Review Reports - Impact of Neuroendocrine Neoplasm-Specific Systemic Treatments on Somatostatin Receptors Expression and Function in Neuroendocrine Tumor Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please explain the mechanism by which etoposide consistently upregulates SSTR2, and some other drugs show opposite effects across cell lines.
Page 5, Lines 219–223: The authors report that SSTR2 mRNA expression is significantly increased following treatment with etoposide, cisplatin, 5-FU, and TMZ, whereas STZ and everolimus decrease expression. However, at the protein level, these changes do not reach statistical significance. (i) How do the authors explain the clear discrepancy between mRNA upregulation and the lack of statistically significant changes at the protein level? (ii) Given that Western blot results are not statistically significant, how do the authors justify the biological relevance of the observed mRNA changes? (iii) Cisplatin shows increased mRNA expression but a trend toward decreased protein levels. Can the authors provide a mechanistic explanation for this inverse relationship?
The three cell lines originate from different anatomical sites: BON-1 and QGP-1 are pancreatic, while MS-18 is derived from a rectal NEC metastasis. The drug-specific and cell-line-dependent responses may reflect organ-specific biology. This is an important conceptual point that should be explicitly addressed in the Discussion, particularly since clinical NEN management differs significantly by primary tumor site. The authors should clarify whether they conclude that the differential responses are organ-specific.
Everolimus is an mTOR inhibitor with a fundamentally different mechanism of action from the cytotoxic chemotherapeutics included in this study. Its effects on SSTR expression likely operate through different signaling pathways (e.g., mTOR–PI3K axis effects on transcription factor activity). This mechanistic distinction is not discussed and should be addressed explicitly.
In the Discussion (lines 327–329), the authors suggest that differential SSTR2/SSTR5 responses to cisplatin between cell lines reflect different underlying mutations in regulatory pathways. This is speculative and must be supported by a cited reference.
Multiple bands are visible in the Western blots for SSTR2 and SSTR5. The authors do not explain non-specific bands. Each band should be labeled according to expected molecular weight, and non-specific bands should be acknowledged and discussed.
The rationale for using n=6 biological replicates for qRT-PCR but only n=3 for Western blot analysis is not explained.
The protein quantification bar graph (Fig. 1C) has a y-axis scale that renders the bars for several treatment conditions nearly invisible, particularly for conditions near baseline. The scale should be adjusted or a broken axis used so that all bars are clearly readable.
The IHC images show heterogeneous cell morphology—cells appear in clusters in some panels and scattered in others, with variable cell sizes. This variability likely reflects differences in cytospin preparation density and should be acknowledged. Staining intensity contrast is also poor in several panels. The authors should: (a) standardize cell density across cytospins; (b) improve image contrast and color balance; (c) provide quantitative scoring of staining intensity (e.g., H-score or semi-quantitative scale); and (d) expand the IHC results description in the text.
In Section 2.4 (Methods), the radioligand used throughout is correctly described as ⁶⁸Ga-DOTATOC. However, in the Discussion (lines 352–353), the authors refer to [¹²⁵I-Tyr³] octreotide as the functional assay. This is a clear factual inconsistency that must be corrected throughout the manuscript.
Italicized SSTR2 for the gene, roman SSTR2 for the protein.
The reference to Rogoll et al.'s 2025 findings in the Results section (lines 214–216) should be moved to the Introduction or Discussion.
The abstract states that 5-FU, STZ, and everolimus “generally downregulated SSTR2.” However, the results show that 5-FU actually upregulated SSTR2 in BON-1 cells. The abstract should more carefully reflect the cell-line-specific heterogeneity of these effects.
Several corrections are required: (i) Line 213–214: “The results can are presented” → “The results are presented”; (ii) Line 246: “whereas everolimus and TMZ induced upregulated” → “...induced upregulation”; (iii) p-value formatting is inconsistent— "p<0.001" should be “p<0.001” throughout; (iv) Section heading 3.1: “System therapeutics” should read “Systemic therapeutics.”
The keyword “system therapeutics” should be corrected to “systemic therapeutics.”
The explanation that 68Ga-DOTATOC has lower affinity for SSTR5 vs. SSTR2 (citing Reubi et al. 2000) should be introduced earlier in the Discussion when SSTR5 data are first interpreted, rather than appearing as a late parenthetical remark.
Check the inconsistent units: µM vs µm.
(Lines 115–146): Typo: “Belgia” → Belgium; Pen/Strep repeated twice (Line 126–127)

Comments on the Quality of English Language

The overall language is acceptable. However, a thorough proofread is recommended to correct remaining grammatical inaccuracies before the final version is submitted.

