A study on tumor microenvironment: from metabolism to immunotherapy.

Tumors contain various noncancerous cells, collectively often termed the tumor stroma and, together with factors such as the extracellular matrix, oxygen levels, and pH, they make up the tumor microenvironment (TME )). Many studies showed that within TME, tumor and stromal cells engage a bi directional crosstalk mediated by cytokines and growth factors. A new described mechanism for intercellular communication involves intercellular transfer of extracellular vesicles (EVs )), such as exosomes and microvesicles (MVs), different for size and biogenesis. Many cell types in TME are capable to release different classe s of EVs which are involved in development and progression of cancer and are of relevance to diseases of various sorts. We found that some type of mesodermal origin cells such as fibroblast, monocytes and macrophages can transfer proteins and lipids via MVs to cancer cells . Monocytes and macrophages are able to transfer proteins via MVs, in fact, after co culture with cancer cells and labeled T2k b or J774A.1 cells, we observe an increase of fluorescence in cancer cells due to proteins transfer from monocytes/macrophages to recipient cells while the reverse transfer is negligible. Among differential expressed proteins, it has been found MHC I, a heterodimer of a class I heavy chain and β2 microglobulin capable to bind endogenous peptides and display them to cells surface for the recognition by CD8+ lymphocytes. In particular, MVs de rived from normal monocytes contain only β2 microglobulin while MVs derived from activated monocytes contain both HLA I and β2 microglobulin. According to this data we hypothesize that activated monocytes, preloaded with a specific antigen, can transfer pMHC I to cancer cells make them recognizable by cytotoxic T cells, so that a physiological phenomenon can be used for a new type of cancer imm unotherapy. Alongside I also investigated the role of bisphosphoglycerate mutase in cell proliferation and the involvement of extracellular vesicles in the transfer of biomass. BPGM has a well known role in red blood cells where it is highly expressed and participates in the Luebering Rapoport pathway while little is known about its role in maintaining the glycolytic flux. This enzyme has mutase activity that converts 1,3 BPG, to 2,3 BPG and phosphatase activity, converting 2,3 BPG to 3 phosphoglycerate (3 PG). In this way the cells have an energetic cost bypassing the step of ATP formation. We investigated the role of 2 3 biphosphoglycerate mutase (BPGM) in the Rapoport Lubering shunt to better understand its involvement in proliferation of normal and cancer cell lines. We observed that BPGM mRNA level differs in different cells line and also the protein expression is different based on the presence of proliferation inducing factors or the physiological state. After BPGM silencing cancer cells showed a slow down prolife ration and a different metabolic profile. We took into account DU145 and PC3 that had the higher level of BPGM and we found out a drastically decrease in glucose uptake and increase in CO 2 production. The high glucose uptake typical of proliferating tumor cells is necessary for allowing a high rate of biomass synthesis. BPGM, shunting glycolytic pathway, reduces the ATP production efficiency allowing the high glucose influx rate necessary to produce anabolic metabolites. In fact, by silencing BPGM expressio n, we increase the efficiency of glycolytic pathway in term of ATP production, but we drastically reduce both the glucose uptake and the cell proliferation rate. In conclusion BPGM is a key enzyme of the metabolic setting of proliferating tumor cells.