In this year, Guangxi-U-China has conducted the engineering of 3 parts including scFv-Fc, SIRPαD1-Fc and acid inducible P-asr promoter for the designed “TuTaBa” bacteria. The three engineered parts are designed for specific targeting, phagocytic promotion, and acid inducible expression of intended functional genes. We have conducted the extraction of full-length RNA from breast cancer tumor tissues, reverse transcriptase-polymerase chain reaction (RT-PCR), generation of cDNA for gene cloning, construction of recombinant plasmid and translation in E. coli. Nissle 1917. Their full functions will be characterized in 2023.

Female nude model mice BALB/c that carry with transplanted 4T1 mammary carcinomas have been used for the extraction of full-length RNA samples. Figure 1 shows the electrophoretic images of extracted RNA with the optimized experimental protocols.

Figure 1. Gel electrophoretic imaging of extracted RNA with regular(A) and optimized protocol (B).

Then resultant RNA samples are further subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and generate cDNA for gene cloning. Figure 2 shows the electrophoretic image of cloned DNA and engineered bacteria.

Figure 2. Gel electrophoretic imaging of DNA and engineered bacteria from the 4^th DNA.

Gel pieces containing intended DNA were excised and subjected to further experiments for the recovery of DNA from gel slices with a gel dissolution method. Then conduct a DNA ligation to fuse the insert to the recipient plasmid. Competent cells DH5alpha are used for the transformation. Figure 2 shows that engineered bacteria were only obtained from the 4^th band DNA at this time.

We have demonstrated activities of RNases are different in normal and tumor tissues. Determination of RNases activities are important not only for the extraction of full-length RNA but also for the understanding of the biological roles of RNases.

Conventional methods involve the extractions of RNases from tissues by which the spatial distribution of enzymatic activities cannot be obtained. An in situ mass spectrometric imaging is established to visualize the activities of RNases in different regions of the tissue. Normal and cancerous tissues are sliced at 10 μm with a freezing microtome. Then either a piece of synthesized marker or endogenous small fragments of RNA can be used as references. Tissue slices are uniformly sprayed and covered with a solution of DHB (2, 4-dihydroxybenzoic Acid). A Nd³⁺:YAG pulsed laser beam (355 nm) scans across the tissue slice with a pixel size of 20 μm. DHB is used as a matrix material that can absorb laser energy, facilitate soft ionization and protect samples from direct laser ablation. RNases present in tissues cause the degradation of intact references into fragments. Detected intensities of the fragments of references are related with the activities of RNases. Figure 3 shows the mass spectrometric imaging of detected fragments in normal and cancerous tissue slices. It is shown there are heterogenous distribution of detected fragments GGA, GGG and CAG in tissue slices of both BALB/c 4T1 and BALB/c mice models.

Figure 3. Mass spectrometric images of three fragments in different tissue slices.

In summary, we developed a new method for the visualization of activities of RNases based on the detection of small degraded fragments. However, detected fragments of RNA may result from not only in vivo RNases but also in vitro degradation.

A high-performance liquid chromatographic (HPLC) method was developed in this project for the monitoring the dynamics of glucose and lactate metabolism in three different cell lines including MCF 10A, MCF7 and MDA-MB-231. These cells were treated with 3 inhibitors of key regulators glucose transporters, lactate transporters and lactate dehydrogenase. Samples have been collected at different growth time under the treatment with different inhibitors. Because the culture medium contains lots of chemicals, samples were loaded on a reversed phase HPLC C18 column and eluted with a mobile phase containing 30% acetonitrile and 70% water. The pH of the mobile was adjusted as 2 with HCl. Figure 4 shows the chemical structures of glucose and lactate. The detector was set at 210 nm and 285 nm for lactate and glucose, respectively.

Figure 4. Chemical structures of glucose and lactate

Because glucose does not have functional groups for ultraviolet absorption, it was converted to 5-hydroxymethylfurfural in 4.5 mol/L HCl solution at 100°C for 14 minutes as shown in Figure 5. Figure 6 and Figure 7 show the chromatograms of lactate and converted glucose, respectively.

Figure 5. Conversion of glucose to 5-hydroxymethylfurfural.

Figure 6. Chromatogram of lactate that was detected at 210 nm

Figure 7. Chromatogram of glucose that was converted to 5-hydroxymethylfurfural and detected at 285 nm.

A series of standard samples containing known amounts of glucose and lactate was quantified with HPLC. Figure 8 (A) and (B) shows the plots of peak areas vs quantities of lactate and glucose, respectively.

Figure 8. Plots of peak areas vs quantities of lactate (A) and glucose (B).

In this year, we have mainly set up the analytical procedure. Analysis of samples collected at different growth time under the treatment with different inhibitors have not been completed.

Our project developed a mass spectrometric imaging technique for spatial metabolome analysis of normal and cancerous mice models. It enables the localization of metabolomic changes in different tissue regions. Regular methods are based on the extraction of metabolites from the whole tissues, in which the spatial distribution information is lost. Spatial metabolomics enabled with mass spectrometric imaging (Figure 9) generates big hyper-spectral imaging data that provide rich information on disease progress. Tumor breast tissues (BALB/c 4T1) are sliced as 10 μm in a freezing microtome and then covered with a matrix material called 9-aminoacridine for mass spectrometric imaging in negative ion mode.

Figure 9. Mass spectrometric imaging with matrix assisted laser desorption ionization (MALDI)

Figure 10 shows the spatial distribution of representative metabolites on a tissue slice that clearly reveal the differences in tumorous and precancerous regions.

Figure 10. Mass spectrometric imaging of metabolites in a tissue slice from BALB/c 4T1 mouse model.

It shows that saturated fatty acids C16:0, C18:0, C20:0 and monounsaturated fatty acid C18:1are highly abundance in the tumorous region. There are highly heterogenous distribution of metabolites in different regions. In comparison, the spatial distribution of an unknown small molecule at m/z 232.943 is relatively uniform across the whole slice.

We have also applied the mass spectrometric imaging technique to the proteomic analysis of normal and tumor tissues of BALB/c mice models. Because the ionization and dissociation of intact proteins are difficult in the mass spectrometer, in situ digestion with trypsin was conducted before the tissue slices were covered with a matrix called α-cyano-4-hydroxycinnamic acid (CHCA) for laser irradiation. Mass spectrometric imaging was performed in the positive ion mode and tryptic peptides are detected. Figure 11 shows the spatial distribution of different peptides on tissue slices of BALB/c and BALB/c 4T1 mice, respectively.

Figure 11. The spatial distribution of peptides on tissue slices of BALB/c and BALB/c 4T1 mice.