A surgical orthotopic approach for studying the invasive progression of human bladder cancer

Abstract

The invasion of bladder cancer into the sub-urothelial muscle and vasculature are key determinants leading to lethal metastatic progression. However, the molecular basis is poorly understood, partly because of the lack of uncomplicated and reliable models that recapitulate the biology of locally invasive disease. We developed a surgical grafting technique, characterized by a simple, rapid, reproducible and high-efficiency approach, to recapitulate the pathobiological events of human bladder cancer invasion in mice. This technique consists of a small laparotomy and direct implantation of human cancer cells into the bladder lumen. Unlike other protocols, it does not require debriding of the urothelial lining, injection into the bladder wall, specialized imaging equipment, bladder catheterization or costly surgical equipment. With minimal practice, the procedure can be executed in <10 min. Tumors develop with a high take rate, and most cell lines exhibit local invasion within 4 weeks of implantation.

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Fig. 1: Schematic illustration comparing the development of normal human bladder cancer to the development of bladder cancer in mice after this protocol is performed.
Fig. 2: Orthotopic inoculation of bladder cancer cells in a recipient NOD-SCID mouse.
Fig. 3: End-stage bladder cancer xenograft growth as observed with different methods.
Fig. 4: Xenograft models and their key histological features.
Fig. 5: Main histological features of the xenografted tumors.

Data availability

The datasets generated during the current study are included in this published article and its SupplementaryInformation files.

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Acknowledgements

This work was supported by a University of Michigan Rogel Cancer Center Research Grant to E.T.K.; Department of Urology Research funds to M.L.D.; NIH R01CA154252 to M.L.D.; the European Egyptian Pharmaceutical Industries (EEPI) research collaboration with M.L.D. and, the Genmab research collaboration with M.L.D. In addition, EEPI provided salary funds to L.E.-S., K.C.D., L.J.B. and M.L.D., as well as consulting fees to L.E.-S. and M.L.D. Genmab provided salary support for G.L.H. and A.L.C. G.L.H. was also supported by the Postdoctoral Translational Scholars Program of the Michigan Institute for Clinical and Health Research (UL1TR002240), NIH. We thank J. Escara-Wilke for her assistance with the banked samples utilized in this study, E. Breij for her conceptual advice, and M.C. Winkler, who aided with the surgical setup and preliminary stages of the research.

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Authors

Contributions

G.L.H., A.L.C., L.E.-S. and K.C.D. conceived different aspects of the experimental design and implemented the surgical technique with contributions from E.T.K. and M.L.D. The luciferase construct was developed by E.V.A., and K.C.D. labeled the cancer cells with the luciferase tag. G.L.H., A.L.C., L.E.-S., K.C.D., L.J.B., A.L.H., A.E. and A.C. performed the surgical experiments. G.L.H., A.L.C., K.C.D., L.J.B., A.H., H.L.B., A.E. and A.C. collected, analyzed and interpreted data. G.L.H., A.L.C. and H.L.B. conducted the MI and LVI examinations. M.L., P.L.P., A.M.U., E.T.K. and M.L.D. provided conceptual advice. A.M.U. contributed to the histological analysis and provided the bladder cancer specimens. M.L., E.T.K. and M.L.D. provided reagents, materials and mice. G.L.H., A.L.C., K.C.D., L.J.B., H.L.B., E.T.K. and M.L.D. contributed to the manuscript preparation.

Corresponding authors

Correspondence to Evan T. Keller or Mark L. Day.

Ethics declarations

Competing interests

EEPI provided salary support to L.E.-S., K.C.D., L.J.B. and M.L.D., and consulting fees to M.L.D. and L.E.-S. M.L.D. has received research funds from EEPI as part of a sponsored research agreement. L.E.-S. is currently an employee of EEPI. Genmab provided research funds to M.L.D. and salary support to G.L.H. and A.L.C. Neither EEPI nor Genmab participated in the development, design, execution or analysis of the experiments, or the publication of the manuscript. The remaining authors declare no competing interests.

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Related link

Key references using this protocol

Han, A. L. et al. Oncogene 36, 5243–5251 (2017): https://www.nature.com/articles/onc2017149

Integrated supplementary information

Supplementary Figure 1 Required surgical supplies and setup.

Main tools, equipment, and reagents necessary for successful orthotopic inoculations. a, Set of surgical tools to be autoclaved in a cassette for instruments (see also i). From left to right: ceramic-coated cupped forceps, Adson forceps, tissue forceps, Stevens needle holder, micro-dissecting angled forceps, pointed forceps, and iris scissors. b, Povidone iodine scrub solution, 7.5%. c, Isoflurane with anti-drip top. d, Germinator 500, bead sterilizer for surgical tools. e, Autoclaved cotton swabs, sterile gauze pads, alcohol swabs. f, Sterile towel drape. g, One milliliter tuberculin slip tip syringe and 30½ gauge needle. h, Ophthalmic ointment. i, Minimum required surgical setup.

Supplementary Figure 2 Pre-surgical hair removal.