Author Response

Responses to the reviewer I

We thank the reviewers for their constructive and insightful comments, which helped to improve the quality of our manuscript. We have carefully revised the manuscript accordingly and addressed all comments point-by-point below.

Reviewer: Please explain the mechanism by which etoposide consistently upregulates SSTR2, and some other drugs show opposite effects across cell lines.

Response: We thank the reviewer for this important comment.

We agree that the mechanisms underlying the observed SSTR2 modulation are not fully understood. To address this point, we have expanded the Discussion to include a concise mechanistic framework. Specifically, we now highlight that SSTR2 expression is regulated at transcriptional, epigenetic, and post-translational levels and is closely linked to neuroendocrine differentiation.

Regarding etoposide, we note that its consistent effect may be related to activation of the DNA damage response, including p53-associated pathways. However, as no direct mechanistic link between etoposide and SSTR2 regulation has been established, we have carefully phrased this as a plausible interpretation.

The revised text has been added to the Discussion.

Page 5, Lines 219–223: The authors report that SSTR2 mRNA expression is significantly increased following treatment with etoposide, cisplatin, 5-FU, and TMZ, whereas STZ and everolimus decrease expression. However, at the protein level, these changes do not reach statistical significance. (i) How do the authors explain the clear discrepancy between mRNA upregulation and the lack of statistically significant changes at the protein level? (ii) Given that Western blot results are not statistically significant, how do the authors justify the biological relevance of the observed mRNA changes? (iii) Cisplatin shows increased mRNA expression but a trend toward decreased protein levels. Can the authors provide a mechanistic explanation for this inverse relationship?

Response: We thank the reviewer for this insightful comment.

(i) The discrepancy between mRNA upregulation and the lack of statistically significant changes at the protein level may reflect post-transcriptional and post-translational regulatory mechanisms. In addition, the relatively short treatment durations and the dynamic nature of protein synthesis may have limited the detection of significant protein changes within the experimental timeframe.

(ii) Despite the lack of statistical significance at the protein level, we consider the mRNA findings biologically relevant, as they were consistent across independent experiments and were supported by functional readouts. Notably, etoposide-induced SSTR2 mRNA upregulation was paralleled by a significant increase in ⁶⁸Ga-DOTATOC uptake across all cell lines, indicating that transcriptional changes translated into functional receptor availability.

(iii) The inverse trend observed for cisplatin, with increased mRNA but reduced protein levels, may be explained by enhanced protein degradation or impaired translation under cellular stress conditions. Cisplatin-induced DNA damage is known to activate stress responses that can suppress global protein synthesis or promote proteasomal degradation, thereby uncoupling mRNA and protein expression.

We have added a corresponding explanation to the Discussion to clarify these aspects.

The three cell lines originate from different anatomical sites: BON-1 and QGP-1 are pancreatic, while MS-18 is derived from a rectal NEC metastasis. The drug-specific and cell-line-dependent responses may reflect organ-specific biology. This is an important conceptual point that should be explicitly addressed in the Discussion, particularly since clinical NEN management differs significantly by primary tumor site. The authors should clarify whether they conclude that the differential responses are organ-specific.

Response: We thank the reviewer for this important comment.

We agree that the anatomical origin of the cell lines may potentially contribute to differences in treatment response. However, in our dataset, we did not observe a consistent response pattern separating pancreatic (BON-1, QGP-1) and rectal (MS-18) models. Instead, responses varied in a drug- and cell line–specific manner.

Therefore, our findings may argue against a purely organ-specific effect and rather support a context-dependent interpretation, likely influenced by intrinsic cellular characteristics such as differentiation status and baseline SSTR expression. We have clarified this point in the Discussion.

