BMP antagonist CHRDL2 enhances the cancer stem cell phenotype and increases chemotherapy resistance in Colorectal Cancer

  1. Eloise Clarkson
  2. Annabelle Lewis

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Abstract

Chordin-like-2 (CHRDL2) is a secreted BMP antagonist, with overexpression and genomic variants associated with colorectal cancer (CRC) risk. BMP signalling in the normal intestinal epithelium operates in opposition to the WNT signalling pathway, which maintains stem-cells and self-renewal. Elevated WNT signalling leads to expansion of the stem cell compartment and hyperproliferation, defining characteristics of CRC.

Here, we explored the impact of CHRDL2 overexpression on CRC cells and normal intestinal organoids to investigate whether CHRDL2’s inhibition of BMP signalling intensified WNT signalling, and enhanced the cancer stem-cell phenotype.

CHRDL2 increased cell survival during chemotherapy and irradiation with associated activation of DNA damage response pathways. Organoids treated with secreted CHRDL2 exhibited elevated levels of stem cell markers and reduced differentiation. RNA-seq analysis revealed that CHRDL2 increased the expression of stem cell markers and well-established cancer-associated pathways. We suggest that CHRDL2 overexpression can augment the stem-cell potential of CRC and normal intestinal cells.

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    Reply to the reviewers

    We thank the reviewers for taking the time to read and comprehensively evaluate our manuscript. We are pleased that, overall, they recognize the quality of our data and that it supports our conclusions. We are grateful for their comments, insights and advice and have revised the manuscript accordingly as described in the point-by-point response below. We believe that the revised manuscript is substantially improved by some experimental additions, additional replicates, improved analysis and increased clarity. Some key enhancements are as follows:

    Previously we had found increased expression of the WNT pathway following CHRDL2 treatment, using RNA seq. We have now demonstrated this experimentally using the cellular levels and localisation of β-catenin. Previously we had shown that overexpression of CHRDL2 increased resistance to common chemotherapy treatments, as well as irradiation in colorectal cell lines. We have now shown that cells surviving treatment show a further reduction SMAD1/5/8 phosphorylation indicating a selection for CHRLD2 high cells during the treatment. We have also demonstrated a decrease in chemotherapy sensitivity in intestinal organoids treated with secreted forms of CHRDL2.

    1. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Reviewer #1

    __Evidence, reproducibility and clarity __

    Clarkson and Lewis present data suggesting that overexpression of Chordin like 2 (CHRDL2) can affect colorectal cancer cell responses to chemotherapy agents, possibly by modulating stem-cell like pathways. I have the following comments:

    1. Fig. 1J-it is standard to show the images of cell migration-this is important here, given the modest effect of CHRDL2 overexpression here.

    We have now included 3 replicate control and CHRDL2 overexpressing cell images in this figure panel to support the quantification in the graph.

    Fig. 2A-the very small error bars for most of the data on the curves suggests these are n=1 experiments with multiple technical replicates to generate the error bars. Please clarify. The legend says n=3 with ANOVA analysis but no significance detected. Please clarify.

    All experiments in this figure were done with 5 technical replicates per experiment, this was replicated at least three times to give n=3 biological replicates. The error bars represent the standard error of the mean of these 3 biological replicates as stated in the legend. Some data points showed very little data variation, hence the small error bars. Raw data is available if requested.

    1. Fig. 2B-given the overlapping error bars here, how can there be a pWe have removed this representation of the data as it combined many different experiments with variable cell types and chemotherapeutics and it was difficult to carry out meaningful statistics. An overview of the data can be better seen in table form as shown in the revised figure 2B.

    Fig. 2G-did the authors try to estimate the concentration of CHRDL2 in the conditioned medium? Which cell line was used to generate this CM?

    Conditioned media was harvested from the matching transgenic cell lines with inducible CHRDL2. eg RKO cells were treated with media collected from doxycycline induced transgenic RKO cells whereas CaCO2 cells were treated with media from CaCO2 cells. The concentration of doxycycline was represented by ++ for 10ug/ml, the same notation we have used for directly induced cells treated with 10ug/ml dox.

    We did not try to quantify the absolute concentration of CHRDL2 but we have shown the relative amount on a Western blot normalised with a ponceau stain (quantification now included in supplementary figure 1).

    We have clarified our description of this experiment, inserting the following statement, "Conditioned media was harvested from corresponding cell lines with the inducible CHRDL2 transgene and the parental control cells. Induction of CHRDL2 to generate conditioned media was carried out using the same concentration and duration of doxycycline treatment as the cells in figure 2A. "

    Fig. 5-what is the potential mechanism for gene expression changes in response to CHRDL2 overexpression? Is it all due to BMP inhibition? More mechanistic detail would be welcome here.

    *We have suggested other pathways involved in these functional effects based on our RNA seq data but at the moment it is not possible to say whether any changes are independent of BMP signaling. CHRDL2 is relatively understudied and as yet there is not much literature supporting BMP independent actions of CHRDL2. However, we have added some discussion and reference to an article suggesting interactions between CHRLD2 and YAP (Wang et al., 2022) including the following statement on page 17: *"While the changes in BMP and WNT signaling shown in our GSEA analysis suggest that the effects of CHRDL2 in our system work directly through inhibition of BMP, it is not possible to rule out that some pathways are affected by BMP independent actions of CHRLD2. Indeed, Wang et al, suggest that CHRDL2 can directly alter phosphorylation and activity of YAP in gastric cancer cell lines, which merits further exploration (Wang et al., 2022)"

    Significance

    Unclear whether genetically engineered inducible overexpression has any real physiological significance but we all use cell models so this is OK.

    Reviewer #2

    __Evidence, reproducibility and clarity __

    Summary: In the manuscript entitled "BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer" the authors demonstrated that Chordin-like 2 (CHDRL2), a secreted BMP antagonist, promotes a chemo-resistant colorectal cancer stem cell phenotype through the inhibition of BMP signaling. The authors took advantage of both 2D engineered colorectal cancer (CRC) cells and healthy murine 3D organoid systems. Specifically, the authors showed a decreased proliferation rate and reduced clonogenic capability upon overexpression of CHRDL2 in established human colon cancer cell lines. Subsequently, they identified a chemo-resistant phenotype upon standard therapies (5FU, Oxaliplatin and Irinotecan) in CHDRL2 overexpressing cells by performing MTS assay. The authors showed that this chemo-resistant phenotype is associated with ATM and RAD21 activation, supporting an induction of DNA damage signaling pathway. Of note, the authors assessed that the exposure of 3D murine organoid to CHRDL2 resulted in a stem-like phenotype induction accompanied by a reduction of the differentiated counterpart. From RNA-seq data analysis emerged the upregulation of genes associated to stemness and DNA repair pathways in CHRDL2 overexpressing cells.

    Major comments:

    1. In the first paragraph of the result section authors assessed that "Colorectal adenocarcinoma cell lines were deliberately chosen to encompass a range of CHRDL2 expression levels and genetic mutations", without showing qRT-PCR or WB data on the differential expression levels of CHRDL2 in a panel of immortalized CRC cell lines. Authors should include these data to better support their choice.

