Possibility of SARS-CoV-2 Infection in the Metastatic Microenvironment of Cancer
Round 1
Reviewer 1 Report
Why did authors refered to patients enrolled so long time ago?
How is it possible that authors received ethical approval in august 2019 if covid-19 spread from December 2019 till now?
The aim of the article is not clear. please add at the end of the introduction section
What is the rationale behind examing women with ovarian cancer and bronchiolar cells in mice?
Discussion is not well structured. First section should contain a brief summary of main results shown in the manuscript. Secondly an internal consistency of the results should be discussed. Thirdly a comparison with previous studies should be analysed and lastly limits and strengths of the work should be added. Please revis consistently.
Conclusions are missing. Please consider to add some considerations in terms of public health impact of your results
Author Response
Reviewer 1:
We would like to thank Reviewer 1 for their time considering our manuscript and their constructive comments.
Comment 1: Why did authors refer to patients enrolled so long time ago?
Answer 1: We appreciate the reviewer’s comment and replied to it with the following content.
In case of Japanese female
The patients enrolment occurred a while ago because the prevalence of ovarian cancer in Japanese women by age 70 is approximately 0.012%. Furthermore, the prevalence of high grade serous ovarian cancer, in which metastasis and recurrence are frequently observed, is 0.00408%. Thus, the prevalence of high grade serous ovarian cancer is very low. Therefore, it is not easy to collect excised specimens of many ovarian cancers, particularly samples obtained following an appropriate process allowing their use for clinical and basic research.
Comment 2: How is it possible that authors received ethical approval in August 2019 if covid-19 spread from December 2019 till now?
Answer 2: We appreciate the reviewer’s comment and replied to it with the following content:
The content of the clinical research plan, which I submitted to the editorial department of MDPI, the publisher of medical journals, is ethically approved by the Institutional Review Board (IRB). The date of ethical approval by the Central Institutional Review Board is July 9, 2019. The date of ethical approval by the IRB is stated in the clinical research plan.
Comment 3: The aim of the article is not clear. please add at the end of the introduction section
Answer 3: We agree with the reviewer's comments. We considered the issues pointed out by the reviewer and added a paragraph at the end of the introduction section in the revised manuscript as shown below:
Previous clinical studies have shown that SARS-CoV-2 infection rates and COVID-19 severity are higher in cancer and in people with a history of cancer than they are in healthy individuals. The specific reasons for these observations have not been clarified. Previously, we have shown that, in lung metastases of ovarian cancer, alveolar epithelial cells adjacent to ovarian cancer cells transform into alveolar epithelial stem-like cells7. ACE2 is strongly expressed in alveolar epithelial stem-like cells5. The present study aims to confirm the expression of ACE2 in alveolar epithelial stem-like cells adjacent to ovarian cancer cells in lung metastases of ovarian cancer and to investigate the binding of SARS-CoV-2 spike glycoprotein to alveolar epithelial stem-like cells.
- 5. Salahudeen AA, Choi SS, Rustagi A, et al. Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature 2020; 588: 670-675. doi: 10.1038/s41586-020-3014-1
- 7. Hayashi T, Sano K, Aburatani H, Yaegashi N, Konishi I. Initialization of epithelial cells by tumor cells in a metastatic microenvironment. 2020 39(12): 2638-2640. doi: 10.1038/s41388-020-1171-1.
Comment 4: What is the rationale behind examining women with ovarian cancer and bronchiolar cells in mice?
Answer 4: We appreciate the reviewer’s comment and apologize for the confusion. Indeed, in this study, we did not perform experiments using mouse tissues. The tissue used in this study was lung metastatic tissue of ovarian cancer excised from patients with ovarian cancer.
Comment 5: Discussion is not well structured. First section should contain a brief summary of main results shown in the manuscript. Secondly an internal consistency of the results should be discussed. Thirdly a comparison with previous studies should be analyzed and lastly limits and strengths of the work should be added. Please revise consistently.
