Proteomics and image screening data of cellular secretomes and their biological effects: Comparing the signals sent by cardiac stromal cells and dermal fibroblasts in culture

The study of the secretome of different cell types has gained prominence over the years due to its role in understanding the cell microenvironment and possible uses in acellular therapies. Approaches in this field include proteomic characterizations of the secretomes as well as evaluating their potential to induce cell and tissue responses. Here, we present the mass spectrometry proteomics data from a characterization of the secretome of cardiac resident stromal cells (CRSCs) and dermal fibroblasts in order to compare their compositions. To evaluate the potential for cell proliferation, differentiation, migration, and adhesion, in vitro assays were performed and analyzed using a high-content imaging system. For each assay, specific analysis strategies were developed to quantify the generated data. These datasets provide insights into the differences and similarities between secretomes from different cell sources. It also describes methodologies for analyzing images from different in vitro assays using high-throughput automated imaging.


a b s t r a c t
The study of the secretome of different cell types has gained prominence over the years due to its role in understanding the cell microenvironment and possible uses in acellular therapies. Approaches in this field include proteomic characterizations of the secretomes as well as evaluating their potential to induce cell and tissue responses. Here, we present the mass spectrometry proteomics data from a characterization of the secretome of cardiac resident stromal cells (CRSCs) and dermal fibroblasts in order to compare their compositions. To evaluate the potential for cell proliferation, differentiation, migration, and adhesion, in vitro assays were performed and analyzed using a high-content imaging system. For each assay, specific analysis strategies were developed to quantify the generated data. These datasets provide insights into the differences and similarities between secretomes from different cell sources. It also describes methodologies for analyzing images from different in vitro assays using high-throughput automated imaging.  Table   Subject Biological sciences Specific subject area Cell niche, Cell biology and differentiation, Proteomic characterization. Type of data Table, Figure  How

Value of the Data
• The data provide a proteomics characterization of human ventricle-derived cardiac resident stromal cells (vCRSCs) and dermal fibroblast secretomes, as well as an evaluation of their ability to influence cellular behaviors, which can be useful in identifying the best source of cells for soluble factors to be used in potential future acellular therapies. • The data can be useful for other groups working on secretome characterization. In addition, the strategy of analyzing data obtained from a high-content screening system is a tool that can be used in a wide variety of studies. • Further analysis and additional assays can be performed, allowing comparison with other secretomes, both in terms of protein composition and functionality. • A detailed description of the analysis method for images obtained from a high-content screening system can be used for efficiently evaluating cardiac differentiation, cell proliferation, and migration.

Data Description
The dataset presented here contains the raw proteomic data from conditioned medium (CM) obtained from human ventricle-derived cardiac resident stromal cells (vCRSC) from three donors as well as from normal neonatal human dermal fibroblasts (NHDF-neo) from three independent cell cultures [ 1 , 2 ]. In addition, we showed the analysis of high-content screening system data obtained from cell proliferation, differentiation, adhesion, and migration assays. The schematic design of experiments is depicted in Fig. 1 .
The proteomic raw data of the vCRSC-derived CM from donor 1 (vCM1), donor 2 (vCM2), and donor 3 (vCM3), the CM derived from dermal fibroblasts (fCM1, 2, and 3) and the nonconditioned medium (nCM1 and 2) as well as the proteomic analysis from MaxQuant are available in PRIDE repository under accession number PXD026451 [3] . More details can be found in Table 1 .  We evaluated CMs in vitro potential to interfere with cell processes: we performed cell proliferation, differentiation, adhesion, and migration assays in H9c2 rat cardiomyoblasts and human umbilical vein endothelial cells (HUVEC). The analysis of the cell phenotypes was addressed by the Operetta CLS TM high-content analysis system and the Harmony software (PerkinElmer).
Proliferation and cardiac differentiation assays were performed with H9c2 cells that were kept in the CMs for seven and 15 days. Cellular proliferation was quantified based on the Ki67 staining presented in the nuclei of cells. Cardiac differentiation was confirmed by staining cells with the anti-cardiac troponin I (cTnI) antibody and quantifying the stained area. Using an in vitro wound healing assay, H9c2 cells and HUVEC were treated with CMs and monitored over 24 h, with images acquired every 6 h, to evaluate the change of the open area. Finally, the cell adhesion capacity was verified at early H9c2 and HUVEC culture times: 10, 20, and 40 min after plating with CMs.
The data generated after image analysis with Harmony v.4.5 or 4.8 software (PerkinElmer), as well as the images of the analyzed plates (one for each time and for each experiment) and a file indicating the treatments/ well and the wells that were excluded from the analysis are available at Mendeley Data Repository (doi: 10.17632/72vcr9c6rk.1 ) [4] . These data are separated into folders, one for each assay. Table 2 highlights information about the in vitro functional assays, as the controls used, and presents a brief description of files deposited in Mendeley Data Repository. More details of the exclusion criteria and the analysis methodology in Harmony software (PerkinElmer) were described in the Experimental Design, Materials and Methods section.
Upon reaching 80% confluence, the vCRSC and HDF cultures were washed three times with 1x phosphate buffer saline (PBS), following which Dulbecco's MegaCell ® supplemented only with 0.1 mM BME, 1% NEAA, 2 mM L -glutamine, and 100 IU/mL penicillin, and 0.1 mg/mL streptomycin was added to culture flasks. This medium remained in contact with the cells for 16-20 h (overnight). Subsequently, the CM was collected, and the cells were again incubated with complete Dulbecco's MegaCell ® medium (with FBS and bFGF) for 6-8 h. The process of PBS washing and adding medium without FBS and bFGF was then repeated. This was done for a total of three days (three consecutive CM collections). On each day, the collected medium was centrifuged: first at 1620 xg for 5 min and then at 40 0 0 xg for 20 min, both at 4-8 °C. The CM of each of the three days was pooled and then stored at −80 °C until use. This was done for each of the three vCRSC donors and HDF replicates. In parallel, the non-conditioned medium (nCM) was also stored (Dulbecco's MegaCell ® supplemented only with 0.1 mM BME, 1% NEAA, 2 mM L -glutamine, and 100 IU/mL penicillin, 0.1 mg/mL streptomycin). Fig. 2 summarizes this process.

