F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.13609.1 Research Article Articles Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells

[version 1; peer review: 1 approved with reservations, 1 not approved]

 Purnamawati Purnamawati Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4804-3785 1 Pawitan Jeanne Adiwinata Data Curation Formal Analysis Methodology Project Administration Resources Supervision Validation Visualization Writing – Review & Editing https://orcid.org/0000-0002-6551-5238 2 3 Rachman Andhika Supervision Writing – Review & Editing 4 Wanandi Septelia Inawati Conceptualization Data Curation Formal Analysis Methodology Project Administration Resources Supervision Validation Visualization Writing – Review & Editing https://orcid.org/0000-0002-7963-8853 a 5 Doctoral Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia Department of Histology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia Stem Cell Medical Technology Integrated Service Unit, Universitas Indonesia, Jakarta, Indonesia Department of Internal Medicine, Division Hemato-oncology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia Department of Biochemisty and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia septelia.inawati@ui.ac.id

No competing interests were disclosed.

 28 2 2018 2018 7 249 15 2 2018 Copyright: © 2018 Purnamawati P et al. 2018 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Nowadays, umbilical cord- and adipose-derived stem cells (UCSCs and ASCs) are the most common sources of mesenchymal stem cells (MSCs). As part of the tumor microenvironment, MSCs are known to communicate with cancer cells via their secretomes. Increased activity of aldehyde dehydrogenase-1 (ALDH1) has been widely used as a common intrinsic stemness marker in normal and cancer stem cells. Our study aimed to elaborate on the effect of UCSC and ASC secretomes on the expression of ALDH1A3, as one of the important variants of ALDH1, TGF-β1 and TGF-β receptor type I (TβRI) in human breast cancer stem cells (BCSCs).

Methods: UCSCs and ASCs were cultured in serum-free α-MEM media under standard conditions for 24 hours. The conditioned medium (CM) containing secretomes of UCSCs and ASCs were collected and added 50% (v/v) to the cultured of human BCSCs for 72 hours. The mRNA expressions of ALDH1A3, TGF-β1, and TβRI were determined using quantitative Reverse Transcriptase Polymerase Chain Reaction (q-RT-PCR).

Results: We found that CM of UCSCs significantly increased the ALDH1A3 expression of BCSCs in parallel with the increase of TGF-β1 and TβRI expressions. Conversely, CM of ASCs had no significant effect on the ALDH1A3 expression, but significantly decreased TGF-β1 and TβRI expressions of BCSCs. These results contradict our published data on ALDH1A1, which is another important variant of ALDH1, as well as data of the pluripotency markers OCT4 and SOX2 expressions.

Conclusions: UCSC and ASC secretomes have different regulation on ALDH1A3 expression in human BCSCs, which may be related to the autocrine TGF-β1 signaling in modulating cell proliferation and stemness of BCSCs. Further studies are required to evaluate factors involved in the differential effects of UCSC and ASC secretomes that regulate ALDH1A3 expression in relation to autocrine TGF-β1 signaling and aggressiveness of human BCSCs.

 UCSCs ASCs BCSCs ALDH1A3 ALDH1A1 TGF-β1 TβRI pluripotency Universitas Indonesia Publication of this work was supported by the grant of International Indexed Publication for Final Assignment of the Postgraduate Student from Universitas Indonesia Year 2017 (557/UN2.R3.1/HKP.05.00/2017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Mesenchymal stem cells (MSCs) are non-hematopoietic and adherent cells characterized by high CD90, CD105 and CD73, but lack of CD14, CD34 and CD45 expression. When treated with certain differentiation-stimulating factors, these cells will differentiate into adipocytes, chondrocytes, as well as osteocytes. MSCs show clinical feasibility, as studies on multiple animal models that yielded therapeutic efficacy gave rise to a series of clinical trials in a wide range of major diseases 1 . Umbilical cord and adipose tissue are two of the most common sources of MSCs, due to the fact that they can be non-invasively obtained and have minimal risk in terms of immunological and ethical issues. Apart from sharing the common properties of MSCs, umbilical cord-derived stem cells (UCSCs) are reported to have higher proliferation capability than adipose-derived stem cells (ASCs), whereas ASCs are able to differentiate to adipose tissue better than UCSCs 2 . Very recently, we have demonstrated that both ASCs and UCSCs expressed lower ALDH1A1 and OCT-4 than breast cancer stem cells (BCSCs), indicating that both types of MSCs have lower pluripotency compared to BCSCs (Wanandi SI, Purnamawati, Tamara A, Putri KT, Simadibrata D, in press).

