Breast Cancer Tissues and Organoids BioBank: Constitution, Research Activities and Samples Access

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors from South Italy, report on their experience in the initiation of a biobank of patient derived breast cancer and normal breast organoids. 

 

A few comments:

1) How does the protocol for establishment of organoids as written by the authors differ from that which has already been published by other groups? 

2) Were there any modifications made / practical issues / experience from authors to share with the audience?

3) These are all surgical samples. Do all the patients therefore have non-metastatic disease? It would be good to indicate.

4) If so then would these models be an accurate reflection of metastatic disease which is an area where most of the drug development is occuring.

5) Are there plans to molecularly characterise the organoids? Along with the primary tumors from which the organoids are derived. 

Comments on the Quality of English Language

Generally comprehensible however would benefit more from language editing to ensure clarity and coherence. 

Author Response

Dear Reviewer:

We appreciated your constructive criticisms, and we have addressed each of your concerns as outlined below. 

Please, you will find all the tracked revisions in the ‘marked revised’ manuscript file.

My responses to your comments (normal font) are in italic font.

 

• How does the protocol for establishment of organoids as written by the authors differ from that which has already been published by other groups?

Our protocol differs from those already published by other groups mainly in the tissue digestion step, where: a) a mixture of collagenase type 2 (1mg/mL) and Dispase (0.1mg/mL) has been used, while the only collagenase IV (2 mg/mL) has been used by Aggarwal or collagenase III and hyaluronidase by Mazzucchelli et al.; b)  the digestion time was no longer than 120 minutes compared to the 16 hours digestion time used by Mazzucchelli, who used a different enzymes combination. We realized that we had missed Aggarwal reference. Thus, we added it.

Furthermore, after five days of culture, in our protocol we removed from the culture medium not only the Rock inhibitor but also other factors that promote stemness, such as R Spondin 1, neuregulin-1 and Noggin and we included in their place growth factors, such as insulin and hydrocortisone, to support the proliferation of mammary epithelial cells, as we mention in the discussion section in lines 500-503.

 

• Were there any modifications made / practical issues / experience from authors to share with the audience?

            The digestion time of starting tissue specimen is a critical point for the organoid’s establishment. In our experience a digestion time longer than 120 minutes is dangerous for cell survival. Thus, to share with the audience our experience, we added in discussion section, line 489-491of revised manuscript: ‘In our hands, small tissue fragments of 1–3 mm did not require a digestion time longer than 120 minutes with our enzyme combination to get a good quality cell preparation to generate organoids’.

• These are all surgical samples. Do all the patients therefore have non-metastatic disease? It would be good to indicate.

If we mean metastases to other organs, they are certainly negative. If we think about metastases to the lymph nodes, this information has been integrated in Table S1, in supplementary data of the manuscript, by adding TNM staging data of each patient.

 

• If so then would these models be an accurate reflection of metastatic disease which is an area where most of the drug development is occuring.

These models are an accurate reflection of the single patient and thus they are precious for personalized treatments

 

• Are there plans to molecularly characterise the organoids? Along with the primary tumors from which the organoids are derived. 

Yes, there are plans to characterize them molecularly, by an ‘omic’ approach and phenotypically by single cell analysis

 

Comments on the Quality of English Language

Generally comprehensible however would benefit more from language editing to ensure clarity and coherence. 

Our Editing assistants revised the manuscript

 

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, Miranda and colleagues describe the establishment of the Breast Cancer Tissues and Organoids BioBank, a potentially important resource for basic and translational research and drug discovery. There are a number of issues with the biobank and the manuscript which the authors should consider addressing prior to publication:

1.       It may be premature to publicize a resource until it is ready for public access. For example, how do readers and those who would like to utilize the biobank access the relevant information regarding the collection and related data, especially if the collection continues to grow? Is there a plan in place to provide a web-interface which allows users to search the database and apply for access to tissues and organoids? If so, it may be better to wait until the URL is available for review and subsequent publication?

2.       Validation of organoids across passages (as compared to the original patient samples/tissues) is an important part of ensuring the quality of the collection and the relevance of subsequent studies utilizing them. Those results should be included in the description of the collection, rather than just showing some of the examples (Figures 4 and 5).

