Patient Derived Xenograft Models ---Clinical Applications - - PowerPoint PPT Presentation

Chong-xian Pan, MD, PhD, MS

Professor of Medicine and Urology Co-Leader of Cancer Therapeutic Program University of California Davis School of Medicine UC Davis Comprehensive Cancer Center Sacramento, CA, USA Staff Physician VA Northern California Health Care System Mather, CA

Patient Derived Xenograft Models

• --Clinical Applications

DISCLOSURE:

I have financial interest/arrangement or affiliation with Name of Organization Relationship

Accelerated Medical Diagnostics Inc Co-founder and shareholder LP Therapeutics Inc Co-founder and shareholder Pandomedx Inc Co-founder and shareholder

Four patents

• 1. Bladder cancer-specific ligand cQDGRMGFc for imaging detection, immunotherapy and targeted

therapy of bladder cancer (filed. Inventors: Chong-xian Pan, Hongyong Zhang, Kit Lam and Olulanu Aina). US Patent application No. 61/245,492.

• 2. Leukemia stem cell-targeting ligand and methods of use. Ligands containing the LR(S/T) amino acid

motif for targeted therapy and detection of acute myeloid leukemia. (filed. Inventors: Chong-xian Pan and Hongyong Zhang). Patent application No. 14/130,909.

• 3. Porphyrin-based cancer-targeting nanometer-scale micelles for photodynamic diagnosis and therapy

(Inventors: Yuanpei Li, Kit S. Lam, Chong-xian Pan, Tzu-yin Lin). US Provisional patent Application No. 61/736,067.

• 4. Treatment of Drug Resistant Metastatic Prostate Cancer Using Niclosamide. (Inventors: Allen Gao,

Chengfei Liu, Wei Luo and Chong-xian Pan). U.S. Patent Application No. 15/134,228

their corresponding

Introduction

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State-of-the-art technologies:

• omics

Computational biology to identify targets Individualized therapy

Precision Medicine

Tsimberidou et al: RR 12% with matched targeted therapy vs. 5% with unmatched therapy (Clin Cancer Res. 2014; 20:4827) Andre et al. RR 9% plus 21% stable disease with matched targeted therapy in breast cancer (Lancet Oncology, 2014; 15:267)

Tradition Cancer Medicine

Diagnosis and staging Empirical One formula fits all

Patient-derived models of cancer (PDMCs) for precision medicine

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Engraft tumor into NSG mice P0 growth Primary treatment (same as patient) Secondary treatment A P1 growth Secondary treatment B Secondary treatment C

Patient NSG mice

P1 growth P1 growth Primary treatment (same as patient) Primary treatment (same as patient) Secondary treatment D P1 growth Primary treatment (same as patient) Response?

   

Secondary Tx informed by

• utcomes in mice

Relapse Relapse

Deep sequencing Target selection

Remission

Target validation with IHC, IF, western, etc

Prior to relapse

Secondary treatment Primary treatment

Integrative Computational Biology

PDXs for precision medicine

Features and applications of PDXs

Special features:

• PDXs are directly derived from unselected

uncultured clinical specimens

• PDXs are patient-specific
• PDXs and patient cancers have the same genetic

background

• Many identical PDXs can be generated for

repeated studies

• Frequent biopsies can be done to study resistance

mechanisms

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Clinical Characteristics of the donor patients Stages Tumor ID Age (yrs) Stage Surgery Prior chemo Myoinvasive bladder cancer BL0269F 58 pT4 N0 Mx Cystectomy No BL0293F 77 pT2a N2 Mx Cystectomy No BL0307F 78 pT3b N2 Mx Cystectomy No BL0382F 82 pT2 Nx Mx TURBT No BL0428F 70 pT2 Nx Mx TURBT No BL0429F 60 pT4a N3 M1 Cystectomy No BL0479F 78 pT2b Nx Mx Cystectomy YES (carbo/gem/PTX) BL0440F 71 pT4a N2 Mx Cystectomy YES (gem/cis) BL0515F 78 pT3bN0Mx Cystectomy YES (Gem/Cis) BL0545F 70 pT2 N0 Mx Cystectomy No BL0601F 83 pT3 N0 Mx Cystectomy No BL0629F 74 pT3 N0 Mx Cystectomy No BL0645F 75 pT4a N2 Mx Cystectomy YES (MVAC)# BL0648 71 pT4a N2 Mx Cystectomy

