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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Abstract

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Introduction

Despite being a highly complex and inefficient process1, metastasis is a significant contributor to the morbidity and mortality of cancer patients2. In fact, most cancer-related deaths are attributed to metastatic spread of disease3,4. In order for tumor cells to successfully metastasize, they must detach from the primary site, invade through adjoining stroma, intravasate into blood circulation or lymphatics, travel to the capillary bed of a secondary site, extravasate into the secondary tissue, and proliferate or grow to form metastatic lesions....

Protocol

Animal use followed University Laboratory Animal Resources (ULAR) regulations under the OSU Institutional Animal Care and Use Committee (IACUC)–approved protocol 2007A0120-R4 (PI: Dr. Gina Sizemore).

1. Tail-vein injection of breast cancer cells

  1. Preparation of cells and syringe for injection
    1. Plate an appropriate number of cells based on the number of mice and cell concentration to be used.
      NOTE: The number of cells injected and time to the development of m.......

Representative Results

If using unlabeled cells for tail-vein injection, it may be difficult to confirm lung colonization until (1) the time of necropsy if macrometastases can be observed or (2) following histological analysis if microscopic metastases exist. With extensive metastatic lung tumor burden, mice will have labored breathing. As with any tumor study, mice should be carefully monitored throughout the study duration. The use of labeled cells is an easy way to confirm successful tail-vein injection; hence the use of luciferase-tagged M.......

Discussion

As researchers continue to use intravenous injection of tumor cells as an experimental model for metastasis, standard practices to analyze the resulting metastatic tumor burden are lacking. In some cases, significant differences in metastatic tumor burden upon manipulation of particular cell lines and/or use of chemical compounds can be observed macroscopically. However, in other instances, subtle differences in metastatic seeding and growth may be overlooked or misinterpreted without thorough pathological analysis. This.......

Acknowledgements

Representative data was funded through the National Cancer Institute (K22CA218549 to S.T.S). In addition to their assistance in developing the comprehensive analysis method reported herein, we thank The Ohio State University Comprehensive Cancer Center Comparative Pathology and Mouse Phenotyping Shared Resource (Director – Krista La Perle, DVM, PhD) for histology and immunohistochemistry services and the Pathology Imaging Core for algorithm development and analysis.

....

Materials

NameCompanyCatalog NumberComments
alcohol prep padsFisher Scientific22-363-750for cleaning tail prior to injection
dissection scissorsFisher Scientific08-951-5for mouse dissection and lung tissue inflation
DMEM with L-Glutamine, 4.5g/L Glucose and Sodium PyruvateFisher ScientificMT10013CVcell culture media base for MDA-MB-231 and MVT1 cell lines
Dulbecco's Phosphate-Buffered Salt Solution 1xFisher ScientificMT21030CVused for resuspending tumor cells for injection
ethanol (70 % solution)OSUused to minimize mouse's fur during dissection; use caution - flammable
Evan's blue dyeMillipore SigmaE2129used at 1 % in sterile PBS for practice with tail-vein injection method; use caution - dangerous reagent
Fetal Bovine SerumMillipore SigmaF4135cell culture media additive; used at 10% in DMEM
forcepsFisher Scientific10-270for dissection and lung tissue inflation
FVB/NJ miceThe Jackson Laboratory001800syngeneic mouse strain for MVT1 cells
hemacytometer (Bright-Line)Millipore SigmaZ359629for use in cell culture to obtain cell counts
insulin syringe (28 G)Fisher Scientific14-829-1Bfor tail-vein injections (BD 329424)
MDA-MB-231 cellsATCChuman breast cancer cell line
MVT1 cellsmouse mammary tumor cells
needles (26 G)Fisher Scientific14-826-15used to inflate the mouse's lungs
neutral buffered formalin (10%)Fisher Scientific245685used as a tissue fixative and to inflate lung tissue; use caution - dangerous reagent
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) miceThe Jackson Laboratory005557maintained by OSUCCC Target Validation Shared Resource
Penicillin Streptomycin 100xThermoFisher15140163cell culture media additive
sterile gauzeFisher ScientificNC9379092for applying pressue to mouse's tail if bleeding occurs
syringe (5 mL)Fisher Scientific14-955-458used to inflate mouse lung tissue
tail-vein restrainerBraintree Scientific, Inc.TV-150 STDused to restrain mouse for tail-vein injections
Trypan blue (0.4 %)ThermoFisher15250061used in cell culture to assess viability
Trypsin-EDTA 0.25 %ThermoFisher25200-114used in cell culture to detach tumor cells from plate

References

  1. Chambers, A. F., Groom, A. C., MacDonald, I. C. Dissemination and growth of cancer cells in metastatic sites. Nature Reviews: Cancer. 2 (8), 563-572 (2002).
  2. Steeg, P. S. Targeting metastasis. Nature Reviews: Cancer. 16 (4), ....

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