Modulation of Early Neutrophil Granulation: The Circulating Tumor Cell-Extravesicular Connection in Pancreatic Ductal Adenocarcinoma.

Harrys Charles Jacob,Ashok Saluja,John Charles Richard,Vikas Dudeja,Rossana Signorelli,Shweta Lavania,Ashley Middleton,Tyler Kashuv,Ramakrishnan Sundaram,Melinda Boone,Utpreksha Vaish,Ranjitha Boopathy

doi:10.3390/cancers13112727

Abstract

Simple SummaryCirculating tumor cells (CTCs) found in the blood of pancreatic cancer patients show a worse prognosis to therapy if they are seen in clusters of cells with neutrophils or platelets or with other cell types than when they are seen as singlets. We wanted to investigate if there is a secondary mode of communication between the CTCs and neutrophils that causes them to associate. We describe for the first time an extravesicular (EV) mediated communication between CTCs and neutrophils that modulates early transcriptome changes that can cause neutrophils to partially degranulate and form associations. We also identify the protein cargo carried in such EVs and how when added to naïve neutrophils, they can modulate transcriptomic changes in neutrophils partially disarming them to form clusters rather than undergo specialized cell death, which is characterized by release of condensed chromatin (NETs) and granular contents termed as NETosis.Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tumor cells, CTCs) either alone or as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tissue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on companion cells in clusters.

Highlights

Pancreatic cancer is one of the most aggressive cancers, with a 5-year survival rate of less than 10% [1]
Is it a process that is solely based on surface protein interactions [6,7] or soluble mediators, or can the circulating tumor cells (CTCs) communicate by other modes as well? we decided to investigate the possibility of CTCs being able to secrete Extracellular vesicles (EVs) and whether these EVs could be the effectors in this mechanism
In the present study, in order to identify whether the CTCs secreted EVs, we investigated cell lines isolated from PDAC patients with liver metastases and cultured them for 4–5 generations in low attachment plates and low serum containing medium

Summary

Introduction

Pancreatic cancer is one of the most aggressive cancers, with a 5-year survival rate of less than 10% [1]. Published research suggests that it is a multistep process that involves cellular detachment from the primary site, intravasation into circulation, survival in the blood stream and, extravasation intro distant organs [3]. These disseminated circulating tumor cells (CTCs) travel either as single cells or as clusters. This association helps protect the clusters, evade immune attack and shear stress, and facilitate distant metastasis establishment [5] These CTC clusters display a distinct phenotype, different from single cells seen in circulation [6,7], possibly offering them enhanced survival and colony forming potential [7,8]. We hypothesized that the cellular mode of communication could possibly be through Extracellular vesicles (EVs) that are secreted by most cell types

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