Immunotherapeutic Strategies Targeting Breast Cancer Stem Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors present here an important review on strategies to address the issue of breast cancer stem cells. Some edits / comments that may add to the manuscript 

Introduction:

One general comment is whether the authors want to re-word the clinical part (chemo, surgery, radiation) to present a current landscape for the approach to breast cancer treatment, and then dive into the CSC discussion

1. Please review the breast cancer subtypes: HER2 over expressing is HER2+; Luminal B has ER+ PR negative

2. It is a little bit of a generalization to say that conventional therapeutic approaches are insufficient compared to immunotherapy, especially in breast cancer where there has been significant improvement in outcomes with targeted therapies (HER2, ER). While the statement is true in tumors such as melanoma, this is not fully true in breast cancer

3. It would be good to highlight some of the current approvals for immune checkpoint blockade in breast cancer, currently limited to TNBC (early stage based on Keynote 522 and metastatic based on Keynote 355). Atezolizumab approval was revoked

4. Current immunotherapy strategies (immune checkpoint blockers) are not tumor specific. There are efforts to develop personalized approaches (TILs, vaccines ...) but the approved ones in breast cancer are not tumor specific

5. Please check references 17-19 for the hormone therapy statement

6. Consider including data on frequency / method of identification of CSCs either in clinical or preclinical samples. 

7. Are CSCs in different breast cancer subtypes the same, i.e are they driven by similar pathways, mutations, TME interactions? Or are these breast cancer subtype specific.

**CSC Immune Checkpoint

1. The immune checkpoint molecule paragraph (2.1) is a bit confusing to read. It is unclear how the PD-L1 discussion is linked to the mutational burden, and subsequent immunotherapy benefit 

2. For Tumor associated macrophages, is there a link between cancer stem cells and macrophage differentiation (M1/M2) as that may be an interesting target to tilt the microenvironment towards tumor promoting or tumor suppressive TME

**CSC Immune Evasion

1. There is overlap between CSC immune checkpoint and CSC immune evasion sections. It is not clear in this section if the authors are listing all potential ways of immune evasion, or ones specific to cancer stem cells. It would be good to focus on mechanisms stem cells use to evade the immune surveillance 

2. For tumor dormancy, the mechanisms might be very different for HR+ breast cancer compared to others, especially as recurrences in that subtype can happen very late. Is there any data on that?

**Immunotherapeutic Strategies

1. In the adoptive T cell therapy, please check the percentage of patients with micrometastatic disease and risk of overt metastatic disease as the numbers (30 and 50%) do not add up

2. In the discussion of different approaches, are these being evaluated irrespective of breast cancer subtype?

3. There have been HER2 directed vaccines developed and tested in large trials, however with no clear benefit yet. 

**Please ensure throughout the manuscript all abbreviations have been defined

**The authors must be commended for an extensive review of an evolving topic in breast cancer 

Author Response

See attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This review manuscript addresses immunotherapeutic approaches targeting breast cancer stem cells, a topic of great relevance for this type of cancer. The authors address the topic comprehensively, although the information is presented concisely and easily understandable for readers with basic knowledge of it. The manuscript is, in general, well-written and well-structured. The use of technical language is appropriate. The figure is well-designed, making it relatively easy to understand. However, the figure caption is too short and could be slightly more explanatory. The table is clear and concise. In general, the figure and table complement the main text well. This manuscript may interest a wide range of readers, from researchers interested in the molecular pathogenesis of breast cancer and the options it offers for developing new therapies to physicians who treat patients with this type of cancer and wish to include immunotherapy in their therapeutic arsenal.

There are only a few minor issues that need to be addressed.

1. Lines 13-14. Check if the correct wording is “present challenge.”

2. Lines 30-31. Globocan statistics, consulted today, indicate that lung cancer occupies first place worldwide in both incidence and mortality.

Data indicate that lung cancer occupies first place in incidence worldwide.(https://gco.iarc.who.int/media/globocan/factsheets/cancers/39-all-cancers-fact-sheet.pdf). Therefore, it could be more accurate to say, “According to WHO, breast cancer (BC) is the most common type of cancer among women globally, accounting for 2.26 million cases and 685,000 deaths globally in 2020.”