    *We have now included some qRT-PCR in supplementary figure 1 alongside a table of some of the key driver mutations in each cell line. Western blotting of these cells shows only a very low concentration of CHRDL2 protein. As shown in figure 1B in the control columns, no significant protein expression is observed in any line. *

    In Figure 1F, authors described a reduction of cell proliferation in CRC cell lines expressing high levels of CHRDL2 only under low glucose conditions. Why did the authors perform the assay under these conditions? They should better argue this aspect and validated the role of CHRDL2 in metabolism rewiring by performing additional in vitro assays.

    We have removed this aspect of the paper as it does not add significantly to our overall conclusions and we can clearly see the effects of CHRDL2 overexpression under standard growth conditions (Figure 1G).

    The authors should evaluate the role of CHRDL2 in promoting a stem-like phenotype in human colon cancer stem cells freshly isolated from patients and characterized.

    We would very much like to do experiments such as this but it is beyond the scope of this study and will be included in upcoming grant proposals.

    In order to confirm the data obtained on 3D murine organoids system obtained from normal Intestinal Stem Cells, authors should investigate the stemness induction, driven by CHRDL2, also in human intestinal organoids.

    Experiments using human intestinal organoids are currently planned and ethical approval applications and grant proposals are underway for future experiments of this nature.

    The authors should evaluate the oncogenic role of CHRDL2, through the maintenance of stemness, by performing orthotopic or subcutaneous experiments in vivo model.

    Similarly, this is not possible for this manuscript but is planned for the future alongside a transgenic mouse model of inducible CHRDL2 overexpression in the intestine.

    BMPs proteins are part of a very broad protein family. In the introduction section, authors should indicate the specific BMP protein on which CHRDL2 exerts its inhibitory function. Moreover, they should have assessed BMP protein levels in CACO2, LS180, COLO320 and RKO cell lines.

    We have clarified the interactions between CHRDL2 and specific BMPs in the introduction. We have not specifically assessed the BMP protein levels in our cells however we have now included an analysis of expression data from the Cancer Cell Line Encyclopedia in supplementary figure 1 C.

    In first panel, the authors should quantify the secreted levels of CHRDL2 in the media of overexpressing CHRDL2 cell lines.

    We have done this using the ponceau staining as a loading control and the results are displayed (supplementary figure 1).

    In Figure 2D the authors should use the appropriate controls and describe this with more details in results section.

    *In this figure we have used Hoechst staining followed by FACs analysis to identify the cell cycle profile of our CHRDL2 treated cells. We have improved the description of this in the methods section. Appropriate controls for staining, both negative and positive, are used when setting up the analysis for this experiment. The cell cycle profile is calculated using the Novocyte in house software. *We have now included the histogram plots in the main figure to clarify these data in figure 2D.

    In Figure 3A, the authors should have performed the assay by choosing IC50.

    *We attempted these experiments with the IC50 levels, however the high amount of cell death and frequency of apoptotic cells meant that clear images were difficult to obtain. We therefore reduced the concentrations and still had very measurable effects. *

    In Supplementary Fig. 4A-B. the results are unclear. The control cell lines are already chemo resistant.

    Again, we used IC25 levels of the drugs so that our cells were damaged but still live throughout the experiment. This has been explained on page 10.

    The authors should review and add statistical analysis in both main and supplementary figures.

    *We have now added additional details about statistical analysis throughout the figures, legend and main text, showing all significance levels as well as non-significance for each data set.

    Minor comments:

    1. The quality of immunofluorescence and WB images should be implemented, and in the immunofluorescence panels scale bars should be added.

    We have added or improved scale bars on each immunofluorescence image. Western blot images have been improved.

    In the graphical abstract authors reported that CHRDL2 overexpression increase WNT and EMT pathways, without performing any specific assay to demonstrate this. Authors should correct and graphically improve the graphical abstract.

    *This is a good point and we have now carried out Beta-catenin immunofluorescence as a measure of WNT signaling on both our cancer cell lines - showing an increase in nuclear beta-catenin (figure 1J and K), and our organoids - showing an increase in overall levels and cytoplasmic staining (Figure 4 F). In terms of EMT markers we have carried out immunofluorescence on IQGAP1 (Figure 1I). IQGAP1 is significantly upregulated in CHRDL2 cells, reflecting its role in reduced cell adhesion and increased migration. This correlates with our data showing increased cellular migration as well as the increase in EMT related transcription in our RNAseq data. *

    The term "significantly" in the discussion section is inappropriately referred to data showed in the histogram in Figure 1J. Moreover, in Figure 1Jthe authors should delete from the y-axis the term "corrected".

    We have changed significantly to substantially

    The term "significant" in discussion is inappropriately referred to BMI1 expression level if compared to the histogram in Figure 4G.

    We have changed significantly to "a trend to increase"

    In Figure 2C the authors should add the unit of measurement (fold over control) in the table.

    We have done this

    In Figure 4E the authors should add the figure legend reporting OLFM4 protein.

    We have done this

    The authors should include few sentences summarizing the findings at the end of each paragraph.

    We have added short summaries at the start or end of each section to improve the flow of the results section.

    Significance

    General assessment: Overall, the work is aimed to elucidate the role of CHRDL2 already considered a poor prognosis biomarker involved in the promotion of CRC (PMID: 28009989), in promoting stem-like properties. The authors elucidated new additional insights into the molecular mechanisms regulating stemness phenotype induced by the BMP antagonist CHRDL2 in CRC. The authors include in the study a large amount of data, which only partially support their hypothesis. However, this manuscript lacks organization and coherence, making it challenging to follow and read. Numerous concerns need to be addressed, along with some sentences to rephrase in the result and discussion sections.

    Advance: The manuscript reported some functional insights on the role of CHRDL2 in colorectal cancer, but additional data should be added to support authors 'conclusions.

    Audience: The manuscript is suggested for basic research scientists.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)):____ __ Summary BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer Eloise Clarkson et al. The manuscript explored the function of CHRDL2, a BMP antagonist, on colorectal cancer (CRC). The authors found that CHRDL2 overexpression can enhance the survival of CRC cells during chemotherapy and irradiation treatment with elevated levels of stem-cell markers and reduced differentiation. Further RNA-seq analysis revealed that CHRDL2 increased the expression of stem-cell markers, WNT signaling and other well-established cancer-associated pathways. Overall, the manuscript is well-written and presented. I have some suggestions:

    Major points:

    1. The authors assert BMP antagonism was demonstrated by assessing levels of phosphorylated SMAD1/5 (Figure 1G). However, the immunoblotting assay only depicted P-SMAD 1/5 levels and B-ACTIN as internal control. It's suggested to include total-SMAD1/5 immunoblotting as an internal control to further support the claim of BMP antagonism.

    The reviewer is correct that this is the best control. Western blotting has now been performed with total SMAD1 protein expression used as an internal control and this is shown in Figure 1D and Supplementary figure 1F

    The authors argue that CHRDL2 overexpression reduced the proliferation of CRC cell lines, as evidenced by cell proliferation assays. However, from Figure 1E, the reduction in proliferation appears insignificant. It would be beneficial to perform one-way ANOVA tests on each time point for CHRDL2+ and CHRDL2++ with Control in Figure 1E to ascertain significance.

