Tumor Evolution

8/1/2017

Immune Priming with Ultrasound

Chandan Guha, MBBS, PhD Conflicts and Grants - NIH (R01 EB009040) - NCI SBIR grant with Professor and Vice Chair, Radiation Oncology Celldex Therapeutics, Inc. Professor, Urology and Pathology - Project Energy with Director, Einstein Institute of Onco-Physics Johnson & Johnson Montefiore Medical Center Albert Einstein College of Medicine, Bronx, NY [email protected]

Tumor Evolution

Immune Immune Tumor Restoration Surveillance progression Resurgence

Living “with” Diagnosis Tumor • Cancer cure restored Living “with” Primary => Mets immune surveillance Mutated Cells • Ablation with Immune Restoration is curative “Killing” tumor Mutated Cells Tumor Ablation

Immune Escape (Epigenetic loss – Antigen presentation) Suppression (IL10, TGFß, PD-L1) Cooption (Macrophage, Ectopic Lymphoid structure)

Focal Oncology Clinical Adaptive Learning (FOCAL) Cancer Clinic Network

1. Ablative Therapies for local control induces anti-tumoral immunity, which in turn helps local control.

2. Ablative Therapies for systemic immunity: Immune Priming Ablation (IPA) for In Situ Tumor Vaccines a. UPR => ER stress => Antigen Processing / Presentation b. “Eat Me” and DAMP signals c. Reversal of tolerance d. Antigen Presentation (neo-antigens & cryptic antigens)

“Focal Therapy for Systemic Cure”

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Project ENERGY.01: Proposed Study Design

Immuno-Priming Abla on (IPA) Therapies Study 1: 3LL IP only; Biomarker endpoints

Immuno-Priming (IP) Best 2 of all LOFU1-2 HT RP IRE1-2 RFP1-2 Digoxin Metformin Metastatic Model Immuno-Priming - Ablation (IPA) 3LL Lewis Lung Study 2: 3LL SBRT (10-20 Gy) RFA IPA; Biomarker, Immune Profile, Tumor control GEM models and survival endpoints LOFU / I RE / Digoxin RapidCap + + Liver (Ch) LOFU Low Energy / Intensity SBRT (10-20 Gy) RFA Focused Ultrasound KPC Best 2 IPA’s HT Histotripsy

RP Radiation Priming Energy IPA1-2 Study 3: 3LL; 4T1;GEM + 2 Metastatic (3LL, 4T1) IRE Irreversible Electroportaion GM-CSF IPA + DC Act./Check-Point Inhib.; 1 GEM (CaP / KPC) RFP Radiofrequency Priming +/- Biomarker, Tumor control Anti-PD1 SBRT Stereotactic Body Radiation and survival endpoints Therapy RFA Radio Frequency Ablation

Go/No-Go for IRE/RFA Internal R&D (Ethicon) = Go/No-Go for LoFu Licensing External R&D (Electroblate, Varian) NewCo Formation Thorsten Melcher

Autologous in situ tumor vaccines

Treatment Protocol 1

.8 No Treatment 3 weeks • RT (60 Gy) .6 • Surgery

Flt3L (10 µg/day) .4 3 weeks X 10 days C on tro l • RT (60 Gy) • Surgery R T .2 R T + F lt 3L Flt3L (10 µg/day) F lt 3L 3 weeks X 10 days 0 20 4 0 6 0 80 10 0 1 20 140 1 60 1 80 20 0

Sur vi val T ime ( Day s)

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RT + Flt3L Improves Survival of Tumor-bearing C57Bl/6 Mice

1 .8 RT + Flt3L .6 C57Bl/6 mice .4 Control (RT+Flt3L - 55% cured) RT .2 RT + Flt3L Flt3L 0 20 40 60 80 100 120 140 160 180 200 Survival Time (Days)

Immunodeficient Nude mice

(RT+Flt3L - 0% cured) Survival Time (Days)