3. Line 50-53. The author states that “Conventional therapeutic approaches, such as surgery, chemotherapy, radiotherapy, endocrinotherapy, and molecular targeted therapy, are increasingly proving to be insufficient when compared to immunotherapeutic approaches.” The statement overly generalizes the effectiveness of immunotherapy compared to traditional methods. Indeed, conventional therapeutic approaches do not result in tumor eradication in many breast cancer patients and better therapeutic strategies are urgently needed. While immunotherapy has shown promising results in specific subsets of breast cancer patients, it is not universally more effective than all other approaches for all patients. As with other cancer therapies, immunotherapy is not effective in all breast cancer patients. The response rates can vary widely depending on factors such as the type of breast cancer, the presence of specific biomarkers (e.g., PD-L1 expression), and individual patient differences. Furthermore, some patients who initially respond to immunotherapy may develop resistance over time, which is a challenge for all cancer therapies, including immunotherapy^1-5. In this context, the phrase “when compared to immunotherapy…” may suggest to the readers that immunotherapy has proven to be a better treatment for breast cancer than all conventional approaches. Much research is still needed to understand the potential of immunotherapy in breast cancer fully and to optimize its use.  I suggest the authors evaluate alternative wording for this paragraph.

4. In the abstract (lines 17-18), the authors state, "Cancer stem cells represent a rare population, originating from non-malignant stem or progenitor cells…”. However, in lines 111-112, the statement is “CSCs are a rare subpopulation of cancer cells that are presumably derived from stem cells.” This is a more accurate definition since the exact origin of CSCs remains an area of active investigation, with multiple potential sources and mechanisms contributing to their formation^6-8. I suggest rewriting the paragraph in the abstract.

   1   Ge, L. P. et al. ZNF689 deficiency promotes intratumor heterogeneity and immunotherapy resistance in triple-negative breast cancer. Cell Res 34, 58-75 (2024). https://doi.org:10.1038/s41422-023-00909-w

2   Zheng, Y., Li, S., Tang, H., Meng, X. & Zheng, Q. Molecular mechanisms of immunotherapy resistance in triple-negative breast cancer. Front Immunol 14, 1153990 (2023). https://doi.org:10.3389/fimmu.2023.1153990

3   Ma, S. et al. Hypoxia induces HIF1alpha-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy. Nat Commun 13, 4118 (2022). https://doi.org:10.1038/s41467-022-31764-9

4   Li, J. et al. S100A9-CXCL12 activation in BRCA1-mutant breast cancer promotes an immunosuppressive microenvironment associated with resistance to immunotherapy. Nat Commun 13, 1481 (2022). https://doi.org:10.1038/s41467-022-29151-5

5   Hanna, A. & Balko, J. M. Breast cancer resistance mechanisms: challenges to immunotherapy. Breast Cancer Res Treat 190, 5-17 (2021). https://doi.org:10.1007/s10549-021-06337-x

6   Waldum, H. & Slupphaug, G. Correctly identifying the cells of origin is essential for tailoring treatment and understanding the emergence of cancer stem cells and late metastases. Front Oncol 14, 1369907 (2024). https://doi.org:10.3389/fonc.2024.1369907

7   Fukumoto, C., Uchida, D. & Kawamata, H. Diversity of the Origin of Cancer Stem Cells in Oral Squamous Cell Carcinoma and Its Clinical Implications. Cancers (Basel) 14 (2022). https://doi.org:10.3390/cancers14153588

8   Trosko, J. E. On the potential origin and characteristics of cancer stem cells. Carcinogenesis 42, 905-912 (2021). https://doi.org:10.1093/carcin/bgab042

Comments on the Quality of English Language

The quality of the English language is generally reasonable. The authors use technical terms correctly, making the manuscript easy to understand.

Author Response

Please attached file.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors address a very interesting topic, i.e., the possibility of treating breast cancer (BC) with immunotherapy targeting cancer stem cells.

This reviewer's general comment on the article is that it is a compilation of published information. There is little discussion and the reader is not provided with a clear background that should include at least the following:

current treatment landscape of BC (short) and unmet medical need

authorized immunotherapy for BC

description of cancer stem cells, and the rationale for targeting them

any successful (i.e., authorized by FDA and/or EMA) treatment targeting cancer stem cells in any cancer

breast cancer stem cell markers (and targets for immunotherapy)

limitations of breast cancer stem cell markers

experimental (both clinical and preclinical) approaches to immunotherapy against breast cancer stem cells

for clinical trials to be cited, please refer to both registration number on clinicaltrials.gov and respective publication (when available)

this is just a suggestion to improve the readibility of the manuscript 

Author Response

Please see attached file.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

We appreciate the authors clarifying and addressing the comments and suggestions 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have improved the manuscript which extensively addresses a very interesting topic