Nowadays, MSCs are widely used for regenerative therapies due to their multipotent differentiation capacity. On top of that, MSCs are capable to secrete various paracrine signals for tissue regeneration and revascularization, such as chemoattractive, immunomodulatory, angiogenic, anti-apoptotic, and pro-survival factors. Nevertheless, MSC secretomes have also been reported to promote cancer progression and metastasis 3 . Accumulating evidence of cell-cell and paracrine interactions between MSC and cancer cells has indicated that MSCs can either induce or inhibit tumor progression 3– 9 . Secretomes contained in conditioned medium (CM) of MSCs consist of various biologically active factors that have the same effectiveness as MSCs themselves 10 . In addition, secretomes of MSCs are considered safer to use since they do not contain cellular elements, 11 making them free from possible mutations and transformation into cancer-associated fibroblast in a cancer microenvironment 12 .

Normal and cancer stem cells share similar properties, such as self-renewal capacity and differentiation potential into multiple cell types. Activity of aldehyde dehydrogenase (ALDH) superfamily have been widely used as a marker of viable normal stem cells, as well as cancer stem cells 13 . In breast cancer, the existence of ALDH+ BCSCs often correlates with poor prognosis, progression, chemoradiation resistance, and metastasis. Chemotherapy resistance is due to the role of ALDH as a detoxifying enzyme, which mediates detoxification of toxic aldehyde intermediates that are produced in certain chemotherapeutic agents-treated cancer cells, while radioresistance occurs through direct removal of oxygen radicals and indirect production of antioxidant compound nicotinamide adenine dinucleotide (phosphate) 14 .

ALDH1A1 and ALDH1A3, two of ALDH1 superfamily isozymes, have been known to play important roles in the modulation of the retinoic acid signaling pathway, which can either support or suppress cancer growth 14– 16 . In addition, they also serve as markers of stemness in CSCs 13, 17 . The expression of ALDH1A1 was associated with advanced, triple-negative, and poor prognosis breast cancer after neoadjuvant chemotherapy 18, 19 . ALDH1A1 expression was also found to be predictive of tumor responsiveness to cyclophosphamide treatment 20, 21 . Meanwhile, the isoform of ALDH1A3 has been suggested to promote breast cancer progression via retinoic acid signaling 22 .

TGF-β family members play a major regulatory role in various biological and physiological functions 23 . TGF-β physiologically exerts anticancer activities in normal and benign cells by prohibiting cell proliferation and by creating cell microenvironments that inhibit cell motility, invasion, and metastasis. However, along with the development and progression of tumors, various mutations or deletions occur in genes that encode various TGF-β signaling components. These lead to the loss of protective and cytostatic effects of TGF-β, which in turn alters TGF-β signaling to promote cancer progression, invasion, and tumor metastasis 23, 24 . Recently, the mechanism of the TGF-β paradox may have been explained through Erk activation, which will auto-induce TGF-β in the microenvironment 25 . Auto-induction of TGF-β in benign or early stage cancer cells will create negative feedback of TGF-β signaling leading to growth arrest, whereas progression and metastasis in malignant cancer cells will be induced via a positive feedback loop 25 .

Until now, the interaction between MSC secretomes and BCSCs has not been fully understood. Our recent study has indicated that BCSCs treated with secretomes of ASCs expressed ALDH1A1 significantly higher compared to those treated with UCSCs, in accordance to OCT-4 and SOX2 expressions 26 . Those results showed that secretomes of ASCs contribute to pluripotency and viability of BCSCs more than those of UCSCs. To gain a better understanding of the effects of MSC secretomes on BCSCs, we conducted the present study on ALDH1A3 expression in association with TGF-β1 signaling pathways in BCSCs.

 Methods Ethics and specimens

According to the Declaration of Helsinki 1964, this study has been approved by the Health Research Ethics Committee Faculty of Medicine Universitas Indonesia - Cipto Mangunkusumo Hospital (No. 205/UN2.F1/ETIK/2016).

MSC specimens consisting of three ASCs and three UCSCs samples were obtained from HayandraLab and Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital Faculty of Medicine Universitas Indonesia, Jakarta Indonesia, and have been characterized by the expression of stromal cell markers, i.e. CD73, CD90 positive and CD34 negative, as well as multidifferentiation capabilities to osteogenic, chondrogenic and adipocyte lineages as reported in our previous study 26, 27 .

