Until recently the nucleic acid content of platelets was considered to be fully determined by their progenitor megakaryocyte. However, it is now well understood that additional mediators (e.g. cancer cells) can intervene, thereby influencing the RNA repertoire of platelets. Platelets are highly dynamic cells, able to communicate and influence their environment. For instance, platelets have been involved in various steps of cancer development and progression by supporting tumor growth, survival and dissemination. Cancer cells can directly and/or indirectly influence the platelet RNA content, resulting in tumor-mediated 'education' of platelets. Alterations in the tumor-educated platelet (TEP) RNA profile have been described as a novel source of potential biomarkers. Individual platelet RNA biomarkers as well as complex RNA signatures may be used for early-detection of cancer and treatment monitoring. Here we review the RNA transfer occurring between cancer cells and platelets. We explore the potential use of platelet RNA biomarkers as a liquid biopsy biosource, and discuss methods to evaluate the transcriptomic content of platelets.

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Tumor-educated Platelets (TEPs) have emerged as rich biosources of cancer-related RNA profiles in liquid biopsies applicable for cancer detection. Although human blood platelets have been found to be enriched in circular RNA (circRNA), no studies have investigated the potential of circRNA as platelet-derived biomarkers for cancer. In this proof-of-concept study, we examine whether the circRNA signature of blood platelets can be used as a liquid biopsy biomarker for the detection of non-small cell lung cancer (NSCLC). We analyzed the total RNA, extracted from the platelet samples collected from NSCLC patients and asymptomatic individuals, using RNA sequencing (RNA-Seq). Identification and quantification of known and novel circRNAs were performed using the accurate CircRNA finder suite (ACFS), followed by the differential transcript expression analysis using a modified version of our thromboSeq software. Out of 4732 detected circRNAs, we identified 411 circRNAs that are significantly (p-value < 0.05) differentially expressed between asymptomatic individuals and NSCLC patients. Using the false discovery rate (FDR) of 0.05 as cutoff, we selected the nuclear receptor-interacting protein 1 (NRIP1) circRNA (circNRIP1) as a potential biomarker candidate for further validation by reverse transcription–quantitative PCR (RT-qPCR). This analysis was performed on an independent cohort of platelet samples. The RT-qPCR results confirmed the RNA-Seq data analysis, with significant downregulation of circNRIP1 in platelets derived from NSCLC patients. Our findings suggest that circRNAs found in blood platelets may hold diagnostic biomarkers potential for the detection of NSCLC using liquid biopsies.

Despite the diversity of liquid biopsy transcriptomic repertoire, numerous studies often exploit only a single RNA type signature for diagnostic biomarker potential. This frequently results in insufficient sensitivity and specificity necessary to reach diagnostic utility. Combinatorial biomarker approaches may offer a more reliable diagnosis. Here, we investigated the synergistic contributions of circRNA and mRNA signatures derived from blood platelets as biomarkers for lung cancer detection. We developed a comprehensive bioinformatics pipeline permitting an analysis of platelet-circRNA and mRNA derived from non-cancer individuals and lung cancer patients. An optimal selected signature is then used to generate the predictive classification model using machine learning algorithm. Using an individual signature of 21 circRNA and 28 mRNA, the predictive models reached an area under the curve (AUC) of 0.88 and 0.81, respectively. Importantly, combinatorial analysis including both types of RNAs resulted in an 8-target signature (6 mRNA and 2 circRNA), enhancing the differentiation of lung cancer from controls (AUC of 0.92). Additionally, we identified five biomarkers potentially specific for early-stage detection of lung cancer. Our proof-of-concept study presents the first multi-analyte-based approach for the analysis of platelets-derived biomarkers, providing a potential combinatorial diagnostic signature for lung cancer detection.

