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Title:Macrophage migration inhibitory factor - a therapeutic target in gallbladdercancer
Authors:
Tejaswini Subbannayya ([email protected])Pamela Leal Rojas ([email protected])Mustafa A Barbhuiya ([email protected])Remya Raja ([email protected])Santosh Renuse ([email protected])Gajanan Sathe ([email protected])Sneha Maria Pinto ([email protected])Nazia Syed ([email protected])Vishalakshi Nanjappa ([email protected])Arun H Patil ([email protected])Patricia Garcia ([email protected])Nandini A Sahasrabuddhe ([email protected])Bipin Nair ([email protected])Rafael Guerrero-Preston ([email protected])Sanjay Navani ([email protected])Pramod K Tiwari ([email protected])Vani Santosh ([email protected])David Sidransky ([email protected])T S Keshava Prasad ([email protected])Harsha Gowda ([email protected])Juan Carlos Roa ([email protected])Akhilesh Pandey ([email protected])Aditi Chatterjee ([email protected])
Version:2Date:31 July 2015
Author's response to reviews: see over
July 31, 2015
Dr. Dafne Solera
Executive Editor
BMC Cancer
Dear Dr. Solera,
We are submitting a revised version of our manuscript entitled “Macrophage migration
inhibitory factor - a therapeutic target in gallbladder cancer” for consideration for publication
as a research article in your journal BMC Cancer.
We thank the reviewers for their comments, which helped us to improve the quality of the
manuscript. We have now addressed all concerns raised by the reviewer in the revised
manuscript. We have also included a point-by-point response to the reviewer’s comments.
We believe that the data presented in this manuscript will be of significant interest to the
readership of BMC Cancer.
Thank for your consideration of our manuscript
Sincerely,
Aditi Chatterjee, Ph.D. Faculty Scientist
Point-by-point response to the reviewers’ comments
We thank the reviewers for their comments. We have now revised our manuscript to address all of their concerns. Our point-by-point response to the reviewers’ comments is given below:
Reviewer #1:
In this manuscript, Subbannayya and Rojas et al. present Macrophage Inhibitory Factor (MIF) as a potential therapeutic target in gallbladder cancer (GBC). The authors use a unique
approach of coupling high-resolution mass spectrometry with iTRAQ protein labeling to identify that MIF is overexpressed in invasive GBC cell lines in comparison to one non-invasive GBC cell line. Using tumor tissue microarrays (TMAs), the authors next show that
MIF is overexpressed in 72% of the GBC tumors studied. Using in vitro cell culture methods, they next show that specific MIF targeting reduces colony formation and invasive potential of
GBC cell lines. Thus highlighting the potential tumorigenic activity of MIF as well as potential as a therapeutic target. However, the manuscript in its current form cannot be accepted and needs major revision.
We thank the reviewer for providing us feedback on the manuscript.
Major compulsory Revisions
1) What happens in progression to GBC-does cholecystitis precede GBC? In your studied TMAs, was there clinical history of cholecystitis in the GBC patients? Also, for such
comparisons, it is usually ideal that the “n” for both types of cases (GBC and cholecystitis) studied should at least be equal.
In majority of the cases, cholecystitis precedes GBC (Nat Rev Cancer 2004, 4:695-706). In our study, the specimens which were used in the GBC TMAs had a clinical history of
cholecystitis. We agree with the reviewer that for such comparisons, it might be “ideal” to have same number of samples for both GBC and cholecystitis. However, for us, sample availability depends upon the number of operable cases the clinician can provide along with
informed consent of the patient. With these constraints, we were unable to match the number of GBC and cholecystitis cases. However, we have presented data from 16 cholecystitis cases
and 29 GBC cases. All TMAs were verified and scored by a renowned pathologist Dr. Vani Santosh, NIMHANS, Bangalore.
Wistuba II, Gazdar AF: Gallbladder cancer: lessons from a rare tumour. Nat Rev Cancer 2004, 4:695-706.
