Graphical abstract

   graphic file with name fx1.jpg
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Highlights

     * •
       Simple Ewing sarcoma (EwS) cell culture method with increased
       physiological relevance
     * •
       Minimal necessary fetal calf serum supply for EwS cell line
       proliferation
     * •
       Improved transcriptional reflection of proliferation, hypoxia, and
       differentiation
     * •
       EWSR1::ETS transcriptional activity in refined versus standard EwS
       culture condition

Motivation

   Cell culture remains the main platform for modeling Ewing sarcoma (EwS)
   for research purposes. Yet, concerns exist about the limitations of
   standard in vitro techniques to adequately reflect physiological
   conditions. In this study, we refined EwS cell culture conditions to
   increase modeling capacity while ensuring practical and cost-effective
   handling, thereby broadening their applicability within the scientific
   community.
     __________________________________________________________________

   Ceranski et al. report on a refined Ewing sarcoma cell culture method
   with increased physiological relevance that is technically simple and
   cost efficient. The enhanced in vitro modeling has broad applicability
   for improving the validity of experimental results.

Introduction

   Ewing sarcoma (EwS) is an aggressive pediatric bone and soft tissue
   cancer that is genetically characterized by FET::ETS (members of the
   FUS/EWSR1/TAF15 family of genes fused to an E26 transformation specific
   [ETS] transcription factor) fusion oncogenes encoding aberrant
   transcription factors, mostly EWSR1::FLI1.[50]^1 The clinical outcome
   of many patients, especially that of patients with metastatic disease
   at diagnosis or relapse, remains limited.[51]^1^,[52]^2 In fact,
   patients with metastasis or recurrence typically have <30% overall
   survival rates.[53]^1^,[54]^2 There is consensus in the scientific
   community that preclinical EwS disease models with enhanced
   physiological relevance are needed, which may, in turn, lead to the
   identification of more effective therapies.[55]^3^,[56]^4^,[57]^5 The
   most common preclinic EwS tumor model involves the culture of EwS cell
   lines in standard techniques, employing a two-dimensional (2D)
   monolayer setup and traditional cell culture media.[58]^6 However, 2D
   monolayer culture does not represent the architectural and mechanical
   aspects of the EwS physiological tumor niche and has been identified to
   limit the translatability of preclinical research advances into
   improved therapies for patients with EwS.[59]^7^,[60]^8 Consequently,
   various 3D cell culture techniques, advanced scaffold modeling of the
   bone extracellular matrix (ECM), flow perfusion techniques, and
   microfluidic systems have been introduced to improve EwS model
   accuracy.[61]^3^,[62]^4^,[63]^5^,[64]^8 Other approaches for more
   accurate preclinic EwS models include the genetic engineering of EwS
   cells from mesenchymal stem cells,[65]^9 the use of various types of
   xenografted animal models,[66]^10^,[67]^11 and the attempt to create
   genetically engineered mouse models of EwS, which has not been
   successful until today.[68]^12 Yet, from a practical standpoint, the
   complexity of any experimental technique will largely determine its
   experimental throughput, cost effectiveness, broad applicability, and
   efforts needed for data acquisition.[69]^4^,[70]^13 Besides the
   improvements addressing the architectural aspects of the standard EwS
   cell culture technique, evidence suggests that physiological cell
   culture media, such as human plasma-like medium (HPLM)[71]^14 and
   Plasmax,[72]^15 are essential to enhance the fidelity of in vitro
   cultures.[73]^6^,[74]^16^,[75]^17^,[76]^18 Yet, physiological cell
   culture media[77]^14^,[78]^15 have not been implemented for the culture
   of EwS cell lines until now. Furthermore, although it is well known
   that additives such as fetal calf serum (FCS) introduce nutrients,
   growth factors, and hormones at unknown concentrations to the culture
   medium,[79]^6^,[80]^18^,[81]^19^,[82]^20 they are typically applied in
   high and empiric concentrations, which can compromise the
   reproducibility of experiments. In fact, little is known about whether
   EwS cell culture could also be feasible at reduced FCS concentrations
   that would better comply with the 3R principles (reduction, refinement,
   and replacement), thereby contributing to the promotion of animal
   welfare in the experimental space.[83]^19^,[84]^20

   In this paper, we present a refined EwS cell culture condition with
   increased physiological relevance that is yet simple, inexpensive, and
   amenable to large-scale use. We chose a spheroid approach[85]^21 and
   applied a physiologic medium based on the formulations of HPLM[86]^14
   and Plasmax,[87]^15 which mimic nutrient concentrations observed in the
   human plasma, as well as carefully adapted reduced FCS concentrations
   to increase the modeling relevancy of the EwS cell culture. Using
   transcriptome profiling, we show that this refined culture condition
   better mimics the transcriptional activity related to proliferation,
   hypoxia, and differentiation in EwS patient tumors. Moreover, we
   demonstrate that the refined culture condition has a pervasive effect
   on the baseline transcriptomes of EwS cells and significantly impacts
   the transcriptional outputs of EWSR1::ETS fusion oncogenes.

