Moonlight role of PEDF in breast cancer: self-renewal inhibition in breast cancer stem cells

Moonlight role of PEDF in breast cancer: self-renewal inhibition in breast cancer stem cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Moonlight role of PEDF in breast cancer: self-renewal inhibition in breast cancer stem cells Carmen Gil-Gas, Marta Sánchez-Díez, Paloma Honrubia-Gómez, Jose Luis Sánchez-Sánchez, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2636130/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 5 You are reading this latest preprint version Abstract Background : Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies has increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer initiating cells and slow cellular cycle cells, identified by their capacity to retain long labelling (LT+). Methods : In this study, we perform tumour cell culture dose-response assays in adherent and mamospheres cultures. We also probe our conclusion in Xenograft models. Cell culture and in vivo tumours were analysed by immunofluorescence, qPCR and flow cytometry assays showing how stem cell self-renewal modulating proteins, such as PEDF, can statistically significant modify the properties, cell-percentage expressing biomarkers, and carcinogenicity of cancer stem cells (by student t-test). Results : The PEDF signaling pathway could be a useful tool for controlling cancer stem cells’ self-renewal, and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells in vitro culture. We have designed a peptide consisting in the C-terminal part of this protein, which acts by blocking endogenous PEDF in culture cell assays. Conclusion : We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against Docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this PEDF-modified protein produces a significant decrease in the percentage of cancer stem cell expressing markers. Breast cancer Tumour initiating cells PEDF relapse tumoural biomarkers self-renewal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 I. Background The incidence of breast cancer has increased in recent years, due to an aging population[1,2]. In fact, breast cancer is the leading cause of death among females in developed countries, although the implementation of screening tests and the development of anti-neoplasic therapies such as Trastuzumab has increased the probability of a cure in those patients[3,4]. However, relapse rates remain very high in different types of breast cancer[5,6], indicating a need for further study into new pharmacological drugs and diagnostic methods to stop relapses in patients. Relapse is mainly due to the tumour cell population’s resistance, characterised by their capacity for self-renewal, resistance to drugs, and a slow cellular cycle[7,8]. This last characteristic allows for detection of this population, since this produces long retained labelling[9–11]. The auto-renewal capacity of these cells is essential for stem cells to persist throughout the life of the organism. Pigment Epithelium-Derived Factor (PEDF) protein has been previously related to this self-renewal mechanism[12,13]. This protein may induce cellular differentiation and promote apoptosis in a variety of tumour cells[10,11], and it is also able to inhibit tumour proliferation, vascularization, cell migration, and metastasis[14–16] affecting the division of fast tumour cells. In addition, PEDF is a niche-derived regulator of adult neural stem cells[12,17,18] that activates slowly dividing cells without inducing proliferation nor differentiation[12,13]. Together with the fact that tumour-initiating cells (TICs) are capable of self-renewal, this indicates that this protein-signalling pathway could be important not only as an anti-neoplastic agent but also as a regulator of self-renewal in TICs and patient relapse. As has been widely described, PEDF is a pleiotropic molecule, presenting two domains with clearly differentiated functions, an anti-angiogenic part and a second domain with neurotrophic properties, each activating different signalling pathways. By using this fragmentation of the molecule, we are able to take advantage of these domains’ different effects on signalling pathways, including the carboxy-terminal fragment´s inhibition of the TIC population´s crucial self-renewal ability, which thus hinders tumour recurrence. This is why we suggest a new therapeutic mechanism that consists in the co-administration of carboxy-terminal PEDF protein fragments and chemotherapy. In order to detect TICs, four different epitopes were analysed; these epitopes are implicated in the cells’ different processes and have been previously related with cancer stem cells in literature. BCRP1 is a drug transporter from the ABC transporter family, but while it is not as ubiquitous as other family members (such as MDR1) it is commonly expressed in the stem cell population[19,20]. EpCAM is a transmembrane glycoprotein that is involved in cell signalling, migration, proliferation, and differentiation[21,22]. This whole process is closely related to the epithelial-mesenchymal transition, essential in the metastatic mechanism in which TICs could play an important role. CD133 is a pentaspan membrane glycoprotein that has been used as a stem cell biomarker since its discovery in 1999, although its function is still unknown. AC133 is a glycosylated-isoform of CD133, recognized by a specific antibody which has been described as a biomarker for human hematopoietic stem cells and different cancer stem cell models[23–26]. All these proteins have been involved in different metastatic processes and could be a target for new therapies. More interestingly, they can be indicators of the process’ progress. Moreover, they could shed further light into the mechanisms and the signalling pathways of these cells involved in the resistance to chemotherapeutic treatments. One of the cellular mechanisms that may be involved in these resistance processes is anoikis[27–31]. Anoikis is a form of apoptosis that occurs in anchorage-dependent cells and in a niche context, when the cells detach from the surrounding extracellular matrix (ECM) and lose the connection to the surrounding nurse cells[31,32]; this is one of the processes we observed after the combination of chemotherapy and treatment with the carboxy-terminal part of PEDF protein. In this scenario, we have addressed the problem of locating breast tumour-initiating cells, and the study of the self-renewal mechanism by inhibiting its functionality and potential tumour recurrence. We also postulate that the PEDF signalling pathway could be a potential therapeutic target for control of cancer initiating cells’ self-renewal and tumour relapse, as has been postulated in other neoplastic models[33]. II. Methods II.1. CELL CULTURE MCF7 (ATCC® HTB-22™), MDA-MB-231 (ATCC® HTB-26™) and 293 HEK-293 (ATCC® CRL-1573™) cell lines were acquire from ATCC company (Manassas, Virginia, United States). MCF7 are epithelial cells isolated from the breast tissue of a 69-year-old, white, female patient with metastatic adenocarcinoma with Pleural effusion. The MCF7 line retains several characteristics of differentiated mammary epithelium including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes. The cells express the WNT7B oncogene[50]. The MDA-MB-231 cell line was isolated at M D Anderson from a pleural effusion of a patient with invasive ductal carcinoma and is commonly used to model late-stage breast cancer[73]. This cell line is ER, PR, and E-cadherin negative and expresses mutated p53. In microarray profiling, the MDA-MB-231 cell genome clusters with the basal subtype of breast cancer. Since the cells also lack the growth factor receptor HER2, they represent a good model of triple-negative breast cancer. MDA-MB-231 cells are invasive in vitro and when implanted orthotopically produce xenografts that spontaneously metastasize to lymph nodes[73]. The Pa00 cell line was derived in the laboratory from the sample of a patient with metastatic breast cancer with ascitic effusion [47], in which we found a small population of cells that express stem cell cancer markers, as can be seen in the results of the present article. Pa00, MCF7 and MDA-MB-231 cells were maintained in DMEM (Lonza. Basel, Switzerland) 10% FBS (Lonza), 1% Glutamine (Lonza) (0,2M) and 1% Penicillin/streptomycin (Lonza) (100 units+100 ug/10ul) in a 5% CO2 humidified incubator at 37ºC. II.2. STAIN WITH DDAO AND SORTING Cells for cell cycle dynamic assays were plated (100 000 cells). Next, these cells were washed with PBS, disaggregated and then incubated at 37ºC with Cell Trace ® Far-Red-DDAO-SE (DDAO-SE, Molecular probe ref C34564. Eugene, Oregon, United States), at the concentration recommended in the product data sheet. After 10 min, an aliquot of freshly labeled cells (approx. 200 000 cells) was fixed with 0,5% of paraformaldehyde to use as a positive control for the experiment. Then, we centrifuged the cells at 180g for five minutes and we suspended it in the culture medium to grow under standard conditions. Eight days in vitro later, CM-spf cells were disaggregated and sorted by In-fluxTM (Becton Dickinson. Franklin Lakes, New Jersey, United States) sorting equipment depending on their fluorescent retaining labelling level. Positive cells were considered slowly dividing cells, and thus potential tumour initiating cells. II.3. CYTOMETRY ASSAY Cytometry assays were performed with a MACSQuant Analyzer 10 cytometer (Miltenyi Biotec ref 130-096-343. Bergisch Gladbach, Germany). Samples were first washed in PBS and incubated with Miltenyi Biotec FcR blocking Reagent (human), in 100 microliters of sample. The immunostaining was performed per the standard protocol recommended by the commercial houses of the different antibodies. The antibodies and the working dilutions used were as follows: AntiBCRP-FITC 5D3 Chemicon (Temecula, California, United States) and AntiBCRP-PE 5D3 Chemicon incubated for 20 minutes at a 1:10 dilution; while AntiEpCAM- PE Clon HEA-125, AntiAC133-PE Clon AC133, AntiCD133-PE 293C3, Anti CD44-FITC and CD24-PE, all from Miltenyi Biotec, were incubated 10 minutes at a 1:10 dilution. Sorting experiments were performed with a BD Influx™ cell sorter. FITC or PE are used as abbreviation of Fluorescein isothiocyanate and Phycoerythrin respectively. II.4. GENE EXPRESSION For gene expression assays, total RNA from cultured cells was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) and used immediately for reverse transcriptase reactions or stored at 80ºC until use. RNA concentration was measured in a Nanodrop spectrophotometer (Thermo Fisher,Waltham, MA, USA). cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis kit (Fermentas, Waltham, MA, USA). The reaction was prepared according to the manufacturer’s instructions. Quantitative PCR reaction was performed for CDKN1A (also known as p21 inhibitor) using the KiCqStart SYBR Green kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. KiCqStart SYBR Green predesigned primers (Sigma, St. Louis, MO, USA) were employed (H_CDKN1A_1: Forward primer 5' TGTAAAACGACGGCCAGT and Reverse primer 5' CAGGAAACAGCTATGACC). Primers were used in a final concentration of 300 nM, and 10 ng of cDNA were used per well, for a total volume of 10 µl. All cDNA samples were measured in triplicate in a 96-well plate covered with adhesive seals in the thermocycler Roche LightCycler 480 (Roche, Basel, Switzerland). Reactions started with 10 min at 95ºC, followed by 45 cycles of 15 s at 95ºC, 1 min at 60ºC and 10 s at 72ºC. The 2 - D CT method was used for calculating the normalized mRNA expression. Fold increase relation over untreated controls has been used to graphicaly represent the decrease of gene expression. GADPH was used as housekeeping gene in quantitative PCR assay. II.5. XENOGRAFTS FOXn1nu females, at 1 month of age were obtained from Charles River International Laboratories (Wilmington, Massachusetts, United States) to use in these experiments. Animals were housed and bred under 20-25 ºC, 50-60% humidity and a 12 hour light-dark cycle. All experiments were performed in accordance with relevant guidelines and regulations and the animals were treated in accordance with the approval of the local ethics committee (University of Castilla-La Mancha PI081746). The experiments were performed as previously described[30]. In short, untreated cells (control) and treated cells (8nM PEDF or CTE-PEDF) suspended in PBS were injected subcutaneously into both flanks of immunocompromised mice. A total of 1200, 5000 or 3*10 6 cells (depending of the experiment) were injected in a final volume of 200 uL of a 1:1 dilution with MatrigelTM Basement Membrane Matrix (Becton Dickinson) with a 25 gauge-needle. The experimental unit is a single animal and a total of 28 animals were used. The mice were randomly assigned to the control and treatment groups via tail numbering that was also used as the order for successive xenograft injections. In the first experiment with animals (Figure 1) a total of 11 animals were used; with four animals in the control group and seven animals in the treatment group. For the second animal experiment (Figure 5) a total of nine animals were used, with three animals in each group (control, PEDF or CTE-PEDF treated injected cells). For the last animal experiment (Figure 6) a total of eight animals were used, with two injections each (one per flank, treated cells in the right flank and untreated cells in the left flank). There is an extensive track record in the published literature of using athymic nude mice and mouse xenograft models with human tumour cell lines to increase understanding of the factors affecting tumour growth[87]. The number of mice in each experiment was chosen based on this well-detailed experience, including experience in PDX (patient derived xenograft) models[88]. Possible confounding factors between groups were minimized by placing the mice from each group in different boxes (3-4 animals per cage, corresponding to animals of the same experimental group: control or treatment). All investigators responsible for the animal experiments in the team were aware of the group assignments at the different phases of the experiment (during assignment, conduct of the experiment, evaluation of results and data analysis). Tumour growth was monitored weekly with a caliper. The final tumour volume was calculated as V= 2 x L1 x L2 x π/6. Tumours were mechanically and chemically dissociated with collagenase and trypsin at 37ºC. They were then washed with PBS and seeded in DMEM medium, 10% FBS, 1% glutamine (200 mM), 1% Penicillin/streptomycin, 0.5% EGF and 0.04% FGF in a 5% CO2 humidified incubator at 37ºC 12h before the start of other experiments. The outcome measures assessed in the different experiments were: tumour size over time, markers expressed by tumour cells, and rates of resistance in dose-response assays in cell culture of these xenograft cells. The criteria used to include and exclude both animals (during the experiment) and data points (during the analysis) were established a priori . Specifically, those mice that suffered harassment or mistreatment by their peers in the cage for reasons beyond the control of the caretakers and showed significant deterioration or premature deaths not directly linked to the experiment were to be excluded from the study. Those animals would be mentioned although not considered for cellular and molecular analyses. However, no such circumstance was observed throughout the experiments and therefore no mice were excluded in these experiments. II.6. PEDF and Cter-PEDF, CTE-PEDF PRODUCTION PEDF and the carboxy-terminal part of PEDF protein (Cter-PEDF) were cloned into pcDNA™3.1/myc-His A, B, & C Mammalian Expression Vectors (InvitrogenTM. Carlsbad, California, United States) following the protocol previously described by Sánchez-Sánchez and coworkers[89]. After cloning, vectors were checked by sequencing (3130 Applied Biosystems. Foster City, California, United States). Serine 227 was mutated into a glutamate (E) residue using ser227glu-dw (5’-CCA AGT AGA AAT CCT CGA GCT CAG TCT TTC TGG AGT-3’) and ser227glu-up (5-GTT TGA CTC CAG AAA GAC TGA GCT CGA GGA TTT CTA-3) primers. After mutagenesis, the Cter-PEDF peptide with this glutamic change was named CTE-PEDF. Proteins were produced by HEK-293-T cells after transfecting with phosphate calcium. Conditioned mediums were collected after 3 DIV, quantified by western-blot using anti-c-myc mouse monoclonal IgG1 (Santa Cruz Biotechnology. Dallas, Texas, United States), and anti-phosphoserine clone 4A4, (Millipore. Burlington, Massachusetts, United States) and stored at -20ºC or purified using GE Healthcare Life Sciences™ HisTrap™ FF Crude columns (Thermo Fisher Scientific. Waltham, Massachusetts, United States). II.7. CHRONIC TREATMENT WITH PEDF AND CTE-PEDF Cells were treated with PEDF or CTE-PEDF at a final concentration of 8 nM in the medium. The acute treatment consists of a single treatment: two hours of peptide exposure before chemotherapy treatment. Chronic treatment consists of six peptide treatments week (culture medium with CTE once per week), totalling six weeks of peptide exposure before chemotherapy treatment. II.8. ACUTE TREATMENT WITH PEDF AND CTE-PEDF Cells were plated in a 24-well plate at a final concentration of 15 000 cells/well in a total volume of 200 microliters. Half of the plate was plated with CTE-PEDF medium. The next day, radiotherapy to 2 Gy, 4 Gy and 6 Gy was applied by a Siemens PRIMUS X6MV LINAC. Field size 10 × 10 cm2, depth 10 cm, SSD = 100 cm, Siemens PRIMUS X6MV. II.9. DOSE-RESPONSE CURVE Cells were plated in a 24-well plate at a final concentration of 15 000 cells/well in a total volume of 200 microliters or 200 cells/well in a 96-well plate for sorted cells. Half of the plate was plated with CTE-PEDF medium. The next day, the drug was added in decreasing concentrations 4 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM. These were cultured for 3 days under drug exposure in in vitro conditions, and then developed with an MTT assay or methyl purple assay. II.10. MTT ASSAY The cell culture media was removed, after which 100 microliters/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added. After 30 minutes of incubation, the supernatant was removed, and the precipitated crystals were dissolved in 100 microliters of DMSO. The plate was read in a spectrophotometer at 540 nm. II.11. METHYL PURPLE ASSAY This method[33] is used to quantify surviving cells. 