Answer 5: We agree with the reviewer's comments. We considered the issues pointed out by the reviewer and remodeled the entire discussion in the revised manuscript as shown below:
(First section should contain a brief summary of main results shown in the manuscript.)
Previous clinical studies have shown that SARS-CoV-2 infection rates and COVID-19 severity rates are higher in cancer patients and in people with a history of cancer than it is in healthy individuals. The specific reasons for these observations have not been clarified. Previously, we showed that, in lung metastases of ovarian cancer, alveolar epithelial cells adjacent to ovarian cancer cells transform into alveolar epithelial stem-like cells7. Alveolar epithelial stem-like cells have been reported to strongly express ACE25. The present work aimed to confirm the expression of ACE2 in alveolar epithelial stem-like cells adjacent to ovarian cancer cells in lung metastases of ovarian cancer and to investigate the binding of the SARS-CoV-2 spike glycoprotein to alveolar epithelial stem-like cells. Our IHC analyses showed the expression of ACE2 in alveolar epithelial stem-like cells adjacent to ovarian cancer cells in lung metastases of ovarian cancer. We also demonstrated the binding of SARS-CoV-2 spike glycoprotein to alveolar epithelial stem-like cells.
(Secondly an internal consistency of the results should be discussed.)
A high 30-day all-cause mortality has been reported in patients with cancer and COVID-19. Various factors contributing to COVID-19 severity in cancer patients have been identified. A previous report has shown that SARS-CoV-2 infects alveolar and bronchiolar epithelial cells11. Within the pulmonary metastatic niche, alveolar epithelial cells adjacent to metastatic cancer cells are differentiated into alveolar epithelial stem-like cells. Our experiments showed that, although the expression of ACE2 was not strong in normal alveolar epithelial cells, ACE2 was clearly expressed in alveolar epithelial stem-like cells. We also observed a binding of the alveolar epithelial stem-like cells to the RBD of the SARS-CoV-2 spike glycoprotein. Therefore, SARS-CoV-2 infection of stem cells and/or epithelial progenitor cells present in the metastatic niche in patients with cancer likely is a factor contributing to COVID-19 severity.
(Thirdly a comparison with previous studies should be analyzed)
We examined the environmental niche of lung metastases of ovarian cancer. However, the infection rate of SARS-CoV-2 and the severity of COVID-19 also increase in patients with other cancer types. The results showing that ovarian cancer cells form a metastatic niche near the alveolar stem cells are reminiscent of a previous finding demonstrating that prostate cancer cells metastasizing to the bone settle near the stem cells in the bone marrow, promoting the development of a metastatic environment that supports tumor growth12. A recent report also described cancer-associated parenchymal cells that show stem cell-like characteristics, the expression of lung progenitor markers, multilineage differentiation potential, and self-renewal activity13.
(lastly limits and strengths of the work should be added.)
To obtain accurate histopathological information of positive or negative SARS-CoV-2 infection in tissues from COVID-19 cancer patients, histopathological experiments with metastatic tissues of cancer patients infected with SARS-CoV-2 or with COVID-19 symptoms must be performed. So far, treatment aimed at reducing lung metastases has been limited to surgical treatment. However, in clinical practice, lung metastases have been reduced using immune checkpoint inhibitors and/or poly ADP-ribose polymerase inhibitors. Furthermore, the efficacy of the anti-S100A4 antibody drug in suppressing the metastatic ability of malignant tumors in other organs, including ovarian cancer, has been investigated9,14,15.
A longer follow-up is needed to better understand the effect of COVID-19 on the treatment outcomes of patients with cancer, including on the ability to continue specific cancer treatments. In such a significant intersection of cancer medicine and infectious diseases, the prevention of de novo niche formation of metastatic disease might constitute a novel strategy for the clinical treatment of COVID-19.
