cancers Article Induction of Lysosome Membrane Permeabilization as a Therapeutic Strategy to Target Pancreatic Cancer Stem Cells Timothy P. Cash 1 , Sonia Alcalá 2,3,4 , María del Rosario Rico-Ferreira 5 , Elena Hernández-Encinas 5 , Jennifer García 5 , María Isabel Albarrán 5 , Sandra Valle 2,3,4 , Javier Muñoz 6 , Sonia Martínez-González 5 , Carmen Blanco-Aparicio 5 , Joaquín Pastor 5,† , Manuel Serrano 1,7,8,† and Bruno Sainz, Jr. 2,3,4, *,† 1 Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain; tim.cash@senolytx.com (T.P.C.); manuel.serrano@irbbarcelona.org (M.S.) 2 Department of Cancer Biology, Instituto de Investigaciones Biomédicas “Alberto Sols” (IIBM), 28029 Madrid, Spain; sonia.alcala@uam.es (S.A.); san.valle@predoc.uam.es (S.V.) 3 Department of Biochemistry, Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain 4 Chronic Diseases and Cancer, Area 3—Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), 28034 Madrid, Spain 5 Experimental Therapeutics Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain; ricoferreiram@gmail.com (M.d.R.R.-F.); mehernandez@cnio.es (E.H.-E.); jgarocha@externas.sescam.jccm.es (J.G.); mialbarran@cnio.es (M.I.A.); smartinezg@cnio.es (S.M.-G.); cblanco@cnio.es (C.B.-A.); jpastor@cnio.es (J.P.) 6 Proteomics Unit–ProteoRed-Instituto de Salud Carlos III, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain; jmunozpe@cnio.es 7 Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain 8 Catalan Institution for Research and Advanced Studies (ICREA), 08028 Barcelona, Spain * Correspondence: bsainz@iib.uam.es † Co-senior authors.  Received: 21 June 2020; Accepted: 2 July 2020; Published: 4 July 2020  Abstract: Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC. Keywords: compound library; cancer stem cells; pancreatic ductal adenocarcinoma; lysosomal membrane permeabilization; patient-derived xenografts Cancers 2020, 12, 1790; doi:10.3390/cancers12071790 www.mdpi.com/journal/cancers Cancers 2020, 12, 1790 2 of 24 1. Introduction It is now generally accepted that the majority of solid tumors contain a small sub-population of highly plastic, tumorigenic and chemoresistant cells, known as cancer stem cells (CSCs) or cancer-initiating cells, which are the drivers of tumor evolution, metastasis and tumor relapse [1,2]. Thus, from a clinical perspective, targeting and eliminating the CSC compartment would ensure a more effective tumor eradication [2–5]; however, to date, only a handful of therapeutics specifically targeting CSCs have advanced to clinical trials (reviewed in Saygin et al. [6]). While direct, cell-based chemical screenings of CSCs have the potential to detect new anti-CSC inhibitors, such platforms are plagued by numerous technical and biological limitations [7]. For example, the use of markers expressed on the cell surface of CSCs, side-population or intracellular enzymatic activities (e.g., ALDH1 activity) have been used to isolate and enrich for CSCs, but these methods are limited due to 1) the lack of clearly defined CSC-specific markers 2) CSC phenotypic heterogeneity, 3) the propensity of enriched CSCs to undergo asymmetric division to restore the original cellular distribution of CSCs and non-CSCs prior to enrichment (i.e., CSC plasticity), and 4) the technical limitation of isolating large numbers of highly purified CSC populations. Thus, long-term cultures of purified CSCs isolated based on the expression of cell surface markers or enzymatic activity are technically unsustainable and not amenable to compound screening. Alternative phenotypic/functional approaches based on methodologies such as in vitro sphere cultures have also been utilized, but such approaches are labor-intensive, the heterogeneity of the cultures cannot be controlled for, and spheres are inherently difficult to manipulate making this approach very low-throughput. We took an alternative approach and describe in this study a small-scale screen using mouse embryonic stem cells (mESCs) as a surrogate system for CSCs to identify small-molecules with anti- “stem” properties. Unlike CSCs, mESCs are readily and homogeneously propagated making them amenable to screening, but at the same time share relevant key properties with CSCs in that they 1) are highly plastic and give rise to diverse cellular lineages 2) express markers of pluripotency (e.g., Klf4, Oct3/4, Sox2 and Nanog) and 3) have tumorigenic potential. Thus, mESCs, despite being obviously different from CSCs, represent a suitable surrogate model for identifying inhibitors of CSCs properties, including self-renewal, the latter of which is governed by transcriptional subprograms described to be similar to that of ESCs [8,9]. Using the aforementioned approach, we identified a class of molecules with preferential CSC killing activity. In characterizing the mechanism of action of these molecules, we identified a general strategy to target CSCs: lysosomal membrane permeabilization (LMP) activity, a well-described mechanism of action for a wide range of chemotherapeutics. LMP causes the release of lysosomal contents (e.g., cathepsins, proteases and other hydrolases) to the cytosol, a potentially lethal process as released lysosomal proteases—primarily cathepsins—can affect important cellular organelles (e.g., mitochondria) and degrade essential proteins causing lysosomal-dependent cell death (LDCD) [10,11]. Several reports have documented changes in lysosomes during cancer progression and metastasis, including changes in lysosomal size and volume [12], increased membrane fragility [13], trafficking [14] and composition [15]. Moreover, a correlation has been shown between the metastatic capacity and aggressiveness of tumor cells and high lysosomal cysteine cathepsins activity, such that these cells are especially sensitive to LDCD [16]. Thus, there has been much interest in understanding lysosomes and lysosome-associated proteins as potential therapeutic targets in cancer, and as such, therapies targeting tumor lysosomes to induce LDCD are under development [17]. Indeed, several classes of LMP-inducing drugs that preferentially target cancer cell lysosomes have been described [18,19]. Moreover, there is evidence indicating that glioblastoma and leukemic CSCs present an enhanced susceptibility to LMP-inducing agents [19,20]. Herein we have identified and characterized a new LMP-inducing molecule that shows potent anti-cancer activity. Specifically, we show that the specificity of LMP-inducing agents, in general, can reach beyond that of just cancer cells, as we show that CSCs are more sensitive to LMP drugs than their non-CSC counterparts and these, in turn, are more sensitive Cancers 2020, 12, 1790 3 of 24 than normal cells. Thus, the lysosome can be considered a new Achilles’ heel of CSCs, and the compounds described in this study are attractive candidates to be further developed for clinical use. 2. Results 2.1. Identification of Novel Compounds That Compromise CSC Viability We screened a chemical library of 640 compounds designed to represent a diverse chemical space (see Materials and Methods). Following 30-hour treatment with 5 µM of each compound, we assessed mESCs viability by a standard MTS assay and identified 12 compounds that decreased the number of viable cells by at least 50%, belonging to 2 classes (Figure 1A,B). One class (Series 2) comprised seven compounds previously identified to directly inhibit PI3K and/or mammalian target of rapamycin (mTOR) kinase activity in vitro, and which we revalidated herein (Figure S1A). The role of the PI3K/mTOR pathway in both mESC and CSC biology is well described [21–26] and, therefore, the identification of these compounds validates the effectiveness of the screen. The second class (Series 1) did not show activity against PI3K/mTOR in vitro (Figure S1A) and were structurally similar imidazo [1,2-b]pyridazine-3-carboxylic acid (3-trifluoromethoxy-phenyl) amide derivatives with the 6-position substituted with different “amino-fragments” (Figure 1C). To validate and further prioritize the hits from Series 1, we generated viability dose response curves in mESCs for the top three hits. Consistent with the relative activities determined from the initial screening, compounds (Cpd) 1–3, showed significant activity against mESCs viability with half maximal effective concentrations (EC500 s) of 1.33 µM, 2.05 µM and 1.50 µM, respectively (Figure 1D). Next, we evaluated the effects of Cpd 1 and 3 in human CSCs by first assessing their cytotoxicity on the PDX-derived PDAC culture PDAC1. These cells were grown in adherence or as spheres, where the latter enriches for the CSC population in vitro. While both Cpds 1 and 3 decreased the viability of PDAC1 cells, the effect was more pronounced on CSC-enriched spheres compared to cells grown as adherent monolayers (Figure 1E and Figure S1B). Since Cpd 1 showed the most potent activity, it was selected for follow-up assays on a panel of PDX-derived cultures. Confirming activity across multiple primary cultures, Cpd 1 at a 2.5 µM dose showed similar and preferential anti-CSC activity against three additional PDAC cell lines when cultured as spheres vs. adherent monolayers (PDAC 2–4) (Figure 1F). To rule out the possibility that the effects observed were due to culture conditions (2D adherent