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Article: Cytokine production and the effects of oclacitinib in three canine mast cell tumour cell lines

de Mello Souza, Carlos H. / Shiomitsu, Keijiro / Hwang, Benjamin

Veterinary dermatology. 2022 Apr., v. 33, no. 2

2022

Abstract: BACKGROUND: Cytokines are capable of manipulating the tumour microenvironment supporting tumour growth. Interleukin (IL)‐8 and monocyte chemoattractant protein (MCP)‐1, shown to be produced by various tumours, can negatively affect prognosis. The ... ...

Abstract	BACKGROUND: Cytokines are capable of manipulating the tumour microenvironment supporting tumour growth. Interleukin (IL)‐8 and monocyte chemoattractant protein (MCP)‐1, shown to be produced by various tumours, can negatively affect prognosis. The production of cytokines by canine mast cell tumours (MCT) has not been reported. HYPOTHESIS/OBJECTIVES: We hypothesise that MCT cell lines produce IL‐8 and/or MCP‐1 in addition to other cytokines, and that their production can be modulated by the Janus kinase (JAK) inhibitor oclacitinib. This pilot study aims to investigate the production of IL‐8, MCP‐1 and nine additional cytokines in three canine MCT cell lines, and determine the effects of oclacitinib on their production. METHODS AND MATERIALS: Reverse transcriptase‐PCR was used to detect the expression of IL‐8 and MCP‐1 mRNA in three MCT cell lines (CoMS, CM‐MC1 and VI‐MC1). The supernatant of the cell lines was evaluated for the presence of 11 cytokines [IL‐2, ‐6, ‐7, ‐8, ‐10, ‐15 and ‐18, and MCP‐1, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interferon (IFN)γ and tumour necrosis factor (TNF)α] by enzyme‐linked immunosorbent assay (ELISA). The IC₅₀ of oclacitinib was identified for each cell line. ELISA was performed again to compare changes in IL‐8 and MCP‐1 in treated cell lines versus untreated controls. RESULTS: Interleukin‐8 and MCP‐1 were produced by all MCT cell lines tested. Oclacitinib significantly decreased the release of IL‐8 in the CoMS cell line and of MCP‐1 in CoMS and VI‐MC1 in clinically relevant concentrations. Furthermore, oclacitinib significantly decreased the proliferation of all three cell lines. CONCLUSIONS: Interleukin‐8 and MCP‐1 are produced by canine MCT cell lines. Modulation of their production is possible with oclacitinib.
Keywords	cell lines ; chemoattractants ; dogs ; enzyme-linked immunosorbent assay ; granulocyte-macrophage colony-stimulating factor ; interferons ; interleukin-2 ; interleukin-8 ; mast cells ; monocytes ; neoplasm cells ; neoplasms ; non-specific protein-tyrosine kinase ; prognosis ; reverse transcriptase polymerase chain reaction ; tumor necrosis factors ; veterinary medicine
Language	English
Dates of publication	2022-04
Size	p. 159-e46.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	2011122-8
ISSN	1365-3164 ; 0959-4493
ISSN (online)	1365-3164
ISSN	0959-4493
DOI	10.1111/vde.13046
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Cytokine production and the effects of oclacitinib in three canine mast cell tumour cell lines.

de Mello Souza, Carlos H / Shiomitsu, Keijiro / Hwang, Benjamin

Veterinary dermatology

2021 Volume 33, Issue 2, Page(s) 159–e46

Abstract: Background: Cytokines are capable of manipulating the tumour microenvironment supporting tumour growth. Interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1, shown to be produced by various tumours, can negatively affect prognosis. The ... ...