Everolimus is an mTOR inhibitor with a fundamentally different mechanism of action from the cytotoxic chemotherapeutics included in this study. Its effects on SSTR expression likely operate through different signaling pathways (e.g., mTOR–PI3K axis effects on transcription factor activity). This mechanistic distinction is not discussed and should be addressed explicitly.

Response: We thank the reviewer for this important comment.

We agree that everolimus, as an mTOR inhibitor, differs fundamentally from cytotoxic agents in its mechanism of action. While chemotherapeutics primarily induce DNA damage, everolimus acts through inhibition of the mTOR pathway, affecting cellular growth, metabolism, and protein translation.

Its effects on SSTR expression may therefore be mediated through modulation of mTOR-dependent signaling pathways, including downstream effects on transcription factor activity and protein synthesis. This distinct mechanism may contribute to the heterogeneous and cell line–dependent effects observed in our study.

We have added a corresponding statement to the Discussion.

In the Discussion (lines 327–329), the authors suggest that differential SSTR2/SSTR5 responses to cisplatin between cell lines reflect different underlying mutations in regulatory pathways. This is speculative and must be supported by a cited reference.

Response: We thank the reviewer for this important comment.

We agree that this statement is speculative. As no direct evidence for specific underlying mutations is available in our study, we have revised the sentence to a more cautious interpretation and removed the unsupported claim. Alternatively, we now refer more generally to cell line–specific differences in regulatory mechanisms without attributing them to defined mutations.

The Discussion has been updated accordingly.

Multiple bands are visible in the Western blots for SSTR2 and SSTR5. The authors do not explain non-specific bands. Each band should be labeled according to expected molecular weight, and non-specific bands should be acknowledged and discussed.

Response: We thank the reviewer for this comment. The occurrence of multiple bands for SSTR2 and SSTR5 is a known feature of these antibodies and has been described previously, likely reflecting receptor glycosylation.

We have revised the figures to include molecular weight markers and labeled the bands corresponding to the expected sizes of SSTR2 and SSTR5. Non-specific bands are now acknowledged in the figure legends. Quantification was consistently performed using the bands at the expected molecular weights.

The rationale for using n=6 biological replicates for qRT-PCR but only n=3 for Western blot analysis is not explained.

Response: We thank the reviewer for this comment. The different numbers of biological replicates reflect methodological considerations, as qRT-PCR is less resource-intensive, allowing n=6, whereas Western blot analysis was performed with n=3 independent replicates, which is commonly accepted.

Importantly, we consider the radioligand uptake assays as the most functionally relevant readout, integrating receptor expression and availability, and these showed consistent results across experiments.

This has been clarified in the revised Methods section.

The protein quantification bar graph (Fig. 1C) has a y-axis scale that renders the bars for several treatment conditions nearly invisible, particularly for conditions near baseline. The scale should be adjusted or a broken axis used so that all bars are clearly readable.

Response: We thank the reviewer for this helpful comment. The y-axis scaling of Fig. 1C has been adjusted to improve the visibility and readability of all treatment conditions, particularly those close to baseline.

The IHC images show heterogeneous cell morphology—cells appear in clusters in some panels and scattered in others, with variable cell sizes. This variability likely reflects differences in cytospin preparation density and should be acknowledged. Staining intensity contrast is also poor in several panels. The authors should: (a) standardize cell density across cytospins; (b) improve image contrast and color balance; (c) provide quantitative scoring of staining intensity (e.g., H-score or semi-quantitative scale); and (d) expand the IHC results description in the text.

Response: We thank the reviewer for these helpful suggestions. We acknowledge that variability in cell distribution is inherent to cytospin-based preparations and have now stated this in the manuscript. Representative images with more consistent cell density were selected, and contrast and color balance were uniformly adjusted.

In addition, we implemented a semi-quantitative scoring approach (0–3), assessed across multiple cytospin areas (n = 3 per condition) by two independent observers. The results are provided in the Supplementary Material. Given the exploratory nature of the IHC analysis, a semi-quantitative approach was chosen. The Results section has been expanded accordingly.

