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INDOLEAMINE 2,3 INDOLEAMINE 2,3 DIOXYGENASE (IDO) DIOXYGENASE (IDO) EXPRESSION IN LUNG EXPRESSION IN LUNG CANCER CANCER KERENIDI N. KERENIDI N. Respiratory Department, Respiratory Department, University hospital of Larissa Larissa

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INDOLEAMINE 2,3 INDOLEAMINE 2,3 DIOXYGENASE (IDO) DIOXYGENASE (IDO) EXPRESSION IN LUNG EXPRESSION IN LUNG CANCER CANCER

KERENIDI N. KERENIDI N. Respiratory Department, Respiratory Department, University hospital of University hospital of Larissa Larissa

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  • Tumor

Tumor – –immune response immune response

  • Immune escape mechanisms

Immune escape mechanisms

Munn H and Mellor L 2004; Trends Mol. Med. 10:15-18

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Indoleamine Indoleamine 2,3 2,3 Dioxygenase Dioxygenase -

  • IDO

IDO

  • L

L-

  • tryptophan N

tryptophan N-

  • formylkynurenine

formylkynurenine

IDO

  • cytosolic monomeric hemoprotein

cytosolic monomeric hemoprotein

  • 403aa long (MW 45,324KD)

403aa long (MW 45,324KD)

Tone 1989 Tone 1989

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IDO IDO functions functions

  • antimicrobial defence mechanism

antimicrobial defence mechanism

  • maternal tolerance toward the allogeneic

maternal tolerance toward the allogeneic fetus fetus

  • suppresses T

suppresses T-

  • cell responses to MHC

cell responses to MHC-

  • mismatched allografts and to autoantigens in

mismatched allografts and to autoantigens in animal models of disease animal models of disease

  • role in immune escape of tumor cells

role in immune escape of tumor cells

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IDO expression IDO expression

  • Expressed constitutively at high levels in placenta,

Expressed constitutively at high levels in placenta, gut and epididymis, and at lower levels in spleen, gut and epididymis, and at lower levels in spleen, lymph nodes and thymus lymph nodes and thymus

  • in the lung IDO is expressed at basal levels

in the lung IDO is expressed at basal levels

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IDO and Lung cancer IDO and Lung cancer

most human tumors most human tumors constitutively constitutively express IDO express IDO

Uyttenhove C 2003; Nature Med.

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IDO is expressed by IDO is expressed by : :

  • Cancer cells themselves

Cancer cells themselves

  • Cells in the infiltrating zone

Cells in the infiltrating zone ( (macrophages macrophages, DCs) , DCs)

  • Cells in tumor

Cells in tumor-

  • draining lymph nodes

draining lymph nodes

IDO and cancer IDO and cancer

Subsets of cells express IDO in lung eosinophil granulocytes (NSCLC)

Astigiano S et al Astigiano S et al 2005 2005, , Neoplasia

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  • Low tryptophan concentration

Low tryptophan concentration

  • D

Downstream metabolites

  • wnstream metabolites

(L (L-

  • kynurenine,

kynurenine, 3 3-

  • hydroxyanthranilic acid)

hydroxyanthranilic acid)

How does IDO promote immune How does IDO promote immune escape escape

Hwu P et al. 2000; J Immun 164:3596-99, Woo E et al 2001;Cancer Research 61:4766–72, Mellor A et al. 2002; J. Immun. 168: 3771-76, Munn H and Mellor L 2004; Trends Mol. Med. 10:15-18

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AIM: AIM:

  • To investigate the expression of IDO

To investigate the expression of IDO

  • in lung cancer cell lines

in lung cancer cell lines

  • surgically

surgically resected resected lung cancer tissues lung cancer tissues

  • autologous

autologous non malignant samples non malignant samples

  • Correlations of IDO expression with

Correlations of IDO expression with clinicopathological clinicopathological parameters parameters

