Cancer immunoediting from immunosurveillance to tumor escape in microvillus-formed niche: a study of syngeneic orthotopic rat bladder cancer model in comparison with human bladder cancer

Abstract

Cancer cells can develop an attenuated immunogenicity and/or create an immunosuppressive microenvironment to prevent tumor eradication by host immune system, the so-called "cancer immunoediting" hypothesis. The aim of the present study was to find evidence for this hypothesis by using a rat orthotopic bladder cancer model. Fisher rats were inoculated with AY-27 cells (a Fisher rat bladder cancer cell line). Cultured cancer cells, rat and human bladder cancer tissues, and publicly available microarray data from human bladder cancer were analyzed by means of bioinformatics and morphology. Results showed that 12 of 24 differentially expressed pathways were concordant in connection to cell cycle and proliferation between rats and humans (both non-muscle-invasive and muscle-invasive tumors) and that 11 of the 24 pathways, including major histocompatibility complex, were related to host immunosurveillance with activations of T cells and natural killer cells in rats. The altered pathways and morphogenesis of this rat model corresponded more closely with those of human muscle-invasive rather than non-muscle-invasive tumors. A unique ultrastructure displaying microvillus-formed niches was found in small areas within the tumor of both rats and humans. These niches were interconnected with desmosomes between cancer cells and without infiltration of lymphocytes. The expression of E-cadherin, selectins, PGP9.5, vascular endothelial growth factor, caspase-3, CD133, Oct-4, nestin, CD3, and CD45RA was lower in the tumor than in the adjacent normal epithelium. We suggest that the microvillus-formed niche that harbors a few implanted cancer cells might be the compartment that prevents the tumor eradication by the host immune system.

Figure 1

PCA of gene expression patterns (A) and histological diagnosis (B) of normal bladder (N), bladder cancer (B; 2 weeks after inoculation of AY-27 cells), and bladder cancer AY-27 cells in culture (C). Note that the gene expression profiles of the normal bladder and the bladder cancer are distinctly different; however, the magnitude of change is even greater between the bladder cancer and the cancer cells in panel A. Magnifications are the same as in panel B.

Figure 2

Diagram showing MHC I and II pathways in the bladder cancer tissue 2 weeks after inoculation. Upregulated genes (P < .01; red) and unchanged genes (yellow). Note: No downregulated genes.

Figure 3

Histopathology of rat (A–D) and human (E–F) bladder cancer. (A) Inflammation with lymphocyte infiltration (indicated by arrow) within the mucosa and lamina propria 1 week after inoculation. (B) Carcinoma in situ (asterisk) surrounded by lymphocytes 2 weeks after inoculation. Note also: Many small blood vessels (arrow) in the lamina propria. (C and D) Carcinoma in situ (asterisks) 3 weeks after inoculation. Lymphocyte aggregations (arrows) in the lamina propria in panel C and surrounding the tumor in panel D. (E and F) Carcinoma (asterisks) surrounded and/or infiltrated by lymphocytes (arrows). Hematoxylin-eosin-safron stain: bars, 100 µm (A–D) and 50 µm (E and F).

Figure 4

Ultrastructure of rat (A–D) and human (E–H) bladder cancer cells and tissues (asterisks). Note: AY-27 cancer cells in the culture display microvilli (arrow) (A). Most cancer cells in the bladder (at 3 weeks after inoculation) do not display microvillus-formed mesh (B), but occasionally, small areas of the cancer cells display microvilli (C). (D) Higher magnification of panel C showing desmosomes (arrows) connecting cells. Most cancer cells in patients at stage of T2–T4 do not display microvilli (E and F), but occasionally, small areas of the cancer cells display microvillus-formed mesh (arrow) (G). (H) Higher magnification of panel G showing desmosomes (arrows) connecting cells. Bar, 500 nm.

Figure 5

Immunostained photographs of rat bladder cancer with antibodies to PCNA (A), caspase-3 (B), E-selectin (C), L-selectin (D), P-selectin (E), E-cadherin (F), PGP9.5 (G), VEGF (H), CD3 (I), CD133 in adjacent normal tissue (J), CD133 in tumor tissue (K), CD45RA (L), Oct-4 in adjacent normal tissue (M), Oct-4 in tumor tissue (N), and nestin (O). The tumors are indicated by asterisks, whereas the adjacent normal tissues by arrows. Bar, 100 µm.

Figure 6

Two models for CSC niche. (A) A normal stem cell niche is “hijacked” by CSC. CSCs generate tumor (cancer cells, CCs) on the basis of their self-renewal properties and enormous potential. The niche is made of surrounding cells (e.g., fibroblasts, endothelial cells, macrophages, mast cells, and other cells) as well as extracellularmatrix and is associated with microvasculature and innervations. Immunosurveillance (e.g., by lymphocytes, T) takes place against CCs but not CSCs that underwent immunoediting. (B) CSCsmake their own niche by forming microvillus mesh through desmosomes. The immunosurveillance takes place against CCs and the outer layer of the niche. The niche is not associated with microvasculature and innervations.