Open Access

Human Soluble TRAIL Secreted by Modified Lactococcus lactis Bacteria Promotes Tumor Growth in the Orthotopic Mouse Model of Colorectal Cancer

Katarzyna Kaczmarek
Katarzyna Kaczmarek
, 
Jerzy Więckiewicz
Jerzy Więckiewicz
, 
Ivo Que
Ivo Que
, 
Adrianna Gałuszka-Bulaga
Adrianna Gałuszka-Bulaga
, 
Alan Chan
Alan Chan
, 
Maciej Siedlar
Maciej Siedlar
 and 
Jarek Baran
Jarek Baran
   | Jan 06, 2024
Archivum Immunologiae et Therapiae Experimentalis's Cover Image
About this article
Previous Article
Next Article
Cite
Article Category: Original
Published Online: Jan 06, 2024
Page range: -
Received: Jul 05, 2023
Accepted: Nov 30, 2023
DOI: https://doi.org/10.2478/aite-2024-0002
Keywords
© 2023 Katarzyna Kaczmarek et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig 1.

L.lactis(hsTRAIL+) bacteria given orally stimulate the growth of tumor in orthotopic model of human CRC. a Experimental groups and treatment options. The mouse orthotopic model of human CRC was developed by engrafting of HCT116 Red-FLuc cells into the cecum of SCID mice. Two weeks after the implantation, animals were subjected to BLI to verify the presence and growth of the tumor, then divided into six experimental groups (n=8 and n=11 for mock control), treated from “Day 0” as following: Group I (Mock control) – vehicle (10% skimmed milk in PBS + ZnSO4 + aprotinin + nisin, see Materials and Methods); Group II – L.lactis(“empty” vector); Group III – L.lactis(hsTRAIL+); Group IV – MetF (250 mg/kg); Group V – combined therapy with L.lactis(hsTRAIL+) and MetF; Group VI – combined therapy with L.lactis(“empty” vector) and MetF. All treatments were given by gastric gavage, in the presence of nisin at a dose of 50 ng/mL. b Timeline of the therapy model. Growth of the primary tumor and development of metastases were regularly monitored for 38 days from the beginning of the experiment (“Day 0”) by BLI measurements. All treatments were administered via gastric gavage, once a day for 37 consecutive days. On Day 38, animals were subjected to BLI, then sacrificed. c Growth of CRC during the period of experiment. Tumor growth (BLI) in cecum was monitored at days indicated, as described in Materials and methods. Each point represents the mean for n=8–11 animals. The bars indicate the mean value ± SEM. Statistical analysis was performed using two-way ANOVA, with Tukey’s multiple-comparisons post-hoc test. ***p<0.001. d Representative whole-body bioluminescence images of the growth of HCT116 Red-FLuc tumors, performed at indicated time points after intraperitoneal (i.p.) administration of D-luciferin. Bar: photons/second.
L.lactis(hsTRAIL+) bacteria given orally stimulate the growth of tumor in orthotopic model of human CRC. a Experimental groups and treatment options. The mouse orthotopic model of human CRC was developed by engrafting of HCT116 Red-FLuc cells into the cecum of SCID mice. Two weeks after the implantation, animals were subjected to BLI to verify the presence and growth of the tumor, then divided into six experimental groups (n=8 and n=11 for mock control), treated from “Day 0” as following: Group I (Mock control) – vehicle (10% skimmed milk in PBS + ZnSO4 + aprotinin + nisin, see Materials and Methods); Group II – L.lactis(“empty” vector); Group III – L.lactis(hsTRAIL+); Group IV – MetF (250 mg/kg); Group V – combined therapy with L.lactis(hsTRAIL+) and MetF; Group VI – combined therapy with L.lactis(“empty” vector) and MetF. All treatments were given by gastric gavage, in the presence of nisin at a dose of 50 ng/mL. b Timeline of the therapy model. Growth of the primary tumor and development of metastases were regularly monitored for 38 days from the beginning of the experiment (“Day 0”) by BLI measurements. All treatments were administered via gastric gavage, once a day for 37 consecutive days. On Day 38, animals were subjected to BLI, then sacrificed. c Growth of CRC during the period of experiment. Tumor growth (BLI) in cecum was monitored at days indicated, as described in Materials and methods. Each point represents the mean for n=8–11 animals. The bars indicate the mean value ± SEM. Statistical analysis was performed using two-way ANOVA, with Tukey’s multiple-comparisons post-hoc test. ***p<0.001. d Representative whole-body bioluminescence images of the growth of HCT116 Red-FLuc tumors, performed at indicated time points after intraperitoneal (i.p.) administration of D-luciferin. Bar: photons/second.

Fig 2.

