Synergistic Immunomodulatory Effect of Synbiotics Pre and Postoperative Resection of Pancreatic Ductal Adenocarcinoma
Phase: PHASE4
Conditions: Pancreatic Ductal Adenocarcinoma
Sponsor: Theodor Bilharz Research Institute

Introduction 
Pancreatic cancer (PC) is a highly aggressive malignant tumor that ranked as the 
fourth leading cause of cancer-related deaths in the Europe and United States (Hu 
et al., 2021). Considering all stages combined, PC has the poorest survival rates 
among all types of cancer, with only an 11% survival rate. It is projected to become 
the second leading cause of death by the year 2030 (Siegal et al., 2022). In Egypt, 
pancreatic cancer caused 2,906 deaths in 2020, accounting for 0.54% of total deaths, 
and with a mortality rate that varies by province, with northern districts having 
approximately 2.85 times higher rates than southern districts. This variation may be 
attributed to exposure to different environmental factors, as the northern regions 
have the highest rates of soil and water pollution in the country (Baum et al., 2020). 
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic 
cancer, accounting for nearly all cases of pancreatic malignancies (Sarantis et al., 
2020). PDAC is defined as a tumor microenvironment that is highly stromal and 
exhibits weak immunogenicity. This microenvironment facilitates tumor evolution 
and plays a role in the development of resistance to therapy (Mizrahi et al., 2020).  
Surgical resection is the most commonly used strategy for PDAC treatment followed 
by adjuvant therapy but with long-term limited effectiveness (Wood et al., 2022).  
Hence, it is crucial to investigate novel therapeutic approaches and supportive 
treatments that can improve the quality of patients' lives (Kim et al., 2021). Recently, 
there has been a growing focus on exploring the association between pancreatic 
cancer and different aspects of the gut microbiota (Pushalkar et al., 2020; 
Sammallahti et al., 2021). Consequently, the manipulation of the gut microbiota 
and the restoration of its diversity and balance might have a substantial impact on 
the management of this disease (Mendez et al., 2021).  
 

According to the consensus statement by the International Scientific Association for 
Probiotics and Prebiotics (ISAPP), probiotics are defined as living microorganisms 
that confer health benefits when consumed in adequate quantities (Varela-Trinidad 
et al., 2022). Within the field of cancer research, probiotics have demonstrated the 
ability to bolster the immune response by attracting diverse immune cells, regulating 
inflammation, enhancing the integrity of the gut barrier, and exerting direct 
antitumor effects. These effects are achieved through mechanisms such as the 
production of antimicrobial peptides and the inhibition of cancer cell proliferation 
(Śliżewska et al., 2020; Azad et al., 2018). The majority of research investigating 
the potential benefits of probiotic consumption on pancreatic cancer has been 
conducted using animal models. However, further investigation is necessary to fully 
comprehend its inhibitory role in the progression of pancreatic cancer. This requires 
additional exploration through preclinical and clinical studies (Chen et al., 2020).  
     Prebiotics are a substrate that is selectively utilized by host microorganisms, 
conferring a health benefit, such as mannan oligosaccharides, conjugated linoleic 
acids, polyunsaturated fatty acids, oligosaccharides such as fructooligosaccharides, 
inulin, galactooligosaccharides (Gibson et al., 2017). Prebiotics have also been 
shown to play a beneficial role in reducing the risk of inflammation and exhibiting 
antitumor effects (Mahdavi et al., 2021).  On the other hand, Synbiotics encompass 
the combination of probiotics and prebiotics, providing a potential synergistic effect 
in modulating the gut microbiota and promoting overall health (Markowiak and 
Śliżewska, 2017). In the context of cancer therapy, synbiotics may exert their effects 
through 
mechanisms 
such 
as 
immunomodulation, 
augmentation 
of 
chemotherapeutic effectiveness, and mitigation of treatment-related side effects 
(Singh et al., 2023). 
 

Recent studies and a meta-analysis have suggested that the inclusion of 
probiotic/synbiotic supplements is associated with a significant reduction in the risk 
of postoperative infectious complications in individuals who have undergone 
surgery (Veziant et al., 2022).  However, further research is needed to validate these 
findings, as there may be publication bias and limitations in the quality of evidence 
that could influence the results (Chen et al., 2022). 
The objective of this study was to assess the synergistic effect of synbiotics 
(combination of probiotics and prebiotics) compared to probiotics alone in providing 
a significant anti-tumor immunomodulation effect in PDAC patients, as well as their 
impact on postoperative complications and outcomes. 
 
