An Old New Friend: Folliculo-Stellate Cells in Pituitary Neuroendocrine Tumors

Comments 1: Introduction: The authors need to state what purpose and what kind of research was conducted in this study.

Response 1: Thank you for pointing this out, we have added the requested information at the end of the Introduction chapter [page 2 lines 67-69].

“The current study is a retrospective observational analysis aiming to evaluate the characteristics and distribution of FS cells within PitNETs, in relation to their specific subtypes, as defined according to current immunohistochemical classification.”

Comments 2: Materials and Methos: Fixing solution should be indicated. The number that passed the ethics review should be listed.

Response 2: Agree. We have, accordingly, revised the Materials and Methods to emphasize these points. The ethics committee approval number was listed on page 2 line 74 and fixing solution was listed on page 2 line 78.

“The study has the approval of the University of Medicine and Pharmacy “Victor Babes” Ethics Committee (Ethics Approval no.102/03.10.2022 rev 2025).”

“tissue fixation was performed using 10% neutral buffered formalin”

Comments 3: Results: Scale bars, not objective lens magnification, should be included in all figures. And figures should be shown without digital zoom. Figure 2 is not clear and should be replaced.

Response 3: Agree. We have, accordingly, revised these points.

•            In the attached microscopy folder scale was added to original slide images. Figure 2 images have been replaced with another case showing clear immunopositivity for both GH and ACTH, offering a clear cytoplasmic reaction for each hormone. 

•            In the article body, Figure 1 has been replaced with a cropped version of the original, including the scale.

•            In the article body, Figure 2 images have been substituted with new cropped originals.

•            In the article body, Figure 3 images have been replaced by the same originals, now containing the scale.

•            In the article body, Figure 4 has been replaced with a cropped version of the original, including the scale.

Comments 4: Results: Double and triple staining data with hormones or S100 or GFAP is needed.

Response 4: Regarding double and triple immunostaining with pituitary hormones and GFAP/S100, the technical possibilities are detailed in the 'Materials and Methods' section. The immunohistochemistry equipment currently available does not support double or triple staining involving antibodies that share the same subcellular localization—in this case, cytoplasmic. Practically, such staining would result in overlapping cytoplasmic signals, which would be visually inconclusive due to the lack of contrast. Moreover, an additional limitation arises from the fact that both the antibodies used for pituitary hormones and the one targeting GFAP are anti-human, and thus derived from the same species. This would lead to cross-reactivity between the primary antibodies, further compromising the specificity and interpretability of the staining.

Comments 5: This is a repetition of what was said in the Introduction and should say more about the tumorigenesis of FS cells.

Several studies on FS cells and vascular endothelial cell differentiation have already suggested a link (Horiguchi et al. DOI:10.1038/s41598-018-23923-0 and DOI 10.1007/s00418-020-01862-0).

Response 5: We sincerely thank you for highlighting such valuable and insightful references, which have significantly contributed to improving the quality of our manuscript. After carefully reading the 2 articles we have decided to link our study in the context of the results presented there. Additionally, we have integrated three additional references, one that covers in depth the process of tumorigenesis and two other that focus on the complexity of FS cells [page 8 lines 266-291].

 “In a study by Koyama et al. it was demonstrated that folliculo-stellate-like (FS-like) cells significantly contribute to pituitary tumorigenesis by promoting tumor growth in vivo. Using a murine model, the authors co-implanted GH-producing MtT/S tumor cells with FS-like TtT/GF cells into nude mice, observing that only the combination led to successful tumor formation and enhanced growth hormone secretion, whereas MtT/S cells alone failed to form tumors. Histological examination revealed that FS-like cells surrounded tumor nests, suggesting that their supportive microenvironment, likely through paracrine signaling, plays a critical role in facilitating neuroendocrine tumor proliferation and progression [38]. Moreover, it was mentioned that FS cells, similar to other adenohypophyseal cell types originate from Rathke’s pouch and possess pluripo-tent characteristics, enabling them to perform numerous functions including endocrine regulation, angiogenesis, and modulation of immune responses. The expression of the pituitary-specific transcription factor Ptx1 in both hormone-producing progenitor cells and FSCs further supports the common developmental origin [39,40].

In addition to FS cells, blood vessels constitute a fundamental component of the TME. Our analysis revealed significant interactions between GFAP positive cells and vascular structures. In 27 PitNETs, FS cells were predominantly located in vascular re-gions. Furthermore, in certain instances, strong associations were observed between FS cells and endothelial cells.

Previous studies have suggested the interplay between FS cells and blood vessels. Horiguchi et al. identified a subpopulation of adult pituitary progenitor cells expressing both S100β and SOX2, which exhibit plasticity and multipotency. These cells were iso-lated from the adult rat anterior pituitary using CD9 as a membrane marker. In vitro analyses demonstrated their potential to differentiate into endothelial cells through bone morphogenetic protein (BMP) signaling pathways. Additionally, it was shown that a subpopulation of CD9/S100β/SOX2-positive pituitary progenitor cells express the chemokine CX3CL1 and can differentiate into endothelial cells [41, 42].

Given all the facts mentioned above, as well as the ability of FS cells to produce various paracrine factors such as VEGF and basic fibroblast growth factor [5], their role in blood vessel development may offer valuable insights into understanding tumor physiopathology and progression.”

4. Response to Comments on the Quality of English Language

Point 1: Several abbreviations not spelled out are seen, and several that are spelled out but not using abbreviations (FC cells)

Response 1: Thank you for pointing this out. We have revised the abbreviations by removing the FC cells and bFGF abbreviations from the text as they were not necessary and have replaced in all article parts the following:

Folliculo-stellate cells -> FS cells

Tumoral microenvironment -> TME

Pituitary neuroendocrine tumors -> PitNETs

Glial fibrillary acidic protein -> GFAP

Vascular endothelial growth factor -> VEGF