    *We now have repeated this experiment to reduce variability and have also provided two-way ANOVA analysis between Control and CHRDL2+ and Control and CHRDL2++. One-way ANOVA at timepoint 96hr also provided with details in the figure legend. *

    The findings indicating that overexpressing CHRDL2 can confer resistance to chemotherapy in CRC cells (Figure 2A-C) are noteworthy. To deepen the understanding of BMP signaling in cancer stemness and the molecular underpinning of CHRDL2 antagonism, additional western blot assays on P-SMAD1/5 with CHRDL2 overexpression and drug treatment are recommended.

    *Western blotting of P-SMAD1/5 upon cells treated with IC50 5FU has now been performed in figure 2C (in the same experiment as the revised panels in figure 1D). The data suggest that CHRDL2 overexpressing cells able to survive chemotherapy have higher levels of P-SMAD1/5 reduction compared to that of untreated cells, strongly suggesting that chemotherapy treatment acts to select the cells with the highest CHRDL2 expression. We thank reviewer 3 for this suggested experiment and have included further discussion on this on page 8. *

    The assertion that extrinsic CHRDL2 addition diminishes differentiation and enhances stem-cell numbers in an intestinal organoid model is intriguing. As BMP signaling inhibition contributes to intestinal cell stemness, incorporating additional layers for BMP antagonism of CHRDL2 on intestinal organoids through immunoblotting or real-time quantitative PCR for treated organoids would augment the conclusions.

    As stated in the response to reviewer 2, we have investigated Beta-catenin in our organoids following CHRDL2 treatment using immunofluorescence and find that the levels are increased with the staining shifting from the membrane to the cytoplasm and nucleus (Figure 4F).

    The authors claim CHRDL2 overexpression can decrease BMP signaling based on GSEA analysis (Figure 5E). However, the GSEA results did not demonstrate the downregulation of BMP signaling. Reanalysis of this GSEA analysis is warranted.

    *We agree with this point and have changed the description of this result since the gene set covers both positive and negative regulators of the BMP pathway. We cannot conclusively say from this RNAseq data set that BMP signaling is "downregulated", however since SMAD phosphorylation is increased and nuclear beta-catenin is increased, overall we suggest that the changes we see are likely to represent the effects of decreased BMP signaling along with increased WNT signaling. *

    Minor Points:

    6.Provide the threshold/cutoff values chosen for differential expressed genes (DEGs) in CHRDL2+ and CHRDL2++ RNA-seq compared with control cells. Explain the minimal overlap between CHRDL2 LOW and CHRDL2 HIGH DEGs. Consider presenting all DEGs in CHRDL2 LOW and CHRDL2 HIGH compared with control cells in one gene expression heatmap for better visualization.

    We have now provided the cutoff values for the DEGs in the legend for figure 5 (PThe minimal overlap of DEGs in the low and high expressing cells is an interesting point. We hypothesize that this may be related to the different effects of intermediate vs high levels of WNT signaling that occurs in colon cancer cells, frequently discussed in the literature as the "Just right hypothesis" (Lamlum et al. 1999, Albuquerque et al., 2002, Lewis et al., 2010). However, we haven't included this in the discussion as it merits further exploration. However, we have mainly focused on specific genes that are modified in both data sets, which are more likely to be the direct result of CHRDL2 modification. *

    After DEGs analysis, perform Gene Ontology (GO) analysis on these DEGs to further investigate possible gene functions rather than selectively discussing some genes, enhancing understanding of CHRDL2 functions in CRC cells.

    We have carried out this analysis using a variety of tools and have now included a Gene Ontology Panther analysis as supplementary figure 7. We have included a comment on this in the text on page 14 saying "Gene ontology analysis supports these findings with enrichment in biological processes such as cellular adhesion, apoptosis and differentiation. "

    Conduct similar experiments in both 2D culture and organoid systems, if feasible, to provide more comprehensive insights into CHRDL2's oncogenic roles in CRC tumor progression.

    *We have now performed chemotherapy treatment on our organoid systems, and have found that organoids with extrinsic CHRDL2 addition have a higher survival rate after chemotherapy compared to a control (Figure 4H and I). *

    Label significance (*, **, ***, and n.s.) for every CRC cell line treated with CHRDL2 in Figure 2D, 2F, 2J, 4G, 5D, and 5F.

    We have done this

    Label the antibodies with different colors used for immunofluorescence in the figure text in Figure 4E.

    We have done this

    • Include replicate dots for the Control group in the bar plots in Figure 1F and 2B.

    We have done this

    • Add scale bars in Figure 3A and correct similar issues in other figures if applicable.

    We have done this

    *13.Correct grammar and punctuation mistakes throughout the manuscript. For example:

    We have done this and further proofread our revised manuscript

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P *We have now added additional details about statistical analysis throughout the figures, legend and main text, showing all significance levels as well as non-significance for each data set.

    Reviewer #3 (Significance (Required)):

    The current study presents compelling evidence demonstrating that BMP signaling antagonist CHRDL2 enhances colon stem cell survival in colorectal cancer cell lines and organoid models. Further validation through CRC mouse models could offer invaluable insights into the clinical relevance and therapeutic implications of CHRDL2 in colorectal cancer.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer Eloise Clarkson et al. The manuscript explored the function of CHRDL2, a BMP antagonist, on colorectal cancer (CRC). The authors found that CHRDL2 overexpression can enhance the survival of CRC cells during chemotherapy and irradiation treatment with elevated levels of stem-cell markers and reduced differentiation. Further RNA-seq analysis revealed that CHRDL2 increased the expression of stem-cell markers, WNT signaling and other well-established cancer-associated pathways. Overall, the manuscript is well-written and presented. I have some suggestions:

    Major points:

    1. The authors assert BMP antagonism was demonstrated by assessing levels of phosphorylated SMAD1/5 (Figure 1G). However, the immunoblotting assay only depicted P-SMAD 1/5 levels and B-ACTIN as internal control. It's suggested to include total-SMAD1/5 immunoblotting as an internal control to further support the claim of BMP antagonism.
    2. The authors argue that CHRDL2 overexpression reduced the proliferation of CRC cell lines, as evidenced by cell proliferation assays. However, from Figure 1E, the reduction in proliferation appears insignificant. It would be beneficial to perform one-way ANOVA tests on each time point for CHRDL2+ and CHRDL2++ with Control in Figure 1E to ascertain significance.
    3. The findings indicating that overexpressing CHRDL2 can confer resistance to chemotherapy in CRC cells (Figure 2A-C) are noteworthy. To deepen the understanding of BMP signaling in cancer stemness and the molecular underpinning of CHRDL2 antagonism, additional western blot assays on P-SMAD1/5 with CHRDL2 overexpression and drug treatment are recommended.
    4. The assertion that extrinsic CHRDL2 addition diminishes differentiation and enhances stem-cell numbers in an intestinal organoid model is intriguing. As BMP signaling inhibition contributes to intestinal cell stemness, incorporating additional layers for BMP antagonism of CHRDL2 on intestinal organoids through immunoblotting or real-time quantitative PCR for treated organoids would augment the conclusions.
    5. The authors claim CHRDL2 overexpression can decrease BMP signaling based on GSEA analysis (Figure 5E). However, the GSEA results did not demonstrate the downregulation of BMP signaling. Reanalysis of this GSEA analysis is warranted.