versus 3D spheres), we treated 7-day-old spheres re-plated as adherent 2D cultures with Cpd 1 for only 24 h so that they retain their CSC properties (Figure S1C). As expected, the higher activity of the compound for CSCs was independent of the type of culture condition (Figure S1D). Cancers 2020, Cancers 12,12, 2020, 1790 1790 4 of 425of 24 Figure 1. Identification of novel compounds that compromise cancer stem cell (CSC) viability. (A) Analysis Figure of1. relative (rel.) cell Identification viability of novel for mousethat compounds embryonic compromise stemcancer cells (mESCs) stem cell following 30 h treatment (CSC) viability. (A) with 5 µM of Analysis of each of the relative (rel.)640 cellcompounds viability forcontained within thestem mouse embryonic chemical library. Red cells (mESCs) line indicates following 30 h 50% viability treatment cutoff. with 5 µM(B) Top 12 of each compounds of the that increased 640 compounds containedthe percentage within of non-viable the chemical library. Redcellsline by at least 50% and the series to which they belonged. (C) Chemical structure of indicates 50% viability cutoff. (B) Top 12 compounds that increased the percentage of non-viable cellsthe structurally similar series by atofleast a -imidazo[1,2-b]pyridazine-3-carboxylic 50% and the series to which they belonged. acid (3-trifluoromethoxy-phenyl)-amide (C) Chemical structure of the structurally derivatives similar with series ofatathe substitution -imidazo[1,2-b]pyridazine-3-carboxylic 6-position with several “amino-fragments”. acid (3-trifluoromethoxy-phenyl)-amide (D) Viability curves performed on derivatives mESCs treatedwith substitution with increasingatconcentrations the 6-position with several “amino-fragments”. of compounds (Cpd.) 1, 2 and 3(D) forViability curves in 30 h. Indicated performed red on mESCshalf are the calculated treated with increasing maximal concentrations (EC effective concentration of compounds 50 ). (E) (Cpd.) Viability 1, 2 curvesand 3 for 30 h. on performed Indicated PDAC1 in red 2D adherent are monolayers the calculated or half maximaltreated 3D spheres effective withconcentration (EC50). (E) Viability increasing concentrations curves of compound performed Cpd. 1 for 30onh.PDAC1 Indicated adherent in red2D aremonolayers or 3D the calculated ECspheres treated with increasing concentrations 50 . (F) Analysis of rel. cell viability ± standard of compound Cpd. 1 for 30 h. Indicated in red deviation for PDAC2, PDAC3 and PDAC4 adherent 2D monolayers are the calculated EC50. (F) orAnalysis 3D spheresof rel. following cell viability30 h treatment with 2.5 µM of Cpd. 1. Cancers 2020, 12, 1790 5 of 25 ± standard Cancers 2020, 12, 1790 deviation for PDAC2, PDAC3 and PDAC4 adherent 2D monolayers or 3D spheres 5 of 24 following 30 h treatment with 2.5 µM of Cpd. 1. 2.2. 2.2.Compound Compound1 Impairs thethe 1 Impairs Self-Renewal andand Self-Renewal Tumor Initiating Tumor Capacity Initiating of PDAC Capacity CellsCells of PDAC by Depleting the CSC by Depleting the Compartment CSC Compartment Having Havingobserved observeda apreferential preferentialcytotoxic cytotoxiceffect onon effect human humanPDACPDACCSCs, CSCs,wewenext nextasked askedwhether whether Cpd 1 affects key CSCs properties and biological functions. First, we confirmed by flow cytometry Cpd 1 affects key CSCs properties and biological functions. First, we confirmed by flow cytometry that Cpd 1 preferentially targets the CSC compartment by analyzing the percentage of bulk that Cpd 1 preferentially targets the CSC compartment by analyzing the percentage of bulk and and CD133+ cells undergoing CD133+ apoptosisapoptosis cells undergoing 24 and 4824h and post48treatment (Figure 2A–C). h post treatment (Figure Indeed, when CD133+ 2A–C). Indeed, cells when CD133+ were analyzed separately, we observed that Cpd 1 induced significantly more apoptosis in cells were analyzed separately, we observed that Cpd 1 induced significantly more apoptosis in the the PDAC2 CD133+ PDAC2population comparedcompared CD133+ population to the bulktopopulation 24 and 4824 the bulk population h post treatment and 48 (Figure 2C). h post treatment (Figure 2C). Figure Figure 2.2.Compound Compound 11 preferentially targetsCD133+ preferentially targets CD133+CSCs. CSCs. (A,B) (A,B) Representative Representative flowflow cytometry cytometry plots plots of the of the percentage percentage of early of early apoptosis apoptosis (Annexin-V+), (Annexin-V+), latelate apoptosis apoptosis (Annexin-V+/DAPI+) (Annexin-V+/DAPI+) and and dead dead (DAPI+) (DAPI+) cellscells in the in the total total PDAC2cell PDAC2 cellpopulation population (top)(top) oror within within the theCD133-positive CD133-positivepopulation population (bottom) (bottom) treated with Cpd. 1 at 2.5 µM. Cells were analyzed (A) 24 h and(B) treated with Cpd. 1 at 2.5 µM. Cells were analyzed (A) 24 h and (B)4848h h post treatment post treatment initiation. initiation.(C) (C)Fold-change Fold-changeinintotaltotalapoptosis apoptosis(early(earlyand andlatelateapoptosis) apoptosis)forforeach eachgroup group and andtreatment treatment shown shown inin panels panels AA and andB,B, (Student’s (Student’s t-test). * p* < t-test). p <0.05; ***p p<0.001; 0.05;*** <0.001;ns, ns,not notsignificant. significant. We next compared the decline in CD133-positive or autofluorescent-positive [27] CSC populations We next compared the decline in CD133-positive or autofluorescent-positive [27] CSC in cultures treated with Cpd 1 or gemcitabine, the standard of care chemotherapeutic for PDAC and to populations in cultures treated with Cpd 1 or gemcitabine, the standard of care chemotherapeutic for which CSCs are inherently resistant [28]. Adherent cultures of PDAC2 or PDAC4 were treated for 3 PDAC and to which CSCs are inherently resistant [28]. Adherent cultures of PDAC2 or PDAC4 were days with gemcitabine or Cpd 1 at 2.5 µM or 1.25 µM (Figure 3A–D). We first assessed the capacity treated for 3 days with gemcitabine or Cpd 1 at 2.5 µM or 1.25 µM (Figure 3A–D). We first assessed of each treatment to induce apoptosis (Figure 3A) and modulate the percentage of CD133-positive or autofluorescent-positive CSCs (Figure 3B) in PDAC4 cells by flow cytometric analysis. While all Cancers 2020, 12, 1790 6 of 25 Cancers 2020, 12, 1790 6 of 24 the capacity of each treatment to induce apoptosis (Figure 3A) and modulate the percentage of CD133-positive or autofluorescent-positive CSCs (Figure 3B) in PDAC4 cells by flow cytometric three treatments analysis. While induced all three apoptosis, treatmentstreatment induced with gemcitabine apoptosis, preferentially treatment targeted preferentially with gemcitabine the non-CSC (CD133-negative or autofluorescent-negative) population evident targeted the non-CSC (CD133-negative or autofluorescent-negative) population evident by the increase in the percentage by the CD133-positive or autofluorescent-positive cells following treatment. In increase in the percentage CD133-positive or autofluorescent-positive cells following treatment. contrast, treatment withIn both doses of Cpd 1 led to both an increase in apoptosis and a significant decrease contrast, treatment with both doses of Cpd 1 led to both an increase in apoptosis and a significant in the percentage of CD133-positive decrease or autofluorescent-positive in the percentage of CD133-positive cells, or indicating again selectivecells, autofluorescent-positive elimination indicating of PDAC again CSCs (Figure 3A,B). We then assessed the effect of gemcitabine or Cpd 1 on self-renewal selective elimination of PDAC CSCs (Figure 3A,B). We then assessed the effect of gemcitabine or Cpd by assessing sphere-forming 1 on self-renewal capacity and, as expected, by assessing found thatcapacity sphere-forming while gemcitabine either didfound and, as expected, not affect thatsphere while formation gemcitabine in the case either didof not PDAC2affectorsphere increased it in the formation incase of PDAC4 the case (Figure of PDAC2 3C,D), Cpd or increased 1 decreased it in the case of sphere PDAC4 forming (Figurecapacity 3C,D), at Cpdboth doses tested 1 decreased and informing sphere both PDAC lines at capacity (Figure both 3C,D). Finally,and doses tested in order to in both determine if Cpd 1 impacts tumor initiating capacity, we treated sphere-derived PDAC lines (Figure 3C,D). Finally, in order to determine if Cpd 1 impacts tumor initiating capacity, PDAC2 cells for 3 days with either gemcitabine we treated sphere-derived or Cpd PDAC2 1 at cells 1.25 µM, for 3and days subsequently subcutaneously with either gemcitabine or Cpdinjected non-treated 1 at 1.25 µM, and and treated cells in vivo. Four weeks post injection we observed that while subsequently subcutaneously injected non-treated and treated cells in vivo. Four weeks post injectioncells pre-treated with gemcitabine gave rise to larger tumors, Cpd 1-treated cells exhibited a complete we observed that while cells pre-treated with gemcitabine gave rise to larger tumors, Cpd 1-treated inhibition in tumor formation (Figure cells exhibited 3E,F). This a complete reduced in inhibition tumor-initiating tumor formation capacity correlates (Figure withreduced 3E,F). This the inhibitory effects of tumor-initiating Cpd 1 on CSCs seen in vitro (Figure 3A–D). capacity