Comment 6: Conclusions are missing. Please consider adding some considerations in terms of public health impact of your results
Answer 6: We agree with the reviewer's comments. We modified the revised manuscript accordingly:
Conclusion
Previous clinical studies have shown that SARS-CoV-2 infection rates and COVID-19 severity rates are higher in cancer patients currently being treated and people with a history of cancer than in healthy individuals. The specific reasons for the high SARS-CoV-2 infection rate and COVID-19 aggravation rate in cancer patients have not been clarified. Infection of SARS-CoV-2 into the niche of metastatic lesions in cancer patients may be one of the reasons for the higher rate of SARS-CoV-2 infection and COVID-19 severity compared to healthy individuals.
Comment 7: (x) English language and style are fine/minor spell check required
Answer 7: The revised manuscript was carefully proofread by a native English speaker.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Possibility of SARS-CoV-2 infection in metastatic microenvi- 2 ronment of cancer
Language and syntax need extensive revision
S-100A4 is used as specific ovarian cancer marker and while a few evidences might have correlated its circulating levels to ovarian cancer, it is generally not considered the most prominent marker for this malignancy, either circulating or tissue-based
The patient’s cohort selected is extremely old and most probably does not reflect the current characteristic of ovarian cancer cohorts (in residual post-surgery, PFS and OS). Besides, the data in table 1 are general and not specific (referring to a range for age at diagnosis seems too vague).
Were the tumor sections revised and re assigned to high-grade according to the new two-tier system?
Was the FIGO stage reviewed and re assigned according to the new 2013 criteria?
The methodology is poorly descripted, I understand there is a supplementary but the main text should contain at least the types of staining performed (IHC? IF? Both?)
Results are inconsistent, and with an extensive section that refers to other publications.
Are the authors showing that RBD of the spike protein for SARS COV 2 applied onto the tissue sections, attaches to the ACE2 receptor? Or do they mean that they have performed staining for this domain of the spike protein of SARS COV 2? This is poorly explained
Are the authors implying that epithelial ovarian cancer cells, metastatic to the lungs, express ACE2? This finding seems a bit inconsistent, metastatic cells tend to keep their primary sites markers, not acquire the ones of the tissue they metastasize at. If the authors believe that, they should provide stronger evidence for that. If they instead mean that the two markers are expressed in the same area by different cells it should be better explained. (Lines 140 and 141)
The authors introduce in Results the concept of “normal areas” they should define what they mean by normal areas in the sections, in Materials and Methods.
Author Response
Reviewer 2:
We would like to thank Reviewer 2 for their time considering our manuscript and their constructive comments.
Comment 1: Language and syntax need extensive revision
Answer 1: The revised manuscript was carefully proofread by a native English speaker.
Comment 2: S-100A4 is used as specific ovarian cancer marker and while a few evidences might have correlated its circulating levels to ovarian cancer, it is generally not considered the most prominent marker for this malignancy, either circulating or tissue-based
Answer 2: We appreciate the reviewer’s comment. We suspect that the reviewer may be misunderstanding the link between S100A4 and ovarian cancer malignancy. Many research institutes have been investigating the association between circulating S100A4 and ovarian cancer malignancy worldwide. A clinical study has demonstrated the clinical relevance of circulating MACC1 and S100A4 transcripts for ovarian cancer as shown below.
Link T, Kuhlmann JD, Kobelt D, Herrmann P, Vassileva YD, Kramer M, Frank K, Göckenjan M, Wimberger P, Stein U. Clinical relevance of circulating MACC1 and S100A4 transcripts for ovarian cancer. Mol Oncol. 2019 May;13(5):1268-1279. doi: 10.1002/1878-0261.12484.
