Efficacy of tumor-targeting Salmonella typhimurium A1-R in combination with anti-angiogenesis therapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX) and cell line mouse models

Abstract

The aim of the present study was to examine the efficacy of tumor-targeting Salmonella typhimurium A1-R treatment following anti-vascular endothelial growth factor (VEGF) therapy on VEGF-positive human pancreatic cancer. A pancreatic cancer patient-derived orthotopic xenograft (PDOX) that was VEGF-positive and an orthotopic VEGF-positive human pancreatic cancer cell line (MiaPaCa-2-GFP) as well as a VEGF-negative cell line (Panc-1) were tested. Nude mice with these tumors were treated with gemcitabine (GEM), bevacizumab (BEV), and S. typhimurium A1-R. BEV/GEM followed by S. typhimurium A1-R significantly reduced tumor weight compared to BEV/GEM treatment alone in the PDOX and MiaPaCa-2 models. Neither treatment was as effective in the VEGF-negative model as in the VEGF-positive models. These results demonstrate that S. typhimurium A1-R following anti-angiogenic therapy is effective on pancreatic cancer including the PDOX model, suggesting its clinical potential.

Figure 1. mRNA expression of VEGFA (A), VEGFR1 (B) and VEGFR2 (C) in pancreatic cancer cell lines

mRNA expression was determined with real-time RT-PCR. MiaPaCa-2 significantly expressed VEGFA more than other cell lines (p < 0.001) except for BxPC-3 (p = 0.558) (A). MiaPaCa-2 significantly expressed VEGFR2 more than other the cell lines (BxPC-3: p = 0.005, Capan-1: p < 0.001; Hs766T: p = 0.005; and Panc-1: p = 0.006) (C). VEGFR1 expression was not detected in MiaPaCa-2 and Capan-1 cell lines (B). Data for each treatment are represented as the mean ± SD. ** p < 0.01.

Figure 2. Efficacy of S. typhimurium A1-R on pancreatic cancer cell lines

(A) Confocal imaging of MiaPaCa-2 and Panc-1 pancreatic cancer cells infected with S. typhimurium A1-R in vitro. S. typhimurium A1-R infection was detected in both pancreatic cancer cell types after 60 min. S. typhimurium A1-R replicated in the cells after 120 min. S. typhimurium A1-R showed the ability to infect and induce apoptosis in both cell types after 24 hr. Scale bars: 100 μm (pre); 50 μm (60 and 120 min); 25 μm (24 hr). (B and C) MiaPaCa-2 and Panc-1 were treated with S. typhimurium A1-R. Clonogenic assays show that S. typhimurium A1-R significantly reduced colony formation of both pancreatic cancer cell lines compared to the control groups in vitro (MiaPaCa-2: p = 0.001 and Panc-1: p < 0.001). ** p < 0.01.

Figure 3. Efficacy of BEV treatment on pancreatic cancer cell lines which have different levels of VEGFA and VEGFR2 expression

(A) Schema of treatments on subcutaneous tumors. Based on RT-PCR results, MiaPaCa-2 was determined to be VEGFA-positive and VEGFR2-positive. Panc-1 was determined as VEGFA-negative and VEGFR2-negative. In order to determine the efficacy of BEV on pancreatic cancer cell lines with different levels of VEGFA and VEGFR2 expression, subcutaneous tumors from MiaPaCa-2 and Panc-1 cells were grown in nude mice and randomized to 3 groups as described in the Materials and Methods. (B) BEV significantly reduced the growth of the MiaPaCa-2 tumor compared to the control on Day 22 (p < 0.001). Both BEV → GEM and BEV → S. typhimurium A1-R treatments significantly reduced MiaPaCa-2 tumor growth compared to the control group on Day 43 (p = 0.001). BEV → S. typhimurium A1-R significantly reduced the MiaPaCa-2 tumor growth compared to BEV → GEM (p = 0.037). (C) BEV did not reduce the tumor growth of Panc-1 compared to the control on Day 22, but both BEV → GEM and BEV → S. typhimurium A1-R treatment significantly reduced tumor growth compared to the control on Day 43 (BEV → GEM: p = 0.023; BEV → S. typhimurium A1-R: p = 0.026). * p < 0.05.