correlates with the inhibitory effects of Cpd 1 on CSCs seen in vitro (Figure 3A–D). Figure 3. Cont. Cancers 2020, 12, 1790 7 of 24 Cancers 2020, 12, 1790 7 of 25 Figure 3. Series 1 compounds impair self-renewal and tumor initiating capacity of PDAC cells by Figure 3. Series 1 compounds impair self-renewal and tumor initiating capacity of PDAC cells by depleting the CSC compartment. (A) Left: Representative flow cytometry plots of the percentage of depleting the CSC compartment. (A) Left: Representative flow cytometry plots of the percentage of early apoptosis (Annexin-V+), late apoptosis (Annexin-V+/DAPI+) and dead (DAPI+) cells in PDAC2 early cells apoptosis treated (Annexin-V+), with 0.1 late apoptosis µg/mL gemcitabine (GEM) (Annexin-V+/DAPI+) or Cpd. 1 at 1.25 or 2.5andµM.dead (DAPI+) Apoptosis wascells in PDAC2 determined cells treated with 0.1 µg/mL gemcitabine (GEM) or Cpd. 1 at 1.25 or 2.5 72 h post treatment initiation. Right: Sum of early apoptosis, late apoptosis and dead cell percentagesµM. Apoptosis was determined 72 h post treatment initiation. Right: Sum of early apoptosis, late for each treatment. (B) Left: Representative flow cytometry plots of CD133-PE or autofluorescence apoptosis and dead cell (Fluo) expression (percentage) in PDAC4 cells 72 h following treatment with 0.1 µg/mL GEM or Cpd. 1or percentages for each treatment. (B) Left: Representative flow cytometry plots of CD133-PE at autofluorescence 1.25 or 2.5 µM. Right: (Fluo) expression Percentage (%)(percentage) in PDAC4 cells 72 h following of CD133 or Autofluorescent-positive treatment cells ± standard with 0.1 deviation µg/mL GEM or Cpd. 1 at 1.25 or 2.5 µM. Right: Percentage (%) of CD133 or Autofluorescent-positive for each treatment. (n = 4–6 per sample; Student’s t-test). (C–D) Number of 1st generation spheres/mL ± cells ± standard standard deviationdeviation for (C) for each or PDAC2 treatment. (D) PDAC4(n =cultures 4–6 per treated sample;forStudent’s with 0.1t-test). µg/mL(C–D) GEM or Number Cpd. 1of at 1.25 or 2.5 µM (n = 6; Student’s t-test). (E) Images of tumors obtained 10 weeks after injection of 5for 1st generation spheres/mL ± standard deviation for (C) PDAC2 or (D) PDAC4 cultures treated × with 0.1 µg/mL 103 PDAC4 GEMtreated spheres or Cpd.for1 at3 1.25 daysorwith 2.5 µM 0.1 (n = 6; Student’s µg/mL t-test).1(E) GEM or Cpd. or Images Cpd. 2 atof 1.25 tumorsµMobtained (n = 6 10 weeks after injection of 5 × 10 3 PDAC4 spheres treated for 3 days with 0.1 µg/mL GEM or Cpd. 1 injections per treatment). (F) Summary of weights of tumors shown in (E), (Student’s t-test). *p < 0.05; **por 7) retained revealed by the lackactivity with EC of activity 500 s below of Cpds 19 and1021. whereas non-basic µMNoteworthily, compounds all compounds (pKa in this < 7)with series had a nobasic activity, even at 50 µM (Figure S2). Thus, as an example, Cpd 16 bearing profile (pKa > 7) retained activity with EC50′s below 10 µM whereas non-basic compounds (pKa a piperidine secondary amine moiety, < 7) had showed good no activity, even potency at 50 µM (EC 50 = 3.26 (Figure S2).µM) but as Thus, its an corresponding example, Cpd analog Cpd 15,awhere 16 bearing the piperidine basicity was removed by the protection of the N-atom with a tert-butyl secondary amine moiety, showed good potency (EC50 = 3.26 µM) but its corresponding analog Cpd carbamate group, completely lost 15,activity. where the Thebasicity non-basic Cpds was 13 andby removed 18 the withprotection a hydroxylofgroup in the 6-position the N-atom fragmentcarbamate with a tert-butyl instead ofgroup, a basiccompletely amine were lostalso inactive. activity. Similar behavior The non-basic Cpds 13was and observed for Cpd 19 18 with a hydroxyl substituted group with a in the 6-position non-basic fragment instead of a basic amine were also inactive. Similar behavior was observed for Cpdof19 tetrahydropyranyl ring. Furthermore, the non-basic cyclohexyl derivative 21, an analog Cpd 16 where the substituted withbasic a NH moiety of non-basic piperidine ring was tetrahydropyranyl replaced ring. with a CH2the Furthermore, - group was alsocyclohexyl non-basic inactive, reinforcing derivativethe 21,importance an analog of ofCpd a basic center (basicity) 16 where the basic NH for the activity moiety of this class of piperidine of compounds. ring was replaced with a CH2- group was also inactive, reinforcing the importance of a basic center (basicity) fortechniques Next, we initiated target engagement and target deconvolution tasks using label-free the activity and chemical of this class ofprobes, respectively, in order to identify the molecular target(s) responsible for the compounds. observed anti-CSC Next, phenotype we initiated targetofengagement Series 1 compounds. and targetCpd 2 was selected deconvolution tasksas using a starting Cpd for label-free both of techniques these activities primarily due to its easier synthetic accessibility. Before and chemical probes, respectively, in order to identify the molecular target(s) responsible forproceeding, however, we first the tested the anti-CSC observed anti-CSCactivity phenotypeof Cpd 2. We observed of Series 1 compounds. that Cpd Cpd2,2 like was Cpd 1, reduced selected the percentage as a starting Cpd for both of CD133+ of thesecells and tumor activities growth primarily dueintonude mice synthetic its easier (Figure S3accessibility. and Figure 3E,F),Beforehighlighting proceeding,its anti-CSC however, we properties. Next, we performed a kinome screen to determine if Cpd 2 interacted first tested the anti-CSC activity of Cpd 2. We observed that Cpd 2, like Cpd 1, reduced the percentage with kinases, some of CD133+ cells and tumor growth in nude mice (Figure S3 and Figure 3E,F), highlighting its anti- CSC properties. Next, we performed a kinome screen to determine if Cpd 2 interacted with kinases, Cancers 2020, 12, 1790 8 of 24 Cancers 2020, 12, 1790 8 of 25 of some whichofare which known are to known to be involved be involved in CSCin CSC biology biology and if targeted and if targeted canpotential can have have potential anti-CSC anti-CSC activity. activity. The in vitro binding activity of Cpd 2 against a panel of 468 The in vitro binding activity of Cpd 2 against a panel of 468 kinases (KINOMEscan , San Diego,kinases (KINOMEscan ® ®, San CA, Diego, CA, USA) at 1 µM concentration was assessed. The compound turned out to be highly USA) at 1 µM concentration was assessed. The compound turned out to be highly selective, showing a selective, showing a selectivity index S(35) of 0.01. Only 4 kinases were significantly bound: HASPIN selectivity index S(35) of 0.01. Only 4 kinases were significantly bound: HASPIN (30% signal versus (30% signal versus control), the non-receptor tyrosine-protein kinase 2 (TYK2) (12%), control), the non-receptor tyrosine-protein kinase 2 (TYK2) (12%), serine/threonine-protein kinase serine/threonine-protein kinase pim-3 (PIM-3) (18%) and PIM-1 (5%). After dose response pim-3 (PIM-3) (18%) and PIM-1 (5%). After dose response characterization using kinase orthogonal characterization using kinase orthogonal assays, the compound showed significant activity for PIM- assays, the compound showed significant activity for PIM-1,-3 and HASPIN (IC50 = 98,of129 1,-3 and HASPIN (IC50 = 98, 129 and 915 nM, respectively) and very weak inhibition and 915 nM, TYK2 (IC50 = respectively) 65 µM) (Figure and S4A veryandweak inhibition Table of TYK2we S1). However, 50 = 65 µM) (ICdiscarded (Figureand PIM-1,-3 S4A and Table HASPIN S1).relevant as the However, wetargets discarded PIM-1,-3 and HASPIN as the relevant targets of Cpd 2 against of Cpd 2 against CSCs because compound Cpd 1 is not a PIM-1,-3 nor a HASPIN inhibitor CSCs because compound Cpd(IC150is> not 10 µMa PIM-1,-3 nor a HASPIN for all; Figure inhibitor S4 B). These results(IC 50 > 10to pointed µM for all; Figure a non-kinase S4B). target as These resultsfor responsible pointed the to anti-CSC a non-kinase target effects as responsible of Series 1 compounds.for the anti-CSC effects of Series 1 compounds. Next, Next, a chemical a chemicalproteomics proteomicsapproach approach based based on on the the design designand anduse useofofPhoto-Affinity Photo-Affinity Labeling Labeling (PAL) chemical probes derived from Cpd 2 was attempted. To this end, we (PAL) chemical probes derived from Cpd 2 was attempted. To this end, we designed an irreversible designed an irreversible PALPAL chemical chemicalprobe—Cpd probe—Cpd25—based 25—based on onCpd Cpd22and anditsits non-basic non-basic amide amide inactive inactive analogue analogue Cpd Cpd 26. A 26. so-called “minimalist A so-called “minimalistlinker” linker”with withaaphotoactivable photoactivablediaziridine diaziridine moiety moietyandand anan alkyne alkynereporter reporter tagtag waswas incorporatedinto incorporated intoCpd Cpd2.2.The The diazirine diazirine group group of of 25 25would wouldallow, allow,after afterincubation incubation with withcells and cells and short UV irradiation, the generation of highly reactive carbene species, which short UV irradiation, the generation of highly reactive carbene species, which would react covalently would react covalently with with those those proteins proteins bound bound or very or very proximal proximal to the to the probe. probe. On