Abstract
Metastasis-associated in colon cancer 1 (MACC1) and S100 calcium-binding protein A4 (S100A4) are prominent inducers of tumor progression and metastasis. For the first time, we systematically tracked circulating serum levels of MACC1 and S100A4 transcripts in the course of surgery and chemotherapy and analyzed their clinical relevance for ovarian cancer. MACC1 and S100A4 transcripts were quantified in a total of 318 serum samples from 79 ovarian cancer patients by RT-qPCR and digital droplet PCR, respectively. MACC1 and S100A4 transcripts were significantly elevated in serum of ovarian cancer patients, compared to healthy controls (P = 0.024; P < 0.001). At primary diagnosis, high levels of MACC1 or S100A4 correlated with advanced FIGO stage (P = 0.042; P = 0.008), predicted suboptimal debulking surgery and indicated shorter progression-free survival (PFS; P = 0.003; P = 0.001) and overall survival (OS; P = 0.001; P = 0.002). This is the first study in ovarian cancer to propose circulating MACC1 and S100A4 transcripts as potential liquid biopsy markers.
Comment 3: The patient’s cohort selected is extremely old and most probably does not reflect the current characteristic of ovarian cancer cohorts (in residual post-surgery, PFS and OS). Besides, the data in Table 1 are general and not specific (referring to a range for age at diagnosis seems too vague).
Answer 3: We appreciate the reviewer’s comment. However, we had to comply with the restrictions concerning items described in clinical research reported in academic journals.
Indeed, in accordance with international rules, we cannot provide detailed information such as the patient's medical condition or age as this study did not require an information disclosure as in clinical studies or trials. Therefore, we cannot provide more details in the new Table 2. Our manuscript is not a case report. The contents of medical manuscripts published in academic journals, must comply with international standards (see text below from the IRB, which is reported from Central Ethics Review Board at National Hospital Organization, Tokyo Japan).
The manuscript must not contain too much identifying information and we cannot include information allowing the patient/study participant or their family, friends, or neighbors to be identified (e.g., age, precise location, date of hospital admission, family relationships, potentially identifying medical conditions, current detailed medical condition, and past medical history).
Finally, this study is not a clinical study or clinical trial. In our investigation, tissues of ovarian cancers or lung metastases of ovarian cancers excised by surgical treatment from patients with ovarian cancer were used as samples. Therefore, PFS and OS data are not used because we did not examine the antitumor effects of new drugs.
International guideline for medical ethics for Clinical Research
The manuscript must not contain too much identifying information.
Research members need to remove all information that would allow the patient or their family, friends or neighbors to identify them (e.g., age, precise location, date of hospital admission, family relationships, past medical history, etc.).
- Precise ages should be replaced with an age range, e.g., in their 60’s (5-year range minimum).
- Exact dates should be removed.
- Sample/patient IDs should be removed if known to anyone (e.g., hospital staff or patients themselves) outside the research group. If removal of sample/patient IDs is not possible, they should be replaced with identifiers that cannot reveal the identity of the study subjects.
Please note that in addition to the above changes Institutional Review Board (IRB) require that there be less than three indirect identifiers remaining in your manuscript and supplemental information (if present) for the study to be approved for posting on medical journals. Examples of indirect identifiers are:
- Age range
- Exact dates of patient related events
- Ethnicity
- Religion
- Details about family and family history
- Occupation
- Geographical area
- Potentially identifying medical conditions.
The research manuscript will not be approved for posting on Medical Journal if in ethics declaration by Institutional Review Board (IRB), research member does not include the above-mentioned information about ethical oversight of research work.
Comment 4: Were the tumor sections revised and re assigned to high-grade according to the new two-tier system?
Answer 4: Yes, several surgical pathologists participated in our study. They processed the excised specimens properly.
Comment 5: Was the FIGO stage reviewed and re assigned according to the new 2013 criteria?
Answer 5: The advanced stages of ovarian cancer were classified by the International Federation of Gynecology and Obstetrics (FIGO) into four advanced stages, stage I to stage IV. In the treatment guidelines for ovarian, fallopian tube, and peritoneal cancers in Japan, the advanced stage of surgery classification is described based on the FIGO 2014 classification. However, the medical evidence are similar to that of the FIGO 1988 classification. We classified the stage of surgery for ovarian cancer according to the treatment guidelines for ovarian cancer, fallopian tube cancer, and peritoneal cancer in Japan.
