CHRDL1 Regulates Stemness in Glioma Stem-like Cells

Glioblastoma (GBM) still presents as one of the most aggressive tumours in the brain, which despite enormous research efforts, remains incurable today. As many theories evolve around the persistent recurrence of this malignancy, the assumption of a small population of cells with a stem-like phenotype remains a key driver of its infiltrative nature. In this article, we research Chordin-like 1 (CHRDL1), a secreted protein, as a potential key regulator of the glioma stem-like cell (GSC) phenotype. It has been shown that CHRDL1 antagonizes the function of bone morphogenic protein 4 (BMP4), which induces GSC differentiation and, hence, reduces tumorigenicity. We, therefore, employed two previously described GSCs spheroid cultures and depleted them of CHRDL1 using the stable transduction of a CHRDL1-targeting shRNA. We show with in vitro cell-based assays (MTT, limiting dilution, and sphere formation assays), Western blots, irradiation procedures, and quantitative real-time PCR that the depletion of the secreted BMP4 antagonist CHRDL1 prominently decreases functional and molecular stemness traits resulting in enhanced radiation sensitivity. As a result, we postulate CHRDL1 as an enforcer of stemness in GSCs and find additional evidence that high CHRDL1 expression might also serve as a marker protein to determine BMP4 susceptibility.


Introduction
Glioblastoma (GBM) is highly aggressive and the most common brain tumour in adults. According to WHO criteria, it is classified as a grade 4 astrocytoma [1,2], and despite an intensive treatment regimen consisting of maximally possible surgery, followed by radiochemotherapy using the alkylating agent Temozolomide, the median survival barely exceeds one year [3][4][5][6]. One key characteristic of GBM is its highly infiltrative growth, which makes relapses virtually unavoidable [7]. It is hypothesised that recurrences are further supported by tumour cells that can transiently obtain a stem-like phenotype. These glioma stem-like cells (GSCs) [8][9][10] or tumour-initiating cells are thought to be able to replenish the entire tumour and are considered particularly resistant to conventional therapy, including radiotherapy and chemotherapy [11][12][13]. It has been further shown that these cells reside in specific niches, namely hypoxic or perivascular areas, and express proteins associated with stemness, whereas they lack protein expression associated with differentiated neuronal cells [12,[14][15][16][17][18].

Sphere Formation Assays
To measure the sphere-forming ability, an assay (SFA) based on Gilbert et al. [34] was performed. Briefly, 500 and 1000 dissociated NCH644 and GS-5, respectively, were seeded in 96 well plates and measured after 7 days. Images were acquired using a Tecan Spark plate reader and analysed using a self-developed macro via FIJI (v1.52p) [35], which enhanced contrasts and identified sphere area and properties as described previously [29].

Cell Cycle Analysis
A total of 150,000 cells were seeded in a 6-well plate for 24 h. For analysis, the medium was removed, the cells were washed with PBS, and a single cell suspension was prepared using Accutase-incubation for 10 min at 37 • C. The cells were next harvested in FACS tubes and were washed with 100 µL cold PBS. An amount of 3 mL of ice-cold 70% ethanol was added dropwise to the cells by simultaneous vortexing, followed by incubation for 2 h on ice. The FACS tubes were centrifuged for 3 min at 1000 rpm to form pellets. For analysis, cells were incubated for 5 min at room temperature with 50 µL RNAse A (Qiagen, 20 µg/mL) and followed by 150 µL Propidium Iodide (Sigma-Aldrich, 50 µg/mL) for 30 min at room temperature. The flow cytometric determination of the cell cycle was performed by counting 20,000 cells on an Accuri C6 (Becton Dickinson, Franklin Lakes, NJ, USA) operated through BD Accuri C6 software (Version: 1.0.264.21; Becton Dickinson) and by measuring no more than 500 events/s.
The quantification of Western Blot images was performed using the raw files with Image Studio Lite (Version 5.2, LI-COR Biosciences) by manual selection of the regions of interest. The signal values of the target proteins were first normalised to the housekeeping proteins and afterward to the respective control condition, which was set to 1.