LC-MS/MS
Thirty micrograms of protein from each sample were mixed with 4x SDS-PAGE sample buffer (160 mM Tris-HCl pH 6.8, 4% SDS, 10% b-mercaptoethanol, 24% glycerol, and 0.02% bromophenol blue) to final buffer concentration of 1x and were resolved in 10% SDS-PAGE. Then, SDS-PAGE lanes were sliced and underwent in gel trypsin digestion. The samples were analyzed at the mass spectrometry facility RPT02H/Carlos Chagas Institute -Fiocruz Paraná. The peptides were analyzed in triplicate (for vCM3 and nCM1 samples) and in duplicate (for other samples) by LC-MS/MS in an Easy-nLC 10 0 0 online with a LTQ Orbitrap XL ETD (Thermo Scientific). The chromatography was performed in a C18 column (30 cm length, 75 μm I.D., 1.9 μm particle) with a flow of 250 nL/min and a linear gradient of 5-40% acetonitrile in 0.1% formic acid and 5% DMSO for 2 h. The MS data were acquired in DDA mode, with the MS1 full scan performed in the orbitrap (60,0 0 0 resolution) and the MS2 in the linear trap quadrupole, where the top 10 most intense ions were subjected to CID fragmentation. Three consecutive run batches, spaced in time, were carried out using the same experimental protocol and equipment, according to the following scheme: vCM3 and nCM1 in parallel; vCM1, vCM2, fCM1, and nCM2 in parallel; fCM2 and fCM3 in parallel.

Data analysis
The raw data from LC-MS/MS (from all the samples) were analyzed in MaxQuant software, version 1.6.1.0 [ 6 , 7 ]. The default parameters were used, including trypsin as protease, car- Table 4 Experimental design for the in vitro assays and configuration for image acquisition in Operetta CLS TM high-content analysis system (Perkin Elmer).

Assay
Wound healing Adhesion Proliferation Differentiation

Immunofluorescence
The cell cultures were washed once with PBS and then fixed with 4% paraformaldehyde. Cells were subsequently permeabilized with 0.5% Triton X-100 for 30 min, blocked with 1% bovine serum albumin (PBS/BSA 1%) for 1 h, and incubated with primary antibody (specific for each assay) for 1 h at room temperature or overnight at 4 °C. Next, cells were washed with PBS, incubated with Alexa Fluor ® 488 Goat Anti-Rabbit (IgG) secondary antibody for 1 h and, after PBS washes, 4 ,6-diamidino-2-phenylindole (DAPI) was added for 10 min to stain the nuclei.
Image acquisition was performed in Operetta CLS TM high-content analysis system (PerkinElmer). Image analysis was carried out with the Harmony software (PerkinElmer). The images from all the functional assays were obtained with a 20x Air, NA 0.4 objective, in nonconfocal mode, binning = 2, with laser power ranging from 20% (Brightfield) to 50-75% (fluorescent channels), with exposure time and focus adjusted for each assay. Table 4 summarizes the experiment design and the image channels used for imaging acquisition for each assay.

