1. Macrophage
repolarization and its
effects on Tumor
TO TAME AN ORGAN

2. 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.

3. 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.

4. 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.

6. 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.

8. 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.

10. 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.

11. 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.

12. 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.

14. 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.

15. How does HRG skew TAMs away
from
M2-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.

16. 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

17. HRG reduces hypoxia and
normalizes vessels.

18.  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

19.  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.

20. *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

21. 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)