1) The study examines ways to reprogram tumor-associated macrophages towards an anti-tumor phenotype by investigating the effects of genotoxic stress and DNA damage pathway proteins on macrophage function and behavior. 2) Experiments show that treatment with the chemotherapeutic agent mafosfamide directly increases the phagocytic activity of macrophages and induces changes in their morphology. 3) Downregulation of the p53 protein leads to reduced phagocytosis by macrophages, while activating p53 with nutlin-3a has a similar effect to genotoxic stress in increasing macrophage phagocytosis. This suggests p53 plays a key role in modulating macrophage function in response to stress signals.

1. Reprogramming of tumor-associated macrophages in the leukemic
microenvironment
Tatiana Erlikh1,2
Supervisors: Christian Pallasch2, Daniela Vorholt2, Hans van Oostveen1
1Vrije Universiteit Amsterdam, the Netherlands
2Department I of Internal Medicine, University Hospital Cologne, Germany
Swartz, M.A., et al., Tumor Microenvironment Complexity: Emerging Roles in
Cancer Therapy. Cancer Research, 2012. 72(10): p. 2473-2480.
Lewis, C.E., and Pollard, J.W. Distinct role of macrophages in different tumor
microenvironments. 2012 Cancer Res. 66, 605–612.
Steidl, C., et al., Tumor-Associated Macrophages and Survival in Classic
Hodgkin's Lymphoma. New England Journal of Medicine, 2010. 362(10): p.
875-885.
Li L, Ng DS, Mah W, Almeida FF, Rahmat SA, Rao VK et al. A unique role for
p53 in the regulation of M2 macrophage polarization. Cell Death Differ2014
Vassilev, et al. In vivo activation of the p53 pathway by small-molecule
antagonists of MDM2. 2004 Science 303, 844–848.
Pallasch, C.P. et al., Sensitizing Protective Tumor Microenvironments to
Antibody-Mediated Therapy. 2014 Cell
Special thanks to Michael Hallek, Michael T. Hemann, Peter Bruno, Günter Krauze, Elisa Göckeritz, Christoph Göttlinger, Astrid Schauss, Christian Jüngst, Inge van Wijk, Jennie Souissa
Background
Tumor microenvironment plays a crucial role in the development of malignant
processes and represents one of the main problems in the occurrence of
drug-refractory malignancies (Schwartz, 2012). One of the key players in the
tumor microenvironment are macrophages, designated as tumor-associated
macrophages (Lewis and Pollard, 2006). Recent investigations have
indicated that the tumor-associated macrophages are directly involved in
creating a permissive microenvironment and are critically important in
providing pro-tumor signals in leukemic malignancies, including B cell
lymphoma, chronic lymphocytic leukemia and myeloid leukemias (Steidl et al,
2010). We postulate that reprogramming of tumor-associated macrophages
towards an activated phenotype can be of essential benefit in the treatment of
leukemic malignancies. Our study is aimed at finding mechanisms that are
involved in the altered phagocytic activity of macrophages and investigating
conditions that can stimulate anti-tumor phenotype of these cells.
Objectives
Ø Examine possible ways to foster the anti-tumor activity of tumor-
associated macrophages
Ø Determine the effect of genotoxic stress with frontline
chemotherapeutic drugs on the functional behavior of macrophages
Ø Investigate the role of DNA damage pathway key players in the
changes occuring to macrophages after genotoxic
c
Figure 1: Schema of
the macrophage
reprogramming
strategies in the bone
marrow
chemoresistant niche
Figure 2: Combination therapy of mofosfamide/
alemtuzumab fosters antibody-directed macrophage
engulfment of leukemic cells
Fig 2:
A) ADCP (Antibody-dependent phagocytosis) assay with J774A.1 murine
macrophage cell line. Macrophages were incubated with the selected
chemotherapeutic agent (Mafosfamide, Bendamustine, Fludarabine)
for 24h. The optimal dosage for genotoxic stress was determined by
viabillity assays with the selected agents. After 24h, the drug was
washed away and macrophages were co-cultured with hMB cells
(humanised murine leukemic B cell line) for 16h. The antibody-
mediated phagocytosis was induced by monoclonal antibody
Alemtuzumab.
B) ADCP assay with thioglycollate elicicted primary murine macrophages.
For the expirement set up see fig.2A .
A
B
ADCP Fludarabine n= 3
M
-hM
B
-
M
+hM
B
-
M
+hM
B
+
M
-hM
B
+
0
20
40
60
80
%antibodyspecificphagocytosis
ADCP Mafosfamide n=6
C
trlM
afo
on
hm
b
M
afo
on
M
acrophage
0
5
10
15
20
25
***
**
%antibodyspecificphagocytosis
ADCP Mafosfomide n=3
M
-hM
B
-
M
+hM
B
-
M
+hM
B
+
M
-hM
B
+
0
20
40
60
M-hMB-
M+hMB-
M+hMB+
M-hMB+
%antibodyspecificphagocytosis
ADCP Bendamustine n=2
M
-hM
B
-
M
+hM
B
-
M
+hM
B
+
M
-hM
B
+
0
20
40
60
80
M-hMB-
M+hMB-
M+hMB+
M-hMB+
%antibodyspecificphagocytosis
Results
Figure 3: Cyclophosphamide, but not other
chemotherapeutic agents have a direct effect on the
phagocytic capacities of macrophages. The effect is not
soluble-factor driven
Fig 3:
A) Phagocytic capacities of J774A.1 macrophages (normalized to control
100%) under treatment with different chemotherapeutic agents.
Macrophages were incubated with IgG beads and analyzed by FACS.
