Characteristics of circulating tumor cells in organ metastases, prognosis, and T lymphocyte mediated immune response

Figures & data

Figure 1 Organ metastases.

Figure 2 (A) ECTCs possessing only epithelial molecular markers (EpCAM and cytokeratin 8/18/19) were stained with red immunofluorescence. (B) EMCTCs having both epithelial and mesenchymal molecular markers were stained with both green and red immunofluorescent dyes. (C) MCTCs occupying only mesenchymal molecular markers (vimentin and Twist) were stained with green immunofluorescence. (Bars =2.5 µm).

Abbreviations: ECTCs, epithelial circulating tumor cells; EMCTCs, epithelial–mesenchymal circulating tumor cells; EpCAM, epithelial cell adhesion molecule; MCTCs, mesenchymal circulating tumor cells.

Figure 3 Twenty-seven out of 31 patients were detected with CTCs. The levels of EMCTCs predominated within the three CTC subpopulations among 16 patients and the levels of EMCTCs were equivalent to ECTCs count or MCTCs count among three patients.

Abbreviations: CTCs, circulating tumor cells; ECTCs, epithelial circulating tumor cells; EMCTCs, epithelial–mesenchymal circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; NSCLC, non-small cell lung cancer.

Table 1 CTCs and the general characteristics

Characteristics	Number	TCTCs	ECTCs	EMCTCs	MCTCs
Age (years)
<65	21	5 (1.5, 10)	0 (0, 1)*	3 (0.5, 8)	0 (0, 1)
≥65	10	7 (3.5, 15)	2 (0, 4.5)*	4 (0, 8)	0.5 (0, 3.25)
Gender
Female	17	5 (2.5, 7.5)	0 (0, 4.5)	3 (0, 6)	0 (0, 2.5)
Male	14	7.5 (1.5, 12.5)	1 (0, 1.25)	5 (0.75, 8.25)	0 (0, 1.25)
Smoking
Yes	11	4 (0, 12)	0 (0, 1)	4 (0, 8)	0 (0, 0)
No	20	6.5 (3, 9.5)	1 (0, 3.5)	3.5 (0.25, 6.0)	0 (0, 2.75)
Family history of cancer
Yes	3	12 (2, 19)	1 (1, 2)	9 (1, 18)	0 (0, 1)
No	28	5.5 (2, 8)	0 (0, 2)	3.5 (0, 6)	0 (0, 2)
Untreated
Yes	23	4 (1, 8)*	1 (0, 2)	2 (0, 4)*	0 (0, 1)
No	8	9 (6.25, 17.75)*	0.5 (0, 1)	7 (6, 10.25)*	0 (0, 2.75)

Notes:

* P<0.05; statistical description: median (figures outside the parentheses) and interquartile range (figures in the parentheses).

Abbreviations: CTCs, circulating tumor cells; ECTCs, epithelial circulating tumor cells; EMCTCs, epithelial–mesenchymal circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; TCTCs, total CTCs.

Table 2 CTCs and disease-related characteristics

Characteristics	Number	TCTCs	ECTCs	EMCTCs	MCTCs
Pathologic type
Adenocarcinoma	26	5.5 (1.75, 12.25)	0.5 (0, 2)	4 (0, 8.25)	0 (0, 1.25)
Adenosquamous	3	8 (2, 10)	1 (0, 1)	6 (1, 8)	0 (0, 3)
Location
Right lung	19	7 (1, 8)	0 (0, 1)	2 (0, 8)	0 (0, 1)
Left lung	12	5.5 (3.25, 13)	1 (0, 3.5)	4.5 (0.75, 9.75)	0 (0, 2.75)
Tumor stage
III	5	1 (0, 7)	0 (0, 0)*	1 (0, 7)	0 (0, 0)
IV	24	7 (3, 12.75)	1 (0, 2)*	4 (1, 8.75)	0 (0, 2)
T
1	2	4 (1, 7)	3 (1, 5)	1 (0, 2)	0 (0, 0)
2	2	6.5 (3, 10)	0.5 (0, 1)	4 (2, 6)	2 (1, 3)
3	5	2 (0, 27)	0 (0, 16)	1 (0, 10)	0 (0, 1.5)
4	20	7.5 (3.25, 12.75)	0.5 (0, 2)	4.5 (1, 8.75)	0 (0, 1.75)
M
M0	5	1 (0, 7)	0 (0, 0)*	1 (0, 7)	0 (0, 0)
M1a	2	6.5 (0, 13)	0 (0, 0)*	5.5 (0, 11)	1 (0, 2)
M1b	/	/	/	/	/
M1c	22	7 (3, 12.5)	1 (0, 2.5)*	4 (1, 8.25)	0 (0, 2.25)
N
N0	2	3.5 (0, 7)	1 (0, 2)*	2 (0, 4)	0.5 (0, 1)
N1	0	/	/	/	/
N2	10	3.5 (0, 8.5)	0 (0, 0.25)*	3 (0, 6.5)	0 (0, 3.25)
N3	17	7 (3.5, 13.5)	1 (0, 4.5)*	6 (1, 11)	0 (1, 1.5)

