Identification and Characterisation of Canine Osteosarcoma Biomarkers and Therapeutic Targets

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Jackson-Oxley et al. investigated overexpressed mRNAs in canine osteosarcoma tissue, and checked expression of four proteins, GPR64, TOX3, MMP-12, and FOXF1 by IHC.

1. The title of the manuscript should be "Identification and Characterization of Canine Osteosarcoma Biomarkers and Therapeutic Targets."
2. The previous paper (Simpson et al. Cancers 2020) shows that the overexpression of 25 genes was detected by RNAseq, then qRT-PCR analysis and immunostaining confirmed positive expression of MMP3, SLC2A1 and DKK3 proteins in OSA tissue. About the four genes (GPR64, TOX3, MMP-12, and FOXF1), overexpression of mRNAs are described, but RT-PCR data or IHC seem not described. The reviewer could not understand how the authors selected these four genes.
3. In the Results section, cBioPortal data show that mRNA overexpression of the four genes seems not so frequent. The authors should show the frequency of patients with overexpression or amplification of four genes clearly.
4. In the discussion section, the previous data about canine and human patients are mixed. The authors should show how the results of this manuscript would contribute to canine and human medicine separately.

Author Response

Reviewer 1.

Jackson-Oxley et al. investigated overexpressed mRNAs in canine osteosarcoma tissue, and checked expression of four proteins, GPR64, TOX3, MMP-12, and FOXF1 by IHC.

Thank you for your positive comments and for your valuable time and suggestions. We have provided a track changes version and a ‘clean’ version of the manuscript so you can see changes too.

 

Comment 1: The title of the manuscript should be "Identification and Characterization of Canine Osteosarcoma Biomarkers and Therapeutic Targets."

Response 1: Thank you, we have amended this as per your suggestion.

 

Comment 2: The previous paper (Simpson et al. Cancers 2020) shows that the overexpression of 25 genes was detected by RNAseq, then qRT-PCR analysis and immunostaining confirmed positive expression of MMP3, SLC2A1 and DKK3 proteins in OSA tissue. About the four genes (GPR64, TOX3, MMP-12, and FOXF1), overexpression of mRNAs are described, but RT-PCR data or IHC seem not described. The reviewer could not understand how the authors selected these four genes.

Response 2: Our present samples were clinical samples from patients, as such ethics only allowed for the actual tumour tissue to be excised and used for research - clinical cases from formalin fixed, paraffin embedded tissues with ethics allowing us to take limited sections for the present study, once clinical reports had been finalised. We have analysed the same samples from our previously published study using clinically relevant H-scoring, which is not clinically used in conjunction with RT-PCR, but full IHC was therefore conducted as part of the present paper as H-Scoring involves firstly conducting IHC and then scoring.

Regarding the selection of the genes/proteins of interest. Our apologies, we did not make this clear in our manuscript. At the end of the introduction we presented two paragraphs explaining why these four were selected. Based on previous research we have shown their overexpression, associations with OSA and other cancers in people and dogs, and highlighted potential drugs which could target them or their pathways – which means clinical testing of these drugs may start sooner than for example if we needed to develop drugs de novo. We have added a few sentences and words in though to really clarify this for the reader.

‘The present study expands on previous work conducted by our research group by identifying and characterising expression of biomarkers that have the potential to be therapeutic targets and/or prognostic factors in canine OSA. GPR64, TOX3, MMP-12 and FOXF1 RNA expression were previously shown to be expressed at significantly higher levels in canine OSA tissues compared to matched non-tumour bone [29]. These are of interest due to their associations with either OSA and/or other cancers in people, and because some already have potential therapeutic agents which could be developed for use in OSA. GPR64 is a member of the adhesion G protein-coupled receptor family (aGPCRs). Mutations in human aGPCRs have been evidenced within several cancer types including OSA and bone cancer cell lines, but also in various bone disorders [30-32]. It is also notable that overexpression of GPR110 and GPR56 correlate with poorer survival rates in people with OSA [33, 34]. TOX3 has been identified as a transcriptional factor of the protein transporter ATP Binding Cassette Subfamily G Member 2 (ABCG2), which has enhanced ABCG2 expression in colorectal cancer [35]. ABCG2 has been shown to be expressed highly in human OSA, contributing to significantly shorter overall patient survival times compared with normal ABCG2 expression levels [36]. A number of drugs indirectly inhibit TOX3 making it a valuable potential target in cancer therapy [37, 38]. MMP-12 overexpression has been human lung adenocarcinoma (LUAD), colorectal cancer, hepatocellular carcinoma (HCC) and OSA [39-43]. Potential therapeutic agents include all-trans retinoic acid (ATRA) which can reduce MMP-12 secretion suppressing OSA cell migration [43], Aderamastat (FP-025), a selective MMP-12 inhibitor, and AZD1236 a dual MMP-9/MMP-12 inhibitor, which have not yet used in cancer [44-46]. High expression levels of FOXF1 have been observed in human OSA cell lines and tumours and associated with lung metastasis [47]. It has also shown both anti- and protumour effects, especially in breast cancer in people [48, 49]. This research all indicates that GPR64, TOX3, MMP-12 and FOXF1 are highly relevant as potential biomarkers and targets in canine and human OSA. The aims of the present study were to determine GPR64, TOX3, MMP-12 and FOXF1 expression patterns utilising immunohistochemistry and H-scoring in OSA tissues from OSA canine patients. Additionally, analysis was conducted to determine whether differences in sex or anatomical location of the tumour affected protein expression. cBioPortal analysis was also conducted to determine whether genetic alterations were present in human patients with OSA.’

