Integrated therapies based on immunotherapy through macrophage polarization

Project objectives

The most recent findings point to the tumor stem cell component as the primary driver of progression and chemoresistance in lung cancer. Specifically, it emerges that these characteristics are only partially due to cell-intrinsic genetic or epigenetic modifications, instead attributing them predominantly to the interplay between tumor stem cells (CSCs) and the microenvironment. Tumor-Associated Macrophages (TAMs) represent the most abundant cell component within this microenvironment. Although they possess potentially high cytotoxic and antitumor activity when they adopt the so-called M1 polarization, they are in most cases "hijacked" by the tumor to adopt an M2 configuration, which, conversely, is able to preserve the stem-like characteristics of tumor cells. Re-educating these cells to resume an antitumor function would help break a vicious cycle that, in clinical practice, inevitably leads to the loss of efficacy of currently available treatments, paving the way for integrated therapeutic approaches combining conventional therapies and immunotherapy. Optimizing these approaches, however, requires new investigative tools that allow for a greater understanding of the mechanisms of tumor/immune system interaction, given the inadequacy of conventional in vivo and in vitro techniques.

The cells-on-chip approach, which uses microfluidic techniques and cell coculture to reconstitute and control the cellular microenvironment while maintaining compatibility with state-of-the-art analysis and microscopy systems, enables the simultaneous study of cellular interactions, both single cells and populations, and represents the cutting edge of in vitro and ex vivo models. A biochip is therefore used to coculture tumor cells and immune cells in lung cancer, evaluating, in the highly controlled setting of the chip, the effects of integrated therapy on CSC survival.

Materials and methods

Tumor cells collected from patient pleural aspirates, from which the stem cell compartment (EPCAM+, CD133+) is isolated, are used in combination with a human macrophage line, engineered to express GFP-Mannose Receptor fusion protein following M2 polarization and, through stable silencing of IRF4, to be refractory to M2 activation. A chip developed and validated by a project partner (CNR-IFN, Rome) is used to study tumor/immune system interactions. The CSCs are maintained in spheroid culture on a chip in the presence of TAM (GFP-MR+), and time-lapse microscopy allows for the following to be assessed:

1. the kinetics and extent of macrophage infiltrate within the tumor

2. TAM polarization in the M1/M2 direction, through quantitative, spatial and temporal fluorescence analysis

3. CSC resistance to chemotherapy drug treatment associated with the presence of TAMs (wildtype or IRF4-silenced).

Expected results

The project aims to investigate crosstalk between CSCs and TAMs as a cause of increased chemoresistance in CSCs, with the goal of developing new and improved integrated approaches based on chemo- and immunotherapy. Expected outcomes of the project are:

1. Temporal-spatial characterization of crosstalk between CSC and TAM subpopulations

2. Understanding the protective mechanism of M2 TAMs against CSCs in response to chemotherapy

3. Evaluation of the effect of IRF4 silencing on macrophage stabilization at M1 with consequent increased toxicity towards CSCs.

Start and end date

June 2013 - June 2015

Project Manager

Prof. Daniele Santini - Scientific Coordinator and Project Manager

Coordinating institution of the project

Università Campus Bio-Medico di Roma

Funding source(s).

G. Berlucchi Foundation for Cancer Research