Moreover Dr. Ikuo Konishi is coauthor of this study and is a qualified professional insuring the proper grading of the cancer stages. Dr. Ikuo Konishi is the director of the National Hospital Organization Kyoto Medical Center and is also an emeritus professor at the Faculty of Medicine, Kyoto University. He is a specialist in obstetrics and gynecology and a former director of the Japanese Society of Obstetrics and Gynecology. He is also the director of the Asian Gynecology Association. Dr. Ikuo Konishi is the Advisory Committee of the World Obstetrics and International Federation of Gynecology and Obstetrics (FIGO) Gynecology Association Oncology Committee. Dr. Ikuo Konishi also is International Member of the Female Genital tumors classification on World Health Organization (WHO).
Comment 6: The methodology is poorly descripted, I understand there is a supplementary but the main text should contain at least the types of staining performed (IHC? IF? Both?)
Answer 6: The materials and methods section of the manuscript already describes the types of staining performed as shown in the text below in red:
- Antibodies and immunohistochemistry (IHC)
IHC staining of S100A4, cluster of differentiation 90 (CD90 or Thy1), ACE2, and the RBD of the SARS-CoV-2 spike glycoprotein was performed on tissue sections from pulmonary micrometastases of patients with high-grade serous ovarian cancer. Tumor tissue sections were incubated with the appropriate primary antibodies at 4°C overnight. For the staining of the RBD of the SARS-CoV-2 spike glycoprotein, tumor tissue sections were incubated with 10 ng of recombinant RBD of the spike protein (Sino Biological Inc. Beijing, China) at 4°C overnight. After this incubation, the sections were incubated with the mouse monoclonal antibody recognizing the RBD of SARS-CoV-2 spike glycoprotein at 4°C overnight.
IHC-stained sections were visualized under a confocal microscope (Leica TCS SP8, Wetzlar, Germany) according to the manufacturer’s procedure. Photographs of the normal alveoli and bronchioles areas (Bron.) as well as of metastases areas (Met.) were taken from tissue sections of lung metastases resected from a patient with ovarian cancer as shown in Figure 2B. Then, the expression levels of each factor were calculated using fluorescent color. These IHC experiments on human tissue sections were performed using standard procedures at Shinshu University (Matsumoto, Nagano, Japan) and the National Hospital Organization Kyoto Medical Center (Kyoto, Kyoto Japan) in accordance with the institutional guidelines (approval no. M192). The supplementary materials contain the list of primary or secondary antibodies used in the experiments and the detailed materials and methods.
Comment 7: Results are inconsistent, and with an extensive section that refers to other publications.
Answer 7: We suspect that the reviewer may be confusing the introduction and result sections of the manuscript.
Niches promoting metastatic colonization have been previously investigated using models with human-in-mouse ovarian cancer xenograft in immunodeficient mice. For example, CD34-positive lineage ovarian cancer stem-like cells sorted using the side population procedure were injected into the mammary fat pads of BALB/c nu/nu mice8. CD90, also known as Thy1, is used as a marker for several stem cells9. S100A4, a member of the S100 calcium-binding protein family secreted by ovarian cancer cells, supports the tumorigenesis by stimulating angiogenesis9 (Supplementary Table 1). Pathological examinations have shown the existence of S100A4-negative and CD90-positive stem-like cells in vimentin-positive normal neighboring alveolar epithelial cells9. Similarly to this previous observation, we found that the initialization of mimicry represented incomplete differentiation of normal alveolar epithelial cells toward the stem-like lineage in pulmonary micrometastases of patients with ovarian cancer (Figure 1A).