Systemic Effects of Primary Tumor Irradiation

Kaplan & Meier Survivorship Function 1 B - RT C - Surgery .8 E - S + Flt3L D1 - RT + Flt3L-10 .6 D2 - RT + Flt3L-30

.4 Surgery + Flt3L .2

0 0 40 80 120 160 Survival Time (Days)

Sample size: 29 patients

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Nitin Ohri

Sample size: 29 patients Ablative SBRT dose fractionation • 34 Gy x 1 Fx • 18 Gy x 3 Fx • 10 Gy x 5 Fx

Study Subjects

# Demographics Disease Burden Prior Treatment(s) SBRT Clinical Course Right lung squamous cell carcinoma with 50 Gy in 5 Progression at 6 73 year-old 1 multiple lung masses Carboplatin + gemcitabine fractions to weeks, death at 4 Korean male and bilateral RLL mass months mediastinal adenopathy Chemoradiotherapy for Right lung 34 Gy in 1 Partial response 55 year-old localized disease, 2 adenocarcinoma, with fraction to at 2 months, Hispanic female nivolumab for metastatic bilateral lung nodules LUL nodule stable at 4 months disease Chemoradiotherapy for Right lung squamous 50 Gy in 5 80 year-old localized disease, cell carcinoma with fractions to Partial response 3 Caucasian carboplatin + gemcitabine spine and pelvic right lung at 2 months female for metastatic disease, metastases mass nivolumab Right lung 73 year-old Carboplatin/ + 50 Gy in 5 adenocarcinoma with 4 Caucasian pemetrexed, maintenance fractions to PET/CT on 4/27 mediastinal adenopathy female pemetrexed RLL mass and liver metastasis

Patient 2

Right lung mass reduced from 5.0 cm (maximum SUV 10.7) to 2.1 cm (maximum SUV 6.5) on first post- treatment PET/CT Target Lesion Total Glycolytic Activity (TGA): 1.0 cc  0.3 cc Other lesions’ TGA: 44.7 cc  4.5 cc

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Patient 3

Improvement or resolution of most of the osseous foci

Target Lesion Total Glycolytic Activity (TGA): 49.1 cc  6.2 cc Other lesions’ TGA: 130.2 cc  45.4 cc

Patient 3

Target Lesion Total Glycolytic Activity (TGA): 49.1 cc  6.2 cc Other lesions’ TGA: 130.2 cc  45.4 cc

Energy activated in situ Tumor Vaccines POC Studies Energy Tumor Models End Points Limitation Immunotherapy Single Flt3L • Lewis Lung 1. Primary Tumor • Murine Ectopic Fraction + / - 3LL in C57/Bl6 Growth Transplantation SFRT • BN1LNE Liver 2. Metastases Models CD40L 25-60 Gy (HCC) in 3. Survival 4. Immune assays • RT Dose Balb/c • Fractionation 20 Gy TLR9 • Lewis Lung PTG, Mets, Survival • RT to Draining agonist 3LL in C57/Bl6 Lymph Node 10 Gy Listeria- • TRAMPC1 PTG and Immune PSA TPSA23 Assays • Break Tolerance ADXS31- Prostate to self antigens 142 Cancer • Small Animal 20Gy x 3 PD1-Fc • Lewis Lung PTG, Mets, Survival Treatment 3LL in C57/Bl6 LOFU HIFU • TPSA23 PTG, Immune • Lack of Immune Prostate Assays Surveillance and carcinogenic LOFU SBRT • B16 Melanoma PTG, Mets, Survival environment (10 Gy x 3) Immune Assays

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Acoustic priming

ULTRASOUND -- ADVANTAGES

1 W/cm2 2000 W/cm2 • US Can do both Imaging and Treatment

TM • Quick Tissue Destruction • Bloodless • Precise and Accurate • Non-sterile environment

Focal Zone HIFU Beam Peripheral Zone

Diagnostic (Imaging) Beam HIFU directed harmlessly across skin and rectum toward the tumor

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Therapeutic Ultrasound as an autologous in situ tumor vaccine