 

 

 

Comment 3: In the Results section, cBioPortal data show that mRNA overexpression of the four genes seems not so frequent. The authors should show the frequency of patients with overexpression or amplification of four genes clearly.

Response 3:

In the results we have written the percentage of genetic alterations and specified the number of patients with overexpression or amplification for each gene eg

‘The cBioPortal search revealed that 3% of n=159 human OSA tissues had GPR64 genetic alterations (Figure A2). Two patients had missense mutations of unknown significance and three patients had high GPR64 mRNA expression.’

In addition, we have presented Figure A2 which also shows the cBioPortal results. We have now added an extra written section in the discussion highlighting the importance of heterogeneity and subtypes and of the differing genetic alterations seen in people:

‘Rothzerg and coauthors [98] highlighted the importance of investigating each subtype, expression profiles and their underlying pathophysiologies, and of developing new biomarkers to facilitate development of novel treatments and help detect and monitor dis-ease. Although work is ongoing in both canine and human OSA to fully appreciate the different subtypes, understanding the potential biomarkers we have presented, and others, in different subtypes and cell types is valuable future research. Furthermore, OSA and their metastases are heterogeneous, with differing TMEs, cell types, expression levels of genes and proteins, genomic mutations, which can lead to therapeutic challenges such as drug resistance and increased metastasis [16, 99]. This further highlights the need for personalized, multi-target therapies, biomarkers in both human and canine OSA. The canine specimens in the present study have shown large ranges of proteins within the tumours, supporting their heterogeneity in terms of GPR64, TOX3, MMP-12 and FOXF1 expression. In addition, the genetic alteration rates of the four genes of interest in people are variable.  Our cBioPortal results revealed that 3-7% of n=159 human OSA tissues had GPR64, TOX3, MMP-12 and/or FOXF1 genetic alterations including missense mutations, deep deletions, high mRNA expression and amplifications. These findings likely reflect both the heterogeneity of OSA and the differing subtypes present in the cohort and highlight the need for personalized medicine and more advanced diagnostic and detection methods.’

 

Comment 4: In the discussion section, the previous data about canine and human patients are mixed. The authors should show how the results of this manuscript would contribute to canine and human medicine separately.

Response 4: Thank you. Our discussion hoped to blend concepts and research to better understand each biomarker separately, as little has been conducted in dogs or OSA in general we have brought in evidence from all sources and ensured we have reported which species each piece of work pertains to. We do see the value in summarising this for the reader though in terms of separating people and dogs. Therefore, we have summarised this at the end of the discussion too as below.

‘The present study investigated Rottweiler patients, but research into other breeds, and indeed other species, could be valuable in understanding the clinical applications of GPR64, TOX3, MMP-12 and FOXF1 biomarkers or therapeutic targets. Research has shown that they are expressed in a range of human cancers and our work has shown that genetic alterations are present in 4-7% of an OSA human patient cohort. As all four proteins are expressed in a range of cancers as such they could potentially serve as multi-cancer biomarkers. Expression could differ between species and between cancer types, therefore individual verification for each protein, within each species and cancer would be necessary, as with all biomarkers. In people there are also a range of drugs targeting the proteins of interest which provides possible routes for clinical trials in OSA patients. In dogs, we have shown GPR64, TOX3, MMP-12 and FOXF1 expression via quantitative H-scoring and described qualitative staining patterns and investigated differences be-tween anatomical location of the bone tumours and in both sexes. Future work to develop and validate clinical tests and understand potential expression differences between individuals, subtypes, and cell types, in different breeds, would help advance this research into pathology laboratories. Different cancer types could also be investigated in dogs, given the more extensive evidence present in human cancers of GPR64, TOX3, MMP-12 and FOXF1 expression. Given that H-scoring in veterinary medicine is not as standard as in human medicine, there is more work to be conducted in this area in general too. Likewise work progressing treatments in OSA, perhaps repurposing present drugs, would offer more insights into treating this complex cancer in dogs.’