BCSCs (ALDH+) were obtained from Cell Culture Laboratory for Cancer Stem Cells, Department Biochemistry and Molecular Biology Faculty of Medicine Universitas Indonesia, Jakarta Indonesia. The BCSC specimen has been isolated by ALDEFLUOR™ assay for subsequent downstream assessment (Fluorescence Activated Cell Sorting in Tsukuba University, 2015).

 Cell cultures

MSCs were grown in Minimum Essential Medium Alpha (α-MEM) supplemented with 10% FBS, while BCSCs were grown in non-serum DMEM/F12 medium. Both cell cultures were supplemented with 1% penicillin-streptomycin and 1% amphotericin. Cells were incubated under standard conditions (5% CO 2 and 37°C). The media were replaced every 3 days and cells were subcultured when confluence was obtained.

 Preparation of MSC-conditioned medium (MSC-CM)

Early-passage human MSCs (P3-P5) were grown to 70–80% confluence in α-MEM medium with 10% FBS. Culture medium was removed and the cells were washed three times by PBS 1x to remove any residual serum. The cells were then grown in non-serum α-MEM medium under standard conditions for 24 hours. MSC-CM was centrifuged at 200xg for 10 min to remove cell debris and filtered using 0.22 μm filters. Subsequently, CM was stored at -20°C and used within 3 days. To prepare the CM for each experiment, CM was diluted with DMEM/F12 to obtain a 50% (v/v) concentration.

 Incubation of BCSCs (ALDH+) with MSC-CM and viability assay

BCSCs (ALDH+) were harvested and counted using an automatic cell counter (Luna ®). About 5 x 10 5 cells with more than 90% viability were grown in non-serum DMEM/F12 medium. After 24 hours, the BCSC medium was replaced with 50% (v/v) MSCs-CM. After 72 hours of incubation, cells were harvested and counted using Tryphan blue exclusion dye assay. Cells that were grown in 50% (v/v) non-serum α-MEM medium were used as a control. The experiment was performed in triplicate.

 RNA isolation and qRT-PCR assay

Isolation of total RNA was performed according to the manufacturer’s instruction (TriPure Isolation Reagent ®, Roche). The RNA concentration was measured using a MicroDrop spectrophotometer (Thermo Scientific Skanlt Software for Varioskan™ Flash Multimode Reader). Quantitative reverse transcriptase PCR was performed using SYBR Green and reverse transcriptase enzyme (One-Step qRT-PCR Kit KAPA™SYBR®FAST). The cycling conditions were 5 minutes at 42°C for cDNA synthesis, 5 minutes at 95°C for inactivation of reverse transcriptase enzyme, then 40 cycles consisting of 30 seconds at 95°C for double stranded denaturation and 20 seconds at annealing gene temperature optimized for annealing stage, 20 seconds at 72°C for elongation stage. 18S rRNA was used as an internal control. The normalized fold expression was obtained using the 2 -ΔΔCT (Livak) method. Primers used for qRT-PCR were obtained from IDT ® ( Table 1).

Primer sequences used in the qRT-PCR.
Gene Primer Forward Primer Reverse Amplicon Length (bp)
ALDH1A3 5’-CGA CCT GGA GGG CTG TAT TA-3’ 5’-TGG TGA AGC ACA CGA CGT T-3’ 104
TGF-β1 5’-GCC TTT CCT GCT TCT CAT GG-3’ 5’-CTC CGT GGA GCT GAA GCA ATA-3’ 105
TβRI 5’- ACT TCC AAC TAC TGG CCC TTT-3’ 5’-AGA TGC AGA CGA AGC ACA CT-3’ 100
18S rRNA 5’- AAA CGG CTA CCA CAT CCA AG-3’ 5’-CCT CCA ATG GAT CCT CGT TA-3’ 155
Statistical analysis

All relative gene expression data were analyzed using unpaired Student’s t-test, SPSS 20 and presented as mean ± standard error.

 Results Morphologies of MSCs and BCSCs

BCSCs showed sphere formation within 2–3 days after plating ( Figure 1a). UCSCs and ASCs used in this study adhered within hours after plating and displayed spindle-fibroblast-like morphology ( Figure 1b–c). Both MSCs gradually fused into a single layer after cell confluence has been reached. Multidifferentiation capability of ASCs and UCSCs have been verified in our previous study 26, 27 .