Abstract	Background: Cytokines are capable of manipulating the tumour microenvironment supporting tumour growth. Interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1, shown to be produced by various tumours, can negatively affect prognosis. The production of cytokines by canine mast cell tumours (MCT) has not been reported. Hypothesis/objectives: We hypothesise that MCT cell lines produce IL-8 and/or MCP-1 in addition to other cytokines, and that their production can be modulated by the Janus kinase (JAK) inhibitor oclacitinib. This pilot study aims to investigate the production of IL-8, MCP-1 and nine additional cytokines in three canine MCT cell lines, and determine the effects of oclacitinib on their production. Methods and materials: Reverse transcriptase-PCR was used to detect the expression of IL-8 and MCP-1 mRNA in three MCT cell lines (CoMS, CM-MC1 and VI-MC1). The supernatant of the cell lines was evaluated for the presence of 11 cytokines [IL-2, -6, -7, -8, -10, -15 and -18, and MCP-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ and tumour necrosis factor (TNF)α] by enzyme-linked immunosorbent assay (ELISA). The IC Results: Interleukin-8 and MCP-1 were produced by all MCT cell lines tested. Oclacitinib significantly decreased the release of IL-8 in the CoMS cell line and of MCP-1 in CoMS and VI-MC1 in clinically relevant concentrations. Furthermore, oclacitinib significantly decreased the proliferation of all three cell lines. Conclusions: Interleukin-8 and MCP-1 are produced by canine MCT cell lines. Modulation of their production is possible with oclacitinib.
MeSH term(s)	Animals ; Cell Line ; Cytokines/genetics ; Cytokines/metabolism ; Dog Diseases/drug therapy ; Dogs ; Pilot Projects ; Pyrimidines ; Sulfonamides ; Tumor Necrosis Factor-alpha
Chemical Substances	Cytokines ; Pyrimidines ; Sulfonamides ; Tumor Necrosis Factor-alpha ; oclacitinib (99GS5XTB51)
Language	English
Publishing date	2021-12-09
Publishing country	England
Document type	Journal Article
ZDB-ID	2011122-8
ISSN	1365-3164 ; 0959-4493
ISSN (online)	1365-3164
ISSN	0959-4493
DOI	10.1111/vde.13046
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Article ; Online: RNA in situ hybridisation as a molecular diagnostic technique targeting IBA-1 and CD204 in canine histiocytic sarcoma.

Engelien, Julia L / Lejeune, Amandine T / Dark, Michael J / Milner, Rowan J / Shiomitsu, Keijiro

Veterinary medicine and science

2022 Volume 8, Issue 4, Page(s) 1400–1408

Abstract: Background: Canine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA-1, ionised calcium-binding adapter molecule 1, and CD204, a ... ...

Abstract	Background: Canine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA-1, ionised calcium-binding adapter molecule 1, and CD204, a macrophage scavenger receptor, have been shown to be specific immunohistochemical markers helpful in distinguishing HS from other tumour types with similar morphological features. Objectives: This study was performed to demonstrate the use of RNA in situ hybridisation (ISH) technology allowing single-molecule RNA visualisation in formalin-fixed paraffin-embedded (FFPE) tissues as a molecular tool for the diagnosis of canine HS. Methods: Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis for IBA-1 and CD204 were performed to correlate gene expression and protein expression of these two markers in the histiocytic sarcoma DH82 cell line. RNA-ISH for IBA-1 and CD204 was performed on the DH82 cell line to validate the RNA-ISH probes. RNA-ISH and immunohistochemistry (IHC) were performed in clinical HS FFPE samples to demonstrate mRNA and protein expression of IBA-1 and CD204. FFPE archived samples of canine round cell tumours, melanoma and anaplastic sarcoma were used as negative controls. Results: RNA-ISH and IHC showed moderate to strong expression for IBA-1 and CD204 in the neoplastic cells in both the canine DH82 cell line and the archived canine HS samples. RNA-ISH and IHC showed scattered positive staining in the control tumours samples, consistent with macrophagic infiltration. Conclusion: RNA-ISH for CD204 and IBA-1 appeared to have a high specificity and sensitivity in our samples and may be an additional valuable diagnostic technique in identifying HS.
MeSH term(s)	Animals ; Biomarkers ; Dog Diseases/diagnosis ; Dog Diseases/pathology ; Dogs ; Histiocytic Sarcoma/diagnosis ; Histiocytic Sarcoma/pathology ; Histiocytic Sarcoma/veterinary ; Immunohistochemistry ; Molecular Diagnostic Techniques/veterinary ; Neoplasms/veterinary ; RNA
Chemical Substances	Biomarkers ; RNA (63231-63-0)
Language	English
Publishing date	2022-03-26
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2819409-3
ISSN	2053-1095 ; 2053-1095
ISSN (online)	2053-1095
ISSN	2053-1095
DOI	10.1002/vms3.795
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Characterization of expression and prognostic implications of transforming growth factor beta, programmed death-ligand 1, and T regulatory cells in canine histiocytic sarcoma.

