Naturally-Occurring Invasive Urothelial Carcinoma in Dogs, a Unique Model to Drive Advances in Managing Muscle Invasive Bladder Cancer in Humans

Abstract

There is a great need to improve the outlook for people facing urinary bladder cancer, especially for patients with invasive urothelial carcinoma (InvUC) which is lethal in 50% of cases. Improved outcomes for patients with InvUC could come from advances on several fronts including emerging immunotherapies, targeted therapies, and new drug combinations; selection of patients most likely to respond to a given treatment based on molecular subtypes, immune signatures, and other characteristics; and prevention, early detection, and early intervention. Progress on all of these fronts will require clinically relevant animal models for translational research. The animal model(s) should possess key features that drive success or failure of cancer drugs in humans including tumor heterogeneity, genetic-epigenetic crosstalk, immune cell responsiveness, invasive and metastatic behavior, and molecular subtypes (e.g., luminal, basal). Experimental animal models, while essential in bladder cancer research, do not possess these collective features to accurately predict outcomes in humans. These key features, however, are present in naturally-occurring InvUC in pet dogs. Canine InvUC closely mimics muscle-invasive bladder cancer in humans in cellular and molecular features, molecular subtypes, immune response patterns, biological behavior (sites and frequency of metastasis), and response to therapy. Thus, dogs can offer a highly relevant animal model to complement other models in research for new therapies for bladder cancer. Clinical treatment trials in pet dogs with InvUC are considered a win-win-win scenario; the individual dog benefits from effective treatment, the results are expected to help other dogs, and the findings are expected to translate to better treatment outcomes in humans. In addition, the high breed-associated risk for InvUC in dogs (e.g., 20-fold increased risk in Scottish Terriers) offers an unparalleled opportunity to test new strategies in primary prevention, early detection, and early intervention. This review will provide an overview of canine InvUC, summarize the similarities (and differences) between canine and human InvUC, and provide evidence for the expanding value of this canine model in bladder cancer research.

Keywords: animal models; bladder cancer; cancer prevention; dog; immunotherapy; targeted therapy; transitional cell carcinoma; urothelial carcinoma.

Figure 1

Canine invasive urothelial carcinoma (InvUC). Canine InvUC often produces papillary lesions extending into the lumen of the urethra (as seen in the cystoscopic image in A) and bladder (as seen on post mortem specimen in B,C), along with deep invasion into the bladder wall. For comparison, in (A), the inset demonstrates the normal appearance of this region of the urinary tract in the absence of cancer. In (C), note the transmural growth in the entire bladder (please see the 2* on the right side of the panel), hydroureter (thin arrow), and hydronephrosis (thick arrow) caused by obstruction of the ureteral orifice by tumor growth in the bladder. An adjacent iliac lymph node (dark dot) is also infiltrated by this neoplasm. The photomicrograph in (D) (H&E 40X) is typical of high-grade InvUC. There is lack of normal cell maturation and marked nuclear atypia with some binucleated and multinucleated cells, and mitotic figures (arrows). Note the presence of cytoplasmic vacuoles within neoplastic cells, a common but not unique finding to InvUC. Canine InvUC is locally aggressive and metastasizes to distant sites in more than 50% of cases. Note metastases to the lung (E) and liver (F). The gross appearance of metastases range from single to multiple nodules that can become confluent as observed in the lung (E).

Figure 2

Basal and luminal subtypes in canine invasive urothelial carcinoma. RNA-seq data from canine InvUC (n = 33) and normal bladder mucosal samples (n = 4) were normalized using TMM and DESeq concurrently using Strand NGS (Strand, Bengaluru, India). Statistical analyses were conducted using edge R (on TMM normalized data) and DESeq2 (on DESeq normalized data) with p corr ≤ 0.05 and ≥2-fold change from each analysis. These two lists of differentially expressed genes were pooled as described previously (59). A prediction model reported earlier was employed to assign luminal and basal subtypes (59). Supervised hierarchical clustering was performed using genes that assign basal and luminal subtypes in human InvUC (17). Two distinct groups were identified as basal (n = 15) and luminal (n = 18).

Figure 3

Immunohistochemical detection of T lymphocytes with an antibody to CD3 in canine invasive urothelial carcinoma. In (A) all areas examined (intraepithelial, tumor stroma, and peritumoral) contain CD3 positive cells. In (B) a detail of the triphasic pattern of CD3 expression is noted. In (C) only the peritumoral lymphoid infiltrate expresses CD3 in this tumor. In (D) the tumoral stroma contains numerous CD3 positive lymphocytes, but the tumor epithelium is negative. TE, tumoral epithelium; TS, tumoral stroma; PT, peritumoral stroma; Small arrow, intraepithelial T-lymphocytes; Large arrow, tumoral stroma T lymphocytes.

Figure 4

Canine invasive urothelial carcinoma (InvUC) samples display gene expression patterns classifying the tumors as immune infiltrated (immune “hot”) or non-immune infiltrated (immune “cold”). A list of immune signature genes known to be upregulated in T-cell inflamed human InvUC samples were used (170) to visualize the immune patterns that exist in canine InvUC. Normalized intensity values were used for supervised hierarchical clustering using Euclidean distance metrics and Ward's linkage algorithm as a distance metric. Note the predominantly high expression of immune genes in the right cluster of the canine InvUC samples (n = 15, 45%) classifying them as immune “hot” (immune infiltrated).

Figure 5

Single cell RNA-seq analysis of canine invasive urothelial carcinoma. Unsupervised clustering of the canine InvUC sample was performed (Seurat package, Satija Lab). The cells segregated into seven different clusters. Putative cell type assignment was based on marker gene expression and abundance within the cluster. The gene expression heatmap focused on the CD45+ cells (immune cells) shows the cell clusters with putative immune cell type assignments on x-axis and top 10 marker genes in each cluster on Y-axis. The split dotplot on right shows the intensity (dot color) and percentage of cells expressing (dot size) 13 marker genes (x axis) analyzed in InvUC tissue across clusters before and after treatment (y axis). This type of data can be used to study mechanisms and generate new hypotheses. For example, GPR183 which increases in cluster 1 cells, is known for its role in lymphoid organ development and positioning of activated CD4 T cells in lymphoid follicles, but its role in the immune state of InvUC has not been elucidated (174).