Pan-tumour analysis of COX-2 expression in dogs.

Cyclooxgenase-2 (COX-2) is associated with inflammatory microenvironment and tumour progression. COX-2 expression was reported in canine tumours, and anti-COX treatment showed therapeutic effects in selected tumour types. Currently, direct comparisons between different tumour types or reports were impossible due to varying evaluation protocols. Additionally, COX-2 expression in relatively uncommon tumours were yet to be evaluated. Here, we analysed COX-2 expression across various tumour types in dogs in a consistent protocol, aiming to revisit accumulated evidence in the field and report novel candidate tumours for anti-COX therapy. COX-2 expression in 32 histological types of tumours, which consisted of 347 samples in total, was investigated using immunohistochemistry followed by the Belshaw's method scoring (range: 0-12). More than the half of the samples expressed COX-2 in mast cell tumours, transitional cell carcinoma in the urinary tract, squamous cell carcinoma, liposarcoma, and melanoma, with COX-2 median scores ranging from 1-8. On the other hand, <20% tissues expressed COX-2 in the half of tumour types investigated. Overall COX-2 positive rate was 27%. In conclusion, the results confirmed COX-2 expression in the well-known COX-2-expresing tumour types and suggested novel candidate tumours for anti-COX-2 therapy. At the same time, overall COX-2 expression was low, and inter- and intra-histology heterogeneity was apparent. This study will provide a foundation reference for future research in canine tumours.

Molecular features of luminal breast cancer defined through spatial and single-cell transcriptomics.

BACKGROUND: Intratumour heterogeneity is a hallmark of most solid tumours, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing on patient-derived xenografts (PDXs) to profile spatially resolved cell populations within oestrogen receptor-positive (ER+ ) breast cancer and to elucidate their importance in oestrogen-dependent tumour growth. METHODS: Two PDXs of 'ER-high' breast cancers with opposite oestrogen-mediated growth responses were investigated: oestrogen-suppressed GS3 (80-100% ER) and oestrogen-dependent SC31 (40-90% ER) models. The observation was validated via single-cell analyses on an 'ER-low' PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by a public ER+ breast cancer single-cell dataset and protein-based dual immunohistochemistry (IHC) of SC31 examining important luminal cancer markers (i.e., ER, progesterone receptor and Ki67). The translational implication of our findings was assessed by clinical outcome analyses on publicly available cohorts. RESULTS: Our space-gene-function study revealed four spatially distinct compartments within ER+ breast cancers. These compartments showed functional diversity (oestrogen-responsive, proliferative, hypoxia-induced and inflammation-related). The 'proliferative' population, rather than the 'oestrogen-responsive' compartment, was crucial for oestrogen-dependent tumour growth, leading to the acquisition of luminal B-like features. The cells expressing typical oestrogen-responsive genes like PGR were not directly linked to oestrogen-dependent proliferation. Dual IHC analyses demonstrated the distinct contribution of the Ki67+ proliferative cells toward oestrogen-mediated growth and their response to a CDK4/6 inhibitor. The gene signatures derived from the proliferative, hypoxia-induced and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the oestrogen-responsive signature showed better prognoses, suggesting that this compartment would not be directly associated with oestrogen-dependent tumour progression. CONCLUSIONS: Our study identified the gene signature in our 'proliferative' compartment as an important determinant of luminal cancer subtypes. This 'proliferative' cell population is a causative feature of luminal B breast cancer, contributing toward its aggressive behaviours.

Identification and characterization of a proliferative cell population in estrogen receptor-positive metastatic breast cancer through spatial and single-cell transcriptomics.