B) Phagocytic capacities of J774A.1 macrophages cultured for 24h in
conditioned media generated from stressed J774A.1 macrophages
with respective chemotherapeutic agent
n=5 n=1
A B
Phagocytosis assays J774A.1
0
uM
2,5
uM
m
afos
5
uM
m
afos
10
uM
m
afos25
uM
flud50
uM
flud
50
uM
bend
0
50
100
150
200
250
0 uM
2,5 uM mafos
5 uM mafos
10 uM mafos
25 uM flud
50 uM flud
50 uM bend
**
%increasephagocytosis
Phagocytosis assay with supernatant (16-12)
J774A 18-12
0
uM
5
uM
m
afos
25
uM
flud
50
uM
bend
0
20
40
60
0 uM
5 uM mafos
25 uM flud
50 uM bend
%phagocytosis
Figure 7. Macrophages show an altered morphology
under treatment with mafosfamide
control 5 µM mafosfamide
Fig 7:
A) Flow cytometric assessment of the granularity and the size of J774A.1
macrophages untreated (1), compared to mafosfamide treatement (2).
B) Confocal microscopy imaging. Investigation of the morphology of
macrophages under different conditions. Phagocytosis assay protocol was
followed and cells were fixed with paraformaldehyde 4% after 16h of
incubation with IgG PE-labelle beads (red). Macrophage are GFP positive
(green).
1) Control (empty vector) without beads.
2) Control (empty vector) incubated with beads for 16h
3) Control (empty vector) treated with mafosfamide and incubated with
beads for 16 hours.
4) p53 KD without beads.
5) p53 KD incubated with beads for 16h
6) p53 KD treated with mafosfamide and incubated with beads for 16 h
A
1) 3 )
4)
2)
6)5)
1) 2)
B
Figure 6: p53 downregulation leads to insufficient antibody-
mediated and baseline phagocytosis
Fig 6:
A) Baseline phagocytic capacities of
J774A.1, transfected with a p53
knockdown compared to empty
control. There is a significant
decrease in the phagocytic
capacities of p53-deficienct
macrophages.
B) Antibidy mediated phagocytosis of
J774A.1, transfected with a p53
knockdown compared to empty
control. Antibody-mediated
phagocytosis is also affected
em
pty
p53
0
10
20
30
40
50
*
%antibodyspecificphagocytosis
em
pty
p53
0
20
40
60
80
*
%beadsphagocytosis
n=2 n=1
m
afos
nutlin
dox
0
1
2
3
4
5 empty
p53
foldincreasein%BCGphagocytosis
em
pty
cntrlem
pty
dox
p53
cntrl
p53
dox
0
10
20
30
40
*** ***
%BCGphagocytosis
em
pty
cntrl
em
pty
m
afos
em
pty
nutlin
0
10
20
30
40
50
***
***
%BCGphagocytosis
p53
cntrlp53
m
afosp53
nutlin
0
5
10
15
20
25
*
**
%BCGphagocytosis
Phagocytosis assays J774A.1 empty
cntrl5
uM
m
afos10
uM
nutlin
0
20
40
60
80
100
***
**
%phagocytosis
cntrlem
pty
nutlin
em
pty
p53
control
p53
nutlin
0
10
20
30
40
50 *
%antibodyspecificphagocytosis
Fig 3:
A) Flow cytometric analysis of size and granularity of macrophages in the
BCG-based assay. Macrophages were incubated with dsRED-positive
mycobacteria and phagocytosis activity was assessed by the fraction of
macrophages with the engulfed bacteria. Under treatment with nutlin 3a
(2), macrophages show a significantly altered morphology, resembling the
morphology of macrophages after genotoxic stress.
B) Phagocytic activity of J774A.1 macrophages under treatment with
mafosfamide and nutlin 3a. Treatment with nutlin 3a significantly increases
phagocytosis of beads.
C) Phagocytic activity of J774A.1 macrophages evaluated in the BCG assay.
Treatment with nutlin 3a increases englufment of mycobacteria.
D) Phagocytic activity of J774A.1 p53 deficient macrophages in the BCG
assay. The effect of nutlin-3a and mafosfamide can be still observed which
suggests that the effect is not entirely p53-dependent.
E) The fold increase in phagocytosis of mycobacteria in wild type and p53
KD J774A.1 in BCG assay. The increase in phagocytosis of wild type
macrophages under treatment with mafosfamide, nutlin and doxorubicin is
higher than in p53 deficient cells.
F) Antibody-mediated phagocytosis of J774A.1 wild type and p53 KD. In this
assay, treatment with nutlin-3a did not have an effect on the phagocytic
capacity of p53-deficient macrophages which underlines the importance of
p53 for the effect of nutlin-3a.
G) Phagocytic capacity of p53 KD and control under treatment with alkylating
agent doxorubicin. It significantly increases phagocytosis of mycobateria.
H) Phagocytosis of beads is also increased under treatment with doxorubicin.
Notably, macrophages with the p53 KD still respond to the treatment with
mafosfamide and increase their phagocytic activity which
Figure 7: Nutlin 3a significantly increases phagocytosis and
resembles the effect of mafosfamide on the morphology
change in macrophages.
cntrlem
pty
D
ox
50
uM
em
pty
p53
cntrl
p53
dox
50
uM
0
20
40
60
80
100
%phagocytosis
n=2
n=1n=1
Conclusions
Ø Genotoxic stress with mafosfamide increases the antibody-dependent and baseline phagocytosis in macrophages
Ø Mafosfamide induces a change in morphology of macrophages, their size and granularity.
Ø p53 plays an important role in phagocytosis; Activation of p53 with nutlin 3a has the same effect on macrophages as
genotoxic stress; knock down of p53 leads to the insufficient phagocytosis
n=1
n=1n=1 n=1
1) 2)A B
C D E
F G H