On the the otherother hand, hand, the terminal the terminal alkyne alkyne group group of 25 could be used for subsequent conjugation to suitable reporters of 25 could be used for subsequent conjugation to suitable reporters (rhodamine-N3 or biotin-N3 ) (rhodamine-N 3 or biotin- N3) using using bio-orthogonal bio-orthogonal click click chemistry. chemistry. These These conjugated conjugated molecules molecules were were thenthen used used totodetermine determinethe the localization of the probe in cells by imaging techniques and in pull-down experiments to identify localization of the probe in cells by imaging techniques and in pull-down experiments to identify probe-linked cellular protein targets [29]. The PAL probe 25 was synthesized following standard probe-linked cellular protein targets [29]. The PAL probe 25 was synthesized following standard procedures (Figure 4A). Next, the effect of Cpds 2 and 25 (and the inactive Cpd 26) on PDAC4 CSC procedures (Figure 4A). Next, the effect of Cpds 2 and 25 (and the inactive Cpd 26) on PDAC4 CSC viability was assessed side by side, and the activity of probe 25 was comparable or even better than viability was assessed side by side, and the activity of probe 25 was comparable or even better than that that of Cpd 2 (EC50 = 2.62 and 7.17 µM, respectively, Figure 4B), whereas, as expected, Cpd 26 was of Cpd 2 (EC50 = 2.62 and 7.17 µM, respectively, Figure 4B), whereas, as expected, Cpd 26 was inactive. inactive. Figure 4. Cont. Cancers 2020, 12, 1790 Cancers 2020, 12, 1790 9 of 24 9 of 2 Figure 4. Chemical probes of Series 1 demonstrate interaction with lysosomes. (A) Synthesis of PAL probe Cpd. 25. Figure 4. Chemical (B) Viability curvesprobes of Series performed 1 demonstrate on PDAC4 interaction 3D spheres treatedwith withlysosomes. increasing(A) Synthesis of PAL probe concentrations of Cpds. 2 orCpd. 25.30(B) 25 for Viability in h. Indicated curves red areperformed on PDAC4 the calculated 3D Fluorescence EC50 . (C) spheres treated with increasing concentrations (via analysis of Rhodamine-conjugated of Cpds. 2 or 25 for Cpd. click chemistry) 30 h. Indicated in redinare 25 localization the calculated PDAC4 EC50. (C) Fluorescence cells. Cells analysis were additionally stained withofanRhodamine-conjugated anti-lysosome-associated (viamembrane click chemistry) proteinCpd. 25 localization 2 (LAMP2) antibodyin PDAC4 cells. Cells were additionally and DAPI. LAMP2 localization stained with an Alexa-fluor was visualized anti-lysosome-associated membraneantibody 488-labelled secondary protein 2 (LAMP2) antibody and DAPI. (10×). (D) Representative LAMP2 amplified imageslocalization was visualized of Rhodamine with localization withanLAMP2-positive Alexa-fluor 488-labelled vesicles secondary antibody (Adobe® Photoshop®(10×). (D) Representative CS5 extended v11.0, Sanamplified Jose, CA, images USA). of Rhodamine localization with LAMP2-positive vesicles (Adobe Photoshop CS5 extended v11.0, San Jose, CA, USA). ® ® Probe 25 was then used to determine intracellular localization. Specifically, 7-day-old PDAC2 spheres re-plated as adherent Probefor 2524was h were thentreated used towith Cpd 25, exposed determine to UVlocalization. intracellular to crosslink Specifically, the probe to 7-day-old PDAC its potential putative targetsre-plated spheres and/or proximal proteins, as adherent for 24and subsequently h were treated withconjugated to rhodamine Cpd 25, exposed to UVvia to crosslink the prob click chemistry to visualize the probe. Confocal microscopy imaging revealed that Cpd 25 concentrated to its potential putative targets and/or proximal proteins, and subsequently conjugated to rhodamin in vesicular structures reminiscent via click of lysosomes. chemistry to visualizeCo-staining the probe. of cells formicroscopy Confocal the lysosome-associated imaging revealed that Cpd 2 membrane protein concentrated 2 (LAMP2), ain marker of lysosomes, vesicular confirmed that structures reminiscent Cpd 25 concentrated of lysosomes. Co-stainingclose to for the lysosome of cells LAMP2-positive structures, associated appearing membrane to protein localize2within lysosomes (LAMP2), a marker (Figure 4C,D). confirmed that Cpd 25 concentrate of lysosomes, close to LAMP2-positive structures, appearing to localize within lysosomes (Figure 4C,D). 2.4. Compound 2 Induces Lysosomal Membrane Permeabilization Given that the2.4. Compoundof localization 2 Induces Cpd 2 to Lysosomal lysosomes Membrane appeared Permeabilization to be required for its activity, we considered that it might Given be inducing that thelysosomal membrane localization of Cpdpermeabilization (LMP) as atomechanism 2 to lysosomes appeared be required to for its activity, w reduce CSC viability. It is known that LDCD-mediated cell death can be produced by lysosomotropic considered that it might be inducing lysosomal membrane permeabilization (LMP) as a mechanism drugs. The accumulation to reduce of these CSC agents viability.in the lysosome It is known isthat dependent on their lipophilicity LDCD-mediated cell death and can be produced b basicity, both of which are essential for their interaction with the lysosomal membrane, lysosomotropic drugs. The accumulation of these agents in the lysosome is dependent on the protonation in the acid lysosomal environment, lipophilicity and and subsequent basicity, both of retention which are within lysosomes essential [30]. As for their described interaction with the lysosoma above, the anti-CSCmembrane, activity of Cpd 2 also depends on its basicity (pKa = 10.01) and its protonation in the acid lysosomal environment, and subsequent retention withilipophilicity (cLogP = 4.04), which falls within lysosomes [30].the As accepted describedrangeabove, forthe lysosomotropic anti-CSC activity agents. The2same of Cpd behavioron its basicity (pK also depends was observed for other active analogues in this series (Figure S2). Therefore, to test this hypothesis, = 10.01) and its lipophilicity (cLogP = 4.04), which falls within the accepted range for lysosomotropi we first confirmed that agents.the effect The sameof Cpd 2 on PDAC2 behavior was CSC survival observed for was partially other active prevented analogues(≈in3-fold) this series (Figure S2 by the addition of Therefore, Bafilomycin-A to test this hypothesis, we first confirmed that the effect of Cpd 2 onthe (Figure 5A), a V-ATPase inhibitor, which increases the pH of PDAC2 CSC surviva lysosome (less acidic) thereby, was partially presumably prevented reducing (≈ 3-fold) the accumulation by the addition of of Cpd 2 in lysosomes. Bafilomycin-A (Figure 5A), a V-ATPase inhibito Next, we explored whichwhether increasesCpd 2 could the pH of theinduce lysosomecanonical characteristics (less acidic) of LMP by reducing thereby, presumably using a the accumulatio standard assay involving loading cells of Cpd 2 in lysosomes. with acridine orange (AO), a fluorescence dye used to stain acidic vacuoles. Re-plated PDAC2-derived sphere cultures were loaded with AO and subsequently Next, we explored whether Cpd 2 could induce canonical characteristics of LMP by using treated with either Cpd 2 or negative standard assay control Cpd 21,loading involving and AO fluorescence cells with acridinewasorange visualized (AO), byafluorescence fluorescence dye used to stai microscopy. As expected, untreated cells showed a vesicular pattern acidic vacuoles. Re-plated PDAC2-derived sphere cultures were loaded of AO staining in thewith far-red AO and subsequentl channel, reflecting treated accumulation in lysosomes, with either Cpd 2 orand a diffuse negative cytosolic control Cpdand 21, nuclear and AOstaining in the was visualized b fluorescence green channel, reflecting AO’s known fluorescence microscopy.abilityAstoexpected, bind nucleic acids cells untreated (Figure 5B). Treatment showed a vesicularof cells of AO staining i pattern with 5 µM of Cpd the2 greatly reduced lysosomal staining in the far-red channel far-red channel, reflecting accumulation in lysosomes, and a diffuse cytosolic within just 2 h andand nuclear stainin significantly increased the overall signal in the green channel, a phenomenon indicative in the green channel, reflecting AO’s known ability to bind nucleic acids (Figure 5B). Treatment o of LMP (Figure 5B). As expected, cells with cells 5 µMtreated of Cpdwith the non-active 2 greatly reducedCpd 21 hadstaining lysosomal the same in pattern the far-red of staining channel within just 2 h an in both the red andsignificantly green channels as untreated increased cells,signal the overall even at in athe dose of 10channel, green µM, indicating that the indicative of LM a phenomenon (Figure 5B). As expected, cells treated with the non-active Cpd 21 had the same pattern of staining i Cancers 2020, 12, 1790 10 of 24 Cancers 2020, 12, 1790 10 of 25 both activity ofthe Cpdred2and greenCSC against channels as untreated viability cells, even and lysosomal at a dose integrity of 10linked. were µM, indicating that the activity Flow cytometric analysis of Cpd 2 against CSC viability and lysosomal integrity were linked. Flow cytometric analysis performed on PDAC2 cells from a parallel experiment quantitatively confirmed these results at 6 h performed on PDAC2 cells from a parallel experiment quantitatively confirmed