Morphologies of BCSCs (a), UCSCs (b), and ASCs (c). About 1x10 5 cells were plated in each well of a 12-well plate and were grown under standard conditions as described under Materials and Methods. After 2–3 day incubation, cell morphology was observed under inverted microscope at 100x magnification. BCSCs, breast cancer stem cells; UCSCs, umbilical cord-derived stem cells; ASCs, adipose-derived stem cells; CM, conditioned medium.

http://dx.doi.org/10.5256/f1000research.13609.d194562

Expression of ALDH1A3 gene in BCSCs treated with CM of UCSCs and ASCs ( <xref ref-type="fig" rid="f2">Figure 2</xref>)

Treatment of UCSC-CM to BCSCs could significantly increase the relative expression of ALDH1A3 gene (1.79 times, p=0.001) compared with the control cells grown in 50% (v/v) non-serum α-MEM medium. In contrast, ASC-CM had no significant effect on ALDH1A3 gene expression in BCSCs (1.18 times, p=0.316) compared with the control cells. Nevertheless, the effect of ASC- was significantly lower than that of UCSC-CM (p=0.024) These results indicate that ALDH1A3 was distinctly expressed in BCSCs treated with UCSC- and ASC-CM.

Relative expression of ALDH1A3 mRNA in BCSCs. Human BCSCs were treated with 50% (v/v) UCSC-CM and ASC-CM, respectively. As a control, BCSCs were treated in 50% (v/v) non-serum α-MEM medium. After 72-hour incubation, total RNA was isolated and quantitative reverse transcriptase PCR was performed to determine ALDH1A3 mRNA expression levels in human BCSCs.The Cq obtained was normalized to 18S rRNA and control cells. Data is presented as mean ± SE. Significance differences are considered at *p<0.05; **p<0.01 (Student’s t-test). BCSCs, breast cancer stem cells; UCSCs, umbilical cord-derived stem cells; ASCs, adipose-derived stem cells; CM, conditioned medium.

http://dx.doi.org/10.5256/f1000research.13609.d194563

ALDH1A3 Cq was used to calculate ALDH1A3 mRNA expression levels using Livak formula as demonstrated in Figure 2 (Control: 50% (v/v) a-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells).

 Expression of TGF-β1 and TβRI genes in BCSCs treated with CM of UCSCs and ASCs ( <xref ref-type="fig" rid="f3">Figure 3</xref>)

In attempt to analyze the effect of MSC-CM on TGF-β signaling, we determined the relative mRNA expressions of TGF-β1 and its receptor, TβRI. UCSC-CM significantly increases the relative expression of TGF-β1 (1.72 times, p=0.003) and TβRI (1.54 times, p=0.000). In contrast to this data, ASCs-CM significantly decreases the expression of TGF-β1 (0.64, p=0.003) and TβRI (0.76, p=0.014) in BCSCs compared with controls. Additionally, UCSC- and ASC-CM showed differential effects on either TGF-β1 (p=0.000) or TβRI (p=0.000) expression of BCSCs, suggesting that UCSC and ASC secretomes may be different in content and levels of growth factors, thereby influencing differential regulation of TGF-β1 and TβRI expressions.

Relative expression of TGF-β1 (A) and TβRI (B) mRNA in BCSCs.

Human BCSCs were treated with 50% (v/v) UCSC-CM and ASC-CM, respectively. As a control, BCSCs were treated in 50% (v/v) non-serum α-MEM medium. After 72-hour incubation, total RNA was isolated and quantitative reverse transcriptase PCR was performed to determine TGF-β1 and TβRI mRNA expression levels in human BCSCs.The Cq obtained was normalized to 18S rRNA and control cells. Data is presented as mean ± SE). Significance differences are considered at *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). BCSCs, breast cancer stem cells; UCSCs, umbilical cord-derived stem cells; ASCs, adipose-derived stem cells; CM, conditioned medium.

http://dx.doi.org/10.5256/f1000research.13609.d194564

TGF-b1 Cq was used to calculate TGF-b1 mRNA expression levels using Livak formula as demonstrated in Figure 3A (Control: 50% (v/v) a-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells).

http://dx.doi.org/10.5256/f1000research.13609.d194565

TbRI Cq was used to calculate TbRI mRNA expression levels using Livak formula as demonstrated in Figure 3B (Control: 50% (v/v) a-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells).

http://dx.doi.org/10.5256/f1000research.13609.d194566

18S rRNA Cq was used to calculate ALDH1A3, TGF-b1, and TbRI mRNA expression levels using Livak formula (Control: 50% (v/v) a-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells).

