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1. WO2020107120 - HYDROGEL BIOMIMETIC FOR INVASIVE DISEASES

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[ EN ]

CLAIMS

1 . An extracellular biomimetic for culturing diseased cells, comprising: hydrogel matrix,

a first extracellular matrix protein-mimetic peptide crosslinked to the hydrogel matrix, said first extracellular matrix protein-mimetic peptide being responsive to a first substance released by diseased cells upon invasion into the extracellular biomimetic, and

at least one modulating agent enabling cell invasion independent from said first substance.

2. The extracellular biomimetic according to claim 1 , wherein the hydrogel matrix comprises hyaluronate or hyaluronic acid, modified with furanyl functional groups.

3. The extracellular biomimetic according to claim 2, wherein the furanyl functional groups are furan, or furan substituted with alkyl-, aryl-, or electron-donating functional groups.

4. The extracellular biomimetic according to any one of claims 1 to 3, wherein the modulating agent is at least one viscoelastic component forming reversible crosslinks within the hydrogel matrix.

5. The extracellular biomimetic according to claim 4, wherein the component comprises any one of comprises methyl cellulose, alginate crosslinked with calcium cations, amphiphilic block polymers, amphiphilic block polypeptides, coiled-coil peptides, reconstituted basement membrane protein extract, laminin, or collagen.

6. The extracellular biomimetic according to claim 4 wherein the viscoelastic polymer is methyl cellulose having any one of aldehyde, ketone, hydrazine and thiol functional groups.

7. The extracellular biomimetic according to any one of claims 4 to 6, wherein the first extracellular matrix protein-mimetic peptide is further immobilized to the viscoelastic polymer.

8. The extracellular biomimetic according to any one of claims 4 to 7, further comprising a second extracellular matrix protein-mimetic peptide immobilized to the hydrogel matrix and/or the viscoelastic polymer.

9. The extracellular biomimetic according to claim 8 wherein the second extracellular matrix protein-mimetic peptide is present in the amount of less than about 1000 mM.

10. The extracellular biomimetic according to claim 9 wherein the second extracellular matrix protein-mimetic peptide is present in the amount of about 25 pM to about 250 pM.

1 1. The extracellular biomimetic according to any one of claims 8 to 10 wherein the second extracellular matrix protein-mimetic peptide is any one or combination of vitronectin-mimetic peptide and fibronectin-mimetic peptide.

12. The extracellular biomimetic according to any one of claims 1 to 1 1 , wherein the first substance released by diseased cells is an enzyme.

13. The extracellular biomimetic according to claim 12, wherein the enzyme is matrix metalloproteinase (MMP).

14. The extracellular biomimetic according to claim 13, wherein the first extracellular matrix protein-mimetic peptide is maleimide-modified collagen l-derived peptide crosslinker degradable by the MMP.

15. A cell culture kit comprising the extracellular biomimetic according to any one of claims 1 to 14 and diseased cells.

16. The cell culture kit according to claim 15 wherein the diseased cells are from any invading cells, such as any one of the lung, brain, breast, prostate, skin, liver, colon, pancreas, thyroid, bone, muscle, human pluripotent stem cells and their subsequently differentiated cells.

17. The cell culture kit according to claim 15 wherein the diseased cells comprise cells isolated from lung cancer patients or derived from human pluripotent stem cells (hiPSCs) to model lymphangioleiomyomatosis (LAM).

18. The cell culture kit according to claim 15, wherein the diseased cells comprise hiPSC-derived smooth muscle cells (SMCs) that model lymphangioleiomyomatosis (LAM-SMCs).