2) In the studies that previously identified CD44, MMP1 and CDH1, were the methods or cell lines used different? Not sure why MIF was not identified. Please elaborate in the results or
discussion section.
Yes, the methods and cell lines used by the other studies were different. Hirata et al., 2006 (Oncology 2006, 71:102-110) have used 2 out of the seven cell lines we have used to probe expression of CDH1 by Western blotting.
Those studies report differential expression of CD44, MMP1 and CDH1 based on
immunohistochemistry and Western blotting where only these molecules were investigated.
We carried out an unbiased global profiling study where we identified and quantified MIF and found it to be differentially expressed. MIF has not been associated or reported in any
form to be associated with GBC before. Hence, we focused on this molecule and have studied its potential to act as a novel therapeutic target in GBC. As suggested by the reviewer, we
have included this under the “Results” section. Page 15, line 368-370. Hirata K, Ajiki T, Okazaki T, Horiuchi H, Fujita T, Kuroda Y: Frequent occurrence of
abnormal E-cadherin/beta-catenin protein expression in advanced gallbladder cancers and
its association with decreased apoptosis. Oncology 2006, 71:102-110.
3) Lines 392-395 and 405- there isn’t any formal statistics represented in the text, legends or in the graph. Either show formal statistics or remove from the word “significant”. Statistics
should be presented for Fig 3 and 4.
As suggested by the reviewer, we have now provided statistics for Fig. 3 and 4. 4) Table 2: Is there an implication if MIF is more cytoplasmic than extracellular?
Literature evidence as well as our IHC data indicates that MIF is both cytoplasmic and
extracellular. The implication of this needs further investigation. However, its potential as a target remains unaffected as demonstrated by our study.
5) In the introduction, there is mention about therapies for GBC, how does MIF and the proteins that it networks with, would affect the current therapies or targeted therapies for
GBC. A focused discussion on therapeutic potential of MIF combined with current therapies would add value to these results. Please also discuss how MIF as a target fits/might fit with current immunotherapies under development etc.
Studies by Schoenfeld et al., has shown that blocking of cytotoxic T-lymphocyte-associated
antigen-4 (CTLA4) with antibody-based therapy elicits a humoral response against MIF, which in turn, decreases the expression of TEK tyrosine kinase, endothelial (TEK) and matrix metalloproteinase-9 (MMP9) (Cancer Res 2010, 70: 10150–10160). There are no studies
that report MIF as a target for immunotherapy in cancer. However, targeted MIF therapy might be effectively combined with antibody-based therapy to improve patient outcome in
GBC. We have discussed this under the “Discussion” section Page 21, Line 499-505. Schoenfeld J, Jinushi M, Nakazaki Y, Wiener D, Park J , Soiffer R, Neuberg D, Mihm M,
Hodi FS, Dranoff G: Active immunotherapy induces antibody responses that target tumor
angiogenesis. Cancer Res 2010, 70: 10150–10160.
6) Either the introduction or discussion needs mention if the MIF-therapeutics are clinically validated (ISO-1 and 4-IPP) or only experimentally tested.
MIF therapeutics (ISO-1 and 4-IPP) have only been tested experimentally (Cancer Res 2008,
68:7253-7257; Mol Med 2009, 15:1-10 and our current study). ISO-1 has been tested in pre-clinical studies. We are not aware of any clinical studies that have tested ISO-1 and 4-IPP.
Winner M, Meier J, Zierow S, Rendon BE, Crichlow GV, Riggs R, Bucala R, Leng L, Smith N, Lolis E, et al: A novel, macrophage migration inhibitory factor suicide substrate inhibits
motility and growth of lung cancer cells. Cancer Res 2008, 68:7253-7257.
He XX, Chen K, Yang J, Li XY, Gan HY, Liu CY, Coleman TR, Al-Abed Y: Macrophage
migration inhibitory factor promotes colorectal cancer. Mol Med 2009, 15:1-10.
