Toll-like receptor 5 engagement modulates tumor development and growth in a mouse xenograft model of human colon cancer

Abstract

Background & aims: Toll-like receptor (TLR)-dependent signaling was proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Here, we investigated whether TLR5-dependent signaling modulates colonic tumor development in mouse xenograft model of human colon cancer.

Methods: The expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human colon cancer cells (DLD-1). Nude mice were subcutaneously implanted with MyD88-knocked down (KD), TLR5-KD, or control cells (n = 16) to examine the pathophysiology of tumor xenografts. Protein microarray assessed the differential expression of cytokines in these tumors. Leukocyte infiltration and tumor angiogenesis were assessed by immunohistochemistry with antibodies against neutrophil (Gr-1, 7/4) or macrophage-specific antigens (CD68, F4-80) and the vascular endothelial cell marker CD31, respectively. Tumor xenografts from DLD-1 cells were treated with flagellin (5.0 microg/kg, 1 injection/every 2 days for 3 weeks), and tumor regression and histopathology were examined.

Results: Lack of MyD88 or TLR5 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenografts of human colon cancer. In contrast, TLR5 activation by peritumoral flagellin treatment substantially increased tumor necrosis, leading to significant tumor regression. Tumors from MyD88-KD or TLR5-KD cells revealed the reduced production of neutrophil attracting chemokines (epithelial cell-derived neutrophil-activating peptide-78, macrophage-inflammatory protein alpha, and interleukin-8). Consequently, neutrophil infiltration was dramatically diminished in MyD88- or TLR5-KD xenografts, whereas tumor-associated macrophage infiltration or angiogenesis was not changed.

Conclusions: TLR5 engagement by flagellin mediates innate immunity and elicits potent antitumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate colonic tumors.

Figure 1. Generating MyD88-KD or MyD88-WT DLD-1 cells

(A) DLD-1 cells were stably transfected with a construct encoding shRNA against human MyD88 or a control vector. Since these vectors encode GFP fusion protein to confirm stable expression of an exogenous gene, stably transfected cells were identified by fluorescence microscopy. (B) Endogenous MyD88 expression was successfully silenced in several clones (clone number 2, 3, and 4). C, wild type control cells. (C) Lack of MyD88 expression blocked flagellin- or IL-1-induced NFκB-reporter activation, whereas, in wild type control cells, flagellin or IL-1 strongly stimulated NFκB activity. TNFα, stimulating MyD88-independent pathways, is still able to induce NFκB activation in MyD88-KD cells. RLA, relative luciferase activity. (D) Silencing MyD88 expression blocked IL-8 and MIP3α expression in response to flagellin measured by ELISA.

Figure 2. Blocking TLR5/MyD88-dependent signaling substantially enhanced the tumor growth in mouse xenografts model of human colon cancer

(A) Multi-spectral fluorescence (upper panel) was used for in vivo imaging of tumor xenografts from MyD88-KD or MyD88-WT DLD-1 cells(1.0 × 10⁶) and gross appearance of xenografts at day 21 was shown. Tumor volume of MyD88-KD and MyD88-WT xenografts was measured. * P = 0.014, n=16. (B) Tumor xenografts from TLR5-KD or TLR5-WT DLD-1 cells (1.6 × 10⁶) at day 21 were shown. Tumor volume of TLR5-KD or TLR5-WT xenografts was measured. * P = 0.006, n=16. (C and D) Tumors from MyD88-KD (C), TLR5-KD (D), or its control DLD-1 cells were excised at day 21 and tumor weight was evaluated. Each index in the ruler represents one mm.

Figure 3. Tumor necrosis and leukocytes infiltration were dramatically reduced in MyD88- or TLR5-knocked down tumor xenografts

(A and B) Tumor sections from MyD88-KD (A), TLR5-KD (B), or its control DLD-1 cells were stained with H&E (upper or lower images represent lower or higher magnification, respectively) to show necrotic or viable non-necrotic area. Percent necrotic surface area was measured using the Image-J software. Scale bar, 50 µm. Horizontal bar in graph represents median.

Figure 4. Neutrophil attracting cytokines were reduced in tumor xenografts from MyD88-KD cells

(A) MyD88-KD tumors showed the reduced expression of chemokines involved in attracting leukocytes. Equal amounts of total protein extracts (300 µg) from tumor xenografts (n=4/group) were used for human cytokine micro-array analysis (upper panel). Density of each spot was determined to analyze the expression ratio of each cytokine (MyD88-KD to MyD88-WT). Cytokines with significantly altered expression are presented (lower panel). (B) To confirm altered cytokine expression evaluated by micro-array analysis, we measured the level of ENA-78, MIP3α, or IL-8 protein by ELISA.

Figure 5. Neutrophil, not macrophage, infiltration was diminished in TLR5 or MyD88 deficient tumors

(A and B) Immunohistochemistry with antibodies against the neutrophil specific markers 7/4 and Gr-1 (A) or macrophage specific markers CD68 and F4-80 (B) visualized the infiltration of neutrophils or macrophages in tumors. (C) Micro-vessels in tumors were visualized by immunohistochemistry with antibody against PECAM-1/CD31 (Upper or lower panels indicate lower or higher magnification, respectively). Scale bar, 50 µm.

Figure 6. Peritumoral treatment with flagellin suppresses tumor growth

(A) Two days after DLD-1 cells(1.5 × 10⁶) were subcutaneously injected into nude mice, flagellin (5.0 µg/kg in 150µl) was administered around the tumor site (one injection/every 2 days for 3 weeks) followed by measuring tumor volume. * P = 0.0015, n=16. (B) Tumor xenografts were excised at day 21 and tumor weight was measured. (C) H&E staining of tumor sections showed more prevalent tumor necrosis in flagellin-treated tumors (upper or lower image represents lower or higher magnification, respectively). Scale bar, 50 µm. Horizontal bar in graph represents median. Veh, Vehicle; Fla, Flagellin.