br for cancer diagnosis positron emission
for cancer diagnosis, positron emission tomography with the radio-pharmaceutical 18-Fluorodeoxyglucose (FDG-PET), is based on this singular metabolism of tumors, which forces them to consume more glucose than the surrounding tissues .
Despite the importance of this altered metabolism, there is a lack of molecular and functional studies that approximate to what is really happening in vivo. We know now that tumors are not uniform masses of
Abbreviations: OXPHOS, Oxidative phosphorylation; ROS, Reactive oxygen species; CAFs, Cancer associated fibroblasts; MIMP, Mitochondrial inner membrane potential
E-mail addresses: [email protected] (A. Cruz-Bermúdez), [email protected] (M. Provencio). 1 These authors contributed equally.
cells. Besides the transformed cells, many cell types coexist, including fibroblasts, endothelial or immune cells, which results in what is known as tumor microenvironment. Following this line of thought, over the last years many studies focusing on the microenvironment have been conducted. This interest relies on the fact that diﬀerent cell types communicate with each other, changing their behavior and thus con-ditioning tumor metabolism , tumor progression , and treatment response . Therefore, additional studies on cancer metabolism are needed taking into account this complexity, since most studies have focused exclusively on analysis of isolated tumor cell lines. It is likely that the discrepancy between cancer basic knowledge and its clinical application is due to underestimation of the microenvironment com-ponent of tumors.
One of the most important components of the tumor micro-environment are the cancer associated fibroblasts (CAFs) , identified by the expression of proteins such as α-smooth muscle GS9620 (α-SMA) and fibroblast activation protein (FAP) . The role of CAFs at meta-bolic level has been recently described as the Reverse Warburg eﬀect. According to this model, tumor cells within the microenvironment would activate the fibroblasts present by diﬀerent factors and would control them, taking advantage of their metabolism. Although this phenomenon is still not completely understood, it seems to implicate a decrease on mitochondrial function in CAFs with the ensuing increase on glycolysis. This metabolic shift would lead to a release of highly energetic metabolic substrates to the microenvironment that would be used by the tumor cells through an increased OXPHOS function [10–13].
We have focused on lung cancer because it is a major public health problem. Lung cancer is the most common cancer worldwide in terms of incidence and it is also the leading cause of cancer-related death in the world despite new targeted therapies based on their mutational profile have been incorporated [14–16]. Approximately one-third of patients with non-small cell lung cancer (NSCLC) are diagnosed with locally advanced (stage III) disease. These patients experience poor overall survival due to the presence of metastatic diseases that are not ade-quately detected. Therefore, many patients are subjected to aggressive localized treatments, such as surgery or radiotherapy, when they al-ready present advanced distant disease .
Usually lung tumors have a variable component of mesenchymal-parenchyma composed mainly of fibroblasts and collagen fibers . Importantly, in spite of the clinical relevance of lung cancer (in which FDG-PET is a key diagnostic tool  and the fibroblasts are an im-portant component of tumors ) the role of the microenvironment in metabolic reprogramming has not been studied.
Our results on stage IIIA NSCLC patients show a positive correlation between the grade of fibrosis and the glycolysis phenotype of the tumor. In vitro studies confirm that, a metabolic reprogramming involving ROS and TGF-β signaling occurs in lung cancer cells and fibroblasts in-dependently of α-SMA induction and suggest that specific attributes among tumor cells and fibroblasts may modify or hamper this phe-nomenon.
2. Materials and methods
A cohort of 12 stage IIIA adenocarcinoma patients was studied. All studies were carried out using the Formalin-Fixed Paraﬃn Embedded tissue (FFPET) from diagnostic biopsies. All the experiments carried out in this study complied with current Spanish and European Union laws and the principles outlined in the Declaration of Helsinki. The study and experimental protocols were approved by the Hospital Universitario Puerta de Hierro Ethics Committee and written informed consent was obtained from all the patients recruited.
The fibrosis grade was evaluated in the hematoxylin-eosin diag-nostic slides at x50 magnification. A low (< 1/3 desmoplastic stroma) or high (> 1/3 desmoplastic stroma) score was assigned to each sample by three independent anatomical pathology technicians. Vimentin and cytokeratin immunostaining of tumor biopsies using specific antibodies were carried out to confirm the correct identification of tumor cells and fibroblasts on the hematoxylin-eosin samples.