Development of an in vitro assay for screening programmed death receptor-1/programmed cell death ligand 1 monoclonal antibody therapy in dogs.

Immunomodulatory antibody drugs that modulate the function of immune checkpoint molecules, such as programmed death receptor-1 (PD-1) and programmed cell death ligand 1 (PD-L1), have been established as new cancer treatments in human medicine. In recent years, there have also been reports on antibodies that inhibit immune checkpoint molecules in dogs, and clinical trials using such antibodies for canine cancer have been gradually increasing in number. Because inhibitory antibodies restore T-cell function by inhibiting the binding of PD-1 on T cells and its ligand PD-L1, the quality of antibody function has been evaluated using activated T cells or peripheral blood mononuclear cells isolated from healthy dogs; however, the assays and dogs used significantly vary. Therefore, in the present study, we developed a reporter gene assay using reporter cells (Jurkat/NFATluc/cPD1) and effector cells (CTAC/OKT3/cPDL1). Jurkat/NFATluc/cPD1 were generated by introducing both of the NFAT-responsive luciferase gene as a marker of T-cell signaling and canine PD-1, into a human T lymphoid cell line, Jurkat. CTAC/OKT3/cPDL1 were generated by introducing single-chain FV (scFV) of anti-human CD3 antibody (OKT3) and canine PD-L1 into a canine thyroid carcinoma cell line, CTAC. Ligation of PD-1 on Jurkat/NFATluc/cPD1 via binding of PD-L1 on CTAC/OKT3/cPDL1 suppressed NFAT luciferase activity induced by CD3 ligation by scFV of OKT3. The addition of anti-canine PD-1 and PD-L1 antibodies, both of which were previously developed in our laboratory, restored this suppression with high sensitivity, although the anti-human PD-L1 antibody atezolizumab induced a very weak restoration. This assay is an useful method for functionally evaluating the inhibition of canine PD-1 and PD-L1 binding.

Establishment of a histiocytic sarcoma cell line and anti-tumor effect of bortezomib in the African pygmy hedgehog (Atelerix albiventris).

The African pygmy hedgehog (Atelerix albiventris) is known to have a high incidence of tumor. However, investigating the tumors of this species has been constrained by the limited availability of research materials such as cell lines and genome information. In this study, we successfully established a novel cell line from a histiocytic sarcoma (HS) of an African pygmy hedgehog, allowing us to conduct a drug screening. We investigated using FDA-approved drug library screening to determine which anticancer drug this tumor cell line is sensitive to, and as a result of apoptosis experiments, bortezomib among the three proteasome inhibitors was found to induce cell death of cancer cells by significantly increasing caspase-3 cleavage (P<0.01). Thus, we elucidated that the proteasome inhibitors, particularly bortezomib, exhibit anti-tumor effects on a cell line derived from an HS in an African pygmy hedgehog through a mechanism comparable to that described in human tumors. This study reports the first characterized cell line from the African pygmy hedgehog and also highlights the potential utility of bortezomib as an anti-tumor treatment for HS in this species.

The Density of CD8+ Tumor-infiltrating Lymphocytes Correlated With Akt Activation and Ki-67 Index in Canine Soft Tissue Sarcoma.

BACKGROUND/AIM: The activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been implicated in canine soft tissue sarcoma (STS) and may serve as a prognostic marker. This study investigated the correlation between PI3K/Akt activation in tumor cells and tumor-infiltrating lymphocytes (TILs). MATERIALS AND METHODS: A total of 59 STS samples were labeled via immunohistochemistry to calculate the density of TILs, including CD3+ T cells, CD8+ T cells, CD20+ B cells, and FOXP3+ regulatory T cells. RESULTS: Forty-eight samples (81.3%) had intra-tumoral TILs with a high density of CD3+ T cells (mean: 283.3 cells/mm2) and CD8+ T cells (mean: 134.8 cells/mm2). Conversely, CD20+ B cells (mean: 73.6 cells/mm2) and FOXP3+ regulatory T cells (mean: 9.2 cells/mm2) were scarce. The abundance of CD3+/CD8+, CD3+/CD20+, and CD8+/CD20+ TILs were highly correlated in multivariate analyses (r=0.895, 0.946, and 0.856, respectively). Nonetheless, TIL density was unrelated to clinicopathological parameters (sex, age, tumor location, breed) and tumor grade. The abundance of CD8+ T cells was positively correlated with the activation of PI3K/Akt, indicating that samples with high levels of phospho-Akt and phospho-S6 tend to have a higher CD8+ T cell density (p=0.0032 and 0.0218, respectively). Furthermore, TIL density was correlated with the Ki-67 index, a tumor proliferation and growth marker. Samples with a high Ki-67 index had a significantly higher abundance of CD3+ T cells, CD8+ T cells, and CD20+ B cells (p=0.0392, 0.0254, 0.0380, respectively). CONCLUSION: PI3K/Akt pathway activation may influence the infiltration of CD8+ T cells within the tumor microenvironment in canine STS. Prospective studies involving a higher number of cases are warranted to confirm these findings.

