Модели костных метастазов

Summary

Животные модели часто используются для изучения раковых метастаз в костях. В этом протоколе мы опишем два распространенных метода инокуляции опухоли для исследования костного метастазирования, и кратко описать некоторые из анализов используются для мониторинга и количественной оценки этих моделей.

Abstract

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality¹. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy’s group in San Antonio², that has since been transfected for GFP expression and re-cloned by our group³. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis², in certain instances intratibial⁴ or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception ^5,6.

This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Protocol

1. Сотовые обслуживание Многие кости метастатического подклонах MDA-MB-231 существуют, которые имеют переменную склонность к колонизации костей и роста. Для наших экспериментов мы используем клон происходит от GFP с метками MDA-MB-231 разработан в UTHSCA 2. Этот конкретный суб-клон образ…

Discussion

Исследование индуцированного опухолью болезни костей полагаются на нескольких животных моделях из которых два наиболее часто используемых внутрисердечной и intratibial прививок. Во многих случаях внутрисердечной является лучшим вариантом для изучения метастазы в кости, однако она не пр?…

Materials

Name of the reagent	Company	Catalogue number
MDA-MB-231 human breast cancer cells	Originally ATCC-Internally cloned and selected
DMEM	GIBCO	11885
PBS	Mediatech Inc	21-040-CV
FBS	Atlas Biological	F-0050a
Trypsin	GIBCO	15400
Faxitron	Faxitron
Maestro	CRi
μCT scanner	Scanco
Athymic Nude Mice	Harlan	Fox nu -/-
Insulin Syringes (300 μl, 28.5 g)	Becton Dickinson	309300
IVIS	Caliper
Irradiated Rodent Diet	Teklad	2920x