Murphy, Jacqueline D / Shiomitsu, Keijiro / Milner, Rowan J / Lejeune, Amandine / Ossiboff, Robert J / Gell, Jessy Castellanos / Axiak-Bechtel, Sandra

Veterinary immunology and immunopathology

2023 Volume 257, Page(s) 110560

Abstract: Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-β), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this ... ...

Abstract	Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-β), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-β, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope®). After validation was performed, RNAscope® on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO® software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-β and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope® method was validated, TGF-β H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-β dots/cell. In summary, RNAscope® is a valid technique to detect TGF-β and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-β and PD-L1. Increased TGF-β and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-β, PD-L1, and Tregs effect on prognosis.
MeSH term(s)	Animals ; Dogs ; Prognosis ; B7-H1 Antigen ; Transforming Growth Factor beta ; Histiocytic Sarcoma/veterinary ; T-Lymphocytes, Regulatory ; Forkhead Transcription Factors/metabolism ; Dog Diseases/metabolism
Chemical Substances	CD274 protein, human ; B7-H1 Antigen ; Transforming Growth Factor beta ; Forkhead Transcription Factors
Language	English
Publishing date	2023-02-13
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	754160-0
ISSN	1873-2534 ; 0165-2427
ISSN (online)	1873-2534
ISSN	0165-2427
DOI	10.1016/j.vetimm.2023.110560
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Article ; Online: Characterization of expression and prognostic implications of GD2 and GD3 synthase in canine histiocytic sarcoma.

Murphy, Jacqueline D / Axiak-Bechtel, Sandra / Milner, Rowan J / Lejeune, Amandine / Ossiboff, Robert J / Gell, Jessy Castellanos / Shiomitsu, Keijiro

Veterinary immunology and immunopathology

2023 Volume 259, Page(s) 110594

Abstract: GD2 and GD3 are disialoganglioside oncofetal antigens important in oncogenesis. GD2 synthase (GD2S) and GD3 synthase (GD3S) are needed for GD2 and GD3 production. The objectives of this study are to validate the use of RNA in situ hybridization (RNAscope® ...

Abstract	GD2 and GD3 are disialoganglioside oncofetal antigens important in oncogenesis. GD2 synthase (GD2S) and GD3 synthase (GD3S) are needed for GD2 and GD3 production. The objectives of this study are to validate the use of RNA in situ hybridization (RNAscope®) in the detection of GD2S and GD3S in canine histiocytic sarcoma (HS) in vitro and optimize this technique in canine formalin-fixed paraffin-embedded (FFPE) tissues. A secondary objective is to evaluate the prognostic significance of GD2S and GD3S on survival. Quantitative RT-PCR compared GD2S and GD3S mRNA expression between three HS cell lines followed by RNAscope® in fixed cell pellets from the DH82 cell line and FFPE tissues. Variables prognostic for survival were determined with Cox proportional hazard model. RNAscope® was validated for detection of GD2S and GD3S and optimized in FFPE tissues. GD2S and GD3S mRNA expression was variable between cell lines. GD2S and GD3S mRNA expression was detected and measured in all tumor tissues; there was no association with prognosis. GD2S and GD3S are expressed in canine HS and successfully detected using the high throughput technique of RNAscope® in FFPE samples. This study provides the foundation for future prospective research of GD2S and GD3S utilizing RNAscope®.
MeSH term(s)	Animals ; Dogs ; Prognosis ; Gangliosides ; Cell Line, Tumor ; Histiocytic Sarcoma/veterinary ; Sialyltransferases/genetics ; Sialyltransferases/metabolism ; RNA, Messenger/genetics ; Dog Diseases/diagnosis
Chemical Substances	Gangliosides ; Sialyltransferases (EC 2.4.99.-) ; RNA, Messenger
Language	English
Publishing date	2023-04-10
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	754160-0
ISSN	1873-2534 ; 0165-2427
ISSN (online)	1873-2534
ISSN	0165-2427
DOI	10.1016/j.vetimm.2023.110594
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Article: C-kit, flt-3, PDGFR-β, and VEGFR2 expression in canine adrenal tumors and correlation with outcome following adrenalectomy