Subbannayya, T., Rojas, P. L., Barbhuiya, M. A., Raja, R. Renuse, S., Sathe, G., Pinto, S. M., Syed, N., Nanjappa, V., Patil, A. H. et al: Macrophage migration inhibitory factor – a
therapeutic target in gallbladder cancer. (Our current study)
7) The data is largely dependent on one control non-invasive cell line. The data for protein
detection compares 3 invasive cell lines to one non- invasive cell line with only technical replicates. In such a case, a minimum control should be use of another non- invasive cell line or an “experimental” replicate of the first set.
We are limited by the availability of only one non-invasive GBC cell line which was used in
this study. Our mass spectrometry data was obtained for each cell line in duplicate, i.e. “experimental” replicate.
8) As MIF is differentially expressed in each cell line, a supplementary figure showing knockdown western blot or PCR for all the cell lines tested in Fig 3 and 4.
As suggested by the reviewer, we have now provided an immunoblot showing the knockdown of MIF in all GBC cell lines used in this study. See, Additional file 9a.
9) Figure 1- 1A or 1B can easily be moved to supplementary making room for a formal
representation of your supplementary data. A graphical/pictorial representation of the major mechanisms/pathways upregulated and/or downregulated in your iTRAQ screen should be presented. A comparison could be made with your non- invasive cell lines where valid. This
would complement figure 1C. If the TCGA has any somatic data on GBC, a formal comparison could be added here as well of commonly mutated/affected
mechanisms/pathways etc. The proteomic data represents a differential expression of each protein in the invasive cell
lines compared to the non-invasive cell lines. The numbers in Figure 1c represents the proteins overexpressed/downregulated in the indicated cell lines compared to the non-
invasive cell line, TGBC24TKB. As suggested by the reviewer, we have carried out Ingenuity Pathway Analysis to obtain a list of top canonical pathways in our data. This has been included in the “Methods” section (Page 11, Line 250-253) and discussed under the
“Results” (Page 16, Line 382-388) section and a graphical representation has been provided under Additional file 6d.
There is no somatic data on gallbladder cancer in the TCGA database. However, we compared our proteomic data with the whole exome data of gallbladder cancer from a study
by Li et al (Nat Genet. 2014, 46:872-876.). We identified 744 genes to be common between these two datasets, irrespective of their expression pattern in our proteomics data. Of these
744 genes, 8 genes belonged to the MIF nexus identified in our study. MIF was not reported by Li et.al.,. The co-receptor of MIF, CD44 was reported to harbor a somatic mutation (G>A) (Nat Genet. 2014, 46:872-876). The implications of these findings in GBC are known
and needs further investigations, which is not within the scope of this manuscript.
Li M, Zhang Z, Li X, Ye J, Wu X, Tan Z, Liu C, Shen B, Wang XA, Wu W, et al: Whole-exome
and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in
the ErbB pathway. Nat Genet. 2014, 46:872-876.
10) In a supplementary figure, to support your results from the iTRAQ screen, a western blot analysis in your 4 cell lines should validate a few of the listed – CD44, MMP1, CDH1, CALD1, PKP2, DSC2.
Earlier studies have reported the overexpression of CD44, MMP1 and downregulation of
CDH1 in gallbladder cancer (Arch Pathol Lab Med 2000, 124:212-215; Jpn J Clin Oncol 2011, 41:1086-1093; Oncology 2006, 71:102-110). Our studies recapitulate these findings confirming the validity of our quantitative proteomic data. Further, each of these molecules
has independent measurements in all three cell lines used in the study which upholds our inference based on proteomics. We are confident that Western blot will show the same. We do
not have resources to buy antibodies for these many proteins to carry out Western blots. Ylagan LR, Scholes J, Demopoulos R: Cd44: a marker of squamous differentiation in
adenosquamous neoplasms. Arch Pathol Lab Med 2000, 124:212-215.
