Review for 10.1186/s12876-023-02724-4 (2023)

Title: CHFR promotes metastasis of human gastric carcinoma by activating AKT and ERK via
NRF2- ROS axis
Version: 2 Date: 30 Jan 2023
Reviewer's report:
He et al. demonstrated that CHFR tumor suppressor inhibits gastric cancer cell proliferation
but promotes cell migration and invasion. They supported their hypothesis by using 2-different
stable gastric cancer cell lines expressing CHFR and an in vivo xenograft model. This work
demonstrated that CHFR plays a significant role in gastric cancer cell progression, migration,
and invasion. Importantly, they also provided the intracellular mechanism underlying the role
of CHFR in regulating cell proliferation, invasion, and migration. The result of the study may be
of interest to readers of the BMC Gastroenterology. There are some concerns about the format
of the manuscript and the failure to provide sufficient information. Also, the manuscript could
be enhanced by performing an additional in vitro experiment (do EdU instead of MTT for cell
proliferation assay).
1. Format:
- Please check with the journal policy whether the authors can use an abbreviation in the title.
- Please spell out the full term of each abbreviation at its first appearance. For example, write
“Gastric cancer (GC)” at the beginning of the introduction section and then use GC as an
abbreviation. Please review this throughout the manuscript.
2. Introduction:
- “Out previous research reported…invasion in vivo.” I do NOT see any citation here.
3. Materials and methods:
- Please provide more information about the cell lines (SGC-7901 and AGS). What are those
cell lines, and how are they (genetically) different? Whether they have an abnormal CHFR
expression? Are they representing the characteristics of the gastric cells found in GC patients?
- Please provide more detailed information about the antisera, including the full name of
the manufacturer (CST -> Cell Signaling Technology), manufacturer information (city-state-
country), and the catalog number of each antibody.
- How many mice were used in either Xenograft or lung metastasis experiments? Were the AGS
cells successfully injected to the right flank in Balb/c mice without extracellular matrix, such
as Matrigel?
Also, how many mice showed lung metastasis with AGS cells administration through the tail
vein?
- How old were the mice at the time of each experiment? What was the average weight of the
mice before and after the lentiviral injection? How did the authors determine the number of AGS
cells injected?
- Statistics: Did authors perform at least 3 independent (i.e., 3-cohorts) in vivo experiments?
Also, some figures (such as Figure 1B) have two factors (% and time)-this type of data must be
analyzed by 2-way ANOVA, not by the student’s t-test.
- What was the yield (efficiency) of the lenti-CHFR to each SGC-7901 and AGS cell line? Did
all cells show 100% efficiency in transfecting the vectors?
- Transwell assay: I am unsure if adding a Matrigel to the transwell membrane can differentiate
the cell invasion experiment from the invasion assay. It looks like the same type of experiment.
Please add more information about each experiment with appropriate citations.
- The authors seem to be utilized flow cytometry (Figure 2A), but detailed information (such as
the model name, manufacturer, software, and antibody) is missing.
Figure 1. Measuring the cell proliferation using the MTT assay is insufficient since this assay
reflects the cell survival rate (we don’t know about the toxicity of the vector or the lenti-CHFR).
It would be helpful to convince the reader if authors do the proliferation assay using EdU or any
other assay that can exclusively measure cell proliferation.
Figure 2C. Please indicate which data were obtained from either SGC-7901 or AGS cell lines.
Also, the AKT level of SGC-7901 cells (?) that were treated with lenti-CHFR seems to be
increased as well as p-AKT, while the b-actin expression level is not changed. This is not shown
in the right panel (which supposedly has data from the AGS cell line). The author should explain
this discrepancy.
Figure 3B. H2O2 administration significantly decreased cell invasion vs. control, and this was
restored by the lenti-CHFR. In this case, the correct interpretation of this data should be “ectopic
expression of CHFR increased GC cell invasion in a normal or ROS-induced conditions.” In
this case, the CHFR increases the rate of GC cell invasion in a ROS-independent manner.
Figure 3C. I do not see error bars for pCDH on both bar charts.
Figure 4A-B. I do not see error bars for lenti-control on both bar charts.