ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.


Statistics
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Animals and other organisms
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Laboratory animals
Female 6-to 8-week-old NU-Foxn1nu nude mice (Envigo, Spain). Mice were housed according to the following guidelines: a 12 h light/12 h dark cycle, with no access during the dark cycle; temperatures of 65-75°F (~18-23°C) with 40-60% humidity; a standard diet with fat content ranging from 4% to 11; sterilized water was accessible at all times; for handling, mice were manipulated gently and as little as possible; noises, vibrations and odors were minimized to prevent stress and decreased breeding performance; and enrichment was always used per the facility's guidelines to help alleviate stress and improve breeding. These details are also included in the article.

Wild animals
The study did not involve wild animals.

Field-collected samples
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Ethics oversight
Mice were housed according to institutional guidelines and all experimental procedures were performed in compliance with the institutional guidelines for the welfare of experimental animals approved by the Universidad Autónoma de Madrid Ethics Committee (CEI 60-1057-A068) and La Comunidad de Madrid (PROEX 335/14) and in accordance with the guidelines for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS).
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Population characteristics
For the human serum samples used in this study, no identifying information was obtained or used. The only information provided and taken into consideration was the tumor stage. Patients with Resectable tumors (n = 14), locally-advanced tumors (n = 17) and metastasis (met) (n = 19) patients. Also included was serum from healthy individuals (n = 21) that were neither ago or sex matched.

Recruitment
Patients were not recruited for this specific study, but rather samples were retrospectively obtained from collections already established by the the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002, ISCIII Biobank Register No. B.0000678) or by Dr.

October 2018
Alfredo Carrato from the "Collection of samples of the Familial Pancreas Cancer Registry". Samples used are non-identifiable, as detailed above. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Invitrogen™ Attune™ NxT software, version 3.1.1 was used for cytometry data collection, BD FACSDiVa software was used for sorting data collection, and FlowJo 9.3 software (Tree Star Inc., Ashland, OR) was used for flow cytometry image preparation and analysis.
Cell population abundance Cancer stem cells represent a small percentage of the total cell population (<10%) and often times purity is not confirmed due to low cell numbers recovered post sorting. When confirmed using the Invitrogen™ Attune™ NxT, purity is typically between 80-90%.

Gating strategy
Generally, cells was first gated on FSC-Area/FSC-Height to remove aggregates. Dead cells were removed by gating in DAPInegative cells versus FSC-Area. Debris free, live, single cells were gated using FSC-Area and SSC-Area. Surface antigen gating was performed on the live, single, debris free cell population. Gates were determined based on negative controls, unstained controls or IgG controls. A figure exemplifying all of the gating strategies used and controls is provided in Supplementary Figure 17.
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