Role of AXL in invasion and drug resistance of colon and breast cancer cells and its association with p53 alterations

Table 1 Cell lines sources and maintenance

Cell line	Source	Media	FBS %	Additives
MCF7	France1	DMEM	10%	1% P/S
1001	France	DMEM	10%	1% P/S
ZR-75-1	Helsinki2	RPMI 1640	10%	1% P/S + Sodium pyruvate (1 mmol/L)
RKO	Horizon3	RPMI 1640	10%	1% P/S + Sodium pyruvate (1 mmol/L)
RKO.p53-/-	Horizon	RPMI 1640	10%	1% P/S + Sodium pyruvate (1 mmol/L)
HCT116	Horizon	RPMI 1640	10%	1% P/S + Sodium pyruvate (1 mmol/L)
HCT116.p53	Horizon	DMEM	10%	1% P/S + 50% F12
HeLa	France	DMEM	10%	1% P/S
MDA-MB-361	France	DMEM	10%	1% P/S
MDA-MB-231	France/ATCC	DMEM	10%	1% P/S + 50% F12 + insulin (10 mg/mL in 25 mmol/L HEPES) + hydrocortisone (0.5 μg/mL)
CAL-51	Helsinki	DMEM	20%	1% P/S
T47D	France	DMEM	10%	1% P/S + 50% F12 + EGF (20 ng/mL)
BT-549	Helsinki	RPMI 1640	10%	1% P/S + Sodium pyruvate (1 mmol/L) + insulin (10 mg/mL in 25 mmol/L HEPES)
SW480	Helsinki	DMEM	10%	1% P/S + 50% F12 + insulin (10 mg/mL in 25 mmol/L HEPES) + hydrocortisone (0.5 μg/mL) + EGF (20 ng/μL)

¹France: Kind Gift from Professor Salem Chouaib, Institute Gustave Roussy, France;

²Helsinki: Kind gift from Professor Paivi peltomaki; Department of Medical Genetics, Helsinki University, Finland;

³Horizon Discovery, Cambridge, United Kingdom.

Table 2 Cell lines origin and p53 status

Cell lines	Cell line description1	p53 status1
Breast cancer cell lines
MCF7	Adenocarcinoma of the mammary gland, derived from 69-year-old female, cells were obtained from metastatic site; pleural effusion	Wild-type[34]
1001	1001 is derived from its parental MCF7 (MCF7/R-A1; which are cells exposed to increasing dose of recombinant TNF, transfected by p55 TNF receptor cDNA, Mutation in R280K)[35]	TNF resistant associated with loss of p53 function[35]
CAL51	Adenocarcinoma isolated from a malignant pleural effusion of a 44-year-old female with metastatic breast cancer, normal karyotype with genetic stability[36]	Wild-type[37]
ZR-75-1	Ductal carcinoma of the mammary gland, derived from 63-year-old female, cells were obtained from metastatic site: ascites	Wild-type
MDA-MB-361	Adenocarcinoma of the mammary gland, derived from 40-year-old female, cells were obtained from metastatic site; Brain	Wild-type
T47D	ductal carcinoma of the mammary gland, derived from 54-year-old female, cells were obtained from metastatic site; pleural effusion	Heterozygous mutant.
MDA-MB-231	Adenocarcinoma of the mammary gland, derived from 51-year-old female, cells were obtained from metastatic site; pleural effusion.	Homozygous mutant
BT549	Ductal carcinoma of the mammary gland, derived from 72-year-old female, cells were obtained from mammary gland.	Homozygous mutant
Colon Cancer Cell lines
RKO	poorly differentiated colon carcinoma cell line developed by Michael Brattain	Wild-type.
RKO-p53	RKO parental cell line double negative mutant for p53	Homozygous knock out2
HCT 116	Colorectal carcinoma of Adult Male derived from primary tumor site	Wild-type
HCT 116.p53	HCT116 parental cell line with hemizygous p53 mutation and knock out of the homologous p53	R284w/-2
SW480	Dukes' type B, colorectal adenocarcinoma, derived from 50-year-old male, cells obtained from primary tumor site	Homozygous mutant
Positive control for EMT
HeLa	Cervical adenocarcinoma of 31-year-old female derived from primary tumor site	Wild-Type but HPV inactivated[34]

¹The cell line origin and the p53 status were from ATCC data sheets/website, the Cancer Cell Line Encyclopedia or the indicated references;

²As per the source/supplier: Horizon Discovery, Cambridge, United Kingdom.

Table 3 Summary results of proteins investigated

Cell lines	p53 status	AXL	E-Cadherin
Positive control
HeLa	HPV inactivated	Highest	Negative
Colon
HCT116	Wild-Type	Negative	High
RKO	Wild-Type	High	Negative
HCT116.p53	Mutant	High	Lowest
RKO.p53-/-	Mutant	Highest	Negative
SW480	Mutant	Highest	High
Breast
MCF7	Wild-Type	Negative	Low
MDA-MB-361	Wild-Type	Negative	Highest
ZR-75-1	Wild-Type	Negative	Highest
CAL-51	Wild-Type	Negative	Low
1001	Mutant	Highest	Negative
MDA-MB-231	Mutant	High	Negative
T47D	Mutant	Negative	Highest
BT-549	Mutant	Lowest	Negative

Table 4 AXL mRNA expression from our microarray of colon cancer cell lines[21]

	RKO	HCA7	KM12	LoVo	DLD1	HCT116	SW48	LIM 1215
P53 mutation	WT	mut	mut	WT	WT	WT	WT	WT
AXL expression1	+	-	-	-	-	-	-	-

¹Based on Affymetrix probe 202685_s_at considering a cut off value of 70 to differentiate negative from positive.