Results

Establishing a refined culture condition for EwS cell lines with increased
physiological relevance

   To improve the physiological relevance of the EwS standard culture
   condition, we addressed three technical aspects in the refined EwS
   culture condition: medium composition, FCS concentration, and growth
   architecture ([88]Figure 1A). In the first step, two established
   physiological media, HPLM[89]^14 and Plasmax,[90]^15 were combined to
   generate a physiological medium, hereafter called HPLMax
   ([91]Figure 1B; [92]Table S1; see [93]STAR Methods). EwS cell lines
   were considered adapted after 4 weeks of culturing in HPLMax and were
   then phenotypically characterized. In a second step, to evaluate the
   minimal necessary FCS concentration that did not compromise EwS cell
   viability, we performed a resazurin proliferation assay for five
   selected EwS cell lines cultured in Roswell Park Memorial Institute
   (RPMI) 1640 medium with FCS concentrations ranging from 10% (standard)
   to 1%. The human EwS cell lines A-673, MHH-ES-1, SK-N-MC, TC-71 (all
   EWSR1::FLI1 positive), and TC-106 (EWSR1::ERG positive) were selected
   for study since they are widely used in EwS research and reflect the
   two major EWSR1::ETS fusions found in ∼95% of patients.[94]^1 Of note,
   all cell lines were previously engineered to harbor doxycycline
   (dox)-inducible short hairpin RNAs (shRNAs) to target the respective
   FET::ETS fusions.[95]^22 Moreover, all cell line models were
   characterized in depth beforehand via multi-omics analyses,[96]^22 and
   information on their status concerning frequent EwS secondary
   mutations[97]^1^,[98]^23^,[99]^24 is given in [100]Figure S1A.
   Interestingly, for all five tested EwS cell lines, FCS supplementation
   could be reduced to 7% without significantly decreasing their
   proliferation ([101]Figures 1C and [102]S1B). In fact, 7% FCS
   supplementation resulted in even slightly accelerated proliferation for
   some cell lines (SK-N-MC/TR/shEF1 and TC-71/TR/shEF1; see also
   [103]Figure S1B). While we have chosen 7% FCS because this was the
   concentration under which we did not detect a significantly reduced
   proliferation for all cell lines tested ([104]Figure 1C), it should be
   noted that some EwS cell lines continued to proliferate even under way
   lower FCS concentrations, such as SK-N-MC/TR/shEF1 cells, which did not
   display a significantly reduced signal in the resazurin assay as
   compared to 10% FCS even in 2% FCS ([105]Figure 1B). The 7% FCS
   optimized condition was further validated by manual cell counting using
   standardized hemocytometers and trypan blue exclusion methods in both
   RPMI 1640 medium and the physiological HPLMax medium: all EwS cell
   lines demonstrated no significant change of their doubling time in
   either medium when changing the FCS concentration from 10% to 7%
   ([106]Figure 1D). Moreover, manual cell counting demonstrated no
   notable change in EwS cell viability with 10% versus 7% FCS
   supplementation in either medium ([107]Figure 1E). We concluded that a
   7% FCS supplementation is optimal for these EwS cell lines to provide
   the required growth factors while allowing a 30% reduction of this
   animal product and its entailed drawbacks for
   experimentation.[108]^6^,[109]^18^,[110]^19^,[111]^20 Although
   proliferation rates of all EwS cell lines remained stable within both
   media (RPMI 1640 medium and HPLMax) despite the 30% decrease in FCS
   supplementation ([112]Figure 1D), we did observe a slower proliferation
   in the five EwS cell lines when comparing the doubling time in HPLMax
   to RPMI 1640 medium under an equal medium exchange frequency
   ([113]Figure 1D). This result is consistent with the fact that
   conventional media were designed to maximize the growth rates of
   cultured cells[114]^14^,[115]^15 and similar results have been
   previously reported for the proliferation of hematological and
   epithelial cancer cell lines in HPLM.[116]^14^,[117]^25 Concerning the
   viability of the five EwS cell lines, similar dead cell counts were
   found in RPMI 1640 medium and HPLMax ([118]Figure 1E). Regarding the
   EwS cell line morphology, no striking changes were observed in vitro
   between culture in RPMI 1640 medium and HPLMax ([119]Figures 1F and
   [120]S1C). Lastly, the traditional 2D monolayer culture was replaced
   with a spheroid approach ([121]Figure 1G) by adjusting a published
   protocol for agar-coated, low-attachment spheroid culture[122]^21 (see
   [123]STAR Methods). This allows for optimized mimicking of the tumor
   architecture ([124]Figure 1G), cell-cell interactions, and spatial
   gradients of oxygen[125]^26 and nutrients,[126]^4 which are all
   essential components of the tumor microenvironment.[127]^4^,[128]^27
   The architectural fidelity of the EwS spheroids cultured in the refined
   condition could be demonstrated by histological comparison with an
   in vivo EwS xenograft ([129]Figure 1G).

