Abstract B06: Impact of intratumoral microenvironment and epithelial cells crosstalk in pancreatic carcinogenesis
Authors: Leca Julie , Secq Véronique , Nigri Jérémy , Martinez Sébastien , Rubis Marion , Lavaut Marie Noelle , Dusetti Nelson , Loncle Céline , Garcia Stéphane , Chan Philippe , Benard Magalie , Iovanna Juan Lucio , Brun Christine , Vasseur Sophie , Tomasini Richard 
Published: October 2015

Abstract
Pancreatic adenocarcinoma (PDAC) is a cancer particularly resistant to current therapies. One possible reason consists in its cellular composition that limits access to drugs. Indeed, non-tumor cells, mainly activated fibroblasts (stellate cells) and immune cells, display 80% of the tumor mass composing the microenvironment or stroma. The stellate cells have the ability to secrete a lot of protein and extensive amounts of extracellular matrix which contributes to the formation of a fibrotic shield around epithelial cells, making those tumors highly hypoxic. Despite these knowledge the precise role of stromal components, and specifically stellate cells, in PDAC is poorly understood. In order to develop a simultaneous therapy targeting both tumoral and stromal cells, we investigate specific molecules implicated in the dialogue between those compartments to analyze their impact on pancreatic carcinogenesis.
In this study, we used a laser microdissection approach of human PDAC stromal and epithelial compartment, followed by mass spectrometry and bioinformatics analysis (statistics/interactome) to compare protein composition and networks within both compartments. We found the presence of a proteic complex composed of three proteins, expressed only in stromal compartment, related to lipid and intracellular trafficking. To validate such data, we confirmed the specific stromal expression of each protein, on human PDAC samples, and found that our three targets were expressed mainly in stellate cells. However before to investigate the dialogue between epithelial and stromal cells, we designed an in vitro model, based on co-culture of stromal cells: immune and stellate cells, which take into account the multi-cellular composition of such tumors and added PDAC relevant metabolic stresses: hypoxia and nutrient starvation. To improve our model, we used primary stellate cells that we extracted from freshly resected human PDAC tumor pieces. We added pancreatic tumoral cells in our model to investigate dialogue between stromal and tumoral cells. Our data show that under nutrient and hypoxic stresses, co-cultures from stellate and immune cells provide a proliferative and pro-migratory advantage to tumor cells. Moreover, and only in these same specific condition, the proteic complex identified is formed in stellate cells. So, we hypothesized that a specific “lipid related process” is developed by stellate cells in order to subvert the metabolically deprivated tumor cell.
To confirm such hypothesis, we designed shRNA against the three components of the complex to impair its formation and activity. Interestingly, we showed in vitro that, decreased expression of one protein can destabilize the complex in stellate cells and impair the proliferative and pro-migratory advantages to tumor cells. In parallel, in vivo, we designed an intra-pancreatic co-injection model of tumor and stromal cells in mice. Our experiments suggest that co-injection of stellate cells with tumoral cells increases tumorigenesis, an advantage which is lost if we co-inject stellate cells that carry a shRNA against anyone of the three targets. This is correlated with a decreased survival of tumoral cells within those tumors, stained for apoptotic and proliferative markers as wells as measured for their necrotic areas.
At present, in order to confirm this protein complex as a potential therapeutic target as adjuvant to Gemcitabine or Folfirinox, we need to deepen the mechanisms of communication between stellate and tumoral cells and more specifically its impact on exosome processes. Altogether, our data suggest that if we prevent aids setting up by stellate cell, through destabilization of this complex within stellate cells, it could sensitize tumor cells by decreasing their survival abilities and certainly enhances their response to chemotherapies (under investigation).
Citation Format: Julie Leca, Véronique Secq, Jérémy Nigri, Sébastien Martinez, Marion Rubis, Marie Noelle Lavaut, Nelson Dusetti, Céline Loncle, Stéphane Garcia, Philippe Chan, Magalie Benard, Juan Lucio Iovanna, Christine Brun, Sophie Vasseur, Richard Tomasini. Impact of intratumoral microenvironment and epithelial cells crosstalk in pancreatic carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B06.