 Discussion

Recently, the utilization of secretomes contained within MSC-CM has begun to be performed on various rejuvenation therapies. However, the possible consequences that may arise due to the interaction between biologically active factors (secretomes) and cancer cells has not yet been elucidated. As with MSCs, their secretomes have also been reported to either suppress or promote growth and development of cancer 3 . These contradictory effects may be due to different composition of secretomes, with one of the causes being the fact that they are obtained from different MSC sources 4, 28 .

In this study, we found that secretomes within UCSC-CM have a higher ability to promote ALDH1A3 gene expression in BCSCs, indicating the higher paracrine signaling activity of UCSC secretomes when compared to those of ASCs ( Figure 2). This result is in contrast with the effect of MSC secretomes on ALDH1A1 expression of BCSCs demonstrated in our previous study, which revealed that ALDH1A1 mRNA expression was significantly reduced by UCSC-CM and increased by ASC-CM 26 . In that study, we also found that ALDH1A1 expression is in line with the expression of OCT-4 and SOX2. Therefore, we suggest that unlike ALDH1A1, ALDH1A3 does not prominently contribute to the pluripotency of BCSCs. This has been verified by in silico analysis that showed that there is no direct interaction between ALDH1A3 and pluripotency markers, OCT4, SOX2, NANOG, and KLF4 (in press; Wanandi SI, Purnamawati, Tamara A, Putri KT, Simadibrata D). That study has also indicated that ALDH1A1 expression levels in MSCs were more similar to OCT4 rather than to ALDH1A3 levels, suggesting the role of ALDH1A1 on pluripotency.

In the present study, we highlighted that the effects of UCSC and ASC secretomes on ALDH1A3 were consistent with the expressions of TGF-β1 and its receptor, TβRI, in BCSCs ( Figure 3). In spite of that, the effects of UCSC on ALDH1A3, TGF-β1, and TβRI expressions were opposite to those of ASC secretomes, in which UCSC up-regulated, while ASC secretomes down-regulated those gene expressions. Previous studies have reported that different MSC sources have different growth factor contents and levels 4, 28 . Very recently, we have also demonstrated that UCSCs and ASCs expressed different levels of either ALDH1A1 or ALDH1A3 (in press; <Wanandi SI, Purnamawati, Tamara A, Putri KT, Simadibrata D>). These two isozymes of ALDH1 have newly been confirmed to have differential functional roles in facilitating aggressiveness of human breast cancer cells 29 . ALDH1A1 suppresses proliferation, metastatic properties and therapy resistance of breast cancer cells, whereas ALDH1A3 has predominant effect on ALDH activity. These results supported our previous study that presented the increase of viability and pluripotency in ASC secretomes-treated BCSCs in line with the increase of ALDH1A1 26 .

Moreover, the current study also underlines the effect of UCSC and ASC secretomes on TGF-β1 autocrine signaling in BCSCs, as revealed by TGF-β1 and its receptor, TβRI expressions ( Figures 3A and B). We suggest that secretomes of UCSCs and ASCs presumably contained growth factors including TGF-β1 that could auto-induce TGF-β1 signaling in BCSCs. Due to TGF-β1 paradox, tumor proliferation could be stimulated or inhibited either via differential ERK depending on relative level of TGF-β1 present in tumor microenvironment 25 . A plausible explanation of TGF-β1 autocrine signaling induction in our BCSCs is as a cellular homeostasis against reduced cell viability and pluripotency due to enhanced ALDH1A3 and diminished ALDH1A1 expression after supplementation with UCSC secretomes 26, 28 .

In conclusion, the differential effects of UCSC and ASC secretomes on ALDH1A3 expression in human BCSCs may be related to autocrine TGF-β1 signaling, as opposed to ALDH1A1 which regulates BCSC viability and pluripotency. In depth studies are further required to elaborate signaling factors within UCSC and ASC secretomes that specifically regulate ALDH1A1 and ALDH1A3 expressions in relation to TGF-β1 autocrine signaling and its impact on the aggressiveness of BCSCs.

