Diaphorobacter nitroreducens synergize with oxaliplatin to reduce tumor burden in mice with lung adenocarcinoma

ABSTRACT Lung adenocarcinoma (LADC) is the most common lung cancer and the leading cause of cancer-related deaths globally. Accumulating evidence suggests that the gut microbiota regulates the host response to chemotherapeutic drugs and can be targeted to reduce the toxicity of current chemotherapeutic agents. However, the effect of Diaphorobacter nitroreducens synergized with oxaliplatin on the gut microbiota and their impact on LADC have never been explored. This study aimed to evaluate the anti-cancer effects of D. nitroreducens, oxaliplatin, and their combined treatment on tumor growth in tumor-bearing mice. The composition of gut microbiota and the immune infiltration of tumors were evaluated by using 16S rRNA gene high-throughput sequencing and immunofluorescence, respectively. The inhibitory effect of the combination treatment with D. nitroreducens and oxaliplatin was significantly stronger than that of oxaliplatin alone in tumor-bearing mice. Furthermore, we observed that the combination treatment significantly increased the relative abundance of Lactobacillus and Akkermansia in the gut microbiota. Meanwhile, the combination treatment significantly increased the proportions of macrophage but decreased the proportion of regulatory T cells in the LADC tumor tissues of mice. These findings underscored the relationship between D. nitroreducens and the gut microbiota-immune cell-LADC axis, highlighting potential therapeutic avenues for LADC treatment. IMPORTANCE Oxaliplatin is widely used as an effective chemotherapeutic agent in cancer treatment, but its side effects and response rate still need to be improved. Conventional probiotics potentially benefit cancer chemotherapy by regulating gut microbiota and tumor immune infiltration. This study was novel in reporting a more significant inhibitory effect of Diaphorobacter nitroreducens on lung adenocarcinoma (LADC) cells compared with common traditional probiotics and validating its potential as an adjuvant therapy for LADC chemotherapy in mice. This study investigated the impact of D. nitroreducens combined with oxaliplatin on the gut microbiota and immune infiltration of tumors as a potential mechanism to improve anticancer effects.

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Sincerely, Michaeline Albright
Editor mSystems I would like to express my gratitude to the authors for their diligent work in elucidating the roles of Diaphorobacter nitroreducens in synergy with oxaliplatin to reduce the tumor burden of lung adenocarcinoma.However, several areas in the article require improvement, as outlined below: 1. Some words are misspelled and need correction.
Instead, it is suggested to use "tumor-bearing mice." 3. The statement about alternatively activated M2 macrophages is ambiguous and needs clearer articulation.
4. Many statements lack proper references, such as the one mentioning the association between high intratumoral infiltration of CD3+ and CD8+ cells and prognosis.
5. Consider using a more concise expression, such as "CD3+CD8+," unless the authors intended to convey something different.
6.The statement about current studies focusing mainly on the preventative potential of conventional probiotics is weak; consider revising to emphasize the role of microbiota in treatment.
7. Proper credit is not given for information obtained from the literature.It is crucial to acknowledge the intellectual owners of statements.It is a grave ethical issue in the scientific community to use other information without acknowledging it.Some of these are; a) D. nitroreducens is an aerobic, Gram-negative bacterium belonging to phylum Proteobacteria, family Comamonadaceae.
b) Currently, an accumulating number of clinical trials using combination therapies are being conducted to find enhanced sensitization methods.
c) Recently, it was increasingly recognized that the composition of gut microbiota 108 was critical for the progression of cancer treatment d) Tumor immune cell infiltration refers to the infiltration of immune cells from blood to the tumor tissue, which can be separated from the tumor tissue.
e) Immune cells (macrophages and T cells) may represent the primary sources of pro-inflammatory TNFα.
f) In mice treated with probiotics, the overall structure of intestinal villi was restored, and this phenomenon was also found in our study.
g) Additionally, gut microbiota facilitates a plethora of chemotherapyinduced immune and inflammatory responses.
h) It has been reported that Bifidobacteria modified tumor-specific T-cell induction and increased T-cell numbers in tumor microenvironment in patients treated with immunomodulator.