Figure 1.

   [130]Figure 1
   [131]Open in a new tab

   Establishing a refined culture condition for EwS cell lines with
   increased physiological relevance

   (A) Overview on the EwS in vitro standard and refined techniques.

   (B) Workflow to create HPLMax (see also [132]Table S1).

   (C) Proliferation assessment by resazurin cell viability assays in EwS
   cell lines in RPMI 1640 medium with FCS supplementation from 10% to 1%.
   Values were normalized to the proliferation in 10% FCS. Pooled data of
   five representative cell lines are shown, with each dot representing n
   ≥ 5 biologically independent replicates of one cell line. Vertical bars
   represent means and whiskers the standard error of the mean (SEM).
   ∗∗p < 0.01 and ∗∗∗p < 0.001 (see also [133]Figure S1B).

   (D) Doubling time of five EwS cell lines in RPMI 1640 medium and 10%
   FCS (left bar), RPMI 1640 medium and 7% FCS (second bar from left),
   HPLMax and 10% FCS (second bar from right), and HPLMax and 7% FCS
   (right bar) assessed by trypan blue counting of viable cells. Each dot
   represents a single biologically independent experiment. Vertical bars
   represent means and whiskers the SEM. One-way ANOVA with Tukey’s
   multiple comparisons test; ∗∗p < 0.01 and ∗∗∗∗p < 0.0001; n.s.,
   non-significant.

   (E) Dead cell counts using trypan blue exclusion method in RPMI 1640
   medium and 10% FCS (left bar), RPMI 1640 medium and 7% FCS (second bar
   from left), HPLMax and 10% FCS (second bar from right), and HPLMax and
   7% FCS (right bar). Values are the percentage of the total cell count
   (viable and dead cells) in the respective sample. Each dot represents a
   single biologically independent experiment. Vertical bars represent
   means and whiskers the SEM. One-way ANOVA with Tukey’s multiple
   comparisons test; n.s., non-significant.

   (F) Bright-field microscopy images of TC-71/TR/shEF1 cell lines grown
   in RPMI 1640 medium and 10% FCS (left) and in HPLMax and 7% FCS
   (right). Scale bars: 200 μm, 10× magnification (see also
   [134]Figure S1).

   (G) TC-71/TR/shEF1 spheroids grown in agar-coated T25 flasks with
   HPLMax and 7% FCS for 8 days. Light microscopy image (left), scale bar:
   125 μm, in 4× magnification. Hematoxylin and eosin (H&E) staining of
   TC-71/TR/shEF1 spheroids grown as described above in 20× magnification,
   scale bar: 100 μm (middle), and 40× magnification (right), scale bar:
   100 μm.

   (H) H&E staining of mice in vivo xenografts of TC-71 wild-type cells in
   20× magnification, scale bar: 100 μm (left), and 40× magnification
   (right), scale bar: 100 μm.

