Characterization of isolated E. coli strains
Four bacteria species were isolated from patients’ tumor and, using a combination of biochemical assay and MALDI ToF spectroscopy, identified as E. coli.
For the tumor-associated E. coli strains the data on antibiotic sensitivity and biochemical enzyme activity were obtained and compared with standard probiotic M17 strain. Unlike M17, all the tumor-associated E.coli strains were resistant to at least one of the antibiotics tested. Three out of four strains (Col-101, Col-102, Col-103) showed resistance to ampicillin, and one (Col-93) - to amoxicillin (Fig. 1A).
Analysis of the biofilm activity showed that three tumor-associated strains (Col-93, Col-101, Col-103) increased biomass and synthesized a matrix less actively compared to M17 strain (Fig. 1B). One strain (Col-102) had statistically higher reproduction rate and produced the same volume of matrix as M17.
Biochemical activity of the strains has fluctuations in some amino acids and especially in sucrose (Supl.1). Most of the strains associated with colorectal cancer were unable to utilize sucrose, which is typically observed in only a few E.coli that can are typically slow growing or pathogenic strains [15].
The differences in the proportion and composition of the synthesized metabolites were found between tumor-associated and the probiotic strains. The proportion of dominant metabolites (mg/ml) was similar in all strains (Fig. 1, C), tumor-associated and control. While the balance of minor components (µg/ml) was different for the tumor-associated E.coli strains compared to M17 control (Fig. 1, D).
We found statistically higher levels (p ≤ 0.05) of butyric, fumaric, maleic, and glycolic acids in tumor-associated strains’ metabolites compared to probiotic strain M17 (Table 2). The amount of malic acid was increased in Col-101 metabolites more than 5 times compared to other strains and M17. Pyruvic acid level was higher in tumor-associated strains, but statistical significance was shown only for Col-102 and Col-103. The level of valeric acid was higher for Col-101, Col-102 and Col-103 metabolites (p ≤ 0.05).
Daily production of short-chain fatty acids (SCFAs) such as 2-oxybutyrate, propionate, α-Ketoglutaric acid (AKG) and isobutyric acid by tumor-associated strains was lower compared to probiotic strain M17 (Table 2). The production of propionic, malonic, 2-hydroxyglutaric, 2-oxobutyric a and isobutyric acids was dramatically, > 2–5 times decreased for all tumor-associated strains.
Extremely low level of α-ketoglutaric acid (AKG) was found for Col-103 strain (> 90-fold decrease) as well as for other tumor-associated strains. Lactic acid concentration was lower in Col-93 and Col-103 strains. Succinic acid was decreased in all patients’ strain metabolites. No changes were observed for isovaleric acid and glyoxylic acid.
Table 1. Patient’s sample characteristics.
Sample
|
Age
|
Sex
|
TNM classification
|
Tumor type
|
Localisation
|
Grade
|
Col-93
|
76
|
F
|
T3N1М0
|
Adenocarcinoma
|
Sigmoid colon
|
G2
|
Col-101
|
73
|
F
|
рT4bN1bМ1c
|
Adenocarcinoma
|
Sigmoid colon
|
G2
|
Col-102
|
78
|
M
|
рT4bN1bМ0
|
Adenocarcinoma
|
Sigmoid colon
|
G2
|
Col-103
|
76
|
M
|
рT3N0М0
|
Adenocarcinoma
|
Transverse colon
|
G1
|
Table 2
Daily production of the selected metabolites by E.coli strains (* p ≤ 0.05 with M17).
