Supporting Information

For

In Vitro Systems Toxicology Assessment of a Candidate Modified Risk Tobacco Product Shows

Reduced Toxicity Compared to a Reduced Cigarette

by

Ignacio Gonzalez-Suarez*, Florian Martin, Diego Marescotti, Emmanuel Guedj, Stefano Acali, Stephanie Johne, Remi Dulize, Karine Baumer, Dariusz Peric, Didier Goedertier, Stefan Frentzel,

Nikolai V. Ivanov, Carole Mathis, Julia Hoeng, Manuel C. Peitsch

Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland* Corresponding author

Table S1. List of biological networks and sub-networks.

Table S2. Selected AE, TPM and GVP concentrations for 3R4F and THS 2.2 used for the exposure

of NHBE cells to generate gene expression data.

Figure S1. Carbonyl levels in AE and GVP from THS 2.2 aerosol and 3R4F smoke.

Figure S2. Cell viability across fractions.

Figure S3. HCS results in NHBE cells exposed for 4h or 24h to AE from 3R4F smoke and THS 2.2

aerosol.

Figure S4. HCS results in NHBE cells exposed for 4h or 24h to TPM from 3R4F smoke and THS

2.2 aerosol.

Figure S5. HCS results in NHBE cells exposed for 4h or 24h to GVP from 3R4F smoke and THS

2.2 aerosol.

Figure S6. Cell cycle analysis results in NHBE cells exposed for 24h to AE, TPM and GVP from

3R4F smoke and THS 2.2 aerosol.

Figure S7. Biological Impact Factor (BIF).

Figure S8. NPA heatmaps for NHBE cells exposed for 4 h to one common dose of 3R4F smoke and

THS 2.2 aerosol fractions.

Networks SubnetworksCalcium

Cell cycle

Cell interaction

Clock

Epigenetics

Growth factor

Hedgehog

Jak Stat

Mapk

Notch

Nuclear receptors

PGE2

Wnt

Drug metabolism response

Endoplasmic reticulum stress

Hypoxic stress

NFE2L2 signaling

Osmotic stress

Oxidative stress

Caspase cascade

ER stress-induced apoptosis

MAPK signaling

NFkB signaling

PKC signaling

Prosurvival mitochondrial signaling

TNFR1F signaling

ATG induction of autophagy

mTOR signaling

Nutrient transporter signature

Components afecting TP53 activity

Components affecting TP63 activity

Components affecting TP73 activity

DNA damage to G1-S checkpoint

DNA damage to G2-M checkpoint

Double-strand break response

Inhibition of DNA repair

TP53 transcriptional signature

Fas activation

Gene signature

Oncogene-induced senescence

Regulation by tumor suppressors

Regulation of p16INK expression

Replicative senescence

Stress-induced premature senescence

Transcriptional regulation of the SASP

Epithelial barrier defense

Epithelial proinflammatory signaling

Mucus hypersecretion

Tissue damage

Senescence

Inflammation

Cell

Proliferacion

Cell stress

Apoptosis

Autophagy

DNA damage

Necroptosis

Table S1

Table S1. List of biological networks and sub-networks

3R4F 100* 25 13

THS2.2 350 100 25

3R4F 100* 25 13

THS2.2 350 100 25 13

3R4F 32* 25 13

THS2.2 38 32 25

CONCENTRATION (puff / l)