Background: Intratumor heterogeneity is a hallmark of most solid tumors, including breast cancers. We applied spatial transcriptomics and single-cell RNA-sequencing technologies to profile spatially resolved cell populations within estrogen receptor-positive (ER + ) metastatic breast cancers and elucidate their importance in estrogen-dependent tumor growth. Methods: Spatial transcriptomics and single-cell RNA-sequencing were performed on two patient-derived xenografts (PDXs) of "ER-high" metastatic breast cancers with opposite estrogen-mediated growth responses: estrogen-suppressed GS3 (80-100% ER) and estrogen-stimulated SC31 (30-75% ER) models. The analyses included samples treated with and without 17ß-estradiol. The findings were validated via scRNA-seq analyses on "ER-low" estrogen-accelerating PDX, GS1 (5% ER). The results from our spatial and single-cell analyses were further supported by the analysis of a publicly available single cell dataset and a protein-based dual immunohistochemical (IHC) evaluation using three important clinical markers [i.e., ER, progesterone receptor (PR), and Ki67]. The translational implication of these results was assessed by clinical outcome analyses on public breast cancer cohorts. Results: Our novel space-gene-function study revealed a "proliferative" cell population in addition to three major spatially distinct compartments within ER + metastatic breast cancers. These compartments showed functional diversity (i.e., estrogen-responsive, proliferative, hypoxia-induced, and inflammation-related). The "proliferative ( MKI67 + )" population, not "estrogen-responsive" compartment, was crucial for estrogen-dependent tumor growth, leading to the acquisition of luminal B features. The cells with induction of typical estrogen-responsive genes such as PGR were not directly linked to estrogen-dependent proliferation. Additionally, the dual IHC analyses demonstrated the distinct contribution of the Ki67 + proliferative cells toward estrogen-mediated growth and their response to palbociclib, a CDK4/6 inhibitor. The gene signatures developed from the proliferative, hypoxia-induced, and inflammation-related compartments were significantly correlated with worse clinical outcomes, while patients with the high estrogen-responsive scores showed better prognosis, confirming that the estrogen-responsive compartment would not be directly associated with estrogen-dependent tumor progression. Conclusions: For the first time, our study elucidated a "proliferative" cell population distinctly distributed in ER + metastatic breast cancers. They contribute differently toward progression of these cancers, and the gene signature in the "proliferative" compartment is an important determinant of luminal cancer subtypes.

Mechanism-Driven and Clinically Focused Development of Botanical Foods as Multitarget Anticancer Medicine: Collective Perspectives and Insights from Preclinical Studies, IND Applications and Early-Phase Clinical Trials.

Cancer progression and mortality remain challenging because of current obstacles and limitations in cancer treatment. Continuous efforts are being made to explore complementary and alternative approaches to alleviate the suffering of cancer patients. Epidemiological and nutritional studies have indicated that consuming botanical foods is linked to a lower risk of cancer incidence and/or improved cancer prognosis after diagnosis. From these observations, a variety of preclinical and clinical studies have been carried out to evaluate the potential of botanical food products as anticancer medicines. Unfortunately, many investigations have been poorly designed, and encouraging preclinical results have not been translated into clinical success. Botanical products contain a wide variety of chemicals, making them more difficult to study than traditional drugs. In this review, with the consideration of the regulatory framework of the USFDA, we share our collective experiences and lessons learned from 20 years of defining anticancer foods, focusing on the critical aspects of preclinical studies that are required for an IND application, as well as the checkpoints needed for early-phase clinical trials. We recommend a developmental pipeline that is based on mechanisms and clinical considerations.

Postmortem magnetic resonance imaging findings of tricuspid atresia with ventricular and atrial septal defects and subvalvular pulmonic stenosis in a Japanese native Noma horse.

The necropsy of a 2-day-old Noma horse that died of weakness showed an enlarged cardiac base and a narrow cardiac apex, suggesting cardiac malformation. The excised heart underwent imaging to investigate its luminal structure. On three-dimensional magnetic resonance imaging, the right atrium and right ventricle were discontinuous. The right atrium communicated with the left atrium and the left ventricle communicated with the right ventricle. The lumen narrowed near the pulmonary artery valve. Since the same findings were observed on gross examination, the foal was diagnosed with tricuspid atresia with ventricular and atrial septal defects, along with subvalvular pulmonic stenosis.

Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium.