• HIFU = High Intensity Focused Ultrasound • MRgFUS = MR-guided Focused Ultrasound • TULSA = Transurethral ultrasound ablation

• LOFU = Low intensity (energy) focused ultrasound (LOFU coined by Guha group) • SST = Sonic Stress Therapy • APT – Acoustic Priming Therapy

LOFU parameters

Condition Condition #2 Condition #3 Condition #4 Condition #5 #1 Duty Cycle (%) 1 25 50 75 100

Power (W) 32 16 8 4 2 Time (ms) 1000 625 1250 2500 5000 Thermal 0.32 2.5 5 7.5 10 Energy (J) Peak Negative Pressure 8.14 6.08 4.58 3.34 2.46 (MPa)

Thermal Energy

Mechanical Energy

The “sonic stress” of LOFU

Gene function Genes affected by LOFU treatment 1. Protein Folding DNAJB1, HSPH1, HSPE1, HSPB1, HSPD1, HSPA4L, CRYAB, HSPA6, HSPA7, HSP90AA1, HSP90AA4P, DNAJA4, FKBP4, LGSN, PTGES3 2. Cell cycle regulation IER5, JUN,CACYBP, GPRC5A, RRAD, WEE2

3. Cytokines IL8 4. Receptors CSF2RB, IL7R, NPR1, RXFP2, FLT4, ITGA2

5. Cytoskeleton integrity FAM101B, TCP1 6. Transcriptions ATF3, ANKRD1, EYA4, KAT2A 7. Transporters SLC22A2, SLC22A16, RHAG 8. Apoptosis regulation NLRC4, ANGPTL4, BAG3 9. Peptidase NAALADL1, MEP1A, PLOD2

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Gene Ontology

Figure 2 Gene ontology GoTerm network. After LOFU treatment, the gene response showed extensive upregulation of genes that are related to unfolded protein.

Gene Expression with qRT-PCR 10.000 9.000 8.000 7.000 6.000 5.000 4.000 Control 3.000 Treated 2.000 1.000

0.000

IL8

JUN

ATF3

HSPE1

HSPB1

HSPA6 HSPA7

HSPD1

HSPH1

CSF2RB

HSPA4L

DNAJB1

DNAJA4

ANKRD1

HSP90AA1 HSP90AA4P Figure 4 qRT-PCR of select genes from the RNA sequencing. Genes were selected from pathways highlighted in the KEGG pathway analysis and validated using qRT-PCR. The data correlated well with the RNA sequencing results. HSPA6 and HSPA7 were highly regulated to 200+ and 25+ fold respectively.

Increase in Hsp70 mRNA expression after LOFU treatment

140.00

120.00 Hspa1a e

g 132.37

n 100.00 a Hspa1b

h 80.00 C

80.77 d

l 60.00

o F

40.00

A N

R 20.00 m 0.00 Hsp expression -20.00 80 Hspa1b 70

e Hspb1-1 s

a 60 Hspb1-2

e r

c 50 Hspd1

n i

Hsph1 d

l 40 o

f Hsp90aa1 30

A Dnajb5 N

R 20 m 10 0 0 5 10 15 20 25 30 Time post-LOFU (h)

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Immunomodulation of tumor cells

Surface Calreticulin (CRT) Intracellular HSP70

In vitro Effect of LOFU on Cell Surface Markers

Summary of in vitro data under JJI-ENERGY ➢ LoFu is non-ablative ➢ LoFu induces “sonic stress” on cancer cells ➢ Sonic stress signature is consistent across cell types and indicative of immune stimulation

3 Watt LoFu 5 Watt LoFu

3LL 4T1 TPSA23 3LL 4T1 TPSA23

6h 24h 6h 24h 6h 24h 6h 24h 6h 24h 6h 24h

HSP70

ER Stress CRT

BiP

MHC I Co-Stimulatory CD86

Death Fas Receptors CD40

Inhibitory PD-L1

0 5 10 20 30 No change TBD

SRS Ch-RT

LOFU

Induce Cell death

Calreticulin membrane Translocation

HSP activation “danger” signal HMGB1 release TL4 binding “eat me” signal

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Treat the tumor draining lymph node (TDLN – immune privilege site)