Du X, Wang S, Lu J, Cao Y, Song N, Yang T, Dong R, Zang L, Yang Y, Wu T, Li J: Correlation between MMP1-PAR1 axis and clinical outcome of primary gallbladder
carcinoma. Jpn J Clin Oncol 2011, 41:1086-1093.
Hirata K, Ajiki T, Okazaki T, Horiuchi H, Fujita T, Kuroda Y: Frequent occurrence of
abnormal E-cadherin/beta-catenin protein expression in advanced gallbladder cancers and
its association with decreased apoptosis. Oncology 2006, 71:102-110.
11) Figure 3B and C, on a review of the quantification of colony survival data, it does not look like having increased expression of MIF specifically affects colony formation. For e.g.:
3Bii, OCUG-1 has the highest expression of MIF according to your western blot but the colony formation is almost equal to the control line in the control siRNA condition. While NOZ line has more colonies formed in control siRNA condition. Since some of your cell
lines are more invasive, they might not form proper colonies. Maybe plotting these data on a different scale will be more helpful to represent these data. Also reviewing results from Fig
3c (i), the colony forming ability is disrupted but the cells are still growing in some pictures, it would be advisable to report results from MTT/CTB survival assays. This might also answer questions on role of MIF in cell survival-apoptosis.
We agree with the reviewer that MIF levels do not seem to influence colony forming ability of
the cells. We would like to clarify that we have not claimed this in our manuscript. As suggested by the reviewer we have now plotted the colony forming ability and the invasive ability of the GBC cells and represented it under Additional File 9c.
As suggested by the reviewer, we have now included the MTT data of ISO-1 and 4-IPP as
Additional file 10a and 10b. MTT assay is a mixed assay which conflates growth and cell death parameters. MTT is a tetrazolium salt that is reduced by fully functioning mitochondria and results in a change of color from yellow to purple (Proc Natl Acad Sci U S A 1997,
94:514-519). MTT assay measures the loss of mitochondrial function, however its presence does not necessarily measure proliferative index of the cells (Proc Natl Acad Sci U S A 1997,
94:514-519). Hence, toxicity assays such as colony formation, that measures a cell’s ability
to undergo multiple cycles of cell proliferation on treatment with inhibitor, is needed (Biotechniques 2001, 30:1118-1120 and Cancer Res 1988, 48:589-601). Studies by Link et
al., 2013, reports a significant decrease (not complete wipe out) in the number of colonies upon inhibitor treatment (5 µM) however the cellular survival remains unaffected at the same
concentration as was indicated by MTT assay (PLoS One 2013, 8:e57709). A similar result has been reported by Wang et al., 2014 using breast cancer cells (Nat Commun. 2014, 5: 5086).
In our data, we do not observe a significant cell death in GBC cells in presence of 5 µM 4-
IPP or 50 µM ISO-1. However, we do observe a significant decrease in the colony forming ability and invasive ability (and not a complete wipe out) of the GBC cells in presence of MIF inhibitors.
Yakes FM, Van Houten B: Mitochondrial DNA damage is more extensive and persists
longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci U S A 1997, 94:514-519.
Lamb JR, Goehle S, Ludlow C, Simon JA: Thymidine incorporation is highly predictive of
colony formation and can be used for high-throughput screening. Biotechniques 2001,
30:1118-1120. Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo
JG, Shoemaker RH, Boyd MR: Feasibility of drug screening with panels of human tumor
cell lines using a microculture tetrazolium assay. Cancer Res 1988, 48:589-601.
Link A, Balaguer F, Shen Y, Lozano JJ, Leung HC, Boland CR, Goel A: Curcumin
modulates DNA methylation in colorectal cancer cells. PLoS One 2013, 8:e57709.
Wang W, Qin JJ, Voruganti S, Srivenugopal KS, Nag S, Patil S, Sharma H, Wang MH, Wang
H, Buolamwini JK, Zhang R: The pyrido[b]indole MDM2 inhibitor SP-141 exerts potent
therapeutic effects in breast cancer models. Nat Commun 2014, 5: 5086.