Methods 
Ethical approval  
The protocol of this study was approved by the Institutional Review Board (IRB) of 
Theodor Bilharz Research Institute (PT.652, 2021).  The human subjects study were 
enrolled according to REC-TBRI’s ethical standards and the 1964 Helsinki 
Declaration. Written informed consent forms were obtained from all participants.   
 
 
Study design and patients 
This single-blind randomized control study was conducted on patients with PDAC 
who underwent tumor resection at the General Surgery Department of Theodor 
Bilharz Research Institute Hospital from 2021 to 2023.  The data of all patients, 
including laboratory and pathological data, were collected from the electronic 

medical records of the TBRI hospital. All cases underwent Ct pancreatic protocol 
with EUS (endoscopic ultrasound) to exclude local vascular invasion before surgery. 
The inclusion criteria included patients with primary PDAC, with complete 
pathological and follow-up data, without long-distance metastasis, without chronic 
diseases, and without any treatments before the surgery. The exclusion criteria 
included the patients who suffered from other tumors or other chronic diseases or 
died from accidental death or other diseases, lack of pathological and follow-up data, 
and long-distance metastasis before the surgery. Once the informed consent was 
obtained, the included patients were randomized, using randomization software, into 
placebo, probiotic, and synbiotic receiving groups. All included patients were 
blinded for the intervention.  
 
Probiotic and synbiotics treatment program  
The probiotic and prebiotic medications utilized in this investigation are 
commercially available FDA-approved forms and have a well-established safety 
record. Throughout the administration process, both the patient and the nursing staff 
were obligated to report any possible side effects or unfavorable incidents. The study 
was halted if a patient withdrew their consent or if any significant adverse events 
related to the medication administration occurred. The probiotic agent 25 Billion 
CFU (Nowfoods, USA), composed of ten strains of bacteria (Lactobacillus 
acidophilus, Bifidobacterium lactis, Lactobacillus planta Lactobacillus paracasei, 
Bifidobacterium breve, Streotococcus thermophiles, Lactobacillus salivarius, and 
Bifidobacterium longum), was taken in a dosage of two capsules once daily, two 
weeks (oral route) before the surgery and continued postoperatively for one month 
(first day post-operative by diluting the capsules in 50ml sterile water and given 
through feeding jejunostomy tube).  For the synbiotic group, the same probiotic drug 

was used in addition to taking Inulin 1000mg  (Herbamama USA) two capsules once 
daily. For the placebo group, the same regimen was applied using a placebo drug.  
 
Blood samples collection  
For all studied groups, blood samples were collected at the baseline, 14 days pre-
operative (Pre-14d), on the operation day (OP-0d), and 14 and 30 days post-
operative (PO-14d and PO-30 respectively). Blood was collected into BD 
Vacutainer tubes and allowed to clot for 30 minutes. Afterward, it was centrifuged 
for 15 minutes at 1300 rpm. The separated serum was then stored in a -80°C freezer 
until analysis.  
 
Histopathological preparation and technique 
Following tumor resection, received pancreatic samples were submitted to the 
Pathology Department, TBRI for gross and microscopic examination. Pancreatic 
tissue was fixed for 24 hours in 10% neutral buffered formalin solution, then 
dehydrated in ascending grades of ethyl alcohol (70%- 90%- 100%), then processed 
in xylene and paraffinized in wax for preparation of formalin-fixed paraffin-
embedded blocks. Sections of 4 um thick were cut and spread on positively charged 
glass slides for better adherence, then stained by Hematoxylin and Eosin (H&E) for 
evaluation of histopathological changes.  
 
Immunohistochemical (IHC) technique  
One paraffin-embedded block was selected from each case and was cut into 4 μm 
sections. unmasking for antigens was performed with 10 mM sodium citrate buffer, 
pH 6.0, at 90°C for 30 minutes, then incubated in 0.03% hydrogen peroxide for 10 
minutes at room temperature to remove endogenous peroxidase activity, followed 

by adding the blocking serum (0.04% BSA, A2153, Sigma-Aldrich, Shanghai, 
China, and 0.5% normal goat serum X0907, Dako Corporation, Carpinteria, CA, 
USA) for 30 minutes at room temperature. The sections were incubated with an anti-
CD8 polyclonal antibody (YPA2235, Biospes, Chongqing, China), and anti- IFNγ 
polyclonal antibody (YPA2285, Biospes, Chongqing, China), at dilution of 1:100 
for overnight at 4°C. Slides were incubated with ImmunoDetector DAB HBR 
detection system (BioSB, Santa Barbra, USA). Finally, staining was developed with 
3,3’- diaminobenzidine (DAB) solution, and sections were counterstained with 
hematoxylin, dehydrated with graded ethanols, and mounted. Negative controls were 
carried out in which PBS was used instead of the primary antibody 
 
Immunohistochemical interpretation and scoring  
Scoring of CD8 and IFNγ immunostaining was performed blindly to the patients’ 
clinicopathological data. CD8+T cells and IFNγ staining was interpreted as positive 
when >10% of cells showed distinct nuclear staining. The mean percentage of 
positive cells was calculated in 10 randomly selected fields at high magnification 
(x400). Interpretation of stained slides was done using a light microscope (Scope 
A1, Axio, Zeiss, Germany). Photomicrographs were taken using a microscope 
camera (AxioCam, MRc5, Zeiss, Germany). 
 