19. The cell culture kit according to any one of claims 15 to 18, wherein the diseased cells comprise cells treated with one or any combination of inhibitors selected from the group consisting of those that inhibit:

ABL1

ADENOSINE DEAMINASE

AKT3

ALK

ANDROGEN

AROMATASE

AURORA KINASE

BCL-2

BRAF

BRD

BTK

CALCINEURIN

CCR5

CDK

CXCR

CYTOCHROME P450

DAGK

DNA METHYLTRANSFERASE DNA TOPOISOMERASE EGFR

EPH

ERK

Fibroblast Growth Factor Receptors

FARNESYLTRANSFERASE

FLT

FRAP

GSK3

HDAC

HEAT SHOCK PROTEIN

HEDGEHOG

IRE1

ITGB1

JAK2

KDR

KINESIN-LIKE SPINDLE PROTEIN

KIT

LCK

LIMK1

LYN

MAP2K

MDM2

P38B

P70S6K

PARP

PDGFR

PI3K

PKC

PLK1

PIM2

PROTEASOME

RAF1

Rho-associated protein kinase

RET

Src

SIRT2

SPHINGOSINE KINASE TAN KYRAS E TUBULIN WNT,

or one or any combination of agonists selected from the group consisting of:

GLUCOCORTICOID

PKM2

PROGESTERONE

RXR

S1 P RECEPTOR.

20. The cell culture kit according to any one of claims 15 to 19, having 6, 24, 48, 96, 384 or 1536 well plates.

21. A drug screening method comprising:

culturing diseased cells in the extracellular biomimetic according to any one of claims 1 to 14;

quantifying invasion and viability of the diseased cells;

administering candidate drug compounds to the biomimetic; and

identifying compounds that reduce both the invasion and viability of the diseased cells.

22. The method according to claim 21 , wherein the quantifying step comprises measuring the invasion of the diseased cells by staining cells with fluorescent dyes, automated confocal imaging, and automated analysis by an image analysis software program such as custom Image J macros.

23. The method according to claim 21 or 22, wherein the quantifying step comprises measuring the viability of the diseased cells by staining the dead cells with fluorescent dyes, automated microscopic imaging such as confocal imaging, and automated analysis by an image analysis software program such as custom Image J macros.

24. The method according to any one of claims 21 to 23, wherein the diseased cells are from any one of the lung, brain, skin, breast, prostate, liver, colon, pancreas, thyroid, bone, muscle, human pluripotent stem cells and their subsequently differentiated cells.

25. The method according to any one of claims 21 to 24, wherein the diseased cells comprise cells isolated from lung cancer patients or derived from human pluripotent stem cells (hiPSCs) to model

lymphangioleiomyomatosis (LAM).

26. The method according to any one of claims 21 to 24, wherein the diseased cells comprise hiPSC-derived smooth muscle cells (SMCs) that model lymphangioleiomyomatosis (LAM-SMCs).

27. The method according to any one of claims 21 to 26, wherein the diseased cells comprise cells treated with one or any combination of inhibitors selected from the group consisting of those that inhibit:

ABL1

ADENOSINE DEAMINASE

AKT3

ALK

ANDROGEN

AROMATASE

AURORA KINASE

BCL-2

BRAF

BRD

BTK

CALCINEURIN

CCR5

CDK

CXCR

CYTOCHROME P450

DAGK

DNA METHYLTRANSFERASE

DNA TOPOISOMERASE

EGFR

EPH

ERK

Fibroblast Growth Factor Receptors

FARNESYLTRANSFERASE

FLT

FRAP

GSK3

HDAC

HEAT SHOCK PROTEIN

HEDGEHOG

IRE1

ITGB1

JAK2

KDR

KINESIN-LIKE SPINDLE PROTEIN

KIT

LCK

LIMK1

LYN

MAP2K

MDM2

P38B

P70S6K

PARP

PDGFR

PI3K

PKC

PLK1

PIM2

PROTEASOME

RAF1

RET

Rho-associated protein kinase

Src

SIRT2

SPHINGOSINE KINASE TAN KYRAS E TUBULIN WNT,

or one or any combination of agonists selected from the group consisting of:

GLUCOCORTICOID

PKM2

PROGESTERONE

RXR

S1 P RECEPTOR.

28. The method according to any one of claims 21 to 27, carried out in a cell culture kit having 6, 24, 48, 96, 384 or 1536 well plates.

29. The method according to claim 28, wherein each plate of the cell culture kit contains both diseased cells and control cells.