Macrophage repolarization and its effects on TumorTO TAME AN ORGAN

Tumors and Cancer Tumor or neoplasm is a vesicular bulging in particular tissues

due to uncontrolled cell proliferation. While cancer is by definition malignant, a tumor can be benign, pre-malignant, or malignant, or can represent a lesion without any cancerous potential whatsoever.

Benign tumors do not invade other tissues. They are rarely a threat to life unless they compress vital structures or are secreting harmful chemicals in the bloodstream.

Malignant tumors can invade other organs, spread to distant locations (metastasis) and become life-threatening.

The problem :1 The growth of blood vessel network in newly formed tissues

from already existing vessels is called angiogenesis. This process is promoted by expression of VEGF.

VEGF expression is promoted by hypoxia and β-catenin. PKG downregulates β-catenin. In its absence there is an

incessant expression of VEGF causing formation of irregularly shaped, hyper-permeable blood vessels which slowly thicken due to accumulation of debris and plasma. This keeps tumor in a constant state of hypoxia.

The misconception The discovery of VEGF led to the belief that by blocking the

action of this protein , the supply of food and oxygen to the tumor can be cut which will make the tumor starve and thereby eliminate it.

A drug named “avastin” was developed which was able to block the functions of VEGF. This was considered the “silver bullet” treatment.

But this was not successful as anti-angiogenic ‘‘vessel pruning’’ strategies like this can worsen this situation by aggravating hypoxia.

The Problem : 2 Tumor blood vessels have perivascular detachment, vessel

dilation, and irregular shape. They are not smooth like normal tissues, and are not ordered sufficiently to give oxygen to all of the tissues.

Also , abnormal vasculature in tumor cells impedes the delivery of chemo/immuno-therapeutic agents. This causes a state of hypoxia which initiates a series of enzyme activity and increases metastasis.

This also prevents the proper delivery of chemotherapeutic agents thus protecting the tumor.

The inconsistency In non-progressing or regressing tumors, TAMs are biased to

a classic macrophage activation,M1-like program, characterized by pro-inflammatory activity

In malignant tumors, TAMs resemble alternatively activated macrophages (M2-type), that increase angiogenesis and tumor cell intra/ extravasation and growth; they suppress antitumor immunity by preventing activation of dendritic cells, cytotoxic T lymphocytes, and natural killer cells.

The Solution The need to normalize the tumor vessels was felt (R. K. Jain,

2005) which attracted attention to abnormalities in vascular endothelial cells and pericytes. (which was marked to be the presence of RG5 by Hamzah et al, 2008)

Rolny et al suggested that targeting abnormal polarization of TAMs can normalize tumor vessels.

TAMs usually exhibit M2 like phenotype and secrete cytokines like IL-10, CCL-17,CCL-22 and proangiogenic factors like VEGF and PIGF.

HRG TAMs consist of distinct subsets, which coexist in

tumors, adapt to the changing microenvironment, and can be re-educated by immunoregulatory cues.

This has primed interest in developing therapies, aimed at skewing TAMs to an M1-like phenotype. Nonetheless, only few molecules have been identified to orchestrate this process so far.

HRG (histidine-rich glycoprotein) is one of them.

HRG HRG is a multidomain plasma protein synthesized by

hepatocytes and has important function in regulation of tumor angiogenesis and immunity.

The increase of oxygenation in HRG+ tumors caused by vascular normalization seems to provide a stimulus for polarizing TAMs away from M2-like type, which could further sustain the normalized vasculature.

HRG- TAM interaction Exposure of TAMs to HRG downregulated the M2 markers such

as MRC1, Arg1, IL10, and CCL-22 and simultaneously elevated M1 markers such as IL6 and CXCL-9. Accordingly, tumor-infiltrated CD8+ T cells, natural killer (NK) cells, and dendritic cells (DCs) increased and their functions improved in HRG+ tumors

Thus we are able to increase immune surveillance in the tumor and control its growth without aggravating hypoxia i.e. without increasing metastasis.

The repolarization of TAMs increases the vessel normalization which gives an additional edge over anti-angiogenic agents alone.

How does HRG skew TAMs away fromM2-like phenotype? HRG serves as an antagonist to Fcγ receptors which are

expressed in macrophages. Stromal accumulation of autoantibodies in premalignant skin,

through their interaction with activating FcγRs, regulate recruitment, composition, and bioeffector functions of leukocytes in neoplastic tissue, which in turn promote neoplastic progression and subsequent carcinoma development. (andreu et al ,2010)

Thus blocking fcγR skew TAMs towards M1-like configuration.

HRG reduces PIGF HRG reduced PlGF production by TAMs by downregulating it’s

gene. The deletion of PlGF in macrophages phenocopied antitumor and

vascular normalization effects of HRG treatment. HRG did not further suppress tumor growth in the absence of

host-derived PlGF. Similar observations were made when analyzing metastasis.

PlGF deficiency altered tumor immunity. However, HRG overexpression did not further affect the infiltration of these cells in PlGF deficient mice

HRG reduces hypoxia and normalizes vessels.

HRG decreased the metastatic index (nodules per gram tumor)

The reduced tumor spread was partly independent of tumor growth inhibition.

PHH3 staining shows proliferating tumor cells.

Slow growth in HRG+ tumors

HRG allows more dendritic cells, Natural killer cells and cytotoxic T lymphocytes in the tumor.

It increases production of factors that repolarize M2-macrophages into M1-macrophages. It also increases the TAM density.

*Doxorubicin intercalates the DNA and blocks polymerase activity. It is generally used in chemotherapy.

Increased expression of HRG increases impact of inflammation and chemotherapy ,decreases metastasis and regulates angiogenesis.

It skews TAMs to allow an inflammatory response.

The Impact

References o “HRG Inhibits Tumor Growth and Metastasis by Inducing Macrophage Polarization and

Vessel Normalization through Downregulation of PlGF” by Rolny et al (2011)

o “Macrophage Diversity Enhances Tumor Progression and Metastasis” by Qian and Pollard (2010)

o “Polarization of Tumor-Associated Macrophages: A Novel Strategy for Vascular Normalization and Antitumor Immunity” by Huang, Snuderl and Jain (2011)