Enzyme-linked immunosorbent assay (ELISA)  
Serum concentrations of IL-10, IL-6, and IL-1B were determined using sandwich-
based enzyme-linked immunosorbent assay (ELISA) kits, following the 
manufacturer's instructions (Sunlong Biotech, China). Briefly, 50 μL of diluted 
samples or standards were incubated in a micro ELISA strip plate that was pre-
coated with antibodies specific to the tested cytokines. Horse radish peroxidase 
(HRP)-Conjugate was employed as a detection antibody. After incubation for 30 

minutes at 37°C and three subsequent washes, TMP substrate was used to visualize 
the HRP-enzymatic reaction. The absorbance of the produced color was measured 
at 450 nm using an ELISA reader (Multiscan TMFC, Thermofisher, USA), and the 
cytokine concentration was then calculated from the standard curve.  
 
Statistical analysis  
Data were analyzed using statistical software package (IBM-SPSS) version 23 
software. Kolmogorov–Smirnov test showed that the raw data were normally 
distributed.  One ANOVA was applied to study the effect of treatment on the studied 
parameters. Two-way ANOVA was applied to study the effect of time and treatment 
on the studied parameters. The least significant difference (LSD) test was used to 
illustrate the statistical differences among the experimental groups. Duncan's test 
was used to illustrate the homogeneity among the different intervals. Data is 
displayed as mean ± standard error of the mean. 
 
 
Results 
Demographic characteristics of the studied patients  
Between December 2021 and April 2023, and following the assessment of 115 
primary eligible patients, 90 patients were randomly allocated into probiotics, 
synbiotics, and placebo groups (Fig. 1). Some patients were primarily excluded 
(n=10) because they denied consent (n=4), participating in another study (n=3) or 
were diagnosed by COVID 19 (n=3). Furthermore, during the follow-up period, we 
missed two patients in the placebo group due to discontinued follow-up, and we lost 
4 patients in the probiotics group due to death (n=2) and discontinued follow-up 
(n=2), while in the synbiotics group, we missed three patients due to discontinued 
follow-up.  

Baseline features homogeneity was displayed in the three groups (Table 1). There 
were no significant differences between the groups regarding demographic 
characteristics including sex, age, history of chronic disease, and body mass index.  
 
 
 
 
 
 
 
 
 

 
Table 1:  Demographic characteristics of participants  
P>0.05: represents an insignificant effect. 
BMI: body mass index 
 
 
Tissue infiltration of CD8+ T cells and IFN-γ expression 
Tumor tissue infiltration of CD8+T cells as well as the expression of IFN γ in 
tissue samples from 90 patients with PDAC were assessed by IHC (Figures 2(a)–
(f)).  It was observed that patients who received synbiotics and probiotics prior 
to surgery exhibited a significant increase in both the proportion of CD8+T cells 
as well as the expression of IFN-γ compared to those in the placebo group (p=0.000). 
Moreover, the expression of IFN-γ as well as CD8+ T cells infiltration was notably 
higher in the synbiotics-treated group in comparison to the probiotics-treated 
group (p=0.013, p=0.049 respectively)  (Table 2 ) 
 
Table 2: Tissue expression of CD8+cells and INF-γ in subjected groups 
 
Variable 
Group 
Mean ± SE 
P-values 
INF-γ  
Placebo 
15.83 ± 2.28 
--- 
Probiotics 
52.50 ± 5.66 * 
P1=0.002 
Synbiotics 
60.50 ± 6.03* 
P1=0.000, P2=0.013 
CD8  
Placebo 
15.83 ± 2.63 
--- 
Probiotics 
54.83 ± 8.32* 
P1=0.005 
Synbiotics 
61.50 ± 6.62* 
P1=0.002, P2=0.049 
*: represents significant differences (P1< 0.05), as compared to the placebo group 
#: represents significant differences (P2< 0.05), as compared to probiotics group  
Variable 
Placebo 
N (%) 
Probiotics 
N (%) 
Synbiotics 
N (%) 
P-value 
 