Establishment of a novel canine soft tissue sarcoma cell line and comparison of its characteristics with other soft tissue sarcoma cell lines.

Soft tissue sarcoma (STS) is a relatively common tumor in dogs. However, very few canine STS cell lines are available. This study aimed to establish a new cell line, STS-YU1, derived from a recurrence of myxosarcoma in an 11-year-old mixed-breed dog. We examined STS-YU1 for in vitro cell proliferation, migration, anticancer drug sensitivity, transcriptome analysis using next-generation sequencing (RNA-seq), and in vivo tumorigenicity in mice and compared it with previously established STS cell lines, MUMA-G and A72. The cell proliferation and migration of STS-YU1 were higher than MUMA-G although MUMA-G only exhibited tumorigenicity in mice. STS-YU1 showed dose-dependent cytotoxicity to anticancer drugs, but with weak effects. RNA-seq analysis revealed the molecular phenotype of STS-YU1 was different from that of a previously reported cell line, A72. Hence, the use of STS-YU1 would help in efficient drug screening against canine STS in vitro.

Identification of hypoxia-induced metabolism-associated genes in canine tumours.

Canine tumours including urothelial carcinoma, lung adenocarcinoma, mammary gland tumour, squamous cell carcinoma, and melanoma have been identified as causes of death, but effective therapies are limited due to insufficient knowledge of the molecular mechanisms involved. Within the tumour microenvironment, hypoxia activates hypoxia-inducible factor 1α (HIF1α) in tumour cells. High HIF1α expression correlates with enhanced glycolysis and poorer outcomes in human cancers. However, the molecular mechanisms underlying hypoxic tumour cells remain elusive in dogs. In our study, we investigated upregulated genes in a canine malignant melanoma cell line during hypoxia using RNA-sequencing analysis. Glycolysis and HIF1 signalling pathways were upregulated in hypoxic melanoma cells. HIF1α knockout melanoma cells revealed that the glycolysis marker MCT4 is regulated by HIF1α activation. Hypoxia induces high lactate secretion due to enhanced glycolysis in canine melanoma cells. Furthermore, we examined monocarboxylate transporter 4 (MCT4) expression in malignant melanoma and eight other types of canine tumour tissues using immunohistochemistry (IHC). Membrane-localized MCT4 protein was mostly detected in urothelial carcinoma and lung adenocarcinoma rather than malignant melanoma. We conclude that canine MCT4 protein plays a role in lactic acid efflux from glycolytic cells and may serve as a marker for hypoxia and glycolysis in canine tumours. These findings could inform future therapeutic strategies targeting MCT4.

Proof-of-concept study of the caninized anti-canine programmed death 1 antibody in dogs with advanced non-oral malignant melanoma solid tumors.

BACKGROUND: The anti-programmed death 1 (PD-1) antibody has led to durable clinical responses in a wide variety of human tumors. We have previously developed the caninized anti-canine PD-1 antibody (ca-4F12-E6) and evaluated its therapeutic properties in dogs with advance-staged oral malignant melanoma (OMM), however, their therapeutic effects on other types of canine tumors remain unclear. OBJECTIVE: The present clinical study was carried out to evaluate the safety profile and clinical efficacy of ca-4F12-E6 in dogs with advanced solid tumors except for OMM. METHODS: Thirty-eight dogs with non-OMM solid tumors were enrolled prospectively and treated with ca-4F12-E6 at 3 mg/kg every 2 weeks of each 10-week treatment cycle. Adverse events (AEs) and treatment efficacy were graded based on the criteria established by the Veterinary Cooperative Oncology Group. RESULTS: One dog was withdrawn, and thirty-seven dogs were evaluated for the safety and efficacy of ca-4F12-E6. Treatment-related AEs of any grade occurred in 13 out of 37 cases (35.1%). Two dogs with sterile nodular panniculitis and one with myasthenia gravis and hypothyroidism were suspected of immune-related AEs. In 30 out of 37 dogs that had target tumor lesions, the overall response and clinical benefit rates were 6.9% and 27.6%, respectively. The median progression-free survival and overall survival time were 70 days and 215 days, respectively. CONCLUSIONS: The present study demonstrated that ca-4F12-E6 was well-tolerated in non-OMM dogs, with a small number of cases showing objective responses. This provides evidence supporting large-scale clinical trials of anti-PD-1 antibody therapy in dogs.