12) A pictorial/flowchart representation of MIF nexus is needed to show its role in proliferation etc. In a supple/main table please list your findings of proteins in MIF nexus
proteins that were also upregulated when MIF was upregulated in your screen. Please mention how these findings relate to expression in the non- invasive cell line.
As suggested by the reviewer, a pictorial representation of the proteins related to the MIF nexus identified in our study has been provided in Additional file 7. Different color codes
have been used to depict proteins that are overexpressed, downregulated or unchanged in the invasive cell lines compared to the non-invasive cell line. The specific expression profiles of the molecules depicted under Additional file 2.
13) Fig 3A- the western blot analysis does not correlate MIF expression with non- invasive
and invasive potential clearly. MIF expression in GB-d1 cell line, which according to your text is most invasive, is almost like the non- invasive line. In such cases transcript results should be reviewed.
As well as, if MIF is also extracellular, you might be losing some in your ce ll lysis methods, a western blot also showing expression from membrane prep might be more representative.
This also connects to my earlier question- are there any implications in literature if MIF is extracellular or cytoplasmic?
As shown in Fig 4a and b, targeting MIF decreases the invasive ability of the GBC cells.
However, we did not claim that MIF expression correlates with the invasive ability of the GBC cell lines. This indicates that MIF is important for invasive property of the cell lines, however we do not rule out other factors that may affect the invasive property of cells. In
other words, it is not a linear relationship where MIF levels directly correlate with the invasive ability of cells. There could be additional proteins that regulate invasive property of
these cell lines in addition to MIF. Differences in these other regulators require further investigation which might contribute to the observed disparity and is beyond the scope of this manuscript.
We do not expect to see MIF expression in the membrane prep as literature evidence
indicates that MIF is either extracellular or cytoplasmic. We too had observed the same in our IHC data, as scored by pathologist. We have carried out Western blot using the secretome of all GBC cell lines used in this study (Fig. 3a). We see varied MIF levels in the
secretome of the six GBC cell lines used in this study. Secretome of G-415 did not have detectable amounts of MIF.
Minor Essential Revisions
1) For consistency please abbreviate some of the commonly used terms iTRAQ, Immunohistochemistry (IHC), Macrophage Inhibitory Factor (MIF), Gallbladder cancer
(GBC) from the first time they are mentioned in the text. The abstract and main text sometimes interchangeably uses either the non-abbreviated or abbreviated forms.
This has now been included in the manuscript.
2) For consistency, please use same format – hrs or h, not interchangeably. This has now been included in the manuscript.
3) Lines 145-6- abbreviate gallbladder cancer cell lines.
This has now been included in the manuscript.
4) Line 173- Methods section needs to elaborate on the properties of the cell lines used, if not all at least the 4 used for iTRAQ. If they have any specific mutations, list invasive/non-
invasive potential etc. Also please establish what is the difference between- TGBC24TKB and TGBC2TKB cell lines. This gets very confusing as these cell lines are used without proper listing of why these are used in the study.
As suggested by the reviewer, we have now provided a description of the GBC cell lines used
in this study as Additional file 1. TGBC2TKB and TGBC24TKB are two different GBC cell lines obtained from two different patients. TGBC2TKB has been obtained from a metastatic lymph node of gallbladder carcinoma and is invasive. TGBC24TKB has been obtained from
metastatic ascites of gallbladder carcinoma and was found to be non-invasive.
5) Line 288-290- Consider sentence revision
This has now been revised in the manuscript.
6) Fig1A- how was this experiment done, some elaboration is needed on the staining in
methods/figure legends/results. Also please label each cell line in the different sections of the image.