    Minor Points:

    6.Provide the threshold/cutoff values chosen for differential expressed genes (DEGs) in CHRDL2+ and CHRDL2++ RNA-seq compared with control cells. Explain the minimal overlap between CHRDL2 LOW and CHRDL2 HIGH DEGs. Consider presenting all DEGs in CHRDL2 LOW and CHRDL2 HIGH compared with control cells in one gene expression heatmap for better visualization.

    1. After DEGs analysis, perform Gene Ontology (GO) analysis on these DEGs to further investigate possible gene functions rather than selectively discussing some genes, enhancing understanding of CHRDL2 functions in CRC cells.
    2. Conduct similar experiments in both 2D culture and organoid systems, if feasible, to provide more comprehensive insights into CHRDL2's oncogenic roles in CRC tumor progression.
    3. Label significance (*, **, ***, and n.s.) for every CRC cell line treated with CHRDL2 in Figure 2D, 2F, 2J, 4G, 5D, and 5F.
    4. Label the antibodies with different colors used for immunofluorescence in the figure text in Figure 4E.
    5. Include replicate dots for the Control group in the bar plots in Figure 1F and 2B.
    6. Add scale bars in Figure 3A and correct similar issues in other figures if applicable. 13.Correct grammar and punctuation mistakes throughout the manuscript. For example:

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P < 0.0449)," suggesting increased migratory ability, a hallmark of cancer stem cells."

    Page 8: "CHRDL2 overexpression resulted in an approximate twofold increase in IC50 values compared to control cells (P < 0.001)."

    Page 10: "As seen in Figure 4B, upon the" should be corrected to "Figure 4B."

    1. Specify the statistical methods or estimates used for determining statistical significance.

    Significance

    The current study presents compelling evidence demonstrating that BMP signaling antagonist CHRDL2 enhances colon stem cell survival in colorectal cancer cell lines and organoid models. Further validation through CRC mouse models could offer invaluable insights into the clinical relevance and therapeutic implications of CHRDL2 in colorectal cancer.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In the manuscript entitled "BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer" the authors demonstrated that Chordin-like 2 (CHDRL2), a secreted BMP antagonist, promotes a chemo-resistant colorectal cancer stem cell phenotype through the inhibition of BMP signaling. The authors took advantage of both 2D engineered colorectal cancer (CRC) cells and healthy murine 3D organoid systems. Specifically, the authors showed a decreased proliferation rate and reduced clonogenic capability upon overexpression of CHRDL2 in established human colon cancer cell lines. Subsequently, they identified a chemo-resistant phenotype upon standard therapies (5FU, Oxaliplatin and Irinotecan) in CHDRL2 overexpressing cells by performing MTS assay. The authors showed that this chemo-resistant phenotype is associated with ATM and RAD21 activation, supporting an induction of DNA damage signaling pathway. Of note, the authors assessed that the exposure of 3D murine organoid to CHRDL2 resulted in a stem-like phenotype induction accompanied by a reduction of the differentiated counterpart. From RNA-seq data analysis emerged the upregulation of genes associated to stemness and DNA repair pathways in CHRDL2 overexpressing cells.

    Major comments:

    1. In the first paragraph of the result section authors assessed that "Colorectal adenocarcinoma cell lines were deliberately chosen to encompass a range of CHRDL2 expression levels and genetic mutations", without showing qRT-PCR or WB data on the differential expression levels of CHRDL2 in a panel of immortalized CRC cell lines. Authors should include these data to better support their choice.
    2. In Figure 1F, authors described a reduction of cell proliferation in CRC cell lines expressing high levels of CHRDL2 only under low glucose conditions. Why did the authors perform the assay under these conditions? They should better argue this aspect and validated the role of CHRDL2 in metabolism rewiring by performing additional in vitro assays.
    3. The authors should evaluate the role of CHRDL2 in promoting a stem-like phenotype in human colon cancer stem cells freshly isolated from patients and characterized.
    4. In order to confirm the data obtained on 3D murine organoids system obtained from normal Intestinal Stem Cells, authors should investigate the stemness induction, driven by CHRDL2, also in human intestinal organoids.
    5. The authors should evaluate the oncogenic role of CHRDL2, through the maintenance of stemness, by performing orthotopic or subcutaneous experiments in vivo model.
    6. BMPs proteins are part of a very broad protein family. In the introduction section, authors should indicate the specific BMP protein on which CHRDL2 exerts its inhibitory function. Moreover, they should have assessed BMP protein levels in CACO2, LS180, COLO320 and RKO cell lines.
    7. In first panel, the authors should quantify the secreted levels of CHRDL2 in the media of overexpressing CHRDL2 cell lines.
    8. In Figure 2D the authors should use the appropriate controls and describe this with more details in results section.
    9. In Figure 3A, the authors should have performed the assay by choosing IC50.
    10. In Supplementary Fig. 4A-B. the results are unclear. The control cell lines are already chemoresistant.
    11. The authors should review and add statistical analysis in both main and supplementary figures.

    Minor comments:

    1. The quality of immunofluorescence and WB images should be implemented, and in the immunofluorescence panels scale bars should be added.
    2. In the graphical abstract authors reported that CHRDL2 overexpression increase WNT and EMT pathways, without performing any specific assay to demonstrate this. Authors should correct and graphically improve the graphical abstract.
    3. The term "significantly" in the discussion section is inappropriately referred to data showed in the histogram in Figure 1J. Moreover, in Figure 1Jthe authors should delete from the y-axis the term "corrected".
    4. The term "significant" in discussion is inappropriately referred to BMI1 expression level if compared to the histogram in Figure 4G.
    5. In Figure 2C the authors should add the unit of measurement (fold over control) in the table.
    6. In Figure 4E the authors should add the figure legend reporting OLFM4 protein.
    7. The authors should include few sentences summarizing the findings at the end of each paragraph.

    Significance

    General assessment:

    Overall, the work is aimed to elucidate the role of CHRDL2 already considered a poor prognosis biomarker involved in the promotion of CRC (PMID: 28009989), in promoting stem-like properties. The authors elucidated new additional insights into the molecular mechanisms regulating stemness phenotype induced by the BMP antagonist CHRDL2 in CRC. The authors include in the study a large amount of data, which only partially support their hypothesis. However, this manuscript lacks organization and coherence, making it challenging to follow and read. Numerous concerns need to be addressed, along with some sentences to rephrase in the result and discussion sections.

    Advance: The manuscript reported some functional insights on the role of CHRDL2 in colorectal cancer, but additional data should be added to support authors 'conclusions.

    Audience: The manuscript is suggested for basic research scientists.

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    Referee #1

    Evidence, reproducibility and clarity

    Clarkson and Lewis present data suggesting that overexpression of Chordin like 2 (CHRDL2) can affect colorectal cancer cell responses to chemotherapy agents, possibly by modulating stem-cell like pathways. I have the following comments:

    1. Fig. 1J-it is standard to show the images of cell migration-this is important here, given the modest effect of CHRDL2 overexpression here.
    2. Fig. 2A-the very small error bars for most of the data on the curves suggests these are n=1 experiments with multiple technical replicates to generate the error bars. Please clarify. The legend says n=3 with ANOVA analysis but no significance detected. Please clarify.
    3. Fig. 2B-given the overlapping error bars here, how can there be a p<0.01 between the groups?
    4. Fig. 2G-did the authors try to estimate the concentration of CHRDL2 in the conditioned medium? Which cell line was used to generate this CM?
    5. Fig. 5-what is the potential mechanism for gene expression changes in response to CHRDL2 overexpression? Is it all due to BMP inhibition? More mechanistic detail would be welcome here.