Background: Platelets are active players in hemostasis, coagulation and also tumorigenesis. The cross-talk between platelets and circulating tumor cells (CTCs) may have various pro-cancer effects, including promoting tumor growth, epithelial-mesenchymal transition (EMT), metastatic cell survival, adhesion, arrest and also pre-metastatic niche and metastasis formation. Interaction with CTCs might alter the platelet transcriptome. However, as CTCs are rare events, the cross-talk between CTCs and platelets is poorly understood. Here, we used our established colon CTC lines to investigate the colon CTC-platelet cross-talk in vitro and its impact on the behavior/phenotype of both cell types.Methods: We exposed platelets isolated from healthy donors to thrombin (positive control) or to conditioned medium from three CTC lines from one patient with colon cancer and then we monitored the morphological and protein expression changes by microscopy and flow cytometry. We then analyzed the transcriptome by RNA-sequencing of platelets indirectly (presence of a Transwell insert) co-cultured with the three CTC lines. We also quantified by reverse transcription-quantitative PCR the expression of genes related to EMT and cancer development in CTCs after direct co-culture (no Transwell insert) with platelets.Results: We observed morphological and transcriptomic changes in platelets upon exposure to CTC conditioned medium and indirect co-culture (secretome). Moreover, the expression levels of genes involved in EMT (p &lt; 0.05) were decreased in CTCs co-cultured with platelets, but not of genes encoding mesenchymal markers (FN1 and SNAI2). The expression levels of genes involved in cancer invasiveness (MYC, VEGFB, IL33, PTGS2, and PTGER2) were increased.Conclusion: For the first time, we studied the CTC-platelet cross-talk using our unique colon CTC lines. Incubation with CTC conditioned medium led to platelet aggregation and activation, supporting the hypothesis that their interaction may contribute to preserve CTC integrity during their journey in the bloodstream. Moreover, co-culture with platelets influenced the expression of several genes involved in invasiveness and EMT maintenance in CTCs.

Tumor microenvironment (TME) plays a critical role in cancer progression and response to therapies. Therapeutic intervention strategies for cancer treatment can induce inflammation, resulting in changes in the TME. To investigate the response of cancer cells to chemotherapy in the context of an inflammatory microenvironment, we studied the effects driven by the chemotherapeutic drug doxorubicin, and the inflammatory cytokine TNFá. We previously demonstrated using expression microarrays experiments (validated also through qPCR) that the Doxo+TNFá combined treatment determined a strong up-regulation of migration-related genes as well as increased motility in breast cancer cells (MCF7). We confirmed the synergistic activation of a group of 6 different genes upon Doxo+TNFá combined treatment in different cell line models coming from different cancer types (A549-lung, U2OS-osteosarcoma). Moreover, we demonstrated that this effect was only partially p53-depenedent but strongly Doxo-dependent using p53 mutated MDA-MB-231 breast cancer cells or knocking-out p53 in MCF7 or U2OS cells through the cutting edge technology CRISPR/Cas9. We also demonstrated that the combined Doxo+TNFá treatment was not only able to disrupt the 3D architecture of mammary acini observed with MCF10A primary cells grown within matrigel, but also to stimulate the tube-forming potential of Human Umbilical Vein Endothelial Cells (HUVEC). Furthermore, a signature of Doxo+TNFá highly synergistic genes (DT-29) was shown to exhibit prognostic value for luminal breast cancer patients, with adverse outcome correlating with higher relative expression (based on Kaplan-Meier plotter tool). We further used available experimental data sets (RNA-seq measurements from Gene Expression Omnibus) both from breast cancer cell lines (luminal-like vs basal-like breast cancer cells) and breast cancer patients (ER positive vs Triple Negative Breast Cancer, TNBC and vs healthy adjacent tissues). The expression of DT-29 gene signature was analyzed and compared among the different groups of samples. The most statistically relevant genes differentially expressed among the different groups were involved in the STAT3-driven pathways and were prevalently highly expressed in TNBC patients and cell lines. In order to demonstrate a STAT3-dependent regulation of up-regulated genes upon Doxo+TNFá combined treatment, we pharmacologically inhibited STAT3 using Stattic or we knocked-out STAT3 using CRISPR/Cas9 in MCF7, MDA-MB-231 and U2OS cells. Results demonstrated a STAT3-dependent up-regulation for some of the selected genes and STAT3 inhibition resulted also in a reduced migration potential particularly in U2OS cells. We propose that the combined treatment with Doxo+TNFá can lead to the activation of specific gene expression programs that may impact on cancer phenotypes and potentially modify the efficacy of cancer therapy. Citation Format: Federica Alessandrini, Laura Pezze, Francesca Precazzini, Silvia D’Ambrosi, Dennis Pedri, Lia Pinto, Yari Ciribilli. Chemotherapy and inflammation show a p65- and STAT3-dependent pro-tumorigenic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 706.

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