Table 1.
Patient characteristics.
Fig 1.
Biosynthesis of α-gal epitopes on pancreatic ductal adenocarcinoma (PDAC) tumor lysates and normal pancreatic tissue lysates assessed by Western blot analysis.
Pig kidney membrane was employed as the positive control for α-gal epitope expression. High expression of α-gal epitopes was observed in both processed normal tissue and PDAC tumor lysates. No expression of α-gal epitopes was detected in both unprocessed normal tissue and PDAC tumor lysates.
Fig 2.
ELISA analysis of anti-PDAC cell IgG production induced by tumor lysate vaccination.
(a) Anti-PANC-1 IgG production, (b) anti-MIAPaCa-2 IgG production, (c) anti-BxPC-3 IgG production. Vaccination with α-gal(+) PDAC-ly resulted in marked increase in the production of anti-PDAC cell IgG compared with α-gal(-) PDAC-ly vaccination. Vaccinations with either α-gal(-) or α-gal(+) N-ly did not elicit a significant anti-PDAC cell IgG response. Representative data from ten experiments with similar results are shown. ELISA results represent one or three data sets from n = 10 mice/group (one data set: naïve KO mice, α-gal(-) or α-gal(+) N-ly; three data sets: α-gal(-) or α-gal(+) PDAC-ly).
Fig 3.
Anti-PDAC cell IgG subclasses production induced by tumor lysate vaccination.
(a) Induced anti-PANC-1 IgG subclasses. (b) Induced anti-MIAPaCa-2 IgG subclasses. (c) Induced anti-BxPC-3 IgG subclasses. Representative data from five experiments with similar results are shown. ELISA results represent one data set from a group of 10 vaccinated mice.
Fig 4.
Anti-MUC1 IgG and anti-mesothelin IgG production induced by tumor lysate vaccination.
(a) ELISA for anti-MUC1 IgG production. (b) ELISA for anti-mesothelin IgG production. Representative data from ten experiments with similar results are shown. ELISA results represent one or three data sets from n = 10 mice/group (one data set: naïve KO mice, α-gal(-) or α-gal(+) N-ly; three data sets: α-gal(-) or α-gal(+) PDAC-ly).
Fig 5.
B cell and T cell expansion in response to tumor lysate vaccination.
ELISPOT assays for (a) anti-MUC1 Ab secreting B cells and (b) MUC1-specific activated T cells and in vitro and in vivo depletion of CD8+ T cells, detected as IFN-γ secreting lymphocytes. ELISPOT assays for (c) anti-mesothelin Ab secreting B cells and (d) mesothelin-specific activated T cells and in vitro and in vivo depletion of CD8+ T cells, detected as IFN-γ secreting lymphocytes. Data represent the mean ± SD of five independent splenocyte preparations; bars, SD. Statistical analyses were performed using Student’s t-test. N.S.: not significant.
Fig 6.
In vivo tumor growth and survival of adoptive transferred NOD/SCID mice challenged with live PANC-1 cells.
(a) Size of subcutaneous tumors of NOD/SCID mice to which splenocytes were adoptively transferred from normal pancreatic tissue lysate vaccinated KO mice. □: α-gal(+) N-ly vaccinated KO mice, △: α-gal(-) N-ly vaccinated KO mice. The tumor sizes of five individual recipients in each group after adoptive transfer are shown. (b) Size of subcutaneous tumors of NOD/SCID mice to which splenocytes were adoptively transferred from PDAC tumor lysate vaccinated KO mice. ●: α-gal(+) PDAC-ly vaccinated KO mice, ○: α-gal(-) PDAC-ly vaccinated KO mice. The tumor sizes of five individual recipients in each group after adoptive transfer are shown. S: sacrifice, †: cancer death. (c) Survival curves of adoptive transferred NOD/SCID mice after tumor cell challenge with live PANC-1 cells. The curves were generated by the Kaplan-Meier method and assessed by the log-rank test.
Table 2.
The in vivo antitumor response against live PANC-1 cells in adoptive transferred NOD/SCID mice.
Table 3.
In vivo kidney and liver function before/after tumor lysate vaccination in high anti-Gal KO mice.
Fig 7.
α-gal(+) PDAC-tumor lysate vaccination induced the infiltration of lymphocytes (CD4+ and CD8+ T cells and macrophages) into subcutaneous PANC-1 tumors.
No infiltration of lymphocytes was observed in the α-gal(-) N-ly/α-gal(+) N-ly vaccinated groups and α-gal(-) PDAC-ly vaccinated group. However, infiltration by substantial numbers of both CD4+ and CD8+ T cells and macrophages was observed in the α-gal(+) PDAC-ly vaccinated group. H&E stained sections of the α-gal(+) PDAC-ly vaccinated group demonstrate severe destruction of PDAC cells elicited by effector cell infiltration. Representative images of five individual recipients in each group are shown. Scale bars = 100 µm.
Fig 8.
Antibody production against PANC-1 cells and MUC1 peptide induced by tumor lysates originating from patients treated with or without neoadjuvant chemoradiotherapy.
(a) Anti-PANC-1 IgG production induced by tumor lysate vaccines generated from patients treated with or without NACRT. (b) Anti-MUC1 IgG production induced by tumor lysate vaccines generated from patients treated with or without NACRT. +: not significant between α-gal(+) PDAC-ly treated with NACRT vaccinated group and α-gal(+) PDAC-ly treated without NACRT vaccinated group. Representative data from five experiments with similar results are shown. ELISA results represent two data sets from n = 5 mice/group.
Fig 9.
Immunohistological findings of original PDAC tumors obtained from patients treated with or without neoadjuvant chemoradiotherapy.
H&E stained sections of PDAC tumors clearly demonstrate viable PDAC cells in the tumor treated without NACRT. However, grade IIa destruction of PDAC cells (Evans classification) was detected in the tumor treated with NACRT. Expression of MUC1 and mesothelin was observed in PDAC tumors treated with or without NACRT. The expression levels of these TAAs were similar between PDAC tumors treated with or without NACRT. Representative images of four individual patients are shown. Scale bars = 100 μm.