The refined EwS culture condition preserves cell line characteristics but
alters transcriptional programs related to proliferation, differentiation,
and metabolism

   To further characterize the EwS cells in the refined culture condition
   and validate this setup, we performed DNA microarray gene expression
   analyses in four EwS cell lines (MHH-ES-1/TR/shEF1, SK-N-MC/TR/shEF1,
   TC-71/TR/shEF1, and TC-106/TR/shEERG) that enable a dox-inducible and
   shRNA-mediated knockdown of either EWSR1::FLI1 or EWSR1::ERG,
   respectively.[135]^22 An experimental overview is given in
   [136]Figure 2A. Briefly, the Ewing Sarcoma Cell Line Atlas (ESCLA)
   transcriptome experiment from our laboratory in standard culture
   conditions[137]^22 was recapitulated in the refined cell culture
   with/without the addition of dox for 96 h. Gene expression data were
   generated on the same platform (human Affymetrix Clariom D arrays),
   jointly preprocessed, and normalized with the former data[138]^22 to
   generate a harmonized dataset for a direct comparison of refined and
   standard culture conditions. Exploratory data analysis using
   principal-component analysis (PCA) demonstrated that the captured
   variation between all samples was mainly driven by three aspects
   ([139]Figures 2B, [140]S2A, and S2B): the employed culture condition
   (PC1), the specific characteristics of the individual cell lines
   (PC2–PC4), and the dox-dependent EWSR1::ETS expression levels (PC5).
   This illustrates that the applied culture technique fundamentally
   affects transcriptional programs of EwS cell lines, yet their principal
   characteristics are preserved in the refined setup. Next, to further
   explore the effects of the culture conditions on baseline RNA
   transcription, we focused on samples without EWSR1::ETS knockdown (no
   dox). Pre-ranked fast gene set enrichment analysis (fGSEA) with
   differentially expressed genes (DEGs) between the refined and standard
   culture conditions followed by weighted gene correlation network
   analysis (WGCNA) uncovered reduced cell cycle- and
   proliferation-associated signatures as well as enrichment of ribosomal,
   translational, and spliceosomal processes in the physiologically
   refined condition ([141]Figures 2C and 2D; [142]Table S2). Furthermore,
   we observed a strong dysregulation of canonical hallmark and generic
   gene signatures involved in the regulation of differentiation
   (epithelial-to-mesenchymal transition [EMT]), hypoxia, and metabolism
   in the refined setup ([143]Figure 2D). These data are in keeping with
   the results from the functional in vitro proliferation assays
   ([144]Figure 1G) and the oxygen gradients that are a key feature of
   spheroid cultures.[145]^4^,[146]^26 Altogether, these results indicate
   that the refined culture condition does not alter principal EwS cell
   line characteristics but impacts transcriptional programs related to
   proliferation, differentiation, metabolism, and hypoxia.

Figure 2.

   [147]Figure 2
   [148]Open in a new tab

   The refined EwS culture condition preserves cell line characteristics
   but alters transcriptional programs related to proliferation,
   differentiation, metabolism, and hypoxia

   (A) Experimental workflow to generate transcriptome datasets for EwS
   cell lines in refined culture conditions (pink and orange). The same
   experiment was previously performed for the Ewing Sarcoma Cell Line
   Atlas (ESCLA) in standard culture conditions by Orth et al.[149]^22
   (blue and light blue). Data were analyzed via joint processing (see
   [150]STAR Methods).

   (B) Principal-component analysis (PCA) of the normalized data from
   (A) revealing captured variation driven by culture condition (PC1) and
   cell line characteristics (PC2). For captured variation by PC3–PC5, see
   [151]Figures S2A and S2B.

   (C) Weighted correlation network analysis (WGCNA) based on pre-ranked
   fast gene set enrichment analysis (fGSEA) of differentially expressed
   genes (DEGs) from (A) using the average rank of expression fold change
   (FC). Displayed network analyses were based on significant gene sets
   with a maximum false discovery rate (|FDR|) of 0.05 and a minimal
   normalized enrichment score (|NES|) of 2. See also [152]Table S2.

   (D) Enrichment plots from fGSEA of DEGs in refined versus standard
   condition from (A). padj, Bonferroni-adjusted p value. C2 and hallmark
   gene sets from the Human MSigDB Collections. See also [153]Table S2.

   (E) Pairwise correlation plot based on PCA of gene expression profiles
   from (A) (n = 12 in each condition) and EwS primary tumors (n =
   50)[154]^28 after preprocessing, normalization, and batch correction
   for both datasets. See also [155]Figure S2C.

   (F) Heatmaps depicting NESs of single-sample GSEA (ssGSEA) with gene
   expression profiles from (A) (n = 12 in each condition) and EwS primary
   tumors (n = 117).[156]^29 Signatures were derived from C2 and hallmark
   gene sets from the Human MSigDB Collections. See also [157]Figure S2D.

