Team:Worldshaper-Wuhan/Implementation

<!DOCTYPE html> Proposed Implementation

Cell-free system

Circulating tumor cells (CTCs)

Reference

Proposed Implementaion

Breast cancer is the most common cancer among women and is the second cancer frequently occurring worldwide of newly-diagnosed cancers. As mentoned in our HP part, even if the detection methods for breast cancer are already mature enough, for most of the time, breast cancer patients examined and confirmed by the hospital are already in the middle or advanced stage. One of the reason is that some health screening process does not include breast cancer detection.since all health check-up packages require blood draws, if we can also test for breast cancer simply by drawing blood, then breast cancer testing can easily be attached to nearly all health examination packages, we are trying to design a method: Breast cancer detection can be done by simple blood test.

Proposed implementation: Cell-free system

Previous study by Guofeng Bian and Aijun Chen showed that The change trend of miR-155 expression level in serum is consistent with the change trend of miR-155 expression level in tissues. And the miR-155 in serum can reflect the occurrence of resonance miR-155 may be used as a tumor marker for early diagnosis.[1] Therefore, we do not need to collect the patient's cell tissue, only extract the patient's serum and detect the content of mir-155 to meet the non-invasive requirements and achieve the purpose of rapid diagnosis of breast cancer. Non-invasive, fast and accurate early diagnosis can help patients with early detection and early treatment. Through the use of CHO (Chinese hamster ovary cell), a mammalian cell-free expression system, it has rapid and accurate post-transcriptional modification function, high amplification, and expression capacity of exogenous recombinant genes, and high tolerance of shear force and osmotic pressure [2].

Fig.1 Cell-Free Protein Synthesis [3]

We propose that the miR-155 biosensor we desinged can be added into the cell-free expression system together with serum samples isolated from breast cancer patients. After incubation, the GFP fluorescence would be valued by quick visual detection or plate reader (SpectraMax i3) which indicates the expression of miR-155.

The complete process of our proposed implementation is as follows:

Step1: extract and purify patient‘s serum to obtain sample

Step2: patient sample + miR-155 biosensor ---add into cell-free system

Step3: incubate for 90min at 30℃

Step4: Fluorescence signal detection

Step5: calculate the expression level of miR-155

Fig.2 The complete process of our proposed implementation

Another we want to do is to evaluate the effect of chemotherapy and radiotherapy in breast cancer patients. Since Resistance to chemotherapy and radiotherapy remains the major factor for treatment failure and death in breast cancer patients. Thus, there is an urgent search for new, non-invasive, biomarkers to evaluate the effect of chemotherapy and radiotherapy in breast cancer patients.

A growing number of studies highlights the role of miR-155 in breast cancer drug resistance development. Previous study by Jiangcheng Zuo et.al shows that miR-155 is an effective therapeutic target of breast cancer. Their study also shows that Inhibition of miR-155 rendered MDA-MB-231 cells more sensitive to Doxorubicin, which resulted in an increase of inhibition rate from 20.23% to 68.72%. The expression of miR-155 not only was a therapeutic target but also was associated with cancer stem cell formation and Doxorubicin sensitivity. Their results underscore the importance of miR-155 as a therapeutic target and combination of doxorubicin and miR-155-silencing would be a potential way to cure breast cancer. [4]

Doxorubicin is considered to be the most effective agent in the treatment of breast cancer patients. Unfortunately, resistance to this agent is common, representing a major obstacle to successful treatment. The identification of novel biomarkers that are able to predict treatment response may allow therapy to be tailored to individual patients. [5]

Therefore, we come up with the assuming that by using mir-155 as the biomarkers of the breast cancer patient to predict treatment response to individual patients.

In our experiment, we use GFP as the signal marker for mir-155 detection, and detect the difference in mir-155 expression between tumor cells and normal cells by the strength of the GFP signal.

Our study shows that:

1. the GFP value of cells transfected with pEGFP-miR-155-sponge-1 could show the different expression of miRNAs in cells.

2. pEGFP-miR-155-sponge-1 could act as a monitor to detect the expression of miR-155.

3. We find that the value of fluorescence is dependent on the copy numbers of miR-155 in cells. Which means that the Fluorescence value is changes with the expression level of miR-155.

Base on the study above，we proposed another implementation：Circulating tumor cells (CTCs)

In our experiment, MDA-MB 231 breast cancer cells and MDA-MB 468 breast cancer cells were used to measure the expression of miR-155 by using miR-155 sensor.

This system will help doctors diagnose patients with breast cancer earlier and evaluate the effect of chemotherapy and radiotherapy in the future. Now, the application is just stay in the laboratory stage and will be applied in practice in the future. We look forward to the prospects of the future application.

Primary tumor and/or metastasized sites release a small number of tumor cells into the blood circulation. Circulating tumor cells (CTCs) are tumor cells that are shed from the primary tumor, flowing through the bloodstream and circulate throughout the body. Numerous studies in the past decade have shown that CTCs may be used as a marker to predict disease progression and survival in metastatic and possibly even in early-stage cancer patients. CTCs could be isolated based on physical and biochemical methods. The isolation and purification of tumor cells also was used immunomagnetic bead-based cell isolation methods.

Fig.3 Clinical applications of cell-free DNA analysis. cfDNA can be used in (1) diagnosis (2,3) to detect residual disease after surgery, (4) to monitor the response to therapy and (5) follow-up, and (6) to detect resistance. cfDNA, cell-free DNA; CTC, circulating tumor cells. [6]

We can isolate CTCs from breast cancer patients by different methods, for example the products of VyCAP (https://www.vycap.com/), and culture tumor cells in the lab. The plasmids of pEGFP-miR-155-sponge-1 can be transfected into CTCs by using electroporation transformation or infected with recombinant adenovirus. After transfection, cells will be measured the value of GFP fluorescence by plate reader (SpectraMax i3). The value of GFP fluorescence represents the expression of miR-155, which can be evaluate the effect of chemotherapy and radiotherapy in breast cancer patients.Also, we can use this method for breast cancer treatment by dialysis of miR-155 in the blood.

Next, we will gradually verify our proposed implementation through experiments.

Reference：

[1] Study on the correlation between the expression levels of Mir-21 and MiR-155 in blood and tumor tissues of breast cancer patients. Bian Guofeng, Chen Aijun. Hainan Medical Journal2012,23(18) [ISSN] 1003-6350 [DOI] 10.3969/j.issn.1003-6350.2012.18.001)

[2] https://zhuanlan.zhihu.com/p/163206925 )

[3] Yi，Jiao，Yang，Liu，Dan，Luo，Wilhelm，T，S，Huck，Dayong. Microfluidic-Assisted Fabrication of Clay Microgels for Cell-Free Protein Synthesis. ACS Applied Materials & Interfaces. )

[5] Smith L, Watson MB, O'Kane SL, Drew PJ, Lind MJ, Cawkwell L. The analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays. Mol Cancer Ther. 2006 Aug;5(8):2115-20. doi: 10.1158/1535-7163.MCT-06-0190. PMID: 16928833. )

[6] Giulia Siravegna & Alberto Bardelli. Genotyping cell-free tumor DNA in the blood to detect residual disease and drug resistance. Genome Biology volume 15, Article number: 449 (2014)