Fibroblasts have been shown to be one of the essential players for mammary gland organization. Here, we identify two major types of mouse mammary gland fibroblasts through single-cell RNA sequencing analysis: Dpp4 + fibroblasts and Dpp4 - fibroblasts. Each population exhibits unique functional characteristics as well as discrete localization in normal mouse mammary glands. Remarkably, estrogen, a crucial mediator of mammary gland organization, alters the gene expression profiles of fibroblasts in a population-specific manner, without distinct activation of estrogen receptor signaling. Further integrative analysis with the inclusion of five other publicly available datasets reveals a directional differentiation among the mammary gland fibroblast populations. Moreover, the combination with the mouse mammary epithelium atlas allows us to infer multiple potential interactions between epithelial cells and fibroblasts in mammary glands. This study provides a comprehensive view of mouse mammary gland fibroblasts at the single-cell level.

Detection of indoleamine 2,3-dioxygenase 1-expressing cells in canine normal and tumor tissues.

Cancer immunotherapy is a novel cancer treatment for canine tumors. Indoleamine 2,3-dioxygenase 1 (IDO1) is overexpressed in some human tumors and inhibits antitumor immunity. In this study, we comprehensively evaluated expression pattern of IDO1 and the nature of IDO1-expressing cells in canine normal and tumor tissues. In normal tissue samples, IDO1 expression was detected only in the lymph nodes, spleen, tonsil tissues, and colon tissues. In contrast, IDO1-positive tumor cells were observed in several tumor tissue types. This is the first study to evaluate IDO1 expression in canine normal and tumor tissues, and the results suggest that IDO1 is a promising target for novel cancer immunotherapy in dogs with tumors.

Expression of podoplanin in various types of feline tumor tissues.

Podoplanin is expressed in various human tumors where it promotes tumor progression, epithelial-mesenchymal transition, and distant metastasis. Podoplanin is also expressed in cancer-associated fibroblasts and induces tumor malignancy. The objective of this study was to evaluate podoplanin expression in various types of feline tumor tissues. Immunohistochemical analysis revealed that podoplanin was expressed in cells of 13/15 (87%) squamous cell carcinomas and 5/19 (26%) fibrosarcomas. Moreover, cancer-associated fibroblasts expressed podoplanin in most tumor types, including 18/21 (86%) mammary adenocarcinoma tissues. Our findings demonstrate that various types of feline tumor tissues expressed podoplanin, indicating the importance of the comparative aspects of podoplanin expression, which may be used as a novel research model for podoplanin biology.

White button mushroom interrupts tissue AR-mediated TMPRSS2 expression and attenuates pro-inflammatory cytokines in C57BL/6 mice.

White button mushroom (WBM) is a common edible mushroom consumed in the United States and many European and Asia-Pacific countries. We previously reported that dietary WBM antagonized dihydrotestosterone (DHT)-induced androgen receptor (AR) activation and reduced myeloid-derived suppressor cells (MDSCs) in prostate cancer animal models and patients. Transmembrane protease serine 2 (TMPRSS2), an androgen-induced protease in prostate cancer, has been implicated in influenza and coronavirus entry into the host cell, triggering host immune response. The present study on C57BL/6 mice revealed that WBM is a unique functional food that (A) interrupts AR-mediated TMPRSS2 expression in prostate, lungs, small intestine, and kidneys through its AR antagonistic activity and (B) attenuates serum pro-inflammatory cytokines and reduces MDSC counts through its immunoregulatory activity. These findings provide a scientific basis for translational studies toward clinical applications of WBM in diseases related to TMPRSS2 expression and immune dysregulation.

Exploring the Biological Activity and Mechanism of Xenoestrogens and Phytoestrogens in Cancers: Emerging Methods and Concepts.