   Lastly, we compared the microarray-based gene expression profiles from
   the EwS cells in standard and refined culture conditions (both no dox;
   [158]Figure 2A) with those from 50 EwS patient tumors.[159]^28 PCA
   revealed that in PC1 and PC2, which account for the largest proportion
   of variance ([160]Figure S2C), gene expression profiles of EwS cells
   from the refined condition clustered closer with EwS tumors than the
   profiles of EwS cells from the standard culture ([161]Figure 2E).
   However, this pattern was not maintained across PC2–PC5
   ([162]Figure 2E). To quantify the concordance between the two in vitro
   conditions and the tumor samples, we used pairwise Pearson
   correlation-based distance calculations between all samples. While the
   overall distances between the tumor samples and EwS cells from the
   refined condition were significantly smaller than those between tumor
   samples and EwS cells from the standard condition (one-sided Wilcoxon
   test of two sets of pairwise distances, p < 9.266951e−43), the change
   in the absolute values was less prominent. Hence, we hypothesized that
   the refinements made to the EwS standard culture condition enhanced the
   physiological relevance of this model for specific pathophysiological
   aspects rather than globally for any aspect. To uncover which might be
   the precise features of EwS patient tumors that are better mimicked in
   the refined condition, we conducted single-sample GSEAs (ssGSEAs) with
   the gene expression data from both EwS in vitro conditions and an
   extended and well-curated EwS tumor cohort[163]^29 ([164]Figures 2F and
   [165]S2D). The normalized enrichment scores (NESs) of transcriptional
   signatures related to cell cycle progression, hypoxia, and EMT of EwS
   patient tumors were better represented by EwS cells grown in the
   refined condition ([166]Figures 2F and [167]S2D). We also observed an
   improved reflection of a signature comprising the top 1,000 most highly
   expressed genes in EwS patient tumors in the refined versus standard
   EwS culture conditions ([168]Figure S2D). Collectively, these results
   suggest that the transcriptomes of EwS cells cultured in the refined
   condition better represent EwS patient tumor transcriptomes regarding
   signatures for proliferation, hypoxia, and EMT.

The refined EwS culture condition shapes the transcriptional output of
EWSR1::ETS

   Since oncogenic EWSR1::ETS transcription factors are the most prominent
   genetic characteristic of EwS,[169]^1 we subsequently investigated
   their transcriptional activity in the refined culture condition in
   comparison to the standard condition. In both culture conditions,
   dox-induced and shRNA-mediated knockdown of the fusions for 96 h
   resulted in a strong downregulation of a well-established
   EWSR1::ETS-related gene signature[170]^30
   (“Kinsey_targets_of_EWSR1::FLI1-fusion_down,” NES < −3;
   [171]Figure 3A), which was in agreement with the efficient knockdown of
   the fusion at the mRNA and protein levels, as shown in [172]Figures 3B
   and 3C, for the representative EWSR1::FLI1-positive cell model
   SK-N-MC/TR/shEF1. Similar observations were made in the
   EWSR1::ERG-positive cell model TC-106/TR/shEERG ([173]Figure S3). Of
   note, the EWSR1::ETS mRNA and protein levels in the refined culture
   condition (no dox) did not exhibit remarkable changes compared to the
   standard culture condition in either cell line (no dox;
   [174]Figures 3B, 3C, and [175]S3), suggesting that the above-described
   transcriptional alterations regarding cell proliferation, hypoxia, and
   differentiation in the refined culture setup are not primarily due to
   altered EWSR1::ETS expression levels. To further characterize the
   EWSR1::ETS transcriptional activity in our refined cell culture
   condition, we performed ssGSEA with our previously established core
   signature of EWSR1::ETS-induced genes[176]^22 on transcriptome data of
   the EwS cell line samples in both culture conditions (no dox) and the
   EwS patient cohort.[177]^29 Strikingly, as shown in [178]Figure 3D, the
   mean NESs of the EwS cells from the refined condition better reflected
   the mean NESs of primary EwS tumors (no significant difference, p =
   0.51) than those of the standard condition (p = 0.035).

Figure 3.

   [179]Figure 3
   [180]Open in a new tab

   The refined EwS culture condition shapes the transcriptional output of
   EWSR1::ETS

   (A) Enrichment plots from fGSEA of DEGs between EWSR1::ETS-high versus
   EWSR1::ETS-low (as indicated by color)-expressing EwS cell lines in
   standard (top) and refined (bottom) conditions. C2 gene set from the
   Human MSigDB Collections.

