Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Summary

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Abstract

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

Introduction

One in eight women will be diagnosed with breast cancer in her lifetime, and yet despite multiple treatment options breast cancer is the second leading cause of cancer-related deaths in American women¹. These women are not dying from the primary tumor in their breast. Instead, the metastatic burden is responsible for the mortality of this disease as it commonly spreads to the lung, bone, brain, liver, and lymph nodes². Because of this, breast cancer models need to evaluate metastasis to contribute to curbing the mortality of this disease. The 4T1 murine breast cancer model is a superb protocol to accomplish this. The met....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Virginia Tech and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Performing this protocol requires permission from the appropriate institutions and adherence to all appropriate guidelines.

1. Cell Culture

1. Make complete culture media (RPMI + 10% Fetal Bovine Serum +1% Pen Strep). Revive 4T1 cells according to ATCC Protocol¹⁰ and incubate at 37 °C and 5% CO₂ in a T-25 flask until confluent. Change media the day after reviving to r....

Results

This method contains simple adjustments from the Pulaski and Ostrand-Rosenberg 4T1 protocol³ and can be visualized in Figure 1. When 3 separate researchers manually counted metastatic colonies for 12 lung plates (1:10 dilution), the results were very inconsistent between different counters (Figure 2A). All researchers were directed to “count the metastatic colonies that appear as blue dots”, yet the inconsistencies demonstrate the issue w.......

Discussion

As demonstrated, manually counting the metastatic colonies on each lung plate can be an inaccurate and imprecise method to quantify lung metastasis, demonstrating the need for a better means of quantification (Figure 2). Histopathological analysis differed slightly from both manual counting and Fiji-ImageJ analysis (Figure 2B and 4D), likely because the H&E slides are not a representative sample of the entire organ. The protocol harvests an .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthesia chamber	See comments	See comments	Use approved materials in your institution's policies
Anesthetic agent	See comments	See comments	Use approved materials in your institution's policies
BALB/c Female Mice	The Jackson Laboratory	000651
Blunt scissors	Roboz	RS-6700
Calculator	Any	Any
Camera	Any	Any	Minimum of 8 megapixels
Centrifuge	Any	Any	Needs to be capable of 125 x g and 300 x g
CO2 euthanasia setup	See comments	See comments	Use approved materials in your institution's policies
Cold room, refrigerator, cold storage	Any	Any
Computer with Fiji-ImageJ	Any	Any	Needs to be capable of running Fiji-ImageJ
Counting Chamber	Fisher Scientific	02-671-10
Curved scissors	Roboz	RS-5859
Distilled water	Any	Any
Elastase	MP Biomedicals	100617
Electronic scale	Any	Any
Fetal Bovine Serum (FBS)	R&D Systems	S11150
Forceps	Roboz	RS-8100
Ice	N/A	N/A
Incubator	See comments	See comments	Needs to be capable of 5% CO2 and 37 °C
Methanol	Fisher Scientific	A412SK-4
Methylene blue	Sigma-Aldrich	03978-250ML
Penicillin Streptomycin	ATCC	30-2300
Pins or needles	Any	Any	For pinning down mice during necropsy
Plastic calipers	VWR	25729-670
RMPI-1640 Medium	ATCC	30-2001
Rocker or rotating wheel	Any	Any
Sharp scissors	Roboz	RS-6702
Sterile disposable filter with PES membrane	ThermoFisher Scientific	568-0010
T-150 Flasks	Fisher Scientific	08-772-48
T-25 Flasks	Fisher Scientific	10-126-10
T-75 Flasks	Fisher Scientific	13-680-65
Tri-cornered plastic beaker	Fisher Scientific	14-955-111F	Used to weigh mice
Trypan blue	VWR	97063-702
Trypsin-EDTA	ATCC	30-2101
Type IV collagenase	Sigma-Aldrich	C5138
3.5 cm tissue culture plates	Nunclon	153066
1 mL syringe	BD	309659
1.7 mL microcentrifuge tubes	VWR	87003-294
10 cm tissue culture plates	Fisher Scientific	08-772-22
12 well plate	Corning	3512
15 mL centrifuge tube	Fisher Scientific	14-959-70C
1X Dulbecco's Phostphate Buffered Saline (DPBS)	Fisher Scientific	SH30028FS
1X Hank’s Balanced Saline Solution (HBSS)	Thermo Scientific	SH3026802
27 g 1/2 in needles	Fisher Scientific	14-826-48
4T1 (ATCC® CRL­2539™)	ATCC	CRL-2539
50 mL centrifuge tube	Fisher Scientific	14-959-49A
6-Thioguanine	Sigma-Aldrich	A4882
70 μM cell strainer	Fisher Scientific	22-363-548
70% ethanol	Sigma Aldrich	E7023	Dilute to 70% with DI water