Xenoestrogens and phytoestrogens are referred to as "foreign estrogens" that are produced outside of the human body and have been shown to exert estrogen-like activity. Xenoestrogens are synthetic industrial chemicals, whereas phytoestrogens are chemicals present in the plant. Considering that these environmental estrogen mimics potentially promote hormone-related cancers, an understanding of how they interact with estrogenic pathways in human cells is crucial to resolve their possible impacts in cancer. Here, we conducted an extensive literature evaluation on the origins of these chemicals, emerging research techniques, updated molecular mechanisms, and ongoing clinical studies of estrogen mimics in human cancers. In this review, we describe new applications of patient-derived xenograft (PDX) models and single-cell RNA sequencing (scRNA-seq) techniques in shaping the current knowledge. At the molecular and cellular levels, we provide comprehensive and up-to-date insights into the mechanism of xenoestrogens and phytoestrogens in modulating the hallmarks of cancer. At the systemic level, we bring the emerging concept of window of susceptibility (WOS) into focus. WOS is the critical timing during the female lifespan that includes the prenatal, pubertal, pregnancy, and menopausal transition periods, during which the mammary glands are more sensitive to environmental exposures. Lastly, we reviewed 18 clinical trials on the application of phytoestrogens in the prevention or treatment of different cancers, conducted from 2002 to the present, and provide evidence-based perspectives on the clinical applications of phytoestrogens in cancers. Further research with carefully thought-through concepts and advanced methods on environmental estrogens will help to improve understanding for the identification of environmental influences, as well as provide novel mechanisms to guide the development of prevention and therapeutic approaches for human cancers.

White Button Mushroom (Agaricus bisporus) Interrupts Tissue AR-TMPRSS2 Expression and Attenuates Pro-inflammatory Cytokines in C57BL/6 Mice: Implication for COVID-19 Dietary Intervention.

Transmembrane protease serine 2 (TMPRSS2), an androgen-induced protease associated with prostate cancer, is one putative receptor for coronavirus entry into host cells, where triggering aggressive inflammatory cytokine storm and possibly death in COVID-19 patients. We previously reported that dietary white button mushroom (WBM) antagonized dihydrotestosterone (DHT)-induced androgen receptor (AR) activation and reduced myeloid-derived suppressor cells (MDSCs) in prostate cancer animal models and patients. The present study on C57BL/6 mice revealed that WBM is a unique food that A ) interrupts DHT induced AR-TMPRSS2 expression in putative COVID-19 targeted organs through its AR antagonistic activity and B ) attenuates serum pro-inflammatory cytokines which have been implicated in COVID-19 pathogenesis. We hereby propose WBM intake as a potentially low-cost, efficient, and safe dietary intervention to mitigate COVID-19.

BRAFV595E Mutation Associates CCL17 Expression and Regulatory T Cell Recruitment in Urothelial Carcinoma of Dogs.

Regulatory T cells may serve as targets in cancer immunotherapy. A previous study showed that the chemokine CCL17 and the receptor CCR4 play roles in regulatory T cell recruitment in canine urothelial carcinoma. In this article, we show that the BRAFV595E mutation is associated with tumor-produced CCL17 and regulatory T cell infiltration in dogs with urothelial carcinoma. In comparison with healthy dogs, dogs with urothelial carcinoma showed increased CCL17 mRNA expression in the bladder and elevated CCL17 protein concentration in urine. Immunohistochemistry showed increased levels of Foxp3+ regulatory T cells in the tumor tissues of urothelial carcinoma. The density of Foxp3+ regulatory T cells was positively correlated with CCL17 concentration in urine, indicating that CCL17 is involved in regulatory T cell recruitment. Moreover, tumor-infiltrating regulatory T cells and urine CCL17 concentration were associated with poor prognosis in dogs with urothelial carcinoma. The number of tumor-infiltrating regulatory T cells, CCL17 mRNA expression, and urine CCL17 concentration in cases with BRAFV595E mutation were higher than those in cases with wild-type BRAF. In vitro, high CCL17 production was detected in a canine urothelial carcinoma cell line with BRAFV595E mutation but not in an urothelial carcinoma cell line with wild-type BRAF. Dabrafenib, a BRAF inhibitor, decreased CCL17 production in the cell line with BRAFV595E mutation. These results suggest that BRAFV595E mutation induced CCL17 production and contributed to regulatory T cell recruitment in canine urothelial carcinoma.

Phase I/II Clinical Trial of the Anti-Podoplanin Monoclonal Antibody Therapy in Dogs with Malignant Melanoma.

Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed on tumor cells. PDPN is known to be linked with several aspects of tumor malignancies in certain types of human and canine tumors. Therefore, it is considered to be a novel therapeutic target. Monoclonal antibodies targeting PDPN expressed in human tumor cells showed obvious anti-tumor effects in preclinical studies using mouse models. Previously, we generated a cancer-specific mouse-dog chimeric anti-PDPN antibody, P38Bf, which specifically recognizes PDPN expressed in canine tumor cells. In this study, we investigated the safety and anti-tumor effects of P38Bf in preclinical and clinical trials. P38Bf showed dose-dependent antibody-dependent cellular cytotoxicity against canine malignant melanoma cells. In a preclinical trial with one healthy dog, P38Bf administration did not induce adverse effects over approximately 2 months. In phase I/II clinical trials of three dogs with malignant melanoma, one dog vomited, and all dogs had increased serum levels of C-reactive protein, although all adverse effects were grade 1 or 2. Severe adverse effects leading to withdrawal of the clinical trial were not observed. Furthermore, one dog had stable disease with P38Bf injections. This is the first reported clinical trial of anti-PDPN antibody therapy using spontaneously occurring canine tumor models.

Author Correction: Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PDPN Is Expressed in Various Types of Canine Tumors and Its Silencing Induces Apoptosis and Cell Cycle Arrest in Canine Malignant Melanoma.

Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed. It is also known to be linked with several aspects of tumor malignancy in some types of human tumors, including invasion, metastasis, and cancer stemness. However, there are few reports on the expression of dog PDPN (dPDPN) in canine tumors, and the association between dPDPN and tumor malignancy has not been elucidated. We identified that 11 out of 18 types of canine tumors expressed dPDPN. Furthermore, 80% of canine malignant melanoma (MM), squamous cell carcinoma, and meningioma expressed dPDPN. Moreover, the expression density of dPDPN was positively associated with the expression of the Ki67 proliferation marker. The silencing of dPDPN by siRNAs resulted in the suppression of cell migration, invasion, stem cell-like characteristics, and cell viability in canine MM cell lines. The suppression of cell viability was caused by the induction of apoptosis and G2/M phase cell cycle arrest. Overall, this study demonstrates that dPDPN is expressed in various types of canine tumors and that dPDPN silencing suppresses cell viability through apoptosis and cell cycle arrest, thus providing a novel biological role for PDPN in tumor progression.

Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma.

Cancer-promoting inflammation is an important event in cancer development. Canine urothelial carcinoma (cUC) overexpresses prostaglandin E2 (PGE2) and has a unique sensitivity to cyclooxygenase 2 (COX2)-inhibiting therapy. In addition, majority of cUC harbour BRAFV595E mutation. However, mechanisms underlying aberrant PGE2 production in BRAFV595E cUC patients remain unclear. Drug screening revealed that inhibition of RAF/MEK/ERK pathway, p38 and JNK pathway reduced PGE2 production in cUC cells. By pharmacological inhibition of the multiple components in the pathway, activation of the ERK MAPK pathway was shown to mediate overexpression of COX2 and production of PGE2 in BRAFV595E cUC cells. In silico gain-of-function analysis of the BRAF mutation also implicated involvement of mutation in the process. The positive association between ERK activation and COX2 expression was further validated in the clinical patients. Moreover, it was also suggested that p38 and JNK regulates PGE2 production independently of ERK pathway, possibly through COX2-dependent and COX1-/COX2- independent manner, respectively. In conclusion, this study demonstrated that activation of ERK induces production of PGE2 in BRAFV595E cUC cells, which is also independently regulated by p38 and JNK. With its unique vulnerability to COX-targeted therapy, BRAFV595E cUC may serve as a valuable model to study the tumour-promoting inflammation.

Foxp3+ Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs.

