiple functions to better mimic the complex in vivo microenvironment. Based on this micro platform, multi-cancer cell spheroids were formed, the guiding relationship of single-cell migration and collective cell migration with epithelial-mesenchymal transition (EMT) were demonstrated, and the temporal-spatial controlled manipulation of 3D invasive migration of cancer cells was realized. This programmable and adaptable micro platform addresses the current limitation of constructing an artificial 3D microenvironment by combining microfluidics and soft matter technology to supply an ingenious strategy for 3D cancer cell invasion researches. It takes a new step towards mimicking dynamically changing tumor microenvironment and exhibits wide potential applications in cancer research, bio-fabrication, and drug screening to allow for improved personalized treatments of cancer patients. Moreover, it opens a new avenue for recreating 3D in vivo microenvironment and will inspire more future research in this arena.
In addition, we have studied the role of co-culture and interstitial flow (IF) on cancer cell migration and invasion of cancer spheroids, which provide mechanistic insights about in vivo conditions on cancer cell invasion coupled with TME key features (e.g. ECM stiffness, TGF-beta, and shear forces). We showed that TME features can be mimicked precisely and robustly in laboratory conditions (in vitro conditions) by combining microfluidics and soft matter technologies. This work does not only provide fundamental insights into the cancer cell invasion through complex TME but also shows how to describe cell migration mode through complex TME and combine different techniques to characterize TME features with cancer cell migration/invasion.