3.       What is the plan moving forward? The manuscript describes an initial collection of 27 samples but not all samples will yield organoids successfully and those organoids may or may not retain the same characteristics and markers as the source tissues. Are there different phases or collection goals for 100 samples, 200 samples, and for each subtype?

4.       For this and any biobank utilized for research, the accompanying patient information and clinical data for each sample and derived organoids are just as important as the specimens. While the authors refer to some of them in the text and the included the patient questionnaire in the supplement, it might be more helpful to the reader to have a table or figure showing specifically what information and data will be collected and shown for each sample. Relatedly, treatments received and subsequent follow-up on recurrence and death are also key data that would be helpful to researchers but were not mentioned in the text regarding a plan for follow-up collection and curation of these types of data in updates for samples already in the collection.

5.       For the ER-positive samples, it is known that ER-positive breast cancer cell lines can lose their responsiveness to estrogen stimulation over multiple passages, even with expression of ER. This caveat should be mentioned in proposing validation of estrogen response in the ER-positive organoids.

6.       In lines 487-489, the authors mentioned removing stromal cells. They may want to reconsider doing so since they represent key components of the tumor microenvironment and may, in fact, increase the biological and clinical relevance of the organoids.

7.       The quality of writing is good overall but there are a few specific instances which require correction or clarification: In line 65, the authors refer to “immuno-phenotype.”  This might be misunderstood to be phenotype related to tumor immunity or immune cell infiltration in the tumors. Perhaps “immuno-staining of clinical markers” might be a more apt description? In line 306, “man patient” should be revised to “male patient.” In line 369 “About tumor tissues samples 19%...” should be revised to “For tissue samples, 19%...”  In line 460, “being PDOs amenable to long-term expansion” needs to be revised. In line 462 “Interesting” should be revised to “Interestingly.”

Author Response

Dear Reviewer,

W appreciated your constructive criticisms and we have addressed each of your concerns as outlined below. 

Please, you will find all the tracked revisions in the ‘marked revised’ manuscript file.

Our responses to the your comments (normal font) are in italic font.

 

In this manuscript, Miranda and colleagues describe the establishment of the Breast Cancer Tissues and Organoids BioBank, a potentially important resource for basic and translational research and drug discovery. There are a number of issues with the biobank and the manuscript which the authors should consider addressing prior to publication:

1. It may be premature to publicize a resource until it is ready for public access. For example, how do readers and those who would like to utilize the biobank access the relevant information regarding the collection and related data, especially if the collection continues to grow? Is there a plan in place to provide a web-interface which allows users to search the database and apply for access to tissues and organoids? If so, it may be better to wait until the URL is available for review and subsequent publication?

About the first point, it is possible that the term ‘open access’ in the title of the manuscript led to a misunderstanding about the public biobank data access. The samples will be accessible after requesting as described into the website, while about the linked data access, as indicated in lines 434-435 ‘only authorized personnel had access to the database…...’ of the revised manuscript.

To avoid misunderstanding, we changed in the Title ‘open access’ with ‘samples access’.

 

About the second point, we have planned to provide a web-interface, where users can get information about the type/phenotype of samples collected into the biobank, and apply for access to tissues and organoids, but it will not allow users to search the database.

 

2. Validation of organoids across passages (as compared to the original patient samples/tissues) is an important part of ensuring the quality of the collection and the relevance of subsequent studies utilizing them. Those results should be included in the description of the collection, rather than just showing some of the examples (Figures 4 and 5).

Thank you for this point, we integrated the figures 4 and 5 with a Table S3, in supplementary data including the immune-histochemical characterization of all organoids of the biobank.

 

3. What is the plan moving forward? The manuscript describes an initial collection of 27 samples but not all samples will yield organoids successfully and those organoids may or may not retain the same characteristics and markers as the source tissues. Are there different phases or collection goals for 100 samples, 200 samples, and for each subtype?

The goal is to reach a total number of 500 samples, among which at least 100 samples derived from patients diagnosed with triple negative breast cancer.

 

4. For this and any biobank utilized for research, the accompanying patient information and clinical data for each sample and derived organoids are just as important as the specimens. While the authors refer to some of them in the text and the included the patient questionnaire in the supplement, it might be more helpful to the reader to have a table or figure showing specifically what information and data will be collected and shown for each sample. Relatedly, treatments received and subsequent follow-up on recurrence and death are also key data that would be helpful to researchers but were not mentioned in the text regarding a plan for follow-up collection and curation of these types of data in updates for samples already in the collection.