    Significance

    Unclear whether genetically engineered inducible overexpression has any real physiological significance but we all use cell models so this is OK.

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    Reply to the reviewers

    We thank the reviewers for taking the time to read and comprehensively evaluate our manuscript. We are pleased that, overall, they recognize the quality of our data and that it supports our conclusions. We are grateful for their comments, insights and advice and have revised the manuscript accordingly as described in the point-by-point response below. We believe that the revised manuscript is substantially improved by some experimental additions, additional replicates, improved analysis and increased clarity. Some key enhancements are as follows:

    Previously we had found increased expression of the WNT pathway following CHRDL2 treatment, using RNA seq. We have now demonstrated this experimentally using the cellular levels and localisation of β-catenin. Previously we had shown that overexpression of CHRDL2 increased resistance to common chemotherapy treatments, as well as irradiation in colorectal cell lines. We have now shown that cells surviving treatment show a further reduction SMAD1/5/8 phosphorylation indicating a selection for CHRLD2 high cells during the treatment. We have also demonstrated a decrease in chemotherapy sensitivity in intestinal organoids treated with secreted forms of CHRDL2.

    1. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Reviewer #1

    __Evidence, reproducibility and clarity __

    Clarkson and Lewis present data suggesting that overexpression of Chordin like 2 (CHRDL2) can affect colorectal cancer cell responses to chemotherapy agents, possibly by modulating stem-cell like pathways. I have the following comments:

    1. Fig. 1J-it is standard to show the images of cell migration-this is important here, given the modest effect of CHRDL2 overexpression here.

    We have now included 3 replicate control and CHRDL2 overexpressing cell images in this figure panel to support the quantification in the graph.

    Fig. 2A-the very small error bars for most of the data on the curves suggests these are n=1 experiments with multiple technical replicates to generate the error bars. Please clarify. The legend says n=3 with ANOVA analysis but no significance detected. Please clarify.

    All experiments in this figure were done with 5 technical replicates per experiment, this was replicated at least three times to give n=3 biological replicates. The error bars represent the standard error of the mean of these 3 biological replicates as stated in the legend. Some data points showed very little data variation, hence the small error bars. Raw data is available if requested.

    1. Fig. 2B-given the overlapping error bars here, how can there be a pWe have removed this representation of the data as it combined many different experiments with variable cell types and chemotherapeutics and it was difficult to carry out meaningful statistics. An overview of the data can be better seen in table form as shown in the revised figure 2B.

    Fig. 2G-did the authors try to estimate the concentration of CHRDL2 in the conditioned medium? Which cell line was used to generate this CM?

    Conditioned media was harvested from the matching transgenic cell lines with inducible CHRDL2. eg RKO cells were treated with media collected from doxycycline induced transgenic RKO cells whereas CaCO2 cells were treated with media from CaCO2 cells. The concentration of doxycycline was represented by ++ for 10ug/ml, the same notation we have used for directly induced cells treated with 10ug/ml dox.

    We did not try to quantify the absolute concentration of CHRDL2 but we have shown the relative amount on a Western blot normalised with a ponceau stain (quantification now included in supplementary figure 1).

    We have clarified our description of this experiment, inserting the following statement, "Conditioned media was harvested from corresponding cell lines with the inducible CHRDL2 transgene and the parental control cells. Induction of CHRDL2 to generate conditioned media was carried out using the same concentration and duration of doxycycline treatment as the cells in figure 2A. "

    Fig. 5-what is the potential mechanism for gene expression changes in response to CHRDL2 overexpression? Is it all due to BMP inhibition? More mechanistic detail would be welcome here.

    *We have suggested other pathways involved in these functional effects based on our RNA seq data but at the moment it is not possible to say whether any changes are independent of BMP signaling. CHRDL2 is relatively understudied and as yet there is not much literature supporting BMP independent actions of CHRDL2. However, we have added some discussion and reference to an article suggesting interactions between CHRLD2 and YAP (Wang et al., 2022) including the following statement on page 17: *"While the changes in BMP and WNT signaling shown in our GSEA analysis suggest that the effects of CHRDL2 in our system work directly through inhibition of BMP, it is not possible to rule out that some pathways are affected by BMP independent actions of CHRLD2. Indeed, Wang et al, suggest that CHRDL2 can directly alter phosphorylation and activity of YAP in gastric cancer cell lines, which merits further exploration (Wang et al., 2022)"

    Significance

    Unclear whether genetically engineered inducible overexpression has any real physiological significance but we all use cell models so this is OK.

    Reviewer #2

    __Evidence, reproducibility and clarity __

    Summary: In the manuscript entitled "BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer" the authors demonstrated that Chordin-like 2 (CHDRL2), a secreted BMP antagonist, promotes a chemo-resistant colorectal cancer stem cell phenotype through the inhibition of BMP signaling. The authors took advantage of both 2D engineered colorectal cancer (CRC) cells and healthy murine 3D organoid systems. Specifically, the authors showed a decreased proliferation rate and reduced clonogenic capability upon overexpression of CHRDL2 in established human colon cancer cell lines. Subsequently, they identified a chemo-resistant phenotype upon standard therapies (5FU, Oxaliplatin and Irinotecan) in CHDRL2 overexpressing cells by performing MTS assay. The authors showed that this chemo-resistant phenotype is associated with ATM and RAD21 activation, supporting an induction of DNA damage signaling pathway. Of note, the authors assessed that the exposure of 3D murine organoid to CHRDL2 resulted in a stem-like phenotype induction accompanied by a reduction of the differentiated counterpart. From RNA-seq data analysis emerged the upregulation of genes associated to stemness and DNA repair pathways in CHRDL2 overexpressing cells.

    Major comments:

    1. In the first paragraph of the result section authors assessed that "Colorectal adenocarcinoma cell lines were deliberately chosen to encompass a range of CHRDL2 expression levels and genetic mutations", without showing qRT-PCR or WB data on the differential expression levels of CHRDL2 in a panel of immortalized CRC cell lines. Authors should include these data to better support their choice.

    *We have now included some qRT-PCR in supplementary figure 1 alongside a table of some of the key driver mutations in each cell line. Western blotting of these cells shows only a very low concentration of CHRDL2 protein. As shown in figure 1B in the control columns, no significant protein expression is observed in any line. *

    In Figure 1F, authors described a reduction of cell proliferation in CRC cell lines expressing high levels of CHRDL2 only under low glucose conditions. Why did the authors perform the assay under these conditions? They should better argue this aspect and validated the role of CHRDL2 in metabolism rewiring by performing additional in vitro assays.

    We have removed this aspect of the paper as it does not add significantly to our overall conclusions and we can clearly see the effects of CHRDL2 overexpression under standard growth conditions (Figure 1G).

    The authors should evaluate the role of CHRDL2 in promoting a stem-like phenotype in human colon cancer stem cells freshly isolated from patients and characterized.

    We would very much like to do experiments such as this but it is beyond the scope of this study and will be included in upcoming grant proposals.