Age category (year) < 50 
17(56.7) 
12 (40) 
16 (53.3) 
0.393 
> 50 
13 (43.3) 
18 (60) 
14 (46.7) 
Gender 
Male 
21 (70) 
17 (56.7) 
24 (80) 
0.147 
Female 
9 (30) 
13 (43.3) 
6 (20) 
Disease history 
None 
25 (83) 
27 (90) 
26 (86) 
0.210 
Diabetes 
2 (7) 
1 (3) 
2 (7) 
Hypertension 
3 (10) 
2 (7) 
2 (7) 
BMI (Mean ± SE) 
 
22.40 ± 1.85 23.00 ± 1.27 22.00 ± 1.96 
0.919 

 
 

 
 
Association between clinicopathological parameters and tissue expression of 
INF-γ and CD8+T cells 
On studying the association between the expression of INFγ and CD8 infiltration in 
the tumor tissues, and the clinicopathological parameters in the preoperative studied 
groups, we observed that pancreatic tissue exhibited a significant upregulation in the 
expression of INF-γ and CD8+T   cells infiltration in both synbiotics and prebiotics 
treated groups compared to the placebo group. Moreover, as compared to the 
probiotic group, significant increases in the expression of INFγ and CD8 in the 
synbiotic group were obtained. In probiotics treated group, a significant elevation in 
the expression of INFγ was observed in female patients (p=0.011) younger than 50 
years old compared to males, while in probiotics and synbiotics groups there were 
significant elevations in the expression of INF-γ with the pN-stage (P=0.002, 
P=0.025) as well as TNM stage (P=0.000, P=0.025), respectively. In the Synbiotic 
group, the significantly (P=0.036) higher pN-stage as well as TNM stage is 
proportional to the greater expression of CD8 (Table 3).  
 
 
 
 
 
 
 
 
 
 

Table 3: The effect of clinicopathological parameters on the expression of INFγ and CD8+T cells in the tumor tissues of all groups 
pT-stage; pathological tumor stage, pN-stage; pathological lymph node stage, TNM: tumor lymph nodes metastasis stage. Data are displayed as 
mean ± standard error. *, #: significant differences as compared to the placebo (P2<0.05) and probiotic (P3<0.05) groups. In each group, ¶: 
significant differences (P1<0.05), as compared to values at age <50 or Males or pT2 or pN1 or Stage II. 
 
 
Clinicopathological parameters 
INFγ 
CD8 
Placebo 
Probiotic 
Synbiotic 
Placebo 
Probiotic 
Synbiotic 
Age category 
< 50  
13.19 ± 0.74 
27.50 ± 2.26* 
35.89 ± 1.76*# 
23.88 ± 0.77 
32.25 ± 1.56* 
42.78 ± 2.40*# 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.001 
--- 
P2=0.000 
P2=0.000, P3=0.000 
> 50  
13.65 ± 0.90 
26.47 ± 2.01* 
36.75 ± 1.34*# 
26.15 ± 0.96 
31.40 ± 1.30* 
46.50 ± 1.50*# 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.000 
--- 
P2=0.005 
P2=0.000, P3=0.000 
p value 
P1=0.703 
P1=0.735 
P1=0.714 
P1=0.083 
P1=0.677 
P1=0.188 
Gender 
Male  
12.43 ± 0.55 
23.43 ± 1.97* 
36.60 ± 1.56*# 
23.48 ± 0.76 
32.29 ± 1.34* 
46.60 ± 2.06*# 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.000 
--- 
P2=0.000 
P2=0.000, P3=0.000 
Female  
14.87 ± 1.11 
30.69 ± 1.74*¶ 
36.42 ± 1.42*# 
27.47 ± 0.85¶ 
31.23 ± 1.49 
44.68 ± 1.67*# 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.004 
--- 
P2=0.064 
P2=0.000, P3=0.000 
p value 
P1=0.063 
P1=0.011 
P1=0.939 
P1=0.001 
P1=0.602 
P1=0.491 
pT-stage 
pT2 
12.50 ± 0.61 
27.50 ± 1.88* 
35.92 ± 1.75*# 
23.00 ± 0.70 
33.57 ± 1.02* 
46.77 ± 1.82*# 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.000 
--- 
P2=0.000 
P2=0.000, P3=0.000 
pT3 
14.05 ± 0.87 
26.31 ± 2.37* 
36.94 ± 1.33*# 
26.50 ± 0.86 
29.85 ± 1.60 
44.19 ± 1.82*# 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.000 
--- 
P2=0.069 
P2=0.000, P3=0.000 
 
