Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

Summary

Here, we present a reliable and straightforward two-dimensional (2D) coculture system for studying the interaction between tumor cells and bone marrow adipocytes, which reveals a dual effect of melanoma cell-derived factors on the bone marrow adipocytes differentiation and also poses a classic method for the mechanistic study of bone metastasis.

Abstract

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

Introduction

Bone metastases are widespread among advanced cancer patients, but a curative treatment is still unavailable. Beyond specializing in storing energy as fat, adipocytes can support tumor growth and metastasis in bone marrow and other organs^1,2,3,4,5,6. Moreover, adipocytes play an essential role in regulating cancer cell biology^7,8,9,10 and metabol....

Protocol

NOTE: All cells used in this protocol should be grown for at least three generations after thawing from frozen stock cells.

1. Harvest Melanoma Cell-derived Factors

1. Preparations
   1. Obtain B16F10 cells and a mouse melanoma cell line.
      NOTE: For this protocol, a mouse melanoma cell line was obtained from the Stem Cell Bank of the Chinese Academy of Sciences.
   2. Make a complete medium for the B16F10 cell culture (100 mL). Use Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/mL of penicillin, and 50 µg/mL of streptomycin.
   3. M....

Results

In the bone marrow, adipocytes can appear in the tumor microenvironment^{1,13,33,34,35} at an early stage for supporting tumor progression through soluble factors or activating osteoclastogenesis^6,12,36, especially in the context of obesi.......

Discussion

Cocultures with inserts have been widely used to study cell-to-cell interactions. The 2D coculture system is an effective way to observe how the two parts crosstalk in vitro, by which we here showed two different cancer cell-driven effects on bone marrow adipocytes. Many labs have utilized this method to investigate the crosstalk between adipocytes and cancer cells^6,12,27,39.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen Inc.	11965092
Fetal Bovine Serum	Invitrogen Inc.	16000–044
Phosphate Buffered Saline	Invitrogen Inc.	14190-144
Insulin	Sigma-Aldrich	91077C
3-isobutyl-1-methyl-xanthine	Sigma-Aldrich	I5879
Dexamethasone	Sigma-Aldrich	D4902
Oil Red o	Sigma-Aldrich	O0625
24-well plate	Corning	CLS3527
Transwell insert	Millipore	MCHT24H48
Penicillin/Streptomycin	Invitrogen	15140-122
isopropanol	Sigma-Aldrich	I9516
0.25% trypsin	Thermo Scientific	25200056
hemocytometer	Bio-Rad	1450016
Culture incubator	Thermo Scientific
50 mL falcon	Corning	CLS430828
Clean Bench	Thermo Scientific	-
Microscopy	Olympus	-
200 μL pipet tips	BeyoGold	FTIP620
1,000 mL pipet tips	BeyoGold	FTIP628