L.lactis(hsTRAIL+) bacteria survive in the gut after oral administration and MetF supports the persistence of bacteria in primary CRC. a Cecum with corresponding fragment of colon was isolated, homogenized and plated on agar Pertri dishes in the presence of Cm10, as a selection marker. Dishes were cultured for 48 h at 30°C. Shown are bacterial colonies on agar Petri dishes. b After 48 h of incubation at 30°C, the number of grown colonies was counted and compared between groups. c L.lactis(hsTRAIL) grown from the colon homogenates was proven by PCR. Legend: An arrow – hsTRAIL-cDNA sequence insert (438 bp); M – size marker (bp); Ctrl+ – positive control; Ctrl− – negative control (details in Materials and Methods). Legend: y axis – number of bacterial colonies (c.f.u.)/primary tumor area. The bars indicate mean values ± SEM (n=6). Statistical analysis was performed using one-way ANOVA test, with Tukey’s multiple comparisons post-hoc test. *p<0.05.
L.lactis(hsTRAIL+) bacteria survive in the gut after oral administration and MetF supports the persistence of bacteria in primary CRC. a Cecum with corresponding fragment of colon was isolated, homogenized and plated on agar Pertri dishes in the presence of Cm10, as a selection marker. Dishes were cultured for 48 h at 30°C. Shown are bacterial colonies on agar Petri dishes. b After 48 h of incubation at 30°C, the number of grown colonies was counted and compared between groups. c L.lactis(hsTRAIL) grown from the colon homogenates was proven by PCR. Legend: An arrow – hsTRAIL-cDNA sequence insert (438 bp); M – size marker (bp); Ctrl+ – positive control; Ctrl− – negative control (details in Materials and Methods). Legend: y axis – number of bacterial colonies (c.f.u.)/primary tumor area. The bars indicate mean values ± SEM (n=6). Statistical analysis was performed using one-way ANOVA test, with Tukey’s multiple comparisons post-hoc test. *p<0.05.

Fig 3.

Oral administration of L.lactis(hsTRAIL+) bacteria in the presence of nisin results in local secretion of hsTRAIL in the colon. The figure shows representative images from paraffin sections of colons, resected 17 h after the last treatments, subjected to IHC analysis for hsTRAIL. White arrows indicate the presence of hsTRAIL. Scale bar: 50 μm. White dashed lines denote the areas that were subsequently magnified and corrected for light intensity, sharpness, and contrast with the same parameters for all magnified areas.
Oral administration of L.lactis(hsTRAIL+) bacteria in the presence of nisin results in local secretion of hsTRAIL in the colon. The figure shows representative images from paraffin sections of colons, resected 17 h after the last treatments, subjected to IHC analysis for hsTRAIL. White arrows indicate the presence of hsTRAIL. Scale bar: 50 μm. White dashed lines denote the areas that were subsequently magnified and corrected for light intensity, sharpness, and contrast with the same parameters for all magnified areas.

Fig 4.

Continuous production of hsTRAIL in the gut leads to infiltration of CRC by CD206+-macrophages. a Representative images of paraffin sections of colons, subjected to IHC analysis for the presence of M2-macrophages, defined as CD206+ cells (black arrows). Scale bar: 50 μm. b Representative images of paraffin sections of the primary tumor in cecum, subjected to IHC analysis for the presence of M2-macrophages (CD206+ cells, yellow arrows). Scale bar: 50 μm. White dashed lines denote the areas that were magnified and corrected for light intensity, sharpness, and contrast with the same parameters for all magnified areas.
Continuous production of hsTRAIL in the gut leads to infiltration of CRC by CD206+-macrophages. a Representative images of paraffin sections of colons, subjected to IHC analysis for the presence of M2-macrophages, defined as CD206+ cells (black arrows). Scale bar: 50 μm. b Representative images of paraffin sections of the primary tumor in cecum, subjected to IHC analysis for the presence of M2-macrophages (CD206+ cells, yellow arrows). Scale bar: 50 μm. White dashed lines denote the areas that were magnified and corrected for light intensity, sharpness, and contrast with the same parameters for all magnified areas.

Fig 5.

hsTRAIL does not affect the MCP-1 production by CRC cells in primary tumor tissue. Representative images of paraffin sections of primary tumor in cecum subjected to IHC analysis for the presence of MCP-1. Black arrows indicate the presence of MCP-1. Scale bar: 50 μm. White dashed lines denote the areas that were subsequently magnified and corrected for light intensity, sharpness, and contrast with the same parameters for all magnified areas.
hsTRAIL does not affect the MCP-1 production by CRC cells in primary tumor tissue. Representative images of paraffin sections of primary tumor in cecum subjected to IHC analysis for the presence of MCP-1. Black arrows indicate the presence of MCP-1. Scale bar: 50 μm. White dashed lines denote the areas that were subsequently magnified and corrected for light intensity, sharpness, and contrast with the same parameters for all magnified areas.

Fig 6.

hsTRAIL reduces the depth of colon crypts. a Representative images of paraffin sections of colons, subjected to H&E staining for the histopathological analysis. Scale bar: 100 μm. b Analysis of the depth of the colon crypts after 37 consecutive days of respective treatments. Crypt Depth (CD) was calculated using paint.net (version 4.1.6). The bars indicate mean values ± SEM (n=6). Statistical analysis was done using one-way ANOVA test, with Tukey’s multiple comparisons post-hoc test. *p<0.05, **p<0.01.
hsTRAIL reduces the depth of colon crypts. a Representative images of paraffin sections of colons, subjected to H&E staining for the histopathological analysis. Scale bar: 100 μm. b Analysis of the depth of the colon crypts after 37 consecutive days of respective treatments. Crypt Depth (CD) was calculated using paint.net (version 4.1.6). The bars indicate mean values ± SEM (n=6). Statistical analysis was done using one-way ANOVA test, with Tukey’s multiple comparisons post-hoc test. *p<0.05, **p<0.01.
eISSN:
1661-4917
Language:
English
Publication timeframe:
Volume Open
Journal Subjects:
Medicine, Basic Medical Science, Biochemistry, Immunology, Clinical Medicine, other, Clinical Chemistry
Journal RSS Feed