Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma

The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models.Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2 Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2 KO or KCL2 KO and KPCL2 KI or KCL2 KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation. Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment.Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic diseaseg JCL-G received support from a ’la Caixa’ Foundation (ID 100010434) fellowship (LCF/BQ/DR21/11880011). This study was supported by ISCIII FIS grants PI18/00757 and PI21/01110 (BSJ) and PI18/00267 (LG-B), and grants from the Spanish Ministry of Economy and Innovation SAF2016-76504-R (ACan and FP), PID2019-111052RB-I00 (FP), PID2019-104644RB-I00 (GM-B), a Ramón y Cajal Merit Award RYC-2012–12104 (BSJ) and ISCIII, CIBERONC, CB16/12/00446 (ACar) and CB16/12/00295 (ACan and GM-B), all of them co-financed through Fondo Europeo de Desarrollo Regional (FEDER) ’Una manera de hacer Europa’; a Fero Foundation Grant (BSJ); a Coordinated grant (GC16173694BARB) from the Fundación Científica Asociación Española Contra el Cáncer (FC-AECC) (BSJ); a Miguel Servet award (CP16/00121) (PS); a DFG, German Research Foundation Grant—Project no: 492 436 553 (KG); and a Max Eder Fellowship of the German Cancer Aid (111746) (PCH

Selective interaction of PEGylated polyglutamic acid nanocapsules with cancer cells in a 3D model of a metastatic lymph node

Background Metastases are the most common reason of cancer death in patients with solid tumors. Lymph nodes, once invaded by tumor cells, act as reservoirs before cancer cells spread to distant organs. To address the limited access of intravenously infused chemotherapeutics to the lymph nodes, we have developed PEGylated polyglutamic acid nanocapsules (PGA-PEG NCs), which have shown ability to reach and to accumulate in the lymphatic nodes and could therefore act as nanotransporters. Once in the lymphatics, the idea is that these nanocapsules would selectively interact with cancer cells, while avoiding non-specific interactions with immune cells and the appearance of subsequent immunotoxicity. Results The potential of the PGA-PEG NCs, with a mean size of 100 nm and a negative zeta potential, to selectively reach metastatic cancer cells, has been explored in a novel 3D model that mimics an infiltrated lymph node. Our 3D model, a co-culture of cancer cells and lymphocytes, allows performing experiments under dynamic conditions that simulate the lymphatic flow. After perfusion of the nanocarriers, we observe a selective interaction with the tumor cells. Efficacy studies manifest the need to develop specific therapies addressed to treat metastatic cells that can be in a dormant state. Conclusions We provide evidence of the ability of PGA-PEG NCs to selectively interact with the tumor cells in presence of lymphocytes, highlighting their potential in cancer therapeutics. We also state the importance of designing precise in vitro models that allow performing mechanistic assays, to efficiently develop and evaluate specific therapies to confront the formation of metastasisThe authors acknowledge financial support given by the Carlos III Health Institute (ISCIII) and European Regional Development Fund (FEDER) (CP12/03150, PIE13/00024 and PI15/00828), ERA-NET EuroNanoMed 2009 (Lymphotarg PI09/2670) and EuroNanoMed 2013 (053 NICHE). The first author also acknowledges a fellowship received from the Fundación Ramón Domínguez, Spain. Abellan-Pose also acknowledges a fellowship from the Biomedical Sciences and Health Technologies Doctoral School-University of Santiago de Compostela (Spain)S

Molecular Profiling of Circulating Tumour Cells Identifies Notch1 as a Principal Regulator in Advanced Non-Small Cell Lung Cancer

