2. Basic
Terminologies
o Macrophages: These are specialized cells involved in the detection,
destruction of bacteria and other harmful organisms.
o Tumor: It is an abnormal growth of body tissue. It can be malignant or
benign.
o Morphogenesis: The process that cause a cell, tissue or organism to
develop its shape.
o Metastasis: It is the pathogenic agent’s spread from an initial site to
many sites (cancerous tumor).
o Hypoxia: It is the condition in which there is decreased oxygen state in
the body.
o Angiogenic: The growth of new blood vessels that tumor needs to grow
and is caused by the release of chemicals by tumor and host cells near
tumor.
o mTOR: Mechanistic target of rapamycin is the major regulator of
anabolic and catabolic processes in response to nutrients.
o REDD1:Regulated in development and DNA response 1 is a gene that
induces anti-apoptotic response during stress.
4. Heterogeneity Of
Macrophages
Heterogeneity of macrophages has been
long recognized as the result of plasticity
and versatility of these cells to different
stimuli. In tumors, macrophages are
involved in blood and lymphatic vessel
formation, cancer cell invasion,
dissemination and in immune
suppression.
5. Tumor Associated
Macrophages (TAMs)
TAMs with in hypoxic environment of the
tumor acquire a angiogenic, invasive and
tolerogenic environment. They do so by
strong alterations in the expressions of
metabolic genes as they are forced to
adapt their metabolism to low oxygen
tension in order to meet their energy
requirements.
7. The Mechanistic Target
Of Rapamycin (mTOR)
It is a key nutrient and energy sensor that
on the basis of nutrient availability
regulates metabolic processes such as
glycolysis, de novo lipogenesis, protein
synthesis and transcription.
8. Causes Of
Induction
Of REDD1
Hypoxia leads to the induction of
REDD1 and this REDD1 inhibits or
blocks the function of mTOR.
Other than hypoxia, REDD1 is also
induced by stress conditions
which are major features in cancer
like oxidative stress, ER stress,
DNA damaging agents etc.
11. Hypothesis
On the basis of knowledge that hypoxia and stress
conditions specifically control mTOR activity through
REDD1 regulation, researchers hypothesized that
REDD1/mTOR axis is a central relay that links
macrophage metabolism to the immunosuppressive
properties of hypoxic TAMs.
13. • A large body of evidence in several tumors has proved that shortage of oxygen
occurs due to GLUT1.
• In case of Lewis lung carcinomas, arthrotopic E0771 breast cancer, PyMT
mammary tumors confirmed that GLUT1 and hypoxia probe pimonidazole
strongly overllaped.
• GLUT1 high(hypoxic) macrophage and GLUT1 low(normoxic) macrophage from
LLC,E0771 and PyMT tumors shows that tha mRNA level of REDD1 is the highest
in GLUT1 high.
• We also that in the staining of TAM the REDD1 was much higher in hypoxic areas
14.
15. REDD1 Deletion in TAMs Promotes
Tumor Vessel Normalization and
Inhibits Metastasis
16. • 1st of all WT mice were taken and it is injected with KO BM cells.
• In vitro, hypoxic, but not nor-moxic, REDD1 KO TAMs displayed increased
phosphorylation of 4E-BP1 and S6.
17.
18.
19. Model 1
KOWT VS LLC
In 2nd case implanted LLC tumors
subcutaneously.
20. Model 2
KOWT VS E0771
In 2nd case injected E0771 breast cancer cells
orthotopically.
21. Model 3
KOWT VS PyMT
we reconstituted 4-week-old PyMT mice in a C57BL/6
background with WT and REDD1 KO BM cells.
22. Results
LLC tumor blood vessels in WT/WT mice had a disorganized, dilated
appearance, with many protrusions.
In contrast, the tumor vasculature in KO/WT mice displayed a smooth
endothelial alignment and reduced vessel diameter.
Similar features of vascular normalization were found in the orthotopic E0771
breast cancer model as well as in the PyMT-driven spontaneous breast cancer
model after deletion of REDD1 which prevented the Metastasis.
25. • Hypoxic TAMs showed an increase in Glycolysis.
• By deleting Redd1,Gylcolytic flux further increased in an mTOR-
dependent manner.
• Fatty acid oxidation(FAO) was decreased in hypoxic REDD1 KO
TAMs
• Glucose (GO) and Glutamine (QO) oxidation remain unchanged.
26. • In term of ATP production,
• hypoxic WT TAMs, equal balance in glycolytic versus mitochondrial
contribution to the ATP pool.
• In hypoxic REDD1 KO TAMS, glycolysis was the main contributor to
the ATP pool whereas ATP molecules from Fatty acid oxidation was
reduced.
28. • ATP linked mitochondrial oxygen consumption reached the lowest levels in
hypoxic REDD1 KO TAMs, depending on mTOR
• In normoxic TAMs, REDD1 deficiency did not alter the glycolysis or the
contribution of other major fluxes to the ATP pool.
• With the increase in glycolysis, hypoxic REDD1 KO TAMs showed highest
levels of GLUT1,with increase in glucose uptake.
• GLUT1 expression and glucose uptake was enhanced in hypoxic REDD1
KO TAMs-L as compared with hypoxic WT TAMs-L
30. Increase Glycolysis In REDD1 Ko Teams
Cause Tumor Vessel Normalization By
Competing For Glucose
31. Increase Glycolysis In REDD1 Ko Teams Cause Tumor
Vessel Normalization By Competing For Glucose
• In order to observe metabolic changes in hypoxic TAMs which influence on their
phenotype, RNA sequencing is performed on wt and (REDD1 Ko CD1b+ F4/80+
GLUT1 high) using FACS( fluorescence-activated cell sorting) from LLC tumer.
• In general only 5 genes were differentially expressed.
34. In order to observe Glucose uptake is cause
of observing phenotype or not ?
• The gene REDD1 and Pfkfb3 were deleted in macrophages, as a result glycolysis
increased and oxygen consumption become decreased.