5 000 cells/well were seeded in 24-well plates, in a final volume of 250 µL. The next day, cells were treated with increasing doses of chemotherapeutic agents and stored in a humidified incubator at 37ºC, 5% CO2 for 4 days. Then, cells were fixed with 0.5% glutaraldehyde (Sigma. St. Louis, Missouri, United States) for 10 minutes. Next, cells were stained with 0.1% crystal violet for 20 minutes. After several washes with PBS, 10% acetic acid was used to solubilize the sample. Finally, a spectrophotometric reading was performed at a wavelength of 590 nm. The IC50 value was determined as the dose necessary to eliminate 50% of the cell population, obtained through logarithmic regressions made with the DE.0 plus v 1.0 program. II.12. HISTOLOGY Cryostat sectioning slides were performed using a cryostat (Microm HM 550, Thermo Scientific). Tumour samples were fixed with formaldehyde at 4%, placed in sucrose 30% overnight, included into Tissue-Tek® OCT™ (Sakura ® Finetek USA. Torrance, California, United States) and then frozen with liquid nitrogen. Slides (12µm) were stained with hematoxylin and mounted with Dako Ultramount Aqueous Permanent Mounting Medium (Agilent Technologies. Santa Clara, California, United States) and analyzed with a Leica-DMRXA-photomicroscope (Leica. Wetzlar, Germany). II.13. ANALYSIS OF CELL MORPHOLOGY 500 000 treated and control cells were seeded in p100 plates. After 24 hours, cells were incubated for 30 minutes with Hoechst (5µg/mL). Ten random microscopy images were taken (Motic AE31.Barcelona, Spain) using an ultraviolet light. Analysis of the cytoplasmic area and the separation between cells was analyzed using the Image J application (https://imagej.nih.gov/ij/). II.14. IMMNUNO ASSAYS 500,000 treated and control cells were seeded on p100 plates with glass coverslips on the bottom to allow removal of the cells once grown. After chronic treatment (4 weeks) with PEDF, the cells were fixed with 1% paraformaldehyde and stained with specific BCRP1 (MAB4155A4, Sigma-Aldrich) and CD133 (MAB4399-I, Sigma-Aldrich) antibodies according to the manufacturer's instructions. At the end of immunolabeling the cells were incubated for 30 minutes with Hoechst (5µg/mL). Fluorescence microscopy images were taken with a Motic AE31 microscope equipped with an ultraviolet lamp. II.15. STATISTICAL ANALYSIS Data was analyzed with R software 3.5.1 version (https://www.r-project.org/). The statistical analysis was carried out using Mann-Whitney U tests (one tailed, significance level=0.05). The data is expressed as the mean plus the standard error (SE). At least n=3 independent experiments were performed for every assay. The results obtained are considered statistically significant when p<0.05(*), p<0.01(**) and p<0.005(***). Cell data was first transformed into a quadratic variable to improve data homogeneity. Then, normality was tested using the Sapiro-Wilk test, Q-Q plots, and Levene’s test for homogeneity of variance. Next, groups were compared using non-parametrical Kruskall-Wallis test by ranks and Wilcoxon’s not paired test to analyze data in pairs. To test the hypotheses, an outcome measure of significance with a 95% confidence interval was used to determine the sample size, based on the hypothesis of treatment-to-control ratio for tumor growth inhibition studies[90] Finally, logistic regression was used to model the relationship between the number of cells and progression. The odds ratio was used to strengthen the association between variables (Confidence Interval 95%). III. Results III.1. Long-term label retaining cells exhibit characteristics of cancer stem cells, in cancer cell lines and in patient cells. MDA-MB-231 and MCF7 cell lines exhibit a cell population characterized by long retained labelling growing either in adherent or mammosphere conditions (Figure 1A). A population of 0.9 ± 0.3 % and 3 ± 1% of long-term label retaining cells is present when the MDA-MB-231 cell line is grown in an adherent monolayer and in mammosphere conditions, respectively. This result is also seen for the MCF7 (Figure 1B) cell line where the percentage of long labelling cells is 3.32% and 6.28% in monolayer culture and mammosphere conditions, respectively. Ascitic fluid cells from patients with a metastatic adenocarcinoma also present long-term label retaining cells after 8 days in culture (Pa00 cells, Figure 1C). A methyl purple assay proves that LT- cells show the same growth rate as our control cells but have a higher growth rate than LT+ cells (Figure 1C). After a dose-response assay with docetaxel, LT+ cells are more resistant to chemotherapy than control or LT- cells (Figure 1C). IC50 value is double in the case of LT+ cells compared to control or LT- cells (vertical lines in Figure 1D). Docetaxel treatment eradicates 53 ± 5% of LT- cells at 2 nM, while only 27 ± 3% of LT+ were affected by the same concentration. At high doses of chemotherapy, the growth curve returns to control levels, however these are higher than the doses applicable in patients due to their undesirable side effects. Finally, Pa00 LT+ and LT- sorted cells were injected in nude mice to study the carcinogenicity of those populations in vivo. Eleven animals were used in this experiment. Four animals (two flank injections per animal, eight xenografts in total) were used in the control group and in the LT- group while only three animals were used in the LT+ group (five xenografts due to the low number of LT+ cells) for this experiment. The results show that the tumour volume is similar when injecting LT- cells and control non-separated cells but smaller when injecting LT+ cells (Figure 1E). Furthermore, Pa00 LT- cells formed tumours with a frequency of 0.75, while the frequency with LT+ cells is 1. (Figure 1F). In short, LT+ cells show a lower growth rate and they are cells more resistant to chemotherapy than LT- or control non-separated cells. In addition, in vivo assays reveal a decrease in the frequency of tumour formation in LT- cells when compared to LT+. III.2. PEDF modulate CSC properties, producing an increase of drug resistance and LT+ cells proportion. PEDF chronic treatment produces morphological changes in size and cytoplasm shape of Pa00 cells in culture (Figure 2A). We registered an increment of 170 µm2 at the cytoplasmic area in PEDF treated cells (730±80 µm2 in PEDF treated and 560±30 µm2 in control medium), what it could be indicating is alterations in the cytoskeleton related to the tumourigenicity of these cells. However, there are no significant differences between the nucleus size with and without treatment (Figure 2B). We have also studied the growing pattern of chronically PEDF-treated patient-derived cells. This slower growth rate of treated cells has been quantified as 37±1% fewer cells in treated compared to untreated PEDF cell cultures (Figure 2C). This effect translates into a lower tumour volume when the cells are injected in nude mice (Figure 2D); showing close to a 40% decrease in tumour xenograft volume from treated cells compared to controls (Figure 2D). The histological analysis showed that PEDF treated tumours present smaller necrotic areas than control xenografts. Looking at the micrographs (Figure 2E) for the tumours from untreated cells, we can see large acellular areas (arrows in figure 2E) compatible with acellular necrotic spaces.. In contrast, the micrographs for tumours from PEDF-treated cells do not show these same areas, as the nuclei and cytoplasm of the cells are better preserved. PEDF treatment also produces compact growth of the tumoural cells which present dense cytoplasm and a compact external matrix compared to the control tumours, which show acellular areas with cell detritus (Figure 2E). In the micrographs with immunofluorescence staining for BCRP1 and CD133 markers (Figures 2H-K) we again observe a lower number of nuclei in the controls than in the PEDF treatments, with larger acellular areas concordant with areas of necrosis and cell death as in panoramic Figure 2E. In these controls there is a greater abundance of cells in different mitotic phases indicated by white arrows (Figures 2H and 2J).These cells were also checked in a dose-response assay, and despite their low growth rate, PEDF treated cells exhibit higher IC50 values and resistant populations, as 47±8% of PEDF treated cells survived, which means there were 16% more resistant cells than in the control where just 31±2% of the cells survive at the maximum physiologic dose tested. This result implies higher drug resistance to docetaxel as compared to control cells (Figure 2F). We carried out the next assay to prove that the slow growth observed in vitro and in vivo is due to the appearance of a higher number of slow-cycle cells after PEDF treatment. The quantification of LT+ cells after 3DIV (days in vitro ) revealed a significant increase after PEDF treatment compared to the control (Figure 2G), with nearly a three-fold increase in cell number. To sum up, PEDF treated cells show changes in cytoplasm size, decreased cell cycle kinetics, increased drug resistance, and the capacity to produce tumours with not only a lower growth rate but also lower tumour volume and fewer necrotic areas. III.3. Cter-PEDF counteracts the effects of native PEDF, decreasing the resistance in tumours and inducing anoikis and depleting CSC. As the truncated protein CTE-PEDF does not exhibit the same effects in stem cells as the full-size PEDF protein, we decided to check if this peptide would counteract the effects of the native protein also in our cancer model. This protein presents phosphorylation sites that are important for the protein’s function. In order to maintain the negative charge, a glutamate was introduced in position 227 of the protein’s sequence, where a serine was previously located. This modification leads to the CTE-PEDF protein (Figure 3A). We applied CTE-PEDF (200ng/ml) in vitro treatment to patient cultured cells PA00. This experiment shows an anoikis effect (Figure 3B) and morphological changes that can be measured by cytoplasmic and nuclear areas and cell distances (Figure 3C). Nuclear and cytoplasmic areas are reduced 29% (Nuclear area: control 110±2 µm 2 , CTE 81±2.34 µm 2 ; Cytoplasmic area control: 556±18 µm 2 , CTE: 394±17 µm 2 ) while cell distances double their size (control 12±1 µm, CTE: 23.0±0.2 µm). Finally, treated cells divide faster but survive less than untreated cells in culture (Figure 3D). The next approach was a dose-response assay with docetaxel as previously performed with native PEDF treatments. The result demonstrates that CTE-PEDF treated cells are less resistant (squareds in Figure 3E) than control cells (circles in Figure 3E). Only the CTE-PEDF treatment produces a 20% reduction of the initial population. The IC50 value is double in the control group when compared to CTE-PEDF treated cells (vertical lines in Figure 3E). The results demonstrate that CTE-PEDF produces anoikis in cancer patient cells in culture and reduces the drug resistance to Docetaxel. III.4. CTE-PEDF and Cter-PEDF depletes percentage of CSC expressing markers. To consider whether the decrease in resistance and anoikis is produced because of a reduction in CSC number and CSC expression markers, Pa00 cells were injected with Cter-PEDF or PBS acute treatment, in nude mice. Resulting tumours were dissected and dissociated to study stemness marker expression by flow cytometry (Figure 4A). Four different epitopes, previously related to cancer stem cells, were analysed: BCRP1, EpCam, CD133 and AC133. Cter-PEDF treatment produces a significant reduction in all studied markers in patient tumour cells Pa00 (Figure 4B). The same experiment was performed with cells chronically treated during three weeks in culture with CTE-PEDF. CTE-PEDF has a negative charge in the glutamic 227 and is more stable than Cter-PEDF as the latter has a phosporylable serine 227 residue which can lose the negative charge when phosphorylated. The same reduction in marker expression was observed (Figure 4C). In vivo assays with xenografts of PEDF and CTE-PEDF chronically treated cells also show the influence of these proteins in the cancer stem cell population and resistant population. Immunofluorescence assays show differences in expression of tumour stem cell related markers comparing the population of cells treated with PEDF to those treated with CTE-PEDF. 3*106 cells/flank were injected in three animals per group: control, PEDF and CTE-PEDF treated cells, with tumours appearing one week after the injection in every case. Nine animals were employed in this experiment, 3 animals per group, 2 flank injections per animal. In both cases (PEDF and CTE-PEDF treated cells injected) there was a modification of the expression pattern of the BCRP1 and CD133 proteins. When exposed to chronic PEDF treatment there is an increase in cells positive for these markers, while the opposite is observed after chronic treatment with CTE (Figure 5). When treated with CTE fewer cells are positive for BCRP1 and CD133 compared to controls or cells treated with PEDF. Figure 5C shows the relationship between the effect of PEDF and CTE on tumour cells, with the IC50 of docetaxel altered in both cases. PEDF treatment produces more resistant cells than the control whereas CTE treatment decreases the IC50 of docetaxel, which coincides with the hypothesis that this treatment increases sensitization to the drug, due to a higher division rate that thus prevents tumour cell survival. Cells treated with PEDF, in addition to higher expression of markers, also show a higher concentration of LT+ cells (Figure 6A and B). These LT+ cells confer more chemoresistance to the culture (Figure 6C). We therefore set out to combat the effect of PEDF in vivo using the CTE peptide, which reduces marker expression and chemoresistance, as we have already stated. To correlate the reduction in cancer stem cell marker expression in Pa00 cells (Figure 4) with decreased tumourigenicity (function) xenograft tests were carried out by injecting BCRP1 positive cells previously treated with CTE. The results show that PEDF increases LT+ cells (Figure 6A and B). These LT+ form more resistant tumours (Figure 6C). Positive cells were injected at different concentrations, with their respective positive control of Cter-PEDF peptide treatment. Eight mice were used in this experiment, making use of both flanks: the right flank for injection of untreated control cells and the left flank for treated Cter-PEDF cells. Despite the heterogeneity of the in vivo experiments, it can be seen from Figure 6D that in most cases the tumours from treated cells appear later than the untreated tumours. Even when injecting cells that had been previously treated with CTE, after injection the xenograft appears earlier than in control cells. Although tumours formed from cells submitted to chronic CTE treatment are the least resistant of all to