Peripheral blood and tissue assessment highlights differential tumor-circulatory gradients of IL2 and MIF with prognostic significance in resectable pancreatic ductal adenocarcinoma

Figures & data

Table

REAGENT	SOURCE	IDENTIFIER
Antibodies
Rabbit monoclonal Anti-CD3	Abcam	Cat# ab16669 RRID: AB_443425
Mouse monoclonal Anti-CD4	Leica	Cat# CD4-368-L-CE-H RRID: AB_563559
Mouse monoclonal Anti-CD8	Leica	Cat# CD8-4B11-L-CE RRID: AB_442068
Mouse monoclonal Anti-CD20	Leica	Cat# CD20-L26-L-CE RRID: AB_442055
Rabbit monoclonal Anti-CD163	Abcam	Cat# ab182422 RRID: AB_2753196
Mouse monoclonal Anti-NKp46	R&D Systems	Cat# MAB1850-500 RRID: AB_2149153
Mouse monoclonal Anti-FoxP3	Thermo Fisher Scientific	Cat# 14–4777 RRID: AB_467555
Assays
Bio-Plex Cell Lysis Kit	BioRad	Cat# 171304011
Pierce BCA Protein Assay	Thermo Fisher Scientific	Cat# 23227
Bio-Plex Pro Human Cytokine Screening Panel	BioRad	Cat# 12007283
Bio-Plex Pro Human Cytokine ICAM-1	BioRad	Cat# 171B6009M
Bio-Plex Pro Human Cytokine VCAM-1	BioRad	Cat# 171B6022M

Table 1. Patient characteristics

		Subgroup				Total (n = 48)
		Serum IL2		Serum MIF
Characteristic		High (n = 24)	Low (n = 24)	High (n = 24)	Low (n = 24)
Male (n)		12	13	13	12	25
Female (n)		12	11	11	12	23
Age (years)		64.0 ± 2.0	68.7 ± 1.9	68.4 ± 1.6	64.3 ± 2.3	66.3 ± 1.4
Body weight (kg)		73.2 ± 2.4	69.7 ± 2.5	75.3 ± 2.4	68.3 ± 2.3	71.44 ± 1.7
BMI		25.0 ± 0.9	24.0 ± 0.8	25.4 ± 0.5	23.7 ± 0.9	24.5 ± 0.6
Diabetes (n)		8	7	7	8	15
Current glucocorticoids or immunosuppressive drugs (n)		1	0	1	0	1
Cardiovascular comorbidities (n)		14	13	14	13	27
Pulmonary comorbidities (n)		1	4	2	3	5
Renal comorbidities (n)		2	1	2	1	3
Hepatic comorbidities (n)		0	0	0	0	0
Autoimmune comorbidities (n)		1	1	1	1	2
T	T3 (n)	24	23	24	23	47
	T4 (n)	0	1	0	1	1
N	N0 (n)	3	5	3	5	8
	N1 (n)	21	19	21	19	40
M	M0 (n)	22	24	23	23	46
	M1 (n)	2	0	1	1	2
Grade	G2 (n)	13	18	13	18	31
	G3 (n)	11	6	11	6	17
R^a	R0 (n)	1	5	2	4	6
	R1 (n)	22	19	22	19	41
Overall survival (days)		440 ± 72	765 ± 119	471 ± 89	735 ± 111	602 ± 73

Data are shown as mean ± SEM.

BMI = body mass index (calculated as weight in kilograms divided by height in meters squared)

T = stage of primary tumor, N = regional lymph node status,

M = distant metastasis status, R = resection margin status.

^aInformation available on resection margin status from 47 patients.

Figure 1. IL2, MIF and other cytokines are potential diagnostic markers for patients with PDA

(A) Heatmap comparing serum cytokine levels of PDA patients (n = 48) and healthy individuals (n = 30).(B) Scatter dot plot comparing serum cytokine concentration (left: IL2, right: MIF) of PDA patients (n = 48) and healthy individuals (n = 30).*p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0001.IL2 = Interleukin 2MIF = Macrophage migration inhibitory factorPDA = Pancreatic ductal adenocarcinoma

Figure 2. Circulatory IL2 and MIF are associated with poor overall survival

(A) Forest plot of serum cytokine levels and overall survival of PDA patients (n = 48).(B) Kaplan-Meier survival plots of PDA patients with high versus low serum concentration of IL2 (n = 24/n = 24). The median cutpoints were determined to stratify patients into high and low.(C) Kaplan-Meier survival plots of PDA patients with high versus low serum concentration of MIF (n = 24/n = 24). The median cutpoints were determined to stratify patients into high and low.*p ≤ 0.05IL2 = Interleukin 2MIF = Macrophage migration inhibitory factorPDA = Pancreatic ductal adenocarcinoma

Figure 3. Tumor-to-Serum gradient of IL2 and MIF and their relationship to the tumoral immune landscape

(A) Comparison of serum and corresponding tumoral cytokine concentrations (left: IL2, right: MIF) of PDA patients with stratification in low and high serum concentration groups (n = 24/n = 24) (as indicated). The median cutpoints were determined to stratify patients into high and low.(B) Scatter dot plot comparing the tumoral expression of cytokines (left: IL2, right: MIF) of PDA patients with stratification in low and high serum concentration groups (n = 24/n = 24) (as indicated). The median cutpoints were determined to stratify patients into high and low.(C) Scatter dot plot comparing intratumoral quantities of immune cells (as indicated) in PDA patients with stratification in low and high serum cytokine concentration groups (as indicated). The median cutpoints were determined to stratify patients into high and low.*p ≤ 0.05, ***p ≤ 0.0001.IL2 = Interleukin 2MIF = Macrophage migration inhibitory factorPDA = Pancreatic ductal adenocarcinoma

Figure 4. Increased IL2 levels are not associated with systemic inflammation markers

(A) Scatter dot plot comparing serum CRP levels of PDA patients with stratification in low and high serum IL2 groups (n = 24/n = 24). The median cutpoints were determined to stratify patients into high and low.(B) Scatter dot plot comparing serum WBC levels of PDA patients with stratification in low and high serum IL2 groups (n = 24/n = 24). The median cutpoints were determined to stratify patients into high and low.IL2 = Interleukin 2CRP = C-reactive proteinPDA = Pancreatic ductal adenocarcinomaWBC = White blood cell

DATA AVAILABILITY STATEMENT

All data are available upon request from the authors. All data relevant to the study are included in the article.