• No. AdenoCa

Non-myo- invasive bladder cancer BL0262F 64 pTa High TURBT No BL0364F 76 pTa Low TURBT No BL0381F * 60 pTa High TURBT No BL0398F * 60 pT1 No Mx Cystectomy No BL0470F 55 pTa Nx Mx TURBT No BL0591F 65 pTis N0 Mx Cystectomy No BL0606F 77 pT1Nx Mx TURBT No BL0622F 63 pTis cystectomy BL0674F 54 pT1N0Mx cystectomy NO

36 bladder cancer PDXs

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PDXs for precision medicine

Characterization of PDXs

10 20 30 40 50 60 70 80 90 100 All Variants Variants w/o dbSNPs Nonsynonymous+InDels Nonsynonymous Indels

Percentage Conserva on Variant Type

BL0429 BL0440

Genetic Alterations

Fidelity of morphology Conservation of genetic aberrations (92-97%)

BL0293-Passage 6-S.C. BL0293-Patient BL0440-Patient BL0440-Passage 6-S.C. BL0440-Passage 4-orthotopic BL0293-Passage 4-orthotopic

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Applications of PDMCs

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Screening for effective targeted therapy

Robertson et al. Cell. 2017; 171:540

1 6 12 15 5 10

Vehicle Lapatinib (30 mg/kgB.W.) Sorafenib (20 mg/kgB.W.) +Lapatinib (30 mg/kgB.W.)

Drug treatment (Days)

Ponatinib (10 mg/kg B.W.)

Tumor Ratio

5 10 15 20 10 20 30

Vehicle

Drug treatment (Days)

BEZ235 (30 mg/kg)

PDX:BL0269

Tumor Ratio

Screening for effective chemotherapy

BL0269 Tumor Response

10 20 30 40 50 500 1000 1500 2000

Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE)

BL0293 Tumor Response

20 40 60 80 500 1000 1500 2000 Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE)

BL0382 Tumor Response

• 20

20 40 60 80 500 1000 1500

Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE)

BL0515 Tumor Response

• 10

10 20 30 40 50 500 1000 1500 2000

Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE) BL0440 Tumor Response

20 40 60 80 500 1000 1500 2000 2500 Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE)

Re-purposing of FDA approved drugs

10 20 30 40 50 10 20 30 40

Vehicle Palbociclib days tumor size ratio

Palbociclib: CDK4/6 inhibitor ERBB2 FGFR3

1 6 11 14 19 23 10 20 30

Vehicle BGJ398 (30 mg/kg, B.W.)

Drug treatment (Days)

Lapatinib (30 mg/kg, B.W.)

Tumor Ratio

Lapatinib: ERBB2/3 inhibitor

Elucidation of resistance mechanisms Overcoming resistance

1 4 8 12 15 17 5 10 15 20

Vehicle FGFR3 inhibitor

Drug treatment (Days)

PDX: BL0293

Tumor Ratio

Biopsy

Re-transplant and expansion

Biopsy

Applications of PDMCs

• Drug development

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Bladder cancer-specific PLZ4-nanoparticles (Kit Lam, MD, PhD)

Treatment OS (days) WBC (K/ml) PBS

11

3.96±1.40 PTX 10mg/kg

27

1.16±0.19 PLZ4-NP-PTX 10 mg/kg

24

2.03±0.81* PLZ4-NP-PTX 30 mg/kg

>70

1.08±0.28

5 10 15 20 25 30 35 40 45 50 0.0 2.5 5.0 7.5 10.0

PBS PTX 10 mg/Kg PLZ4-NM-PTX 10mg/Kg PLZ4-NM-PTX 30mg/Kg

Days After First Treatment Tumor ratio

In vivo anti-tumor activity In vivo delivery: left: Lung; Right: bladder In vitro delivery Nanoparticle