Fig. 1a, depicts the invasive potential of the GBC cells. The experimental details have been described under the methods sections (Page 14, Line 337-347). Fig. 1a has now been
labelled as per the reviewer’s suggestion. 7) Figure 2d (i) and (ii), H & E staining and MIF staining images don’t match, please check
the image. As a supplementary figure or with main figure 2d, p lease also present IHC representative H& E images as well as MIF staining for Cholecystitis case positive staining
for MIF and GBC case negative/weak for MIF. We generally take 5-10 serial cuts from each TMA mother block and store these slides for
validation experiments. These slides are not numbered, hence, we are unable to identify and select consecutive slides. Although 2d (i) and 2d (ii) sections are from the same block, they
represent different cuts that are not consecutive. Unless the cuts are consecutive, there will be some variations in the appearance of the sections. The sections have been scored and images used have been approved by an expert pathologist. The TMAs (both IHC and H&E
stained) were re-examined by the pathologist and it was confirmed that they are the same. We have included additional images of cholecystitis sections and its corresponding H&E at both
10X and 20X magnifications for the reviewer’s eyes only (Fig. A, B, C, D). In addition, we are providing the 10X magnified image of the region shown in 2d (i) and 2d (ii) of the manuscript which clarifies that the sections used in H&E and IHC are the same (Fig. E,F) 2d
(i) and 2d (ii) as provided in the manuscript are at 20X magnification. We have added the same images here too (labeled as G and H) for easy comparison.
Figure 1: Representative sections from cholecystitis tissues (weak staining) –10X
magnification (A) stained with hematoxylin and eosin; (B) probed with anti-MIF
antibody; 20X magnification (C) stained with hematoxylin and eosin; (D) probed with
anti-MIF antibody; 10X magnification of the section represented in the manuscript (E)
stained with hematoxylin and eosin; (F) probed with anti-MIF antibody; 20X
magnification of the sections as represented in Fig. 2d(i) and 2d(ii) of the manuscript
(labeled G and H here) (G) stained with hematoxylin and eosin; (H) probed with anti-MIF
antibody.
As suggested by the reviewer, we have now provided the representative IHC images for weak staining for MIF in GBC and moderate positive staining for MIF in cholecystitis in
Additional file 8. None of the cholecystitis cases showed 3+ staining.
8) Line 374- Add references for role in tumor cell proliferation. This has now been included in the manuscript.
9) Throughout the text/ figure legends please list cell lines used in the same order preferably
by invasive to non- invasive potential, as that is your focus. As suggested by the author, we have now listed the cell lines in the same order throughout the
text and figure legends.
10) Figure 3A; please present a quantitative graph of the western blot, preferably in order of non- invasive to invasive potential of cell lines.
As suggested by the reviewer, we have now provided the quantitative graph of the Western blot under Fig. 2a (ii).
11) Lines 387-388- please show these data. These results would add more interest to groups working with either of these 2 classes of molecules.
As suggested by the reviewer in the major comments, we have now included the MTT assay
results as Additional file 10a and 10b. 12) Lines 400-402- consider removing, as the text that follows is repetitive.
The sentence has now been modified in the manuscript.
13) Line 405/465, how was it calculated that 4-IPP is 10 times more potent?
We apologize for that claim. We do not have sufficient data to support that claim. We have now removed this from our manuscript.
14) Line 431- As MIF inhibits p53-mediated apoptosis; the p53 status of all your cell lines needs mention in text/methods/supple table.
The cell line NOZ has a nonsense mutation in the p53 gene. The p53 status of the other GBC
cell lines used in this study has not been characterized, so we cannot comment on that. 15) Line 457- remove “the”. Abbreviate GBC.
This has now been included in the manuscript.
16) Line 475 – include IHC in list of abbreviations.
This has now been included in the manuscript.
17) Line 734- in legend/methods mention siRNA concentration used.
As suggested by the reviewer, the siRNA concentration has now been mentioned under the
methods section (Page 14, Line 324-325).
18) Line 739- I believe this is invasive potential and not colony forming ability, please correct.