chemotherapy as we have shown previously in Figure 5C, the effect of CTE is also synergistic with radiotherapeutic treatments, as it decreases cancer stem cell viability significantly with CTE treatment and radiotherapy (gray bars) than with radiotherapy with a negative control peptide CTA without negative charge (white bars). Figures 6E and F show the assays performed on cells exposed to different successive radiotherapeutic treatments. To test the effect of our model in these assays, we have performed new experiments with the CTE peptide and also with its homologue CTA (without phosphoryable serine as it is switched to an alanine). These assays mimic the effects seen with CTE combined with chemotherapy, as these cells also become less resistant to radiotherapy (contrary to what is seen with the negative control CTA) (Figure 6F). This could possibly be because of their faster division, causing those cells with a cell cycle regulated by p21 expression to disappear. Looking at expression assays by qPCR (Figure 6E) it is clear that the expression of p21 , responsible for stopping the cell cycle in tumour stem cells, decreases. The cumulative effect of the different cycles of radiotherapy offered to patients results in down-regulation of proteins involved in the cell division of cancer stem cells or in the number of cancer stem cells themselves. This type of molecules linked to the cancer stem cell cycle could be at the basis of the synergistic effects of the PEDF signaling pathway and chemo- and radiotherapeutic treatments. In summary, CTE-PEDF decreases resistance and putative stemness markers involved in self-renewal and patient relapse, demonstrating the interest in these proteins for potential application controlling drug-resistant cell populations in patient samples. IV. Discussion Breast cancer is the most common cancer in women worldwide. We study how to prevent relapse, taking breast cancer as an example since its impact on society reaches an incidence of 15.53 (95% CI = 14.94 to 16.14) per 1000 PY[34]. The tumour subtype influences the risk of recurrence ranging from 13% to 41% depending on the tumour and the state of the nodes of the primary tumour [35,36] as hormone receptor positive and metastatic breast cancer and triple-negative breast cancer. It’s important to distinguish tumour initiating cells, responsible for tumour formation and tumour relapse. For cancer stem cell detection no good spectra of specific markers is available. The expression of membrane-determinants is a gradient during development and maturation of biological process, and even when testing a combination of the different epitopes found in the literature[37–39], it is difficult to find a completely cancer stem cell specific marker. However, tumour initiating cells (TICs) are considered responsible for the slow cell cycle and quiescent characteristics of the tumour. Our goal was to demonstrate that TICs are important for tumour maintenance, resistance and relapse. Later, our objective was to discover a possible new treatment to delay patient relapse. We demonstrate that PEDF (and derivatives) have a moonlighting role in breast cancer development and relapse. This has a potential application in clinical cancer therapies by the co-administration with chemotherapy to improve relapse-cancer treatment. This is the case for CTE-PEDF, which decreases drug resistance [33,40,41]. The co-administration with chemotherapy would lead to a less resistant population because TICs would have lost their stemness characteristics, as we discuss in the following paragraphs. The regulation of breast cancer progression through regulation of breast cancer stem cell like properties has been recently described, probably mediated by the NF-κB cell signaling pathway[42], and the connection between chemoresistance, mesenchymal plasticity and cancer stem cells in metastasis origin[43]. We have shown that MDA-MB-231 and MCF7 breast adenocarcinoma cell lines exhibit a cell population characterized by long retained labelling growing either in adherent or mammosphere conditions, as has been previously described in other tumour cell lines [44–46] and even in PA00 cells[47]. Other researchers have detected similar percentages of stemness and invasiveness of breast cancer cells in these cell lines, which were modified by estrogen through Gli1 activation [48]. Even in mammosphere cultures, we have observed that a high DDAO positive population, of slow cell cycle cells, remains after 8 days in culture; these cells have probably only divided once in this period. This subpopulation could be related to resistant cells maintained by IL-6 in some breast cancer treatments. Also, they could be affected by the inhibitor of the human epidermal growth factor receptor 2 (HER2) Lapatinib [49], and are our focus in this work. Ascitic fluid cells from a patient with a metastatic adenocarcinoma (Pa00 cells) have been used in this paper parallel to commercial cancer cell lines. Ascitic fluid, usually a result of inflammation events, contains malignant cells in up to 97% of patients with a neoplastic diagnosis and is a gold standard for pericardial carcinomatosis detection [50]. Ascitic fluid contains tumour cells that grow in culture conditions [51,52]. Part of these cells also present long retained labelling (DDAO) after 8 days in culture as we have demonstrated previously [47]. We select these cells by flow cytometry sorting according to their high DDAO content, deemed LT+ cells. This experiment showed that LT+ cells grow slower and respond less to chemotherapy than non-separated control cells or LT- cells. We postulated that this is the reason for the resistance observed in some treatments. Similar differences in resistance have also been observed in other colon and breast cancer cell lines [9,33] and even in other more aggressive types of tumours such as a glioblastoma C6 cell line [40,53]. In our hands, the final percentage of resistant cells after maximum physiological doses of docetaxel treatment is also higher in LT+ positive cells than in the rest. This LT+ population that supports higher docetaxel IC50 than LT- or control cells could be the origin of patient relapse. These same LT+ cells could also be related to higher BCRP1 and CD133 marker expression. To investigate this hypothesis, we injected Pa00 LT+ and LT- sorted cells in nude mice to study the tumourigenicity of those populations. We have considered not only the final volume of the xenograft tumours, but also the timing of the tumours’ appearance. LT- cells formed tumours at the same time as control non-separated cells but LT+ Pa00 took longer to form tumours. Accordingly, the percentage of final tumour growth after LT+ cell injection is 1/3 higher than in LT- xenografts. This result, together with the previously shown data, leads us to consider that the difference between these two cell types is the initial tumour capacity. This data correlates with previous data from other research groups which connect chemoresistance, tumourigenicity potential, and slow-cycling in some tumour cells [44,46,54]. Several other markers (such as TROP2) could be involved in this chemorresistance process too, as has been recently identified by our group[55]. All this data supports the hypothesis that LT+ cells present slow cell cycle division, higher chemotherapy resistance, and a higher frequency of tumour formation than LT- cells. These characteristics confirm the idea that LT+ cells could be involved in relapse and metastasis progression in breast cancer. In our hands, PEDF chronic treatment produces a decrease in the growth rate in vitro and in vivo due to an increase in the number of LT+ cells (slow-cycling cells). The data shown in Figure 2 indicates that cells treated with PEDF have more controlled proliferation, with slower self-renewal processes than simple tumour cell division that ends in internal necrosis of the tumours. As a conclusion from the cytoplasm measurements in Figure 1A/B, and the significant differences observed, we hypothesize that these PEDF-treated cells with a higher cytoplasm-to-nucleus ratio slow down their cell cycle. Several proteins have been related to this phenomenon. For example the histone chaperone nucleoplasmin (Npm2) has been identified as a putative nuclear size effector [56]that binds histones and may play a key role in tumour development and progression [57]. Other cytoskeletal proteins have been related to this activity and cytoplasm size (cytomegaly). For example non-muscle myosin IIA (MYH9), is a ubiquitously expressed cytoplasmic myosin that regulates the actin cytoskeleton, cell migration, cell polarization and signal transduction in cancer cells. MYH9+ cells, size-differentiated by flow cytometry, showed strong CSC characteristics, including in vivo tumourigenicity, migration, invasion, cisplatin resistance and positivity for CD133+[58]. These data recorded by other investigators between cytoplasmic size and proteins involved in tumourigenicity will be tested in our system in the near future. And for the moment they bring agreement with the growth the increased cytoplasm/nucleus ratio and the slower growth observed in our experiments. Cytoplasm size can be related to multiple physiological phenomena, but among them quiescence and cycle exit. Although it is difficult to conclude what the increased cytoplasmic area means in our experiments, the data are in agreement with the hypothesis put forward and also with tumourogenicity and proliferation data observed by other authors as argued. The experiment that supports this optimal cellular state as a result of PEDF treatment is shown in Figure 2F, where it is seen that these cells are more resistant to chemotherapeutic pharmacological stresses, and retain vital dyes for longer, due to their slower cell division, as indicated in Figure 2G. Despite their low growth rate, PEDF treated cells show higher IC50 values and resistant populations than untreated control cells, indicating higher resistance to chemotherapy. One possible explanation for this effect could be that the low growth rate lends more time for these cells to repair chemotherapy damage[59,60]. However, another hypothesis could contribute to this stage, such as higher expression of ABC drug transporters in these cells[61,62]. On the other hand, the effect that we attribute to the increase in slowly dividing cells could be due to other effects, such as the inhibition of angiogenesis widely attributed and recently countersigned for the PEDF protein[63]. In addition to this anti-angiogenic effect, PEDF has been reported to have an impact on lymphangiogenesis[64]. Release of PEDF into the tumour microenvironment, among other factors, inhibits vascular growth, while also promoting lymphangiogenesis associated with cancer[65]. Although PEDF has been recognized as an anti-metastatic factor, its role remains controversial due to some conflicting reports indicating a role in metastatic progression in some cases[66], diversifying PEDF’s impact on tumourigenicity, as our results point out. Current studies of the tumour microenvironment (TME), in which PEDF could be involved, offer an overview of the primary functions of each component of the TME in cancer initiation, progression, and invasion[65,67,68]. In MCF7 breast cancer cells, silencing of PEDF has been shown to promote resistance to tamoxifen[69,70]. It could be hypothesized that the PEDF signaling pathway is involved in the binding of the estrogen receptor to its activators and its proliferation activator function. Some peptides derived from PEDF[71] activate the proliferation of progenitor or stem cells, as in our case. Whether our C-ter peptide is involved in the regulation of resistance to tamoxifen will need to be tested in future experiments; this hypothesis would offer new potential uses for that peptide. In contrast, we present here that PEDF treatment increases resistance to docetaxel. PEDF is self-regulated by VEGF and the angiogenesis process. HIF1alpha-independent angiogenesis could be under interference from both PEDF’s anti-angiogenic effect and tumoural cells’ resistance[72]. In culture however, the anti-angiogenic effect of PEDF could be shielded by its self-renewal effect over cancer stem cells. These results could also be explained based on the metabolic pathway in which cytochrome P450 metabolizes both components (PEDF and docetaxel). It has been described that PEDF function can be blocked by fluvoxamine, a P450 inhibitor[73]. P450 polymorphisms present in each of the cell lines and patient cells analyzed could account for these differences. Returning to the possible effectiveness of the peptide CTE-PEDF, treated patient cells showed symptoms compatible with anoikis and morphological changes in culture. The anoikis effect is the apoptosis induced by lost, insufficient, or inappropriate interactions between the cell and the extracellular matrix[74]. Some of the cell signaling pathways involved in cancer progression (such as BMPs) have also been related with proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells[75]. This anoikis effect could be the basis for the interaction of TICs with the surrounding niche, and could be involved in the loss of stemness properties and chemotherapy resistance[76]. Docetaxel resistance decreases when cells are treated with CTE-PEDF. It counteracts the effect of PEDF, which is a niche protein[12] involved in self-renewal and trophic maintenance of pluripotent cells. PEDF is secreted by endothelial cells and is one of the proteins involved in the self-renewal capacity of stem cells. Endothelial cells also play a significant role in tumour progression and metastasis[77,78], and the niche signals could be key to understanding tumour progression, epithelial-mesenchymal transition, and distant metastasis at patient relapse. A significant difference is shown between treated cells and controls, with PEDF or derived peptides, but the protocol followed in this experiment should be standardized to obtain a better result. The molecular subtype used in this work could be influencing the observed effects, however, the same results have been obtained in cell lines of other types of tumours[33,40]. Also we observe differences in the level of expression of proteins that participate in the PEDF signalling pathway justifying the differences between cell lines in the different experiments while maintaining the final effect and the extractable conclusions. In short, the experiments carried out indicate that the carboxyl terminal fragment of the PEDF protein, CTE-PEDF, could be an effective chemosensitizing agent since it is capable of counteracting the effect of the endogenous native PEDF protein as had been demonstrated in neural stem cell populations previously ([12]), and depleting the expression of TIC markers. Expression of markers that had been previously related to cancer stem cells decreases when cells are treated with CTE-PEDF. This effect confirms the idea that CTE-PEDF produces a decrease in resistance to drugs such as docetaxel, because it reduces the number of tumour initiating cells. This result indicates a possible potent application as a future treatment for this protein when co-administered with chemotherapy[79,80]. What we postulate here is that the modification of the PEDF protein, that is, the C-terminal peptide, can affect the cancer root through a mechanism of competition with native PEDF. Previous experiments have shown this competition and that increasing doses of truncated peptide can inhibit the effect of the native protein[12]. Although further studies need to be carried out, these experiments point to a possible new strategy for treatment of cancer relapse. In order to verify the importance of our data we have compared our results with other kinds of treatments. In addition to chemotherapeutic treatments, radiotherapeutic adjuvant treatment is also widely used in breast cancer [81,82]. We have shown that radiotherapeutic treatment decreases cell cycle markers related to stem cell self-renewal, such as the cell cycle protein p21[83,84], which decreases with progressive radiotherapy treatment. All combined, the results of this comparison are of great applicability in resistant tumours, especially in cases of triple negative breast cancer, as these show the worst response to current treatments. There are many experiments to be performed in this regard, and these will be future lines of research in the laboratory as the effect of different concentrations of peptides or even chemotherapy[85]. However, the data obtained so far seems to us of importance, contributing to general knowledge and permitting us to advance in the fight against