Irradiation Procedures
In total, 500 cells/well were seeded into 96-well plates and irradiated the next day. Irradiation (IR) with single doses of 2, 4, or 6 Gy was performed using a linear accelerator with 6 MV photon energy, a 100 cm focus to the isocentre distance, and a dose rate of 6 Gy/min (Synergy, Elekta, Crawley, UK) at the Department of Radiation and Oncology (University Hospital Frankfurt, Frankfurt, Germany).

Immunofluorescence Microscopy/Foci Assay
For DNA damage analyses, 12,000 NCH644 cells/well were seeded on Laminincoated 8-well chamber slides (Falcon, Corning, Amsterdam, NY, USA). Laminin-coating (10 µg/mL, Sigma-Aldrich, L2020) was performed at 4 • C overnight. One day after seeding, the cells were irradiated as indicated and, after an additional 24 h, were fixed with 4% paraformaldehyde for 20 min at RT. The slides were washed with TBS-Tween (0.1%; TBS-T), blocked with 4% BSA in TBS with 0.3% Triton X-100 for 1 h at RT, and the primary antibody incubation occurred at 4 • C overnight. Hereafter, the slides were washed at least three times with TBS-T, and a secondary antibody was diluted 1:500 in TBS-T and incubated for 1 h at RT. After an additional wash step with TBS-T, the slides were mounted with DAPI containing Immunoselect antifading mounting medium (Dianova, Hamburg, Germany) or Fluoroshield with DAPI (Thermo Fisher, Frankfurt, Germany). Images were acquired with an Eclipse TS100 inverted fluorescence microscope (Nikon, Düsseldorf, Germany) operated by NIS Elements AR software (version 3.22, Nikon).

Taqman-Based qRT-PCR
In total, 300,000 cells/well (NCH644) and 600,000 cells/well (GS-5) were seeded into 6-well plates, and cells were collected the following day. Experiments were performed using 3 biological replicates for each treatment condition, while the experiment was repeated three times. RNA was isolated using the ExtractMe Total RNA Kit (Blirt S.A., Gdanks, Poland), and 1-2 µg RNA was used for cDNA synthesis. SuperScript III System (Life Technologies, Darmstadt, Germany) allowed the synthesis of cDNA, with 100 U per sample to be sufficient. The quantitative real-time PCR (qRT-PCR) was performed using Taqman-probes (Applied Biosystems, Darmstadt, Germany) and the Fast-Start Universal Probe Master Mix (Roche) on a StepOne Plus System (Applied Biosystems) in a 20 µL reaction volume.
Ct values were normalised to the TATA box-binding protein (TBP). Fold-change in the gene expression was determined by the 2 −∆∆Ct method.