p value 
P1=0.206 
P1=0.695 
P1=0.642 
P1=0.007 
P1=0.057 
P1=0.329 
pN-stage 
pN1 
13.78 ± 0.75 
21.73 ± 1.91* 
34.07 ± 1.11*# 
24.89 ± 0.90 
33.45 ± 1.72* 
42.57 ± 1.60*# 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.000 
--- 
P2=0.000 
P2=0.000, P3=0.000 
pN2 
13.11 ± 0.92 
30.50 ± 1.62*¶ 
38.73 ± 1.58*#¶ 
25.39 ± 0.97 
30.63 ± 1.12* 
47.93 ± 1.80*#¶ 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.000 
--- 
P2=0.004 
P2=0.000, P3=0.000 
p value 
P1=0.58 
P1=0.002 
P1=0.025 
P1=0.707 
P1=0.162 
P1=0.036 
TNM stage 
Stage II  
13.78 ± 0.75 
20.60 ± 1.71* 
34.07 ± 1.11*# 
24.89 ± 0.90 
32.90 ± 1.80* 
42.57 ± 1.60*# 
 
p value 
--- 
P2=0.001 
P2=0.000, P3=0.000 
--- 
P2=0.000 
P2=0.000, P3=0.000 
Stage III  
13.11 ± 0.92 
30.65 ± 1.53*¶ 
38.73 ± 1.58*#¶ 
25.39 ± 0.97 
31.12 ± 1.16* 
47.93 ± 1.80*#¶ 
 
p value 
--- 
P2=0.000 
P2=0.000, P3=0.000 
--- 
P2=0.002 
P2=0.000, P3=0.000 
 
p value 
P1=0.58 
P1=0.000 
P1=0.025 
P1=0.707 
P1=0.393 
P1=0.036 

Serum concentrations of IL-1 β, IL 6, and IL 10  
Cytokine concentrations at different time points pre and post-tumor resection were evaluated for all subjected groups, 14 days before surgery (Pre-
14D), at the operation day (OP-0d), 14 and 30 days post-operative (PO-14d and PO-30d respectively). In the placebo group, there was no significant 
difference in the IL-1β concentrations at different time points but at PO-30d, where a significant decline was recorded, as compared to the baseline. 
In addition, as compared to the placebo group, a significant decline was observed at OP-0d, PO-14, and PO-30d (P=0.000) in the synbiotics-treated 
group and at PO-14 and PO-30d in the probiotics-treated group (P=0.000). However, the IL-1 β concentrations showed a significant decline in the 
synbiotics group compared with the probiotics group at the OP-0d (P=0.016), PO-14d (P=0.021), and P)-30 (P=0.002) (Table 4, Figure 3A ).  For 
IL-10 assessment, in the placebo group, there was no significant difference in the IL-10 concentrations among all the time intervals. In the 
synbiotics group and probiotics group, a gradual reduction in the IL-10 concentrations was observed from the OP-0d to PO-30d, as compared to 
the baseline (p= 0.002, p=0.000, p=0.000 respectively). Moreover, as compared to the placebo group, on the PO-14d and PO-30d, the IL-10 
concentrations significantly declined (P=0.000). However, as compared to the probiotics group, the IL-10 concentrations showed a significant 
decline at OP-0d (P=0.04) (Table 4, Figure 3B). Regarding IL-6 concentration, there was no significant difference among all time intervals. In the 
probiotic and synbiotics group, a gradual reduction was detected from the OP-0d to PO-30d, as compared to the baseline. In addition, as compared 
to the placebo, on the OP-0d, PO-14d, and PO-30d, the IL-6 concentrations significantly declined (P=0.000) in both groups (p=0.000). However, 
as compared to the probiotic group, the IL-6 concentrations showed a significant decline at PO-14d (P=0.039) (Table4, Figure 3C) 
 
 
 
 

 
Table 4. The concentrations of IL-1β, IL-10 and IL-6 in all the studied groups. Data is displayed as mean ± standard error. 
 
Group 
Sampling time 
Effect of Time 
 
Pre-14d 
OP-0d 
PO-14d 
PO-30d 
IL-1B 
Placebo 
28.43 ± 3.15b 
25.97 ± 3.31b 
24.91 ± 4.08b 
14.60 ± 2.51a 
F3,116=3.39, P=0.02 
Synbiotic 
29.49 ± 3.24c 
10.44 ±1.44b* 
4.47 ± 0.59a* 
2.27 ± 0.23a* 
F3,116=47.22, P=0.000 
 
P1=0.813 
P1=0.000 
P1=0.000 
P1=0.000 
 
Probiotic 
30.88 ± 3.03c 
19.06 ± 2.36b# 
13.42 ± 2.16a*# 
11.05 ± 2.21a# 
F3,116=12.89, P=0.000 
 
P1=0.583, P2=0.754 
P1=0.053, P2=0.016 
P1=0.003, P2=0.021 
P1=0.198, P2=0.002 
 
Treatment 
F2,87=0.153, P=0.858 
F2,87=3.75, P=0.000 
F2,87=14.55, P=0.000 
F2,87=10.76, P=0.000 
 