The information relating to the patient is described in the attached questionnaire and stored into the database together with the other data relating to the medical record and the biological and experimental data of the samples. The table cannot be shown as it will be generated by the biobank user authorized to query the database and released to the interested researcher, following his specific request. This implies the creation of numerous tables based on the information requested every time.

Follow up and related information are planned to be recorded into the database if the patient returns to the same hospital, otherwise it will not be possible. In line 97of revised manuscript, we added: ‘patients back to the hospital’.

 

5. For the ER-positive samples, it is known that ER-positive breast cancer cell lines can lose their responsiveness to estrogen stimulation over multiple passages, even with expression of ER. This caveat should be mentioned in proposing validation of estrogen response in the ER-positive organoids.

We are aware of that. This is the reason we are checking ER-status in the PDOs at different passages and we are noting in our database the obtained data as well as all the procedures to which our biological samples were subjected. This is important not only for the ER loss issue but also for other issues that could arise that we could solve thanks to all the recorded data associated with each biological sample.

 

6. In lines 487-489, the authors mentioned removing stromal cells. They may want to reconsider doing so since they represent key components of the tumor microenvironment and may, in fact, increase the biological and clinical relevance of the organoids.

Thanks a lot for this point. We are aware of that. We are using only a step of filtration through a 100-µm cell strainer as described in ‘PDOs establishment protocol section’ in lines 141-142.

In discussion section, in lines 487-489 we meant that once established, organoids could be enriched, during culturing, removing the components of the tumor microenvironments in order to understand the role of the tumor microenvironment in the biological and clinical relevance of the organoids.

Thus, to make clear this concept, we changed ‘Moreover, once organoids are established, to enrich them from stroma, inflammatory cells and debries during culturing, procedures need to be adjusted, by for example adding differential centrifugation step and/or using cell strainers’. in: ‘Moreover, once organoids are established, we can enrich them from stroma, inflammatory cells and debries during culturing, by adjusting procedures, by for example adding differential centrifugation step and/or using cell strainers. This will provide a further tool to investigate the role of the tumor microenvironment in breast cancer biology and in its progression’.

 

7. The quality of writing is good overall but there are a few specific instances which require correction or clarification: In line 65, the authors refer to “immuno-phenotype.” This might be misunderstood to be phenotype related to tumor immunity or immune cell infiltration in the tumors. Perhaps “immuno-staining of clinical markers” might be a more apt description? In line 306, “man patient” should be revised to “male patient.” In line 369 “About tumor tissues samples 19%...” should be revised to “For tissue samples, 19%...”  In line 460, “being PDOs amenable to long-term expansion” needs to be revised. In line 462 “Interesting” should be revised to “Interestingly.”

Thanks for this point. Yes, we are agreed: ‘cancer immune-phenotype characterization’ can be misunderstood. Thus, in line 65, we have substituted ‘immuno-phenotype’ with ‘immunohistochemical’.

Further, in line 306 ‘man patient’ has been revised to ‘male patient’; in line 369 “About tumor tissues samples 19%...” has been revised to “For tissue samples, 19%...”; in line 460, now line 462, “being PDOs amenable to long-term expansion” has been revised to “PDOs cultures….”; and in line 462, now line 464, “Interesting” has been revised to “Interestingly.”

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes an initial attempt to set up a biobank for breast cancer tissues collected in hospitals in Southern Italy with a laudable goal of using banked organoids to decide among treatment options for donors and patients with similar tumors. The authors note vast heterogeneity in the tumors themselves and the resulting organoid cultures, as well as difficulties in standardization. Given the small initial sample size, no firm conclusions can be obtained. Nevertheless, this work represents an important start. The manuscript can be improved by addressing the following points:

1. How were established protocols integrated in order to generate the incubation conditions and procedures used in this work? The authors should better reference the derivation of these protocols and cite their reasons for choosing the conditions employed. 

2. Is it reasonable to assume that organoid culture conditions developed for healthy breast tissues or a particular type of tumor will suffice for the diverse range of breast tumors encountered? 