Analyte
|
M17
|
Col-093
|
Col-101
|
Col-102
|
Col-103
|
Lactic acid, mg/l
|
138.6 ± 13.3
|
102.2 ± 39.4 *
|
123.8 ± 23.9
|
124.6 ± 8.1
|
112.5 ± 3.7 *
|
Acetic acid, mg/l
|
1.7 ± 0.1
|
2.0 ± 0.8
|
1.9 ± 0.2
|
2.1 ± 0.1 *
|
2.3 ± 0.0 *
|
Succinic acid, mg/l
|
170.8 ± 18.5
|
120.5 ± 40.7 *
|
118.2 ± 11.4 *
|
147.4 ± 2.1 *
|
144.6 ± 4.1 *
|
Pyruvic acid, mg/l
|
8.9 ± 0.9
|
12.6 ± 4.4
|
10.1 ± 1.2
|
11.6 ± 0.6 *
|
12.7 ± 0.2 *
|
Malic acid, mg/l
|
1.8 ± 0.3
|
1.8 ± 0.6
|
9.7 ± 1.3 *
|
0.5 ± 0.03 *
|
0.3 ± 0.02 *
|
Valeric acid, mg/l
|
0.60 ± 0.09
|
0.6 ± 0.2
|
0.8 ± 0.09 *
|
0.8 ± 0.01 *
|
0.8 ± 0.04 *
|
β-Hydroxybutyric acid, µg/l
|
31.4 ± 2.9
|
43.6 ± 19.0
|
35.8 ± 6.1
|
70.0 ± 8.3 *
|
30.2 ± 5.3
|
Propionic acid, µg/l
|
122.7 ± 23.0
|
24.2 ± 8.5 *
|
37.3 ± 4.3 *
|
37.0 ± 2.9 *
|
21.0 ± 2.6 *
|
Isobutyric acid, µg/l
|
12.7 ± 2.4
|
6.1 ± 0.1 *
|
6.1 ± 0.6 *
|
6.9 ± 2.0 *
|
8.5 ± 1.6 *
|
Butyric acid, µg/l
|
19.1 ± 3.8
|
27.3 ± 10.3
|
36.1 ± 4.1 *
|
36.4 ± 4.0 *
|
27.3 ± 1.4 *
|
2-Hydroxyglutaric acid, µg/l
|
47.0 ± 6.1
|
98.7 ± 43.0 *
|
11.9 ± 0.8 *
|
6.9 ± 2.8 *
|
15.2 ± 1.8 *
|
Isovaleric acid, µg/l
|
5.2 ± 1.0
|
4.2 ± 1.9
|
5.9 ± 1.8
|
5.1 ± 1.3
|
6.5 ± 0.5
|
Fumaric acid, µg/l
|
170.1 ± 16.1
|
325.6 ± 114.8 *
|
372.3 ± 53.2 *
|
263.2 ± 27.2 *
|
476.8 ± 15.5 *
|
Maleic acid, µg/l
|
101.8 ± 6.1
|
366.3 ± 229.3 *
|
446.1 ± 59.6 *
|
337.4 ± 15.9 *
|
465.1 ± 63.5 *
|
Glyoxylic acid, µg/l
|
38.9 ± 3.3
|
36.9 ± 11.9
|
42.1 ± 5.2
|
37.8 ± 8.4
|
41.6 ± 7.4
|
2-Oxobutyric acid, µg/l
|
59.0 ± 5.3
|
6.0 ± 2.1 *
|
9.1 ± 3.6 *
|
11.5 ± 0.6 *
|
8.8 ± 0.7 *
|
α-Ketoglutaric acid, µg/l
|
45.2 ± 16.5
|
6.3 ± 5.7 *
|
9.9 ± 1.9 *
|
0.5 ± 0.2 *
|
3.4 ± 1.0 *
|
Glycolic acid, µg/l
|
235.8 ± 35.3
|
264.7 ± 72.9
|
257.9 ± 34.0 *
|
301.5 ± 2.5 *
|
286.4 ± 10.1 *
|
Malonic acid, µg/l
|
11.2 ± 2.8
|
2.0 ± 1.3 *
|
2.9 ± 0.4 *
|
3.0 ± 1.0 *
|
3.3 ± 1.5 *
|
Effect of E.coli metabolites on tumor spheroids growth
The effect of E.coli metabolites on the growth of tumor spheroids was assessed using colorectal cancer cell lines HCT116 and HT29. The SW480 line lacked the ability for spheroid formation.