Notes:

* P<0.05; statistical description: median (figures outside the parentheses) and interquartile range (figures in the parentheses).

Abbreviations: CTCs, circulating tumor cells; ECTCs, epithelial circulating tumor cells; EMCTCs, epithelial–mesenchymal circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; TCTCs, total CTCs.

Table 3 CTCs and organ metastases

Characteristics	Number	TCTCs	ECTCs	EMCTCs	MCTCs
Medium or large pleural effusion
Yes	7	8 (2, 30)	1 (0, 6)	4 (1, 14)	3 (0, 4)*
No	23	5 (1, 8)	0 (0, 2)	4 (0, 8)	0 (0, 0)*
Intra-lung metastases
Yes	24	5.5 (2, 12.75)	1 (0, 2)	4.5 (0.25, 8.75)	0 (0, 1)
No	5	3 (0.5, 8.5)	1 (0, 3)	2 (0, 4)	0 (0, 2)
Homolateral lung metastases
Yes	23	6 (2, 13)	1 (0, 2)	5 (1, 9)	0 (0, 1)
No	6	2.5 (0.75, 7.75)	1 (0, 2.75)	1 (0, 3)	0 (0, 1.5)
Contralateral lung metastases
Yes	15	7 (3, 14)	1 (0, 2)	6 (0, 11)	0 (0, 2)
No	14	3.5 (1, 7.25)	0 (0, 1)	1.5 (0, 6)	0 (0, 0.25)
Extra-lung metastases
Yes	22	7 (3, 12.5)	1 (0, 2.5)*	4 (1, 8.25)	0 (0, 2.25)
No	7	1 (0, 8)	0 (0, 0)*	1 (0, 8)	0 (0, 0)
Bone metastases
Yes	20	7 (3.25, 13.5)	1 (0, 3.5)*	4 (1.25, 8.75)	0 (0, 2.75)
No	9	1 (0, 7.5)	0 (0, 0.5)*	1 (0, 7)	0 (0, 0)
Cranium metastases
Yes	4	5 (2, 24.5)	1 (0.25, 4.75)	2.5 (1, 13)	2 (0, 7)
No	25	6 (2, 11)	0 (0, 2)	4 (0, 8)	0 (0, 1)
Truncal bone (except vertebra) metastases
Yes	10	5 (2.75, 30.5)	1 (0, 5.25)	3.5 (0.75, 9.5)	0.5 (0, 4.25)
No	19	7 (1, 8)	0 (0, 1)	4 (0, 8)	0 (0, 1)
Vertebra metastases
Yes	15	7 (3, 14)	1 (0, 2)*	4 (1, 9)	0 (0, 2)
No	14	5 (0.75, 8)	0 (0, 1)*	3.5 (0, 6.5)	0 (0, 0.5)
Extremities metastases
Yes	6	3.5 (2.75, 11.25)	0.5 (0, 4.5)	2.5 (0.75, 7.75)	0 (0, 2.75)
No	23	7 (1, 12)	1 (0, 2)	4 (0, 8)	0 (0, 2)
Abdominal metastases
Yes	7	5 (2, 32)	1 (0, 6)	5 (1, 14)	0 (0, 8)
No	22	6.5 (1.75, 8.5)	0.5 (0, 2)	3.5 (0, 6.5)	0 (0, 1)
Kidney metastases
Yes	3	5 (1, 30)	1 (0, 6)	5 (0, 16)	0 (0, 8)
No	26	6.5 (2, 10.5)	0.5 (0, 2)	4 (0.75, 8)	0 (0, 1.25)
Adrenal gland metastases
Yes	4	18 (2.5, 44)	0.5 (0, 23.5)	6 (1.75, 12.5)	1.5 (0, 18.75)
No	25	6 (1.5, 9)	1 (0, 2)	4 (0, 7)	0 (0, 1)
Liver metastases
Yes	1	2 (2, 2)	1 (1, 1)	1 (1, 1)	0 (0, 0)
No	28	6.5 (2.25, 11.5)	0.5 (0, 2)	4 (0.25, 8)	0 (0, 1.75)
Brain metastases
Yes	10	9 (6.5, 15.25)	1 (0.75, 2.75)	4 (3.5, 11.75)	0.5 (0, 3)
No	19	4 (1, 8)	0 (0, 2)	2 (0, 6)	0 (0, 1)
Number of organ metastases
<3	17	5 (0.5, 7.5)*	0 (0, 1.5)	3 (0, 6)*	0 (0, 0)*
≥3	12	11 (3.25, 27.25)*	1 (0.25, 3.5)	7 (1.25, 13.25)*	1 (0, 3)*