IND No: 117868

• 1. Photodynamic diagnosis
• 2. Photodynamic therapy
• 3. Photothermal therapy
• 4. Chelation of Gd(III) for MRI
• 5. Chelation of 64Cu for PET
• 6. Chelation of 67Cu for radiation therapy
• 7. Targeted delivery of chemo
• 8. Chelation of gallium for sonodynamic Tx
• 9. Near infrared imaging
• 10. combination of the above

Li et al. Nature Communication. 2014 Lin et al. Biomaterials. 2016

• Smart “9-in-1” PLZ4-nanoporphyrin

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Polyethylen e glycol

PLZ4-NP targets and deliver chelated metal to the cancer site

Applications of PDMCs

• Drug development

PLZ4-nanoporphyrin

PNP 5637-DiO labeled Phase NIRF DAPI (nucleus) Merge Hema3TM BL269 PDX Normal bladder Ur

• Uro

BL269 Ur

• 40x

5 10 15 20 25 30 35 2000 4000 6000

Normal Urothelial BL269

* * ** ** ** **

Human bladder cancer cell line 5637 Human patient-derived xenograft

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Control DOX PNP+L B mode contrast mode Bladder Gross

Photodynamic therapy Photodynamic diagnosis

30-40X difference 2-3X for 5-ALA

Tumor temperature 100 200 30 40 50 60 70 NPs Control

Light on Time (s) Temperature (

• C)

Photothermal therapy

Applications of PDMCs

• Drug development

Pre-IND: 132838

PLZ4-nanotheranostics

• PLZ4-nanoporphrin
• PLZ4-nanoporphyrin kills cancer cells and release tumor antigens;
• Photodynamic therapy (PDT) produces reactive oxygen species

(ROS) which can modify macromolecules, and make them more immunogenic.

• Heat from photothermal therapy (PTT) denatures macromolecules

and makes them more immunogenic.

• PDT is more effective than radiation in potentiate immunotherapy.
• PDT has been used in bladder cancer. But the photosensitizer has

low efficiency, low potent, nonspecific (Cancer : normal ratio: 2-3 times), and high toxicity.

PLZ4-nanoporphrin to potentiate immunotherapy

PLZ4-nanotheranostics

• PLZ4-nanoporphrin to potentiate immunotherapy

Creation of bilateral syngrafts: Cells: MB49 Mice: C57BL/6 Intravenous injection of PNP Photodynamic therapy of the left tumor Monitoring growth of the right tumor

PLZ4-nanotheranostics

• Photothermal and targeted chemotherapy

Ig G PNP + light RMP1-14

RMP 1- 14+PNP+light

10X 40X 4 8 12 4 8 12

PNP+L PBS PNP+L+PD1 PD1 IgG

days Tumor Growth Ratio

Growth of the distant tumor (not treated with light) Photodynamic therapy converts “cold” tumor to “hot” tumor

150 1000

Cancer-specific drug delivery

PLZ4-nanotheranostics

• PLZ4-nanoporphrin to potentiate immunotherapy

PLZ4-nanoporphrin to potentiate immunotherapy

• SV40T/Ras double transgenic mice

20-30 days Palpable mass MRI to confirm Treatment: PD1: 200 µg/mouse, i.p., weekly PNP: i.v., weekly, plus light (0.2 w, 3 min) Tumor measurement: MRI/T2 Treatment:

• 1. Control
• 2. Anti-PD1 Ab
• 3. PNP + light
• 4. Anti-PD1 Ab +

PNP + light

• 1. Clinical

followup

• 2. MRI
• 3. Molecular

correlative studies Mice were obtained from Xue-Ru Wu at New York University.

