Cells-on-chip models for integrated therapies based on immunotherapy through macrophage polarization: overcoming resistance to conventional treatments for stem compartments in larger cell lung cancers in non small cell lung cancers
Project objectives
The most recent findings indicate the tumor stem cell compartment as the main reason for progression and drug resistance in lung cancer. In particular, it emerges these factors are only partly due to the genetic or epigenetic changes that are intrinsic to the cell. Instead, changes are predominantly derived from the interplay between cancer stem cells (CSC) and their microenvironment.
Tumor-Associated Macrophages (TAM) are the most common cells within this microenvironment. Even if they have high potential for cytotoxic and antitumor activity, when they are M1 polarization they are "diverted" by the tumor to assume a type M2 configuration in most cases. On the contrary, the M2 configuration is able to preserve the stem characteristics of tumor cells.
Re-educating these cells to recommence an antitumor function would help break a vicious circle that in clinical reality leads to an inevitable loss in the effectiveness of currently available treatments so opening up the way to integrated therapeutic approaches that combine conventional therapies and immunotherapy. However, given the inadequacy of conventional in vivo and in vitro techniques, new investigative tools are needed that enable a greater understanding of the interaction mechanisms between tumor and immune systems in order to optimize these approaches.
The cells-on-chip approach, which uses microfluidic and co-culture cell techniques to replenish and check cellular microenvironment while maintaining compatibility with the latest analysis systems and microscopy, makes the contemporary study of cellular interactions possible for both single cell and populations. In fact, it represents the frontier for in vitro and ex vivo models. A biochip for the co-culture of cancer cells and immune system cells applied to lung cancer has therefore been used in order to assess the effects of integrated therapy on CSC survival in a highly controlled setting.
Materials and Methods
Cancer cells are taken from patient pleural aspirate. From these the stem cell compartment (EpCAM, CD133) is isolated and , in combination with a human macrophage line, are engineered to express the GFP-Mannose Receptor fusion protein. Then, via stable silencing of IRF4, to be a refractory to M2 activation as a result of M2 polarization.
cocultured with a human macrophage cell line engineered to express the GFP-Mannose Receptor fusion protein as a result of M2 polarization and, through stable silencing of IRF4, to be refractory to M2 activation.
A chip that has already been developed and validated by a project partner (IFN-CNR, Rome) is used to study the tumor / immune system interactions. CSCs are maintained in a spheroidal culture on the chip in the presence of TAM (GFP-MR+). Using time-lapse microscopy is possible to assess:
- The kinetics and the extension of the macrophage infiltration within the tumor.
- The TAM polarization in the M1 / M2 sense, by means of fluorescent analysis in quantity, spatial and temporal terms.
- The resistance of the CSC to treatment with chemotherapeutic drugs associated with the presence of TAM (wildtype or silenced for IRF4).
Expected results
The project aims to investigate the crosstalk between CSC and TAM as a cause of increased CSC chemoresistance with the aim of developing new and improved integrated approaches based on chemo and immunotherapy. The expected project results are:
- Temporal spatial characterization of the crosstalk between CSC and TAM subpopulations
- Understanding the protective mechanism of TAM M2 for CSC against chemotherapy
- Evaluation of the effect of IRF4 silencing on the stabilization of M1 macrophages resulting in increased CSC toxicity
Start/End Dates
June 2013 - June 2015
Principal Investigator
Prof. Daniele Santini - Scientific Coordinator and Principal Investigator
Host Institution
Campus Bio-Medico University of Rome
Source of funding
Fondazione G. Berlucchi per la Ricerca sul Cancro