CHRDL1 Depletion Reduces Stemness of GSCs
To analyze the role of CHRDL1 in maintaining the GSC phenotype, we first established stable CHRDL1-knockdown (KD) cell lines (shCHRDL1) by lentiviral transduction with two different shRNAs directed against CRHDL1. We could show for both NCH644 [27] ( Figure 1A) and GS-5 [28] ( Figure 1A) that a successful KD could be achieved. Pooled data from the quantification of several independent experiments revealed that for both NCH644 and GS-5 ( Figure 1B), significant depletion was achieved. Based on our hypothesis that CHRDL1 maintains stemness, we next investigated the sphere-forming potential of both GSCs via LDAs. For NCH644 shCtrl ( Figure 1C), we observed a stem-cell frequency of 1/21.7, whereas, upon CHRDL1 depletion, this was reduced 15-fold to 1/333.7. Representative pictures for NCH644 and GS-5 are depicted in Figure 1D. Since GS-5 GSCs grow considerably slower, we further analyzed these cells after 1 week ( Figure 1E) for better comparison with NCH644 and to allow for sufficient sphere formation after an additional week ( Figure 1F) of incubation. One week after seeding, GS-5 shCtrl revealed a calculated stem-cell frequency of 1/404, whereas shCHRDL1 GS-5 had a 1.5-fold lower frequency of 1/600. This finding was further underscored after 2 weeks, which showed a stem-cell frequency of 1/75.5 of shCtrl GSCs, while shCHRDL1-GSCs had a 3.3-fold lower frequency of 1/248.7. Overall, we could show that CHRDL1 depletion reduced stemness.
An analysis of the cell cycle after CHRDL1-depletion ( Figure 2) revealed that NCH644 ( Figure 2A) displayed an increase in cells in the G1-phase with a concomitant decrease in Sand G2/M-phases, indicative of G1-arrest due to CHRDL1-depletion. GS-5 cells display similar cell cycle phase distribution irrespective of CHRDL1 depletion ( Figure 2B), which might reflect the overall lower effect size observed using the limited dilution assay. In summary, the inhibition of proliferation might be partially accountable for the observed reduction in the stem-cell-frequency but does not fully explain our results.
Based on these findings, we therefore asked if this functional reduction in stemness was also associated with the reduced expression of stemness-associated genes. For this purpose, we performed Taqman-based qRT-PCR in both NCH644 and GS-5. For NCH644 (Figure 3), we could validate the robust depletion of CHRDL1 mRNA expression upon shRNA-mediated CHRDL-KD ( Figure 3A). Similarly, we observed a prominent reduction in the stemness marker genes OLIG2 ( Figure 3B) and SOX2 ( Figure 3C), indicating reduced stemness. Curiously, SOX9 expression ( Figure 3D) shows a slight but significant induction after CHRDL1 depletion. We further analyzed ZEB1 ( Figure 3E) and ZEB2 ( Figure 3F), which are associated with enhanced stemness, but also with an EMT-like phenotype, and we observed moderate yet significant repression (ZEB1) and induction (ZEB2), respectively. The transmembrane protein CD44 ( Figure 3G) has also been associated with the invasiveness of GBM cells and is considered a stemness marker, and this gene was strongly reduced upon CHRDL1-KD, while the intermediate filament NES (Nestin) is markedly and strongly depleted ( Figure 3H). Finally, we analyzed a set of differentiation-associated genes. Upon CHRDL1 depletion, the glial marker GFAP ( Figure 3I), as well as the neuronal marker NEFL Cells 2022, 11, 3917 6 of 17 (Neurofilament, Figure 3J), were significantly upregulated, whereas MAP2 ( Figure 3K) only displayed a tendency. Lastly, RBFOX3 ( Figure 3L), another neuronal marker, was significantly increased. Collectively these data indicate that CHRDL1-depletion potently blocks several pathways associated with stemness and invasion while inducing a more differentiated cellular state. ure 2B), which might reflect the overall lower effect size observed using the limit tion assay. In summary, the inhibition of proliferation might be partially accoun the observed reduction in the stem-cell-frequency but does not fully explain our Based on these findings, we therefore asked if this functional reduction in s was also associated with the reduced expression of stemness-associated genes. purpose, we performed Taqman-based qRT-PCR in both NCH644 and GS-5. For N (Figure 3), we could validate the robust depletion of CHRDL1 mRNA expressio shRNA-mediated CHRDL-KD ( Figure 3A). Similarly, we observed a prominent re in the stemness marker genes OLIG2 ( Figure 3B) and SOX2 ( Figure 3C), indica duced stemness. Curiously, SOX9 expression ( Figure 3D) shows a slight but sig induction after CHRDL1 depletion. We further analyzed ZEB1 ( Figure 3E) and ZE ure 3F), which are associated with enhanced stemness, but also with an EMT-like type, and we observed moderate yet significant repression (ZEB1) and induction respectively. The transmembrane protein CD44 ( Figure 3G) has also been associa the invasiveness of GBM cells and is considered a stemness marker, and this g strongly reduced upon CHRDL1-KD, while the intermediate filament NES (N markedly and strongly depleted ( Figure 3H). Finally, we analyzed a set of differen associated genes. Upon CHRDL1 depletion, the glial marker GFAP (Figure 3I), as the neuronal marker NEFL (Neurofilament, Figure 3J), were significantly upre whereas MAP2 (Figure 3K) only displayed a tendency. Lastly, RBFOX3 (Figure other neuronal marker, was significantly increased. Collectively these data indic Similar to NCH644, we analyzed the same gene panel for GS-5 GSCs ( Figure 4) and first validated CHRDL1 depletion ( Figure 4A). Furthermore, we confirmed the efficient depletion of the stemness-associated genes OLIG2 ( Figure 4B) and SOX2 ( Figure 4C), which is also accompanied by a significant reduction in SOX9 expression ( Figure 3D). The EMT-associated gene ZEB1 ( Figure 4E) is also significantly decreased, while ZEB2 ( Figure 4F) and CD44 ( Figure 4G Figure 4L) neither show a significant change, although GFAP and MAP2 show a slight tendency towards increased expression. In summary, we could validate our findings of grossly reduced stemness in a second GSC line, further underscoring that CHRDL1 could be a novel master regulator of GBM stemness.  Similar to NCH644, we analyzed the same gene panel for GS-5 GSCs (Figure 4) and first validated CHRDL1 depletion ( Figure 4A). Furthermore, we confirmed the efficient depletion of the stemness-associated genes OLIG2 ( Figure 4B) and SOX2 ( Figure 4C), which is also accompanied by a significant reduction in SOX9 expression ( Figure 3D). The EMT-associated gene ZEB1 ( Figure 4E) is also significantly decreased, while ZEB2 ( Figure  4F) and CD44 ( Figure 4G  Using GS-5 GSCs, we also validated the loss of the protein expression for the stemnessassociated proteins OLIG2, SOX9, and SOX2 via Western Blot ( Figure 5) and observed a robust depletion of these proteins.  Using GS-5 GSCs, we also validated the loss of the protein expression for the stemness-associated proteins OLIG2, SOX9, and SOX2 via Western Blot ( Figure 5) and observed a robust depletion of these proteins.