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript the authors study 4 known biomarkers of interest in OSA in humans and studies them in canine OSA tissues in comparison to non tumor tissues. They compared expression with respect to sex, cytoplasmic vs nuclear expression levels and tumor location. They utilized H-scoring to quantitatively assess protein expression which is a novel testing in canine tumors. Overall the manuscript reads quite well and adds value to the growing literature. A few comments to note as below:

1)  On page 3 the authors state - Both canine and human OSA are usually treated as standard via amputation or limb sparing surgery, often with adjunct chemo/immunotherapy [14, 20] - I don't believe that immunotherapy is a standard of care in OSA at least in humans. MAP + inteferon treatment did not show improved survival. Can the authors further explain?

2) Have these 4 biomarkers been testing in canine other tumor types other than OSA? If yes and found to overexpressed then how will these biomarkers be used as a diagnostic test?    

Author Response

Reviewer 2

In this manuscript the authors study 4 known biomarkers of interest in OSA in humans and studies them in canine OSA tissues in comparison to non tumor tissues. They compared expression with respect to sex, cytoplasmic vs nuclear expression levels and tumor location. They utilized H-scoring to quantitatively assess protein expression which is a novel testing in canine tumors. Overall the manuscript reads quite well and adds value to the growing literature.

Thank you for your valuable comments. We have provided a track changes version and a ‘clean’ version of the manuscript so you can see changes too.

A few comments to note as below:

Comment 1:  On page 3 the authors state - Both canine and human OSA are usually treated as standard via amputation or limb sparing surgery, often with adjunct chemo/immunotherapy [14, 20] - I don't believe that immunotherapy is a standard of care in OSA at least in humans.

MAP + inteferon treatment did not show improved survival. Can the authors further explain?

Response 1: Our apologies on that sentence this has been altered to: Both canine and human OSA are usually treated as standard via amputation or limb sparing surgery, often with adjunct chemo/immunotherapy [14, 20]. Immunotherapy may be offered where standard treatments have failed, especially in clinical trials, and a canine OSA USDA (Product Code 95A7.50) approved personalized T-cell therapy became available in 2025 [21]

Regarding MAP (methotrexate, doxorubicin, and cisplatin) + interferon, we have not studied this in this paper or any other of our works, or referred to it, but of course there are the published rationales for not working including many patients not receiving/continuing the interferon. We might wonder whether given our results it may only work in a subset of patients and as such could be masked in a large cohort but I do not think we have any direct evidence to suggest this so we have not included it in our paper.  

 

Comment 2: Have these 4 biomarkers been testing in canine other tumor types other than OSA? If yes and found to overexpressed then how will these biomarkers be used as a diagnostic test?   

Response 2: Thank you for bringing this up. Throughout the discussion we have stated where each of the genes/proteins are expressed, where known, in other tissues and types of cancers as we think this helps understand the mechanisms, functions and even potential to investigate other cancer types for biomarker potential i.e. for our proteins of interest all four represent potential multi-cancer biomarkers. Approaches in medicine frequently use many biomarkers for each cancer, and osteosarcoma has many biomarkers under investigation but none have been FDA approved yet. We are adding evidence towards potential biomarkers which can be further investigated and may show promise in treatment development, diagnosis, prognosis etc in OSA but possibly other cancers too.

Clinically approved biomarkers are frequently found in many tumour types, a good example is HER2, famously known to be overexpressed in 15-30% of breast cancers but is also overexpressed in gastric, ovarian, endometrial, salivary gland, bladder, lung, and colorectal cancers, plus, although to less extent cervical, pancreatic, and head/neck cancers. We are not claiming to have an OSA specific biomarker, rather that all four of ours are expressed in OSA and therefore, just as with HER2 and many other biomarkers differing cancers, combining knowledge about expression of these in particular tumours may help identify potential treatment regimes or treatment developments, and may help with diagnosis and prognosis, as present biomarkers do for other tumours. To help the reader we have summarised in the discussion that all four genes/proteins could act as multi-cancer biomarkers and in the final summary added about other tumour/cell types.