these results at 6 h (Figure S5A) and 24 h (Figure 5C) post treatment, with Cpd 2 decreasing overall AO mean far-red (Figure S5) and 24 h (Figure 5C) post treatment, with Cpd 2 decreasing overall AO mean far-red fluorescence intensity fluorescence (MFI) intensity in treated (MFI) cells andcells in Cpd 2-treated with Cpd and 21Cpd with having no effect 21 having on MFI. no effect Similar on MFI. affects Similar were affects observed were observed with PDAC1, PDAC3 and PDAC4 cells. We additionally assessed the effect ofWe with PDAC1, PDAC3 and PDAC4 cells with Cpd 2 at 5 µM at 24 h (Figure S5B). additionally Cpd 2 onassessed cytosolicthe effectcathepsin cysteine of Cpd 2activity, on cytosolic anothercysteine standardcathepsin activity, another assay for monitoring standard the release of assaylysosomal for monitoring contents after LMP. Consistent with the microscopy- and cytometry-based results,and the release of lysosomal contents after LMP. Consistent with the microscopy- cytometry-based treatment of results, treatment re-plated of re-plated PDAC2-derived PDAC2-derived spheres spheres with with Cpd 2 increased Cpd cysteine cytosolic 2 increased cytosolic cathepsin activity, cysteine while Cpd cathepsin 21 had activity, no effect while Cpd 21(Figure had no5D).effect (Figure 5D). Figure 5. Compound 2 induces lysosomal membrane permeabilization. (A) Viability curves performed on PDAC2 3D spheres either untreated or pretreated with 100 nM Bafilomycin for 5 h, then treated with increasing concentrations of compound Cpd. 2 for 72 h. Indicated in red are the calculated EC50 . (B) Fluorescence microscopy analysis of Acridine Orange in re-plated PDAC2 spheres. Cultures were Cancers 2020, 12, 1790 11 of 24 loaded with 1 µg/mL acridine orange for 15 min then non-treated (NT) or treated with either Cpd. 2 (5 µM) or Cpd. 21 (10 µM) for 2 h. Fluorescence was captured in the red and green channels to measure the amount of acridine orange staining acidic organelles or nucleic acids, respectively (Nikon Eclipse Ti, 10×). (C) Representative flow cytometric analysis of Acridine Orange (AO) fluorescence in re-plated PDAC2 spheres. Cultures were either non-treated (NT) or treated with 1.25, 2.5 or 5 µM of Cpd. 2 or Cpd. 21, and fluorescence was assessed at 24 h post treatment. (D) Cytosolic cysteine cathepsin activity, expressed as arbitrary units (au), was determined in PDAC2-derived spheres. Spheres were either NT or treated with 1.25 or 2.5 µM of Cpd. 2 or Cpd. 21 for 6 h then permeabilized with 25 µg/mL digitonin (top) to assess cytosolic cathepsin activity or 250 µg/mL digitonin to assess total cellular cathepsin activity (bottom). 2.5. Broad Spectrum of LMP-Inducing Agents Target CSCs In accordance with evidence indicating that glioblastoma and leukemic CSCs present an enhanced susceptibility to LMP-inducing agents [19,20], well-established LMP-inducers mefloquine and siramesine also affected PDAC2 CSC viability with varying potencies (Figure 6A). We next assessed the effect of siramesine (SIR) alongside Cpd 2 on the self-renewal/sphere-forming capacity of PDAC1 and PDAC3 cultures, and observed that both compounds decreased sphere forming capacity at both concentrations tested (Figure 6B). Like Cpd 2, we confirmed that the anti-CSC effect of siramesine is associated with LMP activity (Figure 6C). Based on the sum of these results, we speculated whether pancreatic CSCs might have an increased lysosomal mass compared to non-CSCs, which might account for their increased sensitivity to LMP-inducing agents. Indeed, altered lysosomal biology has been reported in CSCs from other tumor types such as breast and embryonal rhabdomyosarcoma [31,32]. To explore this possibility, we loaded re-plated PDAC2-derived spheres and PDAC2 adherent cell populations with AO and observed a clear increase in lysosomal mass in re-plated PDAC2-derived spheres compared to adherent cells, as evidence by an increased AO signal in the far-red channel relative to the green (Figure 6D). This observation was validated by flow cytometry (Figure 6E), using AO and lysotracker. Moreover, we took advantage of our previously published RNA sequencing data (ArrayExpress: E-MTAB-3808 [33]) to confirm whether CSC-enriched sphere cultures over express a previously reported lysosomal biogenesis gene signature [34] compared to their adherent counterpart cultures. As expected, CSC-enriched sphere cultures showed transcriptional upregulation (Figure S6A) and significant enrichment (Figure S6B) of this gene signature set. Together, these data suggest that Cpd 2 is a novel LMP-inducing agent. 2.6. LMP Inhibits Pancreatic CSC-Mediated Tumorigenesis and Disease Recurrence In Vivo Lastly, we wanted to translate these findings to human patient-derived xenografts (PDX) in vivo. Initial in vivo PK studies of Cpd 2 in BALB/c mice showed high clearance (>100% hepatic blood flow) and low levels in plasma after intravenous administration pointing to high rate of drug elimination (Figure S7A,B). Given the poor PK properties of Cpd 2, the well-known orally bioavailable LMP-inducer siramesine was selected as a tool compound to test the proof of concept that LMP-inducing agents can impact PDAC CSC biology in vivo. It is important to note the good pharmacokinetics of siramesine, non-toxic effects in humans and the well-established mechanism of action as an LMP [35,36]. Moreover, siramesine and Cpd 2 showed, in our hands, comparable anti-CSC activity in vitro (Figure 6B). To this end, we implanted nude mice with PDAC1 or PDAC3 PDXs. Once tumors reached 500 mm3 , mice were randomized to one of the following four treatment groups: Diluent control; Gemcitabine (GEM) (biweekly 125 mg/kg i.p.); siramesine (SIR) (30 mg/kg oral gavage); or GEM + SIR (Figure S7C). Interestingly, for both utilized PDX models, no significant differences were observed for vehicle-treated and SIR single treatment; however, tumors treated with GEM or a combination of GEM + SIR were significantly reduced compared to control tumors and single treatment tumors (Figure 7A). Importantly, flow cytometry analysis of dissociated tumors at day 23 post treatment showed differences in the percentage of cells expressing the CSC markers autofluorescence, CD133 or CD133/CXCR4 following Cancers 2020, 12, 1790 12 of 24 treatment. While not significant, for PDAC1 there was a marked increase in autofluorescent-positive Cancers 2020, 12, 1790 12 of 25 cells with GEM treatment, indicating an enrichment in this population Figure 6. Lysosomal membrane permeabilization (LMP)-inducing agents specifically target CSCs. (A) Figure 6. Lysosomal membrane permeabilization (LMP)-inducing agents specifically target CSCs. (A) Viability curves performed on PDAC2 3D spheres treated with increasing concentrations of mefloquine Viability curves performed on PDAC2 3D spheres treated with increasing concentrations of (top) or siramesine (bottom) for 30 h. Indicated in red are the calculated EC50 . (B) Number of 1st mefloquine (top) or siramesine (bottom) for 30 h. Indicated in red are the calculated EC50. (B) Number generation spheres/mL ± standard deviation for PDAC1 (left) or PDAC3 (right) cultures treated with of 1st generation spheres/mL ± standard deviation for PDAC1 (left) or PDAC3 (right) cultures treated Cpd. 2with or siramesine Cpd. 2 or (SIR), both (SIR), siramesine at 1.25both or 2.5 1.25(nor=2.5 atµM 6; Student’s µM (n = 6; t-test, *** p 2 and 6.5 < pKa < 11) [30]. Specifically, the lipophilicity of these agents is essential to facilitate their interaction with the membranes of and entry into lysosomes and their basic center(s) are crucial for their retention in the lysosome following their protonation by the acidic environment of the organelle (pH: 4.5–5.0) leading to their subsequent accumulation. Based on the similarities in basicity and lipophilicity with siramesine and mefloquine and the localization of our compounds with the lysosome in PDAC CSCs, we hypothesized and experimentally confirmed via numerous approaches that our Series 1 compounds were also acting via an LCDC-based mechanism of action. Moreover, when compared to the known LMP inducer siramesine, Cpd 2 showed equivalent anti-CSC activity, confirming that the Series 1 compounds function by inducing LCDC. The aforementioned points have several implications. Firstly, using an independent and unrelated screening approach, we also identified LMP inducers as potent inhibitors of CSCs. As mentioned above, only two other studies to date have identified that LMP inducers can target CSCs of glioblastoma [20] and AML [19]. Importantly, our study expands this observation to PDAC CSCs and highlights that LMP inducers may represent a potential anti-CSC therapy for many tumor types (both solid and non-solid). Secondly, siramesine showed equivalent anti-CSC activity as Cpd 2 in vitro. Furthermore, when siramesine was used together with gemcitabine in two PDX models, combination treatment efficiently and significantly reduced the CSC compartment, which was validated by flow cytometry and in serial tumorigenesis assays where the CSC frequency was reduced to 1 in 8290. While gemcitabine alone equally reduced PDAC1 and PDAC3 tumor growth in first generation tumors, flow cytometry analysis of these tumors confirmed an enrichment rather