resistance to cancer treatment. To conclude, our hypothesis can be summed up in the schematic below (Figure 7). There has been much discussion in the literature regarding the idea of a countdown mechanism that limits cellular replicative potential by giving it an intrinsic heuristic value [86]. We consider that cancer stem cell depletion is of particular importance and CTE can help us in this pathway. In the summary figure we can see how PEDF enhances the self-renewal of tumour stem cells, akin to what occurs in normal stem cells [12,17]. Molecular markers involved in the cell cycle of cancer stem cells, such as p21, increase their expression after PEDF treatment, thereby indicating an increase in the self-renewal of these cells, as well as their perpetuation and, as a consequence, the chemoresistance of these cell cultures. In contrast, chronic treatment with CTE-PEDF allows the extenuation of the tumour stem cell population, along with an increase in the proliferative rate, but also better response to chemotherapy treatments, since active cycle cells are more susceptible to apoptosis from anti-neoplastic treatments, thus decreasing the harmful rate of resistant cells (Figure 7). V. Conclusions The PEDF stem cell self-renewal modulator protein modifies the carcinogenicity of cancer stem cells and could be a useful tool to control tumoural self-renewal and therefore control patient relapse. We have designed a transgenic peptide derived from PEDF to interfere with the self-renewal capacity of cancer stem cells, inducing anoikis in vivo and reducing resistance in cells from cancer patients. We have also shown that this PEDF-derived peptide produces a significant decrease in cancer stem cell markers, making it a potential tool for delaying patient relapse. List of abbreviations ABC: ATP-binding cassette AI: artificial intelligence AC133: glycosylated epitope of the pentaspan membrane protein CD133, originally identified as a marker for CD34 + hematopoietic stem and progenitor cells. BCRP1: Breast cancer resistance protein (BCRP1), also known as placenta-specific ATP-binding cassette (ABC) protein (ABCP) or ABC G-subfamily member 2 (ABCG2) BMPs: Bone Morphogenetic Proteins C6: glioma cell line CD133: CD133 antigen, also known as prominin-1, is a glycoprotein that in humans is encoded by the PROM1 gene. It has been proposed that it acts as an organizer of cell membrane topology. CDKN1A: Cyclin Dependent Kinase Inhibitor 1A CI: Confidence interval CM-spf: mammospheres CSC: cancer stem cells CTA-PEDF: carboxyterminal of PEDF (Ala227) CTE-PEDF: carboxyterminal PEDF (Glu227) Cter: Cter-carboxyterminal PEDF (comprising amino acids 195-400 of PEDF) DAPI: 4’6-Diamidino-2-phenylindole DDAO: Near-infrared (NIR) red fluorescent probe with excitation wavelength (600-650 nm) and long emission wavelength (λem = 656 nm). DIV: days in vitro DMSO: Dimethyl sulfoxide ECM: Extracellular matrix EGF: epidermal growth factor EMT: epithelial mesenchymal transition EpCAM: Epithelial cell adhesion molecule, also known as CD326. ER: estrogen receptor FGF: fibroblast growth factor GADPH: Glyceraldehyde 3-phosphate dehydrogenase. Gy: gray HEK-293: Human embryonic kidney cell line HER2: Human Epidermal growth factor Receptor 2 HIF1alpha: Hypoxia Inducible Factor IC50: half maximal inhibitory concentration LT+: Long-term label retaining cells MDA-MB-231: Triple-Negative Breast Cancer Cell Line. MDR1: Multi drugs resistance gen MCF7: epithelial cell line isolated from the breast tissue of a patient with metastatic adenocarcinoma. MLY: monolayer MTT: methyl thiazol tetrazolium MYH9: myosin heavy chain non-muscle NF-kB: Nuclear kappa-light-enhancer of active B cell p21: p21 Cip1 (alternatively p21 Waf1), also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 PA00: ascitic cell line derived from a patient with metastatic breast cancer, in our laboratory. PBS: phosphate buffered saline PEDF: Pigment Epithelium-Derived Factor PKA: protein kinase A (EC 2.7.11.11) PR: progesterone receptor PY: Patient Year SOX2: SRY-Box transcription Factor 2 Tert: telomerase protein TIC: tumour-initiating cells TME: Tumour microenviroment TROP2: transmembrane glycoprotein encoded by the Tacstd2 gene overexpressed in cancers. VEGF: vascular endothelial growth factor Declarations Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of CEIC Hospital Clínico San Carlos (protocol code 13/026-E and 12/02/2013)" for studies with human subjects. (1) Title of the approved project: Localization and control of tumor stem cell self-renewal to improve the efficacy of chemotherapy and prevent recurrence. (2) Name of the institutional approval committee or unit: Comité de Ética de Investigación Clínica Hospital Clínico San Carlos. (3) Approval number: C.I. 13/026-E; (4) Date of approval: 12/02/2013. The protocol of animal study was approved by the Ethics Committee of Ethics Committee of Universidad de Castilla La-Mancha (protocol 25/01/2010)” for animal studies. (1) Title of the approved project: Localization of circulating tumor stem cells in blood by surface markers and inhibition of their self-renewal. (2) Name of the institutional approval committee or unit: Comité de Ética de Experimentación Animal de la Universidad de Castilla La Mancha. (3) Approval number: acta25/01/2010; (4) Date of approval: 10/02/2010. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Funding: This work was supported by grant from the Fundación Científica Asociación Española Contra el Cáncer (División provincial de Albacete). C.R-C. was investigator of the Programa Ramon y Cajal from MEC/Spain. M S-D is predoctoral research from the Programa Propio at the Universidad Politécnica de Madrid. The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Author Contributions: CRC: Conceptualization and design of the work, Formal analysis, Funding acquisition, Project administration, Supervision, Writing-original draft and Writing review and editing. CGG: Data curation, Formal analysis, Investigation, Methodology and Writing-original draft. MSD: Formal analysis, Validation, Visualization and Writing review and editing. PHG: Investigation and Methodology. JLSS: Data curation, Formal analysis and Validation. Carmen Belen Alvarez: Methodology. SS: Data curation and Formal analysis. FSSz: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, writing review and editing. Acknowledgments: We thank C.E. Gavira O’Neill for reading the manuscript and English correction. 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Supplementary Files ARRIVE2.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major Revision 30 Nov, 2023 Reviewers agreed at journal 30 Aug, 2023 Reviewers invited by journal 30 Aug, 2023 Editor assigned by journal 09 Mar, 2023 First submitted to journal 08 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2636130","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":229850743,"identity":"0b25913d-40b1-42d5-a374-04c457a97629","order_by":0,"name":"Carmen Gil-Gas","email":"","orcid":"","institution":"Universidad de Castilla-La Mancha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Gil-Gas","suffix":""},{"id":229850744,"identity":"b7070686-5a19-4e1f-a514-e2fdbf3127ef","order_by":1,"name":"Marta Sánchez-Díez","email":"","orcid":"","institution":"Universidad Politecnica de Madrid","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Sánchez-Díez","suffix":""},{"id":229850745,"identity":"c3ba1643-efb3-450b-a75e-b49ca01cddc2","order_by":2,"name":"Paloma Honrubia-Gómez","email":"","orcid":"","institution":"Universidad de Castilla-La Mancha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paloma","middleName":"","lastName":"Honrubia-Gómez","suffix":""},{"id":229850746,"identity":"90f3f3c5-36ea-4e34-a778-1b900ed20c90","order_by":3,"name":"Jose Luis Sánchez-Sánchez","email":"","orcid":"","institution":"SESCAM: Servicio de Salud de Castilla-La Mancha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jose","middleName":"Luis","lastName":"Sánchez-Sánchez","suffix":""},{"id":229850747,"identity":"019ef959-807b-4ae3-bdef-56bed2fe4b45","order_by":4,"name":"Carmen Belen Alvarez-Simón","email":"","orcid":"","institution":"UCLM: Universidad de Castilla-La Mancha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"Belen","lastName":"Alvarez-Simón","suffix":""},{"id":229850748,"identity":"adc06f74-286b-4a25-8db8-a2b3859377ac","order_by":5,"name":"Sebastiá Sabater","email":"","orcid":"","institution":"SESCAM: Servicio de Salud de Castilla-La Mancha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sebastiá","middleName":"","lastName":"Sabater","suffix":""},{"id":229850749,"identity":"6d860716-9155-40dc-b75a-bd2f790fb7e6","order_by":6,"name":"Francisco Sánchez-Sánchez","email":"","orcid":"","institution":"UCLM: Universidad de Castilla-La Mancha","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Sánchez-Sánchez","suffix":""},{"id":229850750,"identity":"7c01aa8a-e367-441f-a5f8-105edf4ee978","order_by":7,"name":"Carmen Ramirez-Castillejo","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-1877-3381","institution":"Universidad Politecnica de Madrid","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Ramirez-Castillejo","suffix":""}],"badges":[],"createdAt":"2023-02-28 01:49:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2636130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2636130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42647788,"identity":"3f0c2912-10ac-4c90-9d03-dbd4bef18c8d","added_by":"auto","created_at":"2023-09-05 14:48:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":229220,"visible":true,"origin":"","legend":"\u003cp\u003eLong-term label retaining cells (LT+) are present in cell lines and culture from patient cells and display cancer stem cells characteristics. (A) MDA-MB-231 cells were stained with DDAO and cultivated 8DIV (days \u003cem\u003ein vitro\u003c/em\u003e) in monolayer (MLY) or as mammospheres (CM-spf). (B) Also MCF7 cell line showed LT+ population and it was higher in mammosphere assays (cytometry assay) than in monolayer. (C) Growing patterns of LT+ and LT- cells from patient Pa00 cells stained and grown 8DIV (400cells/well) and then sorted according to their DDAO content. The number of living cells after 3DIV was checked by methyl purple assay. LT- cells grew similar to control and faster than LT+ cells. (D) Docetaxel dose-response curves for LT+, LT- and control cells. PA00 cells were stained with DDAO and grown for 8DIV, sorted by their content of DDAO and grown with increasing concentration of docetaxel. LT+ cells showed more resistance against docetaxel than LT- and respective IC50 are marked by perpendicular lines. (E) 5000 PA00 cells were injected in nude mice in each case. The tumour volumes are similar when injecting LT- and control non-separated cells, but smaller when injecting LT+ cells. All tumours were palpable at the same time. LT+ tumours grew slowly compared to those in the control or LT- group. (F) Table of number of injected mice and frequencies of tumour formation of the different cell types. (n\u0026gt;3 all experimental group; *p\u0026lt;0,05, **p\u0026lt;0,01). Bar 50µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/43cf5c08088520faf3762791.png"},{"id":42650267,"identity":"7f6b2f81-ac62-465d-a56b-edbe2125fe06","added_by":"auto","created_at":"2023-09-05 15:04:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":872435,"visible":true,"origin":"","legend":"\u003cp\u003ePigmented Epithelium Derived Factor (PEDF) increases the number of LT+ cells and the docetaxel resistance of breast cancer cells. (A) Cells treated with chronic PEDF showed a different morphology than control cells. (B) Quantification of morphological differences induced by PEDF treatment. (C) Growing pattern after 3DIV of PEDF treated cells and control. PEDF chronic treated cells grew slower than control. (D) Docetaxel dose-response curve of PEDF chronic treated cells and control. PEDF chronic treated cells were more resistant against Docetaxel. (E) Histology of PEDF treated tumours and control tumours. PEDF chronic treatment increased cytoplasm density and external matrix. Necrotic areas are bigger in controls compared to PEDF treatment. The arrows show examples of areas compatible with acellular necrotic spaces. (F) Docetaxel dose-response curve of LT+ PEDF treated cells and LT+ untreated cells, n=3. LT+ PEDF chronic treated cells were more resistant against Docetaxel than untreated ones. (G) PEDF-treated cells grew more slowly than control cells, meaning that the dye-retaining population is up to three times larger than with PEDF treatment. (H) BCRP1 marker immunohistochemistry in control cells. (I) BCRP1 marker immunohistochemistry in PEDF treated cells. (J) CD133 marker immunohistochemistry in control cells (K) CD133 marker immunohistochemistry in PEDF treated cells. (n=3 all experiment; *p\u0026lt;0,05, **p\u0026lt;0,01). Bar 50µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/88f07f9a1a7c0b38541354c6.png"},{"id":42649424,"identity":"d2285d0c-867b-4a1a-b760-744019998820","added_by":"auto","created_at":"2023-09-05 14:56:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":352005,"visible":true,"origin":"","legend":"\u003cp\u003eCTE-PEDF induces loss of anchorage and cell death in vivo and reduces resistance against Docetaxel. (A) CTE-PEDF construction with glutamic instead of serine 227. (B) Pa00 cells were treated chronically with CTE (200ng/uL). After a week, they showed an increase in anoikis figures. (C) Quantification of CTE-PEDF induced morphological changes in nuclear and cytoplasmic areas and cell distance. (D) Growing pattern after 3DIV of CTE-PEDF treated cells and control. Treated cells survive less than control cells. (E) Docetaxel dose-response curve of CTE treated cells and control. CTE chronic treated cells were less resistant against Docetaxel. (n=3 all experiment; *p\u0026lt;0,05, **p\u0026lt;0,01). Bar 50µm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/41a1d9268e844190ec4d9ca9.png"},{"id":42647786,"identity":"0e15016b-fdfe-4497-a56b-676d62c9dd2b","added_by":"auto","created_at":"2023-09-05 14:48:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":277132,"visible":true,"origin":"","legend":"\u003cp\u003eCter-PEDF and CTE-PEDF treatments decrease in vivo cancer stem cell markers. (A) Cytometry assay showing the smaller number of CSC in treated cells compared to control. (B) Quantification of positive cells in acute treatment compared to control. (C) Quantification of positive cells in chronic treatment compared to control. (n=3 all experiment (n=3 all experiment; *p\u0026lt;0,05, **p\u0026lt;0,01).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/738b8c45ce25ff88b4c0fb6b.png"},{"id":42647792,"identity":"2afd96d5-81fe-4e2b-b2d6-b17e388c18bc","added_by":"auto","created_at":"2023-09-05 14:48:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1208706,"visible":true,"origin":"","legend":"\u003cp\u003ePEDF and CTE-PEDF treatments modified \u003cem\u003ein vivo\u003c/em\u003e cancer stem cell markers. (A) Examples of Xenograft of the PEDF, CTE and control injected cells. (B) Immunofluorescence of cells positive (arrows) for CD133 and BCRP1 respectively in xenografts of chronic treatments. Immunocytochemistry with hematoxylin is shown at 20x. Immunocytochemistry assays with CD133 and BCRP1 are shown at 40x with DAPI nuclear staining. (C) Differential effect on cell viability of PEDF and CtE-PEDF treatments over Pa00 tumoural cells in culture. (a) Statistically significant differences between Cter and control or PEDF treated cells, p\u0026lt;0,05. (b) Statistically significant differences between PEDF and control or Cter treated cells, p\u0026lt;0,05. Hematoxiline micrographies Bar 5µm. Immunofluorescence micrographies Bar 50µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/1eb084c9b38cef0c63dbadb4.png"},{"id":42647791,"identity":"e5770772-a168-41ad-85a6-3b3f47855d89","added_by":"auto","created_at":"2023-09-05 14:48:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":305187,"visible":true,"origin":"","legend":"\u003cp\u003eLT+ and BCRP1+ cells used in xenograft assays showing the effect of CTE-PEDF treatments \u003cem\u003ein vivo \u003c/em\u003eand the synergy of this effect with chemo and radiotherapy. (A) Cell cytometry of PEDF treated and untreated PEDF cells. LT+ cells are more abundant after PEDF treatment. (B) Quantification of three independent flow cytometry experiments in A. (C) Xenografts resulting from cells positive for stem cell markers and slow-cycling LT+ cells are more resistant tumours when subjected to dose-response assays after dissection and placed in cell culture. (D) Cells expressing the cancer stem cell marker BCRP1 were injected at different cell concentrations. Control negative cells were also injected. In all cases, an assay was performed with and without treatment with the carboxyl end of PEDF (Cter-PEDF). The xenograft tumours of the Cter-treated cells appear time delayed (in green) with respect to their controls (in red). (E) The effect of CTE is also synergistic with radiotherapeutic treatments, as it decreases cancer stem cell viability significantly more with CTE treatment and radiotherapy (gray bars) than with radiotherapy alone or with a negative control peptide (CTA peptide without negative charge, white bars). (F) Consecutive treatment with radiotherapy (8 treatments and 22 treatments at 6 Gy) downregulates cancer stem cell markers such as the \u003cem\u003ep21\u003c/em\u003e mRNA related to cell cycle arrest of these cells. a: statistically significant differences (p\u0026lt;0.1; **) compared to the non-irradiated control. b: statistically significant differences (p\u0026lt;0.01; ***) compared to 6 Gy treatment without CTE.