Knowledge on the molecular mechanisms underlying metastasis colonization in Non-Small Cell Lung Cancer (NSCLC) remains incomplete. A complete overview integrating driver mutations, primary tumour heterogeneity and overt metastasis lacks the dynamic contribution of disseminating metastatic cells due to the inaccessibility to the molecular profiling of Circulating Tumour Cells (CTCs). By combining immunoisolation and whole genome amplification, we performed a global gene expression analysis of EpCAM positive CTCs from advanced NSCLC patients. We identified an EpCAM+ CTC-specific expression profile in NSCLC patients mostly associated with cellular movement, cell adhesion and cell-to-cell signalling mediated by PI3K/AKT, ERK1/2 and NF-kB pathways. NOTCH1 emerged as a driver connecting active signalling pathways, with a reduced number of related candidate genes (NOTCH1, PTP4A3, LGALS3 and ITGB3) being further validated by RT-qPCR on an independent cohort of NSCLC patients. In addition, these markers demonstrated high prognostic value for Progression-Free Survival (PFS). In conclusion, molecular characterization of EpCAM+ CTCs from advanced NSCLC patients provided with highly specific biomarkers with potential applicability as a “liquid biopsy” for monitoring of NSCLC patients and confirmed NOTCH1 as a potential therapeutic target to block lung cancer dissemination.This work was funded by InveNNta (Innovation in Nanomedicine); Operational Programme for Cross-border Cooperation: Spain-Portugal (POCTEP) and European Regional Development Fund (ERDF). Javier Mariscal is recipient of a fellowship from Escola de Doutoramento Internacional Campus Vida of the University of Santiago de Compostela. Laura Muinelo-Romay is supported by ISCIII as Responsible of the Liquid Biopsy Analysis UnitS

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

© The Author(s) 2020Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.We acknowledge and thank Dr. Nuria Malats and Jaime Villarreal from the Spanish National Cancer Research Center (CNIO) for RNA sequencing and analysis, funded by Fondo de Investigaciones Sanitarias (FIS) grant PI18/01347. We thank Patricia Sánchez-Tomero and Marina Ochando-Garmendia for technical assistance and support and Dr. Raúl Sánchez Lanzas for assistance with autophagy experiments. We want to particularly acknowledge the patients and the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002) integrated in the Spanish National Biobanks Network for its collaboration and, in particular, Adrián Povo Retana for macrophage isolation. We would also like to thank the Transmission Electron Microscopy Unit Laboratory, part of the UAM Interdepartmental Investigation Service (SIdI); Coral Pedrero for exceptional help with in vivo experiments; and the laboratories of Dr. Amparo Cano and Dr. José González Castaño for reagents and helpful discussions. S.V. was a recipient of an Ayuda de Movilidad del Personal Investigador del IRYCIS, a mobility grant from the Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain, and a pre-doctoral fellowship from the Comunidad de Madrid, Ayudas Para La Contratación De Investigadores Predoctorales Y Posdoctorales (PEJD-2017-PRE/BMD-5062), Madrid, Spain. This study was supported by a Rámon y Cajal Merit Award (RYC-2012-12104) from the Ministerio de Economía y Competitividad, Spain (to B.S.); funding from la Beca Carmen Delgado/Miguel Pérez-Mateo from AESPANC-ACANPAN Spain (to B.S.); a Conquer Cancer Now Grant from the Concern Foundation (Los Angeles, CA, USA) (to B.S.); a Coordinated grant (GC16173694BARB) from the Fundación Asociación Española Contra el Cáncer (AECC) (to B.S.); FIS grants PI18/00757 (to B.S.), PI16/00789 (to M.A.F.-M.), PI18/00267 (to L.G.-B.; co-financed through Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”); a Miguel Servet award (CP16/00121) (to P.S.); a Max Eder Fellowship of the German Cancer Aid (111746) (to P.C.H.); and the German Research Foundation (DFG, CRC 1279 “Exploiting the human peptidome for Novel Antimicrobial and Anticancer Agents”; to P.C.H.)

The ever-evolving concept of the cancer stem cell in pancreatic cancer

This article belongs to the Special Issue Latest Development in Pancreatic Cancer.Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is the 4th most frequent cause of cancer-related death worldwide, primarily due to the inherent chemoresistant nature and metastatic capacity of this tumor. The latter is believed to be mainly due to the existence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs), which have been shown to have unique metabolic, autophagic, invasive, and chemoresistance properties that allow them to continuously self-renew and escape chemo-therapeutic elimination. As such, current treatments for the majority of PDAC patients are not effective and do not significantly impact overall patient survival (<7 months) as they do not affect the pancreatic CSC (PaCSC) population. In this context, it is important to highlight the need to better understand the characteristics of the PaCSC population in order to develop new therapies to target these cells. In this review, we will provide the latest updates and knowledge on the inherent characteristics of PaCSCs, particularly their unique biological properties including chemoresistance, epithelial to mesenchymal transition, plasticity, metabolism and autophagy.Peer reviewe