than a reduction in the CSC compartment, which resulted in tumor relapse upon serial passage. Siramesine alone had no effect on tumor size, but did reduce the percentage of CD133+ and CD133+ /CXCR4+ in PDAC3 tumors; however, this effect was not sufficient to reduce tumor growth at the dosage used (30 mg/kg). Thus, in the case of LMP inducers, higher doses or targeting both the CSC and the more differentiated bulk tumor cells (e.g., with gemcitabine) would likely be necessary to achieve sustained tumor clearance. It is interesting that Jensen et al. similarly showed that siramesine alone was ineffective in vivo (with 100 mg/kg) using implanted invasive glioblastoma spheroids or gliobastomas PDXs [20]; however, the authors did not use siramesine in combination with standard chemotherapeutics, nor did they examine the CSC compartment or perform serial transplantation analysis. Thus, the ability of siramesine to effectively target glioblastoma CSCs is still unresolved and would benefit from combination therapy-based studies. Lastly, while we did not measure lysosomal size, we did show that PDAC CSCs have increase lysosomal mass. The latter we attribute to increased lysosomal biogenesis, which was confirmed by RNAseq analysis using a previously published lysosomal biogenesis gene signature set [33]. Using this same signature, Sukhai et al. demonstrated a similar phenotype for primitive and progenitor AML CSCs, which they successfully targeted with mefloquine [19]. Taken together, these observations indicate a plausible mechanism to explain the observed differential and specific activity of the LMP inducers siramesine or mefloquine at the level of CSCs, at least for AML and PDAC, respectively. It would not be surprising if increased Cancers 2020, 12, 1790 16 of 24 lysosomal mass is a conserved hallmark of CSCs across different tumor entities, which is necessary for their biological activity and “stem”-ness. Thus, targeting the lysosome may represent a true Achilles’ heel for CSCs in general and should be further explored. 4. Materials and Methods 4.1. CNIO-640 Chemical Library The Experimental Therapeutics Program at the CNIO, ETP-CNIO, owns a chemical library of about 50,000 single compounds built as a result of the consolidation of several sub-libraries selected attending to different criteria such as chemical diversity, kinase-targeted focus, potential to disrupt protein–protein interactions, and the presence of low molecular weight compounds to facilitate fragment-based drug discovery. The drug-likeness of the whole library was also ensured by the application of filters such as “rule of five” [47]. The compounds were selected from commercial origin as well as from internally newly designed and synthesized chemical matter. Representative libraries of the whole 50,000 library with smaller sizes were defined after clustering, based on similarity analysis, and selection of representative compounds from each cluster. A 640-compound library, subject of the current detailed screening campaign, is the minimum size set of compounds representing the chemical ETP-CNIO collection. 4.2. Murine Embryonic Stem Cells Mouse embryonic stem cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 15% Fetal bovine serum (FBS), non-essential amino acids (NEAA), 2-mercaptoethanol, and Leukemia inhibitory factor (LIF) and passaged on gelatinized plates under standard conditions. 4.3. Primary Human Pancreatic Cancer Cells The PDAC patient-derived xenograft (PDX) Panc354 (referred to hereafter as PDAC3) was obtained from Dr. Manuel Hidalgo under a Material Transfer Agreement with the Spanish National Cancer Centre (CNIO), Madrid, Spain (Reference no. I409181220BSMH). PDAC1, PDAC2 and PDAC4 PDXs were established from primary patient resected tumors. Samples were provided by the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002), integrated in the Spanish National Biobanks Network and they were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees (Control no. No. Control: DE-BIOB-73 AC65, RG.BIOB-96 REV, and CI-BIOB-08-10-01 rev), with informed consent and according to Declaration of Helsinki principles. Primary patient tumors were subcutaneously implanted in immunocompromised female 6- to 8-week-old NU-Foxn1nu nude mice (Envigo, Barcelona, Spain) and subsequently passage in vivo to establish PDX PDAC1, PDAC2 and PDAC4. Primary PDX-derived in vitro cultures were established as previously detailed [48]. Briefly, xenografts were minced, enzymatically digested with collagenase (Stem Cell Technologies) for 60 min at 37 ◦ C and after centrifugation for 5 min at 1800 rpm, the cell pellets were resuspended and cultured in RPMI (1:1; #61870-010; Gibco, Waltham, MA, USA) supplemented with 10% FBS (Invitrogen, Waltham, MA, USA), Pen/Strep (1:100; #1500-063; Gibco, Waltham, MA, USA), and Fungizone (1:250; #15290-018; Gibco, Waltham, MA, USA). Primary cultures were tested for Mycoplasma at least every 4 weeks. 4.4. Sphere Formation Assay Pancreatic CSC spheres were generated as previously described [48] in ultra-low attachment plates (Corning) using CSC media: serum-free DMEM/F12 medium (1:1; #21331-020; Gibco, Waltham, MA, USA), supplemented with Pen/Strep (1:100; #1500-063; Gibco, Waltham, MA, USA), Fungizone (1:250; #15290-018; Gibco), L-Glutamine (1:100; #25030-024; Gibco, Waltham, MA, USA), B-27 (1:50; #17504-044; Gibco, Waltham, MA, USA) and basic fibroblast growth factor (FGF-b) (1:5000; Gibco, Waltham, MA, USA). All cell culture was carried out at 37 ◦ C in a 5% CO2 humidified incubator. To quantify spheres Cancers 2020, 12, 1790 17 of 24 of > 40 µm, 1 mL of sample volume was analyzed with a CASY®Cell Counter (Roche Applied Sciences, Mannheim, Germany). The CASY®Cell Counter measuring principle is based on a capillary particle counter with pulse area analysis, that permits the determination of cell count, cell concentration, cell volume (peak), and average cell diameter, specifically diameters of 40–80 µm, 80–120 µm and >120 µm. 4.5. Drug Screening and Cell Viability Assays Murine embryonic stem cells (mESCs) were plated at 20,000 cells per well in gelatinized 96 well plates. Compounds from the 640 compound ETP library were added by a Beckman FX 96 tip to achieve a final concentration of 5 µM in each well, and each compound was tested in duplicate. Cells were treated for a total of 30 h prior to assessing cell viability using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) assay according to manufacturer’s instructions (G3582, Promega, Madison, WI, USA). Dose response curves were carried out on mESCs under the same conditions using indicated compounds. To test the viability of CSCs, 10,000 PDAC cells were plated in spheroid conditions and immediately treated with indicated compounds and concentrations for the duration of the sphere formation assay. For adherent non-CSCs, 20,000 cells were plated in wells of standard tissue culture-treated plates (Corning, Corning, NY, USA) and treated the following day with indicated compounds and concentrations. For re-plated spheres (Figure S1B), 7-day-old spheres were filtered using a 40 µM filter (Fisher Scientific, Hampton, NH, USA), washed once with 1× PBS, and incubated with trypsin 0.05% EDTA at 37 ◦ C (Thermo Fisher Scientific, Waltham, MA, USA) to achieve a single cell suspension (approximately 10–30 min, depending on the PDAC culture). Trypsinized cells were counted and 20,000 cells were plated in wells of standard tissue culture-treated plates (Corning) and treated the following day with indicated compounds and concentrations. 4.6. Flow Cytometry Cells were analyzed with a 4-laser Attune NxT Acoustic Cytometer (ThermoFisher Scientific). Cells and digested tumors (as described in [41]) were resuspended in FLOW buffer (1X PBS; 3 mM EDTA (v/v); 3% FBS (v/v)), and the following fluorescently-tagged antibodies were used to label cells for 30 min at 4 ◦ C: Mouse monoclonal anti-human CD133-PE (1:20, Cat no. 130-110-962, Miltenyi, Bergisch Gladbach, Germany), Mouse monoclonal anti-human CD133-APC (1:20, Cat no. 130-111-080, Miltenyi, Bergisch Gladbach, Germany), mouse monoclonal anti-human C-X-C chemokine receptor type 4- Phycoerythrin (CXCR4-PE) (1:20, Cat no. 130-117-354, Miltenyi, Bergisch Gladbach, Germany), or mouse monoclonal anti-human Epithelial cell adhesion molecule-fluorescein isothiocyanate (EpCAM-FITC) (1:20, Cat no. 130-111-115, Miltenyi, Bergisch Gladbach, Germany). Autofluorescent cells were excited with blue laser 488nm and selected as the intersection with the filters 530/40 and 580/30 as previously described [27]. For lysosomes quantification, Acridine Orange (AO) (A1301, Thermo Fisher Scientific, Waltham, MA, USA) or Lysotracker Deep Red (DR) (L12492, Molecular probes, Life Technologies, Waltham, MA, USA) were used. AO fluorescence was assessed with two combinations of excitation and emission spectra: BL1 (Ex488/Em695/40, equal to the far-red channel (>670 nm)) and BL3 (Ex488/Em695/40, equal to the far-red channel (>670 nm)) Lysotracker DR fluorescence was assessed with RL1 (Ex638/Em670/14). Bafilomycin A1 (100 nM, Calbiochem, San Diego, CA, USA) was included as a positive control for lysosome disruption. For Annexin V staining, floating and attached cells were pooled and resuspended in 1× AnnexinV staining buffer containing AnnexinV-APC diluted 1:20 (Biotium, Freemont, CA, USA) and incubated for 20 min at room temperature prior to flow cytometric analysis. For all assays, DAPI was used to mark and exclude dead cells, and data were analyzed using the software FlowJo v9.3 (Tree Star Inc., Ashland, OR, USA). 