Saturday, June 9, 2012

Targeting Tumor Associated Macrophages

Figure 6(A) Elimination of TAMs results in
a reduction in tumor angiogenesis (1)

In our cancer project "Host Immune System Enhancing Tumor Growth" as you may recall we went over an interesting immunotherapy experiment in which targeted tumor associated macrophages (TAMS) were destroyed by targeting legumain to identify target molecules. Here I will take a more in-depth analysis of this experiment to better understand it's pros and cons.

This experiment found that M2 (pro-tumor) macrophages were found to highly overexpress the stress protein legumain, and the M1 (anti-tumor) did not. This meant that the tumor associated macrophages that promote angiogenesis as well as produce mitogenic factors for metastasis can be specifically targeted and destroyed due to their over-expression of legumain. The gene encoding legumain was fused to the c terminus of mutant polyubiquitin along with Kozak ATG (start codon) and pCMV. Now when this drug was administered the amount of M2 TAMs expressing legumain were  completely abrogated from the tumor microenvironment. This removal of TAMs resulted in major downregulation of growth factors and pro-angiogenic factors. The destruction of the TAMs was explained to be as a result of calling cytotoxic T cells to the tumor to destroy them.

The following Figure (4a) shows how the destruction of TAMs by cytotoxic T cells occured: 

Figure 4: MHC class I antigen–restricted specific CD8+ T cell response against legumain-expressing cells. (A)DNA vaccination enhances expression of costimulatory molecules by DCs. Lymphocytes from Peyer’s patches obtained 3 days after vaccination were stained with FITC-labeled anti-CD11cAb in combination with PE-conjugated anti-CD80, anti–MHC class I, or anti-CD40 Abs. *P < 0.05 compared with control groups.(1)

Lymphocytes were treated with control and the drug and were analyzed for CD11c, CD40, CD80, and MHC class I molecules. CD11c is an integrin found on the surface of dendritic cells, and other immune cells including macrophage. It's function is to signal and regulate many factors such as inflammation and cytoxicity (2). CD40 and CD80, are both costimulatory proteins that are required for antigen presenting cell activation. Antigen presenting cells function to alert the rest of the immune system to the pathogens. MHC class I molecules are major histocompatibility molecules basically provide the immune system with the ability to find out what is happening within our cells from extracellular surface. MHC proteins will detect proteins within the cell and express them as antigens on the cell surface, allowing the immune system to differentiate between self antigens vs. foreign antigens (3). Knowing the properties of the molecules that the cells were stained for we can deduce that the higher the expression of these molecules, the more cytotoxic environment that the cells are in. Ultimately the pLegumain treated immune cells showed a higher expression of these molecules (both PBS and Empty vector serve as controls). This meant that there were more antigen presenting cells present indicating the activation of T cells. The expression was analyzed using FITC labeled molecules, or fluorescent stained molecules to measure the expression. 

Now the Lymphocytes are mentioned to have been obtained from Peyer's patch cells. Peyer's Patch cells or PPC's are located in the small intestine and ileum and function as a type of lymph nodes that house immune system cells such as macrophages and dendrites (4). It is interesting to me that this was performed on immune system cells that were not associated with tumor cells, also that the cells used were not isolated to just macrophages but others as well. It is understood that because the drug is pLegumain, it will in theory only target the M2 macrophages expressing it. Using these cells to analyze the method of TAM depletion is a weakness. This is because there is a very large difference between unaffected immune system cells from lymph nodes and immune cells that are integrated into a tumor microenvironment. Also, the PPC's include both helper and cytotoxic T cells, which are very rarely found active in an agressive tumor microenvironment. Thus the expression of the antigen presenting cell receptors could easily be overexpressed due to the presence of the T cells that would be more prepared to destroy the macrophages. Also because the recptors being tested for are found on dendritic cells as well, this could also be an additional factor to it's overexpression. In order to better understand these effects TAMs should have been stained as opposed to PPC's.

After reading this article my main question was: 
Where else is legumain expressed in the body? What is the probability of pLegumain destroying these cells?

This question needs to be addressed and understood before any actual immunotherapy applications to humans a re proposed because the affecting of this drug on other body cells could be detrimental because t serves as a method for cell destruction.