Design and evaluation of novel theranostic fluorogenic dual probe-prodrug in cancer

Background: In spite of major advances in the diagnosis and treatment of cancer, there remains a paucity of biomarkers for early detection. Legumain is a potential cancer biomarker and a molecular target for imaging and drug targeting. Legumain is a lysosomal protease with a remarkably restricted specificity, as it cleaves only substrate sequences having an asparagine (Asn) at the P1 site. We have previously shown that a legumain substrate probe (SM9), Pro-Ala-Asn~PEG-AQ, is quenched until activated by proteolytic action of legumain. Design of first generation molecular probe SM9 has been extended to a second generation theranostic dual probe-prodrug SM20 (RhoPro-Ala-Asn~Lys-PEG-AQ) that serves as both a diagnostic and therapeutic (theranostic) tool for cancer.Material and Methods: Solution and solid phase peptide methods have been used to create a novel rhodamine-labelled tetra peptide second generation theranostic dual probe-prodrug (SM20) of legumain, which was purified by chromatographic methods and characterised by high resolution mass spectrometry, together with all synthetic intermediates. Fluorescence spectroscopic methods were used to determine the efficiency of FRET in the prodrug (SM20). In vitro fluorimetric assay was developed with humanrecombinant legumain at 370C in MES assay buffer (pH 5.0). The lipophilic character (Log D) of the SM20 was assessed by distribution coefficient measurements. Confocal microscopy studies were performed to investigate the cellular uptake and sub-cellular localization of both SM9 and SM20 and their legumain-mediated cleavage fragments in PC3 prostate cancer cells.Results: The fluorogenic second generation prodrug (SM20) is an efficient FRET substrate and affords good restoration of fluorescence when incubated at 10mM with rh-legumain (40 ng) in legumain assay buffer (pH-5.0; lex 544 nm, lem 585 nm). Cleavage at the designed Asn~Lys (SM20) cleavage ‘hotspot’ was demonstrated at concentrations of legumain in the 5−40 ng range in in vitro metabolism experiments using recombinant legumain. The second generation theranostic prodrug (SM20) is structurally similar to the SM9 probe, with a different aminoanthraquinone quencher, which is lysosomotropic.Measurement of the distribution coefficients have shown that SM20 is more lipophilic than the Lys-PEG-AQ cleavage product from which it is derived. Confocal substrate for sensitive and early detection of legumain and a smart therapeutic agent for cancer. Work is ongoing to optimize the dual probe-prodrug and to extend the studies in vivo

Evaluation of Circulating Tumor Cells and Related Events as Prognostic Factors and Surrogate Biomarkers in Advanced NSCLC Patients Receiving First-Line Systemic Treatment

In the present study we investigated the prognostic value of Circulating Tumour Cells (CTC) and their utility for therapy monitoring in non-small cell lung cancer (NSCLC). A total of 43 patients newly diagnosed with NSCLC were prospectively enrolled. Blood samples were obtained before the 1st, 2nd and 5th cycles of chemotherapy and analyzed using CellSearch technology. Both CTC and CTC-related objects (not morphological standard or broken epithelial cells) were counted. At baseline 18 (41.9%) patients were positive for intact CTC count and 10 (23.2%) of them had ≥5 CTC, while CK positive events were found in 79.1% of patients. The group of patients with CTC ³5 at baseline presented worse PFS and OS than those with &lt;5 CTC (p = 0.034 and p = 0.008, respectively). Additionally, high levels of total CK positive events were associated with poor prognosis in the group of patients with &lt;5 CTC. Regarding therapy monitoring, patients presenting increased levels of CTC during the treatment demonstrated lower OS and PFS rates. All these data supported the value of CTC as a prognostic biomarker and as a surrogate indicator of chemotherapy effectiveness in advanced NSCLC patients, with the additional value of analyzing other “objects” such as apoptotic CTC or CK fragments to guide the clinical management of these patients