The predictive value and role of stromal tumor-infiltrating lymphocytes in pancreatic ductal adenocarcinoma (PDAC)

Figures & data

Figure 1. (A) Stromal TIL negative group (score 0): almost no lymphocytic infiltration into the stroma of surrounding cancer cell nests; (B) Stromal TIL negative group (score 1): low lymphocytic infiltration into the stroma of surrounding cancer cell nests; (C) Stromal TIL positive group (score 2): moderate lymphocytic infiltration into the stroma without tumor cell nest permeation and (D) Stromal TIL positive group (score 3): intense lymphocytic infiltration into the stroma and between tumor cells. (E) and (F): Representative figure of stromal TILs at modetate scores in the same tissue section verified by HE and the expression of CD45 by immunohistochemistry, respectively. (G) and (H): Representative figure of stromal TILs at low scores based on HE stainings in the same tissue section verified by the expression of CD45 by immunohistochemistry. [100 × magnification, H & E-stained sections or IHC sections. (A-E, G); 100 × magnification, IHC section (F, H)]. Abbreviations: TIL = tumor-infiltrating lymphocyte.

Table 1. Comparison of quantitative clinical factors between according to stromal tumor-infiltrating lymphocytes negative group and positive group.

	TILs-Negative		TILs- Positive
Factors	Mean	Standard deviation	Mean	Standard deviation	P value
Age(years)	63.16	10.94	62.93	9.48	0.41
Albumin(g/dL)	40.12	5.41	41.62	5.91	0.73
CEA(ng/mL)	6.05	8.56	3.29	3.78	0.01
AFP(U/mL)	3.08	2.25	2.93	1.38	0.50
CA125(U/mL)	24.89	31.13	16.38	16.00	0.12
CA19-9(U/mL)	468.25	766.40	8322.14	56524.15	0.01
Platelet (× 10^3/mL)	203.42	66.14	215.71	69.26	0.43
Lymphocyte (× 10^3/mL)	1.38	0.56	1.50	0.53	0.54
Neutrophil (× 10^3/mL)	3.98	1.75	3.70	1.17	0.08
Diameter(cm)	4.11	2.38	3.49	1.25	0.09

Abbreviations: TILs = tumor-infiltrating lymphocytes

Table 2. Univariate analysis of clinical characteristics according to stromal tumor-infiltrating lymphocytes.

		TILs-Negative		TILs- Positive
Characteristics		(N = 100)	(%)	(N = 55)	(%)	P value
Gender	Male	60	60	38	69	0.26
	Female	40	40	17	31
Age	<65	54	54	31	56	0.78
	≥65	46	46	24	44
Albumin(g/dL)	<3.5	12	12	6	11	0.84
	≥3.5	88	88	49	89
CEA(ng/mL)	<5	67	67	47	85	0.01
	≥5	33	33	8	15
CA125(U/mL)	<35	77	77	47	85	0.12
	≥35	2	2	7	13
CA19-9(U/mL)	<39	16	16	16	29	0.05
	≥39	84	84	39	71
ABO	A	32	32	23	42	0.56
	AB	14	14	5	9
	B	31	31	17	31
	O	23	23	10	18
Location	Uncinate process	27	27	21	38	0.35
	Head	48	48	23	42
	Body and tail	25	25	11	20
Differentiation	Poor	50	50	25	45	0.59
	Well to moderate	50	50	30	55
Diameter(cm)	<3.5	38	38	30	55	0.05
	≥3.5	62	62	25	45
T staging	1-2	28	28	26	47	0.02
	3-4	72	72	29	53
LN metastasis	Negative	43	43	26	47	0.61
	Positive	57	57	29	53
AJCC	<IIb	41	41	26	47	0.45
	≥IIb	59	59	29	53

Abbreviations: TILs = tumor-infiltrating lymphocytes; LN = lymph node; AJCC = the pathological stage of PDAC was determined according to the American Joint Committee on Cancer (AJCC) 7th Edi.

Table 3. Univariate and multivariate analysis of clinicopathologic variables in relation to liver metastasis and overall survival after curative operation.

