Project title: Role of CCL2 and IL-8 in Pituitary Neuroendocrine Tumor Microenvironment

Description

Marques and his team will use patient tumor samples to study the interaction between chemokines and immune cells as well as the mechanisms by which such crosstalk may promote the growth and invasive behavior of pituitary neuroendocrine tumors.

What question will the researchers try to answer?

Pituitary neuroendocrine tumors (PitNETs) are composed of tumor cells as well as immune system cells like macrophages and neutrophils that may be attracted into tumors by substances produced by cancer cells. These substances, called chemokines, also promote the growth and invasion of tumor cells, thus rendering these tumors more aggressive and difficult to treat. The microenvironment of PitNETs, particularly the chemokine network, remains poorly investigated. More specifically, the significance of CCL2 and IL-8 remains unknown, and their clinical, diagnostic, and therapeutical usefulness has not yet been studied in PitNETs. Dr. Marques will study the role of these substances, and their interaction with immune and tumor cells to determine whether CCL2 and IL-8, macrophages and neutrophils are relevant to PitNET biology, are useful diagnostic and prognostic markers in these tumors, and whether they are potential targets for therapy.

Why is this important?

The study will advance the knowledge of the initiation, progression and aggressiveness of PitNETs, which may ultimately translate into the development of new diagnostic biomarkers and prognostic tools to better manage PitNET and to facilitate the design of novel therapies for aggressive and difficult-to-treat PitNETs. 

What will researchers do?

Dr. Marques and his team will study the expression of CCL2 and IL-8, and two immune cell types attracted to tumors by these substances (macrophages and neutrophils), in PitNET samples. They will correlate the expression levels of CCL2 and IL-8 with clinical features and outcomes in a large group of patients with PitNETs. They will then establish in vitro models of the disease to study the interactions between CCL2/IL-8 and immune cells and their biological effects in pituitary tumor cells in terms of potentiating their ability to grow, move, and invade surrounding tissues. They will also test the effectiveness of anti-CCL2 and anti-IL-8 therapies in vitro (cell culture experiments) and in vivo (models with transplanted human tumors).

How might this improve the treatment of NETs?

This study may identify valuable markers for the biology and aggressiveness of PitNETs, which can be translated into clinical practice by, for example, incorporating such markers in histopathological reports, in risk stratification systems, or in managing algorithms for patients who have PitNET. Moreover, their research may lead to the identification of novel pharmacological targets useful for managing aggressive PitNETs and pave the road for using immunotherapeutic agents in patients who have a PitNET, such as anti-cancer drugs that target CCL2 or IL-8.

What is the next step?

As a preliminary step, they will investigate the role of CCL2 and IL-8 in the PitNET microenvironment in modulating different tumorigenic mechanisms as well as in determining tumor aggressiveness. To accomplish this they will assay their expression on human PitNET samples at both RNA (RT-qPCR) and protein (immunohistochemistry) levels. If CCL2 and/or IL-8 prove to be relevant for PitNET biology and aggressiveness, they will then test the effectiveness of anti-CCL2 and anti-IL-8 therapies in vitro and in vivo to assess whether these may be translated to treat patients who have PitNETs.

Outcomes:

Pituitary neuroendocrine tumors (PitNETs) are composed of tumor cells and immune system cells, like macrophages and neutrophils, that may be attracted into tumors by substances produced by cancer cells. These substances, termed chemokines, also promote the growth and invasion of tumor cells, thus potentially rendering such tumors more aggressive and difficult-to-treat. The microenvironment of PitNETs, particularly the chemokine network, remains poorly investigated. More specifically, the significance of CCL2 and IL-8 (two highly secreted chemokines from pituitary tumor cells) remains unknown, and their clinical, diagnostic and therapeutical usefulness has not yet been studied in PitNETs.

The expression of CCL2 and IL-8 at an RNA level was assessed by RT-qPCR, and at protein level by immunohistochemistry on 86 human PitNET samples, and these expression levels correlated with clinico-pathological features and outcomes of those 86 PitNET patients. Our data suggest that CCL2, but not IL-8, may be relevant for PitNET biology and aggressiveness, or at least be an important biomarker in predicting aggressive or difficult-to-treat PitNETs. Also, we noted that CCL2 expression was negatively correlated with the expression of the gene encoding E-cadherin, a key protein for cell adhesion; thus, CCL2 may contribute to the activation of the so-called epithelial-to-mesenchymal transition pathway, leading to increased migration and invasiveness of pituitary tumor cells. We observed no correlation between the expression of CCL2/IL-8 and macrophages or neutrophils, suggesting that these chemokines might not be the only ones involved in shaping the immune cell landscape of PitNETs, and other factors (potentially other chemokines) might act synergistically in bringing these immune cells into the microenvironment of PitNETs. Data from our in vitro cell line experiments suggest that CCL2 does not stimulate growth/proliferation of pituitary tumor cells (possibly having an opposite effect, i.e. inhibits cell proliferation), nor induce migration, invasion or morphology in pituitary tumor cells.

Taken these findings together, assessing the expression of CCL2 in human PitNETs in routine clinical practice, e.g. in pathology reports, might add some prognostic value as a biomarker predicting more aggressive and difficult-to-treat PitNETs. However, the lack of association between CCL2 and pituitary tumor behavior in our in vitro experiments suggest that there may be no key role of CCL2 in directly and independently determining pituitary tumor cell aggressiveness, thus, it might not justify developing or employing immunotherapy to specifically block or inhibit CCL2.

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