 Data availability

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2018 Purnamawati P et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Dataset 1: Raw unedited images for Figure 1A, 1B, and 1C. DOI, 10.5256/f1000research.13609.d194562 30

Dataset 2: Data for Figure 2 (ALDH1A3 Cq value). ALDH1A3 Cq was used to calculate ALDH1A3 mRNA expression levels using Livak formula as demonstrated in Figure 2 (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d194563 31

Dataset 3: Data for Figure 3A (TGF-β1 Cq value). TGF-β1 Cq was used to calculate TGF-β1 mRNA expression levels using Livak formula as demonstrated in Figure 3A (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d194564 32

Dataset 4: Data for Figure 3B (TβRI Cq value). TβRICq was used to calculate TβRI mRNA expression levels using Livak formula as demonstrated in Figure 3B (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d194565 33

Dataset 5: Data for Figure 2, Figure 3A, and Figure 3B (18S rRNA Cq value). 18S rRNA Cq was used to calculate ALDH1A3, TGF-β1, and TβRI mRNA expression levels using Livak formula (Control: 50% (v/v) α-MEM-treated cells; UCSC-CM: conditioned medium of umbilical cord-derived stem cells; ASC-CM: conditioned medium of adipose-derived stem cells). DOI, 10.5256/f1000research.13609.d194566 34

Acknowledgements

We thank Dr. dr. Novi Silvia Hardiany, M.Biomed (Dept of Biochemistry and Molecular Biology FKUI) dr.Isabella Kurnia Liem, M.Biomed., PA., Ph.D (Cell Medical Technology Integrated Service Unit, RSCM-FKUI), dr. Karina, SpBP-RE (HayandraLab) and Dr. dr. Reza Y. Purwoko, SpKK (Erpour Laboratory) for their generosity in providing BCSCs (ALDH+), USCSc, and ASCs, respectively.