In Section 2.4 (Methods), the radioligand used throughout is correctly described as ⁶⁸Ga-DOTATOC. However, in the Discussion (lines 352–353), the authors refer to [¹²⁵I-Tyr³] octreotide as the functional assay. This is a clear factual inconsistency that must be corrected throughout the manuscript.

Response: We thank the reviewer for pointing out this inconsistency. The functional assays in this study were performed using ⁶⁸Ga-DOTATOC, as correctly described in the Methods section. The reference to [¹²⁵I-Tyr³] octreotide in the Discussion is an error and has been corrected accordingly throughout the manuscript.

Italicized SSTR2 for the gene, roman SSTR2 for the protein.

Response: We have revised the manuscript accordingly and now consistently use italicized SSTR2 to denote the gene and roman SSTR2 for the protein throughout the text.

The reference to Rogoll et al.'s 2025 findings in the Results section (lines 214–216) should be moved to the Introduction or Discussion.

Response: The reference has been adapted accordingly to the reviewers suggestion.

The abstract states that 5-FU, STZ, and everolimus “generally downregulated SSTR2.” However, the results show that 5-FU actually upregulated SSTR2 in BON-1 cells. The abstract should more carefully reflect the cell-line-specific heterogeneity of these effects.

Response: We thank the reviewer for this important comment.

We have revised the abstract to more accurately reflect the cell line–specific heterogeneity of the observed effects. In particular, we no longer describe a general downregulation of SSTR2 by 5-FU, but instead emphasize the substance- and cell line-dependent responses, including divergent effects across models.

The abstract has been updated accordingly.

Several corrections are required: (i) Line 213–214: “The results can are presented” → “The results are presented”; (ii) Line 246: “whereas everolimus and TMZ induced upregulated” → “...induced upregulation”; (iii) p-value formatting is inconsistent— "p<0.001" should be “p<0.001” throughout; (iv) Section heading 3.1: “System therapeutics” should read “Systemic therapeutics.”

Response: We thank the reviewer for these helpful comments.

All suggested corrections have been implemented. Specifically, grammatical errors have been corrected, wording has been revised for clarity, p-value formatting has been standardized throughout the manuscript, and the section heading has been corrected to “Systemic therapeutics.”

The keyword “system therapeutics” should be corrected to “systemic therapeutics.”

Response: The keyword has been corrected from “system therapeutics” to “systemic therapeutics.”

The explanation that 68Ga-DOTATOC has lower affinity for SSTR5 vs. SSTR2 (citing Reubi et al. 2000) should be introduced earlier in the Discussion when SSTR5 data are first interpreted, rather than appearing as a late parenthetical remark.

Response: We thank the reviewer for this helpful suggestion.

We agree that the differential binding affinity of 68Ga-DOTATOC for SSTR2 versus SSTR5 is an important aspect for interpreting our findings. Accordingly, we have moved this explanation to an earlier section of the Discussion where SSTR5 data are first addressed, in order to provide a clearer mechanistic context for the observed differences in radioligand uptake.

Check the inconsistent units: µM vs µm.

Response: We thank the reviewer for this comment.

The units have been carefully checked throughout the manuscript and corrected for consistency where necessary (µM vs. µm).

Reviewer 2 Report

Comments and Suggestions for Authors

Authors evaluated the effects of six systemic agents (cisplatin, etoposide, 5 FU, streptozocin, temozolomide and everolimus) on SSTR2 ans SSTR5 expression ans uptake of Ga-68 DOTATOC in 3 cell lines.

Results are heterogeneous .Etoposide and temozolomide increase SSTR2 expression, the first in alla cell lines, the second only in the first (VPN-1).

Why authors have chosen these systemic therapies?

Which is the real interest in studying SSTR expression in vitro, at present when there are clinical trials published or ongoing with the objective to combine temozolomide or temozolomide+capecitabine with Lu-177 Dotatate in order to increase survival in patients with metastatic NETs?

It would be interesting that authors asses the effects of capecitabine as well as capcitabine+ temozolomide in their cell lines.

It would also be very interesting to asses uptake ofLu-177 Dotatate in he three cell lines.

The main hindback is related to the heterogeneity of cell lines and the fact that cel line do not reproduce patients uptake of sstr2 agonists.