4.7. In Vivo Assays Female 6- to 8-week-old NU-Foxn1nu nude mice (Envigo, Spain) were subcutaneously injected with control or pre-treated 1 × 104 PDAC cells in 50 µL of MatrigelTM (Corning). Tumor growth was monitored bi-weekly for up to 10 weeks. For Siramesine experiments, mice were subcutaneously Cancers 2020, 12, 1790 18 of 24 implanted with PDAC1 and PDAC3 PDXs. After 4 weeks of tumor growth, mice were randomized into treatment groups (5 mice per group). Mice were treated with Siramesine twice a week (30 mg/kg by oral gavage formulated in 0.5% methylcellulose 15 cP in 0.9% NaCl solution) for ~4 weeks. Gemcitabine (GEMZAR® , Eli Lilly, Indianapolis, IN, USA) was administered twice a week (125 mg/kg i.p.) for ~4 weeks. Tumor volumes (mm3 ) were determined twice per week using a digital caliper. Mice were sacrificed at 24 days post treatment initiation and tumors were extracted and analyzed by flow cytometry as described above. For second generation tumor formation, 1000 and 100 cells of indicated treatment groups were resuspended in Matrigel TM and injected subcutaneously in recipient NU-Foxn1nu nude mice and tumor formation was monitored for 10 weeks. Mice were housed according to institutional guidelines and all experiments 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) or by the Instituto de Salud Carlos III Ethics Committee (CBA12_2014-v3) and La Comunidad de Madrid (PROEX 335/14 or PROEX 53/14) 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). 4.8. Pharmacokinetic Studies (PK) Female BABL/c mice, 10 weeks old were used (n = 3 per time point). Compound 2 was formulated in 0.9% NaCl solution for both oral and intravenous (i.v.) injections. Plasma samples were collected following a single i.v. or oral administration of 8 mg/kg at 0.08, 0.16, 0.25, 0.5, 1, 4, 8 and 24 h. The extraction of Cpd 2 from plasma was achieved by solid phase extraction followed by high performance liquid chromatography/tandem mass spectrometry (Agilent 1100, Applied Biosystems API2000) analysis. The amount of inhibitor and the internal standard in each mouse plasma sample were quantified based on calibration curves generated using standards of known concentrations of compound. This assay method was sufficiently accurate for the quantification of Cpd 2 with a limit of detection (LOD) and a low limit of quantification (LLOQ) of 5 ng/mL. Pharmacokinetic parameters were estimated using WinNonlin Version 5.2 software (Pharsight Corp., Mountain View, CA, USA), by fitting both the experimental i.v and oral data to a bicompartimental model. Mice were housed according to institutional guidelines and all experiments were performed in compliance with the institutional guidelines for the welfare of experimental animals approved by the Instituto de Salud Carlos III Ethics Committee (CBA12_2014-v3) and La Comunidad de Madrid (PROEX 53/14), 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). 4.9. KinomeScan The KINOMEscan TM screening platform employs an active site-directed competition binding assay to quantitatively measure interactions between test compound and 468 human kinases and disease relevant mutant variants. These assays do not require ATP and thereby report thermodynamic interaction affinities. The assay was performed at DiscoveRx Corporation. 4.10. Synthesis of Affinity Chemical Probes General Procedure; Chemicals were purchased from Aldrich Chemical Company Ltd., Apollo Scientific Ltd. and TCI Europe N.V. Unless otherwise stated, commercial chemicals, and solvents were used without further purification. Anhydrous dichloromethane (DCM), dioxane, acetonitrile, and TEA (Tris base, acetic acid and EDTA) were purchased from Aldrich in Sure SealTM bottle and kept under nitrogen. Proton (1 H) NMR spectra were recorded on Bruker Avance II 300 using DMSO-d6 as solvent. Chemical shifts are expressed in parts per million (ppm) (δ relative to residual solvent peak for 1 H). Cancers 2020, 12, 1790 19 of 24 Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad) or combination thereof. The high-performance liquid chromatography (HPLC) measurements were performed using a HP 1100 from Agilent Technologies comprising a pump (binary) with degasser, an autosampler, a column oven, a diode-array detector (DAD) and a column Gemini-NX C18 (100 × 2.0 mm; 5 µm particle size). Eluent A, water with 0.1% formic acid; eluent B: acetonitrile with 0.1% formic acid. Gradient 5% to 100% of B within 8 min at 50 ◦ C, DAD. Flow from the column was split to a MS spectrometer. The MS detector was configured with an electrospray ionization source or API/APCI. Nitrogen was used as the nebulizer gas. Data acquisition was performed with ChemStation LC/MSD quad, software. Purities of all reported compounds were greater than 95% based on HPLC chromatograms obtained on an Agilent HP 1100 LCMS system. 6-Chloro-imidazo[1,2-b]pyridazine-3-carboxylic acid (3-trifluoromethoxy-phenyl)-amide (23). Trimethylaluminium (2M in hexanes, 1 mL, 2 mmol) was carefully added to a solution of 3-trifluoromethoxyphenylamine (345 mg, 2 mmol) in dry CH2 Cl2 (9 mL), under N2 , and this mixture was stirred for 30 min. Then, ethyl 6-chloroimidazo[1,2-b]pyridazine-3-carboxylate 22 (400 mg, 1.773 mmol) was added and the reaction was refluxed (40 ◦ C) for 17 h. The reaction was left to cool down, quenched with HCl 0.2 M (10 mL) and extracted with CH2 Cl2 , and washed with brine. The organic layers were dried (MgSO4 ), filtered and concentrated, affording the desired product 23 as a pale brown solid (530 mg, Y: 83%). LC-MS (ESI) RT 4.3 min m/z 357.1, 359.1 [M+ H]+. 6-[(Piperidin-4-ylmethyl)-amino]-imidazo[1,2-b]pyridazine-3-carboxylic acid (3-trifluoromethoxy- phenyl)-amide (24). A solution of compound 23 (194 mg, 0.54 mmol) and 1-Boc-4-(aminomethyl)piperidine (350 mg, 1.632 mmol) in dioxane (7 mL) with triethylamine (0.4 mL) was irradiated in the microwave at 160 ◦ C for 12 h. Water was added and the reaction mixture was acidified to pH 5, then extracted with EtOAc; the organic phase was dried and concentrated to give a residue which was purified by automated chromatography in SiO2 (eluents: EtOAc/MeOH 96:4 to 84:16) to give the corresponding tert-butoxycarbonyl (BOC) protected derivative (186 mg, Y: 64%) as a solid. LC-MS (ESI) rt 4.752 min m/z 535.25 [M+ H]+. The solid in 3 mL of dioxane was treated with 4M HCl/diox (1 mL) and the mixture was stirred at room temperature for 20 h. Ether was added and white solid precipitates were filtered, washed with ether and dried to give compound 24 (194 mg) as a chloridrate salt. LC-MS (ESI) RT 3.093 min m/z 435.15 [M+ H]+. 6-({1-[2-(3-But-3-ynyl-3H-diazirin-3-yl)-ethyl]-piperidin-4-ylmethyl}-amino)-imidazo [1,2-b]pyridazine- 3-carboxylic acid (3-trifluoromethoxy-phenyl)-amide (25). 3H-Diazirine, 3-(3-butyn-1-yl)-3-(2-iodoethyl) (45 mg, 0.096 mmol) was added to a solution of compound 24 (47 mg, 0.19 mmol) and TEA (0.08 mL) in AcCN (1 mL) and the mixture was stirred in a closed vessel at 100 ◦ C for 24 h. Aqueous saturated NaHCO3 was added and the reaction was extracted with a mixture of CHCl3 /iPrOH 1:1. The organic phase was dried and concentrated to give a residue which was purified by automated chromatography in SiO2 (eluents: DCM/MeOH(NH3 ) 9:1) giving 10 mg of 25 (19% yield). LC-MS (ESI) rt 3.559 min m/z 527.3 mainly fragmentation of diazirine ring [M+ H]+. 1 H NMR (300 MHz, DMSO): δ ppm 11.07 (s, 1 H), 8-05 (s, 1 H), 7.91 (d, J = 9.6 Hz, 1 H), 7.82 (m, 1H), 7.67 (m, 1 H), 7.61 (m, 1 H), 7.52 (t, J = 8.1 Hz, 1 H), 7.13 (m, 1 H), 6.93 (d, H = 9.9 Hz, 1 H), 3.29 (m, 4 H, signal under water of DMSO), 2. 79 (m, 3 H), 2.07 (m, 2 H), 1.98 (m, 2 H), 1.78 (m, 4 H), 1.54 (m, 4 H). 6-({1-[2-(3-But-3-ynyl-3H-diazirin-3-yl)-acetyl]-piperidin-4-ylmethyl}-amino)-imidazo[1,2-b] pyridazine-3-carboxylic acid (3-trifluoromethoxy-phenyl)-amide (26). DMAP (20 mg, 0.16 mmol) and EDC (10 mg, 0.05 mmol)) were added to a solution of compound 24 (15 mg, 0.03 mmol) and 3H-Diazirine-3-acetic acid, 3-(3-butyn-1-yl) (10 mg, 0.06 mmol) in dry DCM (1 mL) and the mixture was stirred at room temperature (RT) for 4 h. Solvent was removed and the Cancers 2020, 12, 1790 20 of 24 residue purified by automated chromatography in SiO2 (DCM/MeOH up to 9:1) giving a residue which was purified by HPLC to yield 4 mg of desired compound 26 (22% Yield). LC-MS (ESI) RT 5.65 min m/z 569.3 [M+ H]+. 1 H NMR (300 MHz, DMSO): δ ppm 11.02 (s, 1 H), 8.06 (s, 1 H), 7.92 (d, J = 9.9 Hz, 1 H), 7.87 (m, 1H), 7.70 (m, 1 H), 7.55 (m, 2 H), 7.13 (m, 1 H), 6.93 (d, H = 9.9 Hz, 1 H), 4.35 (m, 2 H), 3.67 (m, 2 H), 2.96 (m, 2 H), 2.80 (m, 1 H), 1.99 (m, 3 H), 1,8 (m, 3 H), 1.61 (m, 2 H). 