PLZ4-nanotheranostics

• PLZ4-nanoporphrin to potentiate immunotherapy

Groups Ear Tag# Date of Death Overall survival Current Status Control #539 #150 #1191 #2032 #1737 01/09/2018 01/10/2018 02/03/2018 02/24/2018 03/22/2018 31 days 28 days 36 days 23 days 37 days Dead Dead Dead Dead Dead Anti-PD1 antibody #536 #542 01/16/2018 01/16/2018 38 days 38 days Dead Dead PDT and PTT #560 #581 #582 #1791 #1783 01/07/2018 02/08/2018 03/16/2018 03/21/2018 04/12/2018 31 days 34 days 55 days 45 days 67 days Dead Dead Dead Dead Dead Anti-PD1 antibody + PDT + PTT #1994 #535 #538 Alive Alive 03/08/2018 52 days 150 days 90 days Study ongoing Study ongoing Dead Mice were obtained from Xue-Ru Wu at New York Univ.

PLZ4-nanotheranostics

• PLZ4-nanoporphrin to potentiate immunotherapy

MRI to evaluate tumor growth

Control Anti-PD1 PNP PDT Anti-PD1 + PNP PDT

Applications of PDMCs

• Biomarker development

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DNA adducts as a biomarker for chemoresistance to alkylating agents:

• Platinum agents (cisplatin, carboplatin and oxaliplatin) kill

cancer cells through induction of DNA damage (adducts)

• Cells with high Pt-DNA adducts will be killed by

chemotherapy, and are chemosensitive.

• We developed a microdosing approach to measuring DNA

adducts after a non-toxic microdose of 14C-drug

Microdosing

• Accelerator Mass Spectrometry (AMS)

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• Carbon-14 dating to determine the age of fossils
• Measure 14C at 10-21 mole in mg-size specimens
• 14C-labeled drug: one drug molecule per cell in 105 cells
• Because of the ultrasensitivity, cells and patients are treated

with one non-toxic microdose of 14C-labeled drug to allow the detection of DNA damage and chemoresistance

*

*

1) DNA 2) Cancer cells

Pt H3N NH3 Diadducts

HOOC HOOC

* +

[*14C]carboplatin

Accelerator Mass Spectrometry (AMS)

Study of chemoresistance

• Microdosing technology

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Low DNA adduct levels correlate with chemoresistance

• Bladder cancer patient-derived xenografts (PDX)

Carboplatin microdosing NSG model comparison

BL0269 BL0293 BL0440 BL0645

**** **** *

relative carboplatin adduct level

BL0269 Tumor Response

10 20 30 40 50 500 1000 1500 2000 Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE)

BL0440 Tumor Response

20 40 60 80 500 1000 1500 2000 2500 Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE)

BL0293 Tumor Response

20 40 60 80 500 1000 1500 2000 Group 1 - Vehicle Group 2 - Cisplatin Group 3 - Gemcitabine Group 4 - Cisplatin & Gem

Days (Day 0 = treatment initiation) Mean tumor volume (mm3+/-SE)

Sensitive

Study of chemoresistance

• Microdosing Clinical Trial

Bladder cancer and NSCLC

Phase 0 study: One microdose (1/100th) of 14C-carboplatin:

1. PK study (drug metabolism); 2. DNA adducts of PBMC. 3. Repair of DNA adducts in cultured PBMC. 4. DNA adducts in bladder cancer specimens from TURBT Off-study therapeutic chemo with platinum chemotherapy

• 1. Evaluate response, and correlate with DNA damage and repair,

PK, cell uptake and efflux,.

• 2. Molecular correlation (such as ERCC and XRCC)

A Phase 0 microdosing trial

Clinicaltrials.gov: NCT01261299; NCT02077998. PIs: Pan

Study of chemoresistance

• Microdosing Clinical Trial

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500 1000 1500 2000

• 20000

20000 40000 60000 80000 100000

#1 micro #1 Tx #2 Micro #3 Micro #4 Micro #5 Micro #6 Micro #7 Micro #8 Micro #8 Tx #9 Micro DNA Damage in PBMC over 24 hours-all patients 500 1000 1500 2000

• 1

1 2 3 4

#1 Micro #2 Micro #3 Micro #4 Micro #5 Micro #6 Micro #7 Micro #8 Micro

• 1. 24 hr is the best time for biopsy/sampling
• 2. Microdosing predicts PK of therapeutic dosing
• 3. High DNA adduct levels correlate to response

Why two pts with low DNA adducts responded? Is this because of the chemo partner drug?