   (B) EWSR1::ETS expression in SK-N-MC/TR/shEF1 cultured in eight
   different culture conditions. GAPDH served as the HKG, and values are
   shown as a percentage of EWSR1::ETS expression in the standard
   condition (2D RPMI 1640 medium and 10% FCS, no dox). n = 3 biologically
   independent experiments. Vertical bars represent means and whiskers the
   SEM. Statistical significance comparisons are to the standard
   condition. One-way ANOVA with Dunnett’s multiple comparisons test;
   ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001; n.s., non-significant.

   (C) EWSR1::ETS expression in SK-N-MC/TR/shEF1 in eight different
   culture conditions. Representative western blot and densitometry values
   (n = 3 biologically independent experiments; AU, area under the curve).
   GAPDH was used as a housekeeping protein.[181]^22 Vertical bars
   represent means and whiskers the SEM. Statistical significance
   comparisons are to the standard condition with one-way ANOVA with
   Dunnett’s multiple comparisons test.

   (D) Violin plot of NESs of single-sample GSEA (ssGSEA) on gene
   expression profiles from [182]Figure 2A (n = 12 in each condition) and
   EwS primary tumors (n = 117)[183]^29 with our previously established
   core signature of EWSR1::ETS-induced genes.[184]^22 Horizontal dashed
   lines represent the first and third quartiles (Q1 and Q3), and solid
   lines indicate the median. ∗p < 0.05; n.s., non-significant.

   (E) Area-proportional Venn diagram showing overlap of DEGs, defined as
   having FDR-adjusted p ≤ 0.05 and |log2 FC| ≥ 1.5, in refined and
   standard culture conditions. Left side: EWSR1::ETS positively
   correlated DEGs; right side: EWSR1::ETS negatively correlated DEGs. See
   also [185]Table S3.

   (F) Gene Ontology (GO) analysis of EWSR1::ETS positively correlated
   DEGs from (E) showing top five enriched GO biological processes for
   each collection of DEGs. The top row (pink) shows DEGs from refined
   culture conditions exclusively, the middle row (violet) shows DEGs that
   are shared in both culture conditions, and the bottom row (blue) shows
   DEGs from standard culture conditions only. See also [186]Table S3.

   (G) Analogously to (F) with EWSR1::ETS negatively correlated DEGs. See
   also [187]Table S3.

   Next, we compared differentially regulated genes under EWSR1::ETS
   knockdown in the two culture conditions for shared EWSR1::ETS
   positively correlated and EWSR1::ETS negatively correlated genes in
   both conditions as well as genes uniquely regulated in either culture
   condition. Strikingly, while there were notable overlaps in
   EWSR1::ETS-regulated genes between the two conditions, there was also a
   large proportion (∼50%) of genes specific to each culture technique
   ([188]Figure 3E). Among the EWSR1::ETS-regulated genes that were shared
   in both cell culture conditions, Gene Ontology (GO) analysis revealed
   that upregulated gene sets in EWSR1::ETS-high conditions (no dox) were
   predominantly associated with cell cycle progression ([189]Figure 3F;
   [190]Table S3). Similarly, proliferation signatures were
   overrepresented among EWSR1::ETS-correlated genes exclusively in
   standard cell culture conditions ([191]Figure 3F; [192]Table S3), which
   corresponds to previous reports that linked higher EWSR1::ETS
   expression in EwS to increased proliferation.[193]^31^,[194]^32 Most
   interestingly, genes that were EWSR1::ETS correlated exclusively in the
   refined culture condition were primarily categorized by GO analysis as
   participating in amino acid membrane transport (e.g., L-alanine and
   leucine import across plasma membrane, GO: 1904273 and 0098713)
   ([195]Figure 3F; [196]Table S3). Hence, this suggests that EWSR1::ETS
   facilitate nutrient transport, which is not apparent under the
   supraphysiological nutrient concentrations present in traditional cell
   culture media. For EWSR1::ETS negatively correlated genes, GO analysis
   retrieved differentiation processes that were reduced through the
   upregulation of EWSR1::ETS in genes overlapping between the two medium
   conditions as well as those unique to either condition ([197]Figure 3G;
   [198]Table S3). This reflects the known role of EWSR1::ETS in
   maintaining a dedifferentiated state.[199]^31 Collectively, our data
   show that the activity of EWSR1::ETS fusions in EwS cell lines is
   shifted under cell culture conditions with increased physiological
   relevance from a hyperproliferative state toward enhanced amino acid
   transport while maintaining a dedifferentiated state. As the reliance
   on extracellular nutrients under nutrient limiting conditions can be an
   exploitable vulnerability in cancer cells,[200]^18 these findings may
   have significant implications for future EwS research that aims for
   accurate discoveries of EWSR1::ETS-regulated genes and drug
   targets.[201]^33