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/960ce65df2ce6e5cbb925253.png"},{"id":42647790,"identity":"3676a90f-ed29-430f-9831-bcfd28d60149","added_by":"auto","created_at":"2023-09-05 14:48:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":198783,"visible":true,"origin":"","legend":"\u003cp\u003eHypothesis of the exhaustion and depletion of tumour stem cells by blocking PEDF self-renewal factor receptors. This results in increased proliferation of chemotherapy-responsive cells and decreased tumour resistance.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/a78e8ef795b083b397181cbd.png"},{"id":42650268,"identity":"4f68b51b-6104-4471-acf5-9987d8e4e087","added_by":"auto","created_at":"2023-09-05 15:04:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3418314,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/463499fe-167e-4a34-aa4c-f4c726e4b6bc.pdf"},{"id":42647793,"identity":"ea7a316e-1d9c-432b-ac0d-e0e025faee3e","added_by":"auto","created_at":"2023-09-05 14:48:25","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":338495,"visible":true,"origin":"","legend":"","description":"","filename":"ARRIVE2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2636130/v1/a9dcb319ee75357292fcc6f2.pdf"}],"financialInterests":"","formattedTitle":"Moonlight role of PEDF in breast cancer: self-renewal inhibition in breast cancer stem cells","fulltext":[{"header":"I. Background","content":"\u003cp\u003eThe incidence of breast cancer has increased in recent years, due to an aging population[1,2]. In fact, breast cancer is the leading cause of death among females in developed countries, although the implementation of screening tests and the development of anti-neoplasic therapies such as Trastuzumab has increased the probability of a cure in those patients[3,4]. However, relapse rates remain very high in different types of breast cancer[5,6], indicating a need for further study into new pharmacological drugs and diagnostic methods to stop relapses in patients. Relapse is mainly due to the tumour cell population\u0026rsquo;s resistance, characterised by their capacity for self-renewal, resistance to drugs, and a slow cellular cycle[7,8]. This last characteristic allows for detection of this population, since this produces long retained labelling[9\u0026ndash;11]. The auto-renewal capacity of these cells is essential for stem cells to persist throughout the life of the organism. Pigment Epithelium-Derived Factor (PEDF) protein has been previously related to this self-renewal mechanism[12,13]. This protein may induce cellular differentiation and promote apoptosis in a variety of tumour cells[10,11], and it is also able to inhibit tumour proliferation, vascularization, cell migration, and metastasis[14\u0026ndash;16] affecting the division of fast tumour cells. In addition, PEDF is a niche-derived regulator of adult neural stem cells[12,17,18] that activates slowly dividing cells without inducing proliferation nor differentiation[12,13]. Together with the fact that tumour-initiating cells (TICs) are capable of self-renewal, this indicates that this protein-signalling pathway could be important not only as an anti-neoplastic agent but also as a regulator of self-renewal in TICs and patient relapse. As has been widely described, PEDF is a pleiotropic molecule, presenting two domains with clearly differentiated functions, an anti-angiogenic part and a second domain with neurotrophic properties, each activating different signalling pathways. By using this fragmentation of the molecule, we are able to take advantage of these domains\u0026rsquo; different effects on signalling pathways, including the carboxy-terminal fragment\u0026acute;s inhibition of the TIC population\u0026acute;s crucial self-renewal ability, which thus hinders tumour recurrence. This is why we suggest a new therapeutic mechanism that consists in the co-administration of carboxy-terminal PEDF protein fragments and chemotherapy.\u003c/p\u003e\n\u003cp\u003eIn order to detect TICs, four different epitopes were analysed; these epitopes are implicated in the cells\u0026rsquo; different processes and have been previously related with cancer stem cells in literature. BCRP1 is a drug transporter from the ABC transporter family, but while it is not as ubiquitous as other family members (such as MDR1) it is commonly expressed in the stem cell population[19,20]. EpCAM is a transmembrane glycoprotein that is involved in cell signalling, migration, proliferation, and differentiation[21,22]. This whole process is closely related to the epithelial-mesenchymal transition, essential in the metastatic mechanism in which TICs could play an important role. CD133 is a pentaspan membrane glycoprotein that has been used as a stem cell biomarker since its discovery in 1999, although its function is still unknown. AC133 is a glycosylated-isoform of CD133, recognized by a specific antibody which has been described as a biomarker for human hematopoietic stem cells and different cancer stem cell models[23\u0026ndash;26].\u003c/p\u003e\n\u003cp\u003eAll these proteins have been involved in different metastatic processes and could be a target for new therapies. More interestingly, they can be indicators of the process\u0026rsquo; progress. Moreover, they could shed further light into the mechanisms and the signalling pathways of these cells involved in the resistance to chemotherapeutic treatments. One of the cellular mechanisms that may be involved in these resistance processes is anoikis[27\u0026ndash;31]. Anoikis is a form of apoptosis that occurs in anchorage-dependent cells and in a niche context, when the cells detach from the surrounding extracellular matrix (ECM) and lose the connection to the surrounding nurse cells[31,32]; this is one of the processes we observed after the combination of chemotherapy and treatment with the carboxy-terminal part of PEDF protein.\u003c/p\u003e\n\u003cp\u003eIn this scenario, we have addressed the problem of locating breast tumour-initiating cells, and the study of the self-renewal mechanism by inhibiting its functionality and potential tumour recurrence. We also postulate that the PEDF signalling pathway could be a potential therapeutic target for control of cancer initiating cells\u0026rsquo; self-renewal and tumour relapse, as has been postulated in other neoplastic models[33].\u003c/p\u003e"},{"header":"II. Methods","content":"\u003cp\u003eII.1. CELL CULTURE\u003c/p\u003e\n\u003cp\u003eMCF7 (ATCC\u0026reg; HTB-22\u0026trade;), MDA-MB-231 (ATCC\u0026reg; HTB-26\u0026trade;) and 293 HEK-293 (ATCC\u0026reg; CRL-1573\u0026trade;) cell lines were acquire from ATCC company (Manassas, Virginia, United States). \u003c/p\u003e\n\u003cp\u003eMCF7 are epithelial cells isolated from the breast tissue of a 69-year-old, white, female patient with metastatic adenocarcinoma with Pleural effusion. The MCF7 line retains several characteristics of differentiated mammary epithelium including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes. The cells express the WNT7B oncogene[50].\u003c/p\u003e\n\u003cp\u003eThe MDA-MB-231 cell line was isolated at M D Anderson from a pleural effusion of a patient with invasive ductal carcinoma and is commonly used to model late-stage breast cancer[73]. This cell line is ER, PR, and E-cadherin negative and expresses mutated p53. In microarray profiling, the MDA-MB-231 cell genome clusters with the basal subtype of breast cancer. Since the cells also lack the growth factor receptor HER2, they represent a good model of triple-negative breast cancer. MDA-MB-231 cells are invasive \u003cem\u003ein vitro\u003c/em\u003e and when implanted orthotopically produce xenografts that spontaneously metastasize to lymph nodes[73]. \u003c/p\u003e\n\u003cp\u003eThe Pa00 cell line was derived in the laboratory from the sample of a patient with metastatic breast cancer with ascitic effusion [47], in which we found a small population of cells that express stem cell cancer markers, as can be seen in the results of the present article. \u003c/p\u003e\n\u003cp\u003ePa00, MCF7 and MDA-MB-231 cells were maintained in DMEM (Lonza. Basel, Switzerland) 10% FBS (Lonza), 1% Glutamine (Lonza) (0,2M) and 1% Penicillin/streptomycin (Lonza) (100 units+100 ug/10ul) in a 5% CO2 humidified incubator at 37\u0026ordm;C.\u003c/p\u003e\n\u003cp\u003eII.2. STAIN WITH DDAO AND SORTING\u003c/p\u003e\n\u003cp\u003eCells for cell cycle dynamic assays were plated (100 000 cells). Next, these cells were washed with PBS, disaggregated and then incubated at 37\u0026ordm;C with Cell Trace \u0026reg; Far-Red-DDAO-SE (DDAO-SE, Molecular probe ref C34564. Eugene, Oregon, United States), at the concentration recommended in the product data sheet. After 10 min, an aliquot of freshly labeled cells (approx. 200 000 cells) was fixed with 0,5% of paraformaldehyde to use as a positive control for the experiment. Then, we centrifuged the cells at 180g for five minutes and we suspended it in the culture medium to grow under standard conditions. Eight days \u003cem\u003ein vitro\u003c/em\u003e later, CM-spf cells were disaggregated and sorted by In-fluxTM (Becton Dickinson. Franklin Lakes, New Jersey, United States) sorting equipment depending on their fluorescent retaining labelling level. Positive cells were considered slowly dividing cells, and thus potential tumour initiating cells. \u003c/p\u003e\n\u003cp\u003eII.3. CYTOMETRY ASSAY\u003c/p\u003e\n\u003cp\u003eCytometry assays were performed with a MACSQuant Analyzer 10 cytometer (Miltenyi Biotec ref 130-096-343. Bergisch Gladbach, Germany). Samples were first washed in PBS and incubated with Miltenyi Biotec FcR blocking Reagent (human), in 100 microliters of sample. The immunostaining was performed per the standard protocol recommended by the commercial houses of the different antibodies. The antibodies and the working dilutions used were as follows: AntiBCRP-FITC 5D3 Chemicon (Temecula, California, United States) and AntiBCRP-PE 5D3 Chemicon incubated for 20 minutes at a 1:10 dilution; while AntiEpCAM- PE Clon HEA-125, AntiAC133-PE Clon AC133, AntiCD133-PE 293C3, Anti CD44-FITC and CD24-PE, all from Miltenyi Biotec, were incubated 10 minutes at a 1:10 dilution. Sorting experiments were performed with a BD Influx\u0026trade; cell sorter. FITC or PE are used as abbreviation of Fluorescein isothiocyanate and Phycoerythrin respectively. \u003c/p\u003e\n\u003cp\u003eII.4. GENE EXPRESSION\u003c/p\u003e\n\u003cp\u003eFor gene expression assays, total RNA from cultured cells was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany) and used immediately for reverse transcriptase reactions or stored at 80\u0026ordm;C until use. RNA concentration was measured in a Nanodrop spectrophotometer (Thermo Fisher,Waltham, MA, USA). cDNA synthesis was performed using the RevertAid First Strand cDNA Synthesis kit (Fermentas, Waltham, MA, USA). The reaction was prepared according to the manufacturer\u0026rsquo;s instructions. Quantitative PCR reaction was performed for CDKN1A (also known as p21 inhibitor) using the KiCqStart SYBR Green kit (Sigma, St. Louis, MO, USA) according to the manufacturer\u0026rsquo;s instructions. KiCqStart SYBR Green predesigned primers (Sigma, St. Louis, MO, USA) were employed (H_CDKN1A_1: Forward primer 5\u0026apos; TGTAAAACGACGGCCAGT and Reverse primer 5\u0026apos; CAGGAAACAGCTATGACC). Primers were used in a final concentration of 300 nM, and 10 ng of cDNA were used per well, for a total volume of 10 \u0026micro;l. All cDNA samples were measured in triplicate in a 96-well plate covered with adhesive seals in the thermocycler Roche LightCycler 480 (Roche, Basel, Switzerland). Reactions started with 10 min at 95\u0026ordm;C, followed by 45 cycles of 15 s at 95\u0026ordm;C, 1 min at 60\u0026ordm;C and 10 s at 72\u0026ordm;C. The 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003eD\u003c/sup\u003e\u003csup\u003eCT\u003c/sup\u003e method was used for calculating the normalized mRNA expression. Fold increase relation over untreated controls has been used to graphicaly represent the decrease of gene expression. GADPH was used as housekeeping gene in quantitative PCR assay.\u003c/p\u003e\n\u003cp\u003eII.5. XENOGRAFTS\u003c/p\u003e\n\u003cp\u003eFOXn1nu females, at 1 month of age were obtained from Charles River International Laboratories (Wilmington, Massachusetts, United States) to use in these experiments. Animals were housed and bred under 20-25 \u0026ordm;C, 50-60% humidity and a 12 hour light-dark cycle. All experiments were performed in accordance with relevant guidelines and regulations and the animals were treated in accordance with the approval of the local ethics committee (University of Castilla-La Mancha PI081746). The experiments were performed as previously described[30]. In short, untreated cells (control) and treated cells (8nM PEDF or CTE-PEDF) suspended in PBS were injected subcutaneously into both flanks of immunocompromised mice. A total of 1200, 5000 or 3*10\u003csup\u003e6 \u003c/sup\u003ecells (depending of the experiment) were injected in a final volume of 200 uL of a 1:1 dilution with MatrigelTM Basement Membrane Matrix (Becton Dickinson) with a 25 gauge-needle. The experimental unit is a single animal and a total of 28 animals were used. The mice were randomly assigned to the control and treatment groups via tail numbering that was also used as the order for successive xenograft injections. In the first experiment with animals (Figure 1) a total of 11 animals were used; with four animals in the control group and seven animals in the treatment group. For the second animal experiment (Figure 5) a total of nine animals were used, with three animals in each group (control, PEDF or CTE-PEDF treated injected cells). For the last animal experiment (Figure 6) a total of eight animals were used, with two injections each (one per flank, treated cells in the right flank and untreated cells in the left flank). There is an extensive track record in the published literature of using athymic nude mice and mouse xenograft models with human tumour cell lines to increase understanding of the factors affecting tumour growth[87]. The number of mice in each experiment was chosen based on this well-detailed experience, including experience in PDX (patient derived xenograft) models[88]. Possible confounding factors between groups were minimized by placing the mice from each group in different boxes (3-4 animals per cage, corresponding to animals of the same experimental group: control or treatment). All investigators responsible for the animal experiments in the team were aware of the group assignments at the different phases of the experiment (during assignment, conduct of the experiment, evaluation of results and data analysis).\u003c/p\u003e\n\u003cp\u003eTumour growth was monitored weekly with a caliper. The final tumour volume was calculated as V= 2 x L1 x L2 x \u0026pi;/6. Tumours were mechanically and chemically dissociated with collagenase and trypsin at 37\u0026ordm;C. They were then washed with PBS and seeded in DMEM medium, 10% FBS, 1% glutamine (200 mM), 1% Penicillin/streptomycin, 0.5% EGF and 0.04% FGF in a 5% CO2 humidified incubator at 37\u0026ordm;C 12h before the start of other experiments. The outcome measures assessed in the different experiments were: tumour size over time, markers expressed by tumour cells, and rates of resistance in dose-response assays in cell culture of these xenograft cells.