IL-10 
Placebo 
21.42 ± 2.56a 
20.36 ± 2.44a 
22.48 ± 3.22a 
24.79 ± 2.36a 
F3,116=0.504, P1=0.681 
Synbiotic 
19.34 ± 1.98c 
11.31 ± 0.94b* 
7.69 ± 0.49a* 
5.85 ± 0.29a* 
F3,116=27.96, P1=0.000 
 
P1=0.524 
P1=0.002 
P1=0.000 
P1=0.000 
 
Probiotic 
20.31 ± 2.32c 
17.19 ± 2.26b# 
12.81 ± 1.50a* 
8.89 ± 0.77a* 
F3,116=7.48, P1=0.000 
 
P1=0.734, P2=0.765 
P1=0.265, P2=0.04 
P1=0.001, P2=0.084 
P1=0.000, P2=0.14 
 
Treatment 
F2,87=0.205, P2=0.815 
F2,87=5.29, P2=0.007 
F2,87=13.14, P2=0.000 
F2,87=49.69, P2=0.000 
 
IL-6 
Placebo 
18.93 ± 1.74a 
18.85 ± 1.59a 
23.42 ± 1.95a 
22.27 ± 1.56a 
F3,116=1.84, P1=0.143 
Synbiotic 
18.98 ± 1.59c 
11.38 ± 1.05b* 
6.91 ± 0.40a* 
4.79 ± 0.24a* 
F3,116=40.97, P1=0.000 
 
P1=0.984 
P1=0.000 
P1=0.000 
P1=0.000 
 
Probiotic 
18.14 ± 1.56c 
12.42 ± 1.22b* 
9.07 ± 0.75a* 
7.67 ± 0.58a*# 
F3,116=18.02, P1=0.000 
 
P1=0.733, P2=0.718 
P1=0.001, P2=0.578 
P1=0.000, P2=0.218 
P1=0.000, P2=0.039 
 
Treatment 
F2,87=0.083, P2=0.92 
F2,87=9.58, P2=0.000 
F2,87=53.45, P2=0.000 
F2,87=93.06, P2=0.000 
 
*: a significant difference (P1<0.05), as compared to the control group. 
#: a significant difference (P2<0.05), as compared to the symbiotic group. 
 

 
 
 
 
A 

 
 
 
 
 
 
B 

 
 
 
 
 
Fig 3. The concentrations of IL-1B (A), IL-10 (B) and IL-6 (C) in all the studied groups. Data is displayed as mean ± standard error. *: a 
significant difference (P1<0.05), as compared to the control group. #: a significant difference (P2<0.05), as compared to the symbiotic group. 
C 

Post-operative short-term outcome 
Post-operative short-term outcome conditions were evaluated in all groups included in the study (Table 5). Both probiotics and symbiotics groups 
demonstrated a significant reduction in the average number of days until the first bowel movement, in comparison to the placebo group (P=0.000). 
There were no significant differences in the postoperative hospital stay period as well as the days till the return to normal activity. Incidence of 
infectious complications showed significant improvement in bacteremia in probiotics and synbiotics groups (0.047) with a remarkable decrease in 
the synbiotics group as well as the incidence of pneumonia.  Non- infectious complications including anastomotic leakage, diarrhea, and abdominal 
distension, showed significant improvement in synbiotics and probiotics receiving- groups compared to the placebo group (p=0.032, p=0.044, and 
p= 0.42 respectively). Moreover, diarrhea and abdominal distension were particularly noteworthy decreases in the synbiotics group.  
Table 5. Postoperative short-term outcomes 
 
Placebo cases 
(n=30) 
Probiotic cases 
(n=30) 
Synbiotic cases 
(n=30) 
P-Value 
Bleeding (%) 
1/30 (3.3%) 
1/30 (3.3%) 
1/30 (3.3%) 
1.000 
Mean Day to First Stool (day) 
5.00 ± 0.13 
4.00 ± 0.09* 
4.00 ± 0.14* 
0.000 
Post-operative stay (day) 
10.00 ± 0.13 
10.00 ± 0.14 
10.00 ± 0.14 
1.000 
Return to usual activity (day) 
14.00 ± 0.13 
14.00 ± 0.17 
14.00 ± 0.08 
1.000 
Incidence of infectious complications (%) 
 
 
 
