identification of key factors mediating crosstalk between macrophages and breast cancer cells文献综述

1

SUMMARY OF THE RESEARCH PROPOSAL

(a)

For scientifically qualified non-expert assessors: (no more than 200 words).

Macrophages are closely associated with tumor cells, helping cancer metastasis (Kitamura et al, 2015). IL4R gene expression was found to be up-regulated in tumor associated macrophages(TAMs) (Anon, 2017). We hypothesize that tumor cells release important factors that cause IL4R upregulation and changes in its downstream signalling in macrophages. During in-vitro experiments, TGF-beta and CXCL12 were identifed to cause these changes in gene expression in macrophages. In this project, we will examine the role of these key factors in-vivo by knocking down corresponding genes and monitoring their effects on macrophages and tumor growth. Expected outcomes are suppressed tumor growth, lower IL4R expression and expression level changes of its downstream genes in macrophages compared to control groups.

Fluoresent-labeled inducible shRNAs will be transducted into luciferase-expressing breast tumor cells to knock down CXCL12 and TGF-beta, respectively. The tumor cells will be injected into mice, and tumor growth will be monitored by in-vivo bioluminescence imaging(BLI). The gene expression level in macrophages will be quantified by RT-PCR and western blot after clinical end point.

The conclusion will lead to better understanding of how macrophages associate with tumor cells. Furthermore, key factors in this pathway may provide potential targets for cancer therapy by inhibiting metastasis.

(b)

For lay readers: (no more than 50 words).

Macrophages are recruited to the tumor microenvironment and play important roles in helping metastasis. My project aims to identify the key factors involved in the crosstalk between macrophages and tumor cells by silencing corresponding genes in tumor cells and monitoring its effects on macrophages and tumor growth.

The honour project aimed to identify key factors mediating crosstalk between macrophages and breast tumor cells based on in-vitro experiments. The expected outcome was expression level changes of IL4R and its downstream genes in macrophages in “knockdown” tumor medium (genes silenced by RNAi), compared with that in “non-knockdown” medium.

This proposal is for following-up in-vivo experiments, in which we knockdown the key genes identified and see its effect on macrophages and tumor growth in living mice.

3.

RESEARCH QUESTION

(a)

What is the research question of your proposal? (no more than 50 words).

It should be specific to your proposal but understandable by non-experts.

What is the key factors released by tumor cells leading to the IL4R gene up-regulation and the expression level changes of its downstream genes in bone marrow-derived macrophages (BMMs)? What is the consequence of knocking down these key factor in living mice?

(b)

Why is this question important?

(no more than 150 words)

Metastasis is the main cause of death in cancer. Macrophages are closely associated with tumor cells in each step of the metastasis cascade (Kitamura et al, 2015). In order to understand the role of macrophages in cancer metastasis, it is important to identify the key factors mediating crosstalk between macrophages and tumor cells. This leads to a better understanding of how macrophages associate with tumor cells and its important role in bone metastasis cascade. Moreover, the key factors in this signalling pathway may potentially be targeted to inhibit the metastasis for cancer treatment.

The macrophage and tumor cell cultures we used in in-vitro experiments were isolated from its natural environment. The results might not accurately predict the conditions in a living mouse model. Therefore it is important to determine its in-vivo role in affecting the macrophages and tumor growth in a living mouse in its natural environment.

(a) Aims of the Project

As a following-up experiment of in-vitro experiments, my project aim to determine the role of the identified key factors in the interactions between macrophages and tumor cells in vivo, and how they affect the tumor growth and metastasis.

(b) Background to the project

Unpublished data in Qianrsquo;s lab showed that the expression of IL4R (interleukin-4 receptor) was up-regulated in the “tumor medium-cultured” bone marrow-derived macrophages (BMMs). As one of the ligands of IL4R, IL-4 is suspectible to cause the IL4R up-regulation.

However,it was found that breast tumor cells do not produce IL-4 (unpublished). Therefore, we hypothesize that other factors were produced by the breast tumor cells, leading to the up-regulation of IL4R gene in BMMs.

To address this question, in-vitro experiments were performed. Candidate genes encoding the suspectible factors (TGF-beta, CXCL12) were knocked down independently by RNAi, and the key factors of regulating the IL4R and its downstream gene expression in macrophages were identified.

(c) Experimental design and methods

We will use fluorescent-labeled inducible shRNAs to knock down certain genes (CXCL12, TGF-beta) expressed in tumor cells (Fellmann et al, 2013). In contrast with using transfection during in-vitro experiments, we will utilize retroviral vectors to deliver the shRNAs as the plasmids can be stably expressed in tumor cells (Fellmann et al, 2013). We will inject luciferase-expressing tumor cells into the tail vein of mice and monitor the tumor growth by in-vivo bioluminescence imaging(BLI) 7 days after injection (Kitamura et al, 2015,2). Upon administration of inducer drug, the targeted genes (such as TGF-beta and CXCL12) are expected be silenced, and this can be monitored by the fluorescence detected in intravital imaging. BLI will be performed twice per week to monitor the tumor growth until the clinical end points (3 weeks) (Kitamura et al, 2015,2). Mice will be killed and tissue will be extracted at the clinical end point. The gene expression will be quantified using RT-PCR amp; qPCR and western blot.

(d) Expected outcomes and Timeline

Expected outcomes:

As tested in vivo, the knockdown of TGF-beta or CXCL12 gene is expected to cause the IL4R gene downregulation and changes in its downstream gene expression level in macrophages, and suppress the breast tumor growth and metastasis. Expected results to be collected are listed below:

1. Fluorescence detected in intravital imaging suggests the successful knockdown.
2. In vivo BLI shows suppressed tumor growth.
3. RT-PCR and western blot show lower expression of IL4R and the expression level changes of its downstream genes in macrophages compared to control groups.

Timeline:

• 6 weeks - Retroviral vector construction
• 6 weeks - Transduction
• 7 months - in-vivo test: Approximately 4-5 rounds, 6 weeks per round

Tumor injection - knockdown induction(1 week) - clinical end point (4 weeks), gene expression level quantification (1 week)

• 2 months - Data analysis and report

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Anon (2017) Findings from S.MP. Vadevoo and Co-Authors Broaden Understanding of Proteomics (IL4 Receptor-Targeted Proapoptotic Peptide Blocks Tumor Growth and Metastasis by Enhancing Antitumor Immunity). Health amp; Medicine Week, p.684.

Fellmann C, Hoffmann T, Sridhar V, Hopfgartner B, Muhar M et al. (2013) An Optimized microRNA Backbone for Effective Single-Copy RNAi. Cell Reports 5: 1704-1713

Kitamura T, Qian B amp; Pollard J (2015) Immune cell promotion of metastasis. Nature Reviews Immunology 15: 73-86

Kitamura T, Qian B, Soong D, Cassetta L, Noy R et al. (2015,2) CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages. The Journal of Experimental Medicine 212: 1043-1059