Visualization of Metastatic Lung Cancer with TiNIR
, Hyun Bo Sim 1,†, Ji Yeon Han 1, Hyeongyeong Kim 1, Dae-Han Park 1, Dong-Jo Chang 2, Sung-Tae Yee 2,*, Young-Tae Chang 3,4,* and Jong-Jin Kim 1,*Round 1
Reviewer 1 Report
Visualization of metastatic lung cancer with TiNIR
The study on “Visualization of metastatic lung cancer with TiNIR” provides good scientific insights about the effectiveness of TIC detecting NIR probes in metastatic cancer cells. The data presented is interesting.
However, I would like to suggest authors to address certain concerns or corrections.
1. Authors should include the description for the TiNIR probe, like the structure, synthesis and mode of interaction for HO2.
2. The expression of HO2 in metastatic tumor mass in vivo model should further be confirmed with other already established metastatic biomarker expressions.
3. Colocalization studies with HO2 expression and TiNIR localization should be done to confirm the detection of HO2 expression by this probe.
4. The selective/ specific interaction of the probe with the HO2 enzyme should further be confirmed by the inhibitory study by using certain competitive HO inhibitors or other compound which specifically interacts and inhibits HO2 enzyme activity.
5. How do the authors finalized the mentioned concentration of probe needed for detection in vitro and in vivo.
6. In page 3 line 136 what does “remove OCT” means?
Minor editing of English language required for resubission.
Author Response
Response to reviewer 1.
The study on “Visualization of metastatic lung cancer with TiNIR” provides good scientific insights about the effectiveness of TIC detecting NIR probes in metastatic cancer cells. The data presented is interesting.
However, I would like to suggest authors to address certain concerns or corrections.
- Authors should include the description for the TiNIR probe, like the structure, synthesis, and mode of interaction for HO2.
Answer: Thanks for the careful checking, we already have reported the detailed information of the probe in a previous paper (J. Am. Chem. Soc. 2019, 141, 14673-14686) which includes the synthesis scheme and structure, and provided reference.
- The expression of HO2 in metastatic tumor mass in vivo model should further be confirmed with other already established metastatic biomarker expressions.
Answer: As suggested by the reviewer, we checked the expression level of other metastatic biomarkers in the migrating cells. We modified the as follows “First we investigated the expression level of vimentin, sanil, slug, and N-cadherin to confirm the metastatic characteristics, and observed up-regulation of those genes.” (line 161-162, 170,173) and materials and methods section (line 98-103)
- Colocalization studies with HO2 expression and TiNIR localization should be done to confirm the detection of HO2 expression by this probe.
Answer: As suggested by the reviewer, we confirmed the co-localization of the TiNIR signal and HO2 protein by using an antibody (Figure 4C) and we described as follows: “To confirm the co-localization of TiNIR and HO2 protein, we treated the HO2 antibody. The TiNIR signal was located in a similar area to the HO2 antibody signal (Figure 4C).” (line 223-225) and materials and methods section (lines 144~149).
- The selective/ specific interaction of the probe with the HO2 enzyme should further be confirmed by the inhibitory study by using certain competitive HO inhibitors or other compounds which specifically interacts and inhibits HO2 enzyme activity.
Answer: We appreciate the reviewer’s helpful suggestion. We already have reported in the previous paper the TiNIR selectivity to the HO2 (J. Am. Chem. Soc. 2019, 141, 14673-14686) using HO2 knockout experiment as follows the figure.
- How do the authors finalized the mentioned concentration of probe needed for detection in vitro and in vivo.
Answer: In the previous paper, we reported the appropriate concentration of TiNIR to utilize in vivo and in vitro. Here we used a non-cytotoxic concentration of 100 nM in vitro and a single injection of 100 μM. (J. Am. Chem. Soc. 2019, 141, 14673-14686)
- In page 3 line 136 what does “remove OCT” means?
Answer: Thanks for the careful checking. OCT has an auto-fluorescence. So to measure accurate probe and antibodies fluorescence, OCT must be removed by washing with PBS before checking signals.