\u003c/p\u003e\n\u003cp\u003eThe criteria used to include and exclude both animals (during the experiment) and data points (during the analysis) were established \u003cem\u003ea priori\u003c/em\u003e. Specifically, those mice that suffered harassment or mistreatment by their peers in the cage for reasons beyond the control of the caretakers and showed significant deterioration or premature deaths not directly linked to the experiment were to be excluded from the study. Those animals would be mentioned although not considered for cellular and molecular analyses. However, no such circumstance was observed throughout the experiments and therefore no mice were excluded in these experiments.\u003c/p\u003e\n\u003cp\u003eII.6. PEDF and Cter-PEDF, CTE-PEDF PRODUCTION\u003c/p\u003e\n\u003cp\u003ePEDF and the carboxy-terminal part of PEDF protein (Cter-PEDF) were cloned into pcDNA\u0026trade;3.1/myc-His A, B, \u0026amp; C Mammalian Expression Vectors (InvitrogenTM. Carlsbad, California, United States) following the protocol previously described by S\u0026aacute;nchez-S\u0026aacute;nchez and coworkers[89]. After cloning, vectors were checked by sequencing (3130 Applied Biosystems. Foster City, California, United States). Serine 227 was mutated into a glutamate (E) residue using ser227glu-dw (5\u0026rsquo;-CCA AGT AGA AAT CCT CGA GCT CAG TCT TTC TGG AGT-3\u0026rsquo;) and ser227glu-up (5-GTT TGA CTC CAG AAA GAC TGA GCT CGA GGA TTT CTA-3) primers. After mutagenesis, the Cter-PEDF peptide with this glutamic change was named CTE-PEDF. Proteins were produced by HEK-293-T cells after transfecting with phosphate calcium. Conditioned mediums were collected after 3 DIV, quantified by western-blot using anti-c-myc mouse monoclonal IgG1 (Santa Cruz Biotechnology. Dallas, Texas, United States), and anti-phosphoserine clone 4A4, (Millipore. Burlington, Massachusetts, United States) and stored at -20\u0026ordm;C or purified using GE Healthcare Life Sciences\u0026trade; HisTrap\u0026trade; FF Crude columns (Thermo Fisher Scientific. Waltham, Massachusetts, United States).\u003c/p\u003e\n\u003cp\u003eII.7. CHRONIC TREATMENT WITH PEDF AND CTE-PEDF\u003c/p\u003e\n\u003cp\u003eCells were treated with PEDF or CTE-PEDF at a final concentration of 8 nM in the medium. The acute treatment consists of a single treatment: two hours of peptide exposure before chemotherapy treatment. Chronic treatment consists of six peptide treatments week (culture medium with CTE once per week), totalling six weeks of peptide exposure before chemotherapy treatment. \u003c/p\u003e\n\u003cp\u003eII.8. ACUTE TREATMENT WITH PEDF AND CTE-PEDF\u003c/p\u003e\n\u003cp\u003eCells were plated in a 24-well plate at a final concentration of 15 000 cells/well in a total volume of 200 microliters. Half of the plate was plated with CTE-PEDF medium. The next day, radiotherapy to 2 Gy, 4 Gy and 6 Gy was applied by a Siemens PRIMUS X6MV LINAC. Field size 10 \u0026times; 10 cm2, depth 10 cm, SSD = 100 cm, Siemens PRIMUS X6MV.\u003c/p\u003e\n\u003cp\u003eII.9. DOSE-RESPONSE CURVE\u003c/p\u003e\n\u003cp\u003eCells were plated in a 24-well plate at a final concentration of 15 000 cells/well in a total volume of 200 microliters or 200 cells/well in a 96-well plate for sorted cells. Half of the plate was plated with CTE-PEDF medium. The next day, the drug was added in decreasing concentrations 4 nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM. These were cultured for 3 days under drug exposure in \u003cem\u003ein vitro\u003c/em\u003e conditions, and then developed with an MTT assay or methyl purple assay.\u003c/p\u003e\n\u003cp\u003eII.10. MTT ASSAY\u003c/p\u003e\n\u003cp\u003eThe cell culture media was removed, after which 100 microliters/well of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added. After 30 minutes of incubation, the supernatant was removed, and the precipitated crystals were dissolved in 100 microliters of DMSO. The plate was read in a spectrophotometer at 540 nm.\u003c/p\u003e\n\u003cp\u003eII.11. METHYL PURPLE ASSAY\u003c/p\u003e\n\u003cp\u003eThis method[33] is used to quantify surviving cells. 5 000 cells/well were seeded in 24-well plates, in a final volume of 250 \u0026micro;L. The next day, cells were treated with increasing doses of chemotherapeutic agents and stored in a humidified incubator at 37\u0026ordm;C, 5% CO2 for 4 days. Then, cells were fixed with 0.5% glutaraldehyde (Sigma. St. Louis, Missouri, United States) for 10 minutes. Next, cells were stained with 0.1% crystal violet for 20 minutes. After several washes with PBS, 10% acetic acid was used to solubilize the sample. Finally, a spectrophotometric reading was performed at a wavelength of 590 nm. The IC50 value was determined as the dose necessary to eliminate 50% of the cell population, obtained through logarithmic regressions made with the DE.0 plus v 1.0 program.\u003c/p\u003e\n\u003cp\u003eII.12. HISTOLOGY\u003c/p\u003e\n\u003cp\u003eCryostat sectioning slides were performed using a cryostat (Microm HM 550, Thermo Scientific). Tumour samples were fixed with formaldehyde at 4%, placed in sucrose 30% overnight, included into Tissue-Tek\u0026reg; OCT\u0026trade; (Sakura \u0026reg; Finetek USA. Torrance, California, United States) and then frozen with liquid nitrogen. Slides (12\u0026micro;m) were stained with hematoxylin and mounted with Dako Ultramount Aqueous Permanent Mounting Medium (Agilent Technologies. Santa Clara, California, United States) and analyzed with a Leica-DMRXA-photomicroscope (Leica. Wetzlar, Germany).\u003c/p\u003e\n\n\u003cp\u003eII.13. ANALYSIS OF CELL MORPHOLOGY\u003c/p\u003e\n\u003cp\u003e500 000 treated and control cells were seeded in p100 plates. After 24 hours, cells were incubated for 30 minutes with Hoechst (5\u0026micro;g/mL). Ten random microscopy images were taken (Motic AE31.Barcelona, Spain) using an ultraviolet light. Analysis of the cytoplasmic area and the separation between cells was analyzed using the Image J application (https://imagej.nih.gov/ij/). \u003c/p\u003e\n\u003cp\u003eII.14. IMMNUNO ASSAYS \u003c/p\u003e\n\u003cp\u003e500,000 treated and control cells were seeded on p100 plates with glass coverslips on the bottom to allow removal of the cells once grown. After chronic treatment (4 weeks) with PEDF, the cells were fixed with 1% paraformaldehyde and stained with specific BCRP1 (MAB4155A4, Sigma-Aldrich) and CD133 (MAB4399-I, Sigma-Aldrich) antibodies according to the manufacturer\u0026apos;s instructions. At the end of immunolabeling the cells were incubated for 30 minutes with Hoechst (5\u0026micro;g/mL). Fluorescence microscopy images were taken with a Motic AE31 microscope equipped with an ultraviolet lamp.\u003c/p\u003e\n\u003cp\u003eII.15. STATISTICAL ANALYSIS \u003c/p\u003e\n\u003cp\u003eData was analyzed with R software 3.5.1 version (https://www.r-project.org/). The statistical analysis was carried out using Mann-Whitney U tests (one tailed, significance level=0.05). The data is expressed as the mean plus the standard error (SE). At least n=3 independent experiments were performed for every assay. The results obtained are considered statistically significant when p\u0026lt;0.05(*), p\u0026lt;0.01(**) and p\u0026lt;0.005(***). Cell data was first transformed into a quadratic variable to improve data homogeneity. Then, normality was tested using the Sapiro-Wilk test, Q-Q plots, and Levene\u0026rsquo;s test for homogeneity of variance. Next, groups were compared using non-parametrical Kruskall-Wallis test by ranks and Wilcoxon\u0026rsquo;s not paired test to analyze data in pairs. To test the hypotheses, an outcome measure of significance with a 95% confidence interval was used to determine the sample size, based on the hypothesis of treatment-to-control ratio for tumor growth inhibition studies[90] Finally, logistic regression was used to model the relationship between the number of cells and progression. The odds ratio was used to strengthen the association between variables (Confidence Interval 95%). \u003c/p\u003e"},{"header":"III. Results","content":"\u003cp\u003eIII.1. Long-term label retaining cells exhibit characteristics of cancer stem cells, in cancer cell lines and in patient cells.\u003c/p\u003e\n\u003cp\u003eMDA-MB-231 and MCF7 cell lines exhibit a cell population characterized by long retained labelling growing either in adherent or mammosphere conditions (Figure 1A). A population of 0.9 \u0026plusmn; 0.3 % and 3 \u0026plusmn; 1% of long-term label retaining cells is present when the MDA-MB-231 cell line is grown in an adherent monolayer and in mammosphere conditions, respectively. This result is also seen for the MCF7 (Figure 1B) cell line where the percentage of long labelling cells is 3.32% and 6.28% in monolayer culture and mammosphere conditions, respectively. \u003c/p\u003e\n\u003cp\u003eAscitic fluid cells from patients with a metastatic adenocarcinoma also present long-term label retaining cells after 8 days in culture (Pa00 cells, Figure 1C). A methyl purple assay proves that LT- cells show the same growth rate as our control cells but have a higher growth rate than LT+ cells (Figure 1C). After a dose-response assay with docetaxel, LT+ cells are more resistant to chemotherapy than control or LT- cells (Figure 1C). IC50 value is double in the case of LT+ cells compared to control or LT- cells (vertical lines in Figure 1D). Docetaxel treatment eradicates 53 \u0026plusmn; 5% of LT- cells at 2 nM, while only 27 \u0026plusmn; 3% of LT+ were affected by the same concentration. At high doses of chemotherapy, the growth curve returns to control levels, however these are higher than the doses applicable in patients due to their undesirable side effects.\u003c/p\u003e\n\u003cp\u003eFinally, Pa00 LT+ and LT- sorted cells were injected in nude mice to study the carcinogenicity of those populations in vivo. Eleven animals were used in this experiment. Four animals (two flank injections per animal, eight xenografts in total) were used in the control group and in the LT- group while only three animals were used in the LT+ group (five xenografts due to the low number of LT+ cells) for this experiment. The results show that the tumour volume is similar when injecting LT- cells and control non-separated cells but smaller when injecting LT+ cells (Figure 1E). Furthermore, Pa00 LT- cells formed tumours with a frequency of 0.75, while the frequency with LT+ cells is 1. (Figure 1F).\u003c/p\u003e\n\u003cp\u003eIn short, LT+ cells show a lower growth rate and they are cells more resistant to chemotherapy than LT- or control non-separated cells. In addition, in vivo assays reveal a decrease in the frequency of tumour formation in LT- cells when compared to LT+.\u003c/p\u003e\n\u003cp\u003eIII.2. PEDF modulate CSC properties, producing an increase of drug resistance and LT+ cells proportion.\u003c/p\u003e\n\u003cp\u003ePEDF chronic treatment produces morphological changes in size and cytoplasm shape of Pa00 cells in culture (Figure 2A). We registered an increment of 170 \u0026micro;m2 at the cytoplasmic area in PEDF treated cells (730\u0026plusmn;80 \u0026micro;m2 in PEDF treated and 560\u0026plusmn;30 \u0026micro;m2 in control medium), what it could be indicating is alterations in the cytoskeleton related to the tumourigenicity of these cells. However, there are no significant differences between the nucleus size with and without treatment (Figure 2B).\u003c/p\u003e\n\u003cp\u003eWe have also studied the growing pattern of chronically PEDF-treated patient-derived cells. This slower growth rate of treated cells has been quantified as 37\u0026plusmn;1% fewer cells in treated compared to untreated PEDF cell cultures (Figure 2C). This effect translates into a lower tumour volume when the cells are injected in nude mice (Figure 2D); showing close to a 40% decrease in tumour xenograft volume from treated cells compared to controls (Figure 2D). The histological analysis showed that PEDF treated tumours present smaller necrotic areas than control xenografts. Looking at the micrographs (Figure 2E) for the tumours from untreated cells, we can see large acellular areas (arrows in figure 2E) compatible with acellular necrotic spaces.. In contrast, the micrographs for tumours from PEDF-treated cells do not show these same areas, as the nuclei and cytoplasm of the cells are better preserved. PEDF treatment also produces compact growth of the tumoural cells which present dense cytoplasm and a compact external matrix compared to the control tumours, which show acellular areas with cell detritus (Figure 2E). In the micrographs with immunofluorescence staining for BCRP1 and CD133 markers (Figures 2H-K) we again observe a lower number of nuclei in the controls than in the PEDF treatments, with larger acellular areas concordant with areas of necrosis and cell death as in panoramic Figure 2E. In these controls there is a greater abundance of cells in different mitotic phases indicated by white arrows (Figures 2H and 2J).These cells were also checked in a dose-response assay, and despite their low growth rate, PEDF treated cells exhibit higher IC50 values and resistant populations, as 47\u0026plusmn;8% of PEDF treated cells survived, which means there were 16% more resistant cells than in the control where just 31\u0026plusmn;2% of the cells survive at the maximum physiologic dose tested. This result implies higher drug resistance to docetaxel as compared to control cells (Figure 2F). We carried out the next assay to prove that the slow growth observed \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e is due to the appearance of a higher number of slow-cycle cells after PEDF treatment. The quantification of LT+ cells after 3DIV (days \u003cem\u003ein vitro\u003c/em\u003e) revealed a significant increase after PEDF treatment compared to the control (Figure 2G), with nearly a three-fold increase in cell number. \u003c/p\u003e\n\u003cp\u003eTo sum up, PEDF treated cells show changes in cytoplasm size, decreased cell cycle kinetics, increased drug resistance, and the capacity to produce tumours with not only a lower growth rate but also lower tumour volume and fewer necrotic areas.\u003c/p\u003e\n\u003cp\u003eIII.3. Cter-PEDF counteracts the effects of native PEDF, decreasing the resistance in tumours and inducing anoikis and depleting CSC.\u003c/p\u003e\n\u003cp\u003eAs the truncated protein CTE-PEDF does not exhibit the same effects in stem cells as the full-size PEDF protein, we decided to check if this peptide would counteract the effects of the native protein also in our cancer model. This protein presents phosphorylation sites that are important for the protein\u0026rsquo;s function. In order to maintain the negative charge, a glutamate was introduced in position 227 of the protein\u0026rsquo;s sequence, where a serine was previously located. This modification leads to the CTE-PEDF protein (Figure 3A). We applied CTE-PEDF (200ng/ml) \u003cem\u003ein vitro\u003c/em\u003e treatment to patient cultured cells PA00. This experiment shows an anoikis effect (Figure 3B) and morphological changes that can be measured by cytoplasmic and nuclear areas and cell distances (Figure 3C). Nuclear and cytoplasmic areas are reduced 29% (Nuclear area: control 110\u0026plusmn;2 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, CTE 81\u0026plusmn;2.34 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e; Cytoplasmic area control: 556\u0026plusmn;18 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e, CTE: 394\u0026plusmn;17 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e) while cell distances double their size (control 12\u0026plusmn;1 \u0026micro;m, CTE: 23.0\u0026plusmn;0.2 \u0026micro;m). Finally, treated cells divide faster but survive less than untreated cells in culture (Figure 3D).\u003c/p\u003e\n\u003cp\u003eThe next approach was a dose-response assay with docetaxel as previously performed with native PEDF treatments. The result demonstrates that CTE-PEDF treated cells are less resistant (squareds in Figure 3E) than control cells (circles in Figure 3E). Only the CTE-PEDF treatment produces a 20% reduction of the initial population. The IC50 value is double in the control group when compared to CTE-PEDF treated cells (vertical lines in Figure 3E).\u003c/p\u003e\n\u003cp\u003eThe results demonstrate that CTE-PEDF produces anoikis in cancer patient cells in culture and reduces the drug resistance to Docetaxel.\u003c/p\u003e\n\u003cp\u003eIII.4. CTE-PEDF and Cter-PEDF depletes percentage of CSC expressing markers.