Figure 4. Microvessel density (MVD) in s.c. pancreatic cancer xenografts in nude mice treated with bevacizumab (BEV)

To determine MVD, sections were stained with an antibody to CD31 as described in the Materials and Methods. MVD was determined by counting the number of CD31-positive vessels in three fields at ×100 magnification of the highest vascular density. The values are the average numbers of microvessels ± S.D. (bars) of five different tumors. (A-D) Representative images of frozen sections stained with anti-CD31 antibody. The number of vessels in MiaPaCa-2 tumors treated with BEV (B) was reduced compared to the control (A). In contrast, there was no difference between Panc-1 tumors treated with BEV (D) and control (C). Scale bars: 200 μm. (E and F) Bar graphs of MVD in pancreatic tumors with and without BEV treatment. BEV significantly reduced the MVD of MiaPaCa-2 compared to the control (p = 0.002) (E), but did not reduce the MVD of Panc-1 (F). ** p < 0.01.

Figure 5. Distribution of GFP-labeled S. typhimurium A1-R bacteria in tumors and organs

Livers and tumors were minced and mixed with PBS, as was blood. The PBS, was plated on LB agar to identify S. typhimurium A1-R in each tissue. Fluorescent S. typhimurium A1-R GFP colonies were observed with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). Representative images of GFP-labeled S. typhimurium A1-R cultured from the tumor and the normal organs (blood and liver) of the mice in the BEV → S. typhimurium A1-R and BEV → GEM groups. GFP-labeled S. typhimurium A1-R bacteria were clearly detected in both MiaPaCa-2 tumors (A) and Panc-1 tumors (B) in the BEV → S. typhimurium A1-R groups. No GFP-labeled S. typhimurium A1-R was detected in other tissues. Scale bars: 10 mm. BF= brightfield

Figure 6. S. typhimurium A1-R treatment following BEV and GEM combination therapy of the MiaPaCa-2-GFP tumor in an orthotopic mouse model

(A) Schema of treatment of orthotopic MiaPaCa-2-GFP tumors. After confirmation of tumor growth by imaging with the OV100, the mice with orthotopic tumors were randomized to 4 groups: (G₁) saline (vehicle/control, 4 weeks); (G₂) GEM (4 weeks); (G₃) BEV (4 weeks) / GEM (4 weeks); and (G₄) BEV (2 weeks) / GEM (2 weeks) → S. typhimurium A1-R (2 weeks). Animals underwent laparotomy on day 36, and the tumors were imaged with the OV100 and weighed and harvested for analysis. (B) Representative images at laparotomy. Upper panels indicate bright-field images (BF) and lower panels indicate GFP fluorescence images. A large primary tumor and many metastases spreading over the entire abdominal cavity were detected in the control group. Many metastases were found in the mice treated with GEM but rarely found in the BEV/GEM and BEV/GEM → S. typhimurium A1-R groups. All regimens significantly reduced tumor weight compared to the untreated control group (G₂: p < 0.001; G₃: p < 0.001; G₄: p = 0.001). BEV/GEM significantly reduced tumor weight compared to GEM (p = 0.038). BEV/GEM → S. typhimurium A1-R significantly reduced tumor weight compared to GEM (p = 0.012) and BEV/GEM (p = 0.029)

Figure 7. S. typhimurium A1-R treatment following BEV and GEM combination therapy on a VEGF-positive pancreatic cancer PDOX

(A) Histological characterization of the PDOX. The PDOX was diagnosed as moderately-differentiated adenocarcinoma with H&E staining, and was strongly stained with an anti-VEGF antibody. Scale bars: 100 μm. (B) Representative images at laparotomy. The areas surrounded by the yellow broken lines indicate the primary tumors, and yellow arrow heads indicate metastasis. A large primary tumor and several metastases were detected in the control group (G₁). A few metastases were found in the mice treated with GEM (G₂) but rarely found in the BEV/GEM (G₃) and BEV/GEM → S. typhimurium A1-R groups (G₄). (C) Bar graphs of the total tumor weight (primary + metastasis) in each group. All regimens significantly reduced the tumor weight compared to the control group (G₂: p = 0.004; G₃: p = 0.002; G₄: p = 0.001). BEV/GEM significantly reduced tumor weight compared to GEM (p = 0.005). BEV/GEM → S. typhimurium A1-R significantly reduced the tumor weight compared to GEM (p = 0.001) and BEV/GEM (p = 0.029)