Development of Dual-Scale Fluorescence Endoscopy for In Vivo Bacteria Imaging in an Orthotopic Mouse Colon Tumor Model

Round 1

Reviewer 1 Report

In this study, Su Woong Yoo et al. report of a dual-scale fluorescence endoscopy method for in vivo bacterial imaging in mouse colon tumor model.

 

I think the topic is relevant considering recent interest in the role of bacteria in colorectal carcinogenesis, as well as treatment. My review is limited by the lack of my expertise in the technical aspects of this research. However, I think the validation of the method appears appropriate.

 

The authors report having taken ethical aspects of this animal research into consideration.

 

The manuscript would benefit from English language editing.

Author Response

In this study, Su Woong Yoo et al. report of a dual-scale fluorescence endoscopy method for in vivo bacterial imaging in mouse colon tumor model. I think the topic is relevant considering recent interest in the role of bacteria in colorectal carcinogenesis, as well as treatment. My review is limited by the lack of my expertise in the technical aspects of this research. However, I think the validation of the method appears appropriate. The authors report having taken ethical aspects of this animal research into consideration. The manuscript would benefit from English language editing.

 

Reply: Thanks for the positive comments. We underwent additional English editing services with reviewer’s suggestion.

Reviewer 2 Report

Woong Yoo et al. aimed to develop an imaging technique for monitoring tumour growth in orthotopic mouse model under microbial therapy, for the purpose of evaluating the distribution of bacteria. The authors propose the use of wide-field fluorescence endoscopic imaging to obtain a tissue-scale imaging, in combination with confocal endomicroscopy to image on cell-level. Imaging modalities aimed at morphological and cellular longitudinal in-vivo monitoring of tumour growth and microenvironment are important and needed tools. However, there are several concerns regarding the broad use of the proposed technique:

Major concerns:

It would be important to demonstrate the applicability of the tool for morphological characterization, vasculature detection, etc., and that the technique can be used to obtain high-sensitivity imaging. It would be important to demonstrate that the bacteria counts can be quantified from in vivo imaging (e.g. using segmentation approaches) and that they correlate with the ex-vivo-obtained bacteria counts from the extracted tumour tissue and adjacent/distant normal colon tissue. Quantification of the off-target signal is necessary. The low signal-to-noise ratio between normal and tumour fluorescent intensity poses challenges for bacteria quantification from these images.  The number of viable bacteria was shown to be higher in the tumour (colon) compared to other vital organs (lung, liver, spleen). Distant and adjacent normal colon tissue should be included for a reference. What is the number of samples used to compare the bacteria counts in extracted tissue from different organs (Figure 5)? Is there a statistically significant difference? Was autofluorescence in different tissue types adjusted for when comparing the bacterial counts? What is the imaging speed (frames per time unit), and how does it compare to current endomicroscopic approaches?

Minor comments:

In line 218, the figure description is not clear. Line 239, referenced Figure 4d, but described Figure 4e.

Author Response

Please, check the attached review comment.

Author Response File: Author Response.docx

Reviewer 3 Report

Early diagnosis and proper treatment monitoring are important are important for the effective treatment of colorectal cancer.  In this regard, in recent times cancer treatment using bacteria has been performed and this is monitored using fluorescence imaging techniques. The fluorescence endoscopic approach is an optimal monitoring method to evaluate the therapeutic response of bacteria in orthotopic colon cancer. In this study, the authors developed dual-scaled fluorescence endoscopy (DSFE) by combining wide-field fluorescence endoscopy (WFE) and confocal fluorescence endomicroscopy (CFEM), and demonstrated its usefulness for evaluating bacterial therapy. They developed the endoscopic probe of DSFE was developed by integrating the CFEM probe into the guide sheath of WFE. Then they monitored colorectal cancer tumor growth and tumors infiltrating the fluorescent bacteria using DSFE. The bacterial distribution of the tumor and organs were imaged and quantitatively analyzed using CFEM. DSFE successfully exhibited fluorescent bacterial signals in an orthotopic mouse colon tumor model. The authors concluded that the DSFE system is a promising method to monitor bacterial therapy in vivo.

The experiments are performed with appropriate controls and the results are significant. I recommend its publication with minor corrections.

Author Response

Early diagnosis and proper treatment monitoring are important are important for the effective treatment of colorectal cancer.  In this regard, in recent times cancer treatment using bacteria has been performed and this is monitored using fluorescence imaging techniques. The fluorescence endoscopic approach is an optimal monitoring method to evaluate the therapeutic response of bacteria in orthotopic colon cancer. In this study, the authors developed dual-scaled fluorescence endoscopy (DSFE) by combining wide-field fluorescence endoscopy (WFE) and confocal fluorescence endomicroscopy (CFEM), and demonstrated its usefulness for evaluating bacterial therapy. They developed the endoscopic probe of DSFE was developed by integrating the CFEM probe into the guide sheath of WFE. Then they monitored colorectal cancer tumor growth and tumors infiltrating the fluorescent bacteria using DSFE. The bacterial distribution of the tumor and organs were imaged and quantitatively analyzed using CFEM. DSFE successfully exhibited fluorescent bacterial signals in an orthotopic mouse colon tumor model. The authors concluded that the DSFE system is a promising method to monitor bacterial therapy in vivo. The experiments are performed with appropriate controls and the results are significant. I recommend its publication with minor corrections.

 

Reply: Thank you for the reviewer’s comprehensive review and recommendation. We underwent additional English editing services with reviewer’s suggestion.

Reviewer 4 Report

I find the article very interesting and would like to congratulate the authors for the work done. However, I have one short comment. In the introduction the bacteria were mentioned as a therapeutic agent and the expectation is raised by the reader that they will be used in that manner, whereas in fact in this study they have been used only as a diagnostic tool. In order not to confuse the reader, I suggest that this section should be modified slightly to point out that bacteria ca be used as a diagnostic as well as therapeutic agent.

Author Response

I find the article very interesting and would like to congratulate the authors for the work done. However, I have one short comment. In the introduction the bacteria were mentioned as a therapeutic agent and the expectation is raised by the reader that they will be used in that manner, whereas in fact in this study they have been used only as a diagnostic tool. In order not to confuse the reader, I suggest that this section should be modified slightly to point out that bacteria ca be used as a diagnostic as well as therapeutic agent.

 

Reply: Thank you for the reviewer’s suggestion. The main focus of our study is to show a real-time visualization of bacteria in the tumor area. We added and modified the sentences which can express the importance of the “visualization” of the therapeutic bacteria in vivo in the 3^rd paragraph of the introduction section as follows:

 

“The visualization of the bacteria is important to understand the biodistribution of the bacteria in vivo. Therefore, the fluorescence imaging techniques, which have characteristics including high-sensitivity, real-time imaging, and easy accessibility, are introduced. Fluorescence imaging enables the monitoring of the bacteria without euthanization of the animal tumor model.”