			Liver Metastasis			Overall Survival
			Univariate analysis	Multivariate analysis		Univariateanalysis	Multivariate analysis
Parameter		N	P value	HR(95% CI)	P value	P value	HR(95% CI)	P value
Gender	Male	98	0.92			0.59
	Female	57
Age(years)	<65	85	0.43			0.11
	≥65	70
Albumin(g/dL)	<3.5	18	0.39			0.89
	≥3.5	137
CEA(ng/mL)	<5	114	0.12			0.00	1	0.04
	≥5	41					1.63(1.04-2.59)
CA125(U/mL)	<35	124	0.91			0.05	1	0.08
	≥35	31					1.619(0.95-2.74)
CA19-9(U/mL)	<39	32	0.11			0.01	1	0.56
	≥39	123					1.15(0.71-1.87)
ABO	A	55	0.13			0.94
	AB	19
	B	48
	O	33
Location	Uncinate process	48	0.80			0.43
	Head	71
	Body and tail	36
Differentiation	Poor	75	0.20			0.16
	Well to moderate	80
Diameter(cm)	<3.5	68	0.20			0.01	1	0.04
	≥3.5	87					1.50 (1.02-2.22)
T staging	1-2	54	0.01	1	0.03	<0.01	1	0.04
	3-4	101		1.67(1.06-2.62)			1.55(1.03-2.33)
LN metastasis	Negative	69	0.15			<0.01	1	<0.01
	Positive	86					1.95(1.32-2.87)
AJCC	<IIb	67	0.08	1	0.33	<0.01	1	0.88
	≥IIb	88		1.234(0.81-1.89)			1.12(0.26-4.74)
Stromal TILs	Positive	100	<0.01	1	<0.01	<0.01	1	<0.01
	Negative	55		2.80(1.75-4.48)			2.70(1.80-4.06)

Abbreviations: TILs = tumor-infiltrating lymphocytes; LN = lymph node; HR = hazard ratio; CI = confidence interval.

Figure 2. Plot of the overall survival for all patients stratified based upon TIL groups. Abbreviations: TIL = tumor-infiltrating lymphocyte.

Figure 3. Plot of the liver metastasis-free survival for all patients stratified according to TIL groups. Abbreviations: TIL = tumor-infiltrating lymphocyte.

Figure 4. Comparison of the monoculture, indirect co-culture, and direct co-culture of the three cell lines (Panc-1, MiaPaCa-2 and AsPC-1) at 3 days. The co-culture (both indirect co-culture, and direct co-culture) with lymphocytes markedly limited the proliferation of pancreatic cancer cell lines, and many cancer cells showed evidence of apoptosis, such effect are much more evident in indirect co-culture. The quantitative results of 6 parallel groups are shown in a histogram, cell counting of both the direct co-culture and the indirect co-culture are significantly lower than monoculture in all three pancreatic cancer cell lines. (* P < 0.05, **P < 0.01).

Figure 5. CCK8 assay to test the function of lymphocytes on pancreatic cancer cells (Panc-1, MiaPaCa-2 and AsPC-1). The co-culture with lymphocytes inhibited pancreatic cancer cell growth. (monoculture: pancreatic cancer cell culture alone, co-culture: pancreatic cancer cells culture with lymphocytes. * P < 0.05).

Figure 6. ELISA experiments to test the possible cytokines secreted from lymphocytes on pancreatic cancer cells (Panc-1, MiaPaCa-2 and AsPC-1). The co-culture with lymphocytes inhibited pancreatic cancer cell growth. Cocultures of lymphocytes and cancer cells at a 4:1 ratio increased the secretion of IL-8 and TNF-alpha in the culture media compared with at a ratio of 1:1, and much higher than lymphocytes or cancer cells alone(A and C). IL-6, IL-1and IL-12 are also secreted significantly, but there were no significant additive effects in the coculture groups (E, H and I). There was no significant change in the TGF-beta, EGF, MMP9 and GM-CSF concentration among the experimental groups (B, D, F and G). (P: Panc-1; M: MiaPaCa-2; A: AsPC-1: D: direct coculture; I: indirect coculture).

Figure 7. Comparison of the TNF-alpha positive and TNF-alpha negative of the three cell lines (Panc-1, MiaPaCa-2 and AsPC-1) at 3 days. TNF-alpha significantly limited the proliferation of pancreatic cancer cell lines (* P < 0.05, **P < 0.01).