Cannabinoids in Combination with Conventional Breast Cancer Therapies: Mechanistic Insights and the Gap to Clinical Translation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript provides a timely and comprehensive narrative review of preclinical and emerging clinical evidence on the combination of cannabinoids with conventional breast cancer therapies. The topic is of clear clinical relevance given the increasing use of cannabinoid-based products among cancer patients and the potential for pharmacological interactions with standard treatments. The authors successfully summarize a wide range of mechanistic studies and highlight important translational gaps, particularly regarding immunotherapy and targeted therapies. However, several claims would benefit from clearer distinction between preclinical and clinical evidence, more cautious interpretation of synergistic effects, and improved consistency in mechanistic framing. Addressing these issues would strengthen the rigor, balance, and translational relevance of the review.

1. Lines 15–18: The claim that cannabinoid use among breast cancer patients is increasing is stated without quantitative prevalence data. Add recent epidemiological statistics or a systematic review to substantiate this assertion.
2. Lines 19–24: The stated aim of the review is broad and descriptive. Clarify a unifying conceptual framework (e.g., sensitization vs antagonism vs toxicity modulation) to guide interpretation of combination effects.
3. Lines 32–36: The abstract implies therapeutic benefit and cardioprotection from cannabinoids, but evidence cited is almost exclusively preclinical. Explicitly state that these findings are derived from in vitro and animal models.
4. Lines 39–42: Mixed effects of cannabinoids with immunotherapy are mentioned without specifying cancer-type heterogeneity. Clarify that negative outcome data largely come from non–breast cancer cohorts.
5. Lines 65–68: The discussion of patient motivations for cannabinoid use is anecdotal. Replace or support with structured survey or observational study data.
6. Lines 70–75: The pharmacological properties of cannabinoids are summarized broadly. Distinguish more clearly between receptor-dependent and receptor-independent mechanisms.
7. Lines 83–87: Mechanistic conclusions about 5-HT1A signaling are extrapolated from limited models. Clarify whether these findings were derived from neuronal, cancer, or mixed systems.
8. Lines 91–103: Cardiotoxicity mitigation is presented as synergistic with chemotherapy. Emphasize that tumor protection and cardiac protection were not evaluated simultaneously in most cited studies.
9. Lines 114–126: The causal link between mitochondrial biogenesis markers and functional cardioprotection is inferred. Recommend tempering language or adding functional outcome measures where available.
10. Lines 134–143: Antitumor synergy is described without reporting drug ratios or synergy metrics. Specify whether combination index, Bliss, or Loewe models were used, or acknowledge this limitation.
11. Lines 145–149: CB2 expression is correlated with survival, but causality is implied. Rephrase to emphasize association rather than mechanistic causation.
12. Lines 168–176: Bioinformatic and proteomic findings are discussed alongside functional studies. Clearly separate hypothesis-generating in silico results from validated biological effects.
13. Lines 188–196: Antagonistic effects of CBD at higher doses are briefly mentioned. Expand this discussion and explicitly warn about dose-dependent reversals of efficacy.
14. Lines 210–214: ERα antagonism by CBD is stated definitively. Clarify whether this effect is ligand-dependent, context-specific, or cell-line restricted.
15. Lines 228–229: Claims that cannabinoids outperform tamoxifen in target engagement are speculative. Tone down language or clearly label this as a hypothesis.
16. Lines 239–247: Clinical data on CBD–tamoxifen interaction are limited in size and duration. Explicitly note sample size limitations and lack of long-term outcome data.
17. Lines 253–262: Differential responses between ER+ cell lines are attributed to PR status without direct experimental confirmation. Rephrase as a hypothesis rather than a conclusion.
18. Lines 285–287: Selectivity toward cancer cells is inferred from a single non-tumorigenic line. Acknowledge the limited scope of normal cell testing.
19. Lines 321–327: The absence of studies combining cannabinoids with HER2-targeted therapy is highlighted but not contextualized. Briefly discuss why this gap is clinically significant.
20. Lines 332–336: Conflicting immunotherapy outcomes are summarized without stratification by cannabinoid type. Explicitly differentiate THC-dominant from CBD-dominant exposure.
21. Lines 356–365: Clinical immunotherapy studies include highly heterogeneous cannabinoid formulations. Emphasize that this heterogeneity limits causal interpretation.
22. Lines 389–400: Immunosuppressive mechanisms are generalized across cannabinoids. Clarify which effects are THC-dominant and which apply to CBD.
23. Lines 408–421: Photodynamic therapy combinations are based on limited in vitro data. Temper translational claims and clearly label this section as exploratory.
24. Lines 442–453: CYP450 inhibition is discussed as a risk, but drug-specific consequences are not detailed. Add examples of breast cancer drugs most likely to be affected.
25. Lines 468–480: Serum binding effects are critical but discussed late. Consider moving or highlighting this limitation earlier in the manuscript.
26. Lines 498–503: The lack of breast cancer–specific immunotherapy data is clearly stated. Strengthen this point by explicitly recommending prospective study designs.
27. Lines 545–557: Regulatory and labeling inconsistencies are mentioned without clinical implications. Clarify how mislabeling could directly affect trial reproducibility and patient safety.
28. Lines 590–607: Conclusions are generally balanced but still optimistic. Reiterate more strongly that no cannabinoid–therapy combination is currently ready for clinical recommendation in breast cancer.