 
Bacteremia 
15/30 (50.0%) 
7/30 (23.3%) 
4/30 (13.3%)* 
0.047 
Wound infection 
2/30 (6.7%) 
1/30 (3.3%) 
1/30 (3.3%) 
0.770 
Pneumonia 
4/30 (13.3%) 
2/30 (6.7%) 
2/30 (6.7%) 
0.578 
Urinary tract infection 
5/30 (16.7%) 
5/30 (16.7%) 
4/30 (13.3%) 
0.919 
Incidence of non-infectious complications (%) 
 
 
 
 
Anastomotic Leakage 
3/30 (10.0%) 
0/30 (0 %)* 
0/30 (0 %)* 
0.032 
Diarrhea 
17/30 (56.7%) 
10/30 (33.3%)*  
8/30 (26.7%)* 
0.044 
Abdominal distension 
16/30 (53.3%) 
10/30 (33.3%)* 
7/30 (23.3%)*  
0.042  
Mortality (n) 
N/P (0 %)  
 N/P (0%) 
N/P (0%) 
0.364 
Data are presented as mean ± standard error and number (percentages). P<0.05: Significant differences according to Chi-squared and One-way ANOVA. 
 

Discussion 
 
    In contrast to the numerous studies conducted on colorectal cancer (CRC) and gastric cancer (GC), there is a lack of research 
exploring the role of synbiotics in the prevention and treatment of other gastrointestinal (GI) cancers, such as pancreatic and 
liver cancer. Only a limited number of published reports have investigated the potential link between probiotics and prebiotics 
in pancreatic ductal adenocarcinoma (PDAC), focusing primarily on the suppression of tumorigenesis and the impact on post-
surgical complications (Abdul Rahman et al., 2023; Rad et al., 2021; Tang et al., 2021). To the best of our knowledge, this 
is the first randomized clinical study to evaluate the immunomodulatory effect and post-surgical complications of oral 
synbiotics (probiotics and inulin) compared to probiotics alone in PDAC patients. Our results indicated that consuming 
synbiotics and probiotics supplements for six weeks (two weeks pre-operative and four weeks post-operative),  significantly 
improved the immune response in the treated groups compared to the placebo group. Immunohistochemical results showed 
that the proportion of CD8+ T cells and the expression of IFN-γ in tumor tissue were significantly higher in patients treated 
with synbiotics or probiotics compared to those treated with a placebo. Additionally, the synbiotics group showed a greater 
elevation in CD8+ T cells and IFN-γ expression compared to the probiotics group. These findings are consistent with the 
results obtained by Mao et al. (2020), who suggested that probiotics could enhance the antitumor immune response of CD8+ 
T cells and IFN-γ+ T cells in the tumor microenvironment in patients with colorectal carcinoma (Mao et al., 2020). In our 
study, we used a ten-strain cocktail of probiotics including Bifidobacterium, Lactobacillus, and Streptococcus bacteria strains 
in addition to inulin fibers prebiotics. Several studies have demonstrated the efficient immunomodulatory effects of such 
strains. For example, Yoon et al. (2021) found that supplementation with B. breve strains reduced tumor growth in mice with 
colon carcinoma by augmenting lymphocyte-mediated anti-cancer immunity (Yoon et al., 2021). Similarly, Lee et al. (2015) 
concluded that Lactobacillus acidophilus strains increased serum IFN-γ, CD4+, and CD8+ cells in mice with induced colon 
cancer (Lee et al., 2015). Other studies have also shown the modulation of the anti-

tumor immune response by using a combination of Bifidobacterium or Lactobacillus 
acidophilus bacteria, leading to the improvement of dendritic cells function, 
infiltration of CD8+ T cells, and reduction of inflammatory cytokines (Sivan et al., 
2015; Gui et al., 2015). 
      In our study, the significant increase in the expression of CD8+ T cells and IFN-
γ in the synbiotics group compared to the probiotics group may be attributed to the 
beneficial effect of consuming inulin prebiotics along with these probiotic strains. 
These results are consistent with various pre-clinical studies that have suggested the 
anti-tumor immune system effect of inulin, particularly when combined with 
different probiotic regimens. For instance, Kassayová et al. (2016) found that 
consuming Lactobacillus plantarum along with inulin prebiotics reduced 
carcinogenesis by inducing apoptosis and diminishing pro-inflammatory cytokines 
in a colon cancer model (Kassayová et al., 2016). Also, Li et al. (2020) used inulin 
and mucin prebiotics in their study on colon cancer in mice and observed that dietary 
consumption of inulin, but not mucin, reduced tumor growth through the induction 
of CD8+ T and CD4+ T cells mediated anti-tumor immune response (Li et al ., 
2020). Boucher et al. (2023) also demonstrated that an inulin-enriched diet along 
with probiotic strains triggered an enhanced CD4+ and CD8+ αβ T cell-mediated 
anti-tumor response and attenuated tumor growth in preclinical tumor-bearing 
mouse models (Boucher et al., 2023). 
      Elevated levels of inflammatory-mediated cytokines such as IL-6 and IL-10 have 
been associated with poor prognosis in patients with pancreatic cancer (Feng et al., 
2018; Lippitz et al ., 2016; Tanaka et al., 2014). In our study, we evaluated the 
concentration of circulating inflammatory-mediated cytokines (IL-10, IL-6, and IL-
1ß) in all groups at different time intervals before and after tumor resection. Our 
results showed a significant decrease in all tested cytokines in both the probiotics 
and synbiotics groups compared to the placebo group. The synbiotics group 
exhibited a more pronounced decline in IL-1ß levels on the day of the operation, 14 
days, and 30 days after surgery. These findings are consistent with Das et al. (2020), 
who discussed the association between poor prognosis of pancreatic cancer and 
elevated concentrations of IL-1ß. They suggested that neutralizing IL-1ß could 
promote intratumoral CD8+ T cell infiltration and function and sensitize pancreatic 
ductal adenocarcinoma to checkpoint immunotherapy (Das et al., 2020). 
Additionally, we found that IL-6 and IL-10 levels gradually decreased on the day of 
the operation and 30 days post-operative compared to the baseline.  The synbiotics 
group showed a significant reduction in IL-6 levels 14 days post-operative and in 