Harding, Kayla / De Mello Souza, Carlos H. / Shiomitsu, Keijiro / Maxwell, Elizabeth / Bertran, Judit

Canadian journal of veterinary research. 2021 Oct., v. 85, no. 4

2021

Abstract: The objective of this retrospective study was to evaluate the expression of receptor tyrosine kinases (RTKs) in canine adrenal tumors and correlate this expression with features of tumor aggressiveness and survival in dogs undergoing adrenalectomy. Forty- ...

Abstract	The objective of this retrospective study was to evaluate the expression of receptor tyrosine kinases (RTKs) in canine adrenal tumors and correlate this expression with features of tumor aggressiveness and survival in dogs undergoing adrenalectomy. Forty-three canine adrenal tumors were evaluated for expression of c-kit, fms-like tyrosine kinase 3 (flt-3), platelet-derived growth factor receptor-β (PDGFR-β), and vascular endothelial growth factor receptor 2 (VEGFR2) using immunohistochemistry. Tumor RTK staining characteristics were compared to normal adrenals. Medical records were reviewed for data regarding patient outcome and tumor characteristics. Expression of c-kit, flt-3, PDGFR-β, and VEGFR2 was detected in 26.9%, 92.3%, 96.2%, and 61.5% of cortical tumors and 0%, 63.2%, 47.4%, and 15.8% of pheochromocytomas, respectively. Expression of RTKs was not significantly increased when compared to normal adrenals and did not correlate with survival after adrenalectomy. Receptor tyrosine kinases are not overexpressed in canine adrenal tumors compared to normal adrenal tissue. Therapeutic inhibition of these receptors may still represent an effective approach in cases where receptor activation is present.
Keywords	adrenalectomy ; dogs ; immunohistochemistry ; neoplasms ; patients ; platelet-derived growth factor ; retrospective studies ; tyrosine ; vascular endothelial growth factor receptor-2 ; veterinary medicine
Language	English
Dates of publication	2021-10
Size	p. 279-284.
Publishing place	Canadian Veterinary Medical Association = Association canadienne des vétérinaires
Document type	Article
ZDB-ID	632814-3
ISSN	0830-9000
ISSN	0830-9000
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Characterization of expression and prognostic implications of GD2 and GD3 synthase in canine histiocytic sarcoma

Murphy, Jacqueline D. / Axiak-Bechtel, Sandra / Milner, Rowan J. / Lejeune, Amandine / Ossiboff, Robert J. / Gell, Jessy Castellanos / Shiomitsu, Keijiro

Veterinary Immunology and Immunopathology. 2023 May, v. 259 p.110594-

2023

Abstract	GD2 and GD3 are disialoganglioside oncofetal antigens important in oncogenesis. GD2 synthase (GD2S) and GD3 synthase (GD3S) are needed for GD2 and GD3 production. The objectives of this study are to validate the use of RNA in situ hybridization (RNAscope®) in the detection of GD2S and GD3S in canine histiocytic sarcoma (HS) in vitro and optimize this technique in canine formalin-fixed paraffin-embedded (FFPE) tissues. A secondary objective is to evaluate the prognostic significance of GD2S and GD3S on survival. Quantitative RT-PCR compared GD2S and GD3S mRNA expression between three HS cell lines followed by RNAscope® in fixed cell pellets from the DH82 cell line and FFPE tissues. Variables prognostic for survival were determined with Cox proportional hazard model. RNAscope® was validated for detection of GD2S and GD3S and optimized in FFPE tissues. GD2S and GD3S mRNA expression was variable between cell lines. GD2S and GD3S mRNA expression was detected and measured in all tumor tissues; there was no association with prognosis. GD2S and GD3S are expressed in canine HS and successfully detected using the high throughput technique of RNAscope® in FFPE samples. This study provides the foundation for future prospective research of GD2S and GD3S utilizing RNAscope®.
Keywords	RNA ; carcinogenesis ; cell lines ; dogs ; gene expression ; hybridization ; immunology ; immunopathology ; models ; prognosis ; sarcoma ; B2M ; FFPE ; GAPDH ; GD2S ; GD3S ; HS ; Iba-1 ; IHC ; ISH ; MI ; MST ; RT-PCR ; Histiocytic sarcoma ; RNAscope® ; GD2 Synthase ; GD3 Synthase
Language	English
Dates of publication	2023-05
Publishing place	Elsevier B.V.
Document type	Article ; Online
ZDB-ID	754160-0
ISSN	1873-2534 ; 0165-2427
ISSN (online)	1873-2534
ISSN	0165-2427
DOI	10.1016/j.vetimm.2023.110594
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Molecular diagnosis using RNAscope