Regulatory T cells (Tregs) can be targeted in cancer immunotherapy. A previous study has shown that the chemokine CCL17 and the receptor CCR4 play a role in Treg recruitment in canine urothelial carcinoma. Here, we describe the association of tumor-infiltrating Tregs with CCL17/CCR4 expression in dogs with other carcinomas. In this study, we investigated 23 dogs with mammary carcinoma, 14 dogs with oral squamous cell carcinoma, 16 dogs with pulmonary adenocarcinoma, and 8 healthy control dogs. Immunohistochemistry showed that Foxp3+ Tregs and CCR4+ cells were increased in the tumor tissues of mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, when compared with the healthy tissues. The number of CCR4+ cells was associated with that of Foxp3+ Tregs. Double immunofluorescence labeling confirmed that most tumor-infiltrating Foxp3+ Tregs expressed CCR4. In vitro, canine carcinoma cell lines expressed CCL17 mRNA. Quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) showed that CCL17 mRNA expression in canine carcinomas was increased approximately 10- to 25-fold relative to that of healthy tissues. These results suggest that the CCL17/CCR4 axis may drive Treg recruitment in a variety of canine carcinomas. CCR4 blockade may be a potential therapeutic option for tumor eradication through Treg depletion.

Overexpression of human epidermal growth factor receptor 2 in canine primary lung cancer.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various human cancers. HER2-targeted therapies showed clinical responses in humans with HER2-positive tumors. The incidence of canine primary lung cancer (cPLC) is increasing, but there are no effective systemic therapies for dogs with late-stage cPLC. HER2-targeted therapy could be an option for cPLC, but HER2 expression in cPLC remains unknown. We evaluated HER2 expression in cPLC. Immunohistochemical analysis revealed that 3 samples (19%) scored 3+; 8 (50%), 2+; 5 (31%); and 1+ and 0 (0%), 0. Of the cPLC tissues, 69% were HER2 positive (scored ≥2+). These data would lead to further evaluation of the role of HER2 in cPLC as a mechanism of malignancy and therapeutic target.

ErbB2 Copy Number Aberration in Canine Urothelial Carcinoma Detected by a Digital Polymerase Chain Reaction Assay.

Urothelial carcinoma (UC) is the most common tumor affecting the urinary bladder of dogs. Protein overexpression of ErbB2 (the canine homolog of HER2) has been observed in dogs with UC. However, no study regarding ErbB2 copy number aberration (CNA) is reported in dogs with UC. In this study, a digital PCR assay for detecting CNA of canine ErbB2 was developed. DNA samples were isolated from 83 formalin-fixed, paraffin-embedded urinary bladder tissues (36 UC, 8 polypoid cystitis, and 39 normal) and 94 urinary sediments (54 UC, 30 nonneoplastic, and 10 normal). The copy number of canine chromosome 8 (CFA8) was used as a control. In the urinary bladder tissues, ErbB2 CNA was detected in 12 of 36 (33%) UC, 2 of 8 (25%) polypoid cystitis, and 0 of 39 (0%) normal controls. In the urinary sediments, ErbB2 CNA was also detected in 19 of 54 (35%) UC; however, no ErbB2 CNA was detected in nonneoplastic diseases or normal controls. The sensitivity and specificity of ErbB2 CNA in urinary sediment for the detection of UC were 35% and 100%, respectively. There was a positive correlation between the copy number ratios of ErbB2 to CFA8 in the urinary bladder tissues and urinary sediments. Our findings indicate that the digital PCR assay of urinary sediments may be a useful, noninvasive method for detecting ErbB2 CNA in dogs with UC.

Immunohistochemical evaluation of HER2 expression in canine thyroid carcinoma.

The human epidermal growth factor receptor 2 (HER2) is expressed in various human cancers including thyroid cancers (TC) and is used as a diagnostic marker and therapeutic target. Canine TC (cTC), the most common endocrine malignancy in dogs, shows a high metastasis rate, and HER2-targeted therapy could be a candidate for treatment. Here, we immunohistochemically evaluated HER2 expression in 21 paraffin-embedded cTC tissues and scored the degree of expression based on intensity and positivity (score: 0-3+). Four samples (19%) scored 3+; 6 (29%), 2+; 7 (33%), 1+; and 4 (19%), 0. Therefore, 48% of the cTC tissues were HER2 positive (scored ≥2+). These data may lead to further evaluation of the role of HER2 in cTC as a mechanism of malignancy and a therapeutic target.