Author Response

We thank the reviewer for the positive evaluation of our manuscript and for the constructive suggestions provided. We have carefully revised the text to improve the distinction between preclinical and clinical evidence, to adopt a more cautious interpretation of reported synergistic effects, and to enhance consistency in mechanistic framing throughout the manuscript. Detailed point-by-point responses to all comments are provided in the attached document.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

After reading the manuscript my major concerns are as follows:

1. The excessive use of abbreviations is a major hindrance for readers, especially, if the list of abbreviation is not attached to the paper. Please, replace the acronyms of drugs with their full names (i.e., Ana, Let, DDP, Exe, AI, Tam, Pem, CBN).

2. Page 4, Section 2.3.: Please, consider the fact that CB2 receptors are preferentially present on immune cells and the activation of CB2 receptors can increase the immunological response against breast cancer cells contributing to a better outcomes in patients, in contrast to those without activation of CB2 receptors. Additionally, by blocking CB1 receptors via CB1 antagonists/ invert agonists, one can modulate the endocannabinoid response allowing for anandamide (an endogenous cannabinoid) to activate CB2 receptors. The role of CB2 receptors in breast cancer is somehow neglected. Please, consider to illustrate the effects of endocannabinoids and phytocannabinoids via CB2 receptors.

3. Figure 2 – please, be precise and mention, which chemotherapeutics produce synergy when combined with cannabidiol (CBD). Not all chemotherapeutic drugs exert synergistic interactions with CBD. There are some drugs producing antagonistic interactions. Please, modify the figure 3 by placing the names of the drugs producing synergy with cannabinoids.

4. Figure 3 – Please, consider to delete this figure in order not to duplicate information already presented in the text of the manuscript. Information placed on this figure is only fragmentary and may cause misunderstanding in potential readers. 

5. Please, consider to enlarge the presentation of interaction between cannabinoids and Immune Checkpoint Inhibitors (for more details see: Dobovišek L, Borštnar S, Debeljak N, Kranjc Brezar S. Cannabinoids and triple-negative breast cancer treatment. Front Immunol. 2024 Aug 8;15:1386548. doi: 10.3389/fimmu.2024.1386548. PMID: 39176080; PMCID: PMC11338791.)

Comments on the Quality of English Language

line 178: citotoxic

line 237: of CBS of CBS

figure 3: anti-inflammator 

Author Response

We thank the reviewer for the careful evaluation of our manuscript and for the constructive comments provided. The manuscript has been revised accordingly. Detailed point-by-point responses to each comment are provided in the attached document.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This review examines a relevant topic, but contains significant methodological and substantive shortcomings that significantly reduce its scientific value and suitability for publication in Cancers.

The article examines a relevant topic, but contains significant methodological and substantive shortcomings - insufficient critical assessment of the evidence base, minimal clinical data, as well as critical gaps in pharmacokinetics with contradictory conclusions without adequate explanation and overly optimistic recommendations. At the moment, I recommend a critical revision, taking into account the critical comments.

1. Section 2.3 discusses the results of *in vitro* studies on the MDA-MB-231 and MCF-7 cell lines without adequately discussing that these models do not reflect the biological complexity of tumors.
2. There is no analysis of the quality of the included studies (there are no inclusion/exclusion criteria, bias risk estimates).
3. The authors do not distinguish between synergism, additive effects and artifacts *in vitro*.
4. Section 2.4 indicates the potential *antagonistic* effect of CBD with DOXO at high concentrations (2.5 mM), but this contradicts the conclusions of Section 2.3, which describes the synergy.
5. In section 4.2, the authors acknowledge the "confounding effects" of CBD with immunotherapy, but do not offer a mechanistic explanation for this paradox.
6. The claim that CBD "can" help with cardiotoxicity (Section 2.2) is based only on murine models; there is no clinical data.
7. 7. The article is positioned as a review for translation into clinical practice, but there is minimal clinical evidence.Only one clinical trial in BC patients (Buijs et al., section 3.2) with a small sample size. The pilot study by Fleege et al. (2025) included only 15 patients. There are no data on long-term safety, pharmacokinetics, and drug interactions in BC patients.
8. Section 6.3.1 discusses the effect of serum on CBD activity, but this discussion is superficial. CBD has a very high binding to plasma proteins (>99%), which means that *in vivo* concentrations of free CBD can be 100+ times lower than *in vitro*.The authors cite Liu et al. (2022) and Karkaci-Salli et al. (2024), but do not discuss whether therapeutic concentrations *in vivo* are achievable at safe doses. There is no discussion of CYP3A4 inhibition of CBD and its effects on the metabolism of DOXO, paclitaxel, and other drugs.
9. The authors acknowledge (section 4.1) that there are no studies on combinations of CBD with HER2 inhibitors (traztuzumab, pertuzumab, lapatinib, neratinib, tucatinib). Unfortunately, this is a critical gap, as HER2+ BC accounts for ~20% of all BC. The authors do not propose hypotheses about possible interactions. There is no discussion about whether CBD can modulate the cardiotoxicity of traztuzumab (an urgent clinical problem).
10.  The conclusion should be more conservative and honestly acknowledge the lack of clinical evidence.

Author Response

We thank the reviewer for the comprehensive assessment of our manuscript and for the constructive critical feedback. The manuscript has been thoroughly revised to strengthen the critical appraisal of the evidence base, to provide clearer differentiation between preclinical and clinical data, and to improve the discussion of pharmacokinetic considerations and translational limitations. We have also adjusted the tone of several sections to ensure a more cautious and balanced interpretation of the available evidence. Detailed point-by-point responses to all comments are provided in the attached document.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am satisfied with the authors’ responses to my previous comments and appreciate the revisions they have made to address the concerns raised. The manuscript is now significantly improved, and I believe it meets the standards for publication. I have no further concerns, and I recommend the paper for acceptance in its current form.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your comments, the manuscript can be published