    In order to confirm the data obtained on 3D murine organoids system obtained from normal Intestinal Stem Cells, authors should investigate the stemness induction, driven by CHRDL2, also in human intestinal organoids.

    Experiments using human intestinal organoids are currently planned and ethical approval applications and grant proposals are underway for future experiments of this nature.

    The authors should evaluate the oncogenic role of CHRDL2, through the maintenance of stemness, by performing orthotopic or subcutaneous experiments in vivo model.

    Similarly, this is not possible for this manuscript but is planned for the future alongside a transgenic mouse model of inducible CHRDL2 overexpression in the intestine.

    BMPs proteins are part of a very broad protein family. In the introduction section, authors should indicate the specific BMP protein on which CHRDL2 exerts its inhibitory function. Moreover, they should have assessed BMP protein levels in CACO2, LS180, COLO320 and RKO cell lines.

    We have clarified the interactions between CHRDL2 and specific BMPs in the introduction. We have not specifically assessed the BMP protein levels in our cells however we have now included an analysis of expression data from the Cancer Cell Line Encyclopedia in supplementary figure 1 C.

    In first panel, the authors should quantify the secreted levels of CHRDL2 in the media of overexpressing CHRDL2 cell lines.

    We have done this using the ponceau staining as a loading control and the results are displayed (supplementary figure 1).

    In Figure 2D the authors should use the appropriate controls and describe this with more details in results section.

    *In this figure we have used Hoechst staining followed by FACs analysis to identify the cell cycle profile of our CHRDL2 treated cells. We have improved the description of this in the methods section. Appropriate controls for staining, both negative and positive, are used when setting up the analysis for this experiment. The cell cycle profile is calculated using the Novocyte in house software. *We have now included the histogram plots in the main figure to clarify these data in figure 2D.

    In Figure 3A, the authors should have performed the assay by choosing IC50.

    *We attempted these experiments with the IC50 levels, however the high amount of cell death and frequency of apoptotic cells meant that clear images were difficult to obtain. We therefore reduced the concentrations and still had very measurable effects. *

    In Supplementary Fig. 4A-B. the results are unclear. The control cell lines are already chemo resistant.

    Again, we used IC25 levels of the drugs so that our cells were damaged but still live throughout the experiment. This has been explained on page 10.

    The authors should review and add statistical analysis in both main and supplementary figures.

    *We have now added additional details about statistical analysis throughout the figures, legend and main text, showing all significance levels as well as non-significance for each data set.

    Minor comments:

    1. The quality of immunofluorescence and WB images should be implemented, and in the immunofluorescence panels scale bars should be added.

    We have added or improved scale bars on each immunofluorescence image. Western blot images have been improved.

    In the graphical abstract authors reported that CHRDL2 overexpression increase WNT and EMT pathways, without performing any specific assay to demonstrate this. Authors should correct and graphically improve the graphical abstract.

    *This is a good point and we have now carried out Beta-catenin immunofluorescence as a measure of WNT signaling on both our cancer cell lines - showing an increase in nuclear beta-catenin (figure 1J and K), and our organoids - showing an increase in overall levels and cytoplasmic staining (Figure 4 F). In terms of EMT markers we have carried out immunofluorescence on IQGAP1 (Figure 1K). IQGAP1 is significantly upregulated in CHRDL2 cells, reflecting its role in reduced cell adhesion and increased migration. This correlates with our data showing increased cellular migration as well as the increase in EMT related transcription in our RNAseq data. *

    The term "significantly" in the discussion section is inappropriately referred to data showed in the histogram in Figure 1J. Moreover, in Figure 1Jthe authors should delete from the y-axis the term "corrected".

    We have changed significantly to substantially

    The term "significant" in discussion is inappropriately referred to BMI1 expression level if compared to the histogram in Figure 4G.

    We have changed significantly to "a trend to increase"

    In Figure 2C the authors should add the unit of measurement (fold over control) in the table.

    We have done this

    In Figure 4E the authors should add the figure legend reporting OLFM4 protein.

    We have done this

    The authors should include few sentences summarizing the findings at the end of each paragraph.

    We have added short summaries at the start or end of each section to improve the flow of the results section.

    Significance

    General assessment: Overall, the work is aimed to elucidate the role of CHRDL2 already considered a poor prognosis biomarker involved in the promotion of CRC (PMID: 28009989), in promoting stem-like properties. The authors elucidated new additional insights into the molecular mechanisms regulating stemness phenotype induced by the BMP antagonist CHRDL2 in CRC. The authors include in the study a large amount of data, which only partially support their hypothesis. However, this manuscript lacks organization and coherence, making it challenging to follow and read. Numerous concerns need to be addressed, along with some sentences to rephrase in the result and discussion sections.

    Advance: The manuscript reported some functional insights on the role of CHRDL2 in colorectal cancer, but additional data should be added to support authors 'conclusions.

    Audience: The manuscript is suggested for basic research scientists.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)):____ __ Summary BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer Eloise Clarkson et al. The manuscript explored the function of CHRDL2, a BMP antagonist, on colorectal cancer (CRC). The authors found that CHRDL2 overexpression can enhance the survival of CRC cells during chemotherapy and irradiation treatment with elevated levels of stem-cell markers and reduced differentiation. Further RNA-seq analysis revealed that CHRDL2 increased the expression of stem-cell markers, WNT signaling and other well-established cancer-associated pathways. Overall, the manuscript is well-written and presented. I have some suggestions:

    Major points:

    1. The authors assert BMP antagonism was demonstrated by assessing levels of phosphorylated SMAD1/5 (Figure 1G). However, the immunoblotting assay only depicted P-SMAD 1/5 levels and B-ACTIN as internal control. It's suggested to include total-SMAD1/5 immunoblotting as an internal control to further support the claim of BMP antagonism.

    The reviewer is correct that this is the best control. Western blotting has now been performed with total SMAD1 protein expression used as an internal control and this is shown in Figure 1D and Supplementary figure 1F

    The authors argue that CHRDL2 overexpression reduced the proliferation of CRC cell lines, as evidenced by cell proliferation assays. However, from Figure 1E, the reduction in proliferation appears insignificant. It would be beneficial to perform one-way ANOVA tests on each time point for CHRDL2+ and CHRDL2++ with Control in Figure 1E to ascertain significance.

    *We now have repeated this experiment to reduce variability and have also provided two-way ANOVA analysis between Control and CHRDL2+ and Control and CHRDL2++. One-way ANOVA at timepoint 96hr also provided with details in the figure legend. *

    The findings indicating that overexpressing CHRDL2 can confer resistance to chemotherapy in CRC cells (Figure 2A-C) are noteworthy. To deepen the understanding of BMP signaling in cancer stemness and the molecular underpinning of CHRDL2 antagonism, additional western blot assays on P-SMAD1/5 with CHRDL2 overexpression and drug treatment are recommended.