Required hair removal procedure one day prior to surgery. Depilatory cream is utilized by our laboratory. A-NOD-SCID mouse is depicted immediately after the hair has been removed and the excess depilatory cream has been washed away. This procedure was approved by the Institutional Animal Care and Use Committee of the University of Michigan following the animal welfare recommendations by the National Institutes of Health.

Supplementary Figure 3 Recommended wound closure tools and abdominal suture procedure.

Incision closure information. a-c, Tools recommended for wound closure. a, Stevens needle holder. b, Adson forceps. c, Coated Vicryl absorbable suture. d, Tissue adhesive. e-l, Protocol utilized by our laboratory to suture the abdominal wall. The procedure is represented in a NOD-SCID mouse. e, Align the sharp end of the needle with the cranial end of the incision. The needle should be oriented such that it is perpendicular to the incision while the needle holders remain parallel to the incision. The sutures are applied from the cranial to the caudal end of the incision. f, Start to suture by inserting the needle from the outside of the muscle layer. Then, insert the needle on the opposite side of the incision from the inside of the muscle. Pull the needle and suture through the muscle layer so that ~2 cm of suture remains on the end of the suture. This small piece will be used to tie the suture. g, Tie three knots to secure the suture in place. The first knot will be double knot. h, The second and third knots will be single knots tied opposite of each other. The suture is now secured in place. i, Pierce the needle through the muscle, Then, grab the muscle on the opposite side and pierce the needle through it. Pull the suture tightly through. Repeat this step. Evenly separate the sutures until the incision is closed. j, Tie the suture closed as in (e) but, using the final loop of the suture. k, Tie one double knot followed by two single, opposite knots to end the suture. l, Gently tighten the suture and trim any excess away. The muscle layer is now fully closed. Sealed the skin closed with tissue adhesive or wound clips (not depicted). This procedure was approved by the Institutional Animal Care and Use Committee of the University of Michigan following the animal welfare recommendations by the National Institutes of Health.

Supplementary Figure 4 Wound progression.

Wound healing patterns observed following surgical xenotransplantation. While some fibrotic tissue is expected, the skin must remain sealed to prevent infection. The recovery period and wound healing process should be completed after 10 days. This procedure was performed in a NOD-SCID mouse and approved by the Institutional Animal Care and Use Committee of the University of Michigan following the animal welfare recommendations by the National Institutes of Health.

Supplementary Figure 5 Troubleshooting and execution problems.

Examples of experimental complications depicted in NOD-SCID mice. a, Improper wound healing in which the abdominal muscle was exposed due to improper closure and excessive retraction of the skin. Built-up of fibrotic tissue is observed at the wound site. b, Histological section showing the consequence of a failed inoculation. A tumor developed on the exterior wall of the bladder. Scale bars, 500 μm (left) and 200 μm (right).

Supplementary Figure 6 Xenograft models in NSG and athymic nude mice.

Representative tumor growth, followed by bioluminiscence imaging, at three weeks post-inoculation of 1 ×106 luciferase-tagged UM-UC-9 cells into the bladder of female NSG and male nude mice. This procedure was approved by the Institutional Animal Care and Use Committee of the University of Michigan following the animal welfare recommendations by the National Institutes of Health.

Supplementary Figure 7 Key histological characteristics of UM-UC-13 and T24 xenograft models.

The xenografted tumors display less aggressive pathobiological features and require a higher inoculum for their development. The figure shows representative histological results after implantation of 5 ×106 UM-UC-13 and 1.5 ×106 T24 human bladder tumor cells into the bladder lumen of NOD-SCID mice. The tumors developed for four weeks until end point. a, Characteristic histological analysis at the invasive edge of the tumor. Cytokeratin 8+ (CK8, clone EP17, Epitomics, 1:100, cat. no. AC-0007) positive cancer cells are observed invading the muscle of the bladder. In general, the mice exhibit muscle-invasive T2 bladder tumors at a solitary focal location. b, Lymphovascular invasion is also observed at lower frequency. CK8 positive tumor cells are detected within CD31 (Dianova, clone SZ31, 1:50, cat. no. DIA-310) and LYVE1 (Abcam, 1:1000, cat. no. ab14917) positive vessels. Scale bars, 50 μm.

Supplementary Figure 8 Sporadic experimental observations.

a, Normal bladder displaying a thin, triple-layered urothelium (insert). b, Non-invasive lesion with engrossment of the bladder urothelium (insert) c, Example of spontaneous sporadic metastasis. A liver metastatic tumor is observed by bioluminescence imaging. The figures display experiments with NOD-SCID mice. Scale bars, 500 μm (left) and 50 μm (right inserts).

Supplementary information

Supplementary Text and Figures and Supplementary Methods

Supplementary Figures 1–8, Processing of murine bladder samples for immunohistological analysis.

Reporting Summary

Supplementary Video 1

Demonstration of the surgical orthotopic procedure to graft human tumor cells into the bladder of an immunodeficient mouse. Step-by-step protocol showing a NOD-SCID mouse undergoing surgery.

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Lorenzatti Hiles, G., Cates, A.L., El-Sawy, L. et al. A surgical orthotopic approach for studying the invasive progression of human bladder cancer. Nat Protoc 14, 738–755 (2019). https://doi.org/10.1038/s41596-018-0112-8

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