8.
Many words or phrases are not spelt out at first mention, pls revise.

9.
You may want to consider pluralizing your figures in naming them, such as Figures 3C and D and not in form of "Figure 3C, D". 10.Please, revise "The gut microbial function changes always accompanied with the alternation of gut microbiota structure."

11.
Your statement "We furtherly double stained macrophages (CD163 and CD68), helper T cell (CD3 and CD4),cytotoxic T cell (CD3 and CD8) and Treg cell (CD4 and FOXP3) and examined cells under an immunofluorescence (IF) (Figure 5 and S3)." Please, consider revising.It is not understood whether the authors were referring to tumor, blood, TDLN or other lymphoid organs, it is important to write clearly.
12. The statement "D.nitroreducens was reported to be one of the top five bacterial species in the normal nonsmoker lung, but not the dominant strain in the lung of cystic fibrosis, COPD and smoker(23)." is misleading.Are the authors referring to the lung as the locale for the microbe?13.Pls, consider revising, the sentence "A recent study also proposed the role of Bifidobacterium infantis in ameliorating oxaliplatin-induced toxicity by inhibiting Th1 and Th17 activation and enhancing cytotoxin regulatory T-cell activity (CD4+CD25+Fox p3+) in rats( 22)", 2017 is not recent!14.Regarding the concluding remark of the "Discussion" before the "Limitation", many questions arise.The question remaining is about other immunosuppressive cells such as TAM, MDSCs.Why not assay cytokine and chemokine profiles in the TME to understand the balance between pro-and anti-inflammatory factors, I think this would be able to explain the TME better than the suppression of Tregs alone.The complex dynamics within the TME are a bit more complex than this explaining with this scope, only macrophage and Tregs!.
15.The references need to be revised.They're not patterned/styled the same way.For example, consider pagination in the refs 6, 8, etc compared to others.
Addressing these points will enhance the clarity, accuracy, and overall quality of the article.

Editor-in-Chief
January 31, 2024 Dear Editor, We appreciate the expert review of our manuscript (mSystems01323-23) entitled "Diaphorobacter nitroreducens synergize with oxaliplatin to reduce tumor burden in lung adenocarcinoma mice".We thank the editors and reviewers for their efforts and scientific insights, and the opportunity to address the comments of the editor and reviewers.Below is a detailed point-by-point response to the editor and reviewer's comments.We are hopeful that the reviewers and editorial board will now find the manuscript suitable for publication in mSystems.thoroughly read through and pay more attention."Response 1: We appreciate the reviewer's careful corrections and suggestions which have improved the paper.The misspelled words in the manuscript have been corrected.

The phrase "...mice bearing subcutaneous A549 cells..." was incorrectly used.
Instead, I would suggest the authors should consider "tumor-bearing mice".Response 2: As the reviewer recommended, we have modified the phrase to "tumor-bearing mice".

The statement "Although alternatively activated M2 macrophages have traditionally been considered a poor prognosis marker (2), current studies have also shown that both M1 and M2 macrophages have good prognostic significance (3)" is ambiguous and needs clearer articulation.
Response 3: As the reviewer recommended, we have modified the statement to "Although M2 macrophages are traditionally thought to contribute to tumor progression ( 2), recent studies have also shown that the infiltration of M2 macrophages is positively associated with favorable clinical outcomes in patients with NSCLC (3)." to indicate the important role of M2 macrophages in NSCLC.4)".I cannot see many studies properly refenced here!Response 4: As the reviewer recommended, we have added appropriate references in the following statements to support them.Statement 1: "Additionally, many studies reported that the high intra-tumoral infiltration of cytotoxic T cells (CD3 + CD8 + ) was associated with good prognosis and improved survival (4,5).In contrast, the increase in the proportion of regulatory T (Treg) cells (forkhead box protein 3-positive, FOXP3 + ) indicated a worse prognosis (6, 7).".Statement 3: "Currently, an increasing number of clinical trials using combination therapies are being conducted to find enhanced sensitization methods (28)(29)(30).".Statement 4: "Increased fatty acid biosynthesis could be a response to the high metabolic demand of cancer cells or an adaptation to reduced efficacy of serum-derived lipids in the tumor microenvironment (47,48)."