\u003c/p\u003e\n\u003cp\u003eTo consider whether the decrease in resistance and anoikis is produced because of a reduction in CSC number and CSC expression markers, Pa00 cells were injected with Cter-PEDF or PBS acute treatment, in nude mice. Resulting tumours were dissected and dissociated to study stemness marker expression by flow cytometry (Figure 4A). Four different epitopes, previously related to cancer stem cells, were analysed: BCRP1, EpCam, CD133 and AC133. Cter-PEDF treatment produces a significant reduction in all studied markers in patient tumour cells Pa00 (Figure 4B). The same experiment was performed with cells chronically treated during three weeks in culture with CTE-PEDF. CTE-PEDF has a negative charge in the glutamic 227 and is more stable than Cter-PEDF as the latter has a phosporylable serine 227 residue which can lose the negative charge when phosphorylated. The same reduction in marker expression was observed (Figure 4C).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e assays with xenografts of PEDF and CTE-PEDF chronically treated cells also show the influence of these proteins in the cancer stem cell population and resistant population. Immunofluorescence assays show differences in expression of tumour stem cell related markers comparing the population of cells treated with PEDF to those treated with CTE-PEDF. 3*106 cells/flank were injected in three animals per group: control, PEDF and CTE-PEDF treated cells, with tumours appearing one week after the injection in every case. Nine animals were employed in this experiment, 3 animals per group, 2 flank injections per animal. In both cases (PEDF and CTE-PEDF treated cells injected) there was a modification of the expression pattern of the BCRP1 and CD133 proteins. When exposed to chronic PEDF treatment there is an increase in cells positive for these markers, while the opposite is observed after chronic treatment with CTE (Figure 5). When treated with CTE fewer cells are positive for BCRP1 and CD133 compared to controls or cells treated with PEDF. Figure 5C shows the relationship between the effect of PEDF and CTE on tumour cells, with the IC50 of docetaxel altered in both cases. PEDF treatment produces more resistant cells than the control whereas CTE treatment decreases the IC50 of docetaxel, which coincides with the hypothesis that this treatment increases sensitization to the drug, due to a higher division rate that thus prevents tumour cell survival.\u003c/p\u003e\n\u003cp\u003eCells treated with PEDF, in addition to higher expression of markers, also show a higher concentration of LT+ cells (Figure 6A and B). These LT+ cells confer more chemoresistance to the culture (Figure 6C). We therefore set out to combat the effect of PEDF \u003cem\u003ein vivo \u003c/em\u003eusing the CTE peptide, which reduces marker expression and chemoresistance, as we have already stated. \u003c/p\u003e\n\u003cp\u003eTo correlate the reduction in cancer stem cell marker expression in Pa00 cells (Figure 4) with decreased tumourigenicity (function) xenograft tests were carried out by injecting BCRP1 positive cells previously treated with CTE. The results show that PEDF increases LT+ cells (Figure 6A and B). These LT+ form more resistant tumours (Figure 6C). Positive cells were injected at different concentrations, with their respective positive control of Cter-PEDF peptide treatment. Eight mice were used in this experiment, making use of both flanks: the right flank for injection of untreated control cells and the left flank for treated Cter-PEDF cells. Despite the heterogeneity of the \u003cem\u003ein vivo\u003c/em\u003e experiments, it can be seen from Figure 6D that in most cases the tumours from treated cells appear later than the untreated tumours. Even when injecting cells that had been previously treated with CTE, after injection the xenograft appears earlier than in control cells. Although tumours formed from cells submitted to chronic CTE treatment are the least resistant of all to chemotherapy as we have shown previously in Figure 5C, the effect of CTE is also synergistic with radiotherapeutic treatments, as it decreases cancer stem cell viability significantly with CTE treatment and radiotherapy (gray bars) than with radiotherapy with a negative control peptide CTA without negative charge (white bars). Figures 6E and F show the assays performed on cells exposed to different successive radiotherapeutic treatments. To test the effect of our model in these assays, we have performed new experiments with the CTE peptide and also with its homologue CTA (without phosphoryable serine as it is switched to an alanine). These assays mimic the effects seen with CTE combined with chemotherapy, as these cells also become less resistant to radiotherapy (contrary to what is seen with the negative control CTA) (Figure 6F). This could possibly be because of their faster division, causing those cells with a cell cycle regulated by p21 expression to disappear. Looking at expression assays by qPCR (Figure 6E) it is clear that the expression of \u003cem\u003ep21\u003c/em\u003e, responsible for stopping the cell cycle in tumour stem cells, decreases. The cumulative effect of the different cycles of radiotherapy offered to patients results in down-regulation of proteins involved in the cell division of cancer stem cells or in the number of cancer stem cells themselves. This type of molecules linked to the cancer stem cell cycle could be at the basis of the synergistic effects of the PEDF signaling pathway and chemo- and radiotherapeutic treatments.\u003c/p\u003e\n\u003cp\u003eIn summary, CTE-PEDF decreases resistance and putative stemness markers involved in self-renewal and patient relapse, demonstrating the interest in these proteins for potential application controlling drug-resistant cell populations in patient samples.\u003c/p\u003e"},{"header":"IV. Discussion","content":"\u003cp\u003eBreast cancer is the most common cancer in women worldwide. We study how to prevent relapse, taking breast cancer as an example since its impact on society reaches an incidence of 15.53 (95% CI = 14.94 to 16.14) per 1000 PY[34]. The tumour subtype influences the risk of recurrence ranging from 13% to 41% depending on the tumour and the state of the nodes of the primary tumour [35,36] as hormone receptor positive and metastatic breast cancer and triple-negative breast cancer. It\u0026rsquo;s important to distinguish tumour initiating cells, responsible for tumour formation and tumour relapse. For cancer stem cell detection no good spectra of specific markers is available. The expression of membrane-determinants is a gradient during development and maturation of biological process, and even when testing a combination of the different epitopes found in the literature[37\u0026ndash;39], it is difficult to find a completely cancer stem cell specific marker. However, tumour initiating cells (TICs) are considered responsible for the slow cell cycle and quiescent characteristics of the tumour. Our goal was to demonstrate that TICs are important for tumour maintenance, resistance and relapse. Later, our objective was to discover a possible new treatment to delay patient relapse. We demonstrate that PEDF (and derivatives) have a moonlighting role in breast cancer development and relapse. This has a potential application in clinical cancer therapies by the co-administration with chemotherapy to improve relapse-cancer treatment. This is the case for CTE-PEDF, which decreases drug resistance [33,40,41]. The co-administration with chemotherapy would lead to a less resistant population because TICs would have lost their stemness characteristics, as we discuss in the following paragraphs. The regulation of breast cancer progression through regulation of breast cancer stem cell like properties has been recently described, probably mediated by the NF-\u0026kappa;B cell signaling pathway[42], and the connection between chemoresistance, mesenchymal plasticity and cancer stem cells in metastasis origin[43].\u003c/p\u003e\n\u003cp\u003eWe have shown that MDA-MB-231 and MCF7 breast adenocarcinoma cell lines exhibit a cell population characterized by long retained labelling growing either in adherent or mammosphere conditions, as has been previously described in other tumour cell lines [44\u0026ndash;46] and even in PA00 cells[47]. Other researchers have detected similar percentages of stemness and invasiveness of breast cancer cells in these cell lines, which were modified by estrogen through Gli1 activation [48]. Even in mammosphere cultures, we have observed that a high DDAO positive population, of slow cell cycle cells, remains after 8 days in culture; these cells have probably only divided once in this period. This subpopulation could be related to resistant cells maintained by IL-6 in some breast cancer treatments. Also, they could be affected by the inhibitor of the human epidermal growth factor receptor 2 (HER2) Lapatinib [49], and are our focus in this work. \u003c/p\u003e\n\u003cp\u003eAscitic fluid cells from a patient with a metastatic adenocarcinoma (Pa00 cells) have been used in this paper parallel to commercial cancer cell lines. Ascitic fluid, usually a result of inflammation events, contains malignant cells in up to 97% of patients with a neoplastic diagnosis and is a gold standard for pericardial carcinomatosis detection [50]. Ascitic fluid contains tumour cells that grow in culture conditions [51,52]. Part of these cells also present long retained labelling (DDAO) after 8 days in culture as we have demonstrated previously [47]. We select these cells by flow cytometry sorting according to their high DDAO content, deemed LT+ cells. This experiment showed that LT+ cells grow slower and respond less to chemotherapy than non-separated control cells or LT- cells. We postulated that this is the reason for the resistance observed in some treatments. Similar differences in resistance have also been observed in other colon and breast cancer cell lines [9,33] and even in other more aggressive types of tumours such as a glioblastoma C6 cell line [40,53]. In our hands, the final percentage of resistant cells after maximum physiological doses of docetaxel treatment is also higher in LT+ positive cells than in the rest. This LT+ population that supports higher docetaxel IC50 than LT- or control cells could be the origin of patient relapse. These same LT+ cells could also be related to higher BCRP1 and CD133 marker expression. To investigate this hypothesis, we injected Pa00 LT+ and LT- sorted cells in nude mice to study the tumourigenicity of those populations. We have considered not only the final volume of the xenograft tumours, but also the timing of the tumours\u0026rsquo; appearance. LT- cells formed tumours at the same time as control non-separated cells but LT+ Pa00 took longer to form tumours. Accordingly, the percentage of final tumour growth after LT+ cell injection is 1/3 higher than in LT- xenografts. This result, together with the previously shown data, leads us to consider that the difference between these two cell types is the initial tumour capacity. This data correlates with previous data from other research groups which connect chemoresistance, tumourigenicity potential, and slow-cycling in some tumour cells [44,46,54]. Several other markers (such as TROP2) could be involved in this chemorresistance process too, as has been recently identified by our group[55].\u003c/p\u003e\n\u003cp\u003eAll this data supports the hypothesis that LT+ cells present slow cell cycle division, higher chemotherapy resistance, and a higher frequency of tumour formation than LT- cells. These characteristics confirm the idea that LT+ cells could be involved in relapse and metastasis progression in breast cancer.\u003c/p\u003e\n\u003cp\u003eIn our hands, PEDF chronic treatment produces a decrease in the growth rate \u003cem\u003ein vitro \u003c/em\u003eand \u003cem\u003ein vivo \u003c/em\u003edue to an increase in the number of LT+ cells (slow-cycling cells). The data shown in Figure 2 indicates that cells treated with PEDF have more controlled proliferation, with slower self-renewal processes than simple tumour cell division that ends in internal necrosis of the tumours. \u003c/p\u003e\n\u003cp\u003eAs a conclusion from the cytoplasm measurements in Figure 1A/B, and the significant differences observed, we hypothesize that these PEDF-treated cells with a higher cytoplasm-to-nucleus ratio slow down their cell cycle. Several proteins have been related to this phenomenon. For example the histone chaperone nucleoplasmin (Npm2) has been identified as a putative nuclear size effector [56]that binds histones and may play a key role in tumour development and progression [57]. \u003c/p\u003e\n\u003cp\u003eOther cytoskeletal proteins have been related to this activity and cytoplasm size (cytomegaly). For example non-muscle myosin IIA (MYH9), is a ubiquitously expressed cytoplasmic myosin that regulates the actin cytoskeleton, cell migration, cell polarization and signal transduction in cancer cells. MYH9+ cells, size-differentiated by flow cytometry, showed strong CSC characteristics, including in vivo tumourigenicity, migration, invasion, cisplatin resistance and positivity for CD133+[58].\u003c/p\u003e\n\u003cp\u003eThese data recorded by other investigators between cytoplasmic size and proteins involved in tumourigenicity will be tested in our system in the near future. And for the moment they bring agreement with the growth the increased cytoplasm/nucleus ratio and the slower growth observed in our experiments. Cytoplasm size can be related to multiple physiological phenomena, but among them quiescence and cycle exit. Although it is difficult to conclude what the increased cytoplasmic area means in our experiments, the data are in agreement with the hypothesis put forward and also with tumourogenicity and proliferation data observed by other authors as argued.\u003c/p\u003e\n\u003cp\u003eThe experiment that supports this optimal cellular state as a result of PEDF treatment is shown in Figure 2F, where it is seen that these cells are more resistant to chemotherapeutic pharmacological stresses, and retain vital dyes for longer, due to their slower cell division, as indicated in Figure 2G. Despite their low growth rate, PEDF treated cells show higher IC50 values and resistant populations than untreated control cells, indicating higher resistance to chemotherapy. One possible explanation for this effect could be that the low growth rate lends more time for these cells to repair chemotherapy damage[59,60]. However, another hypothesis could contribute to this stage, such as higher expression of ABC drug transporters in these cells[61,62]. On the other hand, the effect that we attribute to the increase in slowly dividing cells could be due to other effects, such as the inhibition of angiogenesis widely attributed and recently countersigned for the PEDF protein[63]. In addition to this anti-angiogenic effect, PEDF has been reported to have an impact on lymphangiogenesis[64]. Release of PEDF into the tumour microenvironment, among other factors, inhibits vascular growth, while also promoting lymphangiogenesis associated with cancer[65]. Although PEDF has been recognized as an anti-metastatic factor, its role remains controversial due to some conflicting reports indicating a role in metastatic progression in some cases[66], diversifying PEDF\u0026rsquo;s impact on tumourigenicity, as our results point out. Current studies of the tumour microenvironment (TME), in which PEDF could be involved, offer an overview of the primary functions of each component of the TME in cancer initiation, progression, and invasion[65,67,68]. In MCF7 breast cancer cells, silencing of PEDF has been shown to promote resistance to tamoxifen[69,70]. It could be hypothesized that the PEDF signaling pathway is involved in the binding of the estrogen receptor to its activators and its proliferation activator function. Some peptides derived from PEDF[71] activate the proliferation of progenitor or stem cells, as in our case. Whether our C-ter peptide is involved in the regulation of resistance to tamoxifen will need to be tested in future experiments; this hypothesis would offer new potential uses for that peptide.\u003c/p\u003e\n\u003cp\u003eIn contrast, we present here that PEDF treatment increases resistance to docetaxel. PEDF is self-regulated by VEGF and the angiogenesis process. HIF1alpha-independent angiogenesis could be under interference from both PEDF\u0026rsquo;s anti-angiogenic effect and tumoural cells\u0026rsquo; resistance[72]. In culture however, the anti-angiogenic effect of PEDF could be shielded by its self-renewal effect over cancer stem cells. These results could also be explained based on the metabolic pathway in which cytochrome P450 metabolizes both components (PEDF and docetaxel). It has been described that PEDF function can be blocked by fluvoxamine, a P450 inhibitor[73]. P450 polymorphisms present in each of the cell lines and patient cells analyzed could account for these differences.