The Tumor Microenvironment of Mif Deficient and Wild Type Mouse Injected of Colon Carcinoma

Tumor is an abnormal mass of tissue, which could be solid, or fluid filled. Migration
Inhibitory Factor (MIF) is a ubiquitously expressed cytokine in tumor. High levels of MIF
expression have been observed in several human cancers and expression correlates with
tumor grading and clinical prognosis. This experiment was designed to see the size of
tumor, cell population, arginase and nitric oxide activity and production and presence
of pro and anti-inflammatory cytokine in cell and serum of MIF wild type and MIF- /-
mouse injected of colon carcinoma tumor cell line.


Introduction
Cancer is known medically as a malignant neoplasm, which is a broad group of various diseases that involve unregulated cell growth. The cancer cells can grow and divide without control and are able to invade tissues. Tumor is an abnormal mass of tissue, which could be solid or fluid-field. Tumors can be Benign Macrophage-Migration Inhibitory Factor (MIF) is a ubiquitously expressed cytokine with a variety of mitogenic and pro-inflammatory functions [2] and released by macrophages, T cells, and the pituitary gland during inflammatory responses. There are at least two distinct pro-inflammatory effects of MIF. First, MIF can "override" or counter-regulate the immuno-suppressive effects of steroids [3]. Second, the addition of MIF enhances Tumor Necrosis Factor (TNF) and nitric oxide production by Lipopolysaccharide (LPS)-stimulated macrophages. Conversely, MIF-deficient peritoneal macrophages express less of these inflammatory mediators than wild-type macrophages [4]. Studies Tumor is an abnormal mass of tissue, which could be solid, or fluid filled. Migration Inhibitory Factor (MIF) is a ubiquitously expressed cytokine in tumor. High levels of MIF expression have been observed in several human cancers and expression correlates with tumor grading and clinical prognosis. This experiment was designed to see the size of tumor, cell population, arginase and nitric oxide activity and production and presence of pro and anti-inflammatory cytokine in cell and serum of MIF wild type and MIF-/mouse injected of colon carcinoma tumor cell line. Bacterial LPS is used to stimulate cells for nitric oxide and cytokine assay. The average size of the tumor in eight mice and the cell count was larger in wild type mice. Up on FACS analysis Live gate percentage of cell population of MIF -/-mouse seem to have a bit higher MHCII High (TAMS) and MHCII low (TAMS) than the WT mouse. Median Fluorescence Intensity measured shifts mainly to the left indicating lower or less CD74 expression as compared to the isotype. NO production is higher in tumor cell supernatant stimulated by LPS as compared to the non-stimulated ones but negative for arginase activity. The result of serum cytokine IL-6 and IL-10 concentration is negative. The IL-6 and IL-10 concentration is higher in both wild type and MIF-/-mouse tumor cell supernatant stimulated with LPS as compared to the non-stimulated ones but with no cytokine production in non-stimulated. All the results except the tumor size and the manual cell count are statistically in significant, which might require further study, especially for this particular model.  [5] have indicated MIF as a regulator of cell growth and apoptosis.
MIF expression patterns change during organogenesis and tissue specification and they are influenced by growth inhibition in vivo [6]. High levels of MIF expression have been observed in several human cancers and expression correlates with tumor grading and clinical prognosis [7]. Kleemann et al. [8] have provided evidence for an intracellular function of MIF in the regulation of cell growth. Cell-based genetic screens [9], have demonstrated that MIF interacts with the p53 tumor suppressor by inhibiting p53responsive gene activation and apoptosis. The high frequencies of mutations in the p53 gene that are found in human tumors [10], and the apparent correlation of p53 loss with tumor aggressiveness [11] emphasize the importance of p53 as a ''gatekeeper'' in the development of neoplastic disease. Accordingly, it has been hypothesized that the bypass of p53 regulatory functions by MIF at sites of chronic inflammation might impair the normal response to genetic damage, enhance cell proliferation, and promote the accumulation of oncogenic mutations [12].

Arginase Activity
To determine the arginase activity of the tumor cell, cells were

Nitric Oxide Activity
To determine the nitric oxide production of the tumor cell, cells were collected from both wild type and MIF deficient mice and prepared according to the protocol and the optical density was measured and compared to the standard. Nitrite concentrations in culture supernatants were determined using Griess reagent (Sigma-Aldrich).

Cytokines Assay Using Elisa
Cytokine concentration was determined in the culture

Tumor Size
The average size of the tumor in eight mice was calculated by the formula (Dxd2) x3 .14/6 where it is larger in wild type mice than MIF-/-ones ( Figure 1). However, the size difference is statistically insignificant at p<0.005.

Cell Count
Manual Cell Count: Cell were counted from two wild type and two MIF-/-mouse in neubarcytometer and calculated by the formula nx104 x dilution factor(10 in this case)x1. The computed average number of cells in wild type mouse is higher than MIF-/mouse ( Figure 2).This result is statistically significant with P=0,015 at 5% significant level.    as compared to the isotype (red curve) ( Figure 6).

Arginase Assay
We performed Arginase assay for all (wild type, MIF-/-and MC38 tumor cell line) samples but all samples were negative for the assay. This might be related with our tumor microenvironment which donot have enough mature myeloid cell which are responsible for the production of arginase.

Nitric Oxide Assay
The assay was performed on tumor cell supernatant and MC38

Conclusions
Understanding the role of MIF in neoplastic development and progression is complicated by the pleiotropic influences that MIF plays in many tumorigenic processes. This experiment was conducted to understand the contribution of MIF in tumor development, its effect in tumor micro environment as well as the systemic involvement. Knowledge and detailed investigation of this cytokine might help to design one possible and probably effective target to treat cancer. There for, further study should be performed in detail to unreveal the secrete and to help and combat related issues.