Consider using a more concise expression, such as "CD3+CD8+," when you mean cytotoxic T-cells, please, unless the authors intended to convey
something different, which is required to be clarified here.Response 5: As the reviewer recommended, we have modified the statements as follows.
6.The statement "However, current studies mainly focused on the preventative potential of conventional probiotics.Therefore, it is necessary to explore other potentially beneficial bacteria for synergistic treatment against cancers" is a very weak knowledge gap; consider revising to emphasize the role of microbiota in treatment.The therapeutic/treatment involvement or consideration regarding probiotics has been repeatedly reported, the authors should delve more into the recent literature.Response 6: As the reviewer recommended, we have modified the statement to "Moreover, many potential next-generation probiotics are currently developed using the latest-generation sequencing and bioinformatics platforms ( 14).These bacteria, including Eubacterium limosum (15,16), Enterococcus hirae ( 8), Enterococcus faecium ( 17), Collinsella aerofaciens (18,19) and Burkholderia cepacian ( 20) have demonstrated promising efficacy in promoting the anticancer effects.However, further exploration and evaluation are needed to elucidated the potential role of the microbiota in effectively modulating of anticancer treatment."

h) It has been reported that Bifidobacteria modified tumor-specific T-cell induction and increased T-cell numbers in tumor microenvironment in patients treated with immunomodulator.
Response 7: We appreciate the reviewer's expert insights and effort.As the reviewer recommended, we have added appropriate references in these statements to support them.
8. Many words or phrases are not spelt out at first mention, pls revise.
Moreover, pls, put all acronyms in their correct form.The standard way of abbreviating hematoxylin and eosin stain is "H&E" and not "HE"!Response 8: We appreciate the reviewer's suggestions.As the reviewer recommended, we have defined each abbreviation at first time mention.9.You may want to consider pluralizing naming your figures, such as "Figures 3C and D" and not in form of "Figure 3C, D".Response 9: As the reviewer recommended, we have modified the names of figures.

Please, revise the statement "The gut microbial function changes always
accompanied with the alternation of gut microbiota structure."Response 10: As the reviewer recommended, we have modified the statement to "Altered gut microbiota composition might result in metabolic dysregulation or functional changes."

In your statement "We furtherly double stained macrophages (CD163 and CD68), helper T cell (CD3 and CD4), cytotoxic T cell (CD3 and CD8) and Treg cell (CD4 and FOXP3) and examined cells under an immunofluorescence (IF) (Figure 5 and S3
)." please, consider revising.It is not understood whether the authors were referring to tumor, blood, TDLN or other lymphoid organs, but it is important to write clearly.Response 11: As the reviewer recommended, we have modified the statement to "We furtherly double-stained macrophages (CD163 + CD68 + ), helper T cell (CD3 + CD4 + ), cytotoxic T cell (CD3 + CD8 + ) and Treg cell (CD4 + FOXP3 + ) of subcutaneous tumor tissues and examined the cells using IF (Figures 5 and S3)."

The statement "D. nitroreducens was reported to be one of the top five bacterial species in the normal nonsmoker lung, but not the dominant strain in the lung of cystic fibrosis, COPD and smoker (23)
." is misleading.Are the authors referring to the lung as the locale for the microbe?Response 12: As the reviewer recommended, we have deleted the statement to avoid ambiguity.22)", 2017 is not recent!Response 13: As the reviewer recommended, we have modified the statement to "Bifidobacterium infantis in ameliorating oxaliplatin-induced toxicity by inhibiting helper T cell 1 and helper T cell 17 activation and enhancing cytotoxin regulatory T cell activity (CD4 + CD25 + Foxp3 + ) in rats (41).".