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Material Material

Patient no Gender Age Histology Differentiation pTNM status pStage Tumour volume (mm 3) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 F M M F F M M M M M M M M M M M M M M M M M M F M F M M 56 65 65 48 59 73 55 60 65 74 48 71 61 67 55 79 70 63 79 59 62 59 58 76 40 75 74 59 ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCLC BAC BAC BAC poor moderate poor moderate well moderate moderate poor poor poor poor poor poor moderate poor moderate moderate moderate poor moderate poor moderate poor moderate poor moderate moderate poor T1N1M0 T3N0M0 T1N2M0 T1N0M0 T2N2M0 T2N0M0 T4N2M0 T2N1M0 T3N0M0 T2N0M0 T3N1M0 T2N2M0 T2N0M1 T1N0M0 T2N1M0 T2N0M0 T2N0M0 T3N1M0 T2N0M0 T2N1M0 T2N0M0 T1N1M0 T2N0M0 T2N0M0 T3N1M0 T2N0M0 T2N1M0 T4N0M1 IIA IIB IIIA IA IIIA IB IIIB IIB IIB IB IIIA IIIA IV IA IIB IB IB IIIA IB IIB IB IIA IB IB IIIA IB IIB IV 15 56 15 27 120 52 57 35 55 26 1188 361 54 8 42 14 12 27 37 23 14 13 29 428 225 120 150 31

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Methods Methods

tumor tissue cancer cell lines:

CALU-1, CALU-6, GILI, ONET, SK-LU-1, NCI-H441, NCI-H460, NCI-H596, NCI-H661

RNA extraction cDNA synthesis qReal Time PCR for ABL and IDO normal tissue

2.5 - 11 cm

Reference tissue

(amartoma)

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Methods Methods

  • qReal Time

qReal Time – –PCR PCR

SYBR supermix kit (Invitrogen, SYBR supermix kit (Invitrogen, Paisley UK Paisley UK) )

  • Rotor Gene software

Rotor Gene software

FORWARD 5' GGTCATGGAGATGTCCGTAA 3' REVERSE 5' ACCAATAGAGAGACCAGGAAGAA 3' 50oC 5 s, 95oC 10 min and then 45 cycles of 95oC 15 s and 60oC 1min.

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IDO IDO – –LC Cell lines 3 / 9 LC Cell lines 3 / 9

4.7 4.7 ± ± 11.1 (0.0 11.1 (0.0-

  • 33.9)

33.9)

1.778 4.840

Results

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Patient no I DO Expression by Tum or a I DO Expression by Norm ala

Ratio T/N

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 1 6 .6 6 .5 2 .6 3 .8 3 .3 1 .0 8 .5 6 .8 3 7 .5 3 .9 4 .7 7 .1 6 .6 1 .8 1 .0 1 .0 N/ A 9 .0 2 .9 N/ A N/ A N/ A 1 .9 1 .6 1 .1 7 0 .0 2 .4 1 .5 1 0 .2 4 .4 7 .5 2 .3 1 .7 3 .0 2 .1 4 .1 2 .7 3 .8 1 .2 0 .9 4 .5 0 .3 N/ Ab 1 .4 8 .8 4 .9 2 .2 5 .2 N/ A N/ A 0 .6 2 .0 2 .2 2 .4 0 .8 2 .7 1 ,6 1 ,5 0 ,3 1 ,6 1 ,9 0 ,3 3 ,9 1 ,6 1 4 ,1 1 ,0 3 ,8 7 ,5 1 ,5 6 ,4

  • 0 ,7
  • 1 ,8

1 ,3

  • 3 ,2

0 ,8 0 ,5 2 9 ,1 3 ,1 0 ,5

a copies IDO/100 copies ABL . b Not available.

Results

21 / 24 21 / 24 Tumor Tumor 23 / 27 23 / 27 Normal Normal

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ΚΣ Φ Κ 5 10 15 20

p=0.021 p=0.236 p=0.014

m RNA IDO

The relative expression of IDO in lung cancer cell lines (4.7±11.1) was significantly lower than that of all patients’ tumor samples as well as that of the autologous non affected lung tissues

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10 20 30 40 50 60 70 80 K Φ 1 2 3 4 5 6 7 8 9 10 11 12 26 27 28

