Review Reports - Generation of Heterotypic Primary Human Spheroids from Glioblastoma Resections and (Pre-)Clinical Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present manuscript addresses a topic of great clinical interest. The authors present an innovative approach that aims to complement clinical practice and the oncological management of glioblastoma; however, the following points should be considered:

Clearly state the objective of the publication, reviewing the scope of the commercial products used in the development of the manuscript, which provides its scientific basis and contribution.
Clarify, through inclusion and exclusion criteria, the reason why more than 50 clinical samples are reported but fewer than 10 samples are ultimately analyzed.
Clarify the correlational implications of the histopathological results with the experimental findings for samples processed and those not processed, using the antibodies used in the analysis.
Clarify the lack of both positive and negative controls in all experimental analyses, given that histopathological reports of the analyzed samples are not included as attachments.
Clarify why cell viability assays were not performed alongside the cytotoxicity assays carried out with the selected drugs. In this regard, further analysis is required to address the extrapolations beyond the stated scope of the proposed model.
Expand the methodological explanation, results, and discussion of the spheroid penetration assays in the matrices.
Provide a more detailed analysis of the microscopy images to demonstrate or at least analyze some pattern of cellular populations beyond fluorescence microscopy.

Author Response

We first want to thank the reviewer for the valuable feedback and the time spent reviewing our manuscript.

We rephrased parts of the manuscript, especially the introduction, in order to improve the quality of English language as the reviewer has criticized it. We acknowledge the given comments and want to address them individually:

Clearly state the objective of the publication, reviewing the scope of the commercial products used in the development of the manuscript, which provides its scientific basis and contribution.

We strengthened the aspect using commercial products by adding it to the implications of the main findings section in line 19.

Clarify, through inclusion and exclusion criteria, the reason why more than 50 clinical samples are reported but fewer than 10 samples are ultimately analyzed.

Overall >50 samples were used to establish the protocol and validate the final workflow as it is presented in the manuscript. The aim of this study was to create a protocol suitable for (in best case) all obtained glioblastoma specimens, independent of patient age, sex, or genetic alterations. We suppressed the mention of 50 patient samples in the abstract to avoid any misinterpretation and modified lines 279-280 to make this point more clear.

Clarify the correlational implications of the histopathological results with the experimental findings for samples processed and those not processed, using the antibodies used in the analysis.

Unfortunately, we do not have access to the histopathological results as the study design does not include pathologic images. This is however a relevant issue that we will address in further studies. At the present time, the focus of this methodology paper is the reliable generation of spheroids as a tool for further analyses.

Clarify the lack of both positive and negative controls in all experimental analyses, given that histopathological reports of the analyzed samples are not included as attachments.

As stated above, the histopathological results are not accessible for this study and cannot be added as attachments. Furthermore, we do not see the lack of positive and negative controls in any of the experiments and would therefore kindly ask for clarification of this point.

Clarify why cell viability assays were not performed alongside the cytotoxicity assays carried out with the selected drugs. In this regard, further analysis is required to address the extrapolations beyond the stated scope of the proposed model.

We agree that more analyses, such as viability assays, could have been performed. However, we would like to point out that the list of possible assays is limitless and assays to be performed will fully depend on the research question and lab equipment.

Expand the methodological explanation, results, and discussion of the spheroid penetration assays in the matrices.

We included more explanations in the methodology (lines 208-209) and results (lines 416-417) sections. More details about the assay can be found in the cited references 9, 10, and 11.

Provide a more detailed analysis of the microscopy images to demonstrate or at least analyze some pattern of cellular populations beyond fluorescence microscopy.

Besides immunohistochemistry we performed flow cytometry analysis to further characterize the cellular populations. Both methods are validated in the field and build the basis for cell composition analysis. In this study, our aim was not to characterize different cell types in detail, but to demonstrate the feasibility of both approaches to get insight into cellular distribution of these patient-derived spheroids. We focused on tumor cells and myeloid cells as two highly relevant components of the tumor mass and could observe specific distribution for these two cell types.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled “Generation of heterotypic primary human spheroids from glioblastoma resections and (pre-)clinical applications” describes the development of a standardized protocol for generating primary human spheroids from resected glioblastoma patient tissues. The authors systematically evaluate the generated spheroids across multiple parameters, including invasion efficiency and drug response using clinically relevant compounds. Overall, the manuscript is clearly written, methodologically sound, and provides sufficient background and rationale for each experimental step. The discussion section appropriately acknowledges the study’s limitations and suggests logical context-specific solutions.

