Title: Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal
appearing colonic mucosa from patients with colorectal neoplasia
Version: 1 Date: 09 Nov 2021
Reviewer's report:
Feddersen et al. present interesting data concerning the effects of PGE2 stimulation of colon
mucosa in both control and cancer patients. Furthermore they investigate expression of specific
enzymes and receptors and show differences in some and especially the LOX-pathway.  While
the results are interesting, there are some concerns.
Minor comments
1. For data showing patient data (such as EP-receptor expression) it would be helpful to show
single datapoints instead of bar graphs.
2. Was normality tested for patient data? Some error bars show very high variance and might
indicate non-normal distribution, necessitating other testing strategies. Again, this cannot be
evaluated in the absence of single datapoints.
Major comments
1. Fig. 2: The authors should also show ssc measurements for the other agonists. Similarly,
the proposed fits/ raw values for EP1 and EP3 should be shown. Additionally, although it is
mentioned in the text, the EP2 data are missing.
2. The fact that most antagonists did not prevent EP-receptor activation at all is alarming. It is not
clear to this reviewer how this could be explained. Subtype specific antagonists have been shown
to inhibit receptor signaling in a wide variety of in vivo/ex vivo and in vitro models.  For the
central claim that effects are EP4-receptor mediated, a different antagonist (i.e. ONO-AE3 208)
should also be used, especially as GW-x shows almost equipotent actions on the TP-receptor.
3. It is unclear how the novel data relate to the 2019 publication by the same authors.  Of note,
they find opposing results for EP1, Ep2, and EP3 receptors (the first two being not different  in
the first study, and EP3 being not significantly different in this study). While there might be an
explanation it is certainly no indication that there are “expressional alterations in at least 3 out
of 4 EP receptor subtypes present in normal appearing colonic mucosa from CRN patients”. As
the first study had higher n-numbers, it seems to be more reliable, but further data are needed.
4. Fig 3. Contains some data that only stem from one patient and other experiments have not
been performed at all/did not work. It is hard to justify the inclusion of this data in the study
as it is impossible to interpret.
5. A previous study found no effect of either ep1 or ep3 agonism on colonic mucosa but showed
effects of ep4 and ep2 agonism (•  DOI: 10.1016/j.ejphar.2012.06.020 ). How do the authors
explain the differences? Furthermore, did the authors test the non-selective EP1-3 antagonist
AH6809?
6. It is unclear to this reviewer what receptor high and receptor low means (e.g. Fig.3, Fig.4).
From the given information it seems like that are different methods for modelisation, but this
does not explain why n-numbers are different. It would be helpful to explain this in more detail.
7. To corroborate the differences in LOX-expression, IHC or WB should be performed.
Title: Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal
appearing colonic mucosa from patients with colorectal neoplasia
Version: 1 Date: 09 Nov 2021
Reviewer's report:
Feddersen et al. present interesting data concerning the effects of PGE2 stimulation of colon
mucosa in both control and cancer patients. Furthermore they investigate expression of specific
enzymes and receptors and show differences in some and especially the LOX-pathway.  While
the results are interesting, there are some concerns.
Minor comments
1. For data showing patient data (such as EP-receptor expression) it would be helpful to show
single datapoints instead of bar graphs.
2. Was normality tested for patient data? Some error bars show very high variance and might
indicate non-normal distribution, necessitating other testing strategies. Again, this cannot be
evaluated in the absence of single datapoints.
Major comments
1. Fig. 2: The authors should also show ssc measurements for the other agonists. Similarly,
the proposed fits/ raw values for EP1 and EP3 should be shown. Additionally, although it is
mentioned in the text, the EP2 data are missing.
2. The fact that most antagonists did not prevent EP-receptor activation at all is alarming. It is not
clear to this reviewer how this could be explained. Subtype specific antagonists have been shown
to inhibit receptor signaling in a wide variety of in vivo/ex vivo and in vitro models.  For the
central claim that effects are EP4-receptor mediated, a different antagonist (i.e. ONO-AE3 208)
should also be used, especially as GW-x shows almost equipotent actions on the TP-receptor.