ACE2, a host-side receptor for SARS-CoV-2, was expressed in CD90-positive alveolar epithelial stem-like cells in the pulmonary metastatic niches of patients with high-grade serous ovarian cancer is essential (Figure 1A and Table 1). Furthermore, histopathological analyses showed that the RBD of the SARS-CoV-2 spike glycoprotein bound to ACE2-expressing CD90-positive alveolar epithelial stem-like cells (Figure 1A and Table 1). Based on these findings, SARS-CoV-2 is deemed to infect the alveolar epithelial stem-like cells in pulmonary micrometastases of patients with ovarian cancer.
The pathological examination with an anti-human CD90 monoclonal antibody revealed that CD90 was not expressed in the alveolar and bronchiolar epithelial cells (Figure 1B). However, analyses with an anti-human ACE2 monoclonal antibody showed that bronchiolar epithelial cells expressed ACE2 (Figure 1B, Table 1). Therefore, the binding of SARS-CoV-2 RBD to the ACE2-positive bronchiolar epithelial cells was confirmed in the normal tissue section (Figure 1B).
In the photographs of the normal alveolar and bronchiolar areas as well as the metastases areas, the expression levels of each factor were calculated using fluorescent color. Moreover, the ratios of CD90-positive, ACE2-positive, and RBD-positive cells were determined in these areas. A quantitative analysis showed that the average ratio of CD90-positive cells was higher in the metastatic areas (36.4%) than that in the normal alveoli and bronchioles areas (4.5%) (Figure 2A, Table 1, Supplementary Figure 1). Additionally, the average proportion of ACE2-positive cells in the normal alveolar and bronchiolar areas was 11.8%, and that in the metastatic areas was 27.4% (Figure 2A, Table 1, Supplementary Figure 2). The average ratio of bronchiolar epithelial cells binding to SARS-CoV-2 RBD in normal alveolar and bronchiolar areas was 7.3%, whereas it reached 15.5% in the metastatic areas (Figure 2A, Table 1). Figure 2B shows the normal alveoli and bronchioles areas and metastases areas.
The ratio of ACE2 and RBD double-positive cells to the total number of ACE2-positive cells was 39.3% in the normal alveolar and bronchiolar areas and 43.8% in the metastatic areas (Figure 2C), suggesting that the binding property of RBD to ACE2-positive cells did not significantly change between normal alveolar and bronchiolar areas and the metastatic areas.
Previous clinical studies have shown that SARS-CoV-2 infection rates and COVID-19 severity rates are higher in cancer and in people with a history of cancer than they are in healthy individuals. The specific reasons for the high SARS-CoV-2 infection rate and COVID-19 aggravation in cancer patients have not been clarified. Previously, we have shown that, in lung metastases of ovarian cancer, alveolar epithelial cells adjacent to ovarian cancer cells transform into alveolar epithelial stem-like cells7. ACE2 is strongly expressed in alveolar epithelial stem-like cells5. The present study aims to confirm the expression of ACE2 in alveolar epithelial stem-like cells adjacent to ovarian cancer cells in lung metastases of ovarian cancer and investigate the binding of SARS-CoV-2 spike glycoprotein to alveolar epithelial stem-like cells. We showed that ACE2 was indeed expressed in alveolar epithelial stem-like cells adjacent to the ovarian cancer in the pulmonary micrometastatic niche. Furthermore, the receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein bound to alveolar epithelial stem-like cells.
- 5. Salahudeen AA, Choi SS, Rustagi A, et al. Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature 2020; 588: 670-675. doi: 10.1038/s41586-020-3014-1
- 7. Hayashi T, Sano K, Aburatani H, Yaegashi N, Konishi I. Initialization of epithelial cells by tumor cells in a metastatic microenvironment. 2020 39(12): 2638-2640. doi: 10.1038/s41388-020-1171-1.
Comment 8: Are the authors showing that RBD of the spike protein for SARS-COV 2 applied onto the tissue sections, attaches to the ACE2 receptor? Or do they mean that they have performed staining for this domain of the spike protein of SARS-COV 2? This is poorly explained
Answer 8: Yes, we applied recombinant RBD of the SARS-CoV-2 spike protein onto the tissue sections, and then stained the RBD bound to the ACE2 receptor.