Comments for Authors:

Impact of cellular heterogeneity:
The heterogeneity of cellular composition among patient-derived samples is expected. However, it remains unclear how this variability influences the reproducibility and interpretation of preclinical drug efficacy analyses. The authors are encouraged to discuss this more as the invasion experiments and drug efficiency has been tested with only two patient samples.
Presence of neuronal populations:
While the study emphasizes astrocytic and immune cell components within the spheroids, it would be valuable to clarify whether neuronal cells were also assessed. The presence of neuronal populations could significantly alter spheroid behavior, particularly with respect to invasion dynamics and drug response. Including this information or discussing its potential impact would strengthen the translational relevance of the model. This is particularly relevant because maintenance of neurons in spheroid cultures requires more complex media requirements. The efficiency of survival of neurons in primary glioblastoma spheroids with the standardized protocol is immensely relevant for clinical applications.

Author Response

We first want to thank the reviewer for the valuable feedback and the time spent reviewing our manuscript. We acknowledge the given comments and want to address them individually.

Impact of cellular heterogeneity:
The heterogeneity of cellular composition among patient-derived samples is expected. However, it remains unclear how this variability influences the reproducibility and interpretation of preclinical drug efficacy analyses. The authors are encouraged to discuss this more as the invasion experiments and drug efficiency has been tested with only two patient samples.

We fully agree that the cellular heterogeneity is an important factor to be considered and evaluated when testing drug candidates. Depending on the mechanism of action several cell types will be more affected (e.g. proliferating cells/tumor cells when working with cytostatic drugs) or are mandatory to achieve an effect (e.g. immune cells). We are convinced that our method could be used for exactly this type of analysis as it allows the generation and monitoring of multicellular spheroids. The effects of the cellular composition on drugs efficacies is an important topic which needs to be addressed in specialized studies and can therefore not be covered in this methodology paper. However, we fully agree to the raised point and indicated possible effects due to the cellular composition in lines 451-452.

Presence of neuronal populations:
While the study emphasizes astrocytic and immune cell components within the spheroids, it would be valuable to clarify whether neuronal cells were also assessed. The presence of neuronal populations could significantly alter spheroid behavior, particularly with respect to invasion dynamics and drug response. Including this information or discussing its potential impact would strengthen the translational relevance of the model. This is particularly relevant because maintenance of neurons in spheroid cultures requires more complex media requirements. The efficiency of survival of neurons in primary glioblastoma spheroids with the standardized protocol is immensely relevant for clinical applications.

We again fully agree to the comment, given the impact of neurons on tumor behavior. The protocol we present in our study has not been optimized for the possible maintenance of viable neurons in the spheroids and we did not assess for their presence. We therefore cannot guarantee that the spheroids we generated with the reported protocol are suitable to study neurons associated with the tumor. This, will most likely require to adapt culture conditions to conditions facilitating neuron survival (please see additions in lines 501-503).

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript by Wiener et al. presents a study describing the development of a protocol for generating heterotypic primary glioblastoma spheroids from patient-derived material. The main strength of the work lies in the use of primary human samples and the effort to optimize a reproducible, scalable and accessible protocol. However, several important issues limit the overall impact of the manuscript. Addressing these concerns would significantly strengthen the manuscript.

Detailed comments:

Lines 29-30: “more than 50 patient samples” is mentioned, but no detailed table is provided later. Please include a table summarizing patient characteristics such as MGMT status, age, sex, tumor subtype, etc. Of course not in the abstract, but i.e. in the SI.

Lines 31-33: “comprehensive characterization”, well, the analysis is limited to a small number of markers….

Lines 45-49: The Introduction would benefit from a more critical comparison with existing 3D glioblastoma models.