AE

GVP

TPM

Table S2

Table S2. Selected AE, TPM and GVP concentrations for 3R4F and THS 2.2 used for

the exposure of NHBE cells to generate gene expression data

Figure S1

0

10

20

30

40

50

sbPBS GVP

µg

/ st

ick

FORMALDEHYDE

3R4F P1

0250500750

100012501500

sbPBS GVP

µg

/ st

ick

ACETALDEHYDE

3R4F P1

0

250

500

750

sbPBS GVP

µg

/ st

ick

ACETONE

3R4F P1

0

50

100

150

sbPBS GVP

µg

/ st

ick

ACROLEIN

3R4F P1

0

10

20

30

sbPBS GVP

µg

/ st

ick

BUTYRALDEHYDE

3R4F P1

0

50

100

150

sbPBS GVP

µg

/ st

ick

MEK

3R4F P1

0

25

50

75

sbPBS GVP

µg

/ st

ick

CROTONALDEHYDE

3R4F P1

0

25

50

75

100

sbPBS GVP

µg

/ st

ick

PROPIONALDEHYDE

3R4F P1

A B

C D

E F

G H

Figure S1. Carbonyl levels in AE and GVP from THS 2.2 aerosol and 3R4F smoke: A,

Acetaldehyde; B, Acetone; C, Acrolein; D, Propionaldehyde; E, MEK; F, Crotonaldehyde; G,

Formaldehyde; H, Butyraldehyde. Values are expressed as µg of carbonyl per one 3R4F

cigarette or THS 2.2 stick and represent average and SEM from eight determinations performed on

eight different smoke/aerosol generations on eight different days. * indicates p <0.0001 vs. 3R4F

(reference cigarette) by the paired t-test.

AE AE

AE AE

AE AE

AE AE

Figure S2

Figure S2. Cell viability across fractions. NHBE cells were exposed for 24h to AE, TPM or GVP

fractions from (A) 3R4F smoke or (B) THS 2.2 aerosol and cell viability was measured using a

real-time cellular analysis system. Values represent average ± SEM of at least three independent

experiments. Values are normalized to vehicle control. R.U. relative units.

P1 (GVP)

10 100 10000.0

0.5

1.0

1.5

Concentration (puff/l)

Imp

ed

an

ce (

R.U

.)SbPBS (3R4F)

0.001 0.01 0.1 10.0

0.5

1.0

1.5

Concentration (puff/l)

Imp

ed

an

ce (

R.U

.)

3R4F

1 10 100 1000

Concentration (puff / l)

1.5

1.0

0.5

0.5

Cell

via

bilit

y (

R.U

.)

THS2.2

10 100 1000

Concentration (puff / l)

1.5

1.0

0.5

0.5

Cell

via

bilit

y (

R.U

.)

GVP (3R4F)

0.001 0.01 0.1 10.0

0.5

1.0

1.5

Concentration

Cell v

iab

ilit

y (

R.U

.)

3R4F TPM

1 10 100 10000.0

0.5

1.0

1.5

Puff/L

TPM

GVP

AE

P1 TPM

10 100 10000.0

0.5

1.0

1.5

Puff/L

P1 sbPBS

10 100 10000.0

0.5

1.0

1.5

puff/l

TPM

GVP

AE

A

B

A B

D

C

E

F G H

I J

Figure S3. HCS results in NHBE cells exposed for 4h or 24h to AE from 3R4F smoke and

THS 2.2 aerosol. Values were normalized to the vehicle control and represent average ± SEM of

at least three independent experiments (n ⩾ 3) with independent smoke/aerosol generations on

different days. The error bars represent SEM. ∗ Significant different from the vehicle control.

Figure S3

Figure S4. HCS results in NHBE cells exposed for 4h or 24h to TPM from 3R4F smoke and

THS 2.2 aerosol. Values were normalized to the vehicle control and represent average ± SEM of

at least three independent experiments (n ⩾ 3) with independent smoke/aerosol generations on

different days. The error bars represent SEM. ∗ Significant different from the vehicle control.

A B

D

C

E

F G H

I J

Figure S4

Figure S5. HCS results in NHBE cells exposed for 4h or 24h to GVP from 3R4F smoke and

THS 2.2 aerosol. Values were normalized to the vehicle control and represent average ± SEM of

at least three independent experiments (n ⩾ 3) with independent smoke/aerosol generations on

different days. The error bars represent the SEM. ∗ Significant different from the vehicle control.

