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. 2020 Feb 5;12(2):368.
doi: 10.3390/cancers12020368.

Immune Responses Raised in an Experimental Colon Carcinoma Model Following Oral Administration of Lactobacillus casei

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Free PMC article

Immune Responses Raised in an Experimental Colon Carcinoma Model Following Oral Administration of Lactobacillus casei

Georgios Aindelis et al. Cancers (Basel). .
Free PMC article

Abstract

The role of dietary probiotic strains on host anticancer immune responses against experimental colon carcinoma was investigated. We have previously shown that Lactobacillus casei administration led to tumor growth suppression in an experimental colon cancer model. Here, we investigated the underlying immune mechanisms involved in this tumorgrowth inhibitory effect. BALB/c mice received daily live lactobacilli per os prior to the establishment of a syngeneic subcutaneous CT26 tumor. Tumor volume, cytokine production, T cell differentiation and migration, as well as tumor cell apoptosis were examined to outline potential immunomodulatory effects following L. casei oral intake. Probiotic administration in mice resulted in a significant increase in interferon gamma (IFNγ), Granzyme B and chemokine production in the tumor tissue as well as enhanced CD8+ T cell infiltration, accompanied by a suppression of tumor growth. Cytotoxic activity against cancer cells was enhanced in probioticfed compared to control mice, as evidenced by the elevation of apoptotic markers, such as cleaved caspase 3 and poly (ADPribose) polymerase 1 (PARP1), in tumor tissue. Oral administration of Lactobacillus casei induced potent Th1 immune responses and cytotoxic T cell infiltration in the tumor tissue of tumorbearing mice, resulting in tumor growth inhibition. Thus, the microorganism may hold promise as a novel dietary immunoadjuvant in raising protective anticancer immune responses.

Keywords: Lactobacillus casei; immune responses; oral administration; probiotics; syngeneic murine colon carcinoma.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Oral administration of Lactobacillus casei (LC) suppressed subcutaneous tumor growth in BALB/c mice. (a) Schematic representation of the in vivo tumor model. Female BALB/c mice received daily 109 CFU of live L. casei per os for 13 days (n = 9/10). Control group received PBS (n = 9/10). On day 10, 5 × 106 CT26 cells were injected subcutaneously. Growing tumors were excised 7 days post-inoculation of CT26 cells; (b) Representative photographic presentation of tumors from both groups; (c) Diagram showing mean tumor volume inhibition in probiotic-fed mice (LC) compared to control mice. *, p < 0.05, groups were repeated three times.
Figure 2
Figure 2
Effect of Lactobacillus casei administration on the ex vivo cytokine production. BALB/c mice received daily 109 CFU of live L. casei for 13 days (n = 4/5). Control group received PBS (n = 4/5). On day ten (10) of oral administration, 5 × 106 CT26 cells were injected subcutaneously. PP were excised after euthanasia three (d13 from start) or seven (d17 frorm start) days post-inoculation of CT26 cells. Cytokine levels of (a) IFN-γ, (b) IL-12 and (c) IL-10 were quantified by ELISA in a PP lysate of L. casei (LC)-fed mice as compared to the respective lysate of PBS-fed control mice. Cytokine production was determined at the indicated time points by ELISA following the manufacturer’s instructions. *, p < 0.05, groups were repeated two times.
Figure 3
Figure 3
Effect of Lactobacillus casei administration on the percentage of effector immune cells in the spleen. BALB/c mice received daily 109 CFU of live L. casei per os for 13 days (n = 4/5). Control group received PBS (n = 4/5). On day ten (10) of oral administration, 5 × 106 CT26 cells were injected subcutaneously. Spleen cells were isolated from mice three (d13 from start) or seven (d17 from start) days post CT26 injection. Cells isolated at d17 were analyzed with flow cytometry. Isolated cells from both time points were co-cultured ex vivo with 108 CFU/mL of L. casei for 48 h. (a) Representative dot plots for each subtype of immune cells (CD8+ T cells, CD4+ T cells, NK cells) in the spleen; (b) Frequencies of immune cell subtypes present in the spleen; (c) Cytokine production of spleen cells isolated from probiotic-fed mice at d13 or d17. *, p < 0.05, groups were repeated two times.
Figure 4
Figure 4
Dietary oral administration of probiotic Lactobacillus casei (LC) resulted in distinct immunoadjuvant and pro-apoptotic activities in tumor-bearing mice. Excised tumors from tumor-bearing LC-treated or control (PBS-treated) mice (n = 6) were either fixed in formalin, cut in sections, stained and observed under a microscope or mechanically homogenized. Homogenized tumors were further treated for protein isolation and analysis with ELISA and western blot, or enzymatically digested for cell isolation and flow cytometry analysis. (a) Cytokine production (IFN-γ, IL-12p40, Granzyme B) in the tumor tissue excised from treated animals as compared to tumor tissue from control mice; (b) Representative dot plots showing the percentage of each subtype in TILs; (c) Diagram of the frequency of TILs as percentage of mean infiltration; (d) Western blot analysis for the cleavage of caspase 3 and PARP1. Please note the increase in cleaved caspase 3 and cleaved PARP1 in LC-treated mice as compared to PBS-treated mice; (e) Representative images of tumor sections from control and LC-treated mice, stained for cleaved caspase 3. Please note the increase in the percentage of positive cells for caspase 3 activation in sections originating from animals that received oral administration of live L. casei. *, p < 0.05, groups were repeated three times.
Figure 5
Figure 5
Administration of probiotic Lactobacillus casei (LC) resulted in increased production of various interleukins and chemotactic factors in the tumor. Tumors were homogenized, three tumors per group were pooled, and then analyzed for protein expression with a commercially available proteome profiler array. (a) Images of dot plots. Duplicate dots represent a protein expressed in tumor tissues of LC-fed or PBS-fed mice; (b) Both plots were scanned and signal intensity, normalized against positive controls provided by the manufacturer, was determined. Note the elevated levels of IFN-γ that was previously detected, and the accumulation of interleukins, like IL-1b and IL-16 and chemotactic agents such as CXCL9, CXCL10 and CXCL11 as well as CCL3, CCL4 and CCL5.

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