CHRDL1 Depletion Re-Activates BMP4-Signaling in GSCs
Based on the notion that a high CHRDL1 expression blocks B thereby enforces an enhanced stem-like state of GSCs, we first confirm proficient at relaying BMP4-mediated signals by treating the cells wi human BMP4 ligand ( Figure 6A). This showed us that upon BMP4 tre was highly increased, indicative of active BMP4-signaling. Similarly, NCH644 GSCs ( Figure 6B) also displayed an increased pSMAD1/5 ex ment of NCH644 and GS-5 GSCs with recombinant BMP4 (Figure 6C, duced sphere formation potential in both GSCs, as did CHRDL1 deple of CHRDL1-depleted cells with rBMP4 led to no further enhancemen pared to the solvent-treated shCHRDL1 GSCs, whereas for GS-5, the c further and potentially synergistically reduced the sphere-forming po Figure 5. CHRDL1-depletion reduces stemness-associated marker protein expression. Western Blots of GS-5 GSCs after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1) and measurement of the protein expression of OLIG2, SOX9, and SOX2. CHRDL1 was detected to validate efficient depletion and GAPDH served as a housekeeping protein.

CHRDL1 Depletion Re-Activates BMP4-Signaling in GSCs
Based on the notion that a high CHRDL1 expression blocks BMP4 signaling and thereby enforces an enhanced stem-like state of GSCs, we first confirmed that NCH644 is proficient at relaying BMP4-mediated signals by treating the cells with the recombinant human BMP4 ligand ( Figure 6A). This showed us that upon BMP4 treatment, pSMAD1/5 was highly increased, indicative of active BMP4-signaling. Similarly, CHRDL1-depleted NCH644 GSCs ( Figure 6B) also displayed an increased pSMAD1/5 expression. The treatment of NCH644 and GS-5 GSCs with recombinant BMP4 (Figure 6C,D) resulted in a reduced sphere formation potential in both GSCs, as did CHRDL1 depletion. The treatment of CHRDL1-depleted cells with rBMP4 led to no further enhancement in NCH644 compared to the solvent-treated shCHRDL1 GSCs, whereas for GS-5, the combined approach further and potentially synergistically reduced the sphere-forming potential.