A B

D

C

E

F G H

I J

Figure S5

Vehicle 6 13 25 50 100 200

Polyploid 0.10 ± 0.03 0.08 ± 0.02 0.10 ± 0.01 0.10 ± 0.02 0.07 ± 0.01 0.06 ± 0.01 0.01 ± 0.00

Apoptotic 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.01 0.01 ± 0.00 0.02 ± 0.00 0.05 ± 0.08 0.70 ± 0.06

S-phase 0.08 ± 0.01 0.09 ± 0.03 0.06 ± 0.03 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

G2-M 0.28 ± 0.02 0.27 ± 0.02 0.29 ± 0.02 0.39 ± 0.04 0.38 ± 0.04 0.33 ± 0.05 0.09 ± 0.03

G0-G1 0.54 ± 0.04 0.55 ± 0.04 0.54 ± 0.02 0.48 ± 0.04 0.53 ± 0.04 0.56 ± 0.05 0.20 ± 0.03

Vehicle 35 70 140 200 300 350

Polyploid 0.12 ± 0.00 0.09 ± 0.05 0.09 ± 0.06 0.12 ± 0.08 0.12 ± 0.07 0.13 ± 0.07 0.10 ± 0.05

Apoptotic 0.01 ±0.00 0.02 ±0.02 0.02 ±0.02 0.02 ±0.02 0.05 ±0.06 0.01 ±0.00 0.01 ±0.00

S-phase 0.08 ±0.03 0.11 ±0.07 0.10 ±0.07 0.07 ±0.06 0.04 ±0.04 0.01 ±0.01 0.00 ±0.00

G2-M 0.29 ±0.01 0.28 ±0.04 0.26 ±0.04 0.29 ±0.03 0.34 ±0.01 0.40 ±0.01 0.41 ±0.02

G0-G1 0.50 ±0.02 0.50 ±0.01 0.53 ±0.01 0.51 ±0.03 0.45 ±0.07 0.45 ±0.06 0.47 ±0.04

Vehicle 6 13 25 50 100 200

Polyploid 0.05 ± 0.03 0.07 ± 0.03 0.08 ± 0.03 0.07 ± 0.04 0.05 ± 0.02 0.03 ± 0.03 0.01 ± 0.00

Apoptotic 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.08 ± 0.10 0.32 ± 0.49 0.82 ± 0.03

S-phase 0.10 ± 0.02 0.07 ± 0.03 0.03 ± 0.03 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

G2-M 0.26 ± 0.03 0.26 ± 0.02 0.32 ± 0.08 0.37 ± 0.01 0.32 ± 0.02 0.22 ± 0.17 0.05 ± 0.01

G0-G1 0.58 ± 0.05 0.59 ± 0.04 0.56 ± 0.02 0.54 ± 0.03 0.55 ± 0.07 0.43 ± 0.29 0.12 ± 0.02

Vehicle 35 70 140 200 300 350

Polyploid 0.05 ± 0.02 0.07 ± 0.03 0.08 ± 0.04 0.09 ± 0.04 0.09 ± 0.04 0.08 ± 0.05 0.06 ± 0.03

Apoptotic 0.02 ±0.03 0.02 ±0.02 0.02 ±0.02 0.02 ±0.02 0.02 ±0.02 0.03 ±0.02 0.03 ±0.01

S-phase 0.12 ±0.05 0.10 ±0.05 0.09 ±0.05 0.05 ±0.04 0.02 ±0.02 0.00 ±0.00 0.00 ±0.00

G2-M 0.27 ±0.05 0.27 ±0.03 0.27 ±0.02 0.35 ±0.06 0.39 ±0.02 0.37 ±0.03 0.35 ±0.04

G0-G1 0.53 ±0.02 0.55 ±0.03 0.54 ±0.03 0.49 ±0.01 0.48 ±0.03 0.52 ±0.06 0.56 ±0.06

3R4F (TPM) THS 2.2 (TPM)

Vehicle 8 16 32 42 50 64

Polyploid 0.03 ± 0.01 0.04 ± 0.02 0.05 ± 0.02 0.07 ± 0.03 0.03 ± 0.01 0.01 ± 0.01 0.01 ± 0.00