Cell culture conditions may influence expression of commonly used HKGs

   It is well established that specific experimental conditions (e.g.,
   cell culture techniques) can influence the expression levels of
   housekeeping genes (HKGs), which may introduce a bias into studies
   based on real-time qPCR readouts.[202]^34^,[203]^35 Indeed, when
   assessing our microarray data, we observed that ribosomal protein
   lateral stalk subunit P0 (RPLP0), a routinely used HKG in EwS
   research,[204]^22 exhibited markedly different baseline expression
   levels between the standard and refined cell culture conditions. To
   nominate potential HKGs exhibiting minimal variation across cell
   culture conditions, we employed and adapted an approach described by de
   Jonge et al.[205]^34 (see [206]STAR Methods). As displayed in
   [207]Figure 4A, the top three candidate HKGs were the mitochondrially
   encoded cytochrome c oxidase I (MT-CO1), mitochondrially encoded NADH
   dehydrogenase 1 (MT-ND1), and RNA component of 7SK nuclear
   ribonucleoprotein (RN7SK), a small nuclear RNA (snRNA). Indeed, these
   three genes showed little variation as compared to RPLP0
   ([208]Figure 4B). A comprehensive list of potential HKGs is provided in
   [209]Table S4. These data confirm the rationale that the choice of a
   suitable HKG must be tailored to the specific cell line and applied
   cell culture condition.[210]^34^,[211]^35

Figure 4.

   [212]Figure 4
   [213]Open in a new tab

   Cell culture conditions may influence the expression of commonly used
   HKGs

   (A) Table of the top three housekeeping gene (HKG) candidates and the
   routinely used HKGs GAPDH and RPLP0. HKG candidates were selected
   through the adapted approach from de Jonge et al.[214]^34 Mean
   expression value is defined as mean of the expression values of all
   samples from [215]Figure 2A, including the four different conditions.
   SD, standard deviation; MFC, maximum FC; CV, coefficient of variation.
   See also [216]Table S4 and [217]STAR Methods.

   (B) Log2-transformed gene expression of HKG candidates from the
   microarray data in the four EwS cell lines from [218]Figure 2A.

Discussion

   In this report, we pursue a practical approach to enhance the
   physiological relevance of the standard culture technique for EwS cell
   lines. The refined culture condition was validated through comparative
   transcriptome analyses of gene expression data from four EwS cell lines
   cultured in standard[219]^22 and refined conditions as well as from EwS
   patient tumors.[220]^28^,[221]^29 These analyses indicated that EwS
   cell line characteristics, including EWSR1::ETS activity, were
   preserved between the two culture conditions, while transcriptionally
   altered signatures in the refined versus standard culture conditions
   involved cell cycle progression, hypoxia, metabolism, translation, and
   differentiation (i.e., EMT). Of these, we found that cell cycle
   progression-, hypoxia-, and EMT-related transcriptional signatures of
   EwS cells from the refined condition better represented the respective
   transcriptional activity detected in EwS patient tumors than those from
   EwS cells grown in the standard condition. Notably, hypoxia is an
   important component of the EwS tumor microenvironment[222]^27^,[223]^36
   that is relevant for EwS pathophysiology,[224]^9^,[225]^36 as well as
   the complex interplay of proliferation and differentiation
   processes.[226]^1^,[227]^31^,[228]^32 Hence, we consider the
   herein-presented refined culture condition a suitable model for EwS
   in vitro studies, which might be, due to the improved modeling of
   proliferation, differentiation, and hypoxia, particularly relevant for
   future studies investigating these processes in EwS pathophysiology.

   Our results demonstrate that the employed cell culture conditions
   influence two aspects relevant to EwS research: (1) transcriptional
   programs linked to a variety of cellular processes, such as
   proliferation, translation, differentiation, and metabolism, and (2)
   the activity of the EWSR1::ETS oncogenic transcription factors.
   Regarding the first aspect, our data are consistent with previously
   published studies in that metabolism and proliferation are frequently
   altered when cells are cultured in physiological
   media.[229]^14^,[230]^17^,[231]^37 This is also reflected in our data
   by the upregulation of ribosomal and translational processes when
   changing from standard to refined EwS cell culture conditions since
   translation and metabolism are tightly correlated in both normal and
   cancer cells.[232]^38^,[233]^39 Consequently, it is intriguing to
   further characterize EwS features in the refined and physiologically
   relevant condition by means of extended multi-omics profiling,
   including the proteomic level.