3. It is unclear how the novel data relate to the 2019 publication by the same authors.  Of note,
they find opposing results for EP1, Ep2, and EP3 receptors (the first two being not different  in
the first study, and EP3 being not significantly different in this study). While there might be an
explanation it is certainly no indication that there are “expressional alterations in at least 3 out
of 4 EP receptor subtypes present in normal appearing colonic mucosa from CRN patients”. As
the first study had higher n-numbers, it seems to be more reliable, but further data are needed.
4. Fig 3. Contains some data that only stem from one patient and other experiments have not
been performed at all/did not work. It is hard to justify the inclusion of this data in the study
as it is impossible to interpret.
5. A previous study found no effect of either ep1 or ep3 agonism on colonic mucosa but showed
effects of ep4 and ep2 agonism (•  DOI: 10.1016/j.ejphar.2012.06.020 ). How do the authors
explain the differences? Furthermore, did the authors test the non-selective EP1-3 antagonist
AH6809?
6. It is unclear to this reviewer what receptor high and receptor low means (e.g. Fig.3, Fig.4).
From the given information it seems like that are different methods for modelisation, but this
does not explain why n-numbers are different. It would be helpful to explain this in more detail.
7. To corroborate the differences in LOX-expression, IHC or WB should be performed.
Please review this paper.
user
Title: Nanomolar EP4 receptor potency and expression of eicosanoid-related enzymes in normal
appearing colonic mucosa from patients with colorectal neoplasia
Version: 1 Date: 09 Nov 2021
Reviewer's report:
Feddersen et al. present interesting data concerning the effects of PGE2 stimulation of colon
mucosa in both control and cancer patients. Furthermore they investigate expression of specific
enzymes and receptors and show differences in some and especially the LOX-pathway.  While
the results are interesting, there are some concerns.
Minor comments
1. For data showing patient data (such as EP-receptor expression) it would be helpful to show
single datapoints instead of bar graphs.
2. Was normality tested for patient data? Some error bars show very high variance and might
indicate non-normal distribution, necessitating other testing strategies. Again, this cannot be
evaluated in the absence of single datapoints.
Major comments
1. Fig. 2: The authors should also show ssc measurements for the other agonists. Similarly,
the proposed fits/ raw values for EP1 and EP3 should be shown. Additionally, although it is
mentioned in the text, the EP2 data are missing.
2. The fact that most antagonists did not prevent EP-receptor activation at all is alarming. It is not
clear to this reviewer how this could be explained. Subtype specific antagonists have been shown
to inhibit receptor signaling in a wide variety of in vivo/ex vivo and in vitro models.  For the
central claim that effects are EP4-receptor mediated, a different antagonist (i.e. ONO-AE3 208)
should also be used, especially as GW-x shows almost equipotent actions on the TP-receptor.
3. It is unclear how the novel data relate to the 2019 publication by the same authors.  Of note,
they find opposing results for EP1, Ep2, and EP3 receptors (the first two being not different  in
the first study, and EP3 being not significantly different in this study). While there might be an
explanation it is certainly no indication that there are “expressional alterations in at least 3 out
of 4 EP receptor subtypes present in normal appearing colonic mucosa from CRN patients”. As
the first study had higher n-numbers, it seems to be more reliable, but further data are needed.
4. Fig 3. Contains some data that only stem from one patient and other experiments have not
been performed at all/did not work. It is hard to justify the inclusion of this data in the study
as it is impossible to interpret.
5. A previous study found no effect of either ep1 or ep3 agonism on colonic mucosa but showed
effects of ep4 and ep2 agonism (•  DOI: 10.1016/j.ejphar.2012.06.020 ). How do the authors
explain the differences? Furthermore, did the authors test the non-selective EP1-3 antagonist
AH6809?
6. It is unclear to this reviewer what receptor high and receptor low means (e.g. Fig.3, Fig.4).
From the given information it seems like that are different methods for modelisation, but this
does not explain why n-numbers are different. It would be helpful to explain this in more detail.
7. To corroborate the differences in LOX-expression, IHC or WB should be performed.
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