Shiomitsu, Keijiro / Bechtel, Sandra M / Thompson, Patrick M / Frasca, Salvatore

Canadian journal of veterinary research = Revue canadienne de recherche veterinaire

2020 Volume 84, Issue 4, Page(s) 319–323

Abstract: Immunohistochemistry has been used extensively to evaluate protein expression in clinical and research settings. However, immunohistochemistry is not always successful in veterinary medicine due to the lack of reliable antibody options, poor tissue ... ...

Abstract	Immunohistochemistry has been used extensively to evaluate protein expression in clinical and research settings. However, immunohistochemistry is not always successful in veterinary medicine due to the lack of reliable antibody options, poor tissue preservation, labor-intensive staining, and antigen-retrieval optimization processes. RNAscope
MeSH term(s)	Animals ; Dog Diseases/diagnosis ; Dogs ; Immunohistochemistry/methods ; Immunohistochemistry/veterinary ; In Situ Hybridization/methods ; In Situ Hybridization/veterinary ; Neoplasms/diagnosis ; Neoplasms/veterinary ; RNA, Messenger
Chemical Substances	RNA, Messenger
Language	English
Publishing date	2020-09-02
Publishing country	Canada
Document type	Journal Article
ZDB-ID	632814-3
ISSN	1928-9022 ; 0830-9000
ISSN (online)	1928-9022
ISSN	0830-9000
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Article ; Online: C-kit, flt-3, PDGFR-β, and VEGFR2 expression in canine adrenal tumors and correlation with outcome following adrenalectomy.

Harding, Kayla / De Mello Souza, Carlos H / Shiomitsu, Keijiro / Maxwell, Elizabeth / Bertran, Judit

Canadian journal of veterinary research = Revue canadienne de recherche veterinaire

2021 Volume 85, Issue 4, Page(s) 279–284

Abstract	The objective of this retrospective study was to evaluate the expression of receptor tyrosine kinases (RTKs) in canine adrenal tumors and correlate this expression with features of tumor aggressiveness and survival in dogs undergoing adrenalectomy. Forty-three canine adrenal tumors were evaluated for expression of c-kit, fms-like tyrosine kinase 3 (flt-3), platelet-derived growth factor receptor-β (PDGFR-β), and vascular endothelial growth factor receptor 2 (VEGFR2) using immunohistochemistry. Tumor RTK staining characteristics were compared to normal adrenals. Medical records were reviewed for data regarding patient outcome and tumor characteristics. Expression of c-kit, flt-3, PDGFR-β, and VEGFR2 was detected in 26.9%, 92.3%, 96.2%, and 61.5% of cortical tumors and 0%, 63.2%, 47.4%, and 15.8% of pheochromocytomas, respectively. Expression of RTKs was not significantly increased when compared to normal adrenals and did not correlate with survival after adrenalectomy. Receptor tyrosine kinases are not overexpressed in canine adrenal tumors compared to normal adrenal tissue. Therapeutic inhibition of these receptors may still represent an effective approach in cases where receptor activation is present.
MeSH term(s)	Adrenal Gland Neoplasms/blood ; Adrenal Gland Neoplasms/metabolism ; Adrenal Gland Neoplasms/surgery ; Adrenalectomy/veterinary ; Animals ; Biomarkers, Tumor/blood ; Dog Diseases/blood ; Dog Diseases/metabolism ; Dog Diseases/surgery ; Dogs ; Gene Expression Regulation, Neoplastic ; Immunohistochemistry ; Proto-Oncogene Proteins c-kit/blood ; Receptor, Platelet-Derived Growth Factor beta/genetics ; Receptor, Platelet-Derived Growth Factor beta/metabolism ; Treatment Outcome ; Vascular Endothelial Growth Factor Receptor-2/genetics ; Vascular Endothelial Growth Factor Receptor-2/metabolism ; fms-Like Tyrosine Kinase 3/genetics ; fms-Like Tyrosine Kinase 3/metabolism
Chemical Substances	Biomarkers, Tumor ; Proto-Oncogene Proteins c-kit (EC 2.7.10.1) ; Receptor, Platelet-Derived Growth Factor beta (EC 2.7.10.1) ; Vascular Endothelial Growth Factor Receptor-2 (EC 2.7.10.1) ; fms-Like Tyrosine Kinase 3 (EC 2.7.10.1)
Language	English
Publishing date	2021-10-01
Publishing country	Canada
Document type	Journal Article
ZDB-ID	632814-3
ISSN	1928-9022 ; 0830-9000
ISSN (online)	1928-9022
ISSN	0830-9000
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Identification of the interleukin-8 (CXCL-8) pathway in feline oral squamous cell carcinoma - A pilot study