   Concerning the second aspect, our study provides insight into potential
   alterations of the metabolic landscape in EwS induced by the activity
   of EWSR1::ETS oncoproteins in a physiologically relevant cell culture
   model. We hypothesize that the observed altered expression of genes
   engaged in amino acid membrane transport in physiologic media is
   necessary to sustain the proliferation of high-EWSR1::ETS-expressing
   EwS cell lines[234]^31^,[235]^32 in physiological media. Interestingly,
   the transcriptional regulation of genes involved in amino acid
   metabolic pathways in the context of proliferation in physiological
   media has already been described in other tumor
   entities.[236]^17^,[237]^37 Our data support the idea that altered
   metabolic pathways related to amino acid abundance are a characteristic
   trait of proliferating cells in physiological media. To fully
   characterize which amino acids and metabolic pathways are required for
   the proliferation of high-EWSR1::ETS-expressing EwS cell lines, further
   studies on the interaction of FET::ETS oncogenes and the metabolism in
   EwS cells are needed. Finally, our data illustrate that the applied
   cell culture technique, as a part of the general experimental
   conditions, can influence the expression levels of routinely used
   HKGs[238]^34^,[239]^35—as was the case for RPLP0[240]^22 in this study.

   In summary, our work underlines the necessity to improve the
   physiological relevance of cell culture conditions, provides a
   pragmatic and cost-effective approach for the EwS field to overcome
   typical barriers for establishing these, and contributes a
   transcriptome dataset generated in refined in vitro conditions as a
   valuable resource to the EwS research community.

Limitations of the study

   The technical limitations of our refined culture technique for EwS cell
   lines involve primarily the natural restrictions of any spheroid-based
   technique. The ability of tumor cells to spontaneously form spheroids
   when cultured in low-attachment conditions is a prerequisite for this
   approach.[241]^4 Further restrictions include the variability in size
   and diameter between individual spheres; if cultured on flat-bottom
   surfaces, the lack of a vasculature or dynamic flow system; and, in our
   experimental setup, the absence of ECM components.[242]^4 On that
   account, alternative methods have been developed to mimic the
   interactions of EwS cells with the ECM and mechanical
   forces.[243]^3^,[244]^4^,[245]^5^,[246]^8 These techniques could also
   be combined with physiological media[247]^14^,[248]^15 and the reduced
   FCS concentration that we propose in this report.

   Limitations to our intention to increase the physiological relevance by
   means of refining the standard EwS cell culture condition were mirrored
   in the relatively small improvements in the overall comparison of the
   in vitro gene expression profiles with EwS patient tumors. This might
   be explained in part by the systematic discrepancies between cancer
   cell lines employed for in vitro studies and tumor cells thriving in
   the human body.[249]^40 While we could detect an improved modeling for
   some physiological processes such as proliferation, differentiation,
   and hypoxia in the refined EwS culture, this did not apply for all
   pathophysiological aspects of EwS patient tumors. These considerations
   should guide the selection of appropriate in vitro models for future
   EwS studies.

   Some of the limitations discussed above are a result of our intention
   to keep the cell culture technique simple, feasible, and cost
   effective. It is due to this motivation that our refined cell culture
   condition for EwS cell lines is amenable to routine and large-scale use
   and especially suitable for bulk analyses in a setting with increased
   physiological relevance.

Resource availability

Lead contact

   Requests for resources of this article and any additional information
   should be directed to the lead contact, Thomas G.P. Grünewald
   (t.gruenewald@dkfz-heidelberg.de)

Materials availability

   This study did not generate new unique reagents.

Data and code availability

     * •
       Original Affymetrix transcriptome profiling data have been
       deposited at the GEO and are publicly available under the accession
       code GEO: [250]GSE270118
       .
     * •
       Microscopy data and western blot images reported in this paper will
       be shared by the [251]lead contact upon request.
     * •
       This study did not generate new original code.
     * •
       Any additional information required to reanalyze the data reported
       in this paper is available from the [252]lead contact upon request.
     * •
       In addition, Affymetrix microarray data from our published Ewing
       Sarcoma Cell Line Atlas (ESCLA)[253]^22 (accession code:
       [254]GSE176190
       ) as well as an Affymetrix microarray dataset from multiple human
       tumor samples including EwS[255]^28 and an Affymetrix microarray
       dataset from an EwS patient tumor cohort[256]^29 (accession code
       [257]GSE34620) were used for comparison.

Acknowledgments