4.11. Kinase Assays PIM1/3 kinase activities were measured as previously described in Martínez-Gonzalez et al. [49]. HASPIN activity was measured using the commercial ADP GLO assay (Promega), a homogeneous assay measuring ADP accumulation, as a universal product of kinase activity. The assay was done following general manufacturer recommendations and adapting protein to optimal conditions. Kinase buffer was 15 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 20 mM NaCl, 1 mM egtazic acid (EGTA), 0.02% Tween-20, 10 mM MgCl2 and 0.1 mg/mL BGG (bovine γ-globulin). The assay to measure autophosphorylation of HASPIN (ProQinase, Freiburg, Germany) was done at 150 µM ATP. In order to calculate the IC50 of described compounds, serial 1:5 dilutions were tested. Luminescence counts were read in a Victor instrument (Perkin Elmer) Values were plotted against inhibitor concentration and fit to a sigmoid dose–response curve with activity base from IDBS software. TYK2 kinase assay was performed at ProQinase. 4.12. Immunofluorescence with Click Chemistry Probes Seven-day-old spheres were disaggregated with trypsin and re-plated in chamber slides. One day after plating, cells were treated with 2.5 µM Cpd 25 for 30 min, then media was removed and replaced with ice cold PBS. Cells were exposed to 366 nM UV for 30 min using a hand-held lamp then washed, fixed, permeabilized and washed once again in PBS containing 1% BSA. Click it chemistry was performed using Click-it reaction cocktail from Invitrogen’s Click-it Cell Reaction Buffer Kit (Cat. no. C10269) containing 5 µM Rhodamine-azide for 2 h. Cell were subsequently stained with LAMP2 mouse monoclonal antibody from Abcam and anti-mouse IgG Alexa Fluor 488 and with DAPI. Preparations were mounted and visualized by confocal microscopy (Leica TCS SP5 Confocal Microscope, Wetzlar, Germany). 4.13. Acridine Orange Lysosomal Integrity Assay Re-plated spheres were loaded with 1 µg/mL acridine orange (A1301, Thermo Fisher Scientific) for 15 min then washed 3 times with PBS. Cells were treated with indicated concentrations of compounds for 2 h. Relative intensities of acridine orange binding to nucleic acids versus that contained in lysosomes was observed with an upright fluorescent microscope or via flow cytometry as detailed above. To visualize lysosomal mass in spheres versus adherent cells, acridine orange was added at 1 µg/mL for 15 min and fluorescent signal was observed in absence of compound treatment via flow cytometry as detailed above or with an upright fluorescent microscope (Nikon Eclipse Ti microscope with NIS Elements BR Imaging Software, Amsterdam, Netherlands). 4.14. Measurement of Cytosolic Cathepsin Activity Spheres were re-plated, and then treated the following day with the indicated concentration of compounds for 6 h in CSC media. Cells were subsequently placed on ice and extracted with either 25 µg/mL or 250 µg/mL of digitonin in 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM pefablock, pH 7.5. The extracts were taken and spun down briefly to eliminate floating cells. Cathepsin activity was measured in a 96 well format by measuring cleavage of zFR-AMC (Z-Phe-Arg-AMC. Z: N-carbobenzyloxy; 7-Amino-4-methylcoumarin), as previously described [50]. Cancers 2020, 12, 1790 21 of 24 4.15. RNA Sequencing Illumina RNA sequencing of five primary PDX-derived PDAC cultures grown as either adherent (Non-CSCs) or anchorage-independent spheres (CSCs), in duplicate, was previously performed [33], and raw data was deposited in ArrayExpress accession number: E-MTAB-3808. Differential expression of genes across the different conditions was calculated with Cuffdiff. Heatmaps showing gene expression levels (FPKM, Fragments per kilobase of transcript per million mapped fragments) for the different samples were drawn with Morpheus, (https://software.broadinstitute.org/morpheus) for a subset of selected genes involved in Lysosome Biogenesis [34]. This signature set was uploaded to the Gene Set Enrichment Analysis (GSEA) module of the Genepattern suite from the Broad Institute and false discovery rate (FDR) < 25% was considered statistically significant. 4.16. Statistical Analyses Results are presented as means ± standard error of the mean (SEM) unless stated otherwise. An unpaired two-tailed t test was performed to determine significance. p values < 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism version 6.0 (San Diego, California USA). CSC frequencies and significance were determined using the on-line extreme-limiting dilution assays (ELDA) program (http://bioinf.wehi.edu.au/software/elda/) [51]. 4.17. Data Availability Unique identifiers for publicly available datasets are indicated, a list of figures that have associated raw data can be provided, and there are no restrictions on data availability. 5. Conclusions In conclusion, while CSCs represent the root of most tumors, they are not without their weaknesses. In this study, we screened a chemical library to identify candidate anti-CSC molecules and identified several compounds, belonging to the same series, with potent activity against PaCSCs in several PDX culture models. Interestingly, these compounds acted on PaCSCs via induction of LMP, highlighting a potentially important role for lysosomes in PaCSC biology. The latter is supported by the fact that PaCSCs have increased lysosomal mass, a phenotype that makes them more susceptible to LMP inducers and one that appears to be conserved across CSCs of different tumor entities. While our lead compounds are still far from in vivo application, we show that the well characterize LMP-inducer siramesine can efficiently target and eliminate the PaCSC population in mice implanted with pancreatic PDX tumors, demonstrating the potential clinical application of LMP inducers as a promising anti-CSC therapeutic strategy for PDAC. Thus, inducers of LMP, in general, should be explored as possible therapeutics for CSC-driven tumors. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/7/1790/s1, Figure S1: Compounds preferentially target CSCs, Figure S2: Cellular activity and calculated physico-chemical properties of compounds from Series 1, Figure S3: Compound 2 preferentially targets CD133+ CSCs, Figure S4: Selectivity profile of compound 2, Figure S5: Compound 2 induces lysosomal membrane permeabilization as early as 6 h, Figure S6: PDAC CSCs upregulate genes involved in lysosome biogenesis, Figure S7: In vivo studies; Table S1: Kinase orthogonal assay results. Author Contributions: T.P.C. conceived the study, performed the majority of the experiments, designed experiments, analyzed data, wrote the first draft of the manuscript and co-led the work. M.d.R.R.-F. synthesized compounds and chemical probes. E.H.-E. and J.G. performed P.K. studies and PDX treatments with the compounds. M.I.A. performed biochemical assays. S.A. and B.S.J. designed and carried out experimental work and analyzed data related to the in vitro and in vivo PDAC CSC studies. S.V. analyzed extracted tumors by flow cytometry. J.M. performed the mass spectrometry and subsequent analyses. J.P. and S.M.-G. designed and/or selected appropriate compounds for the study. J.P. additionally contributed to the design of the target deconvolution strategy and formulation of the hypothesis of lysosomotropism as the basis for the mechanism of action of this class of compounds, and collaborated in the design of studies to test the hypothesis. T.P.C., B.S.J., J.P., C.B.-A. and S.M.-G. participated in data analysis. J.P., M.S., B.S.J. acquired funding, participated in drafting and editing the Cancers 2020, 12, 1790 22 of 24 manuscript, and were responsible for the overall conduct of the study. All authors have read and agreed to the published version of the manuscript. Funding: This study was financially supported by a Ramón y Cajal Merit Award (RYC-2012-12104) from the Ministerio de Economía y Competitividad, Spain (B.S.Jr.); funding from The Fero Foundation (B.S.Jr.); a Coordinated grant from the Fundación Asociación Española Contra el Cáncer (AECC) GC16173694BARB (B.S.,Jr.); Fondo de Investigaciones Sanitarias (FIS) grant PI15/01507 and PI18/00757 (B.S.,Jr.), (co-financed through Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”). ETP activities were supported by funds of The Spanish National Cancer Research Centre (CNIO). Work in the laboratory of M.S. was funded by the CNIO, the IRB and “laCaixa” Foundation, and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2017-82613-R), the European Research Council (ERC-2014-AdG/669622) and, Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement of Catalonia (Grup de Recerca consolidat 2017 SGR 282). Acknowledgments: We want to acknowledge the patients and the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002) integrated in the Spanish National Biobanks Network for its collaboration at the level of providing primary patient tumors used to produce the PDX tumors used in this study. We also want to particularly thank Christopher Heeschen for leading the RNAseq study referenced and used in this study. Conflicts of Interest: The authors declare no conflict of interest. References 1. Da Silva-Diz, V.; Lorenzo-Sanz, L.; Bernat-Peguera, A.; Lopez-Cerda, M.; Munoz, P. Cancer cell plasticity: Impact on tumor progression and therapy response. Semin. Cancer Biol. 2018, 53, 48–58. [CrossRef] [PubMed] 2. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. [CrossRef] [PubMed] 3. Hermann, P.C.; Sainz, B., Jr. Pancreatic cancer stem cells: A state or an entity? Semin. Cancer Biol. 2018, 53, 223–231. [CrossRef] [PubMed] 4. 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