Peptide-fluorescent bacteria complex as luminescent reagents for cancer diagnosis

Abstract

Currently in clinic, people use hematoxylin and eosin stain (H&E stain) and immunohistochemistry methods to identify the generation and genre of cancers for human pathological samples. Since these methods are inaccurate and time consuming, developing a rapid and accurate method to detect cancer is urgently demanded. In our study, binding peptides for lung cancer cell line A549 were identified using bacteria surface display method. With those binding peptides for A549 cells on the surface, the fluorescent bacteria (Escherichia coli with stably expressed green fluorescent protein) were served as specific detecting reagents for the diagnosis of cancers. The binding activity of peptide-fluorescent bacteria complex was confirmed by detached cancer cells, attached cancer cells and mice tumor xenograft samples. A unique fixation method was developed for peptide-bacteria complex in order to make this complex more feasible for the clinic use. This peptide-fluorescent bacteria complex has great potential to become a new diagnostic tool for clinical application.

Figure 1. The process of screening and enriching the binding peptide library for cancer cells with bacteria surface display methods.

(A) Schematic representation of peptides screening and selection with bacterial display library. 1. Constructed the library by transforming plasmids into E.coli MC1061; 2. Discarded the bacteria binding with normal cells after pre-incubation; 3. Incubated the mixture of cancer cells and residual bacteria; 4. Analyzed the binding effect of cancer cells with bacteria using FACS; 5. Sorted the binding bacteria to cancer cells by flow cytometry and cultured the binding bacteria in medium; 6. Performed the next round binding bacteria screening; 7. Isolated the binding bacteria to cancer cells and sequenced the peptides displayed on the surface of the bacteria. (B) The progress of enrichment of bacteria binding with cancer cells by FACS in 6 screening rounds.

Figure 2. FACS results of selected monoclonal peptide-fluorescent bacteria binding with A549 cells and HLF cells.

The binding fraction of A549 cells with bacteria (show in the orange gate) was 80% and that of HLF cells was 0.4%.

Figure 3. The results of selected monoclonal peptide-fluorescent bacteria specifically binding with various cells.

(A) Fluorescence microscope images of bacteria clones incubated with cells. A549^△ was incubated with CPX only bacteria and other cells were incubated with selected monoclonal peptide-fluorescent bacteria, the scale bar was 20 µm. (B) FACS results of various cells binding with monoclonal peptide-fluorescent bacteria at ratio 1∶100. (C) Percentage of binding fraction of monoclonal peptide-fluorescent bacteria with various cells from FACS data. Data were mean ± S.D. of at least three independent experiments.

Figure 4. Characterization of binding ability of fixed bacteria.

(A) Percentage of binding fraction of fresh and fixed monoclonal peptide-fluorescent bacteria with various cells from FACS data. Cells binding to fresh bacteria (black) at ratio 1∶100 and binding to fixed bacteria (gray) at ratio 1∶500. (B) The fluorescence microscope images of A549 cells incubating with fresh bacteria and fixed bacteria at ratio 1∶500, at day 1, day 7 and day 30 after fixation and the scale bar was 20 µm. (C) FACS results of A549 cells binding with fresh and fixed monoclonal peptide-fluorescent bacteria at ratio 1∶500 at different time points. (D) Percentage of binding fraction of fresh and fixed monoclonal peptide-fluorescent bacteria with A549 cells from FACS data Cells binding with fresh bacteria and fixed bacteria at ratio 1∶500 at different time points. Data were mean ± S.D. of at least three independent experiments.

Figure 5. The representative images of selected peptide-fluorescent bacteria binding with tissue sections from the mice xenograft.

The scale bar was 20 µm. Data were representative of at least three independent experiments.