CHRDL1 Depletion Sensitizes GSCs towards Radiation Treatment
Next, we reasoned that CHRDL1-depleted cells might be more vulnerable to con tional treatment, such as radiotherapy. For this purpose, we irradiated the cells wit creased doses of X-ray (single doses) and analysed the amount of DNA double-st breaks (DNA-DSB) using immunofluorescent staining against 53BP1 ( Figure 7A). W served significantly more 53BP1-positive foci, indicative of enhanced DNA damag CHRDL1-depleted cells, which were mock irradiated and after irradiation with 2 Gy ter 4 Gy treatment, no significant differences were observed, whereas irradiation w

CHRDL1 Depletion Sensitizes GSCs towards Radiation Treatment
Next, we reasoned that CHRDL1-depleted cells might be more vulnerable to conventional treatment, such as radiotherapy. For this purpose, we irradiated the cells with increased doses of X-ray (single doses) and analysed the amount of DNA double-strand breaks (DNA-DSB) using immunofluorescent staining against 53BP1 ( Figure 7A). We observed significantly more 53BP1-positive foci, indicative of enhanced DNA damage, in CHRDL1-depleted cells, which were mock irradiated and after irradiation with 2 Gy. After 4 Gy treatment, no significant differences were observed, whereas irradiation with 6 Gy sig-nificantly increased the amount of 53BP1 foci compared to non-irradiated GSCs. Hereafter, we reasoned that higher amounts of DNA-DSBs might lead to enhanced growth inhibition and therefore performed sphere formation assays of freshly dissociated GSCs seeded as single cells ( Figure 7B). This approach revealed that in CHRDL1-expressing NCH644 shCtrl cells, every IR dosage significantly impedes sphere formation. CHRDL1 depletion without irradiation (0 Gy) also significantly impedes sphere formation, basically confirming our results shown above. The combination of irradiation and CHRDL1 depletion can further block sphere formation after treatment with 6 Gy, thus, reflecting the enhanced amount of DNA-DSBs. Representative pictures are depicted in Figure 7C. Hereafter, we sought to confirm that this decrease was due to the re-activation of BMP4-signaling and performed a similar experiment using NCH644 cells treated with recombinant BMP4 (Figure 7D), which could confirm the result obtained with the CHRDL1 shRNA. Hence, we observed that BMP4 treatment reduces sphere sizes, similar to irradiation. The combination of both treatments significantly further reduced sphere areas, indicative of a profound sensitization via BMP4-signaling to the irradiation treatment.
Cells 2022, 11, 3917 12 Gy significantly increased the amount of 53BP1 foci compared to non-irradiated G Hereafter, we reasoned that higher amounts of DNA-DSBs might lead to enhan growth inhibition and therefore performed sphere formation assays of freshly dissoci GSCs seeded as single cells ( Figure 7B). This approach revealed that in CHRDL1-expr ing NCH644 shCtrl cells, every IR dosage significantly impedes sphere forma CHRDL1 depletion without irradiation (0 Gy) also significantly impedes sphere mation, basically confirming our results shown above. The combination of irradiation CHRDL1 depletion can further block sphere formation after treatment with 6 Gy, t reflecting the enhanced amount of DNA-DSBs. Representative pictures are depicte Figure 7C. Hereafter, we sought to confirm that this decrease was due to the re-activa of BMP4-signaling and performed a similar experiment using NCH644 cells treated w recombinant BMP4 ( Figure 7D), which could confirm the result obtained with CHRDL1 shRNA. Hence, we observed that BMP4 treatment reduces sphere sizes, sim to irradiation. The combination of both treatments significantly further reduced sp areas, indicative of a profound sensitization via BMP4-signaling to the irradiation tr ment.