– Reprogram tolerogenic DCs (IDO inhibitors) – Inhibit regulatory T cells (Treg) – Reverse T-cell anergy in TDLN – LOFU primary tumor

T cell activation vs. T cell anergy

Full stimulation Re-stimulation CD3 CD28

T cell Activation

+ CD4 T cell Cytokine production Cytokine production Cell Proliferation Cell Proliferation

Re-stimulation Anergic stimulus

T cell Anergy

CD4+ T cell Hyporesponsive Hyporesponsive phenotype phenotype

T cell Activation vs. T cell Anergy

Full Activation Anergic Stimulus

CD28 TCR TCR

Ca Ca Ca MAP kinases Ca Ca

Ca p NFAT p p AP-1 p p NFAT p p p p p NF-kB p p p p p p Co-partners ? p50 p65 NFAT NFAT

Activation-induced genes Anergy-inducing genes

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B16 tumors induced CD4+ T cell anergy

B16 in B6 mice B16-OVA in OT-II mice B16 in Tyrp1 mice

20 30 18 *** ** *** 25 15 * 15 ** ** 20 12

10 15 9

2 (ng/mL)2

- - - 6

IL 10 IL 5 IL 5 3

0 0 0 NoNo TumurTumor T-DLN T-NDLN No Tumor T-DLN T-NDLN NoNo Tumour-Tumor T-DLN T-NDLN

10 8 * 12 * * 10 * 8 6 * 8 6

6 (ng/mL) 4

(ng/mL)

(ng/mL) g 4 g

4 IFN IFN

IFN 2 2 2

0 0 0 No Tumor T-DLN T-NDLN No Tumor T-DLN T-NDLN NoNo Tumour-Tumor T-DLN T-NDLN

LOFU prevents B16-induced CD4+ T cell anergy

30 *

2 (ng/mL)2

- IL 10

NDLN DLN

T cell anergy: transcriptional program

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LOFU prevents B16-induced CD4+ T cell anergy

12 6 4 Grail Itch Cbl-b 3 8 4 2 4 2 1

0 0 0 FoldInduction mRNA Untreated LOFU Untreated LOFU Untreated LOFU

6 5 Grg4 Egr2 4 4 3

2 2 1 0 FoldInduction mRNA 0 Untreated LOFU Untreated LOFU

LOFU can reverse established T cell anergy

250 5 ** 4 200 Control Th1

Anergic Th1 3 150

2 (ng/mL)2 2

- IL 2 (ng/mL)2 100

- 1 IL 50 0 0 DC + + + + Resting Stimulated Anergic T cells − + + + Untr. lysate − − + − LOFU lysate − − − + Tyrp1 CD4 Th1 cells Anergized in vitro

LOFU as immune adjuvant for IGRT

LOFU

IGRT B16-M1 Melanoma

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44440000000 L O FLUO F U

)

3 3

3 RT L O F U + R T

m m

m 3 0 0 m m 333000000

( RT

(

m m m 2 0 0 m 2 0 0

u 220000

V V V 1 0 0 111000000

0 000 000 111000 2 0 2200 3 0 3300 4 04400 555000 6600 0 1 0 2 0 3 0 4 0 5 0 6 0 TTTiiImmeee (((dddaaayyysss))) T im e (d a y s )

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LOFU as adjuvant for IGRT requires T cells

Nude mice 8 0 0 B16 melanoma

U n t 6 0 0

L O F U )

3 L O F U + R T

m m

( R T

e 4 0 0

m u

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V U nt

4 0 0 L O F U ) 2 0 0 3 L O F U + R T

m 3 0 0 RT

(

m 2 0 0 u

0 l

0 2 0 4 0 6 0 o V T im e (d a y s ) 1 0 0

0 0 1 0 2 0 3 0 4 0 5 0 6 0 T im e (d a y s )

LOFU as adjuvant to potentiate effect of RT and improve control of distal metastases

Untreat LOFU

IGRT LOFU+IGRT

CD62L-VE/CD4+VE population in Tumor infiltrating lymphocytes (TILs)

No Treatment

A marked shift from naïve to activated phenotype was observed in the combination treatment TILs.