Notes:

* P<0.05; statistical description: median (figures outside the parentheses) and interquartile range (figures in the parentheses).

Abbreviations: CTCs, circulating tumor cells; ECTCs, epithelial circulating tumor cells; EMCTCs, epithelial–mesenchymal circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; TCTCs, total CTCs.

Figure 4 (A) Kaplan–Meier life-table analysis showed EMCTCs were associated with worsened clinical outcomes. (B) Kaplan–Meier life-table analysis showed MCTCs had negative prognostic value.

Abbreviations: EMCTCs, epithelial–mesenchymal circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; OS, overall survival.

Table 4 Cox regression of overall survival

	Univariate Cox regression		P-value	Multivariate Cox regression		P-value
	HR	95% CI		HR	95% CI
Gender (F=1)	1.025	(0.312, 3.362)	0.968	–	–	–
Age (≥65=1)	3.929	(1.190, 12.971)	0.027*	–	–	–
Smoking	0.987	(0.289, 3.373)	0.983	–	–	–
Tumor history	0.891	(0.114, 6.966)	0.912	–	–	–
Pathologic type	0.357	(0.074, 1.723)	0.200	–	–	–
Lump location	0.708	(0.355, 1.411)	0.326	–	–	–
Number of organ metastases	1.672	(1.059, 2.641)	0.027*	–	–	–
T stage	1.141	(0.517, 2.518)	0.744	–	–	–
N stage	1.179	(0.496, 4.808)	0.709	–	–	–
M stage	3.686	(0.377, 36.058)	0.262	–	–	–
TCTCs	2.133	(1.253, 3.631)	0.005*	–	–	–
[0, 6]
[7, 13]
[14, 20]
[21, +∞]
ECTCs	1.314	(0.636, 2.717)	0.461	–	–	–
[0, 3]
[4, 7]
[8, 11]
[12, +∞)
EMCTCs	1.760	(1.012, 3.060)	0.045*	2.204	(1.099, 4.420)	0.026*
[0, 3]
[4, 7]
[8, 11]
[12, +∞)
MCTCs	2.473	(1.394, 4.387)	0.002*	2.308	(1.166, 4.569)	0.016*
[0, 3]
[4, 7]
[8, 11]
[12, +∞)
Mutation + target therapy	0.172	(0.022, 1.342)	0.093	–	–	–

Note:

* P<0.05.

Abbreviations: CI, confidence index; CTCs, circulating tumor cells; ECTCs, epithelial circulating tumor cells; EMCTCs, epithelial–mesenchymal circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; TCTCs, total CTCs.

Table 5 Correlations of T-cell–related CD and CTCs

	r	P-value
CD3^+
TCTC	−0.321	0.264
ECTC	−0.097	0.749
EMCTC	−0.659	0.01*
MCTC	−0.256	0.376
CD4^+
TCTC	−0.107	0.716
ECTC	−0.057	0.846
EMCTC	−0.24	0.409
MCTC	−0.019	0.948
CD8^+
TCTC	−0.394	0.163
ECTC	−0.216	0.458
EMCTC	−0.713	0.04*
MCTC	−0.262	0.366

Note:

* P<0.05.

Abbreviations: CD, cluster of differentiation; CTCs, circulating tumor cells; ECTCs, epithelial circulating tumor cells; EMCTCs, epithelial–mesenchymal circulating tumor cells; MCTCs, mesenchymal circulating tumor cells; TCTCs, total CTCs.