 Madrigal M Rao KS Riordan NH : A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J Transl Med. 2014;12(1):260. 25304688 10.1186/s12967-014-0260-8 4197270 Hu L Hu J Zhao J : Side-by-side comparison of the biological characteristics of human umbilical cord and adipose tissue-derived mesenchymal stem cells. Biomed Res Int. 2013;1–12. 23936800 10.1155/2013/438243 3722850 Zimmerlin L Park TS Zambidis ET : Mesenchymal stem cell secretome and regenerative therapy after cancer. Biochimie. 2013;95(12):2235–45. 23747841 10.1016/j.biochi.2013.05.010 3825748 Zhu W Huang L Li Y : Mesenchymal stem cell-secreted soluble signaling molecules potentiate tumor growth. Cell Cycle. 2011;10(18):3198–207. 21900753 10.4161/cc.10.18.17638 Ohta N Ishiguro S Kawabata A : Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes. PLoS One. 10(5):e0123756. 25942583 10.1371/journal.pone.0123756 4420498 Doi C Maurya DK Pyle MM : Cytotherapy with naive rat umbilical cord matrix stem cells significantly attenuates growth of murine pancreatic cancer cells and increases survival in syngeneic mice. Cytotherapy. 2010;12(3):408–17. 20345214 10.3109/14653240903548194 2896419 Rhee KJ Lee JI Eom YW : Mesenchymal Stem Cell-Mediated Effects of Tumor Support or Suppression. Int J Mol Sci. 2015;16(12):30015–33. 26694366 10.3390/ijms161226215 4691158 Kudo-Saito C : Cancer-associated mesenchymal stem cells aggravate tumor progression. Front Cell Dev Biol. 2015;3:23. 25883937 10.3389/fcell.2015.00023 4381695 Yagi H Kitagawa Y : The role of mesenchymal stem cells in cancer development. Front Genet. 2013;4:261. 24348516 10.3389/fgene.2013.00261 3842093 Pawitan JA : Prospect of stem cell conditioned medium in regenerative medicine. Biomed Res Int. 2014;2014(2014):1–14965849. 25530971 10.1155/2014/965849 4229962 Tran C Damaser MS : Stem cells as drug delivery methods: application of stem cell secretome for regeneration. Adv Drug Deliv Rev. 2015;82–83:1–11. 25451858 10.1016/j.addr.2014.10.007 4398586 Guan J Chen J : Mesenchymal stem cells in the tumor microenvironment. Biomed Rep. 2013;1(4):517–21. 24648978 10.3892/br.2013.103 3916973 Rodriguez-Torres M Allan AL : Aldehyde dehydrogenase as a marker and functional mediator of metastasis in solid tumors. Clin Exp Metastasis. 2016;33(1):97–113. 26445849 10.1007/s10585-015-9755-9 4740561 Shima H Yamada A Ishikawa T : Are breast cancer stem cells the key to resolving clinical issues in breast cancer therapy? Gland Surg. 2017;6(1):82–88. 28210556 10.21037/gs.2016.08.03 5293654 Bednarz-Knoll N Nastały P Żaczek A : Stromal expression of ALDH1 in human breast carcinomas indicates reduced tumor progression. Oncotarget. 2015;6(29):26789–803. 26305673 10.18632/oncotarget.4628 4694953 Tomita H Tanaka K Tanaka T : Aldehyde dehydrogenase 1A1 in stem cells and cancer. Oncotarget. 2016;7(10):11018–32. 26783961 10.18632/oncotarget.6920 4905455 Allahverdiyev AM Bagirova M Oztel ON : Aldehyde Dehydrogenase: Cancer and Stem Cells. InTech. [Internet] .2012. [cited 2017 Jul 29]; 10.5772/48591 Khoury T Ademuyiwa FO Chandraseekhar R : Aldehyde dehydrogenase 1A1 expression in breast cancer is associated with stage, triple negativity, and outcome to neoadjuvant chemotherapy. Mod Pathol. 2012;25(3):388–97. 22080062 10.1038/modpathol.2011.172 3426278 Zhao D Mo Y Li MT : NOTCH-induced aldehyde dehydrogenase 1A1 deacetylation promotes breast cancer stem cells. J Clin Invest. 2014;124(12):5453–65. 25384215 10.1172/JCI76611 4348941 Marcato P Dean CA Pan D : Aldehyde dehydrogenase activity of breast cancer stem cells is primarily due to isoform ALDH1A3 and its expression is predictive of metastasis. Stem Cells. 2011;29(1):32–45. 21280157 10.1002/stem.563 Raha D Wilson TR Peng J : The cancer stem cell marker aldehyde dehydrogenase is required to maintain a drug-tolerant tumor cell subpopulation. Cancer Res. 2014;74(13):3579–90. 24812274 10.1158/0008-5472.CAN-13-3456 Marcato P Dean CA Liu RZ : Aldehyde dehydrogenase 1A3 influences breast cancer progression via differential retinoic acid signaling. Mol Oncol. 2015;9(1):17–31. 25106087 10.1016/j.molonc.2014.07.010 5528683 Lebrun JJ : The Dual Role of TGFβ in Human Cancer: From Tumor Suppression to Cancer Metastasis. ISRN Mol Biol. 2012;2012: 381428. 27340590 10.5402/2012/381428 4899619 Principe DR Doll JA Bauer J : TGF-β: Duality of function between tumor prevention and carcinogenesis. J Natl Cancer Inst. 2014;106(2):djt369. 24511106 10.1093/jnci/djt369 3952197 Zhang Q Yu N Lee C : Mysteries of TGF-β Paradox in Benign and Malignant Cells. Front Oncol. 2014;4:94. 24860782 10.3389/fonc.2014.00094 4026682 Purnamawati P Pawitan JA Rachman A : Secretomes of Adipose and Umbilical Cord-Derived Stem Cells Affect ALDH1A1 Expression in Breast Cancer Stem Cells. Adv Sci Lett. 2017;23(7):6701–04. 10.1166/asl.2017.9376 Pawitan JA Kispa T Mediana D : Simple production method of umbilical cord derived mesenchymal stem cell using xeno-free materials for translational research. J Chem Pharm Res. 2015;7(8):652–6. Reference Source Heo JS Choi Y Kim HS : Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow, umbilical cord blood, placenta and adipose tissue. Int J Mol Med. 2016;37(1):115–25. 26719857 10.3892/ijmm.2015.2413 4687432 Croker AK Rodriguez-Torres M Xia Y : Differential Functional Roles of ALDH1A1 and ALDH1A3 in Mediating Metastatic Behavior and Therapy Resistance of Human Breast Cancer Cells. Int J Mol Sci. 2017;18(10): pii: E2039. 28937653 10.3390/ijms18102039 5666721 Purnamawati P Pawitan JA Rachman A : Dataset 1 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source Purnamawati P Pawitan JA Rachman A : Dataset 2 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source Purnamawati P Pawitan JA Rachman A : Dataset 3 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source Purnamawati P Pawitan JA Rachman A : Dataset 4 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source Purnamawati P Pawitan JA Rachman A : Dataset 5 in: Effects of umbilical cord- and adipose-derived stem cell secretomes on ALDH1A3 expression and autocrine TGF-β1 signaling in human breast cancer stem cells. F1000Research. 2018. Data Source 10.5256/f1000research.14783.r35664 Reviewer response for version 1 Marcato Paola 1 Referee Department of Microbiology and Immunology, Dalhousie University, Halifax, Canada

Competing interests: No competing interests were disclosed.