    *Western blotting of P-SMAD1/5 upon cells treated with IC50 5FU has now been performed in figure 2C (in the same experiment as the revised panels in figure 1D). The data suggest that CHRDL2 overexpressing cells able to survive chemotherapy have higher levels of P-SMAD1/5 reduction compared to that of untreated cells, strongly suggesting that chemotherapy treatment acts to select the cells with the highest CHRDL2 expression. We thank reviewer 3 for this suggested experiment and have included further discussion on this on page 9. *

    The assertion that extrinsic CHRDL2 addition diminishes differentiation and enhances stem-cell numbers in an intestinal organoid model is intriguing. As BMP signaling inhibition contributes to intestinal cell stemness, incorporating additional layers for BMP antagonism of CHRDL2 on intestinal organoids through immunoblotting or real-time quantitative PCR for treated organoids would augment the conclusions.

    As stated in the response to reviewer 2, we have investigated Beta-catenin in our organoids following CHRDL2 treatment using immunofluorescence and find that the levels are increased with the staining shifting from the membrane to the cytoplasm and nucleus (Figure 4F).

    The authors claim CHRDL2 overexpression can decrease BMP signaling based on GSEA analysis (Figure 5E). However, the GSEA results did not demonstrate the downregulation of BMP signaling. Reanalysis of this GSEA analysis is warranted.

    *We agree with this point and have changed the description of this result since the gene set covers both positive and negative regulators of the BMP pathway. We cannot conclusively say from this RNAseq data set that BMP signaling is "downregulated", however since SMAD phosphorylation is increased and nuclear beta-catenin is increased, overall we suggest that the changes we see are likely to represent the effects of decreased BMP signaling along with increased WNT signaling. *

    Minor Points:

    6.Provide the threshold/cutoff values chosen for differential expressed genes (DEGs) in CHRDL2+ and CHRDL2++ RNA-seq compared with control cells. Explain the minimal overlap between CHRDL2 LOW and CHRDL2 HIGH DEGs. Consider presenting all DEGs in CHRDL2 LOW and CHRDL2 HIGH compared with control cells in one gene expression heatmap for better visualization.

    We have now provided the cutoff values for the DEGs in the legend for figure 5 (PThe minimal overlap of DEGs in the low and high expressing cells is an interesting point. We hypothesize that this may be related to the different effects of intermediate vs high levels of WNT signaling that occurs in colon cancer cells, frequently discussed in the literature as the "Just right hypothesis" (Lamlum et al. 1999, Albuquerque et al., 2002, Lewis et al., 2010). However, we haven't included this in the discussion as it merits further exploration. However, we have mainly focused on specific genes that are modified in both data sets, which are more likely to be the direct result of CHRDL2 modification. *

    After DEGs analysis, perform Gene Ontology (GO) analysis on these DEGs to further investigate possible gene functions rather than selectively discussing some genes, enhancing understanding of CHRDL2 functions in CRC cells.

    We have carried out this analysis using a variety of tools and have now included a Gene Ontology Panther analysis as supplementary figure 7. We have included a comment on this in the text on page 14 saying "Gene ontology analysis supports these findings with enrichment in biological processes such as cellular adhesion, apoptosis and differentiation. "

    Conduct similar experiments in both 2D culture and organoid systems, if feasible, to provide more comprehensive insights into CHRDL2's oncogenic roles in CRC tumor progression.

    *We have now performed chemotherapy treatment on our organoid systems, and have found that organoids with extrinsic CHRDL2 addition have a higher survival rate after chemotherapy compared to a control (Figure 4H and I). *

    Label significance (*, **, ***, and n.s.) for every CRC cell line treated with CHRDL2 in Figure 2D, 2F, 2J, 4G, 5D, and 5F.

    We have done this

    Label the antibodies with different colors used for immunofluorescence in the figure text in Figure 4E.

    We have done this

    • Include replicate dots for the Control group in the bar plots in Figure 1F and 2B.

    We have done this

    • Add scale bars in Figure 3A and correct similar issues in other figures if applicable.

    We have done this

    *13.Correct grammar and punctuation mistakes throughout the manuscript. For example:

    We have done this and further proofread our revised manuscript

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P *We have now added additional details about statistical analysis throughout the figures, legend and main text, showing all significance levels as well as non-significance for each data set.

    Reviewer #3 (Significance (Required)):

    The current study presents compelling evidence demonstrating that BMP signaling antagonist CHRDL2 enhances colon stem cell survival in colorectal cancer cell lines and organoid models. Further validation through CRC mouse models could offer invaluable insights into the clinical relevance and therapeutic implications of CHRDL2 in colorectal cancer.

  6. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer Eloise Clarkson et al. The manuscript explored the function of CHRDL2, a BMP antagonist, on colorectal cancer (CRC). The authors found that CHRDL2 overexpression can enhance the survival of CRC cells during chemotherapy and irradiation treatment with elevated levels of stem-cell markers and reduced differentiation. Further RNA-seq analysis revealed that CHRDL2 increased the expression of stem-cell markers, WNT signaling and other well-established cancer-associated pathways. Overall, the manuscript is well-written and presented. I have some suggestions:

    Major points:

    1. The authors assert BMP antagonism was demonstrated by assessing levels of phosphorylated SMAD1/5 (Figure 1G). However, the immunoblotting assay only depicted P-SMAD 1/5 levels and B-ACTIN as internal control. It's suggested to include total-SMAD1/5 immunoblotting as an internal control to further support the claim of BMP antagonism.
    2. The authors argue that CHRDL2 overexpression reduced the proliferation of CRC cell lines, as evidenced by cell proliferation assays. However, from Figure 1E, the reduction in proliferation appears insignificant. It would be beneficial to perform one-way ANOVA tests on each time point for CHRDL2+ and CHRDL2++ with Control in Figure 1E to ascertain significance.
    3. The findings indicating that overexpressing CHRDL2 can confer resistance to chemotherapy in CRC cells (Figure 2A-C) are noteworthy. To deepen the understanding of BMP signaling in cancer stemness and the molecular underpinning of CHRDL2 antagonism, additional western blot assays on P-SMAD1/5 with CHRDL2 overexpression and drug treatment are recommended.
    4. The assertion that extrinsic CHRDL2 addition diminishes differentiation and enhances stem-cell numbers in an intestinal organoid model is intriguing. As BMP signaling inhibition contributes to intestinal cell stemness, incorporating additional layers for BMP antagonism of CHRDL2 on intestinal organoids through immunoblotting or real-time quantitative PCR for treated organoids would augment the conclusions.
    5. The authors claim CHRDL2 overexpression can decrease BMP signaling based on GSEA analysis (Figure 5E). However, the GSEA results did not demonstrate the downregulation of BMP signaling. Reanalysis of this GSEA analysis is warranted.

    Minor Points:

    6.Provide the threshold/cutoff values chosen for differential expressed genes (DEGs) in CHRDL2+ and CHRDL2++ RNA-seq compared with control cells. Explain the minimal overlap between CHRDL2 LOW and CHRDL2 HIGH DEGs. Consider presenting all DEGs in CHRDL2 LOW and CHRDL2 HIGH compared with control cells in one gene expression heatmap for better visualization.