Carboplatin plasma concentration

Therapeutic [g/mL] Microdose [g/mL] 10 20 30 40 0.0 0.1 0.2 0.3 0.4 patient 1 patient 8 patient 10 p < 0.0001 R2 0.8076

24h PBMC

Bladder Lung 0.0 0.5 1.0 1.5 Responder Non-Responder Carboplatin Monoadducts/108 nt

Study of chemoresistance

• Gemcitabine microdosing study

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• Nucleoside analog
• Incorporation into DNA and block

DNA replication

• Inhibits ribonucleotide reductase
• Combine with platinum for bladder

cancer

• The level of gemcitabine in DNA correlates with cellular sensitivity

to gemcitabine

• Using 3H- or 14C-labeled gemcitabine, the AMS-based microdosing

approach may be able to measure the incorporation of gemcitabine into DNA and identify chemoresistance.

Study of chemoresistance

• Microdosing -Gemcitabine

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BL0269 Survival

20 40 60 80 25 50 75 100 125 Control Carboplatin Gemcitabine Gem/Carbo Days (Day 0 = treatment initiation) % Survival BL0293 Survival

20 40 60 80 25 50 75 100 125 Control Carboplatin Gemcitabine Gem/Carbo Days (Day 1 = treatment initiation) % Survival

BL0440 Survival

20 40 60 80 25 50 75 100 125 Control Carboplatin Gemcitabine Gem/Carbo Days (Day 1 = treatment initiation) % Survival

BL0645 Survival proportions

20 40 60 80 25 50 75 100 125 Control Carboplatin Gemcitabine Gem/Carbo Days (Day 1 = treatment initiation) Tumor Specific Survival

BL0269 BL0293 BL0440 BL0645 5 10 15 20 25

24h Gemcitabine microdosing * ** * **

Gemcitabine Adducts/108 nt

BL0293 [14C]Gemcitabine Microdosing

4h 24h 5 10 15 BL0293 BL0293R p < 0.0001 p = 0.018 Gemcitabine Adducts/108 nt

BL0293 Gemcitabine Response

20 40 60 80 1 2 3 4 5 BL0293 BL0293R PFS [days]: BL0293: BL0293R: 18 3 Days (Day 1 = treatment initiation) Tumor volume ratio

sensitive sensitive Resistant Resistant

Low gemcitabine incorporation in DNA correlated with resistance

PDXs for Personalized therapy

Summary

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• 1. We have established over 30 Bladder cancer PDX

models;

• 2. PDXs retain the morphology and genetic aberrations of

parental patient cancers;

• 3. Deep sequencing identified multiple druggable targets;
• 4. PDX platform can potentially be used for: screening

for effective targeted therapy, chemotherapy, drug re- purposing, replacing the role of serial biopsies to study secondary drug resistance, facilitating drug development, and developing biomarkers;

Acknowledgements

Lab:

Chong-xian Pan. MD, PhD Paul Henderson, PhD (Co-PI) Ai-hong Ma, MD, PhD Hongyong Zhang, DVM, PhD Tzu-Yin (Cindy) Lin, DVM, PhD Maike Zimmermann, PhD Tifffany Scharadin, PhD Weiming Yu, MD Wei Shi MD Shuxong Zeng, MD

Former lab members:

Qilai Long, MD Shuai Jiang, MD, PhD Fuli Wang, MD Sisi Wang, MD, PhD Tao Li, PhD Yanchun Wang, PhD Miaoling He, BA

Financial Support: Dr. de Vere White’s philanthropic funding, R01, DoD, VA Merit grants and others.

Acknowledgements

PLZ4 project

Kit S. Lam, MD, PhD: Yuanpei Li, PhD and their labs

UC Davis-others

Ralph de Vere White, MD Christopher Evans, MD Primo Lara MD Marc Dall’Era MD Stanley Yap, MD Richard Valicenti MD Regina Gandour-Edward, MD Clifford Tepper, PhD

EyePOD PDX

Edward Pugh, PhD Kit S Lam, MD, PHD

PDX Project

Jackson Laboratory:

Edison Liu MD Susie Airhart Carol Bult, PhD James Keck, PhD

Microchamber Project

Alex Revzin Pantea Gheibi

Thank you very much!!! Questions?