Ασθενείς

1 2 3 4 5 6 7 8 9 10 K Φ 13 14 16 18 19 23 24

Ασθενείς

Adeno SCC

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ΑΔ-Κ ΑΔ-Φ ΠΛ-Κ ΠΛ-Φ 5 10 15 20 p=0.046 p=0.945 p=0.075 p=0.998

mRNA IDO I DO

Tumor Normal Patients

8 .4 6 ± 1 5 .1 8 3 .2 8 ± 2 .5 1

Adeno

1 1 .7 5 ± 1 8 .5 2 3 .3 2 ± 2 .5 4

SCC

3 .2 2 ± 2 .9 6 3 .3 2 ± 2 .7 5

Only in ADC the relative expression

  • f IDO was higher in tumor samples

than in non malignant lung tissues. No statistically significant differences were noted between ADC and SCC regarding either the tumor samples

  • r the autologous non affected samples.
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IDO n mean±SD p-value

Age

<70 16 7.07 ± 9.06 0.538 ≥70 8 11.24 ± 23.83

SEX

♂ 19 5.67 ± 8.17 0.078 ♀ 5 19.06 ± 29.10

Smoke history

Non smokers 4 12.55 ± 16.73 0.590 Ex smokers 7 5.18 ± 5.64 Smokers 13 8.96 ± 18.53

Type

Adeno 15 11.75 ± 18.52 0.075 SCC 8 3.22 ± 2.96

Differentiation

G1-G2 11 9.90 ± 20.14 0.772 G3 13 7.24 ± 10.00

T

T1-T2 17 7.90 ± 16.45 0.309 T3-T4 7 9.82 ± 12.59

N

N0 13 10.77 ± 20.28 0.417 N1-N2 11 5.73 ± 4.59

M

M0 22 8.86 ± 15.80 M1 2 4.05 ± 3.60

Stage

I–II 15 10.58 ± 18.99 0.551 III-IV 9 4.93 ± 2.98

Tumor volume

<50m m 3 12 4.40 ± 4.53 0.340 ≥50m m 3 12 12.53 ± 20.62

No significant correlations between IDO expression and clinicopathological parameters were found

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Conclusion: Conclusion:

  • Direct evidence

Direct evidence is provided demonstrating that IDO is provided demonstrating that IDO mRNA can be constitutively e mRNA can be constitutively expressed by lung cancer xpressed by lung cancer cells. cells.

  • The higher

The higher IDO IDO expression observed in patients expression observed in patients’ ’ samples samples can be attributed to the production of the enzyme by can be attributed to the production of the enzyme by

  • ther cells recruited in the tumor microenvironment and
  • ther cells recruited in the tumor microenvironment and

the peri the peri-

  • tumoral lung area and/or to its induction by

tumoral lung area and/or to its induction by soluble factors of tumor origin. soluble factors of tumor origin.

  • Future : IDO inhibition

Future : IDO inhibition (immunotherapy and chemotherapy protocols) (immunotherapy and chemotherapy protocols)

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Collaborators:

Departm ent of I m m unology and Departm ent of I m m unology and Histocom batibility Histocom batibility, UHL , UHL Departm ent of Pathology, UHL Departm ent of Pathology, UHL Thoracic Surgery Department, Thoracic Surgery Department, UHL UHL Thoracic Surgery Department, Thoracic Surgery Department, Athens Athens Medical Medical Center Center Ludwig Institute for Cancer Research, Belgium Ludwig Institute for Cancer Research, Belgium Austin Research I nstitute, Australia Austin Research I nstitute, Australia

Funding:

Hellenic Ministry of Education, PYTHAGORAS, EPAEK II Project Gra Hellenic Ministry of Education, PYTHAGORAS, EPAEK II Project Grant nt EU EU Marie Curie Incoming International Fellowship 021975 Marie Curie Incoming International Fellowship 021975-

  • IRTALUNG

IRTALUNG Phizer Phizer Hellas, Grant in Aid Hellas, Grant in Aid Hellenic Hellenic General Secretariat of R&T General Secretariat of R&T – – ΕΝΤΕΡ ΕΝΤΕΡ2004 2004 Grant# Grant# 04 04ΕΡ ΕΡ 9 9 Hellenic Hellenic General Secretariat of R&T General Secretariat of R&T – – Matching funds Matching funds

Indoleamine 2,3-dioxygenase (IDO) expression in lung cancer. Cancer Biol Ther 2007, 6(8):1258-62.

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THANKS FOR YOUR ATTENTION