3. How long did the investigators try to culture organoids from healthy or tumor tissues? How much could individual cultures be expanded? Were there any notable changes over time in culture?

 

Author Response

Dear Reviewer,

We appreciated your constructive criticisms and we have addressed each of your concerns, as outlined below. 

Please, you will find all the tracked revisions in the ‘marked revised’ manuscript file.

Our responses to your comments (normal font) are in italic font.

 

This manuscript describes an initial attempt to set up a biobank for breast cancer tissues collected in hospitals in Southern Italy with a laudable goal of using banked organoids to decide among treatment options for donors and patients with similar tumors. The authors note vast heterogeneity in the tumors themselves and the resulting organoid cultures, as well as difficulties in standardization. Given the small initial sample size, no firm conclusions can be obtained. Nevertheless, this work represents an important start. The manuscript can be improved by addressing the following points:

1. How were established protocols integrated in order to generate the incubation conditions and procedures used in this work? The authors should better reference the derivation of these protocols and cite their reasons for choosing the conditions employed. 

Our protocol differs from those already published by other groups mainly in the tissue digestion step, where: a) a mixture of collagenase type 2 (1mg/mL) and Dispase (0.1mg/mL) has been used, while the only collagenase IV (2 mg/mL) has been used by Aggarwal or collagenase III and hyaluronidase by Mazzucchelli et al.; b)  the digestion time was no longer than 120 minutes compared to the 16 hours digestion time used by Mazzucchelli, who used a different enzymes combination. We realized that we had missed Aggarwal reference. Thus, we added it.

            The digestion time of starting tissue specimen is a critical point for the organoid’s establishment. In our experience a digestion time longer than 120 minutes is dangerous for cell survival. Thus, to share with the audience our experience, we added in discussion section, line 486-488: ‘In our hands, small tissue fragments of 1–3 mm did not require a digestion time longer than 120 minutes with our enzyme combination to get a good quality cell preparation to generate organoids’.

 

2. Is it reasonable to assume that organoid culture conditions developed for healthy breast tissues or a particular type of tumor will suffice for the diverse range of breast tumors encountered?

Thanks for this point. In our experience, the culture conditions of the organoids need to be adjusted depending on the type of tumor or the type of tissue, whether tumoral or healthy from the same tumor, as also confirmed by the percentage of yield of the organoids. However, biological sample data collected in our database will help address this issue.  For instance, where it is possible, for example when a greater quantity of starting tissue is available, we will use different culture conditions, which we will note in our database, together with the results obtained.

 

3. How long did the investigators try to culture organoids from healthy or tumor tissues? How much could individual cultures be expanded? Were there any notable changes over time in culture?

Thank you for this question, we appreciated it. We have in our biobank PDOs at early and late passage until passage #8. We will keep growing them until they grow. Some of them died at the second-third passage independently if derived from healthy or tumor tissues; and some at the sixth passage. However, we will perform marker expression and karyotype analysis for PDOs at late passages (P10) to ensure their identity and eventual observed changes over time in culture will be noted in our database. This data could be also a reflection of tumor evolution over time and thus need to be recorded.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors responded to the issues raised, but their responses were inadequate.  The main critique stands, that the biobank is not ready. There is no indication that this will be an accessible resource, including access to the relevant clinical data for each of the organoid models. How would potential users know which model to select, based on their specific scientific or clinical question if they cannot access that information. The models and the associated patient and sample information need to be de-identified, as is the norm, but not providing access and not making this information available other than contacting the authors make this a private collection and not a biobank or a public resource. For the comment regarding the loss of estrogen response over multiple passages, just monitoring ER status alone will not be sufficient. Cultured ER-positive cells remain ER-positive but no longer respond to estrogen treatment. At the very least, this should be included as a caveat and discussed in the manuscript. Relatedly, responses to comments which ask for clarification should also be accompanied by corresponding revisions to the manuscript which includes the information and clarifications provided by the authors. 

Author Response

Dear Referee

I appreciated your constructive criticisms. I have addressed each of your concerns below.

My responses to your comments (normal font) are in italic font.

 

The authors responded to the issues raised, but their responses were inadequate. The main critique stands, that the biobank is not ready. There is no indication that this will be an accessible resource, including access to the relevant clinical data for each of the organoid models. How would potential users know which model to select, based on their specific scientific or clinical question if they cannot access that information.