CHRDL1 Is Associated with Poor Prognosis in Glioma and Other Cancers
Lastly, we interrogated the human protein atlas [37] in order to unravel the relevance of CHRDL1 for patient outcomes and observed that high CHRDL1 expression was associated with worse clinical outcomes in glioma ( Figure 8A), while it could also be considered unfavourable in urothelial ( Figure 8B) and renal cancer ( Figure 8C). (C) Representative microphotographs of NCH644 shCtrl or NCH644 shCRHDL1 without irradiation (0 Gy) or after irradiation with 6 Gy; scale bar: 200 µm. * p < 0.05; ** p < 0.01; **** p < 0.0001; twoway-ANOVA with Sidak's multiple comparison tests. (D) Sphere formation of NCH644 treated with solvent or recombinant human BMP4 (25 ng/mL) immediately prior to irradiation with a dose of 4 or 6 Gy. Sphere area was determined 7 days after treatment. * p < 0.05; **** p < 0.0001; Kruskal-Wallis test with Dunn's multiple comparisons test.

CHRDL1 Is Associated with Poor Prognosis in Glioma and Other Cancers
Lastly, we interrogated the human protein atlas [37] in order to unravel the relevance of CHRDL1 for patient outcomes and observed that high CHRDL1 expression was associated with worse clinical outcomes in glioma ( Figure 8A), while it could also be considered unfavourable in urothelial ( Figure 8B) and renal cancer ( Figure 8C).