I suggest that data from clinical trials combining chemotherapy+ Lu-177 DOTATATE might be discussed in the conclusion, as well as clinical trials looking at Ga DOTATOC uptake in patients treated with chemotherapy if any.

Author Response

Reviewer: Authors evaluated the effects of six systemic agents (cisplatin, etoposide, 5 FU, streptozocin, temozolomide and everolimus) on SSTR2 ans SSTR5 expression ans uptake of Ga-68 DOTATOC in 3 cell lines.

Results are heterogeneous .Etoposide and temozolomide increase SSTR2 expression, the first in alla cell lines, the second only in the first (VPN-1).

Why authors have chosen these systemic therapies?

Response: We thank the reviewer for this comment. The selected systemic agents were chosen based on their use in current clinical guidelines, as they represent standard therapeutic options particularly for higher-grade neuroendocrine neoplasms (NEN). By including these clinically established substances with different mechanisms of action, we aimed to assess the potential impact of guideline-relevant treatments on SSTR expression and function. This rationale has been clarified in the revised manuscript.

Response: We thank the reviewer for this important point. While clinical trials combining temozolomide (± capecitabine) with ^177Lu-DOTATATE are ongoing, the underlying biological mechanisms remain insufficiently understood. In particular, it is unclear whether systemic therapies consistently enhance SSTR expression and radioligand uptake, or whether observed effects are partly driven by additive cytotoxicity rather than true receptor upregulation. This could have relevant consequences for optimizing the sequence of e.g. temozolomide and PRRT.

Our in vitro approach enables a controlled, mechanistic analysis of drug- and cell line–dependent effects that cannot be resolved in clinical studies. Notably, we demonstrate that temozolomide does not uniformly increase SSTR2 expression, but can also reduce receptor expression and uptake depending on baseline receptor status.

These findings help to better interpret current clinical trial results and may contribute to optimizing treatment sequencing and patient selection.

It would be interesting that authors asses the effects of capecitabine as well as capcitabine+ temozolomide in their cell lines.

Response: We thank the reviewer for this suggestion. The aim of our study was to systematically evaluate selected guideline-relevant systemic agents across different mechanistic classes, and therefore combination regimens were beyond the scope of this work. Moreover, capecitabine is a prodrug of 5-FU, which was included in our analysis and reflects its biological effects in great extent.

While investigation of CAPTEM may be of interest, our data already indicate that effects on SSTR expression are highly heterogeneous and context-dependent, suggesting that combination effects cannot be assumed. This aspect has been acknowledged in the revised Discussion.

It would also be very interesting to asses uptake ofLu-177 Dotatate in he three cell lines.

Response: We thank the reviewer for this suggestion. While assessment of ^177Lu-DOTATATE uptake would be of interest, the aim of this study was to evaluate receptor expression and function using ^68Ga-DOTATOC as a well-established diagnostic surrogate. ^68Ga-DOTATOC uptake has been shown to correlate with SSTR2 expression and is commonly used to predict PRRT eligibility and response. Therefore, it provides a suitable and widely accepted functional readout in this experimental setting.

The main hindback is related to the heterogeneity of cell lines and the fact that cel line do not reproduce patients uptake of sstr2 agonists.

Response: We thank the reviewer for this important comment. We agree that the heterogeneity of cell lines and the limited ability of in vitro models to reflect patient-derived SSTR uptake represent important limitations. These aspects have already been acknowledged in the Discussion, where we address both the heterogeneity of NEN models and the limited translatability of in vitro findings.

Response: We thank the reviewer for this helpful suggestion. We agree that clinical data on combination strategies are highly relevant. Accordingly, we have expanded the Discussion to include available studies on chemotherapy combined with PRRT (e.g., temozolomide/CAPTEM plus ^177Lu-DOTATATE), which are increasingly being investigated in clinical settings.

However, clinical evidence specifically addressing changes in ^68Ga-DOTATOC/-TATE uptake under chemotherapy in patients remains very limited and largely indirect, with available studies primarily focusing on treatment response rather than receptor modulation per se. This gap further underlines the relevance of our mechanistic in vitro approach.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors replied thoroughly to comments ans suggestions.The manuscript was improved.