 

‘The present study investigated Rottweiler patients, but research into other breeds, and indeed other species, could be valuable in understanding the clinical applications of GPR64, TOX3, MMP-12 and FOXF1 biomarkers or therapeutic targets. Research has shown that they are expressed in a range of human cancers and our work has shown that genetic alterations are present in 4-7% of an OSA human patient cohort. As all four proteins are expressed in a range of cancers as such they could potentially serve as multi-cancer biomarkers. Expression could differ between species and between cancer types, therefore individual verification for each protein, within each species and cancer would be necessary, as with all biomarkers. In people there are also a range of drugs targeting the proteins of interest which provides possible routes for clinical trials in OSA patients. In dogs, we have shown GPR64, TOX3, MMP-12 and FOXF1 expression via quantitative H-scoring and described qualitative staining patterns and investigated differences between anatomical location of the bone tumours and in both sexes. Future work to develop and validate clinical tests and understand potential expression differences between individuals, subtypes, and cell types, in different breeds, would help advance this research into pathology laboratories. Different cancer types could also be investigated in dogs, given the more extensive evidence present in human cancers of GPR64, TOX3, MMP-12 and FOXF1 expression. Given that H-scoring in veterinary medicine is not as standard as in human medicine, there is more work to be conducted in this area in general too. Likewise work progressing treatments in OSA, perhaps repurposing present drugs, would offer more insights into treating this complex cancer in dogs.’

Reviewer 3 Report

Comments and Suggestions for Authors

The paper entitled Identification and Characterisation of Osteosarcoma Biomarkers and Therapeutic Targets is potentially interesting.  This study analyzed proteins that are upregulated in canine osteosarcoma tissues, which has not only improved our understanding of canine bone cancer but also important for identifying potential therapeutic targets and diagnostic biomarkers. The methods used were justified and the conclusions were reasonable.  

 

Some issue

Figure 1: GPR64 H-score analysis. Can you verify these results by PCR or by WB?.

Figure 2. TOX3 H-score analysis. Can you verify these results by PCR or by WB?.

Figure 3. MMP-12 H-score analysis. Can you verify these results by PCR or by WB?.

 Figure 4. FOXF1 H-score analysis. Can you verify these results by PCR or by WB?.

Can you conform these results in human samples? I human, different Subtypes of Osteosarcoma with Histopathological Patterns and Clinical Behaviour  (https://www.mdpi.com/2673-5261/4/2/11) would have different Tumour Microenvironment in Osteosarcoma?  It would be relevant to discuss the expression of these proteins in these Different Subtypes of Osteosarcoma.

The cellular mechanisms of  GPR64, TOX3, MMP-12 and FOXF1 in OS were not fully elucidated? Recent studies have found by single-cell RNAseq analyses (for example ; PMID: 37439349 ) osteosarcoma tissues contain many cell types. It would be informative to discuss how GPR64, TOX3, MMP-12 and FOXF1 may affect these types of cells in  Tumour Microenvironment in Osteosarcoma and be used as biomarkers.  

Author Response

Reviewer 3.

 

The paper entitled Identification and Characterisation of Osteosarcoma Biomarkers and Therapeutic Targets is potentially interesting.  This study analyzed proteins that are upregulated in canine osteosarcoma tissues, which has not only improved our understanding of canine bone cancer but also important for identifying potential therapeutic targets and diagnostic biomarkers. The methods used were justified and the conclusions were reasonable. 

Thank you for your positive comments and for your valuable time and suggestions. We have provided a track changes version and a ‘clean’ version of the manuscript so you can see changes too.

 

Comment 1: Figure 1: GPR64 H-score analysis. Can you verify these results by PCR or by WB?. Figure 2. TOX3 H-score analysis. Can you verify these results by PCR or by WB?. Figure 3. MMP-12 H-score analysis. Can you verify these results by PCR or by WB?. Figure 4. FOXF1 H-score analysis. Can you verify these results by PCR or by WB?.

Response 1: H-Scoring is a very specific type of immunohistochemistry scoring used by pathologists to assess expression in tissue and is not used in conjunction with WB or PCR in the pathology laboratories for this endeavour. Therefore, we have used this technique as it is clinically relevant and shows not only a clinically relevant quantitative expression of the protein but also enables qualitative assessment of the tissue simultaneously, which is not applicable for WB or PCR. We felt this was especially important in OSA as the tissue is heterogeneous and therefore identifying the tissue is essential. This paper is intended as a follow up paper to our original paper which looked at overexpression using RNA-Seq, by assessing the proteins in the tumours using H-Scoring to provide a quantitative and qualitative assessment of protein expression. We have written more in the discussion about the heterogeneity of this cancer though as it certainly makes diagnosis and treatment much more complex and it is important to address.