[LOFU (3W) + RT (10 Gy)] X 3

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CD62L-VE/CD8+VE in Tumor infiltrating lymphocytes (TILs)

No Treatment

LOFU+RT treatment causes a significant increase in activated CD8 T-cells in TILs

[LOFU (3W) + RT (10 Gy)] X 3

of the UPR. Indeed, TUNEL staining demonstrated that LOFU+17AAG inhibits Chaperone Mediated LOFU induced minimal apoptosis over untreated controls. unchanged with LOFU or 17AAG or the combination Autophagy (CMA) in tumor cells. therapy (Figure 4A & C), indicating that macroautophagy Treatment with 17AAG induced asignificnt apoptosis in was not altered with ultrasound therapy. However, LOFU prostate tumors, which was further increased by LOFU alone or 17AAG alone induced the expression of LAMP- (p<0.004) (Figure 3E). Thus, 17AAG-mediated inhibition Degradation of misfolded proteins is mediated by 2A (Figure 4A & B), indicating a compensatory increase in the proteosomal pathway and autophagy. Autophagy has CMA after therapies that increase the burden of misfolded of HSP90 and activation of CHOP by the combination proteins in the ER. Interestingly, the combination of of LOFU+17AAG switched on apoptotic cell death of been implicated in the tumorigenesis process in a context- LOFU+17AAG inhibited the levels of LAMP-2A below prostate tumors. dependent role, where it might provide amino acids and the basal levels seen in these tumors. This suggests that the www.impactjournals.com/oncoscience/ Oncoscience 2014, Vol.1, No.6other essential nutrients to the metabolic pathways of hypoxic tumors that are nutrient deprived [22]. Indeed, an Low intensity focused ultrasound (LOFU) modulates unfolded increase in CMA activity has been described in a wide protein response and sensitizes prostate cancer to 17AAG variety of human tumors and CMA has been implicated

Subhrajit Saha1, Payel Bhanja1, Ari Partanen2, Wei Zhang1, Laibin Liu1, Wolfgang in survival, proliferation, and metastases of tumor cells A. Tomé1,4 and Chandan Guha1,3,4 [23]. Therefore, we quantitated the levels of two key 1 Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York, USA 2 Philips Healthcare, Cleveland, OH, USA proteins participating in autophagy, Beclin, a marker of 3 Department of Pathology, Albert Einstein College of Medicine, Bronx, New rYork, USA macroautophagy, and LAMP-2A lysosomal receptor, a 4 Montefioe Me di cal Cent er , New York, NY, USA Correspondence to: Chandan Guha, email: [email protected] marker of CMA in the tumor tissues of various treatment Keywords: Prostate Cancer, 17AAG, Focused Ultrasound, UPR, ER stress cohorts. As shown in Figure 5, Beclin levels remain Received: April 30, 2014 Accepted: June 02, 2014 Published: June 03, 2014

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT unchanged with LOFU or 17AAG or the combination The hypoxic tumor microenvironment generates oxidative Endoplasmic Reticulum (ER) stress, resulting in protein misfolding and unfolded protein response (UPR). therapy (Figure 4A & C), indicating that macroautophagy UPR induces several molecular chaperones including heat-shock protein 90 (HSP90), which corrects protein misfolding and improves survival of cancer cells and resistance was not altered with ultrasound therapy. However, LOFU to tumoricidal therapy although prolonged activation of UPR induces cell death. The HSP90 inhibitor, 17AAG, has shown promise against various solid tumors, including alone or 17AAG alone induced the expression of LAMP- prostate cancer (PC). However, therapeutic doses of 17AAG elicit systemic toxicity. In this manuscript, we describe a new paradigm where the combination therapy of a non-ablative and non-invasive low energy focused ultrasound (LOFU) and a non-toxic, 2A (Figure 4A & B), indicating a compensatory increase in low dose 17AAG causes synthetic lethality and significant tumoricidal effects in mouse and human PC xenografts. LOFU induces ER stress and UPR in tumor cells without CMA after therapies that increase the burden of misfolded inducing cell death. Treatment with a non-toxic dose of 17AAG further increased ER stress in LOFU treated PC and switched UPR from a cytoprotective to an apoptotic proteins in the ER. Interestingly, the combination of response in tumors resultinga in significnt induction of apoptosis and tumor growth retardation. These observations suggest that LOFU-induced ER stress makes the LOFU+17AAG inhibited the levels of LAMP-2A below ultrasound-treated tumors more susceptible to chemotherapeutic agents, such as 17AAG. Thus, a novel therapy of LOFU-induced chemosensitization may be designed the basal levels seen in these tumors. This suggests that the for locally advanced and recurrent tumors.

