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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ONCOSYSTEMS (Phenotypic characterization of Liver-derived exosomes populations associated with liver metastasis in pancreatic cancers)

Teaser

Summary

Oncologic diseases are multifactorial pathologies involving intricate cell-cell communication networks, in which by interplaying with healthy host cells tumors disseminate pro-tumorigenic messages creating a systemic tumor environment, or macroenvironment, which is key for malignant progression. Thus, deciphering the mechanisms that mediate cell-cell communication between transformed and non-transformed cells is of key significance, as it has the potential to significantly improve the diagnosis and treatment of oncologic patients.
Extracellular vesicles, such as exosomes (exo), are produced by virtually all cells and are emerging cell-cell communication players in physiological and pathological scenarios. As exo can carry and transfer packages of information from cell to cell, locally and to distant cellular targets by traveling though the peripheral circulation, they have been used as liquid biopsies for both oncologic and non-oncologic diseases. We have recently shown that tumor-derived exo can induce microenvironments supportive of tumor growth and metastasis, such as pre-metastatic niches that precedes and support metastatic seeding, by acting in non-tumor cells. However, we still have insufficient information on how to detect the formation of pre-metastatic microenvironments (such as liver pre-metastatic niches â€“ LPMN) in clinical settings. We here proposed that the characterization of stroma-derived exo present in biologic fluids has the potential to offer a non-invasive alternative to detect and characterize tumor-associated microenvironments, such as LPMNs. In addition, we proposed to test whether liver-derived exo play a role in supporting metastatic Pancreatic Cancer (PC) lesions grow in the liver.
For that we proposed to:
- Evaluate whether overall production and content of liver-derived exo are modified during LPMN formation;
- Identify new exosomal biomarkers for the detection of LPMN formation;
- Test whether liver-derived exo interact with PC cells and play a role in supporting liver metastasis.

Work performed

After induction of LPMN in mice with PC-derived exo, liver-derived exo were counted and measured for protein concentration. We found that exo derived from LPMN have 36% higher protein content per exosome (Figure 1). It suggests a potential modification in the molecular composition of liver-derived exo in cases of LPMN setup. The protein composition of exo was characterized by mass spectrometry. We found that 2 proteins were upregulated in LPMN-derived exo and absent in PC exo. We are currently measuring liver- and plasma-derived exo samples collected from mice (n=40, from 4 independent experiments) throughout this project. In addition, upon approval by our ethical committee, plasma samples from PC patients (302 samples from 83 patients) were collected during the period of this project and will be also analyzed.
We also tested whether LPMN-derived exo supports PC liver metastasis. Although in vitro experiments indicated that LPMN-derived exo had higher capability to be incorporated by PC cells (Figure 2a-c) and to induce cell proliferation (Figure 2d) when compared to control liver counterparts, our in vivo experiments did not indicate any clear effect in the liver metastatic burden of PC (Figure 2e-f).
We have also developed a new method for population analysis of exo by high-resolution flow cytometry (dHRFC). It can facilitate and expand the use of exo-based liquid biopsy. We found that by using Carboxyfluorescein Succinimidyl Ester (CFSE) staining we could differentiate vesicular from non-vesicular particles and provide quantitative measurements of sample purity prepared by different isolation methods. By enabling the identification of exo in non-isolated samples, our method accelerates the analysis of exo proteins (3 hours vs 2-3 days). In addition, it allows exo population analysis in less than 5uL of plasma. It not only enables the analysis of hundreds of analytes from a single blood collection, but also permits non-lethal blood collection from mice during an experiment. It facilitates longitudinal studies of exo animal-by-animal and reduces the number of animals per experiment. We are currently testing the application of dHRFC for detection and follow-up of LPMN. Results on the standardization and applications of dHRFC are ready for submission, and results on the application of exo to detect and follow-up LMPN are being tested in pre-clinical and clinical settings and will be submitted for publication by the end of 2019.

Final results

This project was the first to our knowledge to identify a potential biomarker for the identification pre-metastatic microenvironments. This can not only help to predict liver metastatic progression of tumours, but also enable the follow-up of patients treated with therapeutic strategies directed to key molecular components of pre-metastatic niches. In fact, our group is currently testing the potential use of target-specific drugs to prevent and/or revert the formation of pre-metastatic niches. If successful, these studies have the potential to identify patients with risk to develop frequently incurable metastatic disease and to reduce the lethality of oncologic diseases.
Our new dHRFD method developed during this project enables fast analysis of plasma, without the demand for collection of additional volumes of blood for exo studies. In addition, it does not require long and laborious steps for exo isolation. By doing so, it has a great potential to facilitate the study of exo in clinical settings. In fact, we are currently testing the potential application of dHRFC for the analysis of LPMN-associated proteins and other tentative markers in plasma exo of PC patients from our clinical department.
It also represents a precious tool to simplify the identification of novel physiological and pathological cell-cell communication systemic networks involving sEVs in in vivo models. Likewise, it can potentially be used to characterize sEVs heterogeneity and differential packaging of biomolecules during sEVs biogenesis in highly controlled small-scale in vitro systems. Finally, our method has an unexplored potential to the study of sEVs populations in non-pooled clinical samples with intrinsically limited volumes, such as lacrimal, vitreous humour and synovial fluids.