Comments on the Quality of English Language
Minor editing of English language required for resubission.
Answer: we edited the grammar and errors.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This is an interesting and important study that adds significantly to the field and will have a noticeable impact.
The manuscript is well-written and concise.
It seems necessary to correct and supplement the following contents.
1. line 21: Abstract: in vivo edit in italics (in vivo)
2. Line 106: CO2 edit ----Line89 same CO2
3. Line 128: ex vivo edit in italics (ex vivo)
4. Figure 1A: Scale bar add in image
5. Line 164: Figure 1C. X axis add
6. Lines 195, 196: Figure 2. n, p edit in italics---(n, p)
7. Line 198, 211: ex vivo edit in italics (ex vivo )
8. Line 250: in vitro edit in italics (in vitro)
9. Line: 336 ~340: References Number check (correct 37, 38, 39 to 27,28,29)
Author Response
Response to reviewer 2.
This is an interesting and important study that adds significantly to the field and will have a noticeable impact.
The manuscript is well-written and concise.
It seems necessary to correct and supplement the following contents.
- line 21: Abstract: in vivo edit in italics (in vivo)
Answer: As suggested by the reviewer, we have modified the Abstract (line 21)
- Line 106: CO2 edit ----Line89 same CO2
Answer: As suggested by the reviewer, we have modified the results (line 110)
- Line 128: ex vivo edit in italics (ex vivo)
Answer: We have modified the results (line 132)
- Figure 1A: Scale bar add in image
Answer: We have added scale bar. (line 170)
- Line 164: Figure 1C. X-axis add
Answer: We have modified X-axis of Figure 1C. (line 170)
- Lines 195, 196: Figure 2. n, p edit in italics---(n, p)
Answer: We have modified legend of Figure 2. (line 197, 198)
- Line 198, 211: ex vivo edit in italics (ex vivo)
Answer: We have modified the results. (line 200, 204)
- Line 250: in vitro edit in italics (in vitro)
Answer: We have modified the results. (line 260)
- Line: 336 ~340: References Number check (correct 37, 38, 39 to 27,28,29)
Answer: We have modified the References Number. (line 346~350)
Author Response File: Author Response.docx
Reviewer 3 Report
We thank the authors for their contribution "Visualization of metastatic lung cancer with TiNIR" which is interesting.
I have following comments to the manuscript:
1. Please add citations to the lines 62-64.
2. May I confirm that you only took two mice for this study? Please clarify in introduction why only two mice were used?
3. Please clarify the number of animals on the line 122.
4. I believe the lines 149 to 153 don`t belong to the Result section and should be added either in Introduction or in Discussion section.
5. The lines 174 to 178 don`t belong to the Result section and should be added either in Introduction or in Discussion section.
Author Response
Response to Reviewer 3
We thank the authors for their contribution "Visualization of metastatic lung cancer with TiNIR" which is interesting.
I have following comments to the manuscript:
- Please add citations to the lines 62-64.
Answer: We have added citations to the lines. (line 64)
- May I confirm that you only took two mice for this study? Please clarify in introduction why only two mice were used?
Answer: We appreciate your comment. In this study, we used total of 3 mice/group, and indicated in the materials and methods section as follows. “BALB/c nude mice (6-8 weeks, CAnN. Cg-Foxninu/CrlOri, n=3 per group) were purchased from Orient-bio (Seongnam, Republic of Korea)” (line 125-126)
- Please clarify the number of animals on line 122.
Answer: Thank you for your comment. We answered reviewer comment 2.
- I believe the lines 149 to 153 don`t belong to the Result section and should be added either in Introduction or in Discussion section.
Answer: As suggested by the reviewer, we modified the suggested sentence in the discussion section. (line 239~240, 255~259)
- The lines 174 to 178 don`t belong to the Result section and should be added either in Introduction or in Discussion section.
Answer: As suggested by the reviewer, commented paragraph has been removed because it is already similarly mentioned in the introduction and discussion.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Authors have addressed all the comments satisfactorily.