Proliferation and cardiac differentiation analysis
For the proliferation and cardiac differentiation assays, H9c2 cells were cultured with DMEM 10%FBS, DMEM 1%FBS as controls, as well as with vCM1, vCM2, vCM3, fCM, or nCM for seven and 15 days, following which the cells were fixed and immunostained.
For the proliferation assay, 1.2 × 10 4 cells were plated in each well of 24-well plates. The treatments started 24 h after cell plating. The proliferating cells were identified by immunostaining for nuclear protein Ki67 using an anti-Ki67 antibody (Abcam, cat.: ab15580, dilution 1:300) and the Alexa Fluor ® 488 Goat Anti-Rabbit (IgG) secondary antibody in association with nuclear counterstain DAPI. Forty-nine photos were acquired in each well and analyzed individually. Fig. 3 details the image analysis sequence used to quantify the total number of cells and the number of Ki67 + cells in the cultures treated with CM and nCM. Briefly, DAPI staining was used to distinguish the nuclei from cells. Next, using the images obtained from the Alexa 488 channel, we determined the number of cells that had nuclear staining for Ki67 (Ki67 + cells). The analy-   sis from [1] , depicted in Fig. 2 , was generated based on a quantification of the total number of nuclei and the Ki67 + nuclei from 23 out of 49 photos.
To assess cardiac differentiation, 2.5 × 10 3 cells were plated in each well in 96-well plates and with treatments starting after 24 h. The cardiomyocytes presented in the H9c2 cell culture after CM treatment were characterized using the anti-cardiac troponin I antibody (Santa Cruz, cat.: sc-15368, dilution 1:100) and the Alexa Fluor ® 488 Goat Anti-Rabbit secondary antibody. Thirty-six photos were acquired in each well and, using Harmony 4.8 software, they were combined to form only one image for each well (global image). Fig. 4 describes the image analysis procedure for the images generated by the Operetta CLS TM high-content analysis system (PerkinElmer) to determine cardiac differentiation efficiency. Briefly, we trained the Harmony 4.8 software to identify the texture of differentiated cells (Alexa 488/ cTnI + ) from non-differentiated ones (Alexa 488/ cTnI − ). After that, the stained area (cTnI + ) and total nuclei were quantified. The wells that had problems at some stage of immunofluorescence or in the reading on Operetta CLS TM high-content analysis system (PerkinElmer) were excluded from the analysis.

Adhesion and migration analysis
A description of cell plating density and culture times can be found in [1] . In the adhesion assay, after DAPI staining, 49 photos were taken in Operetta CLS TM high-content analysis system (PerkinElmer) for each well and time. The analysis was performed in Harmony 4.5 software (PerkinElmer) and consisted of the identification and quantification of nuclei. The treatments performed for HUVEC cultures were: vCM1, vCM2, vCM3, fCM, nCM, EBM2 complete medium (positive control) and EBM2 without supplements (negative control). The treatments performed for H9c2 cell cultures were: vCM1, vCM2, vCM3, fCM, nCM, DMEM 10% (positive control) and DMEM 1% (negative control).
For wound healing assay, a time-lapse analysis was carried out with Operetta CLS TM highcontent analysis system (PerkinElmer). Before starting the assay, the equipment was set for the standard cell culture condition: 37 °C and 5% CO 2 . The wells were photographed every 6 h over a 24 h period. To cover the entire scratch, 30 photos were acquired with a 20x objective and were combined to form a single image from the whole well (global image) ( Fig. 5 ). Then, using Harmony 4.8 software (PerkinElmer), we measured the open area each time ( Fig. 5 ). Wells that did not exhibit valid values (NaN) at any of the intervals were excluded from the analysis, as were wells in which the initial scratch (time 0 h) was improperly done or outside of the image, or where the percentage reduction in area was negative at more than one point in time. The treatments performed for HUVEC cultures were: vCM1, vCM2, vCM3, fCM, nCM, and EBM2 complete medium (control). The treatments performed for H9c2 cell cultures were: vCM1, vCM2, vCM3, fCM, nCM, and DMEM 10%FBS (control).

Ethics Statements
This study was conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). The research was approved by the ethics committee of the Oswaldo Cruz Foundation (CAAE number: 48374715.8.0 0 0 0.5248 ).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.