Apoptotic 0.01 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.01 ± 0.01 0.34 ± 0.19 0.72 ± 0.05 0.75 ± 0.00

S-phase 0.04 ± 0.02 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

G2-M 0.24 ± 0.02 0.32 ± 0.06 0.37 ± 0.02 0.36 ± 0.02 0.23 ± 0.06 0.11 ± 0.03 0.09 ± 0.00

G0-G1 0.67 ± 0.03 0.64 ± 0.07 0.57 ± 0.04 0.55 ± 0.06 0.39 ± 0.13 0.16 ± 0.02 0.14 ± 0.00

Vehicle 75 150 225 300 375 450

Polyploid 0.04 ± 0.02 0.06 ± 0.04 0.08 ± 0.04 0.08 ± 0.04 0.07 ± 0.04 0.09 ± 0.05 0.07 ± 0.03

Apoptotic 0.01 ±0.01 0.00 ±0.00 0.00 ±0.00 0.01 ±0.01 0.01 ±0.01 0.01 ±0.01 0.17 ±0.27

S-phase 0.05 ±0.02 0.03 ±0.03 0.01 ±0.01 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00 0.00 ±0.00

G2-M 0.25 ±0.04 0.26 ±0.03 0.28 ±0.02 0.32 ±0.02 0.35 ±0.01 0.34 ±0.03 0.30 ±0.09

G0-G1 0.65 ±0.06 0.64 ±0.07 0.63 ±0.05 0.60 ±0.05 0.57 ±0.04 0.56 ±0.06 0.45 ±0.17

3R4F (GVP) THS 2.2 (GVP)

A3R4F (AE) THS 2.2 (AE)

B

C

Figure S6. Cell cycle analysis results in NHBE cells exposed for 24h to AE (A), TPM (B) and

GVP (C) from 3R4F smoke and THS 2.2 aerosol. Values are expressed as cell percentage and

represent the average of three independent experiments with independent smoke/aerosol

generations on different days. Specific values of percentage of cells in different phases (polyploid,

apoptotic, S-phase, G2-M, G0-G1) for each fraction concentration (indicated in bold in puffs/L),

are expressed as average ± standard deviation in the table below each graph. The percentage of

cells in S-phase is highlighted in orange.

Figure S6

GVP AE TPM GVP AE TPM

3R4F THS2.2

GVP AE TPM

THS2.2

100%

80%

60%

40%

20%

BIOLOGICAL IMPACT FACTOR (BIF)

Figure S7. Biological Impact Factor (BIF). Relative BIF values for 3R4F and THS2.2 fractions

at one common dose (25 puffs/L). The percentages give the relative biological impact that is

derived from the cumulated network perturbations caused by the treatment relative to the

reference (3R4F TPM), which is defined as the treatment comparison showing the highest level of

perturbation.

Figure S7

Oxidative Stress

Osmotic Stress

NFE2L2 Signaling

Xenobiotic Metabolism Response

Senescence

Response to DNA Damage

Tissue Damage

Necroptosis

Wnt

PGE2

Nuclear Receptors

Mapk

Jak Stat

Growth Factor

Autophagy

Cell Cycle

25 Puffs / L

Epithelial Immune Activation

Notch

mTOR

hOX

Hedgehog

Calcium

Cell interaction

Epigenetics

Clock

3R4F THS2.2

CEL

L FA

TEP

RO

LIFE

RA

TIO

NC

ELL

STR

ESS

INFL

AM

MA

TIO

N

3R4F THS 2.2

GV

P

AE

TPM

GV

P

AE

TPM

Endoplasmic reticulum stress

Hypoxic stress

Apoptosis

Figure S8. NPA heatmaps for NHBE cells exposed for 4h to one common dose of 3R4F smoke

and THS 2.2 aerosol fractions. Significantly perturbed networks are indicated as *. Darker colors

indicate higher NPA scores.

Figure S8