Discussion
Glioblastoma remains one of the most dismal cancer diagnoses in adults, and current treatment strategies fail to cure the patients. It is hypothesized that this is largely due to stem-like states that can be obtained by some or all residual tumour cells that survive surgery and radiochemotherapy, resulting in more aggressive and ultimately lethal tumours. A better understanding of the underlying mechanisms is crucial to develop targeted and patient-centred approaches that can be based on the molecular knowledge of different tumour types.
BMP4 was shown to enforce the differentiation of GSCs almost two decades ago and has been tested as a "differentiation therapy" [21]. Although this notion has not been fulfilled and the entire concept of stable differentiation has been challenged, particularly due to the presence of different, highly plastic cell states that might be driven by different molecular alterations [8][9][10], the question remains as to how tumour cells evade these stemness-blocking cues. One factor that has been shown recently to be overexpressed in cancers, including GBM, is the secreted BMP4 antagonist CHRDL1 [24,25]. Accordingly, our working hypothesis was that CHRDL1 acts as an enforcer of stemness in GSCs. Within this report, we employed two previously described GSC spheroid cultures [27,28] and depleted them of CHRDL1 using the stable transduction of a CHRDL1-targeting shRNA. This CHRDL1 depletion was accompanied by functional as well as molecular changes that conclusively can be interpreted as stemness blockade. Furthermore, considering that GSCs are known to be more resistant towards conventional therapy, such as radiotherapy, we could further confirm that CHRDL1-depleted cells are indeed sensitized to IR treatment. Interestingly, it has been proposed that IR treatment induces stemness in cancer cells [38,39] via the upregulation of SOX2 [38], among other known stemness factors. SOX2 is one of the major factors regulated by CHRDL1, as shown by us and others [40]. Therefore, it seems likely that CHRDL1 depletion not only counteracts this IR-induced stemness increase but even effectively prevents the stemness traits of GSCs. Similarly, BMP4 treatment would be expected to result in a similar cooperative effect, as has been confirmed by us to our knowledge for the first time in this report. Similarly, it has already been shown that the BMP4 treatment of GBM cells sensitizes them to the current gold-standard chemotherapy with Temozolomide [41], as well as to more targeted agents such as Bevacizumab [42,43]. Very recently, it was shown in diffuse intrinsic pontine glioma, a very aggressive juvenile glioma type, that the activation of BMP signalling enforces cellular differentiation, similar to our findings presented in the current study. Mechanistically it was shown that the upregulation of CHRDL1 expression results in the counteraction of the tumoursuppressive effects of BMP4 and that the depletion of CHRDL1 significantly reduces cell proliferation and sphere formation as well as tumour growth in a xenograft model [40]. The authors also showed that the BMP4 treatment of diffuse intrinsic pontine glioma (DIPG) cells induced a stemness blockade accompanied by decreased SOX2 and OLIG2 expression, further validating our findings. Mechanistically the authors proposed that by the epigenetic upregulation of the tumour suppressor CXXC5, the BMP4-response could be mediated [40]. Considering these studies, one may propose that the (re-) activation of BMP4 signalling might be a suitable strategy to block the stem-like phenotype of adult and juvenile gliomas and thereby sensitize them towards conventional therapy regimes. In our study, we found additional evidence that high CHRDL1 expression might serve as a marker protein to determine BMP4 susceptibility. Whether this mechanism might also be expanded to other tumour entities should also be investigated in future studies. For example, it is known that BMP4 can exert pro-tumourigenic functions in breast cancer, where it mediates migration and invasion, which can be blocked via CHRDL1 [25], whereas CHRDL1 induces the neuronal differentiation of neural stem cells [26]. Accordingly, the origin and/or location of the tumour likely dictates how external cues are perceived.
Additionally, an open question that remains unanswered for now is whether different GBM subtypes, such as proneural, mesenchymal, and classical GBM [44], respond differently to BMP4 treatment/CHRDL1-blockade. We could show that the proneural [45] NCH644 responds moderately to BMP4 treatment while GS-5, which has been classified as a mixture of proneural and mesenchymal [28], display an additional stemness blockade by combined BMP4 treatment and CHRDL1 depletion. However, based on our very restricted sample size, it is difficult to reach a conclusive statement, and the response of different subtypes should be analysed more systematically in future studies.
Curiously, a recent report by Sachdeva et al. [23] took an opposing view from the literature and our recent findings by claiming that BMP4 treatment rather induces quiescence instead of blocking stemness. Similarly, as discussed above, this could be explained by different subtypes reacting differently to this kind of treatment. Moreover, the report by Sachdeva et al. does not necessarily contradict our findings. In their report, they employed a short-term high-dose treatment with BMP4, whereas we analysed cells stably depleted of an upstream regulator of endogenous BMP4. Hence, one plausible hypothesis is that (re-)activating BMP4 signalling first induces a shift towards quiescence/cell cycle arrest, which results in the loss of stemness traits after the continuous activation of BMP4 signalling. Whether this hypothesis remains true and whether the inhibition of BMP4-signaling reestablishes stem-like traits should be answered in future studies. In addition, it cannot be excluded that CHRDL1 also has BMP-independent functions.
Clinically it is interesting to note that CHRDL1 is, according to data from the human protein atlas, associated with worse survival in glioma, as well as in urothelial and renal cancer. One could therefore hypothesize that CHRDL1 has the potential as a prognostic factor and could be considered in routine diagnostics. According to the literature, CHRDL1 is associated with a worse prognosis in oral squamous cell carcinoma [46], where it also regulates metastasis and EMT [47].
Interestingly, astrocytes are also able to secrete CHRDL1 during the physiological process of wound healing after ischemic injury to facilitate the healing process [48]. This leaves the open questions on where CHRDL1 originates in a multicellular tumour and how it affects non-tumour cells. Using more complex model systems, such as an organotypic brain slice culture or even in vivo approaches, these questions could be addressed in future studies, and the possibility of targeting CHRDL1 in a cell-autonomous way, e.g., via targeting antibodies, should be explored.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
No large-scale datasets have been generated. Raw data of the experiments and/or materials can be provided upon reasonable request.