IL-10 levels on the day of the operation compared to the probiotics group. This 
reduction in cytokine levels can be attributed to the synergistic anti-inflammatory 
effect of synbiotics, particularly due to the inclusion of inulin fibers as a prebiotic. 
These results align with other studies that have discussed the anti-inflammatory and 
immunomodulatory effects of inulin prebiotics (Farabegoli et al., 2023; Abreu et 
al., 2022). 
Considering that surgery is the primary determinant of prognosis in PDAC, and 
holds curative potential, our study aimed to investigate the short-term post-operative 
outcomes when probiotics or synbiotics were administered. Limited research has 
been conducted on the supplementation of probiotics or synbiotics in patients 
undergoing pancreaticoduodenectomy. Between 2007 and 2021, only six studies 
with conflicting results were carried out, involving a total of 294 participants (147 
in the control group and 147 in the intervention group). Three studies utilized 
probiotics with limited strains, Folwarski et al. ( 2021),  Diepenhorst et al. ( 2011), 
and  Nomura et al. (2007), and three studies used synbiotics, including Yokoyama 
et al. (2016), Sommacal et al. (2015), and Rayes et al. (2007). In our study, we 
observed a significant reduction in the average number of days until the first bowel 
movement and the incidence of diarrhea in both the synbiotics and probiotics groups 
compared to the placebo group. The reduction was particularly notable in the 
synbiotics group. These findings are consistent with the results obtained by 
Folwarski et al. (2021) and Diepenhorst et al. (2011), who observed that the 
consumption of Lactobacillus strains improved bacterial translocation and 
postoperative outcomes after pancreatoduodenectomy. Furthermore, our results 
showed a decrease in infectious complications in both the probiotics and synbiotics 
groups compared to the placebo group, with a more pronounced improvement 
observed in the synbiotics group. These findings are in line with the research 
conducted by Rayes et al. (2007). In their prospective randomized trial involving 
80 patients who underwent pylorus-preserving pancreatoduodenectomy (PPPD) and 
received lactobacillus and fibers for 30 days, they observed a significant reduction 
in bacterial infection rates following PPPD (Rayes et al., 2007). Sommacal et al. 
(2015) also concluded that perioperative synbiotics supplementation decreases 
postoperative complications in periampullary neoplasms. Their study included 23 
patients who received a cocktail of Lactobacillus strains and fructooligosaccharides 
twice daily for 14 days, resulting in improved hospital stay length and a decreased 
incidence of infection.  Regarding postoperative hospital stay and the time required 
for normal activities, there were no significant differences observed among the 

groups. We believe that this is directly related to our protocol in TBRI, which 
includes routine feeding jejunostomy in all cases and the use of a new technique for 
anastomosis, specifically end-to-side duct to mucosa with an internal stent and two-
layered pancreaticojejunostomies with omental wrapping.  
Conclusion 
This study provided evidence of the significant immunomodulatory impact of 
synbiotics supplementation and its contribution to improving post-operative 
complications in PDAC patients. However, further large-scale studies are necessary 
to explore more pertinent clinical evidence in this field. These studies should involve 
diverse combinations and regimens of probiotics and prebiotics administered over 
extended periods. Additionally, investigating the potential synergies between 
synbiotics and immunotherapeutic drugs is crucial. By conducting such research, we 
can gain a deeper understanding of the topic and obtain more valuable clinical 
insights.