 

The Biobank is ready. We provided in line 382 of the manuscript the website of the biobank (BC-Tiss&Org BioBank, https://www.igb.cnr.it/index.php/bcto-biobank/), where are described the mission and all about the BCTO biobank, enclosed the regulation and how potential users can have access to the samples and their related data, based on the respect of specific ethical and legal criteria.

However, we agree that it may not be clear. Therefore, we added to the manuscript test a new section (section 3.5 BCTO biobank data and samples access, line 461-486) to describe how the users can have access to samples and data.

 

The models and the associated patient and sample information need to be de-identified, as is the norm, but not providing access and not making this information available other than contacting the authors make this a private collection and not a biobank or a public resource.

 

This is not a private collection, but a biobank like other biobanks around the world. To clarify this point, biobank data, in general, cannot be accessed without permission, and this does not mean that it is private collection.

Biobanks collect sensitive data and biological samples from individuals, and to ensure privacy protection and comply with ethical and legal standards, access to this data must be strictly regulated.

I will just clarify some points all biobanks need to care about:

1. Privacy and data protection: Data collected by biobanks often contains sensitive personal, genetic and health information. To protect participant privacy, access is limited to researchers who have approved a research project that meets specific ethical and legal criteria (European General Data Protection Regulation EU GDPR 2016/679).
2. Research ethics: Biobanks operate under strict ethical guidelines. Participants provide informed consent for the use of their data and biospecimens, but such consent is often limited to specific purposes. Researchers must obtain permission before accessing data and must ensure that research is conducted in compliance with ethical regulations.
3. Approval process: To access data from a biobank, researchers usually need to submit an application for approval. This process includes review of the project by ethics committees or competent authorities, which examine the scientific validity, impact on privacy and data security.
4. Controlled access: Biobanks, such as the UK Biobank or the National Institutes of Health, use controlled access systems where approved researchers can use data via secure and cloud platforms. In many cases, access to the data is de-identified, meaning the data is not directly linked to the identity of the participants.

The BCTO biobank, as part of BBMRI-ERIC (Biobanking and BioMolecular resources Research Infrastructure - European Research Infrastructure Consortium), and as other biobanks around the world (UK Biobank, NIH biobank, etc) adopt the same restrictions.

 

For the comment regarding the loss of estrogen response over multiple passages, just monitoring ER status alone will not be sufficient. Cultured ER-positive cells remain ER-positive but no longer respond to estrogen treatment. At the very least, this should be included as a caveat and discussed in the manuscript. Relatedly, responses to comments which ask for clarification should also be accompanied by corresponding revisions to the manuscript which includes the information and clarifications provided by the authors. 

We are aware that over multiple passages, cultured ER-positive cells may lose ER or remain ER-positive but no longer respond to estrogen treatment and thus just monitoring ER status alone will not be sufficient to capture these changes. Therefore, we added to the manuscript test:

in line 421-424 ‘Moreover, since cultured ER-positive PDOs may remain ER-positive but no longer respond to estrogen treatment, we will test their ability to respond to ER and we will note in our database. This data could be also a reflection of tumor evolution over time, relevant under pathological and clinical perspectives, and thus need to be recorded [16].’where we added reference 16 (Malainou, C.P.; Stachika, N.; Damianou, A.K.; Anastopoulos, A.; Ploumaki, I.; Triantafyllou, E.; Drougkas, K.; Gomatou, G.; Kotteas, E. Estrogen-Receptor-Low-Positive Breast Cancer: Pathological and Clinical Perspectives. Curr Oncol. 2023, 30(11):9734-9745.) to underline the role ER changes, under pathological and clinical perspectives, in breast cancer.

and

in line 378-381’ We will perform marker expression and karyotype analysis for PDOs at late passages (P10) to ensure their identity and eventual observed changes over time in culture, such as their ability to respond to estrogen treatment, will be noted in our database.’

Best Regards,

Emilia Caputo, PhD

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for providing the URL and making the revisions. The only missing information on the website is the number of organoid models and the types of BC they represent, for example 27 total models, 56% ER-positive, etc. 

Author Response

Dear referee,

We have added the missing information on the biobank website (https://www.igb.cnr.it/index.php/about-the-bcto-biobank/), under the section About BCTO biobank.