This has now been corrected in the manuscript.
19) Line 521- Do references need a heading? There is a heading required for references as per the journal’s instructions to the author
Discretionary Revisions
1) Your article keywords should include MIF
This has now been included in the manuscript.
2) Table 2 shows 8 GBCs were weaker in MIF staining while 21 were stronger, is there any difference in the clinical pattern in these? If this information is available it will add a new dimension to your results.
The only clinical parameter we know about these tumors is their grade. We do not observe
any correlation between the grade of the tumor and intensity of staining. 3) 4-IPP and ISO-1 - if these downregulate/degrade MIF protein, please show a
supplementary western blot for your experiments.
Previous experiments have already characterized the mode of action of the MIF inhibitors, 4-IPP and ISO-1. Reports by Winner et al., and Al-Abed et al., demonstrates that ISO-1 and 4-IPP affects MIF activity and not its expression (Cancer Res 2008, 68:7253-7257. and J Biol
Chem 2005, 280:36541-36544.) This has been discussed under the “discussion section” of our manuscript. 4-IPP acts as a suicide substrate of MIF by covalently modifying the
catalytically active N-terminal proline. This covalent modification of MIF makes it inactive both in its catalytic and biological functions. ISO-1 is an antagonist of MIF which binds to the hydrophobic catalytic pocket of MIF and inhibits its tautomerase and glucocorticoid
regulating activity. Therefore, both ISO-1 and 4-IPP are associated with a decrease in MIF activity leading to its functional inhibition and not expression.
Winner M, Meier J, Zierow S, Rendon BE, Crichlow GV, Riggs R, Bucala R, Leng L, Smith N, Lolis E, et al: A novel, macrophage migration inhibitory factor suicide substrate inhibits
motility and growth of lung cancer cells. Cancer Res 2008, 68:7253-7257.
Al-Abed Y, Dabideen D, Aljabari B, Valster A, Messmer D, Ochani M, Tanovic M, Ochani K, Bacher M, Nicoletti F, et al: ISO-1 binding to the tautomerase active site of MIF inhibits its
pro-inflammatory activity and increases survival in severe sepsis. J Biol Chem 2005,
280:36541-36544.
Reviewer #2:
In this manuscript, using quantitative proteomic approach, Subbannayya et al. describe
identification of MIF as a marker for cellular proliferation and invasiveness in GBC. Furthermore, authors suggest the potential of MIF as therapeutic target. While the proteomics
and in vitro studies to assess role of MIF in proliferation and invasiveness are sound, there are some comments about approach and data analysis.
We thank the reviewer for providing us feedback on the manuscript.
Major revisions:
1. Why normal gallbladder cell line was not used as a control in the analysis. What is the
rationale for not including one.
Unfortunately, a non-neoplastic gallbladder cell line is not available. In addition, the goal of this study was to identify molecules contributing to tumor aggressiveness which includes the ability of GBC cells to invade. Towards this end, we chose to compare non-invasive and
invasive cell lines in this study.
2. From the methods section and Suppl. Table 3 it is not clear how selection of proteins from means was done. a. What was the ratio cutoff.
A ratio cut-off of 2-fold was used. It is an arbitrary cut-off used to buffer from any inherent
bias. b. Was there any multiple correction step used to address false positives.
False positives were addressed by searching the data against a decoy database to calculate
the false discovery rate (FDR). Peptide spectrum matches (PSMs) at 1% FDR were used for protein identification.
3. It would be very interesting to summarize (using enrichment method) the identified proteins for GO categories rather reporting GO terms for each protein.
As suggested by the reviewer, we have now included the pie charts for primary localization, molecular class and biological process in Additional file 6a, 6b and 6c.
Minor revisions:
1. listing up-regulated and down-regulated proteins (Venn diagrams) in supplementary tables would help reader.
As suggested by the reviewer, we have now listed overexpressed and down-regulated proteins
in Additional file 4 and 5.