 17 7 2018 Copyright: © 2018 Marcato P 2018 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

This results presented by Purnamawati et al., are potentially interesting; however, since the key findings of the paper hinges on the fact that breast cancer stem cells were used in the assays it is difficult to interpret the findings (i.e. no details of the source of cells or proof they are cancer stem cells was provided).

Major concerns.

1. There is no details of the source of the cells, nor was the Aldefluor sorting of the cells shown.

2. Assay were not performed to confirm that cancer stem cells had been isolated (i.e. are these cells more tumorigenic than non-cancer stem cell counterparts?

3. Expression of ALDH1A3 should be compared in in the breast cancer stem cell versus non-cancer stem cell sorted populations to confirm that the Aldefluor+ cells have higher levels of ALHD1A3 than the Aldefluor- cells. 

4. The culture conditions used to form mammospheres were not appropriate (should seed much lower number of cells in ultra-low adherent plates so that no cells are adherent.) Standard media for mammosphere assays in Mammocult media from Stemcell Technologies. Hence it is unclear if these are mammospheres.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

No

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

No

Reviewer Expertise:

NA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

 10.5256/f1000research.14783.r32467 Reviewer response for version 1 Donnenberg Albert D. 1 2 3 Referee School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Competing interests: No competing interests were disclosed.

 29 5 2018 Copyright: © 2018 Donnenberg AD 2018 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

This study investigated the effects of media conditioned by umbilical cord (UC)-derived and adipose-derived stromal cells on "breast cancer stem cells."  The measured outcomes were aldehyde dehydrogenase 1A3 (ALDH1A3), TGF-β1 and TβR1 messenger RNA.  The methodology and data analysis are straight-forward and sound.  The major finding was that culture in the presence of umbilical cord-derived stromal cell conditioned medium resulted in higher expression of ALDH1A3, TGF-β1, and TβR1 mRNA, compared to control medium.  In contrast, adipose stromal cell conditioned medium resulted in slightly lower expression of TGF-β1, and TβR1 mRNA, but this was a modest effect.  The authors conclude that different secretomes of UC- and adipose-derived stromal cells differentially regulate ALDH1A3 expression in breast cancer "stem cells", a point that is well-supported by their results.  They further conclude that differential regulation of ALDH1A3 may be related to autocrine TGF-β1 signalling in modulating cell proliferation and stemness of breast cancer stem cells. This is speculative and based on a partial correlation of expression levels of the three measured mRNAs.  If anything, the data speak against a causal relationship, as adipose-conditioned medium failed to down-regulate ALDH1A3 message, but modestly downregulated TGF-β1, and TβR1 mRNA. To their credit, the authors point out that the present results are inconsistent with their previous published data on ALDH1A1, OCT4 and SOX2 expression. 

These results speak to the difficulty of modeling the interactions of stromal cells and cancer cells. Using freshly isolated human breast cancer cells, flow-sorted on the basis light scatter and expression of CD90, we found that co-injection of adipose stromal cells in a xenograft model increased tumorgenicity of active, but not resting tumor cells. In vitro, coculture of unpassaged breast cancer cells with adipose stromal cells greatly increased their proliferation 1 .  The take-home is that the state of the cancer cells matters as much as the the signals provided by the stromal cells and microenvironment.  To complicate matters further, stromal cells themselves can be polarized into pro- and anti-inflammatory states 2 resulting in different secretomes. ​​​​

At a minimum, the authors should provide detailed information about the preparation of their "breast cancer stem cells", as these are not standard reagents.

Additionally, UC- and adipose-conditioned media have not been characterized for cytokine/chemokine content.  This is relatively simple to do, as multiplexed kits are commercially available and would significantly contribute to a more mechanistic understanding of the data.

A minor consideration: wording such as "nowadays" and "on top of that" are rather colloquial for a scientific publication.​​​

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Cancer cell biology, regenerative medicine, flow cytometry, cGMP cell therapy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References : Regenerative therapy and cancer: in vitro and in vivo studies of the interaction between adipose-derived stem cells and breast cancer cells from clinical isolates. Tissue Eng Part A .2011;17(1-2) : 10.1089/ten.TEA.2010.0248 93-106 20673000 10.1089/ten.TEA.2010.0248 : A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype. PLoS One .2010;5(4) : 10.1371/journal.pone.0010088 e10088 20436665 10.1371/journal.pone.0010088