To clarify this point, we modified the materials and methods section in the revised manuscript and supplementary material as shown below:
For the staining of the RBD of the SARS-CoV-2 spike glycoprotein, tumor tissue sections were incubated with 10 ng of recombinant RBD of the spike protein (Sino Biological Inc. Beijing, China) at 4°C overnight. After this incubation, the sections were incubated with the mouse monoclonal antibody recognizing the RBD of SARS-CoV-2 spike glycoprotein at 4°C overnight.
Comment 9: Are the authors implying that epithelial ovarian cancer cells, metastatic to the lungs, express ACE2? This finding seems a bit inconsistent, metastatic cells tend to keep their primary sites markers, not acquire the ones of the tissue they metastasize at. If the authors believe that, they should provide stronger evidence for that. If they instead mean that the two markers are expressed in the same area by different cells it should be better explained. (Lines 140 and 141)
Answer 9: We appreciate the reviewer’s comment. We suspect that the reviewer may be misunderstanding the results of our research experiments. We are not implying that epithelial ovarian cancer cells, metastatic to the lungs, express ACE2. We demonstrated the expression of ACE2 in alveolar epithelial stem-like cells adjacent to ovarian cancer cells in lung metastases of ovarian cancer and the binding of SARS-CoV-2 spike glycoprotein to alveolar epithelial stem-like cells.
Previous clinical studies have shown that SARS-CoV-2 infection rates and COVID-19 severity are higher in cancer and in people with a history of cancer than they are in healthy individuals. The specific reasons for these observations have not been clarified. Previously, we have shown that, in lung metastases of ovarian cancer, alveolar epithelial cells adjacent to ovarian cancer cells transform into alveolar epithelial stem-like cells7. ACE2 is strongly expressed in alveolar epithelial stem-like cells5. The present study aims to confirm the expression of ACE2 in alveolar epithelial stem-like cells adjacent to ovarian cancer cells in lung metastases of ovarian cancer and to investigate the binding of SARS-CoV-2 spike glycoprotein to alveolar epithelial stem-like cells. We showed that ACE2 was indeed expressed in alveolar epithelial stem-like cells adjacent to the ovarian cancer in the pulmonary micrometastatic niche. Furthermore, the receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein bound to alveolar epithelial stem-like cells. Altogether, these data indicate that cancer patients with pulmonary micrometastases might be more susceptible to SARS-CoV-2. The prevention of de novo niche formation in metastatic diseases might constitute a new strategy for the clinical treatment of COVID-19 in patients with cancer.
- 5. Salahudeen AA, Choi SS, Rustagi A, et al. Progenitor identification and SARS-CoV-2 infection in human distal lung organoids. Nature 2020; 588: 670-675. doi: 10.1038/s41586-020-3014-1
- 7. Hayashi T, Sano K, Aburatani H, Yaegashi N, Konishi I. Initialization of epithelial cells by tumor cells in a metastatic microenvironment. 2020 39(12): 2638-2640. doi: 10.1038/s41388-020-1171-1.
Summary Figure
Comment 10: The authors introduce in Results the concept of “normal areas” they should define what they mean by normal areas in the sections, in Materials and Methods.
Answer 10: We agree with the reviewer's comments. We considered the issues pointed out by the reviewer and modified the materials and methods section in the revised manuscript and supplementary material as shown below:
- Antibodies and immunohistochemistry (IHC)
IHC staining of S100A4, cluster of differentiation 90 (CD90 or Thy1), ACE2, and the RBD of the SARS-CoV-2 spike glycoprotein was performed on tissue sections from pulmonary micrometastases of patients with high-grade serous ovarian cancer. Tumor tissue sections were incubated with the appropriate primary antibodies at 4°C overnight. For the staining of the RBD of the SARS-CoV-2 spike glycoprotein, tumor tissue sections were incubated with 10 ng of recombinant RBD of the spike protein (Sino Biological Inc. Beijing, China) at 4°C overnight. After this incubation, the sections were incubated with the mouse monoclonal antibody recognizing the RBD of SARS-CoV-2 spike glycoprotein at 4°C overnight.