 

Comment 2: Can you conform these results in human samples? I human, different Subtypes of Osteosarcoma with Histopathological Patterns and Clinical Behaviour  (https://www.mdpi.com/2673-5261/4/2/11) would have different Tumour Microenvironment in Osteosarcoma?  It would be relevant to discuss the expression of these proteins in these Different Subtypes of Osteosarcoma. The cellular mechanisms of  GPR64, TOX3, MMP-12 and FOXF1 in OS were not fully elucidated? Recent studies have found by single-cell RNAseq analyses (for example ; PMID: 37439349 ) osteosarcoma tissues contain many cell types. It would be informative to discuss how GPR64, TOX3, MMP-12 and FOXF1 may affect these types of cells in  Tumour Microenvironment in Osteosarcoma and be used as biomarkers. 

Response 2: We absolutely agree with you, we would love to look at human samples but sadly we do not have access to human samples. Although our team have breast and prostate samples in large cohorts of people, these cohorts have taken decades to develop and we have sadly not yet collated these for our OSA work yet. We completely support the development of our potential biomarkers in people though and have suggested this work is progressed in this manner, especially given our mutation rate findings in people (using publicly available databases). We want researchers with large OSA cohorts to look at these potential biomarkers and we believe this would be a fantastic outcome leading on from this paper.

In our discussion we suggested looking at different subtypes but we agree with you it deserves more information. OS is absolutely heterogeneous and it remains one of the reasons this cancer is so complex. We have therefore expanded upon this important fact in the discussion as per your excellent suggestions. In addition, another reviewer asked us to highlight the human mutations we had presented in our results in the discussion. These results support the heterogeneity and different subtypes/environments of each tumour so we have blended this here too.

We had already included this section in our discussion: Some future areas of study in relation to these potential biomarkers and OSA include analysing potential differences in relation to tumour grade, stage, T-substages I and II, invasion status both intramedullary and extramedullary and metastasis.

We have now added the following:

‘Rothzerg and co-authors [98] highlighted the importance of investigating each subtype, expression profiles and their underlying pathophysiologies, and of developing new biomarkers to facilitate development of novel treatments and help detect and monitor disease. Although work is ongoing in both canine and human OSA to fully appreciate the different subtypes, under-standing these potential biomarkers, and others, in different subtypes and cell type is valuable future research. Furthermore, OSA and their metastases are heterogeneous, with differing TMEs, cell types, expression levels of genes and proteins, genomic mutations, which can lead to therapeutic challenges such as drug resistance and increased metastasis [16, 99]. This further highlights the need for personalized, multi-target therapies, biomarkers in both human and canine OSA. The canine specimens in the present study have shown large ranges of proteins within the tumours, supporting their heterogeneity in terms of GPR64, TOX3, MMP-12 and FOXF1 expression. In addition, the genetic alteration rates of the four genes of interest in people are variable.  Our cBioPortal results revealed that 3-7% of n=159 human OSA tissues had GPR64, TOX3, MMP-12 and/or FOXF1 genetic alterations including missense mutations, deep deletions, high mRNA expression and amplifications. These findings likely reflect both the heterogeneity of OSA and the differing subtypes pre-sent in the cohort and highlight the need for personalized medicine and more advanced diagnostic and detection methods.’

 

And in the final summary of the discussion:

 

‘The present study investigated Rottweiler patients, but research into other breeds, and indeed other species, could be valuable in understanding the clinical applications of GPR64, TOX3, MMP-12 and FOXF1 biomarkers or therapeutic targets. Research has shown that they are expressed in a range of human cancers and our work has shown that genetic alterations are present in 4-7% of an OSA human patient cohort. As all four proteins are expressed in a range of cancers as such they could potentially serve as multi-cancer biomarkers. Expression could differ between species and between cancer types, therefore individual verification for each protein, within each species and cancer would be necessary, as with all biomarkers. In people there are also a range of drugs targeting the proteins of interest which provides possible routes for clinical trials in OSA patients. In dogs, we have shown GPR64, TOX3, MMP-12 and FOXF1 expression via quantitative H-scoring and described qualitative staining patterns and investigated differences be-tween anatomical location of the bone tumours and in both sexes. Future work to develop and validate clinical tests and understand potential expression differences between individuals, subtypes, and cell types, in different breeds, would help advance this research into pathology laboratories. Different cancer types could also be investigated in dogs, given the more extensive evidence present in human cancers of GPR64, TOX3, MMP-12 and FOXF1 expression. Given that H-scoring in veterinary medicine is not as standard as in human medicine, there is more work to be conducted in this area in general too. Likewise work progressing treatments in OSA, perhaps repurposing present drugs, would offer more insights into treating this complex cancer in dogs.’