Pls, consider revising, the sentence "A recent study also proposed the role of Bifidobacterium infantis in ameliorating oxaliplatin-induced toxicity by inhibiting Th1 and Th17 activation and enhancing cytotoxin regulatory T-cell activity (CD4+CD25+Fox p3+) in rats (
14. Regarding the concluding remark of the "Discussion" before the "Limitation", many questions arise.The question remaining is about other immunosuppressive cells such as TAM, MDSCs.Why not assay cytokine and chemokine profiles in the TME to understand the balance between pro-and anti-inflammatory factors, I think this would be able to explain the TME better than the suppression of Tregs alone.The complex dynamics within the TME are a bit more complex than this explaining with this scope, only macrophage and Tregs!Response 14: We appreciate the reviewer's expert insights and comments.As the reviewer recommended, we have supplemented the discussion about TAMs as follows: "The tumor microenvironment (TME) possesses various types of immunosuppressive cells including myeloid-derived suppressor cells (MDSCs), Treg cells and tumor-associated macrophages (TAMs) (53).As one of the most abundant cell types present in TME(54), TAMs have been shown to exert important roles in the tumor therapy (55,56).TAMs can be classified into the proinflammatory M1-type and anti-inflammatory M2-type macrophages (57).TAMs can exert a killing effect on tumor cells once activated by .Besides, there also is wide communication between TMAs and other immune compositions, such as Treg cells, cytotoxic T cells, neutrophils, dendritic cells (DCs) and so on (59,60)." 15.The references need to be modified.They're not patterned/styled the same way.For example, consider pagination in the refs 6, 8, etc compared to others.Response 15: As the reviewer recommended, we have modified these references.
We appreciate for Reviewer' warm work earnestly, and hope that the correction will meet with approval.Moreover, we thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.We have uploaded a "Revised Article with Changes Highlighted" file.In addition, due to changes in the author's institute, the name of affiliated address "Department of Oncology, Wuxi Children's Hospital, Wuxi, China" has been replaced with "Department of Oncology, Affiliated Children's Hospital of Jiangnan University, Wuxi, Jiangsu, China" and the order of the first and the second affiliated address has also been adjusted.Once again, thank you very much for your comments and suggestions.
February 29, 2024 1st Revision -Editorial Decision Re: mSystems01323-23R1 (Diaphorobacter nitroreducens synergize with oxaliplatin to reduce tumor burden in mice with lung adenocarcinoma) Dear Prof. Qingjun You: Your manuscript has been accepted, and I am forwarding it to the ASM production staff for publication.Your paper will first be checked to make sure all elements meet the technical requirements.ASM staff will contact you if anything needs to be revised before copyediting and production can begin.Otherwise, you will be notified when your proofs are ready to be viewed.
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7.
Proper credit was not given for information obtained from the literature.It is crucial to acknowledge the intellectual owners of many statements in the article.It is a grave ethical issue in the scientific community to use other investigators' information without acknowledging them.Some of these are implicated sentences are as follow; a) D. nitroreducens is an aerobic, Gram-negative bacterium belonging to phylum Proteobacteria, family Comamonadaceae.b) Currently, an accumulating number of clinical trials using combination therapies are being conducted to find enhanced sensitization methods.c) Recently, it was increasingly recognized that the composition of gut microbiota was critical for the progression of cancer treatment d) Tumor immune cell infiltration refers to the infiltration of immune cells from blood to the tumor tissue, which can be separated from the tumor tissue.e) Immune cells (macrophages and T cells) may represent the primary sources of pro-inflammatory TNFα.f) In mice treated with probiotics, the overall structure of intestinal villi was restored, and this phenomenon was also found in our study.immune and inflammatory responses.

Some words are misspelled and need correction. The authors are advised to
,