ROLE OF THE IMMUNE SYSTEM IN TUMOR PROGRESSION 2

A

Appendices

Contents

Appendix 1: Calculations of proliferation and estimates of size B
Appendix 2: Immune profiling gating strategy E
Appendix 3: Table of chemokine/cytokine expression F
Appendix 4: Mitf expression in LN G
Appendix 5: Alternate inhibitors of HGFR, EGFR and TGF-βR1 H
Appendix 6: Dct expression in lungs of CD8-depleted mice at seven weeks I
Appendix 7: Dct expression in lungs of tumor-bearing MMP9-KO mice J




B

Appendix 1: Calculations of proliferation and estimates of
size

Effect of reduced mitotic index on tumor diameter:
At early time points, tumor growth is assumed to be exponential and described by the
following equation (1):

(1)  = 
0

(



)


Where N is the number of melanoma cells at the end of the experiment, N
0
is the
number of melanoma cells at the onset of the experiment, k is the fraction of
proliferating cells, t is the duration of the experiment (6 or 19 weeks), τ is the duration
of the cell cycle (28h, according to Gordon 1980, Canc Res 40, 4467-4472).

The ratio N
c
/N
d
between the tumor mass in control vs depleted mice is given by:

(2)




� = 
(
[

−

]


)


Where k
c
is the fraction of proliferating cells in the tumor of control mice and k
d
is
the fraction of proliferating cells in the tumor of PMN-MDSC-depleted mice.

For tumors with ellipsoid shape, the longer diameter is proportional to the cubic root
of the cell number. Therefore the ratio between the diameters of tumors from control
and depleted mice is given by:
(3)




� = {
(
[

−

]

)
}
1
3

k
c
and k
d
are deduced from MI, the percentage Ki67
+
cells, and S, the duration of
Ki67 expression during cell cycle (estimated to be 14h based on Lopez 1991
Cytometry 12:42-49) according to:
C


(4)  =  (


�
)

From (3) and (4), one gets:

(5)




� = {
(
[


−

] 

)
}
1
3



We measured MI
c
=0.035 and MI
d
=0.017.

At seven weeks of age, the expected D
c
/D
d
was 1.7 and no significant difference
could be detected experimentally.
At 20 weeks of age, the expected D
c
/D
d
was 5, in good agreement with the 4.7 value
measured experimentally.

Decreased ectopic expression of Dct is not due to reduced cancer cell
proliferation
At early time points, the population of melanoma cells in the lung can be described by
the following equation (1):
(1)  = 
0

(


)


Where N is the number of melanoma cells at the end of the experiment, N
0
is the
number of melanoma cells at the onset of the experiment, k is the fraction of
proliferating cells, t is the duration of the experiment (6 weeks), τ is the duration of
the cell cycle (28h, according to Gordon 1980, Canc Res 40, 4467-4472).




D

It is assumed that Dct expression is proportional to N. Therefore,

(2)





� = 
(
[

−

]

)


Where Dct
c
/Dct
d
is the ratio of Dct expression in the lungs of control vs PMN-
MDSC-depleted mice, k
c
is the fraction of proliferating cells in the lungs of control
mice and k
d
is the fraction of proliferating cells in the lungs of PMN-MDSC-depleted
mice.

(3) 

= 


(


�
)

Where MI
c
is the fraction of Ki67
+
cells in the lung of control animals (1.9%, see
Eyles et al. 2010 J. Clin. Invest.), S is the duration of Ki67 expression during the cell
cycle (estimated to be 14h based on Lopez 1991 Cytometry 12:42-49)

We tested the hypothesis that PMN-MDSC depletion would reduce the proliferation
of disseminated cancer cells by 80% (reduction seen in primary tumor). Therefore

(4) 

= 0.2



By combining (2), (3) and (4), one finds


(5)





� = 3

Therefore, even an 80% inhibition of cancer cell proliferation in the lungs could not
account for the observed 5.5-fold decrease in Dct expression. We therefore favor the
interpretation that the decrease observed in Dct expression is mainly due to reduced
colonization of the lungs by cancer cells rather than reduced proliferation.
E

Appendix 2: Immune profiling gating strategy


Identification of immune subsets in single-cell suspension of RETAAD tumors
The figure shows the strategy used to enumerate immune cells in tumors.


F

Appendix 3: Table of chemokine/cytokine expression

Differential expression of chemokines and cytokines in primary tumors and
metastases
Gene expression was measured by low density qRT-PCR arrays (except for CXCL2
which was done by individual qRT-PCR). Expression values were normalized to
GAPDH and presented as a log
2
(expression in primary tumor/metastases). p-values
were calculated using two-tailed paired t-test. Data was from 11 paired primary
tumors and metastases. Each pair of primary and metastatic tumors was taken from

the same individual mouse.

G

Appendix 4: Mitf expression in LN




PMN-MDSC depletion reduces tumor dissemination
Reduced expression of Mitf in the mandibular LN of mice depleted of PMN-MDSC
(depletion scheme A). Data was from 12 control and 20 PMN-MDSC depleted lymph
nodes. Bars represent mean ± SEM **p<0.01, two-tailed t-test on log transformed
data.




H

Appendix 5: Alternate inhibitors of HGFR, EGFR and
TGF-βR1

Alternative Inhibitors block PMN-MDSC induced EMT
Alternative inhibitors block PMN-MDSC induced EMT in NBT-II cells. AG1478 –
EGFR inhibitor, SGX523 – c-met (HGFR) inhibitor and SB431542 – TGF-βR1
inhibitor.

I

Appendix 6: Dct expression in lungs of CD8-depleted mice at
seven weeks




CD8 depletion does not affect metastases to the lungs at seven weeks
CD8 depletion does not increase tumor cell dissemination to the lungs of seven-week-
old mice. Mice were depleted using rat anti-mouse CD8 depleting antibody (ATCC
TIB-210; provided by Renia L., Singapore Immunology Network, A-STAR). Mice
were administered intraperitoneally 0.25 mg of anti-CD8 antibody or control rat IgG
(Sigma-Aldrich) at one week of age, and then 0.1 mg antibody weekly for seven
weeks. Efficiency of CD8 T cell depletion was monitored by flow cytometry analysis
of blood and spleen. Data was from 11 control and seven CD8-depleted lungs of
seven-week-old mice. Bars represent mean ± SEM ns: p=non-significant, two-tailed t-
test on log transformed data.


J

Appendix 7: Dct expression in lungs of tumor-bearing
MMP9-KO mice




MMP9 does not play a role in dissemination of tumor cells to the lungs
RET mice were crossed with MMP9-KO mice (Jax Laboratoies; FVB.Cg-
Mmp9
tm1Tvu/J

) to obtained RET+ tumor-bearing mice that were deficient of MMP9.
When compared to the control mice that expressed MMP9, no difference in (A)
cutaneous tumor incidence or (B) dissemination to the lung was found Data was from
four MMP9 positive control and six MMP9-KO of 12-week-old mice. Bars represent
mean ± SEM ns: p=non-significant, two-tailed t-test on log transformed data.

A
B