INTRODUCTION propagating ultrasound waves with target tissue[3] . Thermal effects are due to ultrasound absorption and Therapeutic ultrasound is being developed as an conversion to heat through vibrational excitation of image-guided ablative treatment for solid tumors. High- tissue, leading to localized temperature elevation. intensity focused ultrasound e(HIFU) delivers sonic energy Mechanical bioeffects that are unique to HIFU include Figure 5: Tumor growth retardation of murinteo a small, well-defind target region in malignant tissue, acoustic radiation forces and acoustic cavitation[4, 5]. causing a rapid rise in tissue temperature exceeding HIFU may pose a risk to normal tissues, e.g., bones, and human prostate tumors after LOFU+17AAG80 -90oC, thereby inducing instantaneous coagulative blood vessels, and nerves adjacent to target malignant treatment. A. Treatment schema. Palpable tumors were treatende crosis at the focal point. HIFU is quickly emerging as tissue, due to rapid temperature elevation leading to with LOFU every 3-4 days for efiv fractions administered ovearn effective image-guided, minimally invasive treatment nearly instantaneous thermal coagulation at high acoustic two weeks. Animals received 17AAG three times a week durinmgo dality for solid tumors, including prostate cancer (PC) intensities[5]. Furthermore, bubble activity mediated [1, 2]. The biFoloigiucarle e ffe6c:ts oLf OtheFraUpe+ut1ic7 uAltArasGou ndt reatmaecnouts ticr ecdavuitcaetiosn mthaye be induced at high acoustic this time. Tumors were harvested 24 hours after the last fractiornes ults from both thermal and non-thermal/mechanical pressure levels, leading to locally induced stress and high of LOFU. B. RM1 tumor. In C57Bl6 mice, LOFU+17AAGbio effects, wheicxhp rareises iforonm ocfo mpprleoxs tinatetrea ctcioanns coefr steme necreglyl remleaaser, kpoesrssib lyi nre sulting in and assisting thermal combination treatment reduced Ra M1 tumor growth significnt ly RM1 cells. Flow cytometry of isolated aRM1 tumor cells (p<0.004), compared to controls. Note that either LOFU or showed significnt decrease in SCA1 (A & B), CD44 (A & www.impactjournals.com/oncoscience 434 Oncoscience 17AAG alone failed to control atumors significnt ly . LOFU FiguCr),e C D313:3 (AL O& FD)U, a+nd1 7α2Aβ1A inGte grina c(At i&va Et)e cse ll spurfoac-ea poptotic sensitized the effects of a low dose (25mg/kg of body weight) expression on RM1 tumor cells after LOFU+17AAG treatment. 17AAG. C. PC3 tumor. In BalbC nu/nu mice LOFU+17AAG path(Fw) aqRyTs-P CoRf arUrayP fRoll owaend dby hineadt muacpe asn alayspiso sphotwoesdi sth ati n tumor combination treatmenta showed significnt reduction in PC3 cellsL. OAFU +&17 ABA. GW ceomstbeinrnat iobnl otrte aotmf enptP gEroRupK do(Awn)- reagnudla tepse IF2a (B). tumor growth (p<0.007). the mRNA levels of stem cell transcription factors. LOFU+17AAG activates PERK by phosphorylation of PERK (pPERK), which further induces the phosphorylation of eIF2α www.impactjournals.com/oncoscience 439 Oncoscience phosphorylation (peIF2α). C. Real Time-PCR analysis of CHOP LOFU+17AAG reducesmRNA. theTher eexpression was a 25±1.3- foofld prostateincrease in C HOP transcript in LOFU+17AAG treated group, compared to control. D. cancerRea steml Time- PcellCR amarkerrray of RN A isolated from LOFU+17AAG- treated tumors. Heat map analysis showed that LOFU+17AAG Figure 4: LOFU+17AAG treatment inhibits Chaperone treatment group increased the transcript level of apoptotic genes Mediated Autophagy (CMA) in RM1 tumor cells. (A & several folds compared to untreated control or LOFU groups. B) Immunoblot analysis showed several fold down-regulation