IHC-stained sections were visualized under a confocal microscope (Leica TCS SP8, Wetzlar, Germany) according to the manufacturer’s procedure. Photographs of the normal alveoli and bronchioles areas (Bron.) as well as of metastases areas (Met.) were taken from tissue sections of lung metastases resected from a patient with ovarian cancer as shown in Figure 2B. Then, the expression levels of each factor were calculated using fluorescent color. These IHC experiments on human tissue sections were performed using standard procedures at Shinshu University (Matsumoto, Nagano, Japan) and the National Hospital Organization Kyoto Medical Center (Kyoto, Kyoto Japan) in accordance with the institutional guidelines (approval no. M192). The supplementary materials contain the list of primary or secondary antibodies used in the experiments and the detailed materials and methods.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Fine
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The authors perform an interesting analysis of ovarian cancer patients with COVID19 to determine if malignant sites harbor SARS-CoV-2 particles. However, the manuscript does not provide sufficient quantification and details of the patients analyzed to be convincing of the conclusion that ovarian metastases could exacerbate COVID19 in these patients. Furthermore, there are no control staining of COVID19 patients without cancer to validate staining.
It is not clear to the reader that the samples analyzed were from ovarian cancer patients diagnoses with COVID19 until paragraph 2 of the Discussion. Please update the methods to explain exactly what the inclusionary and exclusionary criteria was for the study.
The study is highly underpowered with 4 patients, only 3 of which were alive at the time of sample collection. With that aside though, the authors do not show or indicate that age-matched controls were used as negative control staining or to compare signal intensity between cancer and normal patients. Further, there is no quantification of the immunofluorescent co-localization of the imaging. Is this representative of the entire lung biopsy? Were CD90 patches present in nonmalignant biopsies? How many CD90 patches had RBD presence in COVID patients? was the intesity of RBD different in malignat vs non-malignant samples to justify the hypothesis that microenvironment changes from ovarian cancer increase viral load and therefore disease severity/mortality?
Also, the COVID19 disease course of the patients was not listed aside from ‘vital status’. Can the authors provide more details in a new Table 2?
Author Response
We appreciate and agree with your comment.
- Although our manuscript is Short Communications of preliminary, the editor recommended to submit our manuscript to Current Issues in Molecular Biology.
　2. In addition, the editor invited me to the section editor of The Journal.
For these reasons, I am very confused when we receive such comments from reviewers.
Guideline for Author in Website of MDPI shows as followings:
Short Communications of preliminary, but significant, results will be considered. Quality and impact of the study will be considered during peer review.
Reviewer 2 Report
Data from the World Health Organization indicated a higher COVID-19 mortality and severity rates among cancer patients. In this manuscript the authors investigated whether cancer stem cells from patients suffering metastatic ovarian cancer express ACE2, one of the receptors for SARS-CoV-2, in an attempt to explain the high morbidity and mortality. While the results presented in the manuscript, indicating that metastatic lung cancer cells express ACE2 and CD90 and bind to RBD are appealing, the manuscript lacks controls and the results are not well presented. These issues needs to be addressed in order to support the conclusions. Specifically:
Author Response
We appreciate and agree with your comment.
- Although our manuscript is Short Communications of preliminary, the editor recommended to submit our manuscript to Current Issues in Molecular Biology.
2. In addition, the editor invited me to the section editor of The Journal.
For these reasons, I am very confused when we receive such comments from reviewers.
Guideline for Author in Website of MDPI shows as followings:
Short Communications of preliminary, but significant, results will be considered. Quality and impact of the study will be considered during peer review.