Ackerman, Leah H. / De Mello Souza, Carlos H. / Cortes-Hinojosa, Galaxia / Salute, Marc E. / Stephen, Alexa A. / Anthony, Elizabeth / Shiomitsu, Keijiro / Milner, Rowan J.

Canadian journal of veterinary research. 2022 Jan., v. 86, no. 1

2022

Abstract: The purpose of this pilot study was to detect the presence of interleukin-8 (IL-8) and the potential downstream effects of IL-8 receptor activation in 2 previously characterized feline oral squamous cell carcinoma cell lines (SCCF1 and SCCF2). ... ...

Abstract	The purpose of this pilot study was to detect the presence of interleukin-8 (IL-8) and the potential downstream effects of IL-8 receptor activation in 2 previously characterized feline oral squamous cell carcinoma cell lines (SCCF1 and SCCF2). Interleukin-8 messenger RNA (mRNA) was initially detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A previously validated and commercially available enzyme-linked immunosorbent assay (ELISA) test was used to measure IL-8 production in the supernatant of the 2 cell lines. Western blot was used to detect phosphorylation of proteins (AKT, ERK1/2, JAK2, STAT3, and Src), known to be downstream of interleukin-8 receptor activation. The IL-8 receptor-specific antagonists, Reparixin and SCH527123, were used to identify effects on phosphorylation of these proteins. Interleukin-8 mRNA and protein were detected in both SCCF1 and SCCF2 by RT-PCR and ELISA, respectively. Phosphorylation of ERK1/2, STAT3, and Src was detected in both cell lines. Inhibition of the IL-8 receptor led to a decrease in phosphorylation of Src, but not ERK1/2 or STAT3. In conclusion, feline squamous cell carcinoma cell lines can produce IL-8. Phosphorylation of Src seems, at least in part, a consequence of IL-8 receptor activation. The phosphorylation of ERK1/2 and STAT3, although present, seems independent of IL-8 receptor activation. Due to its potential effects on the tumor microenvironment, in addition to its autocrine effects on Src phosphorylation, the inhibition of the IL-8 receptor may become a beneficial therapeutic tool. Evaluation of the presence of both IL-8 and Src in many cases should elucidate their importance.
Keywords	Western blotting ; autocrine signaling ; cats ; enzyme-linked immunosorbent assay ; interleukin-8 ; messenger RNA ; neoplasm cells ; phosphorylation ; reverse transcriptase polymerase chain reaction ; squamous cell carcinoma ; therapeutics ; veterinary medicine
Language	English
Dates of publication	2022-01
Size	p. 13-19.
Publishing place	Canadian Veterinary Medical Association = Association canadienne des vétérinaires
Document type	Article
ZDB-ID	632814-3
ISSN	0830-9000
ISSN	0830-9000
Database	NAL-Catalogue (AGRICOLA)

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