\u003c/p\u003e\n\u003cp\u003eReturning to the possible effectiveness of the peptide CTE-PEDF, treated patient cells showed symptoms compatible with anoikis and morphological changes in culture. The anoikis effect is the apoptosis induced by lost, insufficient, or inappropriate interactions between the cell and the extracellular matrix[74]. Some of the cell signaling pathways involved in cancer progression (such as BMPs) have also been related with proliferation, anoikis resistance, metastatic migration, and drug resistance of breast cancer cells[75]. This anoikis effect could be the basis for the interaction of TICs with the surrounding niche, and could be involved in the loss of stemness properties and chemotherapy resistance[76]. Docetaxel resistance decreases when cells are treated with CTE-PEDF. It counteracts the effect of PEDF, which is a niche protein[12] involved in self-renewal and trophic maintenance of pluripotent cells. PEDF is secreted by endothelial cells and is one of the proteins involved in the self-renewal capacity of stem cells. Endothelial cells also play a significant role in tumour progression and metastasis[77,78], and the niche signals could be key to understanding tumour progression, epithelial-mesenchymal transition, and distant metastasis at patient relapse. A significant difference is shown between treated cells and controls, with PEDF or derived peptides, but the protocol followed in this experiment should be standardized to obtain a better result. The molecular subtype used in this work could be influencing the observed effects, however, the same results have been obtained in cell lines of other types of tumours[33,40]. Also we observe differences in the level of expression of proteins that participate in the PEDF signalling pathway justifying the differences between cell lines in the different experiments while maintaining the final effect and the extractable conclusions.\u003c/p\u003e\n\n\u003cp\u003eIn short, the experiments carried out indicate that the carboxyl terminal fragment of the PEDF protein, CTE-PEDF, could be an effective chemosensitizing agent since it is capable of counteracting the effect of the endogenous native PEDF protein as had been demonstrated in neural stem cell populations previously ([12]), and depleting the expression of TIC markers. Expression of markers that had been previously related to cancer stem cells decreases when cells are treated with CTE-PEDF. This effect confirms the idea that CTE-PEDF produces a decrease in resistance to drugs such as docetaxel, because it reduces the number of tumour initiating cells. This result indicates a possible potent application as a future treatment for this protein when co-administered with chemotherapy[79,80]. What we postulate here is that the modification of the PEDF protein, that is, the C-terminal peptide, can affect the cancer root through a mechanism of competition with native PEDF. Previous experiments have shown this competition and that increasing doses of truncated peptide can inhibit the effect of the native protein[12]. Although further studies need to be carried out, these experiments point to a possible new strategy for treatment of cancer relapse.\u003c/p\u003e\n\u003cp\u003eIn order to verify the importance of our data we have compared our results with other kinds of treatments. In addition to chemotherapeutic treatments, radiotherapeutic adjuvant treatment is also widely used in breast cancer [81,82]. We have shown that radiotherapeutic treatment decreases cell cycle markers related to stem cell self-renewal, such as the cell cycle protein p21[83,84], which decreases with progressive radiotherapy treatment. All combined, the results of this comparison are of great applicability in resistant tumours, especially in cases of triple negative breast cancer, as these show the worst response to current treatments. There are many experiments to be performed in this regard, and these will be future lines of research in the laboratory as the effect of different concentrations of peptides or even chemotherapy[85]. However, the data obtained so far seems to us of importance, contributing to general knowledge and permitting us to advance in the fight against resistance to cancer treatment.\u003c/p\u003e\n\u003cp\u003eTo conclude, our hypothesis can be summed up in the schematic below (Figure 7). There has been much discussion in the literature regarding the idea of a countdown mechanism that limits cellular replicative potential by giving it an intrinsic heuristic value [86]. We consider that cancer stem cell depletion is of particular importance and CTE can help us in this pathway. In the summary figure we can see how PEDF enhances the self-renewal of tumour stem cells, akin to what occurs in normal stem cells [12,17]. Molecular markers involved in the cell cycle of cancer stem cells, such as p21, increase their expression after PEDF treatment, thereby indicating an increase in the self-renewal of these cells, as well as their perpetuation and, as a consequence, the chemoresistance of these cell cultures. In contrast, chronic treatment with CTE-PEDF allows the extenuation of the tumour stem cell population, along with an increase in the proliferative rate, but also better response to chemotherapy treatments, since active cycle cells are more susceptible to apoptosis from anti-neoplastic treatments, thus decreasing the harmful rate of resistant cells (Figure 7).\u003c/p\u003e"},{"header":"V. Conclusions","content":"\u003cp\u003eThe PEDF stem cell self-renewal modulator protein modifies the carcinogenicity of cancer stem cells and could be a useful tool to control tumoural self-renewal and therefore control patient relapse. We have designed a transgenic peptide derived from PEDF to interfere with the self-renewal capacity of cancer stem cells, inducing anoikis \u003cem\u003ein vivo\u003c/em\u003e and reducing resistance in cells from cancer patients. We have also shown that this PEDF-derived peptide produces a significant decrease in cancer stem cell markers, making it a potential tool for delaying patient relapse.\u003c/p\u003e"},{"header":"List of abbreviations","content":"\u003cp\u003eABC: ATP-binding cassette\u003c/p\u003e\n\u003cp\u003eAI: artificial intelligence\u003c/p\u003e\n\u003cp\u003eAC133: glycosylated epitope of the pentaspan membrane protein CD133, originally identified as a marker for CD34 + hematopoietic stem and progenitor cells.\u003c/p\u003e\n\u003cp\u003eBCRP1: Breast cancer resistance protein (BCRP1), also known as placenta-specific ATP-binding cassette (ABC) protein (ABCP) or ABC G-subfamily member 2 (ABCG2)\u003c/p\u003e\n\u003cp\u003eBMPs: Bone Morphogenetic Proteins\u003c/p\u003e\n\u003cp\u003eC6: glioma cell line\u003c/p\u003e\n\u003cp\u003eCD133: CD133 antigen, also known as prominin-1, is a glycoprotein that in humans is encoded by the PROM1 gene. It has been proposed that it acts as an organizer of cell membrane topology.\u003c/p\u003e\n\u003cp\u003eCDKN1A: Cyclin Dependent Kinase Inhibitor 1A\u003c/p\u003e\n\u003cp\u003eCI: Confidence interval\u003c/p\u003e\n\u003cp\u003eCM-spf: mammospheres \u003c/p\u003e\n\u003cp\u003eCSC: cancer stem cells \u003c/p\u003e\n\u003cp\u003eCTA-PEDF: carboxyterminal of PEDF (Ala227) \u003c/p\u003e\n\u003cp\u003eCTE-PEDF: carboxyterminal PEDF (Glu227) \u003c/p\u003e\n\u003cp\u003eCter: Cter-carboxyterminal PEDF (comprising amino acids 195-400 of PEDF)\u003c/p\u003e\n\u003cp\u003eDAPI: 4\u0026rsquo;6-Diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003eDDAO: Near-infrared (NIR) red fluorescent probe with excitation wavelength (600-650 nm) and long emission wavelength (\u0026lambda;em = 656 nm).\u003c/p\u003e\n\u003cp\u003eDIV: days in vitro\u003c/p\u003e\n\u003cp\u003eDMSO: Dimethyl sulfoxide\u003c/p\u003e\n\u003cp\u003eECM: Extracellular matrix\u003c/p\u003e\n\u003cp\u003eEGF: epidermal growth factor\u003c/p\u003e\n\u003cp\u003eEMT: epithelial mesenchymal transition\u003c/p\u003e\n\u003cp\u003eEpCAM: Epithelial cell adhesion molecule, also known as CD326.\u003c/p\u003e\n\u003cp\u003eER: estrogen receptor \u003c/p\u003e\n\u003cp\u003eFGF: fibroblast growth factor\u003c/p\u003e\n\u003cp\u003eGADPH: Glyceraldehyde 3-phosphate dehydrogenase.\u003c/p\u003e\n\u003cp\u003eGy: gray\u003c/p\u003e\n\u003cp\u003eHEK-293: Human embryonic kidney cell line\u003c/p\u003e\n\u003cp\u003eHER2: Human Epidermal growth factor Receptor 2\u003c/p\u003e\n\u003cp\u003eHIF1alpha: Hypoxia Inducible Factor\u003c/p\u003e\n\u003cp\u003eIC50: half maximal inhibitory concentration \u003c/p\u003e\n\u003cp\u003eLT+: Long-term label retaining cells\u003c/p\u003e\n\u003cp\u003eMDA-MB-231: Triple-Negative Breast Cancer Cell Line.\u003c/p\u003e\n\u003cp\u003eMDR1: Multi drugs resistance gen\u003c/p\u003e\n\u003cp\u003eMCF7: epithelial cell line isolated from the breast tissue of a patient with metastatic adenocarcinoma.\u003c/p\u003e\n\u003cp\u003eMLY: monolayer \u003c/p\u003e\n\u003cp\u003eMTT: methyl thiazol tetrazolium\u003c/p\u003e\n\u003cp\u003eMYH9: myosin heavy chain non-muscle\u003c/p\u003e\n\u003cp\u003eNF-kB: Nuclear kappa-light-enhancer of active B cell\u003c/p\u003e\n\u003cp\u003ep21: p21 Cip1 (alternatively p21 Waf1), also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1\u003c/p\u003e\n\u003cp\u003ePA00: ascitic cell line derived from a patient with metastatic breast cancer, in our laboratory.\u003c/p\u003e\n\u003cp\u003ePBS: phosphate buffered saline\u003c/p\u003e\n\u003cp\u003ePEDF: Pigment Epithelium-Derived Factor \u003c/p\u003e\n\u003cp\u003ePKA: protein kinase A (EC 2.7.11.11)\u003c/p\u003e\n\u003cp\u003ePR: progesterone receptor\u003c/p\u003e\n\u003cp\u003ePY: Patient Year\u003c/p\u003e\n\u003cp\u003eSOX2: SRY-Box transcription Factor 2\u003c/p\u003e\n\u003cp\u003eTert: telomerase protein \u003c/p\u003e\n\u003cp\u003eTIC: tumour-initiating cells \u003c/p\u003e\n\u003cp\u003eTME: Tumour microenviroment \u003c/p\u003e\n\u003cp\u003eTROP2: transmembrane glycoprotein encoded by the \u003cem\u003eTacstd2\u003c/em\u003e gene overexpressed in cancers.\u003c/p\u003e\n\u003cp\u003eVEGF: vascular endothelial growth factor\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement: \u003c/strong\u003eThe study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of CEIC Hospital Cl\u0026iacute;nico San Carlos (protocol code 13/026-E and 12/02/2013)\u0026quot; for studies with human subjects. (1) Title of the approved project: Localization and control of tumor stem cell self-renewal to improve the efficacy of chemotherapy and prevent recurrence. (2) Name of the institutional approval committee or unit: Comit\u0026eacute; de \u0026Eacute;tica de Investigaci\u0026oacute;n Cl\u0026iacute;nica Hospital Cl\u0026iacute;nico San Carlos. (3) Approval number: C.I. 13/026-E; (4) Date of approval: 12/02/2013. The protocol of animal study was approved by the Ethics Committee of Ethics Committee of Universidad de Castilla La-Mancha (protocol 25/01/2010)\u0026rdquo; for animal studies. (1) Title of the approved project: Localization of circulating tumor stem cells in blood by surface markers and inhibition of their self-renewal. (2) Name of the institutional approval committee or unit: Comit\u0026eacute; de \u0026Eacute;tica de Experimentaci\u0026oacute;n Animal de la Universidad de Castilla La Mancha. (3) Approval number: acta25/01/2010; (4) Date of approval: 10/02/2010.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement: \u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement: \u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest: \u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by grant from the Fundaci\u0026oacute;n Cient\u0026iacute;fica Asociaci\u0026oacute;n Espa\u0026ntilde;ola Contra el C\u0026aacute;ncer (Divisi\u0026oacute;n provincial de Albacete). C.R-C. was investigator of the Programa Ramon y Cajal from MEC/Spain. M S-D is predoctoral research from the Programa Propio at the Universidad Polit\u0026eacute;cnica de Madrid. The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions: \u003c/strong\u003eCRC: Conceptualization and design of the work, Formal analysis, Funding acquisition, Project administration, Supervision, Writing-original draft and Writing review and editing. CGG: Data curation, Formal analysis, Investigation, Methodology and Writing-original draft. MSD: Formal analysis, Validation, Visualization and Writing review and editing. PHG: Investigation and Methodology. JLSS: Data curation, Formal analysis and Validation. Carmen Belen Alvarez: Methodology. SS: Data curation and Formal analysis. FSSz: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, writing review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank C.E. Gavira O\u0026rsquo;Neill for reading the manuscript and English correction. COST Action no. CA17118. TRANSCOLONCAN. And the IdISSC Oncology Group.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHulka, B.S.; Moorman, P.G. Breast cancer: hormones and other risk factors. \u003cem\u003eMaturitas\u003c/em\u003e \u003cstrong\u003e2008\u003c/strong\u003e, \u003cem\u003e61\u003c/em\u003e, 203\u0026ndash;13.\u003c/li\u003e\n\u003cli\u003eTrujillo-Mart\u0026iacute;nez, M.; G\u0026oacute;mez-Flores-Ramos, L.; S\u0026aacute;nchez-Zamorano, L.M.; Reynoso-Nover\u0026oacute;n, N.; Grimaldo, L.; Albavera-Hern\u0026aacute;ndez, C.; Flores-Luna, L. Farmacogen\u0026eacute;tica en el c\u0026aacute;ncer de mama: implicaciones de los genes del citocromo p450 en la supervivencia libre de la enfermedad en las mujeres j\u0026oacute;venes. \u003cem\u003eRev. Senol. y Patol. Mamar. - J. 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Biotechnol.\u003c/em\u003e \u003cstrong\u003e2008\u003c/strong\u003e, \u003cem\u003e134\u003c/em\u003e, 193\u0026ndash;201, doi:10.1016/j.jbiotec.2008.01.005.\u003c/li\u003e\n\u003cli\u003eWu, J. Statistical inference for tumor growth inhibition T/C ratio. \u003cem\u003eJ. Biopharm. Stat.\u003c/em\u003e \u003cstrong\u003e2010\u003c/strong\u003e, \u003cem\u003e20\u003c/em\u003e, 954\u0026ndash;964, doi:10.1080/10543401003618983.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Tumour initiating cells, PEDF, relapse, tumoural biomarkers, self-renewal","lastPublishedDoi":"10.21203/rs.3.rs-2636130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2636130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies has increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer initiating cells and slow cellular cycle cells, identified by their capacity to retain long labelling (LT+).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In this study, we perform tumour cell culture dose-response assays in adherent and mamospheres cultures. We also probe our conclusion in Xenograft models. Cell culture and in vivo tumours were analysed by immunofluorescence, qPCR and flow cytometry assays showing how stem cell self-renewal modulating proteins, such as PEDF, can statistically significant modify the properties, cell-percentage expressing biomarkers, and carcinogenicity of cancer stem cells (by student t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The PEDF signaling pathway could be a useful tool for controlling cancer stem cells’ self-renewal, and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells \u003cem\u003ein vitro\u003c/em\u003e culture. We have designed a peptide consisting in the C-terminal part of this protein, which acts by blocking endogenous PEDF in culture cell assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against Docetaxel treatment in cancer patient cells in \u003cem\u003ein vitro\u003c/em\u003e culture. We have also demonstrated that this PEDF-modified protein produces a significant decrease in the percentage of cancer stem cell expressing markers.\u003c/p\u003e","manuscriptTitle":"Moonlight role of PEDF in breast cancer: self-renewal inhibition in breast cancer stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-05 14:48:20","doi":"10.21203/rs.3.rs-2636130/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2023-12-01T00:53:56+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-08-30T16:24:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-08-30T10:35:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-09T08:35:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2023-03-08T12:49:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1039b555-b7b5-42cd-a501-334c4f670e27","owner":[],"postedDate":"September 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2023-12-02T06:57:42+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-05 14:48:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2636130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2636130","identity":"rs-2636130","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}