PDXs for Personalized therapy

Microchamber to complement PDXs

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Drawbacks of PDX:

• Long time to develop P0 PDXs (4-6 mo);
• engraftment rate: 40%
• Expensive

Microchamber organoid cultures to complement PDXs

PDXs for Personalized therapy

Microchamber to complement PDXs

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Glass slide Cell Input/ Media reservoirs (500 µl) Cell culture micro- culture (1 µl)

Microchamber culture to complement PDXs

(Originally developed for culture of hepatocytes)

Microchambers (µC, Alex Revzin) :

• Biocompatible
• Optically transparent
• Excellent oxygen transport
• Accumulation of endogenous factors
• Enhancement of autocrine and

paracrine signals

• Long-term function maintenance of

difficult-to-culture cells (ex. Primary

hepatocytes and mESC (mouse Embryonic Stem Cells))

> 2 mm

Matrigel Suspension Microchamber

75 µm PDMS

PDXs for Personalized therapy

Microchamber to complement PDXs

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BL0908 organoids in microchambers Day 3 Day 9 Day 7 Day 5 Day 1

DAPI Actin Merged Merged Anti-pan cytokeratin DAPI

BL0908 organoids on day 9

Patient specimen PDX

BL0269 BL0440 D3 D5 D7 D9

Ki67 DAPI Merged DAPI Merged Merged DAPI pan Cytokeratin F-actin

PDXs for Personalized therapy

Microchamber to complement PDXs

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a.

1 2 3 4 5 6 7 8 9 D10 D12 D14 D16 D18 D20 D22 D24 D26 D28 D30 Relative growth ratio Culture Duration (days)

BL0269 growth in microchamber

Center Outer rim DAPI F-actin Ki67 Middle Section DAPI pan-Cytokeratin BL0269 (D30)

b.

Cell growth is mainly at the edge of cell mass.

PDXs for Personalized therapy

Microchamber to complement PDXs

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Initial D2 D4 GDC-0941 Gemcitabine Cisplatin

BL0269 (Standard) in Microchambers

b. a.

Efficacy studies

• BL0269 with a PI3K mutation

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Initial D2 D4 Exposure Duration (day) Relative growth ratio

BL0269 (Standard) Drug Response

Control Cisplatin Gemcitabine Cisplatin + Gemcitabine GDC 0941 Triple Combination

* * * * *

PDXs for Personalized therapy

Microchamber to complement PDXs

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0.2 0.4 0.6 0.8 1 1.2 1.4 D10 D12 D14 D16 D16 D18 D20 D22 D22 D24 D26 D28 D28 D30 GDC−0941 Recovery GDC−0941 Recovery GDC−0941 Recovery GDC−0941 1st Exposure 2nd Exposure 3rd Exposure 4th Exposure Relative growth ratio

* * * *

d. e.

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1 2 3 4 5 6 7 8 9 10 11 12 Relative growth ratio Culture Duration (day) S 1 D22 D24 D26 D28 D30 D10

f.

D12 D14 D16 D18 D20 D0 D10 Standard Media +GDC-0941 D12 D16 +GDC-0941 D18 D22 +GDC-0941 D24 Standard Media +GDC-0941 D28 D30 Standard Media Standard Media

Tumor heterogeneity

PLZ4-nanotheranostics

• Summary
• 1. PLZ4 specifically binds to human and dog bladder cancer cells
• 2. Nanomicelles coated with PLZ4 can specifically deliver the

drug load to bladder cancer in vitro and in vivo.

• 3. Micelle formulation of PTX significantly decreases the toxicity

and prolongs the overall survival in mice carrying PDXs.

• 4. PLZ4 nanoporphyrin can be potentially used for PDD, PDT,

PET, MRI, photothermal therapy, radiation, targeted chemotherapy and combination of the above.

• 5. Combination of PNP and immunotherapy