100 100 E. TUNEL staining. Immu100nohistochemical staining showed of SMA marker LAMP2a expression level in combination predominantly tunel positive cells in LOFU+17AAG treatment treatment group. Treatment with either LOFU or 17AAG up- 80 80 80 group, compared to control or LOFU group. Note that 17AAG regulates the LAMP2a expression level. (A & C) Combination 60 alo60ne also induced apoptosis i60n tumor tissue that was augmented treatment of LOFU and 17AAG did not alter the expression 40 40 by LOFU. 40 level of Beclin, a macroautophagy marker.

20 20 20

0 0 0 1 2 3 w4 ww.impactjournals.com/oncoscience 10 10 10 10 10 100 101 102 103 104 0 438 Oncoscience 100 101 102 103 104 SCA1 CD44 CD133 LOFU+17AAG Control

Did the number of cells go down or expression?

Figure 5: Tumor growth retardation of murine and human prostate tumors after LOFU+17AAG treatment. A. Treatment schema. Palpable tumors were treated with LOFU every 3-4 days for efiv fractions administered over two weeks. Animals received 17AAG three times a week during this time. Tumors were harvested 24 hours after the last fraction Figure 6: LOFU+17AAG treatment reduces the 14 of LOFU. B. RM1 tumor. In C57Bl6 mice, LOFU+17AAG expression of prostate cancer stem cell markers in combination treatment reduced Ra M1 tumor growth significnt ly RM1 cells. Flow cytometry of isolated aRM1 tumor cells (p<0.004), compared to controls. Note that either LOFU or showed significnt decrease in SCA1 (A & B), CD44 (A & 17AAG alone failed to control atumors significnt ly . LOFU C), CD133 (A & D), and α2β1 integrin (A & E) cell surface sensitized the effects of a low dose (25mg/kg of body weight) expression on RM1 tumor cells after LOFU+17AAG treatment. 17AAG. C. PC3 tumor. In BalbC nu/nu mice LOFU+17AAG (F) qRT-PCR array followed by heat map analysis showed that combination treatmenta showed significnt reduction in PC3 LOFU+17AAG combination treatment group down-regulates tumor growth (p<0.007). the mRNA levels of stem cell transcription factors. www.impactjournals.com/oncoscience 439 Oncoscience 8/1/2017

Acoustic Priming

Immunotherapy Chemotherapy

Chemosensitization

↑Immunosensitization

Sensitization of ablative therapies Radiosensitization

Tumor HIFU RFA Radiotherapy Cryotherapy

Autologous in situ tumor vaccines

The FOCAL Team

Guha Laboratory Clinical Trials Medical Physics Students.: Rafi Kabariti Assoc.: Patrik Brodin Lorenzo Agoni Nitin Ohri Director: Wolfgang Tome Karin Skalina Madhur Garg Talicia Savage Justin Tang Research Associates: Huagang Zhang Associates: Immune Indranil Basu Immune Tolerance Therapeutics Lisa Scandiuzzi Sanmay Bandyopadhyay Steve Almo Fernando Macian

Thank you!

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