It was found that in the presence of E.coli metabolites the growth of both HCT116 and HT29 spheroids was inhibited compared with a control without metabolites (Fig. 2, A, B). At that, no differences were observed in the effects of M17 and tumor-associated strains.
We also assessed proliferative activity of all cell lines in the presence of metabolites of E.coli strains (Fig. 2, C). A significant increase of the doubling time in the presence of metabolites M17 (p = 0.034) and Col-101 (p = 0.039) was shown for HT29 cells. The metabolites of Col-103 strain extended the doubling time of SW480 (p = 0.042). For other lines, the presence of metabolites only slightly increased the doubling time, without statistical significance.
Effect of E.coli metabolites on cancer cells migration
Metabolites of the probiotic strain M17 did not affect migration of HCT116 cells from the spheroids and slightly (p = 0.035) inhibited migration of HT29 cells compared to control without metabolites (Fig. 3). Tumor-associated E.coli strains metabolites had different effects on migration of HCT116 and HT29 cells. HCT116 cell line showed higher migration activity in the presence of metabolites from all tumor-associated strains compared to control without metabolites and M17 strain (Fig. 3, A). The largest migration area was observed upon exposure to the Col-101 metabolites.
The opposite effects were observed for HT29 cells, whichactively migrated in control without metabolites but inhibited migration in the presence of E.coli metabolites. Of the strains used, M17 metabolites inhibited migration of HT29 cells in a lower degree in comparison with tumor-associated strains metabolites (Fig. 3, B).
Analysis of cell migration in the model of monolayer “wound healing” was performed for the three cell lines HCT116, SW480 and HT29. Two E.coli strains, Col-101 and Col-102, were selected for this test as they demonstrated the most notable effects among other strains obtained from the patients.
Similar to migration from the spheroids, patient-derived E.coli metabolites stimulated migration of HCT116 and SW480 cells and inhibited migration of HT29 cells in the “wound healing” model (Fig. 4). The M17 metabolites did not change migration of HCT116 and SW480 cells and inhibited migration of HT29 cells.
Therefore, the experiments on the cell monolayers and tumor spheroids revealed that tumor-associated E.coli metabolites affected migratory capacity of colorectal cancer cells and could either increase or decrease it depending on the specifics of cancer cells.
Immunocytochemical analysis of the migration-associated markers
To identify the molecular mechanisms through which E.coli metabolites had different effects on colorectal cancer cell lines, the expression levels of E-cadherin and the focal adhesion kinase (FAK) were analyzed using immunofluorescence.
E-cadherin expression statistically decreased in HCT116 and SW480 cell lines upon exposure to metabolites of the Col-101 strain (Fig. 5). The probiotic M17 strain induced a marked increase in E-cadherin level only in HT29 cells, and had no effect on two other cell lines. Since down-regulation of E-cadherin, a major component of adherens junctions, facilitates cell motility and migration, its lower level in HCT116 and SW480 cells correlated with their highest migratory activity upon incubation with Col-101 metabolites.
A significant decrease in FAK expression was noted in HT29 cells under the bacterial metabolites of all tested strains, which explains a decrease of migratory capacity of this cell line. Inhibition of FAK activity is known to decrease cell motility due to suppression of cell-matrix attachment. Of note, the initial FAK activity in HT29 cells was higher compared with other cell lines, suggesting their higher migratory potential. In HCT116 and SW480 cell lines FAK level did not change after incubation with bacterial metabolites (Fig. 5, B).
These results suggest that changes in migratory capacity of colorectal cancer cells under the exposure to tumor-associated E.coli metabolites can be mediated by both the loss of cadherin-based cell − cell adhesions and attenuation of the FAK signaling.