    1. After DEGs analysis, perform Gene Ontology (GO) analysis on these DEGs to further investigate possible gene functions rather than selectively discussing some genes, enhancing understanding of CHRDL2 functions in CRC cells.
    2. Conduct similar experiments in both 2D culture and organoid systems, if feasible, to provide more comprehensive insights into CHRDL2's oncogenic roles in CRC tumor progression.
    3. Label significance (*, **, ***, and n.s.) for every CRC cell line treated with CHRDL2 in Figure 2D, 2F, 2J, 4G, 5D, and 5F.
    4. Label the antibodies with different colors used for immunofluorescence in the figure text in Figure 4E.
    5. Include replicate dots for the Control group in the bar plots in Figure 1F and 2B.
    6. Add scale bars in Figure 3A and correct similar issues in other figures if applicable. 13.Correct grammar and punctuation mistakes throughout the manuscript. For example:

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P

    Page 7: "As seen in Figure 1J, CHRDL2 overexpression significantly increased the number of migrated cells (P < 0.0449)," suggesting increased migratory ability, a hallmark of cancer stem cells."

    Page 8: "CHRDL2 overexpression resulted in an approximate twofold increase in IC50 values compared to control cells (P < 0.001)."

    Page 10: "As seen in Figure 4B, upon the" should be corrected to "Figure 4B."

    1. Specify the statistical methods or estimates used for determining statistical significance.

    Significance

    The current study presents compelling evidence demonstrating that BMP signaling antagonist CHRDL2 enhances colon stem cell survival in colorectal cancer cell lines and organoid models. Further validation through CRC mouse models could offer invaluable insights into the clinical relevance and therapeutic implications of CHRDL2 in colorectal cancer.

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In the manuscript entitled "BMP antagonist CHRDL2 enhances the cancer stem-cell phenotype and increases chemotherapy resistance in Colorectal Cancer" the authors demonstrated that Chordin-like 2 (CHDRL2), a secreted BMP antagonist, promotes a chemo-resistant colorectal cancer stem cell phenotype through the inhibition of BMP signaling. The authors took advantage of both 2D engineered colorectal cancer (CRC) cells and healthy murine 3D organoid systems. Specifically, the authors showed a decreased proliferation rate and reduced clonogenic capability upon overexpression of CHRDL2 in established human colon cancer cell lines. Subsequently, they identified a chemo-resistant phenotype upon standard therapies (5FU, Oxaliplatin and Irinotecan) in CHDRL2 overexpressing cells by performing MTS assay. The authors showed that this chemo-resistant phenotype is associated with ATM and RAD21 activation, supporting an induction of DNA damage signaling pathway. Of note, the authors assessed that the exposure of 3D murine organoid to CHRDL2 resulted in a stem-like phenotype induction accompanied by a reduction of the differentiated counterpart. From RNA-seq data analysis emerged the upregulation of genes associated to stemness and DNA repair pathways in CHRDL2 overexpressing cells.

    Major comments:

    1. In the first paragraph of the result section authors assessed that "Colorectal adenocarcinoma cell lines were deliberately chosen to encompass a range of CHRDL2 expression levels and genetic mutations", without showing qRT-PCR or WB data on the differential expression levels of CHRDL2 in a panel of immortalized CRC cell lines. Authors should include these data to better support their choice.
    2. In Figure 1F, authors described a reduction of cell proliferation in CRC cell lines expressing high levels of CHRDL2 only under low glucose conditions. Why did the authors perform the assay under these conditions? They should better argue this aspect and validated the role of CHRDL2 in metabolism rewiring by performing additional in vitro assays.
    3. The authors should evaluate the role of CHRDL2 in promoting a stem-like phenotype in human colon cancer stem cells freshly isolated from patients and characterized.
    4. In order to confirm the data obtained on 3D murine organoids system obtained from normal Intestinal Stem Cells, authors should investigate the stemness induction, driven by CHRDL2, also in human intestinal organoids.
    5. The authors should evaluate the oncogenic role of CHRDL2, through the maintenance of stemness, by performing orthotopic or subcutaneous experiments in vivo model.
    6. BMPs proteins are part of a very broad protein family. In the introduction section, authors should indicate the specific BMP protein on which CHRDL2 exerts its inhibitory function. Moreover, they should have assessed BMP protein levels in CACO2, LS180, COLO320 and RKO cell lines.
    7. In first panel, the authors should quantify the secreted levels of CHRDL2 in the media of overexpressing CHRDL2 cell lines.
    8. In Figure 2D the authors should use the appropriate controls and describe this with more details in results section.
    9. In Figure 3A, the authors should have performed the assay by choosing IC50.
    10. In Supplementary Fig. 4A-B. the results are unclear. The control cell lines are already chemoresistant.
    11. The authors should review and add statistical analysis in both main and supplementary figures.

    Minor comments:

    1. The quality of immunofluorescence and WB images should be implemented, and in the immunofluorescence panels scale bars should be added.
    2. In the graphical abstract authors reported that CHRDL2 overexpression increase WNT and EMT pathways, without performing any specific assay to demonstrate this. Authors should correct and graphically improve the graphical abstract.
    3. The term "significantly" in the discussion section is inappropriately referred to data showed in the histogram in Figure 1J. Moreover, in Figure 1Jthe authors should delete from the y-axis the term "corrected".
    4. The term "significant" in discussion is inappropriately referred to BMI1 expression level if compared to the histogram in Figure 4G.
    5. In Figure 2C the authors should add the unit of measurement (fold over control) in the table.
    6. In Figure 4E the authors should add the figure legend reporting OLFM4 protein.
    7. The authors should include few sentences summarizing the findings at the end of each paragraph.

    Significance

    General assessment:

    Overall, the work is aimed to elucidate the role of CHRDL2 already considered a poor prognosis biomarker involved in the promotion of CRC (PMID: 28009989), in promoting stem-like properties. The authors elucidated new additional insights into the molecular mechanisms regulating stemness phenotype induced by the BMP antagonist CHRDL2 in CRC. The authors include in the study a large amount of data, which only partially support their hypothesis. However, this manuscript lacks organization and coherence, making it challenging to follow and read. Numerous concerns need to be addressed, along with some sentences to rephrase in the result and discussion sections.

    Advance: The manuscript reported some functional insights on the role of CHRDL2 in colorectal cancer, but additional data should be added to support authors 'conclusions.

    Audience: The manuscript is suggested for basic research scientists.

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Clarkson and Lewis present data suggesting that overexpression of Chordin like 2 (CHRDL2) can affect colorectal cancer cell responses to chemotherapy agents, possibly by modulating stem-cell like pathways. I have the following comments:

    1. Fig. 1J-it is standard to show the images of cell migration-this is important here, given the modest effect of CHRDL2 overexpression here.
    2. Fig. 2A-the very small error bars for most of the data on the curves suggests these are n=1 experiments with multiple technical replicates to generate the error bars. Please clarify. The legend says n=3 with ANOVA analysis but no significance detected. Please clarify.
    3. Fig. 2B-given the overlapping error bars here, how can there be a p<0.01 between the groups?
    4. Fig. 2G-did the authors try to estimate the concentration of CHRDL2 in the conditioned medium? Which cell line was used to generate this CM?
    5. Fig. 5-what is the potential mechanism for gene expression changes in response to CHRDL2 overexpression? Is it all due to BMP inhibition? More mechanistic detail would be welcome here.

    Significance

    Unclear whether genetically engineered inducible overexpression has any real physiological significance but we all use cell models so this is OK.

  9. Version published to 10.1101/2024.01.23.576664v2 on bioRxiv
  10. Version published to 10.1101/2024.01.23.576664v1 on bioRxiv