ROLE OF THE IMMUNE SYSTEM IN TUMOR PROGRESSION 2

H Appendix 6: Dct expression in lungs of CD8-depleted mice at seven weeks .... B Appendix 1: Calculations of proliferation and estimates of size Effect of reduced mitotic index on tumor

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) 𝑁𝑁 = 𝑁𝑁0𝑒𝑒(𝑘𝑘𝑘𝑘𝜏𝜏 )

Where N is the number of melanoma cells at the end of the experiment, N0 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 Nc/Nd between the tumor mass in control vs depleted mice is given by:

𝑁𝑁𝑑𝑑

� = 𝑒𝑒([𝑘𝑘𝑐𝑐−𝑘𝑘𝑑𝑑]𝑘𝑘𝜏𝜏 )

Where kc is the fraction of proliferating cells in the tumor of control mice and kd 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:

𝐷𝐷𝑑𝑑

� = {𝑒𝑒([𝑘𝑘𝑐𝑐−𝑘𝑘𝑑𝑑]𝑘𝑘𝜏𝜏 )}13

kc and kd 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

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

𝐷𝐷𝑑𝑑

� = {𝑒𝑒([𝑀𝑀𝑀𝑀 𝑐𝑐−𝑀𝑀𝑀𝑀 𝑑𝑑] 𝑘𝑘𝑆𝑆 )}13

We measured MIc=0.035 and MId=0.017

At seven weeks of age, the expected Dc/Dd was 1.7 and no significant difference could be detected experimentally

At 20 weeks of age, the expected Dc/Dd 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) 𝑁𝑁 = 𝑁𝑁0𝑒𝑒(𝑘𝑘𝑘𝑘𝜏𝜏 )

Where N is the number of melanoma cells at the end of the experiment, N0 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,

𝐷𝐷𝑐𝑐𝑘𝑘𝑑𝑑

� = 𝑒𝑒([𝑘𝑘𝑐𝑐−𝑘𝑘𝑑𝑑]𝑘𝑘𝜏𝜏 )

Where Dctc/Dctd is the ratio of Dct expression in the lungs of control vs

PMN-MDSC-depleted mice, kc is the fraction of proliferating cells in the lungs of control mice and kd is the fraction of proliferating cells in the lungs of PMN-MDSC-depleted mice

(3) 𝑘𝑘𝑐𝑐 = 𝑀𝑀𝑀𝑀𝑐𝑐 ( 𝜏𝜏 𝑆𝑆� )

Where MIc 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

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

𝐷𝐷𝑐𝑐𝑘𝑘𝑑𝑑

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

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-Mmp9tm1Tvu/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