Lines 97-104: Patient consent and inclusion/exclusion criteria should be described in more detail.

Lines 133-140: Was this strictly standardized across all samples?

Lines 174-199: Validation of segmentation accuracy is not provided.

Lines 252-257: No validation (e.g., comparison to 3D measurements) is provided. This should be discussed as a kind of limitation.

Lines 379-388: Well, quantitative spatial metrics would significantly strengthen this section.

Lines 399-407:This is based on very limited sample size…

Lines 409-416: Complementary assays such as apoptosis markers would strengthen the conclusions. But this is an optional comment.

The limitations section should also include lack of long-term stability, limited cellular characterization and absence of molecular profiling.

Author Response

We first want to thank the reviewer for the valuable feedback and the time spent reviewing our manuscript. We acknowledge the given comments and want to address them individually:

Overall >50 samples were used to establish the protocol and to validate the final workflow as it is presented in the manuscript. The aim of this study was to create a protocol suitable for (in best case) all obtained glioblastoma specimens, independent of patient age, sex, or genetic alterations. The study was therefore not designed to (genetically) characterize glioblastoma specimens. In order to comply with our data safety policy, specific information about tumor characteristics were only given if scientifically necessary.

Lines 31-33: “comprehensive characterization”, well, the analysis is limited to a small number of markers….

We agree that the number of markers is limited and we have therefore removed the word “comprehensive” (line 30). However, we would like to strengthen the point that the aim of this study was to establish a reliable protocol and not to perform in-depth characterization of glioblastoma biology. The chosen markers should represent the main cellular populations in glioblastoma specimen and are therefore a reasonable choice.

Lines 45-49: The Introduction would benefit from a more critical comparison with existing 3D glioblastoma models

We thank the reviewer for this comment and introduced a review about alternative 3D glioblastoma models covering the methods in the field in detail (lines 68-70).

Lines 97-104: Patient consent and inclusion/exclusion criteria should be described in more detail.

The informed consent statement was updated (lines 529-530) and we added a comment on material exclusion (lines 111-112).

Lines 133-140: Was this strictly standardized across all samples?

The short answer is yes. We obtained an automated cell counter “Countess 3” and established a defined counting setup, which has been established using glioblastoma specimens.

Lines 174-199: Validation of segmentation accuracy is not provided.

We used a well-defined pipeline and each analyzed image was quality checked by eye. We agree that image segmentation - especially with varying intensities - is not trivial, but we cannot provide a segmentation accuracy index. If requested we can add exemplary images of the segmentation process as supplements, but we doubt that this will provide further information.

Lines 252-257: No validation (e.g., comparison to 3D measurements) is provided. This should be discussed as a kind of limitation.

We added the word “calculated” in line 329 and added another explanatory sentence to section 2.10 to strengthen the point that this value is based on a mathematical assumption.

Lines 379-388: Well, quantitative spatial metrics would significantly strengthen this section.

We thank the reviewer for this suggestion and agree that a detailed spatial analysis of the spheroids would provide further insights. However, as such an in-depth investigation is beyond the scope of the present study, we have opted for a concise descriptive analysis to maintain the focus of this manuscript.

Lines 399-407:This is based on very limited sample size…

The mentioned section describes the generation of spheroids from one specific patient and is therefore reflecting the patient-specific drug response using several technical replicates.

Lines 409-416: Complementary assays such as apoptosis markers would strengthen the conclusions. But this is an optional comment.

We fully agree. However, our aim was to demonstrate the possible use of the described platform, and for this purpose, we deliberately selected a few assays.

The limitations section should also include lack of long-term stability, limited cellular characterization and absence of molecular profiling.

We fully agree and updated the limitations section to include all mentioned limitations lines 488-505.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author's manuscript was revised.

Author Response

The author's manuscript was revised.

We appreciate the reviewer’s approval of our modifications and thank them for their time and effort.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have revised and improved their work, current version can be accepted.

Author Response

The authors have revised and improved their work, current version can be accepted.

We appreciate the reviewer’s approval of our modifications and thank them for their time and effort.